i m u h i m i2807400862

MULTIPLE ANTIBIOTIC RESISTANCE

IN METHICILLIN-AMINOGLYCOSIDE RESISTANT

STAPHYLOCOCCUS AUREUS

Thesis siubmitted by

PETER ALAN CHRISTOPHER MAPLE

for the Degree of Doctor of Philosophy

in the Faculty of Medicine of the

University of London.

MEDICAL LIBRARY.

ROYAL FREE HOSPITAL

HAMPSTEAD

Department of Medical Microbiology,

Royal Free Hospital School of M e d i c i n e ,

London N W 3 .

ProQuest Number: U552871

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ABSTRACT

Antibiotic susceptibility profiles of 100 strains

of methicillin and gentamicin resistant

Staphylococcus aureus (MGRSA) from 32 centres in 23

countries were determined. This is the first

survey to document the international problem of

multiple antibiotic resistance in MGRSA. Many

differing susceptibility profiles were found, some

strains being sensitive to a range of currently

available antistaphylococcal agents; others were

resistant to many of these agents.

More than 50% of MGRSA studied were non-typable

with the "International Set" of phages. Those

strains which typed, mostly reacted with phage 85

alone, or with other Group III phages as well.

Typing with supplementary phages revealed many of

the non-typable strains to possess Group Ill-

related patterns.

The variety of phage-typing and antibiotic

resistance patterns seen suggests that the

worldwide occurrence of MGRSA is probably not due

to widespread dissemination of a single clone. The

aminoglycoside modifying enzyme APH (2")/AAC ( 6 f) was

found in 44% of strains, while 56% produced APH

(2")/AAC ( 6 T) and APH ( 3 ’)-IV. Gene probing

experiments showed the same gene for APH (2")/AAC

(6') in MGRSA worldwide.

2

Options for treating MGRSA infections are

limited, currently few agents can reliably be used

in place of vancomycin. Fosfomycin and

pristinamycin appear to be promising. For treating

MGRSA carriage, azelaic acid and nitrofurazone may

be useful alternatives to mupirocin. Few, if any,

agents currently under development appear to be

promising alternatives to vancomycin.

Widespread resistance was found to the

fluoroquinolones (eg. ciprofloxacin) in MGRSA.

Experimental studies showed fluoroquinolone

resistance to readily occur. Analysis of resistant

clinical isolates showed the high incidence of

ciprofloxacin resistance in the MGRSA studied

resulted from independent evolution and not cross-

infection. Strains were least able to develop

resistance to ofloxacin, and newer fluoroquinolones

(eg. sparfloxacin) have improved activity against

MGRSA.

Multiple antibiotic resistance in MGRSA is a

major problem which could become significantly

worse should vancomycin resistance develop.

3

Publications arising from this thesis:

1. MAPLE, P.A.C., BRUMFITT, W., HAMILTON-MILLER, J.M.T.(1989). Comparative in vitro activity of vancomycin, teicoplanin, ramoplanin (formerly A16686), paldimycin, DuP 721 and DuP 105 against methicillin and gentamicin resistant Staphylococcus aureus collected worldwide. Journal of Antimicrobial Chemotherapy 23: 517-525.

2. MAPLE, P.A.C., HAMILTON-MILLER, J . M . T . , BRUMFITT, W. (1989). World-wide antibiotic resistance in methicillin- resistant Staphylococcus aureus. Lancet i: 537-540.

3. MAPLE, C.A.P., HAMILTON-MILLER, J.M.T., BRUMFITT, W. (1989). Ciprofloxacin resistance in methicillin- resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 8:622-624.

4. MAPLE, P.A.C., HAMILTON-MILLER, J.M.T., BRUMFITT, W. (1991). Differing activity of quinolones against ciprofloxacin- sensitive and ciprofloxacin- resistant,methicillin- resistant Staphylococcus_______a u r e u s .Antimicrobial Agents and Chemotherapy 35: 345-350.

ABSTRACTS

1. BRUMFITT, W., MAPLE, P.A.C., HAMILTON-MILLER, J.M.T.(1989). Antibiotic Sensitivity Patterns ofMethicillin-Resistant Staphylococcus aureus and their use for Biotyping. Abstract no. 778/PP40, 4th European Congress of Clinical Microbiology, Nice.

2. MAPLE, P.A.C., HAMILTON-MILLER, J.M.T., BRUMFITT, W.(1990). Alternative topical agents to mupirocin for the eradication of staphylococcal carriage. Oralpresentation at the 160th Meeting of thePathological Society of Great Britain and Ireland.

4

CONTENTSfage

TITLE 1

ABSTRACT 2

PUBLICATIONS 4

CONTENTS 5

ACKNOWLEDGEMENTS 17

INTRODUCTION

1.0 Taxonomy and nomenclature of Staphylococcus aureus

1.1 Historical Introduction 18

2.0 The occurrence and pathogenicity of S. aureus

2.1 Distribution of S. aureus in nature 21

2.2 The human Staphylococcus aureus

i. Occurrence 22

ii. Factors influencing the incidence of 23

nasal carriage

2.3 Pathogenicity of Staphylococcus aureus 24

3.0 Staphylococcal infections and their treatment

3.1 The spectrum of staphylococcal infections 26

3.2 Treatment of staphylococcal infections in 27

the pre-antibiotic era

3.3 The antibiotic era 1940-1960

i. The sulphonamides 30

ii. The introduction of penicillin 31

iii. The use of streptomycin, tetracycline, 33

erythromycin and chloramphenicol against S .aureus

iv. The introduction of methicillin 34

v. The efficacy of methicillin and its 35

derivatives

4.0 Staphylococcus aureus resistant to methicillin

4.1 Occurrence of MRSA - 1960-1975 37

4.2 Treatment of MRSA - 1960-1975 39

i. Therapeutic options and the need 39

for combination therapy

ii. Treatment with topical antibiotics 40

iii. Treatment of MRSA infections with 42

penicillinase-resistant penicillins and/or

cephalosporins

4.3 The detection and nature of methicillin 44

resistance

i. Detection 44

ii. Nature of methicillin resistance 46

5.0 Use of gentamicin and the development of resistance

i. Use of gentamicin 49

ii. Development of resistance to gentamicin 51

6.0 Increasing hospital problems due to MGRSA

i. Problems due to MGRSA in Great Britain 53

ii. Problems due to MGRSA in Australia 56

iii. Problems due to MGRSA in the USA 57

iv. Problems due to MGRSA in Europe 60

v. Problems due to MGRSA in other countries 62

OBJECTIVES 64

MATERIALS AND METHODS

1.0 Strains 65

1.1 Strain origins and phage types 65

2 .0 Antibiotics

2.1 Antibiotics used in susceptibility and 72

studies of antibiotic properties

3.0 Media and Reagents

3.1 Media used in susceptibility and other 79

studies of antibiotic properties

3.2 Media used in biotyping studies

i . Egg-yolk agar 79

i i . Ly sed blood DST agar 79

i i i . Milk agar 79

i v . Sheep blood agar 80

v . Tween 80 agar 80

vi . Staph-■typing agar 80

3.3 Antibiotic containing discs for determining

sensitivity profiles and identifying

aminoglycoside-modifying enzymes

i. Antibiotic discs used for antibiotic 81

sensitivity profiles

ii. Antibiotic discs used for identifying 81

aminoglcoside-modifying enzymes

3.4 Materials and reagents used for detection 82

of specific haemolysins

4.0 Buffers used in plasmid isolation studies

i. Buffers used in Takahashi and Nagano method 83

ii. Buffers used in PHLS (Johnson) method 83

iii. Loading buffer 84

5.0 Identification of S. aureus 85

6.0 Detection of methicillin resistance 86

7.0 Determination of susceptibility to antimicrobial

agents

i. Determination of MIC by agar dilution 86

ii. Determination of MIC by broth dilution 87

7

iii. Selection of antibiotic break-points 88

7.1 Determination of bactericidal activity

i. Determination of minimum bactericidal 90

concentration (MBC)

ii. Determination of rate of killing by 90

time-kill curves

7.2 Determination of mutation rates to resistance 91

7.3 Microbiological assay of fluoroquinolones 92

8.0 Development of a system for typing MGRSA

8.1 Phage-typing MGRSA 93

8.2 Identification of aminoglycoside-modifying enzymes

i. Van de Klundert’s method 94

ii. Gene probing aminoglycoside resistance genes 95

8.3 Determination of antibiotic sensitivity profiles 96

8.4 Identification of physical properties of

use in biotyping

i. Identification of haemolysins by titration 96

ii. Haemolysis on sheep blood agar 98

iii. Egg-yolk reaction 99

iv. Tween 80 hydrolysis 99

v. Pigmentation on milk agar 99

8.5 Plasmid isolation by gel electrophoresis

i. Takahashi and Nagano method 101

ii. PHLS (Johnson) method 103

iii. Agarose gel electrophoresis 104

iv. Plasmid-sizing studies 104

APPENDICES

A,B,C Further information on methods used 308

8

RESULTS AND DISCUSSION

1.0 Antibiotic resistance in MGRSA - how 105

serious is the problem?

1.1 Resistance to aminoglycosides in MGRSA 118

1.2 Resistance to tetracyclines in MGRSA 122

1.3 Resistance to macrolides, lincosamides 124

and streptogramins

1.4 Resistance to trimethoprim in MGRSA 127

1.5 Resistance to chloramphenicol in MGRSA 128

1.6 Resistance to rifampicin in MGRSA 129

1.7 Resistance to ciprofloxacin in MGRSA 130

1.8 Resistance to fosfomycin in MGRSA 131

1.9 Resistance to fusidic acid in MGRSA 133

1.10 Resistance to novobiocin in MGRSA 135

1.11 Resistance to bacitracin in MGRSA 136

1 .12 Multiple antibiotic resistance in MGRSA 138

1 .13 Do MGRSA pose a therapeutic problem? 141

2.0 Therapeutic options for the treatment of

MGRSA infections or colonisation

2.1 Antibiotics available for treatment of 143

systemic infections

i. Antistaphylococcal activity of fosfomycin 144

ii. Antistaphylococcal activity of fusidic acid 149

iii. Antistaphylococcal activity of nitrofurantoin 151

iv. Antistaphylococcal activity of novobiocin 152

v. Antistaphylococcal activity of pristinamycin 153

v i . Antistaphylococcal activity of rifampicin 155

vii. Antistaphylococcal activity of teicoplanin 156

and vancomycin9

2.2 New antibiotics active against MGRSA 168

i. Antistaphylococcal activity of daptomycin 168

ii. Antistaphylococcal activity of DuP721 & DuP105 171

iii. Antistaphylococcal activity of paldimycin 172

iv. Antistaphylococcal activity of ramoplanin 173

v. Antistaphylococcal activity of new 14-, 174

15-, and 16- membered macrolides

vi. Antistaphylococcal activity of RP 59500- 175

an injectable streptogramin

vii. The lack of new agents available for 177

use in the treatment of infections due to MGRSA

2.3 Topical antibiotics for treatment of 179

MRSA carriage

i. Antistaphylococcal activity of azelaic 181

acid, nitrofurazone and silver sulphadiazine

compared to mupirocin

3.0 Do fluoroquinolones have a useful therapeutic

role against MGRSA?

3.1 Activity of the present clinically used

fluoroquinolones against MGRSA

i. Inhibitory and bactericidal activity of 190

ciprofloxacin, enoxacin, norfloxacin, ofloxacin

and pefloxacin against MGRSA

ii. Time-kill curves for ciprofloxacin, 193

ofloxacin and pefloxacin against MGRSA

iii. Spontaneous plate mutation rates to 200

resistance for ciprofloxacin, enoxacin,

norfloxacin, ofloxacin and pefloxacin compared

10

to nalidixic acid.

3.2 Fluoroquinolone resistance in MGRSA- development

of resistance in v i t r o , and properties of resistant

strains derived in vitro and from clinical sources

i. Development of fluoroquinolone resistance 202

in vitro during time-kill experiments

ii. Quinolone susceptibility patterns of clinical 210

isolates of ciprofloxacin-resistant MGRSA

iii. Are the reports of high incidences of 216

fluoroquinolone resistance in hospitals due

to the propensity of MGRSA to develop

fluoroquinolone resistance, or are they due to

the epidemic spread of resistant strains?

3.3 New fluoroquinolones with improved 225

activity against MGRSA

GENERAL DISCUSSION AND CONCLUSIONS

4.1 "Barber’s Law - the spread of resistant 229

staphylococci can be controlled either by not giving

antibiotics or by preventing the transmission of

the resistant organisms between persons"

4.2 Antibiotic options for the treatment of 239

MRSA infections and/or colonisation

i. Options available for treatment of 239

systemic infections

ii. Options for treatment of MGRSA carriage 243

4.3 Role of fluoroquinolones against MGRSA 244

11

4.4 The future problem of antibioticresistance in MGRSA

249

REFERENCES 252

APPENDICES 308

A. Influence of antibiotic carry-over in 308

fluoroquinolone MBC and time-kill experiments

B . Development of a biotyping system for MGRSA

i. Use of API STAPH profiles 309

ii. Use of antibiotic susceptibility profiles 310

iii. Determination of haemolysin profiles by 310

microtitre method

iv. Sheep blood haemolysis 312

v. Egg-yolk reaction 312

vi. Tween 80 reaction 313

vii. Pigmentation on Milk agar 313

viii. Plasmid-typing 314

C. Detection and Isoelectric focusing of 317

type SI enzyme

D. Suppliers of strains of MGRSA 319

SUMMARY OF CONCLUSIONS AND SUGGESTIONS FOR 320

FUTURE WORK

INSERTS Papers published

12

List of Tables

Number

I

II

III

IV

V

VIA

VIB

VII

Heading Page

Origins and phage-types of strains 66in study to determine extent ofantibiotic resistance in MGRSA

Antibiotics used in susceptibility and 73 other studies of antibiotic properties

Minimum inhibitory concentrations (MICs) 89 used to classify strains as sensitive, moderately resistant or resistant

Extent of antibiotic resistance 106to currently available antibiotics

Degree of resistance (%) to 23 non 117beta-lactam antibiotics

Inhibitory activity of fosfomycin, 145fusidic acid, nitrofurantoin, novobiocin, pristinamycin, rifampicin and teicoplanin compared to vancomycin against 100 strains of MGRSA from 26 centres in 19 countries

Influence of inoculum size on the 146 activity of fosfomycin, fusidic acid, novobiocin, pristinamycin, rifampicin, teicoplanin and vancomycin against 40 strains of MGRSA

Mutation rates to resistance to 147fosfomycin, fusidic acid, nitrofurantoin, novobiocin, pristinamycin, teicoplanin or vancomycin for 3 sensitive strains of MGRSA

13

VIII MICs and MBCs of teicoplanin and 159vancomycin at different times against 20 strains of MGRSA

X

XI

XII

XIII

XIV

Inhibitory and bactericidal activity 170determined against 80 strains of MGRSA of five new antibiotics possessingnovel chemical structures

Activity of 14-, 15-, and 16- membered 176macrolides, pristinamycin and RP 59500 against an international collectionof MGRSA comprising 13 erythromycin- sensitive strains, 30 inducibly resistant strains and 35 constitutivelyresistant strains

Inhibitory and bactericidal activity 182of azelaic acid, nitrofurazone , and silver sulphadiazine compared to that of mupirocin against 80 strains of MGRSA

Inhibitory and bactericidal activity of 191 clinically used fluoroquinolones compared to nalidixic acid against 160 strains of MGRSA

Pharmacokinetic properties of the 192fluoroquinolones

Fluoroquinolone susceptibilities ofparent and overgrowing strains following exposure to concentrationso f :

A: ciprofloxacinB: enoxacinC: ofloxacin

203204

D: pefloxacin205206

14

XVI

XVII

XVIII

XIX

XX

XXI

List

Number

1

Patterns of quinolone resistance in 212an international collection ofciprofloxacin-resistant MGRSA of clinical origin

Susceptibilities to acrosoxacin, 215fluoroquinolones and nalidixic acid of mutants of strain RFH 10 isolated from time-kill experiments

Phage-types of ciprofloxacin-resistant 219 and sensitive MGRSA from Texas andciprofloxacin-resistant MGRSA from 2 centres in Israel

Antibiotic susceptibility types and 221and biotypes of ciprofloxacin-resistant and sensitive MGRSA from Texas andciprofloxacin-resistant MGRSA from 2 centres in Israel

Susceptibilities of 160 ciprofloxacin- 227sensitive MGRSA and 40 ciprofloxacin-resistant MGRSA to current and futurequinolone agents

Supplementary phage-types of MGRSA 231used in antibiotic resistance studies Plasmid contents and sizes of a 315selection of MGRSA of Worldwide Origins

of figures

Heading Page

Problems due to MRSA in North-East 55Thames Regional Health Authority (RHA) compared to those in Yorkshire RHA from 1986-1990

15

2 Degree of multiresistance in MGRSA strains 139

A. killing curves for fosfomycin at 25 mg/1 161B. killing curves for fusidic acid 162

at 10 mg/1C. killing curves for nitrofurantoin 163

at 32 mg/1D. killing curves for novobiocin at 1.0 mg/1 164E. killing curves for pristinamycin 165

at 4.0 mg/1F. killing curves for rifampicin at 5.0 mg/1 166G. killing curves for strain SA 1 exposed 167

to various concentrations ofteicoplanin and vancomycin

A. killing curves for azelaic acid at 2.5 g/1 186B. killing curves for nitrofurazone at 60 mg/1 186C. killing curves for silver sulphadiazine 187

at 250 mg/1D. killing curves for mupirocin at 4.0 mg/1 187E. killing curves for ramoplanin at 188

1.0 mg/1 and 2.0 mg/1

A. killing curves of ciprofloxacin at 194various concentrations

B. killing curves of enoxacin at 195various concentrations

C. killing curves of ofloxacin at 196various concentrations

D. killing curves of pefloxacin at 197various concentrations

Plate

Plate 1 Growth of a number of different 100strains of MGRSA on Milk agar,Tween 80 agar, Egg-yolk glucose agar and Sheep blood agar

16

ACKNOWLEDGEMENTS

I am very grateful to my supervisors, Professor

W. Brumfitt and Professor J.M.T. Hamilton-Miller for

their support and advice. We are most grateful

to all those microbiologists worldwide who

supplied us with strains of MGRSA.

Three periods of working were spent away from

the Royal Free Hospital. Firstly, I am most

grateful to Dr S.G.B Amyes for allowing me to

spend 3 months at his laboratory in the Old

Medical School, University of Edinburgh. During

this stay I learnt various molecular biological

techniques and isolation and characterisation of

dihydrofolate reductases by isoelectric focussing.

Short periods of time were spent working in the

laboratories of Dr Alan Johnson (Antibiotic

Reference Laboratory) and Dr R. R. Marples

(Staphylococcal Reference Laboratory) at the Central

Public Health Laboratories, Colindale, London. I am

indebted to Dr Johnson for showing me his method

of staphylococcal plasmid isolation, and to Dr

Marples for his considerable help with phage-

typing .

Finally, I would like to thank my family for

their encouragement, perseverence and support during

my studies.

17

1. Taxonomy and Nomenclature of Staphylococcusaureus.

1.1 Historical Introduction,

Cocci were first classified on the basis of

their microscopically observed cellular arrangements

by Billroth in 1874 as part of his treatise on

Coccobacteria septica. Billroth believed that all the

round and rod-shaped bacterial forms were stages in

the development of a plant ("Coccobacteria septica” )

and he used the term "coccos" (seed) to describe the

smallest observed forms of this "plant” . He

differentiated these forms in terms of size or

arrangement into "micrococcos", "monococcos" ,

"diplococcos", "streptococcos", "gliacoccos" etc.

(Bulloch, 1960) .

Sir Alexander Ogston (Ogston, 1882) used the

descriptive term "staphylococcus" in his paper

entitled "Micrococcus Poisoning" to describe cluster

forming cocci observed in certain human pyogenic

abscesses. Ogston proposed this name, which is

derived from the Greek noun s taphy le ("a bunch of

grapes"); to differentiate these organisms from the

chain-forming cocci (streptococci) described by

Billroth. Rosenbach is credited as the first worker

to characterise Staphylococcus. In his original paper

published in 1884 , he initially proposed the names

Staphylococcus pyogenes aureus and Staphylococcus

18

pyogenes albus for orange and white staphylococci

which were indistinguishable from each other except

in colour. Hoewever, as trinomial names are invalid

in bacterial nomenclature, and as Rosenbach used the

binomial Staphylococcus aureus in a later part of

the same paper (Baird-Parker, 1972), Staphylococcus

aureus Rosenbach is accepted as the validly

published name for the nomenclatural type species of

the genus Staphylococcus (Editorial Board, 1958).

In 1906, Winslow and Rogers (Winslow & Rogers, 1906)

classified the staphylococci in the Subfamily

Paracoccaceae (parasites thriving well under anaerobic

conditions). The family was composed of four genera:

Diplococcus, Streptococcus, Aurococcus (staphylococci

producing orange pigment including S . aureus

Rosenbach) and Albococcus (staphylococci producing

porcelain white growth). In the other Subfamily,

Metacoccaceae (facultative parasites or saprophytes

thriving best under aerobic conditions) were the

genera Micrococcus, Sarcina and Rhodococcus. However,

in response to criticism from a number of

colleagues this scheme was modified in 1920 (Winslow

et a l ., 1920) by dispensing with the separate genera

Aurococcus and Albococcus in favour of the genus

Staphylococcus .

During the second quarter of the 20th Century

it was realised that micrococci and staphylococci

were closely related, so much so, that in the 1948

edition of Bergey * s Manual o_f Determinative

19

Bacteriology (Hucker, 1948) the staphylococci were

reclassified under the genus Micrococcus. Conversely,

although Shaw, Stitt and Cowan (1951) believed that

staphylococci and micrococci should belong to the

same genus they preferred the use of the generic

title of Staphylococcus to that of Micrococcus (Shaw

et a l ., 1951). By 1957, there had been yet another

change of view, in that staphylococci were found to

be substantially different from micrococci because of

the ability of the former to anaerobically ferment

glucose. Evans reintroduced the genus Staphylococcus

in the 7th edition of Bergey * s_______ Manual____of

Determinative Bacteriology (Breed et a l . , 1957), and

this distinction has remained until the present day.

Currently (Schleifer, 1986), the family

Micrococcaceae is composed of the genera Micrococcus,

Stomato coccus, Planococcus, and Staphylococcus.

20

2. The Occurrence and Pathogenicity ofStaphylococcus aureus

2.1 Distribution of S. aureus in na t u r e .

Following the reported isolation by Foggie in

1947 of staphylococci from the mouth, nose, vagina

and skin of ewes, Rountree et a l . (1956) were

prompted to screen various domestic and laboratory

animals for S . a ureus. These workers found nasal

carriage in a selection of dogs, guinea pigs, horses

and monkeys, but not in cows, rabbits and sheep, and

they concluded that the staphylococci appeared human

in character because of their similar phage types

to human isolates. Marandon & Oeding (1966, 1967)

established that there were biochemical and antigenic

differences between human and animal strains. Hajek

and Marsalek (1971) proposed that animal strains be

designated into six biotypes for inclusion into the

Baird-Parker classification of staphylococci (Baird-

Parker, 1963). These biotypes included strains from

sources as follows: A human, B swine and poultry, C

cattle and sheep, D hares, E dogs, and F pigeons.

Biotypes E and F were subsequently transferred to

S. intermedius (Hajek, 1976), however, apart from this,

the scheme remains largely unchanged (Parker, 1983).

21

2.2 The Human Staphylococcus aureus

i . Occurrence:

In man, S. aureus has a predeliction for the

anterior nares, although it can be isolated from a

number of other body sites (Williams, 1946).

Frequently, these organisms are commensals in the

healthy host - and their isolation under these

circumstances is referred to (by microbiologists) as

"staphylococcal carriage11. The carriage of S. aureus

in healthy people was first shown by Hallman

(1937). Subsequently, the incidence of nasal carriage

in healthy populations has been studied by a number

of workers (Williams, 1963). Usually, between 35 and

50 per cent of normal adults can be expected to

be nasal carriers of S . aur e u s . Rates of nasal

carriage have been found to be different in various

age groups. Casewell and Hill (1986) have estimated

the following prevalences with age: newborn (less than

5%), babies 2 weeks old (60-70%), 6-24 months (15-

25%), 5-6 year-olds (35-50%), normal adults (30-50%), and

the elderly (20-25%).

Aly et a l . (1977) found in vitro evidence that

S. aureus has a greater affinity for nasal

epithelial cells derived from carriers than from

non-carriers. A genetic predisposition to carriage

has been suggested by the findings of Noble et a l .

(1967) who provided evidence of a familial link.

22

i i . Factors influencing the incidence of nasal

ca rriage.

A number of studies (Williams, 1963, Noble et a l . ,

1964) have shown that exposure to antibiotics and

the hospital environment significantly increases the

rate of nasal carriage of S. a ureus. Casewell & Hill

(1986) estimate prevalences of nasal carriage of 20-

70% for nurses, 40-70% for patients two weeks in

hospital and 80-100% for babies two weeks in

hospital. Noble et a l . (1964) found that patients who

were not nasal carriers of staphylococci on

admission to hospital acquired nasal staphylococci

more often than those admitted carrying

staphylococci, regardless of antibiotic therapy. In

both groups higher incidences of acquisition were

found following antibiotic treatment. These findings

suggested that disruption of normal host flora

predisposes to staphylococcal colonisation. Carriers

of S . aureus have, following admission to hospital,

commonly become colonised with strains different to

those carried prior to admission and it has been

suggested that some strains of hospital staphylococci

have enhanced colonising ability (Williams, 1963).

Tuazon et a l . ( 1975) have reported an increased

rate of carriage compared to control populations in

regularly injecting drug abusers, and also in insulin

injecting diabetics. Patients undergoing long-term

haemodialysis have also been reported to have

23

increased rates of carriage (Kirmani et a l . , 1978).

2.3 Pathogenicity of Staphylococcus aureus.

The production of a staphylococcal infection is

determined by a complex interaction of host and

bacterial factors (Verhoef & Verbrugh, 1981). In

general, overt infection does not occur in the

healthy host with most strains. Predisposing factors

are required, for example skin trauma which can be

due to skin disorders/disease (eg. eczema), or can be

as a result of surgery, accident, or self induced

injuries such as intravenous drug abuse. Other

predisposing factors to infection are neutropenia, the

presence or insertion of foreign bodies, the use of

steroids, or virus induced damage to respiratory

mucosa. Certain genetic (e.g. chronic granulomatous

disease) or pathologically induced (e.g. J o b ’s syndrome)

defects in the h o s t ’s intracellular killing of

bacteria also predispose to infection (Quie et a l . ,

1974) .

Jeljaszewicz (1983) and many microbiologists are of

the opinion that S. aureus are ubiquitous micro­

organisms of rather low virulence and pathogenicity

which possess the capability to cause severe even

lethal disease in the compromised host. This has

not always been so, during the 1950s a particularly

virulent form of S. aureus known as type 80/81

24

spread epidemically amongst hospitals (Rountree &

Freeman, 1955, Hennessey & Miles, 1958; Williams, 1959).

Strain 80/81 caused numerous infections in patients

with none of the above predisposing factors.

25

3. Staphylococcal Infections and their Treatment.

3.1 The Spectrum of Staphylococcal Infections.

S . aureus most often produces acute inflammatory

lesions, often mild and localized at the point of

entry of the bacteria. Such lesions may progress

(especially in compromised patients) to produce

generalized infection. This picture can be

modified by the production at the site of

localized lesions of certain toxins such as

epidermolytic toxin or the toxic shock syndrome

toxins which produce either extensive lesions or

acute toxaemia even in healthy patients. The

introduction of S. aureus into the blood stream

may also produce generalized infection such as

osteomyelitis, perinephric abscesses, septicaemia and

endocarditis. Such introduction can be as a result

of wound sepsis, pneumonia or osteomyelitis, or it

can be due to direct inoculation as in the case

of intravenous drug administration.

Most commonly, S . aureus is associated with the

production of pustular lesions, the most

characteristic of which is the skin boil. These

lesions are usually self limiting, and if this is

not so, the most effective treatment is drainage

of the pus. Following surgery, wound infection with

S . aureus can result in prolonged healing times, or

at the worst, extensive wound breakdown.

26

Staphylococcal pneumonia is a major problem inpatients requiring mechanical ventilation, and it is

a common cause of pneumonia in lungs debilitated

by bacterial or viral infection e.g. post­

influenzal pneumonia.

S. aureus is a major cause of hospital

(nosocomial) sepsis. In a survey of the prevalence

of nosocomial infection in 47 hospitals in 14

countries by the World Health Organisation (Mayon-

White et a l ., 1988) S. aureus was found to be the

most common cause of surgical wound infection (14%)

and skin infection (18%). The opportunities

presented by the hospital environment for the

dissemination of S. aureus (e.g. via the hands of

staff) are numerous and epidemics of infection due

to particular S. aureus strains have occurred. For

example, in a study of the epidemiology and

control of staphylococcal infection in a maternity

hospital, Gillespie et a l . (1958) found there to be

several sources and modes of spread of

staphylococci which made the task of preventing

cross-infection all the more difficult.

3.2 Treatment of Staphylococcal Infections in the

Pre-Antibiotic Era.

Medical bacteriology was first practised in the

last quarter of the 19th Century following the

27

observations of Pasteur and Koch pertaining to the

germ theory of disease. Before this time the

concept of "contagion" had existed, and physicians

had adopted measures such as venesection and

treatment with various herbs and potions (see

Culpeper’s Herbal) to treat the diseased. The

following abridged case report (Paul, 1833) published

in "The Lancet” of 1833 illustrates what nowadays

might be considered as a typical case of post

operative staphylococcal (or streptococcal) sepsis.

"Case of Congenital Fungus Haematodes in whichAmputation of the Thigh was performed in the tenth week of the C h i l d ’s life".

THE LANCET 1833

A male child, seven weeks old, was brought tome by his parents with an enormous swelling ofthe right leg, which had all the characteristic marks of fungus haematodes.... The whole leg being involved in the morbid action of the disease, nothing but amputation above the knee even required the consideration of a moment....On the operation being fin ished...the child sucked almost immediately... and improved daily. The ligatures were all away by the tenth day after the operation,and the greater portion of the stump had united. A few days later the stump became very tense and hot. The next day a blush of erysipelatous redappeared over it and the edges of the woundappeared livid, there was scanty purulent discharge. Following this the erysipelatous inflammation extended to the scrotum and abdomen, and then upthe back and across the abdomen. The childdeveloped a high fever and died.

The surgeon who performed the operation was of

the opinion that the operation was a complete

success at the tenth day and begged the mother

to remain only another week in the hospital.

Following the c h i l d ’s illness, the surgeon learnt

that the c h i l d ’s mother had often taken him to

sit with, and be cradled by a nurse suffering

from aggravated erysipelas. In the surgeons own

words ”To my mind the evidence is here quite

conclusive, that the disease was communicated to

the child by the sick nurse”

Despite the great discoveries made during the

Golden Age of bacteriology from 1875-1925 and the

advances made in medicine with regard to surgical

technique and the need for antisepsis at

operations, by 1935 there were still no treatments

of proven efficacy for acute, invasive

staphylococcal (or streptococcal) infection (Topley and

Wilson, 1936). With this background, it is easy to

appreciate the significance of the introduction of

the first antimicrobial agents - the sulphonamides.

29

3.3 The Antibiotic Era 1940 - 1960

i . The Sulphonamides.

In 1935, a startling chemotherapeutic discovery

was announced by Domagk in Germany. He reported

that injection of the hydrochloride of the dye -

"4- sulphamido-2: 4-diaminoazobenzol" (Prontosil

rubrum) protected mice against streptococcal

infection. In 1936, Colebrook and Kenny (Colebrook &

Kenny, 1936) reported that injection of this dye

had a remarkable curative effect both in mice

infected with haemolytic streptococcus and in human

puerperal infections due to haemolytic

streptococcus. The active principle of Prontosil

rubrum was soon discovered to be p-amino-benzene

sulphonamide - "sulphanilamide", and from this

initial structure many hundreds of analogues were

prepared. Following the introduction into clinical

use in 1937 of sulphanilamide and its subsequent

analogues, the number of deaths in the U.K. due

to puerperal pyrexia in 1935 were halved by 1940

(Barber, 1960) .

Unfortunately, the sulphonamides were to prove

less successful against staphylococcal infections.

Not only was clinical experience in treating

severe staphylococcal sepsis disappointing, bacterial

resistance also readily occurred (Spink, 1954).

30

ii. The Introduction of Penicillin.

In 1929, Fleming reported the production of a

bacteriolytic substance (which he called penicillin)

from a mould contaminating a culture plate of

staphylococcus (Fleming, 1929). Further development of

penicillin was hindered due to problems of

producing sufficient quantities of pure substance.

In 1941, Abraham et a l . reported the results of

the first therapeutic trial with penicillin. They

found penicillin to be of low toxicity, and also

effective in treating staphylococcal and

streptococcal infections (Abraham et al. , 1941). It

was still difficult to produce penicillin in large

quantities, and because of the War, information

concerning the development of penicillin was

restricted (Clarke et a l . , 1949).

From 1942 onwards, increasing supplies of

penicillin became available, and usage of this drug

has had a most profound effect on mortality due

to staphylococcal infections. For instance, a

reduction of mortality from 80% in the pre­

penicillin era to 28% was seen for patients with

staphylococcaemia following the introduction of

penicillin into clinical use at the University of

Minnesota (Spink & Hall, 1945). Penicillin has had a

major and lasting effect on the treatment of

previously fatal diseases such as haemolytic

streptococcal infection, pneumococcal pneumonia,

31

bacterial endocarditis, gas gangrene and a variety

of other infections. However, against staphylococci

the efficacy of penicillin was short-lived, because

of the development of bacterial resistance.

Therapeutic failure due to development of

staphylococcal penicillin resistance during

penicillin treatment was first reported in 1942

(Rammelkamp and Maxon, 1942). According to the

authors the penicillin resistance was not due to

production of a "penicillinase". It had previously

been shown (Abraham et a l . , 1941) that staphylococci

could acquire penicillin resistance in vitro by

serial exposure to increasing concentrations of

penicillin. This "acquired" resistance was believed

to be responsible for the early therapeutic

failures found with penicillin. In 1944, Kirby

reported the isolation of staphylococci naturally

resistant to penicillin, in which resistance was

due to the production of a "penicillinase".

Subsequently, Bondi and Dietz (1945) showed that

penicillin-resistant clinical isolates of S. aureus

did not acquire resistance during therapy, but were

naturally resistant due to the production of

penicillinase.

In 1947, Barber announced that the incidence of

S . aureus resistant to penicillin was increasing at

an alarming rate (Barber, 1947). In a survey

conducted from April-N ovember, 1946 an incidence of

32

12.5% staphylococcal penicillin resistance was

found, which had increased in a second survey

conducted from February to June, 1947 to 38%.

Increasing staphylococcal penicillin resistance was

was also reported from hospitals in the USA

(Finland & Haight, 1953), Australia (Rountree & Thomson,

1949), France (Chabbert & Terrial, 1952), Scandinavia

(Laurell & Wallmark, 1953) and India (Gupta &

Chakravati, 1954). By the late 1940s, staphylococcal

resistance to penicillin had increased to such an

extent that the use of penicillin for treating

infections due to S. aureus virtually had to be

abandoned (Barber & Rozwadowska-Dowzenko, 1948).

iii . The use o_f Streptomycin, Tetracycline,

Erythromycin and Chloramphenicol against S. aureus.

Streptomycin was introduced in 1944, and because

of its activity against Mycobacterium tuberculosis

its use was reserved for the treatment of

tuberculosis. When streptomycin was used for the

treatment of staphylococcal infections resistant

strains rapidly emerged. For example, no

streptomycin-resistant S. aureus were isolated at

the Royal Prince Alfred Hospital in Sydney,

Australia in May 1949, whereas over 20% of S .

aureus isolated in January, 1950 were streptomycin-

resistant. Furthermore, all the streptomycin-resistant

strains were also penicillin-resistant (Rountree ejt

33

a l . , 1951).

Similarly, following the introduction of

chloramphenicol and tetracycline increasing levels

of resistance were found in hospitals in France,

Australia and the USA (Barber, 1960). Once the

toxic effect of chloramphenicol was realised, its

use was restricted, and following this,

staphylococcal resistance to chloramphenicol declined

(Kirby & Ahern, 1953). These experiences with new

antibiotics led to the realisation that there was

a correlation between the extent of antibiotic

usage and development of staphylococcal resistance.

This led to calls that new antibiotics with good

antistaphylococcal activity (e.g erythromycin) be

held in reserve (Barber & Burston, 1955; Beavan &

Burry, 1956). Unfortunately, even when erythromycin

usage was restricted, the ability of S. aureus to

develop resistance to this compound was such that

resistant strains readily appeared (Lepper et a l . ,

1954).

i v . The Introduction of Me thicillin.

By the mid- 1950s, the problems of antibiotic

resistance in S . aureus were so serious that there

was great concern regarding the future effective

use of antibiotics for treating staphylococcal

infections (Barber & Burston, 1955; Finland, 1955).

Furthermore, as the 1950s progressed, there were

34

increasing reports of epidemics of infection

involving particularly virulent ("type 80") strains

(Williams, 1959). A considerable number of the

staphylococci from outbreaks during this period

were multiply resistant to four or more

antibiotics (Leading Article, 1965; Bulger & Sherris,

1968; Ridley et a l ., 1970). In view of the above

problems, the introduction of methicillin in 1960

was most welcome, especially on account of its

resistance to "penicillinase". In association with

methicillin, the implementation of stricter cross­

infection control measures and antibiotic control

policies resulted in a major decrease in the

number of multiple resistant staphylococci isolated

during the 1960s (Bulger & Sherris, 1968; Ridley e_t

a l ., 1970).

v . The Ef f icacy of Methicillin and its

Derivatives.

In the British Medical Journal of September 3,

1960, a series of papers was published detailing

the in v i t r o , in vivo and clinical properties of

a new antibiotic, BRL 1241. BRL1241 was

subsequently named methicillin and commercially

called "Celbenin". Methicillin was resistant to

inactivation by staphylococcal penicillinase. In

terms of antibacterial activity, methicillin was

active against staphylococci and streptococci, but

35

not Gram-negative organisms (Knox, 1960). In clinical

use, methicillin was found to be non-toxic, and was

effective in the treatment of infections due to

penicillin-resistant staphylococci (Douthwaite &

Trafford, 1960; Stewart et_____a l ., 1960) including

staphylococcaemia (Allen et a l ., 1962).

A disadvantage of methicillin was its

instability in the presence of acid, thus it had

to be injected. The introduction of the acid-

stable isoxazolyl penicillins (cloxacillin , oxacillin,

dicloxacillin and flucloxacillin) overcame this

problem (Leading Article, 1964). The isoxazolyl

penicillins possess contrasting properties, for

example flucloxacillin is more active than

methicillin, whereas methicillin is more stable to

staphylococcal penicillinase than flucloxacillin

(Frimodt-Moller et a l . , 1986). Nevertheless, these

contrasting properties are largely self-cancelling,

and such is the efficacy of these compounds that

even more than 25 years since their introduction

they are still recommended for the initial

treatment of severe staphylococcal infections (Garrod

et a l ., 1981; Eykyn, 1988).

36

4.0 Staphylococcus aureus Resistant to Methicillin(MRSA).

4.1 Occurrence of MRSA - 1960-1975.

When methicillin was introduced into clinical

use in 1960, no strains of S. aureus were found

to be resistant to this compound (Thompson et a l .,

1960). However, it was not long until resistant

strains were reported. In 1961 Jevons (Jevons, 1961)

found three strains from a total of 5,440

screened to be methicillin-resistant. These strains

all originated from the same hospital, but

methicillin had only been used to treat one

patient at this institution. There was no evidence

to suggest that the resistant strains had arisen

as a consequence of use of methicillin. Rolinson

(1961) showed that the methicillin resistance in

the strains reported by Jevons was heterogeneous,

that is most cells in a culture appeared

sensitive, however a small proportion were highly

resistant. Furthermore, the highly resistant

organisms did not destroy methicillin by virtue of

production of a methicillin degrading enzyme.

By 1963, Jevons (Jevons et al . , 1963) had

screened 27,479 cultures sent to the Staphylococcus

Reference Laboratory, and this revealed a gradually

increasing incidence of methicillin-resistant S .

aureus (MRSA). In the period Jan-March 1961 the

37

incidence of methicillin resistance was 0.055%, and

from July-September 1962 it was 0.81%. According

to Parker & Hewitt (1970) two independent surveys of

the occurrence of MRSA indicated that there was

a moderate increase until 1963 followed by a

stationary period until 1967. Following 1967 there

was a further increase in MRSA. In 1967 the

incidence of MRSA was 1.0%, however in 1969 it

was 4.1%. These figures refer mainly to isolates

from centres in the U.K.

During the late 1960s, the problems posed by

MRSA were more serious in some countries e.g.

Switzerland, Denmark, and France than others e.g. USA.

For instance, Kayser (1975) reported that from 1966-

1971 around 20% of staphylococcal disease in

hospital inpatients was due to MRSA. In Denmark

between 1966 and 1970 from 10-15% of

staphylococcal strains were methicillin-resistant

(Rosendal et a l . , 1976) . This contrasts with the

USA where the first outbreak of MRSA infection

did not occur until 1967, before which only a few

cases of clinical illness due to MRSA had been

reported (Barrett et a l . , 1968). During the first

half of the 1970s, there was a noticeable decline

in the incidence of MRSA in many countries

(Kayser, 1975; Rosendal et a l . , 1976; Ayliffe et a l . ,

1979).

38

4.2 Treatment of MRSA 1960-1975.

i . Therapeutic options and the need for combination

therapy.

The introduction of methicillin combined with

more rigorous hospital infection and antibiotic

control measures resulted in a reduction of the

multiple antibiotic resistant hospital staphylococci

which had caused so many problems during the late

1950s (Parker et a l . , 1974). Although the frequency

of isolation of MRSA increased during the 1960s

the clinical problems posed by these organisms in

terms of the frequency, severity and options for

the treatment of infections were not comparable to

those presented by the multiple antibiotic

resistant staphylococci of the late 1950s.

MRSA isolated during the 1960s and early 1970s

often displayed a variety of reactions with Group

III phages or they were non- typable (Parker &

Hewitt, 1970; Kayser, 1975). They were nearly always

resistant to streptomycin and tetracycline, and

commonly resistant to erythromycin (Ridley et a l .,

1970; Parker & Hewitt, 1970). Resistance to other

antibiotics usually reflected antibiotic usage

adopted at particular institutions. For instance,

Ridley et al. , (1970) showed that use of

chloramphenicol in just one ward of St Thomas's

39

Hospital resulted in more than half the hospital's

chloramphenicol-resistant S. aureus isolates. From

1966 onwards, this hospital restricted the

prescription of erythromycin-like drugs (e.g

spiramycin, oleandomycin and lincomycin), novobiocin

and sodium fusidate primarily for use in MRSA

infections. Furthermore, use of either erythromycin,

erythromycin-like drugs, novobiocin, rifamycins or

sodium fusidate as monotherapy had often resulted

in the development of bacterial resistance, and

combination therapy using these agents was strongly

recommended (Jensen, 1968; Garrod, 1968; Jensen & Lassen,

1969).

Vancomycin, was known to be effective in the

treatment of severe staphylococcal infections, but

was regarded as too toxic compared to the above

agents, hence during the 1960s it was often kept

as a reserve drug (Garrod, 1968; Kucers, 1972).

i i . Treatment with topical antibiotics.

The topical antibiotics available for use

against MRSA during the first half of the 1960s

were primarily neomycin, bacitracin and Fucidin

(fusidic acid). To prevent the emergence of

resistance, these agents were often used in

combination, for example the cream "Naseptin”

consisted of neomycin sulphate and chlorhexidene

40

hydrochloride. Following the introduction of

neomycin and bacitracin into clinical use during

the early 1950s there were few reports of

resistance for several years. It was believed that

by using the agents in combination with another

antibiotic (e.g. neomycin + bacitracin) or antiseptic

(e.g "Naseptin” ) that even if resistance developed

to one agent the other would prevent growth of

the resistant organism. However, during the early

1960s there were increasing reports of neomycin-

resistant S. a ureus, and by 1965 neomycin-resistant

strains were widespread (Leading Article, 1965).

Lowbury et a l . (1964) suggested that the spread

of neomycin resistance was probably due to the

widespread dissemination of a resistant strain, and

not the development of resistance during individual

treatment episodes . Once in the hospital

environment use of neomycin favoured the spread of

this strain (Mitchell, 1964). In 1965, Rountree &

Beard (1965) reported the spread of S. aureus which

was initially resistant to neomycin, but then

became resistant to bacitracin as well. This has

been attributed to the treatment of patients

harbouring neomycin-resistant strains with neomycin-

bacitracin ointment. Hence, the spread of neomycin-

resistant strains limited the use of such

ointments.

Fusidic acid is an irritant of mucous

membranes, and is consequently inappropriate for

41

topical use against S. aureus nasal carriage.

Staphylococci resistant to fusidic acid have been

isolated from patients receiving topical Fucidin, or

oral fusidic acid therapy (Pattison & Mansell, 1973;

Lowbury et a l ., 1962). Ayliffe et a l . t (1977) have

correlated the amount of fusidic acid used with

the appearance of resistant strains in a burns

unit, hence on restricting topical fusidic acid

usage there was a decline in the number of

fusidic acid resistant strains. In some centres

during the 1960s there was a reluctance to use

fusidic acid topically because of its use for

treating infections due to MRSA. When gentamicin

ointment became available in the late 1960s, and

was found to be more effective than "Naseptin”

(Williams et a l ., 1967) there was considerable usage

of gentamicin (Editorial, 1977) with consequences

which are described later.

iii . Treatment______of_____ MRSA______infections_____ with

penicillinase-resistant__________penicillins__________and/or

cephalosporins.

In 1968 Benner and Kayser reported their

experiences for 26 patients with significant

infections due to MRSA who had been given

intensive therapy with penicillinase-resistant

penicillins and/or cephalosporins. They found that

although 18 of the 26 patients were cured or

42

improved by this therapy, all those patients

infected with highly resistant organisms (oxacillin

MICs of 62.5 mg/1 or greater) died of infection.

Benner and Kayser had believed that treatment of

infections with penicillinase-resistant penicillins

or cephalosporins would be effective because only

a few cells (usually less than 1%) in a population

of MRSA possessed high-level resistance to

methicillin or oxacillin. Following Benner and

Ka y s e r ’s paper, and also other studies by French

workers most microbiologists were left in little

doubt that methicillin is inappropriate for

treating MRSA infections (Leading Article, 1968). In

1970, Acar et a l . (1970) concluded that primary

treatment either with a cephalosporin (cephalothin,

cephaloridine, or cephalexin) or with a cephalosporin

combined with an aminoglycoside (kanamycin or

gentamicin) failed to eradicate staphylococcaemia

after three days treatment.

Despite the evidence that treating

staphylococcal infections with penicillinase-resistant

penicillins or cephalosporins gave less than

optimal results, some workers still saw fit to re­

evaluate the efficacy of these agents in

eradicating MRSA. For example, two trials from the

MRC Industrial Injuries and Burns Unit (Lowbury e_t

a l ., 1977; Kidson et a l ., 1979) concluded that oral

flucloxacillin for treatment of burns eliminated or

reduced MRSA in significantly greater numbers

43

compared to untreated controls. Furthermore,

cefamandole either alone or in combination (with

tobramycin) has been found to be effective in MRSA

infections (Coppens et a l . , 1983; Frongillo et a l . ,

1986). When assessing such findings it is

important to consider that "cure" or "improvement" is

a complex interaction depending upon the severity

of infection, the nature of the infecting organism

and the condition of the host. Many workers still

retain the opinion that penicillinase-resistant

penicillins or cephalosporins are not appropriate

for the treatment of infections due to MRSA

(Hackbarth & Chambers, 1989a).

4.3 The Detection and Nature of Methicillin-

Resistance.

i . Detection

In 1964, Barber reported that the growth of

MRSA on nutrient agar containing methicillin (even

at subinhibitory concentrations) was much less

luxuriant than that seen on nutrient agar plates

containing no antibiotic. Further work showed that

growth in the presence of methicillin was enhanced

by an excess of electrolytes (5% NaCl or 7.5%

( N H ^ ^ S O ^ ) or decrease in agar concentration. In

the same year Sutherland and Rolinson (1964) confirmed

44

Rolinson's earlier investigations showing that

cultures of MRSA consisted of mixed populations in

which the majority of cells were of normal

sensitivity to methicillin with a minority showing

methicillin resistance. Furthermore, the resistance

of MRSA was "intrinsic" and was not due to an

increased ability to inactivate the drug. These

workers also showed the importance of using a

large inoculum to detect methicillin resistance.

Subsequently, it has been shown that temperature,

pH, visible light, growth in anaerobic conditions,

exposure to chelating agents and pre-exposure to

beta-lactam antibiotics all influence the expression

of methicillin resistance (Mathews & Stewart, 1984).

Parker and Hewitt investigated the influence of

temperature on the detection of methicillin

resistance (Parker & Hewitt, 1970) and showed that

initial surveys (e.g. Jevons, 1961) to detect the

prevalence of MRSA under-estimated the true numbers

of these organisms. This was because in earlier

surveys susceptibilities were read after 18 hours

incubation at 37°C, and under these conditions only

a few cells in a culture appear resistant

(Rolinson, 1961, Sutherland & Rolinson, 1964). Annear

(1968) showed that methicillin resistance was much

greater at 30°C, because a greater proportion of

cells expressed methicillin resistance at 30°C

(Dyke, 1969; Parker & Hewitt, 1970).

A great many reports assessing methodologies for

45

detecting methicillin resistance have appeared in

the literature over the years (Hansen & Freedy, 1984;

Jolly & Goldberg, 1989; Mouton et a l ., 1989). In the

USA attempts have been made to introduce standard

tests for determining methicillin resistance

(McDougal & Thornsberry, 1984). However, in the U.K

no such guidelines exist and the techniques used

to conduct and interprate methicillin sensitivity

testing can vary considerably between laboratories.

i i . Nature of Methicillin Resistance

Throughout the 1960s there was considerable

confusion regarding the nature of methicillin

resistance and the genetic factors governing it.

Although many workers (e.g. Rolinson, 1961; Barber,

1964) reported that methicillin resistance was not

due to a "methicillinase” , others suggested that

enzymatic inactivation might be responsible (Stewart

& Holt, 1963; Eriksen & Erichsen, 1963). Dyke (1969) in

a comprehensive survey of the penicillinases of

MRSA found none with an increased efficiency of

hydrolysis of methicillin. Staphylococcal beta-

lactamase has been shown to hydrolyse methicillin

slowly at MIC concentrations of the drug ie.

0.0019% the rate of benzylpenicillin (Hamilton-

Miller & Ramsay, 1967). Lacey & Stokes (1977) have

suggested that staphylococcal beta-lactamase

significantly inactivates flucloxacillin, however, few

46

other workers seem to share this opinion.

During the 1970s it was shown that penicillin

(and other beta-lactam antibiotics) kill bacteria by

inhibiting penicillin-sensitive enzymes involved in

the final stages of peptidoglycan synthesis (Spratt,

1978). In 1980, Brown and Reynolds suggested that

methicillin resistance was due to a modification

of cellular penicillin binding proteins (PBPs), or

the presence of a new PBP with a reduced

affinity for methicillin. Following this observation

a great deal of work has been performed on the

PBPs of S. aureus (Hackbarth & Chambers, 1989b).

Currently, it is believed that methicillin

resistance is due to the production of a modified

protein- P B P 2 f- which is both thermosensitive and

acid-sensitive. Hence, at 30°C P B P 2 T is expressed

in greater amounts than at 37°C, similarly at pH

5.2, PBP 2 f is not expressed (Lyon & Skurray, 1987).

This theory applies to "true methicillin-resistant"

strains. Tomasz et a l . (1989) have suggested that

"borderline" resistance to methicillin may be due

to a number of different types of mechanism such

as hyperproduction of beta-lactamase. Beta- lactamase

hyperproducers can be distinguished from "true" MRSA

because methicillin resistance due to beta-lactamase

hyper-production is lost in the presence of

clavulanate. The explanation that methicillin-

resistance is due to the production of PBP 2* may

be too simplistic. For example, using this theory

47

Madiraju et a l . (1987) could not explain the

influence of NaCl on methicillin resistance.

Neither could Murakami and Tomasz (1989) account for

strains with high homogeneous expression of

methicillin resistance containing the same amounts

of PBP 2' as isolates in which 99.99% of the

cells had MICs of 2.0 or 4.0 mg/1.

Because PBP 2 f is considered to have a reduced

affinity for all beta-lactams, methicillin resistance

results in cross-resistance to all beta-lactam

agents. However, differences in affinities or modes

of action may result in some beta-lactams (e.g.

certain cephalosporins, imipenem) possessing greater

activities against MRSA than others (Chambers &

Sachdeva, 1990). Furthermore, the development of a

beta-lactam with increased affinity for PBP 2 T is

a real possibility e.g. BRL 44154 (Blake et a l . ,

1990).

48

5.0 Use of Gentamicin and the Development ofResistance

i . Use of Gentamicin.

The antimicrobial activity of gentamicin was

first reported in 1963 by a number of workers

who showed it to be a bactericidal antibiotic

possessing broad-spectrum activity (Finland, 1974).

Gentamicin was first given to four patients in

March 1962, and although it proved succesful it

also produced vestibular toxicity (Jackson, 1969).

Subsequent decreases in dosage, and the introduction

of laboratory monitoring of blood levels have

greatly reduced this risk. During the 1960s, the

primary systemic use of gentamicin was in the

treatment of urinary tract infections or

septicaemia due to multiresistant Gram-negative

organisms (Neu, 1974). Comparatively little data was

compiled of gentamicin's activity against

staphylococci, and it was not regarded as amongst

the systemic agents of first choice for the

treatment of staphylococcal infections. In 1966,

Barber and Waterworth reported gentamicin to have

good activity against neomycin-sensitive and

neomycin-resistant hospital staphylococci, and

suggested that use of gentamicin as a spray for

clearing staphylococcal carriage should be

considered.

49

In a trial by White (1964) of the use of

gentamicin as a nasal ointment it was found that

topical application was effective in reducing

carrier rate and dissemination of staphylococci.

Gentamicin was regarded as an excellent agent for

the treatment of skin infections due to its

broad-spectrum activity (including Ps. aeruginosa), and

lack of non-irritant and skin sensitizing effects.

Because of these properties use of gentamicin

ointment steadily increased. For example, at the

Belfast City Hospital gentamicin usage increased

from 15,804 x 15 gram units in 1974-75 to 23,020 x

15 gram units in 1975-1976 (Wyatt et a l . , 1977).

In 1968, at an international symposium on

gentamicin it was reported that gentamicin was

active against multi-resistant strains of MRSA

(Hoeprich, 1969). Furthermore, Waitz and Weinstein

(1969) found that only 4 of 1352 isolates of S .

aureus were not inhibited by 10 mg/l gentamicin.

Despite its activity against multiple-resistant

staphylococci, clinical trials assessing gentamicin

for treating staphylococcal infections produced

contrasting results. Richards et a l . (1971) found

gentamicin to be satisfactory in the treatment of

non life-threatening infections, however reservations

were expressed over gentamicin's efficacy in

serious infections. Klastersky et a l . (1975) reported

gentamicin to be less efficacious than

cephaloridine in the treatment of staphylococcal

50

infections and suggested that gentamicin should not

be used as a first-line drug for such infections.

Because of in vitro observations of synergy

between cephalosporins and aminoglycosides a number

of trials have been performed using such

combinations. Against MRSA cefamandole and

tobramycin were found to be nearly as effective

as vancomycin (Klastersky & Van der Auwera, 1986).

i i . Development of Resistance to Ge ntamicin.

Prior to 1975, staphylococcal resistance to

gentamicin was virtually unheard of (Lacey &

Mitchell, 1969). However, because of the increasing

topical usage of this compound it was predicted

that as with neomycin, resistance would also occur

to gentamicin (Lacey, 1975). Soon after this

prophecy single strains of S. aureus possessing

plasmid-mediated gentamicin resistance were reported

in the U.K and France (Porthouse et a l . , 1976;

Soussy et a l . , 1975). In February 1976, Speller ejt

a l . (1976) reported an outbreak of

colonization/infection at the Westminster Hospital

due to gentamicin-r esis tant S. a u r e u s , and by the

end of the year MRSA resistant to gentamicin had

been reported in the U.K and France (Shanson .et

a l . , 1976; Soussy et a l . , 1976). Whereas the early

1970s had seen the decline of the MRSA, these new

reports of gentamicin resistant MRSA - (MGRSA) were

to herald a new era where once again S. aureus

possessing resistance to many antibiotics was to

become a major problem in hospital medicine.

52

6.0 Increasing Hospital Problems due to M G R S A .

Casewell has suggested that the perception of

the problems caused by MRSA depends upon o n e ’s

historical and geographical viewpoint (Casewell,

1986). Epidemiological data shows that the problems

caused by MRSA are greater in some hospitals than

others. Where outbreaks occur, the institution of

control measures can result in considerable

fluctuations in the number of MRSA isolated over

a few months. Hence, the incidence of MRSA can be

influenced by many factors including the frequency

and time of screening, the inclusion of multiple

isolates from the same patient, and the nature

(burns, surgical etc) of the wards or hospitals

under study.

i . Problems due to MGRSA in Great B r i t a i n .

For the period 1976-1980 few serious problems

due to MGRSA were reported from hospitals in the

United Kingdom, however, in Eire 55 patients in

Dublin hospitals were reported to have had a

MGRSA bacteraemia between 1976 and 1979 (Leading

Article, 1981). Cafferkey et a l . (1985a) showed that

the number of new patients from whom MRSA was

isolated peaked at c. 120 early in 1980 and

then gradually declined to c. 40 in 1984 following

the introduction of effective control measures. The

53

Dublin MRSA was characteristically gentamicin

resistant (Cafferkey et a l . , 1983), whereas in the

U.K the presence of gentamicin resistance has been

more variable (Kerr et a l ., 1990).

In a questionnaire survey conducted for the

period 1982-1983 it was found that although MRSA

were widely distributed in the U.K., they occurred

infrequently in many hospitals and only caused

serious problems in a small number of hospitals,

mainly located in the Thames Regional Health

Authority (Cooke et a l . , 1986). A two region survey

of the occurrence of MRSA in hospitals in the

North East Thames (NETRHA) and Yorkshire Regional

Health Authorities over the last quarter of 1985

showed 190 new cases of colonisation and 136 new

cases of infection in NETRHA compared to 10 and

21 respectively in Yorkshire (Communicable Disease

Report, 1986). The high frequency of MRSA in

hospitals in the South East of England was

attributed to the spread of a single strain

termed "epidemic MRSA" or EMRSA (Marples et a l . ,

1986) .

In 1986, a national surveillance scheme to

detect the prevalence of MRSA was initiated, and

Figure 1 compares the number of new cases of

colonisation or infection in NETRHA and Yorkshire

regions at 6 monthly intervals between 1986 and

1989. From Figure 1 it can be seen that there

is a continuing problem due to MRSA in the South

54

Number of new

patients colonised

or infected with

MRSA in

four week periods

during Jan

or Jun.Data

obtained from

the PHLS

Comm

unicable Disease

Surveillance Centre

61 Colindale

Avenue London

NW9

5EQ —

-

New Cases of MRSAIV)o 09o o oio

CO00CT)

CO00'nJc

3CO8

CO00CO

mCOCOo

33X>oo3TDP)—nCDQ.

133g;CD3CO

OcCD

30 CD >O _

ZTOCOCD

2S 'g;i"CD33X>

oZTmo>CO

pj3CDCO33CD

- CS-o o3 g5 x22 ® o> p>

rr>cCOCOo

55

East, so much so that a combined working party of

the Hospital Infection Society and British Society

for Antimicrobial Chemotherapy issued guidelines in

(Working Party, 1986 and 1990) for the control and

eradication of these organisms.

i i . Problems due to MGRSA in Australia.

Gentamicin resistance rapidly appeared in

Australian strains of MRSA. For example at the

Royal Melbourne Hospital 6% of MRSA were

gentamicin-resistant at the end of 1978, however

one year later more than 70% of isolates were

gentamicin-resistant (McDonald et______ a l . , 1981).

Similarly, at St Vincent's Hospital, Darlinghurst,

New South Wales 32% of MRSA were gentamicin-

resistant in 1978 increasing to 96% in 1981 (King

e t a l . t 1981). From 1979 onwards, teaching hospitals

in Eastern Australia encountered serious problems

due to MRSA (Medical Journal of Australia, 1982).

For example, six university teaching hospitals in

the Melbourne metropolitan area reported MRSA to

comprise 20% to 40% of S. aureus isolates in

1979, however in rural hospitals MRSA seldom posed

a problem (Pavillard e t a l . t 1982). During 1981,

some university teaching hospitals in Melbourne

reported 50% of all S. aureus isolates to be

MRSA, and over a 12 month period (Jan. 1979- Jan

1980) in one representative 700-bed hospital MRSA

56

was isolated from 545 patients (Pavillard et a l .,

1982). The seriousness of this problem attracted

a great deal of publicity. Headlines in the

"popular press" such as ’Killer bug in NSW

hospitals - epidemic warning* and ’Shock killer

infection’s 12 v ictims’ generated increased public

awareness of the problem to the extent of causing

widespread alarm (Blum, 1982).

MRSA continues to be a major problem in

teaching hospitals in Eastern Australia. In 1986-

1987 MRSA comprised c. 25% of S. aureus isolates

from Queensland and Victoria (Turnidge et a l . ,

1989). Genetic analysis has shown that one strain

- "eastern Australian MRSA" has been largely

responsible for the continuing epidemic in Eastern

Australia. It has also been shown that this

strain is closely related to the epidemic MRSA

isolated in South-East England (Townsend et a l . ,

1987) .

iii. Problems due to MGRSA in the U S A .

MRSA resistant to gentamicin first started to

cause problems in hospitals in the USA in 1975

(Crossley et a l ., 1979) . Unlike the situation in

Australia and Great Britain where aminoglycoside-

resistant strains were characterised by resistance

to gentamicin and tobramycin, in the USA there

were outbreaks due to strains possessing a number

57

of different aminoglycoside resistance patterns. For

this reason the Americans have often used the

term MARSA - methicillin-aminoglycoside resistant S .

au r e u s . In the first outbreak of MRSA resistant

to aminoglycosides, 75% of the MARSA were resistant

to tobramycin, amikacin and kanamycin yet

susceptible to gentamicin, sissomicin and netilmicin

(Crossley et a l . , 1979). Similarly, Craven et al

(1981) reported an extensive outbreak over a 16

month period (Sept. 1978- Jan. 1980) in a surgical

building due to tobramycin-resistant MRSA, 30% of

which were sensitive to gentamicin.

In other areas a similar pattern of events

occurred to that experienced in Australia and

Great Britain. Schaefler et _al_. (1981) first

detected MGRSA from hospitals in New York City in

1978, and by the spring of 1980 more than 80% of

MRSA received from hospitals in the New York city

area were gentamicin-resistant. Similarly, Dunkle et

a l . (1981), Graham et a l . (1980), Linnemann et a l .

(1982) and Saroglou et a l . (1980) have all reported

hospital outbreaks due to the emergence of MGRSA.

Haley et al (1982) reviewed the epidemiology of

MRSA infections in United States hospitals up

until 1981, and concluded that the occurrence of

MRSA had reached epidemic proportions. However,

major problems were only experienced by a small

number of large teaching hospitals. Unlike the

situation in Australia and Great Britain there is

no evidence to suggest that the problems in USA

hospitals are due to the spread of a single

epidemic strain.

Since 1981, problems due to MRSA have increased.

In a survey by Wakefield et a l . (1987) of 136

hospital laboratories throughout the United States,

S. aureus isolates reported as resistant to

methicillin ranged from 0% to 52% (percentage of

S. aureus isolates tested) with a mean value of

10%. Unlike the findings of Haley et a l . (1982)

major problems were not restricted to a small

number of large teaching hospitals. 18% of

hospitals in W a k e f i e l d fs survey reported MRSA

isolation rates of at least 20%, although there

was geographic clustering with distinct areas of

very high (mainly in Eastern areas of USA) and

very low (mainly in Western areas of USA) rates

of isolation of MRSA. Furthermore, in certain areas

of the USA, MRSA has spread into the community

(Saravolatz et a l .t 1982; McGowan Jr., 1988) where it

is a particular problem amongst intravenous drug

abusers (Markowitz et a l . t 1983; Craven et a l . ,

1986). In a recent personal communication from

Professor R. A. Weinstein of the Michael Reese

Hospital in Chicago, during 1988 of approximately

26,000 inpatient admissions, 80-90% of patients were

infected with MRSA and over half of these

isolates were derived from the community.

59

iv . Problems due to MGRSA in Europe.

Because there has been no central agency for

the collection of data on MRSA throughout Europe,

it is difficult to assess the problems caused by

MGRSA from 1976 to present. However, reports from

individual countries do reveal problems due to

MRSA of differing extents. In France, MGRSA were

first reported by Soussy et a l . in 1976 and since

this time strains resistant to all aminoglycosides,

including gentamicin, netilmicin and amikacin have

become endemic in many hospitals (Acar & B uu-Hoi,

1988). At the Hopital Saint- Joseph in Paris

during 1988 approximately 80% of MRSA were

resistant to aminoglycosides (Acar & Buu-Hoi, 1988).

The incidence of antibiotic resistance in

Austria, Switzerland and West Germany has been

monitored by the Paul Ehrlich Society (Kresken &

Wiedemann, 1986). The overall rate of isolation of

MRSA was less than 10 %, and no increase was

found between the years 1976- 1984. Only in one

hospital (in Vienna) were problems with MRSA

encontered (Wiedemann & Kresken, 1984) and our

laboratory testing has shown strains from this

hospital to be gentamicin-resistant. In 1988,

Borowski et a l . reported an overall isolation rate

of 17% MRSA for 1283 strains of S. aureus from

15 Polish hospitals, 87% of these MRSA were

gentamicin-resistant.

60

In Italy, the isolation of MRSA has increased

from 6% in 1981 to 26% in 1986 according to

data presented by Schito and Varaldo (1988).

Unfortunately, these authors did not specify the

levels of gentamicin resistance in the different

species studied, however for all strains of

Staphylococcus monitored, gentamicin resistance

increased from 1% in 1981 to 21% in 1986 (Schito

& Varaldo, 1988). According to Varaldo et a l .

(1984), 49% of methicillin-resistant staphylococci

were gentamicin-resistant in 1983, although again

the authors fail to distinguish between levels in

MRSA and other species of methicillin-resistant

staphylococci.

The first report of MGRSA in a Portuguese

hospital was made by Melo Cristino et a l . in

1985. MGRSA first started causing problems in

Spanish hospitals in 1978, for example an outbreak

occurred in a newborn nursery resulting in an

overall hospital rate of MRSA isolation of 23% in

1978 (Trallero et a l ., 1988). In Greece, the first

serious problems due to MRSA appeared in 1978

(Giamarellou et a l ., 1981) and since then MGRSA has

become an important nosocomial pathogen (Kosmidis,

1988).

The experiences in Southern Europe contrast with

those in Scandinavia. For example, in the early

1970s MRSA was a major problem in Danish

hospitals, however, during the late 1970s and

61

continuing into the 1980s problems due to these

organisms have diminished, so as to be virtually

non-existent (Jepsen, 1986; Ipsen & Gahrn- Hansen, 1988).

v . Problems due to MGRSA in other countries.

MGRSA pose considerable hospital problems in

Southeast Asia. For instance, in Hong Kong 25-30%

of all hospital isolates of S. aureus are MRSA,

and these are commonly resistant to gentamicin

(French et a l . , 1988). In Singapore, isolation rates

of MRSA have steadily increased from 12% of all

S. aureus isolates in 1982 to 27% in 1985, these

MRSA are also typically gentamicin-resistant (Grubb

et a l ., 1986).

In Israel, MGRSA has emerged as an important

and common pathogen responsible for serious endemic

and epidemic nosocomial infections. For example,

between 1984 and 1985, in one hospital an

isolation rate of 29% was found (Finkelstein ejt

al. , 1989). In South Africa, MGRSA is also causing

serious hospital problems (Pochee et a l . t 1988;

Coovadia et a l . , 1989). MRSA are also causing

problems in countries in northern Africa eg. Egypt

(Khalifa et a l . , 1989), Ethiopia (Gedebou et a l . ,

1987) and Nigeria (Montefiore et a l . , 1989). High

rates of isolation of MRSA have been reported,

however, it is not stated whether these strains

are gentamicin-resistant or not.

MGRSA is also causing problems in hospitals in

South America. In a Chilean hospital from 1984-

1988 isolations of MRSA have oscillated between 10

and 23%, and these strains are usually gentamicin-

resistant (Monteil et a l ., 1988). In Brazil, MGRSA

is an established problem, for example in hospitals

in Porto Alegre and Rio de Janeiro isolation

rates of 35% - 50% have been found (Marques et_

al. , 1989).

63

Ob jectives

1 . To assess the problem of multiple antibiotic

resistance in methicillin and gentamicin resistant

Staphylococcus aureus worldwide.

2. To consider current and foreseeable antibiotic

therapeutic options in the light of resistance

found at present, and that which might occur in

the future.

As a result of finding widespread ciprofloxacin

resistance early in the above studies, a third

objective was added.

3. To determine possible reasons for the high

incidence of fluoroquinolone resistance found in

MGRSA.

64

MATERIALS AND METHODS

MATERIALS1. Strains

1.1 Strain Origins and Phage T y p e s .

Epidemiologically distinct strains of methicillin

and gentamicin resistant Staphylococcus aureus were

requested from centres worldwide. The origins and

phage types of these strains are shown in Table I.

MGRSA resistant to ciprofloxacin were

specifically obtained from two centres in Israel,

strains IS 1, IS 2, IS 3 were supplied by Professor

D. Merzbach (Rambam Medical Center, Haifa) and strains

IS A - IS 8 were supplied by Dr M. Dan (Edith

Wolfson Medical Center, Holon). Ciprofloxacin-

resistant and ciprofloxacin-sensitive MGRSA (TEX 1 -

TEX 24) from one of the first reported outbreaks

of ciprofloxacin resistance in MRSA (Isaacs et a l . ,

1988) were supplied by Dr R. L. Cohen of the

Veterans Administration Medical Center, Dallas, Texas.

The phage types of these strains are shown in

Table XVII.

Throughout our antibiotic susceptibility studies,

S. aureus NCTC 6571 (strain SOX) was used as a

control. For plate antimicrobial assays of the

fluoroquinolones, Escherichia coli NCTC 10418 was

the indicator strain.

Paulista BZ12

53/85BZ15

85BZ16

NTBZ17

NTBZ18

84

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, (33 P 00 I-* w H- P cr ET w PPu W o 3 cn r+ o o P H- PP H- p P p P p p 3 rt PP P- rt P cp t—1 I-1 p l-t O P Ph-» P P P P cn rt P rt rt

(=L H- - P ET COP < CO P **

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sz: 55 oo 55 5-5 H H Cn H H

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66

Table

Contd.

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3 3

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so ooH3 Cn

so so a so so H H H H H

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< to o

cn oCD &3rt>-4CD

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67

Table I

contd

GERMAN Wittenberg

EG 6

DEMOCRATIC Martin-Luther

EG12REPUBLIC

Universitat

tiw>scnt*a

scW O T ) H-T3 Pn h - i-iU rt H-03 03 CO r t |->

It ) Tj i-rj hrj hr) rtcjN3 h-* I—1 H-* l—■U l -p" LO K3 I—1 U l

s; s; S! 25 OO S5 2; i-3i-3 00■C-OO

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T1 T1 Tl Tl-C> (jO N 3 I— 1

-P~N3M■P-•JLn-c>-Ul00Ul

S3 S3 i-3 H

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O O W T l n 03 oo X •-J

W TJ W c r t H-2 a m p 03 JO> w p r t P H-S3 t - > i-j r t Pi-3 M f W

OO

cCt-h P

3f P CL03 H- 03P < SC Pa. o) o r tCD P 3 H- O(» co c r O O ' s r? r H* H- c CD H- PP 3 r t P P O JO03 03 00 r t r t CDP rr 1 P H-

co co CD OCD 3C P I r tP T * P ■<s r H- P ►t ■o03 P CD pe h - CO coCO 77* H*

03 COp r t

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of

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Prt03PP03rtH-OP

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C/30) H rt v<*T)SP ®HOXi—*Oo

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68

Table I

contd

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a C_ l—l a cn oC3 > H o a os: a > a M 3> > tr- cn M 3H a o rtH M t*

aoacna H3 3 cnC/J H- 3 «o a3 3 a H- ® 3 a a h -H- i-h 'O < O 3 o o 3 >rt 3 o a 3 a 3 3 3 3 OO rtrt H 3 I-J 3 1=) 3 OO a a O

1 o < 3 < H- H* 3 3<=! a cn a H- H- H* rt rt a 3 33 3 3 ^ 3 rt 3 3 3 3 3 rtH- < H- o 3 t-»3 o h-»3 •-t<J 3 < 3 M 33 H- 3 'C OO -►1 rt 3. 33 3 H-H- H-rt rt

a a a C_ 1—1 1—1 1—1l—lM a a a > > C/0s: s: s: T) H i-3 H H t-3 a a a 1-3i-3 O rt

H-1 I—* O tl-J Ui ■p* tO l-i to t—* 00 -o O'* w to I—* W i—* a 3

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69

Table I

contd

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cdtdMn

ooffi r t O Ui dd H- rt

td £c_ cr* pa m 3 H*

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K f td

03 (D £

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70

Table I

contd

TURKEY Capa-Istanbul

Istanbul Medical

Faculty

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Table I

contd

Abbreviations: NT

= not

typable with

International Set

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-O' -O' -O' -O''J Hi \ \^ — -J

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71

For the haemolysin identification studies S .

aureus NCTC 7121, S. aureus NCTC 10345 and S .

aureus NCTC 5664 were used as control organisms.

For short term storage (up to 6 months) strains

were kept on nutrient agar slopes (Southern Group

Laboratories, Lewisham & Southwark), for longer term

storage strains were kept in liquid nitrogen.

2. Antibiotics

2.1 Antibiotics used in susceptibility and other

studies of antibiotic properties.

The antibiotics used in susceptibility, and other

studies of antibiotic properties are shown in

Table II. In Table II the sources of antibiotics,

and the solvents used to dissolve them are given.

Antibiotic stock solutions were formulated according

to batch potency, and all solutions were used

within five hours of preparation.

3. Media and Reagents

3.1 Media used in susceptibility and other studies

of antibiotic properties.

When required strains were subcultured onto 7%

horse blood agar (7% horse blood in Columbia Blood

Agar Base, Oxoid, Basingstoke). For susceptibility

72

CIPROFLOXACIN ciprofloxacin

hydrochloride, lab.

waterstandard-

Bayer AG,

Wuppertal, FRG.

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75

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76

SPARFLOXACIN compound

RP 64206,

lab. standard-

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Seine. NaOH

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77

VANCOMYCIN vancomycin

hydrochloride, lab.

standard- water

Eli Lilly

& Co.,

Indianapolis, In.,

USA.

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78

testing, strains were grown up in peptone water

broth (Southern Group Laboratories).

IsoSensitest Agar (Oxoid, Basingstoke) was used

for all determinations of antibiotic susceptibility

(except for methicillin and paldimycin) by agar

incorporation. Nutrient Agar (Oxoid) was used for

methicillin susceptibility determinations and in

the case of paldimycin, Antibiotic Medium No. 2

(Oxoid) was used because of the improved stability

found for paldimycin at pH 6.5. IsoSensitest Broth

(Oxoid) was used for determination of MICs by

broth dilution studies, and also in kill curve

studies. IsoSensitest Agar was also used in time-

kill and population structure experiments for

viable counting, and in plate antibiotic assays.

3.2 Media used in biotyping studies.

i . Egg Yolk Agar

Egg Yolk Emulsion (Oxoid) was added to molten

(50°C) nutrient agar (Oxoid) to a final concentration

of 10% v/v. The medium was cleared by addition of

1% NaCl, and sterile 40% D-glucose solution was

then added to a final concentration of 1% w/v.

i i . Lysed Blood DST Agar

Lysed horse blood (Tissue Culture Services,

Botolph Claydon, Bucks) was added to DST Agar

(Oxoid) held at 50°C to a final concentration of 7%.

79

i i i . Milk Agar

"Nestles Ideal Evaporated Milk" was added to

molten nutrient agar to produce a final

concentration of 20% w/v.

i v . Sheep Blood Agar

Sheep blood (Oxoid) was added to IsoSensitest

agar held at 48°C to a final concentration of 5%

v/v. This mixture was then layered (c. 10 ml) onto

plates prepoured with 10 ml IsoSensitest agar.

v . Tween 80 Agar

Tween 80 (polyoxyethylene-20-sorbitan mono-oleate)

was added to nutrient agar to a final

concentration of 1% v/v.

v i . Staph-Typing A g a r .

This consisted of 20 g/1 Oxoid Nutrient Broth

no. 2, 5.0 g/1 NaCl and 7.5 g/1 Oxoid Agar no. 1.

After autoclaving (121°C for 15 minutes) the medium

was supplemented with C a C ^ solution to a final

concentration of 1.2 g/1.

80

3.3 Antibiotic-containing discs for determining

sensitivity profiles and identifying aminoglycoside-

modifying enzymes.

i Antibiotic discs used for Antibiotic Sensitivity

Profiles.

The nine antibiotics (disc strengths in

parentheses) used in our antibiotic sensitivity

typing scheme were as follows: amikacin (10 ug) ,

chloramphenicol (10 ug), ciprofloxacin (5 u g ) ,

clindamycin (2 u g ) , neomycin (10 ug), netilmicin (10 ug),

rifampicin (5 ug), tetracycline (10 ug) and trimethoprim

(1.25 u g ) . Netilmicin discs were supplied by Oxoid

(Basingstoke), all the others were supplied by Mast

Laboratories, Merseyside.

ii . Antibiotic discs used for identifying

Aminoglycoside-modifying enzymes.

The antibiotic discs (strengths in parentheses)

used for identifying aminoglycoside-modifying enzymes

were: amikacin (10 u g ) , gentamicin (10 u g ) , kanamycin (30

ug), neomycin (30 ug), netilmicin (30 ug) and sissomicin

(10 ug) . These were supplied in the form of

specially prepared antibiotic disc "rings” by Mast

Laboratories, Merseyside.

81

3.4 Materials and Reagents used for detection ofspecific haemolysins.

The micro-titration assay of Jordens et a l . ,

(1989) was used to detect specific haemolysins.

Human blood (from volunteer), rabbit blood (from

animal house) and sheep blood (in Alsever solution

from Oxoid, Basingstoke) were used. Human blood was

collected in Lithium Heparin LH/10 "Monovette" tubes

(Sarstedt UK Ltd., Leicester) and EDTA KE/2.7

"Monovette" tubes (Sarstedt). Rabbit blood (5 ml) was

collected in 5 ml Alsever solution (glucose 2.05%,

NaCl 0.42% and sodium citrate 0.8% in sterile

distilled water). Rabbit and human blood were used

within 6 days of collection. For use in assays

blood was washed thrice in phosphate buffered

saline (NaCl 8.0 g/1, K2HP04 1.21 g/1 and 0.34 g/1

KH2P0^) supplemented with 1 mM MgSO^.

Heparin ("Monoparin", CP Pharmaceuticals Ltd.,

Wrexham) and bovine blood (fraction 1) fibrinogen (BDH

Chemicals Ltd., Poole) were also used in these

experiments. Assays were performed in "v" well

microtiter plates (Sterilin, Feltham, Middlesex).

82

4.0. Buffers used in plasmid isolation studies.

i . Buffers used in Takahashi and Nagano m e t h o d .

Buffer A. 40 mM TRIZMA base and 20 mM disodium EDTA

in sterile distilled water, adjusted to pH 8.0

with glacial acetic acid.

Buffer B. 3.0 M sodium acetate in sterile

distilled water, adjusted to pH 5.5 with glacial

acetic acid.

Buffer C. 10 mM TRIZMA base and 2.0 mM disodium

EDTA in sterile distilled water, adjusted to pH

8.0 with glacial acetic acid.

Buffer D. 1.0 M sodium acetate, 10 mM TRIZMA base

and 2.0 mM disodium EDTA in sterile distilled

water, adjusted to pH 8.0 with glacial acetic

acid .

i i . Buffers used in PHLS (Johnson) m e t h o d .

Suspending Buffer. 2.5 M NaCl and 50 mM disodium

EDTA, p H 7 .5.

Lysis Buffer. 50 mM Tris- acetate, 50 mM disodium

EDTA, pH 8.0 Brij-58 added to 1% and sodium

deoxycholate added to 0.4%.

83

Tris-Borate Buffer x 20 (for electrophoresis). 216 g

TRIZMA base and 9.3 g disodium EDTA was added to

1.0 litre sterile distilled water, and pH was

adjusted to 9.8 with boric acid.

Buffers were autoclaved at 115°C for 10 mins.

TRIZMA-base, sodium acetate, disodium EDTA and NaCl

(molecular biology grade reagents) and sodium

deoxycholate and Brij-58 were obtained from the

Sigma Chemical Company (Sigma Chemicals), Poole,

Dorset. Glacial acetic acid (AnalaR grade) was

obtained from British Drug Houses (BDH), Poole.

iii. Loading b uffer.

Loading buffer consisted of 0.1% bromocresol

purple and 50% glycerol in water.

84

METHODS

5.0 Identification of S. aureus.

Cultures were identified as S . aureus on the

basis of Gram-stain, catalase production, coagulase

production and "API STAPH" biochemical profile.

Gram-staining and catalase production were performed

by standard laboratory techniques (Cowan & Steel,

1974).

Coagulase production was detected by the tube

coagulase method of Gillespie (Cruickshank et a l . ,

1975). 0.1 ml of 18-24 hour old nutrient broth

culture of bacteria was added to 1 ml of a 1 in

10 dilution of rabbit plasma (Difco Laboratories,

Michigan, USA) in plastic bijoux. The bijoux were

incubated at 37°C and checked at 1 , 3 and 6 hours

for the presence of coagulum (ie. conversion of

plasma to a soft or stiff gel). If negative,

bijoux were left overnight at room temperature and

examined next morning. Weakly positive (S. aureus

NCTC 6571) and negative (S. epidermidis) strains were

run as controls.

API STAPH biochemical test strips were obtained

from API (Montalieu Vercieu, France) and processed

according to the manufacturer’s instructions.

85

6.0 Detection of Methicillin Resistance.

Methicillin resistance was detected by streaking

isolates and appropriate controls onto nutrient

agar (Oxoid). Methicillin (25 ug) strips (Mast

Laboratories, Merseyside) were layed perpendicularly

to the inocula. Following incubation for 40 hours

at 30°C, methicillin-resistant strains grew to

within 3 mm or less of the edge of the strip.

7.0 Determination of Susceptibility to Antimicrobial

A g e n t s .

i . Determination of minimum inhibitory concentration

(MIC) by agar dilution.

Except where stated doubling dilutions of

agent were incorporated into bottles containing

molten IsoSensitest agar (Oxoid, UK) held at 50°C.

A 1:10 to 1:100 ratio of agent to agar was used

to ensure uniform dissolution after mixing. Plates

were immediately poured and were usually used

within 6 hours of pouring. Bacterial isolates were

subcultured from blood agar plates into 3 ml

volumes of peptone water broth (Southern Group

Laboratories, Lewisham & Southwark) which were

incubated for 18-22 hours at 37°C unless otherwise

stated. These broth cultures contained approximately

5.0 x 10® cfu/ml, and using a Multipoint Inoculator86

(Denley Instruments, Billingshurst) which deposited

1.0 ul of broth onto the surface of dried

plates, an initial inoculum of c. 5.0 x 10^ cfu

was delivered. Using dilutions of broth (i.e. 1/100)

the inoculum effect of antibiotics could be

studied .

Inoculated plates were incubated for 24 hours

at 37°C prior to reading. The MIC of an agent

was the lowest concentration at which visible

growth was inhibited. S. aureus NCTC 6571 was used

as a control in all tests.

i i . Determination of minimum inhibitory concentration

(MIC) by broth dilution.

Broth cultures were prepared as stated above.

Doubling dilutions of antibiotic were made in 0.9

ml IsoSensitest broth (Oxoid) in bijoux tubes. 0.1

ml of a 1/100 dilution of broth culture was

added to each tube. Antibiotic-free and organism-

free controls were included in each test. After

24 hours incubation at 37°C the tube containing

the lowest concentration of antibiotic to inhibit

visible growth was taken as the MIC. S. aureus

NCTC 6571 (strain SOX) was used as a control.

87

iii. Selection of Antibiotic break-points.

The MICs of 23 antimicrobial agents against 100

strains of MGRSA were determined in our survey of

antimicrobial resistance (Table IV). Strains were

classified as sensitive (S), moderately resistant ( M ) ,

or resistant (R) to each agent according to the

MICs (see Table III). This classification was made

with different criteria depending on the agent

tested. For fosfomycin breakpoints suggested by an

International Study Group were adopted (Andrews et

al.t 1983).

A number of compounds could be grouped in

relation to the distribution of recorded MICs.

MICs for mupirocin, nitrofurantoin, pristinamycin,

teicoplanin and vancomycin were narrow with a

single peak, and strains were all classified as

sensitive. Separate distributions (bimodal) of MICs

were observed for streptomycin, neomycin, bacitracin,

tetracycline, minocycline, erythromycin, clindamycin,

chloramphenicol and novobiocin. In these instances

strains were classified as sensitive or resistant.

For other antibiotics (trimethoprim, rifampicin,

fusidic acid and ciprofloxacin) three separate

distributions (trimodal) of MICs were found, and

strains were classified as sensitive, moderately

resistant and resistant.

For the aminoglycosides, gentamicin, tobramycin,

netilmicin and amikacin there was no obvious

88

Table III

Minimum inhibitory concentrations (MICs) used to classifystrains as sensitive, moderatelv resistant, or resistant.

AntibioticMIC (mg/1)

Sensitive Moderate Resistant

Gentamicin1 < 1 N/A > 8Tobramycin1 <. 1 N/A > 16Netilmicin1 1 1 2,4 > 8Amikacin1 < 4 8,16 > 32Neomycin 1 4 N/A > 16Streptomycin < 16 N/A > 64Tetracycline < 4 N/A > 32Minocycline 1 4 N/A >. 8Erythromycin < 1 N/A > 8Clindamycin < 0.25 N/A > 16Chloramphenicol 1 16 N/A > 32Trimethoprim 1 0.25 1,2 > 4Rifampicin 1 0.12 1,2 > 16Fusidic acid < 0.5 4 > 16Novobiocin < 0.5 2 N/AFosfomyc in2 < 16 32,64 >128Pristinamycin < 2 N/A N/ACiprofloxacin < 1 2,4 > 16Nitrofurantoin < 32 N/A N/AMupirocin 1 0.5 N/A N/ABac itracin* 1 8 N/A > 32Vancomycin < 2 N/A N/ATeicoplanin < 1 N/A N/A

N/A = not applicable* Bacitracin activity is in international units.1 definitions of sensitivity as suggested by a working party of the British Society for Antimicrobial Chemotherapy (British Society for Antimicrobial Chemotherapy (1988).

2 definition of sensitivity as suggested by an international study group on fosfomycin (Andrews et a l .1983).

89

pattern of distribution, and breakpoints suggested

by the British Society for Antimicrobial

Chemotherapy (1988) were used.

7.1 Determination of bactericidal activity.

i . Determination______ of.______ minimum______ bactericidal

concentration ( M BC).

Minimum bactericidal concentrations (MBCs) were

measured by replicating (with velvet pads) from 24

hour-old MIC plates (initially inoculated with 5.0

x 105 cfu) onto fresh IsoSensitest agar (Elek &

Hilson, 1954). Replica-plates were incubated for 24

hours prior to reading. The transfer efficiency of

this method is believed to be 1% (Elek & Hilson,

1954). Because the MBC is defined as 99.9%

killing, with this method the MBC was the lowest

concentration of agent at which 5 or fewer

colonies grew.

i i . Determination of rate of killing by time-kill

c u r v e s .

Timed killing curves were performed with

selected strains. The strains were grown for 24

hours at 37°C in 10 ml of IsoSensitest broth, and

0.5 ml aliquots were used to inoculate 500 ml

Erlenmeyer flasks containing 100 ml IsoSensitest

90

broth. Antibiotics were then added to the flasks

to achieve the desired concentrations, and 0.5 ml

samples were removed for viable counting. The

flasks were then incubated at 37°C in a rotary

incubator (100 rpm) and sampled at desired time

intervals. Viable counts were performed by

spreading 0.1 ml of sample (or 1 in 10 dilutions

of sample) on IsoSensitest agar plates. After 24

hours incubation at 37°C the numbers of colonies

on plates were counted.

Antibiotic-free control flasks and strain S .

aureus NCTC 6571 were used as controls in these

experiments.

7.2 Determination of mutation rates to r e s i s t a n c e .

Selected strains were shaken at 100 rpm for

18-22 hours in 10 ml IsoSensitest broth at 37°C.

Cultures were centrifuged (3,600 rpm for 12

minutes) and resuspended in phosphate buffered

saline (NaCl 8.0 g/1, K 2HP04 , 1.21 g/1, K H 2P04 , 0.34

g/1). 0.1 ml amounts were spread on IsoSensitest

agar plates containing various concentrations of

agent. The plates were then incubated for a total

of 48 hours at 37°C and read after 24 hours and

48 hours. Colonies which grew were picked off,

reidentified as MRSA and resistance confirmed by

MIC determination. Mutation rates to resistance

were calculated by averaging the results obtained

91

in two separate experiments.

7.3 Microbiological assay of fluoroquinolones

To detect whether fluoroquinolone activity was

lost in time-kill broths as a result of

incubation conditions, or other non-microbiological

factors, fluoroquinolone levels were measured in

IsoSensitest broth after 0, 24 and 48 hours

incubation at 37°C.

Assay plates were prepared by pouring 150 ml

molten IsoSensitest agar into "Nunc" (24 cm x 24 cm

x 1.8 cm) Bio-Assay dishes (A/S Nunc, Denmark) on a

level surface. After cooling and drying, plates

were seeded with approximately 5.0 ml of 1/100

dilution of E. coli NCTC 10418 previously grown for

c. 20 hours in IsoSensitest broth at 37°C. The

agar surface was evenly covered with culture, and

the plates were then dried. 36 x 6 mm wells (6 x 6)

were cut in the agar using a no. 4 size borer.

These holes were filled with either test or

standard solutions according to a random number

pattern. Following incubation for 24 hours at 37°C

zone sizes were measured, and fluoroquinolone

concentrations in the test samples were determined

by interpolation from the standard curve (ie. a

graph of log^Q fluoroquinolone concentration versus

zone diameter).

92

8.0 Development of a system for typing MGRSA.

8.1 Phage-typing of M G R S A .

Phage-typing of the MGRSA was performed by the

author whilst visiting the Staphylococcal Reference

Laboratory (SRL), P H L S , Colindale and also by staff

of the SRL at intermittent periods.

Isolated colonies (not more than 24 hours old)

from blood agar plates were inoculated into 5 ml

volumes of nutrient broth. After incubation at

37°C for 4-6 hours these broths (which should just

be starting to show visible growth) were used to

flood dried plates of "Staph Typing" agar. Excess

culture was pipetted from the plates, which were

left to dry and then inoculated with 0.01 ml of

phage suspension using a multiple-loop applicator.

Plates were then incubated at 30°C overnight.

Next morning the plates were examined for lysis

with a xlO hand lens and the following reactions

were recorded irrespective of the size of plaques

produced.

1-19 plaques +/- weak reaction

20-50 plaques + weak reaction

50 plaques, confluent lysis ++ strong reaction

Phage typing was performed with the

International Set of phages at routine test93

dilution (RTD) and lOOx R T D . Slight variations in

patterns of lysis may be obtained with cultures

of the same organism tested on separate occasions.

Usually strains must differ by at least two strong

reactions to be regarded as epidemiologically

distinct. As an aid to discrimination, we have

also typed strains with complimentary phages (88A,

90, 83C and 932) and also supplementary ("Nuan") phages

(Richardson et a l . , 1988).

8 . 2 Identification______ of_ aminoglycoside-modifying

e nzymes.

i . Van de Klundert's me t h o d .

Dried IsoSensitest agar plates (agar depth 4.0

mm) were seeded with 1.0 ml of a 1/1000 dilution

of 18-hour old culture of organism grown in

IsoSensitest broth at 37°C. Plates were dried and

specially prepared "rings" (Mast Laboratories)

consisting of 6 aminoglycoside-containing paper

discs were placed on their surfaces. Following

incubation at 37°C for 18-22 hours zone diameters

(if any) produced by the discs were measured.

The stepwise determination scheme proposed by

Van de Klundert et a l . (1984) was used to infer

the presence of particular aminoglycoside-modifying

enzymes. Strains produced APH (2")/AAC ( 6 f) + APH

( 3 ’)-IV if inhibition zone diameters of less than

94

18 mm, 19 mm and 20 mm were found to gentamicin,kanamycin and neomycin respectively. If the strains

were sensitive to neomycin (ie. zone diameter of 20

mm or greater) the production of APH (2M )/AAC ( 6 f)

was inferred. Strains sensitive to gentamicin and

sissomicin (ie. zone diameters equal or greater than

18 mm and 21 mm respectively), but resistant to

tobramycin (zone diameter less than 11 mm) produced

the enzyme ANT ( 4 ?,4n ).

i i . Gene probing aminoglycoside resistance g e n e s .

By arrangement with Dr G. Miller of Schering-

Plough Research (Schering-Plough Corporation,

Bloomfield, New Jersey) "dot-blots" prepared by

ourselves were probed. Dot-blots were prepared by

spotting approximately 10 ul of 48 hour-old

cultures (grown in 5 ml Nutrient Broth no. 2 at

37°C and shaken at 100 rpm) onto GeneScreen paper.

The papers were left to dry at room temperature

and then placed for 5 minutes (culture side up) on

Whatman 3MM filter paper saturated with 0.5 M

NaOH. The dot-blot papers were transferred onto

filter paper soaked with 1.0 M Tris pH 7.0, and 5

minutes later they were placed for 3 minutes in

a container with 500 ml of 1.0 M Tris pH 7.0. The

dot-blot papers were then removed and left to dry

at room temperature. Gene probing was then

performed at Schering Research in New Jersey.

95

8. 3 Determination of Antibiotic Susceptibility

P r o f i l e s .

Lysed blood DST agar was seeded with

approximately 10^ cfu of organism (ie. 1 ml of a

1/1000 dilution of 18-22 hour-old culture grown in

peptone water broth at 37°C), and antibiotic-

containing discs (Media & Reagents, 3.3i) were placed

5 on one plate, and 4 on another. After

incubation for 24 hours at 37°C the plates were

read according to the following criteria. Strains

were classified as "sensitive" if the zone diameter

produced by an antibiotic was more than 66% that

produced against S. aureus NCTC 6571. Three digit

code numbers were assembled using this system, and

sensitivity was scored as follows -

netilmicin (1), amikacin (2), neomycin (4) - 1st digit

tetracycline(1) , clindamycin(2), chloramphenicol(4) - 2nd

digit

trimethoprim (1), rifampicin (2), ciprofloxacin (4) - 3rd

digit

8.4 Identification of Physiological Properties of

use in B i o t y p i n g .

i . Identification of Haemolysins by titration.

A micro-titration assay developed by Jordens

96

et a l . (1989) was used to detect and quantifyhaemolysin production in our strains.

Cultures of organisms grown (shaking at 100 rpm)

in 10 ml IsoSensitest broth for 18-22 hours at

37°C were centrifuged down (3,600 rpm for 20

minutes) and the broth supernatant collected. This

was then serially diluted (to 1 in 256) in 100 ul

volumes of phosphate buffered saline (containing 1

mM MgSO^) in a microtitre tray. Dilutions of each

culture supernatant were prepared in triplicate.

Heparin (20 ul of 1000 units/ml) was added to one

set of dilutions and fibrinogen ( 20 ul of 10

rag/ml) was added to another. The plates were then

incubated for 15 minutes at 37°C. The haemolysin

assays were then performed by adding 100 ul of

washed (2% in MgSO^ PBS) human, rabbit or sheep

erythrocytes to the plates. These were then

incubated for 80 minutes at 37°C.

The assays were read using a Titertek (Flow

laboratories) microtitre plate viewer. The highest

dilution showing definite (ie. approximately 50%)

lysis compared to a control using broth instead

of broth supernatant was recorded as the

haemolytic titre of the organism. This was a

subjective assessment based upon appearance of

diffuse haemoglobin in the well (ie. a diffuse red

colouration) and size of the "button" of red cells

at the bottom of the well. Because heparin

inhibits the action of gamma haemolysin and

97

fibrinogen inhibits delta toxin (Jordens et a l .

1989), the titre of individual toxins was

calculated as follows:

alpha-titre = titre of supernate (for rabbit

erythrocytes) not inhibited by fibrinogen.

beta-titre = increase in titre for sheep

erythrocytes after 1 hour at 4°C.

gamma-titre = titre of supernate (for human

erythrocytes) inhibited by heparin.

delta-titre = titre of supernate (for human

erythrocytes) inhibited by fibrinogen.

Three organisms producing known haemolysins i.e

S. aureus strains NCTC 7121 (alpha haemolysin), NCTC

10345 (delta haemolysin) and NCTC 5664 (beta and

gamma haemolysins) were used as controls in these

as s a y s .

i i . Haemolysis on Sheep Blood A g a r .

Sheep blood agar (5%) was inoculated with 18-22

hour-old cultures grown at 37°C using a Denley

Multipoint Inoculator. After 24 hours incubation at

37°C zones of haemolysis (if produced) were

measured. We differentiated strains on the basis

98

of zone size produced, those producing zones of

clearing of greater than 10 mm in diameter were

regarded as positive. A typical sheep blood

haemolysis plate is shown in plate 1.

iii. Egg Yolk React i o n .

We tested our strains for the egg-yolk reaction

by inoculating (with a Denley Multipoint Inoculator)

egg yolk glucose agar with 18-22 hour-old cultures

grown in IsoSensitest broth at 37°C. The plates

were incubated at 37°C for 48 hours and read.

Production of a zone of opacity was taken as a

positive reaction. An example of an egg yolk

glucose agar plate is shown in plate 1.

i v . Tween 80 hydrolysis.

Lipase production was also detected by

inoculating (as in the egg-yolk reaction) strains

onto Tween 80 medium and incubating plates for 48

hours at 37°C. Tween 80 hydrolysis was detected

as a zone of opacity (due to formation of small

crystals) around colonies. A Tween 80 plate is

shown in plate 1.

v . Pigmentation on Milk A g a r .

Lacey's Milk Agar (Lacey et a l . . 1970) was used

99

Plate 1 Growth of a number of different strains of MGRSA on Milk agar, Tween 80 agar, Egg-yolk glucose agar and Sheep blood agar

Milk agar Egg-yolkglucose agar

Tween 80 Sheep bloodagar agar

100

for pigmentation studies. Milk agar plates were

inoculated using a Denley Multipoint Inoculator

with organisms grown for 18-22 hours at 37°C in

IsoSensitest broth. The plates were incubated for

12-16 hours at 37°C and then left at room

temperature for a further 12-16 hours before

reading. An example of a milk agar plate is

shown in plate 1.

8.5 Plasmid isolation by Gel Electrophoresis,

i . Takahashi and Nagano M e t h o d .

This method is a rapid procedure for isolation

of plasmid DNA and its application to

epidemiological analysis (Takahashi & Nagano, 1984). It

involves alkaline lysis and extraction of denatured

chromosomal (linear) DNA and RNA followed by salt

precipitation of chromosomal and other protein

material.

Cells were grown for 12 hours (shaking at 100

rpm) at 37°C in Nutrient broth no. 2 (Oxoid) and

harvested by centrifugation (3,600 rpm for 15

m inutes). Cell pellets were resuspended in 200 ul

buffer A and transferred to "Eppendorf"

polypropylene centrifuge tubes. These were

centrifuged (low-speed) for 4 minutes in a MSE

Micro Centaur centrifuge, buffer A was decanted and

the cell pellet was thoroughly resuspended in 200

101

ul of lysostaphin solution. This contained 12

units/ml lysostaphin (Sigma Chemicals) in 100 mM

NaCl, 40 mM Tris-NaOH, and 50 mM disodium EDTA at

pH 6.9. The tubes were incubated at 37°C for 10

minutes, and then, 400 ul lysing solution (4% sodium

dodecyl sulphate with NaOH added to a final

concentration of 0.2N) was added. The tubes were

gently rotated so as to mix their contents, and

then they were left to stand at room temperature

for 5 minutes. 300 ul of cold (4°C) buffer B was

added and the tube contents mixed by gentle

rotation, then after standing for 5 minutes on ice

the tubes were centrifuged at low-speed (rtp for

5 minutes). The tubes were then stood on ice for

a further 10 minutes, and then centrifuged at low

speed for 5 mins at 4°C. The supernatant was

decanted to new tubes taking care not to carry

over any white flocculate. An equal volume of

chloroform was added to the supernatant, and

following emulsification (by gently inverting 10

times) the tubes were centrifuged at low speed for

5 minutes at 4°C. 500 ul of the upper aqueous

layer was carefully transferred to a new tube to

which 1 ml of cold (-20°C) ethanol was added.

Following mixing and standing on ice for 5 mins

the precipitate was collected by centrifugation at

low speed for 5 mins at 4°C. After drying to

remove traces of ethanol, the pellet was

redissolved in buffer C. For gel electrophoresis

102

25 ul of sample was mixed with 5.0 ul loading

buffer.

i i . PHLS "Johnson" me t h o d .

This method differed from the Takahashi and

Nagano method primarily in that it uses RNase

(bovine pancreas RNase, Sigma Chemicals) and Protease

(Type XIV, Sigma Chemicals) to remove RNA and

protein impurities.

Strains were grown on blood agar overnight, and

thick suspensions were made in Eppendorf tubes

containing 0.25 ml lysis buffer (2.5 M NaCl, 50 mM

EDTA, pH 7.5). Lysostaphin was added to a final

concentration of 10 units/ml. The tubes were

placed in a 37°C water bath for 20 minutes and

then 0.4 ml lysis buffer (see "Materials 4.0ii) was

added. Following mixing the tubes were centrifuged

at high speed (Eppendorf microfuge) for 50 minutes.

The supernatant was carefully decanted into a new

tube to which RNase (final conc. 100 ug/ml) was

added. After incubation at 37°C for 30 minutes

Protease (final conc. 100 ug/ml) was added, and the

tubes were incubated for a further 30 minutes. An

equal volume of cold (-20°C) isopropanol was added

to the tubes which were left overnight at -20°C.

Plasmid DNA was pelleted by centrifuging (high

speed) for 15 minutes, excess isopropanol was

drained off and the tubes left to dry for 30103

minutes. 10 ul loading buffer was added to the

pellet, time was left for it to dissolve and then

agarose gel electrophoresis was performed on these

mixtures.

iii. Agarose gel electrophoresis.

Electrophoresis was performed using a BIO-RAD

DNA sub cell. Tris-acetate pH 7.8 (buffer A) was

used in the Takahashi and Nagano method and Tris-

borate buffer was used for the PHLS method. 0.9%

Agarose (Sigma Chemicals, molecular biology grade)

gels were used. Electrophoresis was usually carried

out overnight. Gels were stained with ethidium

bromide (0.5 ug/ml) for 30 minutes, rinsed in water

and the DNA visualised for photography (with orange

filter) by a shortwave UV transilluminator.

i v . Plasmid sizing studies.

Three plasmids of known size were run in

parallel with the strains to be tested. The

plasmids used (Sykes & Matthew, 1976) were - plasmid R6K

(26 Md), plasmid RP 4 (36 Md) and plasmid R1 (61 Md).

The size of plasmids isolated from test strains

was estimated by interpolation from a graph of

log^Q size (Md) against distance migrated.

104

RESULTS AND DISCUSSION

1. Antibiotic Resistance in MGRSA — how serious is the problem?

As the 1970s progressed, concern over the

problem of multiple antibiotic resistance in S .

aureus declined to such an extent that this

decade has been referred to as the "Decade of

Complacency" (Shanson, 1981). With the emergence of

MGRSA, the 1980s have witnessed increased activity

and debate pertaining to the problems posed by

multi-resistant S . aur e u s . In particular,

strategies for eliminating and controlling MRSA

have received renewed interest from

microbiologists, clinicians, hospital administrators

and pharmaceutical companies alike (Working Party,

1986 and 1990). Because of the differing

geographical incidence of MGRSA, opinions vary as

to their importance, for example, Lacey (1987)

believes that little action is needed to control

these organisms. Because there has been no

international survey to examine the problem of

antibiotic resistance in MGRSA, we have collected

MGRSA from hospitals worldwide and determined

their susceptibility to a wide range of

antibiotics.

In Table IV the extent of resistance to 21

antibiotics is shown for 100 isolates of MGRSA

105

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115

from 32 centres in 23 countries. Table V

summarises these findings in terms of the

percentage of isolates resistant to individual

antibiotics. Although the incidence of antibiotic

resistance in the MGRSA studied has been

quantified, it is intended that our figures

present a qualitative assessment of the extent

of antibiotic resistance in MGRSA. In

epidemiological parlance "incidence" refers to the

number of new cases (or strains) in a specific

population over a defined time period, and

"prevalence" refers to the total number of

current cases (or strains) of an infection in a

defined population at one point in time. Our

data satisfy neither of these requirements. We

wish to show the nature of particular isolates

that are present, or have been present, in

hospitals in different countries and regions, and

hence obtain an indication of the problem of

antibiotic resistance worldwide. Because of the

ability of MGRSA to cross-infect, and the

increasing international transfer of patients

between hospitals, introduction and spread of

multiple-resistant MGRSA from "foreign" hospitals is

already becoming a problem (Carroll et a l ., 1989).

116

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117

1.1. Resistance to Aminoglycosides in MGRSA.

From the hospitals surveyed, we requested

epidemiologically distinct strains of MRSA which

were resistant to gentamicin. In Europe and

Australia the term "MGRSA" is commonly used,

whereas in the USA the term MARSA is preferred.

This is because in the USA, strains of MRSA

sensitive to gentamicin, yet resistant to other

aminoglycosides (e.g. tobramycin) have been reported

on a number of occasions (Crossley et a l . , 1979;

Rimland, 1987). Consequently, in terms of

aminoglycoside resistance patterns, our collection

is biased to those displayed by MGRSA. Two

strains (thought to be gentamicin-resistant by

their senders) which appeared in our tests to be

gentamicin-sensitive but resistant to tobramycin

and other aminoglycosides have been included for

interest.

Strains exhibiting low-level and high-level

gentamicin resistance have been found. High-level

gentamicin resistance was found in many strains.

MGRSA from Australia and England possessed low-

level (MIC less than 32 mg/1) resistance to

gentamicin. Townsend et a l . (1987) have shown that

strains of MGRSA from hospitals in London and

Eastern Australia are very similar, and these

"epidemic" strains characteristically show low-

gentamicin resistance (Cookson £t a l ., 1986;

118

Townsend et a l ., 1984).

Using the method of Van de Klundert et a l .

(1984) the identity of aminoglycoside-modifying

enzymes responsible for aminoglycoside resistance

has been determined. 43% of gentamicin-resistant

strains produced the bifunctional enzyme APH

( 2 ’’)/AAC ( 6 !), whereas 55% of strains produced

A P H ( 2 f,)/AAC ( 6 ’) and the phosphor ylase APH (31)-

IV. These enzymes are unique to staphylococci

(Miller et______ al. , 1980). APH (2’’)/AAC ( 6 f) has 6-

N-acetyltransferase activity combined with an

ability to phosphorylate gentamicin-kanamycin type

antibiotics (Dowding, 1977).

The 3 ’- phosphotransferase - APH ( 3 ’)-IV

determines high levels of resistance to neomycin

B and kanamycins A, B and C, additionally this

enzyme phosphorylates amikacin although this may

not always result in amikacin resistance

(Courvalin & Davies, 1977). A P H (2")/AAC( 6 ’) and

APH(2")/AAC(6 1 ) + APH(3)-IV were ubiquitous in

occurrence, and no marked geographical trends in

their distribution were apparent. The production

of the adenylating enzyme ANT ( 4 T 4 ff) is

responsible for the susceptibility to gentamicin

and resistance to tobramycin seen in the

Australian and Russian MRSA.

The genetic determinants for the production of

APH ( 2 ,?)/AAC ( 6 f) are located on a transposon- Tn

4001 (Lyon et. a_l. , 1987)- which may be

119

chromosomally inserted or plasmid-located, and

there is considerable evidence that this

transposon is disseminated worldwide (Skurray e_t

al. , 1988; Storrs e_t . , 1988). No specific

survey of the aminoglycoside-modifying enzymes

present in MGRSA worldwide has been published,

however Dornbusch et a l . (1990) have found APH

(2 f?)/AAC (6f) to be the most prevalent enzyme in

aminoglycoside resistant staphylococci, whereas APH

( 2 ,f)/AAC ( 6 ’) + APH (3’)-IV was rarely found.

The aminoglycoside-modifying enzyme profiles

found can be used to interpret the incidences

of resistance to the aminoglycosides shown in

Table IV. Tobramycin and kanamycin modification

is mediated primarily by acetylation- AAC (6’),

whereas gentamicin resistance is produced by

phosphorylation by APH ( 2 ff). As both these

enzymes were found in 98 of the strains studied

there is good agreement with the 98% incidence

of gentamicin/kanamycin (data not shown)/tobramycin

resistance. Both amikacin and netilmicin possess

an amido bond at position 6 of the

aminocyclitol ring which is susceptible to

acetylation by AAC (6'). Similar levels of

resistance to gentamicin, amikacin and netilmicin

have been found, however the degree of amikacin

and netilmicin resistance depends upon the

acetylating activity possessed by APH (2f1)/AAC( 6 f )

of individual strains. The presence of APH ( 3 1)-

120

IV mediates high level resistance to neomycinalthough more attention has been paid to the

phosphorylation of amikacin by this enzyme. There

is good correlation between neomycin resistance

and the presence of APH (3’)-IV, however there is

less correlation between amikacin-resistance and

the presence of this enzyme. Devaud et a l .

(1977) have shown the production of APH ( 3 T)-IV

to have little influence on amikacin resistance.

MRSA are characteristically resistant to

streptomycin, and have been so since their first

isolation in the 1960s. MRSA isolated during the

1960s and early 1970s very often had high level

streptomycin resistance. This resistance was due

to chromosomal mutation, and streptomycin MICs of

greater than 10,000 mg/1 were found (Lacey &

Grinstead, 1973). In strains of epidemic MRSA

isolated from Australia and the U.K since the

mid-1970s low-level streptomycin resistance (ie.

MICs of less than 100 mg/1) has been found

(Cookson e t a l . , 1986; Townsend e t a l . , 1984;

Townsend et a l . t 1987). Many of the MGRSA

from Australia and the U.K which we studied

possessed low-level streptomycin resistance. Low-

level streptomycin resistance was also found in

MGRSA from centres in Brazil, Italy, Japan,

Portugal, South Africa, Switzerland and Turkey.

Low-level streptomycin resistance is due to

121

antibiotic modification, and can be plasmid- or

chromosomally-mediated (Lyon & Skurray, 1987).

1 .2 Resistance to Tetracyclines in M G R S A .

The tetracyclines are a family of closely

related antibiotics which act by attaching to

the bacterial 30S ribosomal subunit preventing

protein transcription resulting in inhibition of

protein synthesis. Tetracycline was first

introduced in 1948, and since this time a number

of derivatives have been developed eg. doxycycline

and minocycline.

A c c o r d i n g to Lacey (1975), MRSA

characteristically showed uniform levels (MIC c.

100 mg/1) of plasmid-mediated tetracycline

resistance. More recently, tetracycline resistance

in MRSA has been reported to be chr omosomally

mediated (Townsend et a l ., 1987). We found 83% of

our MGRSA to be tetracycline-resistant

(MICs of 64-256 mg/1). Tetracycline resistance in

our strains was geographically widespread. Most

of the tetracycline-sensitive strains found were

from countries in Southern Europe (Italy and

Greece) and from centres in the USA.

Minocycline may be active against

tetracycline-resistant S. aureus (Minuth et a l . ,

1974). Two types of tetracycline resistance in

S . aureus have been described (Asheshov, 1975).

122

Firstly, there is plasmid-mediated, inducible

resistance to tetracycline where strains appear

t e t r a c y c l i n e - r e s i s t a n t , m i n o c y c l i n e - sensitive.

Alternatively, there is chromosomally-mediated

constitutive resistance in which strains are

resistant to both tetracycline and minocycline.

A range of minocycline MICs (0.25- 64 mg/1) were

found for the MGRSA we studied. 27 strains were

susceptible to 0.25-4.0 mg/1 minocycline, 10 were

susceptible to 8.0 mg/1, and 61 strains were

susceptible to 16-32 mg/1 minocycline.

Bismuth et a l . (1990) have studied the gene

heterogeneity for tetracycline resistance in MRSA

of French origin. An excellent correlation was

found between tetracycline and minocycline MICs

of the strains studied, and their genotypes

determined by DNA-DNA hybridization. According to

these criteria 52% of our tetracycline/minocycline

resistant strains contained tet(K ) and te t ( M ) , and

48% contained tet(M ) . Both of these genotypes

were geographically widespread. Unlike Bismuth e_t

a l . (1990) who found no minocycline-sensitive,

tetracycline-resistant MRSA, 10% of our strains

were of this phenotype.

123

1 . 3 Resistance to Macrolides, Lincosamides andStreptogramins.

The macrolides, lincosamides and streptogramins

are a structurally unrelated group of antibiotics

which act on the 50S ribosomal site to arrest

protein synthesis. The macrolides have large

oxygen-containing ring structures (a macrocyclic

lactone ring) to which sugars are attached, and

the most widely used representative is

erythromycin. The lincosamides are pyrrolidine-

carbohydrate structures of which lincomycin and

(the more active) clindamycin are members. The

streptogramins (or "synergimycins") contain two

components A and B. Component A is a

"depsipeptideM and component B is a macrocyclic

lactone moiety similar to erythromycin. When

combined in certain proportions, components A and

B act synergistically. The antibiotic

pristinamycin is a streptogramin.

Staphylococcal resistance to the macrolide,

lincosamide and streptogramin (MLS) antibiotics can

be due to alteration (methylation) of the

ribosomal target site resulting in decreased

antibiotic binding, or it can be due to

enzymatic inactivation of the antibiotic (Arthur

e t . » 1987). Methylation of ribosomal RNA

results in resistance to the macrolides,

lincosamides and streptogramin B antibiotics and

124

is referred to as MLSg resistance. MLSg

resistance can be constitutive or inducible. In

the latter case resistance is seen to

erythromycin (a highly active inducer), but not

to other MLS antibiotics (poor inducers).

However, in the presence of erythromycin induction

of resistance takes place to the other MLS

antibiotics (Weisblum, 1984).

Organisms with induced MLSg resistance are

resistant to macrolides, lincosamides and the B-

(erythromycin-like) component of the streptogramins.

Constitutively-resistant organisms show the same

resistance pattern as "induced" organisms. Bacteria

exhibiting MLSg resistance are still susceptible

to the A-component of the streptogramin

antibiotics and the synergistic activity between

A and B components appears to be retained.

91% of our MGRSA were erythromycin-resistant.

Nearly all the erythromycin-resistant strains grew

well in the presence of 125 mg/1 erythromycin,

however strains from Brazil showed reduced growth

in the presence of erythromycin at concentrations

of 8.0 mg/1 or greater. Constitutive resistance

to clindamycin was found in 47 strains, and all

these strains grew well in the presence of 32

mg/1 clindamycin. On disc testing for inducible

resistance 44 strains which had appeared

clindamycin-sensitive by MIC were shown to have

inducible clindamycin resistance. Only 9% of

125

strains were completely sensitive to the

mac rolides.

Inducible or constitutive resistance was found

in strains from many different geographical

regions. Initially, "epidemic" MRSA in the U.K and

Australia were typically constitutively resistant

to the MLS antibiotics (Marples et a l . , 1986) ,

however strains possessing inducible resistance

have since occurred (Cookson et a l . , 1986; Kerr e_t

al. , 1990; Townsend et a l . , 1984). We have also

observed this phenomenon. Our Australian strains,

AS 1 , AS 2 and AS 3 were isolated in 1990 and

showed inducible MLS resistance, whereas strains

AS 6 and AS 10 were isolated in 1984 and were

constitutively resistant. MGRSA from the USA were

constitutively resistant to clindamycin, and this

is a common property of many strains from the

USA (Schaefler et a l . . 1984; Hackbarth & Chambers,

1989a). Nevertheless, clindamycin-sensitive MRSA do

occur in the USA (Smith et a l ., 1988).

Constituitive and inducible resistance to the

MLSg antibiotics may be chromosomally or plasmid

mediated, and a transposon (Tn 551) has been

identified which mediates such resistance (Lyon &

Skurray, 1987).

Resistance to pristinamycin has not been

found. Staphylococcal resistance due to enzymatic

inactivation of pristinamycin has been reported

from centres in France (Le Goffic et a l . , 1977),126

but so far, is a rare phenomenon (Duval, 1985).

1 . 4 . Resistance to Trimethoprim in M G R S A .

Trimethoprim resistance in S. aureus may be

chromosomally or plasmid-mediated. The plasmid-

mediated resistance is due to the production of

a novel dihydrofolate reductase (type SI)

possessing a much reduced affinity for

trimethoprim (Young et a l . , 1987). Plasmid-mediated

trimethoprim resistance was first found in

Australian MRSA and was located on plasmid pSKl

which also carried aminoglycoside resistance (Tn

4001) and resistance to quaternary ammonium

compounds (Tennent et a l . , 1985). Trimethoprim MICs

against strains with plasmid-mediated resistance

are characteristically greater than 512 mg/1.

Prior to the appearance of plasmid-mediated

resistance during the early 1980s, trimethoprim-

resistance in MRSA was characteristically low-

level with MICs ranging from 10-250 mg/1.

Chromosomally-mediated overproduction of bacterial

dihydrofolate reductase is responsible for low-

level trimethoprim resistance, and this type of

resistance has been found worldwide (Amyes & Tait,

1989) .

Only low-level resistance was found in the

strains we studied, and our results in terms of

the distribution of MIC values resembles those

127

obtained by Burdeska and Then (1989) who also

investigated trimethoprim susceptibility patterns

in an international collection of MRSA. Like

these workers we found trimethoprim MICs of 64-

128 mg/1 against the majority of trimethoprim-

resistant strains. For smaller numbers of

strains, MICs ranging from 1.0-32 mg/1 were

found. Burdeska and Then also stress that large

differences occur in the incidence of

trimethoprim resistance between centres and we

have also found this trend. No strains resistant

to greater than 512 mg/1 trimethoprim have been

found. Trimethoprim MICs of 512 mg/1 were found

for seven MGRSA (three from Ireland, two from

Australia and two from France). Performance of

isoelectric focusing by Amyes, Tait and Maple (see

Appendix C) using an in-house developed technique

for the selective detection of type SI enzyme

failed to show this enzyme in the aforementioned

s trains.

1.5 Resistance to Chloramphenicol in M G R S A .

Chloramphenicol is a protein-synthesis inhibitor

which acts by binding to the 50S ribosomal

subunit to prevent transpeptidation. Only one

mechanism of chloramphenicol resistance is

believed to occur in S. aureus (and many other

bacterial species as well). This involves the

128

production of chloramphenicol acetyltransferase

which catalyses an acetyl coenzyme A dependent

acetylation of the antibiotic (Shaw, 1984). In S .

aureus chloramphenicol resistance is invariably

associated with a group of small plasmids of

2.9- 5.1 kilobases in size (Tennent et a l . , 1986;

Lyon & Skurray, 1987). Resistance to chloramphenicol

is known to rapidly appear following substantial

usage of the drug and be lost with decreased

usage (Kirby & Ahern, 1953).

41% of the MGRSA strains we studied were

chloramphenicol-resistant. Chloramphenicol resistance

was found in strains from Africa, Asia, Australia

and Europe. Although no chloramphenicol-resistant

MGRSA were found from the USA, resistant strains

have been reported (Locksley e_t a_l. , 1982;

Schaefler et a l ., 1984).

1.6 Resistance to Rifampicin in M G R S A .

Rifampicin binds specifically to bacterial RNA

polymerase, thus inhibiting DNA transcription.

Bacterial resistance to rifampicin is produced by

chromosomal mutations resulting in decreased

antibiotic binding. Mutation to rifampicin

resistance usually results in high-level

resistance (MICs greater than 512 mg/1 for S .

aureus). However, according to Wehrli (1983)

mutation to rifampicin resistance is not an all-129

or-nothing phenomenon. The susceptibility ofrifampicin-resistant mutants can vary , depending

upon the sensitivity of the bacterial RNA

polymerase to the antibiotic, and also on the

interaction of other factors eg. cellular

permeability.

We found four rifampicin-resistant MGRSA (1

from Greece, 3 from Kuwait) to be susceptible to

1.0 mg/1 rifampicin, and 24 MGRSA resistant to

more than 8 mg/1 rifampicin. In terms of

geographic distribution, most of our rifampicin-

resistant MGRSA came from Brazil, France and

Turkey. The development of rifampicin resistance

is governed by usage of the antibiotic itself.

It is often advised that rifampicin be used in

combination with another antibiotic because large

populations of staphylococci contain resistant

variants which can overgrow following rifampicin

monotherapy.

1. 7 . Resistance to Ciprofloxacin in M G R S A .

The first fluoroquinolone to enter widespread

clinical use was ciprofloxacin during the second

half of the 1980s. Bacterial resistance to the

fluoroquinolones was not expected because of

their unique mechanism of action. In our first

survey (Maple et a l . , 1989c) of the prevalence of

ciprofloxacin resistance in MGRSA we found 17%

130

of strains to be ciprofloxacin-resistant. We have

investigated fluoroquinolone resistance in MGRSA

in much greater detail and our findings are

given in section 3.

1 . 8 . Resistance to Fosfomycin in M G R S A .

Fosfomycin has a totally different structure

from those of other clinically used antibiotics.

It is a comparatively simple molecule - cis- 1,2-

epoxypropylphosphonic acid. Fosfomycin inhibits

cell wall synthesis by irreversibly binding to

phosphoenolpyruvate transferase which is essential

for the synthesis of UDP-N-acetylmuramic acid

(Kahan ejt a_l. , 1974) . For binding to

phosphoenolpyruvate transferase to occur, fosfomycin

must traverse the cell membrane which requires

active transport of the antibiotic. The alpha-

glycerophosphate system, which is constitutive in

fosfomycin-susceptible bacteria has been shown to

transport fosfomycin into the cell. An

additional, inducible pathway- the hexosephosphate

system can also transport fosfomycin when it is

induced e.g by glucose-6-phosphate . Phosphate or

glucose inhibit fosfomycin uptake by the glucose-

6-phosphate pathway, and their presence in varying

amounts in different susceptibility testing media

has produced differing MIC values (Goto, 1977).

Bacterial resistance to fosfomycin can result

131

from prevention of fosfomycin uptake by thesepathways as a result of mutation. Fosfomycin

resistance can be of two types (Courtieu et a l .,

1977): certain mutants are sensitive to fosfomycin

in the presence of glucose-6-phosphate (i.e the

hexosephosphate pathway is still operative) whereas

others are totally resistant (i.e both pathways

are inoperative). We have followed international

guidelines for the determination of fosfomycin

susceptibility (Andrews et a l . , 1983) which specify

the incorporation of glucose-6-phosphate into the

susceptibility testing medium, hence only totally

resistant strains are shown in our results.

Another problem concerning the detection of

resistance is that bacterial populations contain

highly-resistant variants which are selected out

when using inocula of greater than 10~* cfu

(Woodruff et a l . , 1977). For the strains against

which fosfomycin MICs of 32 mg/1 or 64 mg/1

were recorded at an inoculum of c. 10^ cfu, on

repeat testing with an inoculum of 10^ cfu

fosfomycin MICs of less than 32 mg/1 were

found. This inoculum effect was not seen with

strains classified as fully resistant ie. MICs

of greater than 64 mg/1. For the 21 strains of

MGRSA which were classified as moderately or

fully fosfomycin-resistant, only those 14 which

were fully resistant can be regarded as genuine

clinically resistant mutants. These strains came

132

from Brazil (5), Italy (1), Federal Republic of

Germany (1), France (3), Spain (1) and Turkey (3).

S. aureus mutates to fosfomycin resistance at

a high frequency. Courtieu et a l . (1977) have

recommended that fosfomycin should be used in

combination with other appropriate antibiotics.

Plasmid-mediated fosfomycin resistance may also

exist, but this has been difficult to prove

because of problems encountered with recipient

strains mutating to resistance (Baquero et a l . ,

1977). In countries where fosfomycin has been

clinically used, there have been widely differing

reports of the incidence of fosfomycin

resistance. For example, Rodriguez et a l . (1986)

reported no fosfomycin resistance in MRSA from

several Spanish hospitals whereas Igari et a l .

(1988) reported over 80% fosfomycin resistance in

MRSA from Japanese hospitals.

1.9 Resistance to Fusidic Acid in MGRSA.

Fusidic acid belongs to a group of several

structurally related, naturally occuring antibiotics

- the ’fusidanes’. Against bacteria, fusidic acid

acts as a protein-synthesis inhibitor by

interfering with the functioning of ribosomally

associated elongation factor G - "translocase"

(Cundliffe, 1972). Two mechanisms of fusidic acid

resistance in S. aureus have been described133

(Chopra, 1976). Resistance may be due to

chromosomal mutation leading to structural

modification of factor G, or plasmid-mediated

exclusion of the antibiotic from the cell.

According to Shanson (1990) the majority of

clinically isolated resistant strains possess the

plasmid-mediated resistance. Reducing fusidic acid

usage can limit the development and spread of

chromosomal fusidic acid resistance, however, this

doesn't apply to plasmid-mediated resistance.

Plasmid-mediated fusidic acid resistance can be

disseminated on plasmids carrying linked

resistance determinants eg. B- lactamase, heavy-

metal, aminoglycoside resistance (Lyon & Skurray,

1987). Consequently, fusidic acid resistance

(plasmid-mediated) can remain stable and even

increase despite reduced usage of the drug.

This may account for the reports of 80% - 100%

fusidic acid resistance in MRSA from Dublin

(Coleman et a l ., 1985; Morgan & Harte-Barry, 1989).

An overall incidence of fusidic acid

resistance of 15% was found, and this was mainly

due to clusters of resistant strains from

centres in Austria, France, Ireland, Switzerland, USA

and the U.K. Phage typing and biotyping of

these strains suggests that strains in the

clusters are unrelated. This suggests that

resistance has spread to strains, and is not a

result of the spread of a single strain.

134

Shanson (1990) has found a low rate ofresistance emergence for acute courses of fusidic

acid monotherapy, which does not correlate with

the higher rates of resistance implicated by in

vitro testing, and he suggests that the plasmid-

mediated form of resistance is the clinically

significant form.

1 . 10 Resistance to Novobiocin in M G R S A .

Novobiocin is a dihydroxy-glycosylated coumarin

derivative discovered in 1955, and used during

the late 1950s and early 1960s for the

treatment of multi-resistant staphylococcal

infections. Although highly active against

staphylococci usage of novobiocin was greatly

curtailed following the introduction of the

penicillinase-resistant penicillins because of the

development of resistance (Kirby et a l . , 1956).

Treatment with novobiocin also gave rise to an

unacceptable incidence of hepatotoxicity and

sensitivity reactions (Bridges et a l ., 1957).

The mechanism of action of novobiocin was not

fully understood until the discovery of DNA

gyrase in 1976. It was then reported that

novobiocin inhibits DNA gyrase to prevent

supercoiling of bacterial DNA (Gellert et a l .,

1976). It is now known that novobiocin acts on

the beta subunits of DNA gyrase whereas the

135

quinolones act on the alpha subunits (Wolfson &

H o o p e r , 1985) .

Only one strain (strain IR 1) showed reduced

novobiocin susceptibility (MIC 2.0 mg/1) in our

survey. Kirby et a l . (1956) have also reported

low-level resistance (less than 5.0 mg/1) occuring

in patients treated with novobiocin.

Strain IR 1 was completely susceptible to the

fluoroquinolones (ciprofloxacin MIC 0.5 mg/1),

which is as expected because of the different

sites of action of these agents.

There have been no reports of plasmid-mediated

novobiocin resistance, however Schaefler (1982) has

reported the phage-mediated transfer of

novobiocin-resistance at high rates between S .

aureus strains. The clinical impact of transfer

of novobiocin resistance in this manner is not

known. The lack of use of novobiocin since the

1960s may account for the low incidence of

resistance we have found. Similarly, Turnidge et

a l . (1989) only reported 1.2% novobiocin resistance

in 4537 isolates of S. aureus screened in a

national survey of resistance in Australian

hospitals.

1.11 Resistance to Bacitracin in MGRSA.

Bacitracin is a peptide antibiotic, first

discovered in 1945 in culture filtrates of a

136

strain of Bacillus licheniformis isolated from a

tibial wound sustained by a young patient-

Margaret Tracy. Commercial bacitracin is a

heterogeneous mixture of related peptides of

which 60-80% is made up of Bacitracin A.

Bacitracin is too toxic for systemic use,

although it has found application in a number

of topical preparations often in combination with

other antibiotics such as neomycin or rifampicin.

According to Jawetz (1961) bacitracin resistance

is infrequently encountered in susceptible

species, and resistance does not emerge rapidly

during clinical infections. Nevertheless, bacitracin

resistance has been reported in S . a u r e u s , for

example, in neomycin-resistant S. aureus which

caused hospital epidemics during the early and

mid-1960s (Leading Article, 1965). We found

bacitracin resistance in only one strain of

MGRSA.

Currently, bacitracin resistance is only found

in sporadic strains of MRSA in the U.K (Kerr ejt

a l ., 1990). Data relating to the overall

prevalence of bacitracin resistance in other

countries is extremely limited, although when

MGRSA are tested for such resistance they are

usually sensitive (Graham et a l ., 1980).

137

Resistance_________to_________Mupirocin,____ Nitrofurantoin,

Pristinamycin, Teicoplanin and Vancomycin in MGRSA

No resistance was found to mupirocin,

nitrofurantoin, pristinamycin, teicoplanin and

vancomycin in the collection of MGRSA studied.

These results will be discussed in the following

sections together with our further studies of

the antimicrobial properties of these compounds.

1.12. Multiple Antibiotic Resistance in M G R S A .

From Table IV the degree of multiple

resistance in individual isolates to 21

antibiotics has been calculated. Our results are

depicted in Figure 2. The most highly multiple

resistant strains (resistant to 13 or 14

antibiotics) originated from Brazil (3 strains),

France (6 strains), Kuwait (1 strain), Turkey (2

strains), and 1 strain each from Austria,

Switzerland and the USA. The least resistant

strains (resistant to 8 or fewer antibiotics)

originated from Hong Kong (3 strains), Italy (3

strains), U.K (5 strains), USA (4 strains) and there

were also single strains from Brazil, Chile, East

Germany, Russia and South Africa.

Few national surveys of multiple antibiotic

resistance in S. aureus have been reported. The

138

Num

ber

of St

rain

s

Figure 2

Degree off Multiresistance in MGRSA Strains

20 -

15 J

imi l l II1

IPI II IIII iil IIIIII

10 J

6 7 8 9 10 11 12 13 14Total of antibiotics tested to which strains

are resistant or moderately resistant

Med micro Maple DrOrl -2 14.11.90

139

sources and nature of isolates examined in such

surveys can greatly influence the prevalences of

resistances seen. For instance, isolates from out­

patients possess much less resistance than

isolates from in-patients (Kayser, 1975).

Furthermore, MRSA tend to possess more resistance

than MSSA. For example, in a national survey of

S. aureus antimicrobial resistance in Australian

teaching hospitals, Turnidge et a l . (1989) found

that gentamicin and novobiocin resistance were

predominantly restricted to MRSA. The types of

hospitals surveyed in studies of the prevalence

of antimicrobial resistance may also significantly

influence the incidence of antimicrobial

resistance found. Haley et a l . (1982) in a survey

of the emergence of methicillin resistance in

the USA reported that such resistance was

virtually restricted to large tertiary referral

and teaching centres, and that the overall

increases in methicillin resistance seen from

1974 to 1981 were due to substantial increases

in only four hospitals.

Within hospitals, the incidence of

antimicrobial resistance can vary widely between

different specialities; in general, high levels of

antibiotic resistance are seen in staphylococci

from dermatological wards, burns and plastic

surgery units where topical antibiotics are

commonly used and the nature of treatments

140

adopted predisposes to cross-infection. The

adoption of antibiotic policy and use of

effective infection control facilities have

considerably reduced the appearance of multi-

resistant strains (Shanson, 1981). With regard to

all these variables, in our survey of multiple

antibiotic resistance in MGRSA we decided that

in order to reduce bias from any one particular

centre it was necessary to look at small

numbers of isolates from a large number of

centres. Using the data thus obtained we could

then consider the overall problem of multiple

antibiotic resistance in MGRSA.

1.13 Do MGRSA pose a therapeutic problem?

Multiple antibiotic resistance in MGRSA is a

variable phenomenon, and so attitudes to the

treatment of infections due to these organisms

may vary from centre to centre. We have found

numerous patterns of resistance to the antibiotics

studied. Although the degree of multiple

antibiotic resistance observed ranged from 6 to

14 antibiotics, the choice of effective recognised

chemotherapy for systemic infections in many

instances was restricted to rifampicin and

fusidic acid, or to vancomycin. For the highly

multi-resistant strains vancomycin was often the

only available recognised therapeutic option.

141

Little resistance was found to fosfomycin and

novobiocin, and no resistance was found to

nitrofurantoin, pristinamycin and teicoplanin.

Currently, knowledge of the in vitro and in vivo

activity of these agents against MGRSA is

limited. Disturbing levels of ciprofloxacin

resistance were also found, and a re-evaluation

of the use of fluoroquinolones against MGRSA is

necessary. Because of these problems, vancomycin

is still regarded as the antibiotic of choice

for treating infections due to MRSA (Milatovic,

1986) .

142

2. Therapeutic Options for the Treatment of MGRSA Infections or Colonization.

2.0 Current Therapeutic Options for the Treatment

of Infections or Carriage Due to M G R S A .

2.1 Antibiotics Available for Treatment of Systemic

Infections

Clindamycin (Smith et a l . t 1988) and trimethoprim

(Elwell et al. , 1986) have been used successfully

to treat infections due to MRSA. Unfortunately,

staphylococcal resistance greatly limits the use of

these agents. For instance, Markowitz et a l . (1983)

was reluctant to use clindamycin to treat MRSA

infections because of the rapid development of

resistance. Trimethoprim (frequently combined with

sulphame thoxazole in the USA) is as effective as

vancomycin in the treatment of serious MRSA

infections, but widespread trimethoprim resistance

prevents more extensive use of this treatment

regimen (Hackbarth & Chambers, 1989a). Less resistance

was found to chloramphenicol. Rapid development of

resistance following chloramphenicol use combined

with fears over its toxicity, and lack of efficacy

against MRSA (Cafferkey et a_l. , 1985) make

chloramphenicol an unattractive therapeutic option.

Fosfomycin, fusidic acid, nitrofurantoin, novobiocin,

pristinamycin, rifampicin and teicoplanin appear to

143

be antistaphylococcal agents of potential value in

the treatment of MRSA infections. Little resistance

was found to these agents, and relative to

vancomycin they are less toxic. The inhibitory

activities of fosfomycin, fusidic acid,

nitrofurantoin, novobiocin, pristinamycin, rifampicin

and teicoplanin compared to vancomycin against 100

strains of MGRSA of diverse origins are shown in

Table Via. In Table VIb the influence of inoculum

size on inhibitory activity is shown, and in Table

VII the spontaneous rates of mutation to

resistance to low and high concentrations of

antibiotic are given. In figure 3, the bactericidal

activity of the antibiotics with time is

portrayed.

i . Antistaphylococcal Activity of Fosfomycin.

The activity of fosfomycin against MGRSA is

substantially decreased on increasing inoculum size

(Table VIb). Goto (1977) has found likewise.

Fosfomycin was bactericidal at concentrations two

to four times MIC in replica-plating studies.

Graninger et a l . (1984) and Lau et a l . (1986) have

also reported fosfomycin to be bactericidal. In

time-kill studies (figure 3a), fosfomycin initially

reduced the inoculum (10^ cfu) 10 to 100-fold,

however regrowth by resistant mutants then took

p l a c e .

144

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146

Table VIIMutation Rates to Resistance to Fosfomycin, FusidicAcid , Nitrofurantoin. Novobiocin , Pristinamycin,Teicoplanin or Vancomycin for 3 Sensitive Strains of MGRSA.

Antibiotic

Fosfomycin + G6P*

C o n c n . (mg/1)

100500

Mutation rates after 48h incubn

RLG

6.0 x 10-7 0

AUS3 RM1

1.5 x 10~6 5.3 x 10"70 0

Fosfomycin 100- G6P 500

1.1 x 10"6 1.5 x 10“ 7

1.8 x 10“60

1.21 . 0

x 10“ 6 x 10"6

Fusidic acid 2 10

6.8 x 10“ 7 4.0 x 10~8 1.5 x 10"71.9 x 10"7 1.3 x 10"8 4.0 x 10~8

Nitrofurantoin 60 300

Novobiocin

Pristinamycin

Rifampicin

Teicoplanin

Vancomycin

4204

202

10

5 25

525

00

7.6 x 10-9 0

00

7.6 x 10"82.3 x 10~8

00

00

00

2.5 x 10~8 0

00

7.7 x5.1 x

00

00

10” 810“ 8

00

00

00

3.4 x 10"72.2 x 10"7

00

00

* G6P = glucose-6-phosphate

0 = no mutant colonies grew from an inoculum of approximately 5.0 x 109 cfu.

147

High spontaneous rates of mutation to resistancewere determined (Table VII) for fosfomycin in the

absence of glucose-6-phosphate. Phenotypically, the

mutant colonies varied in size between pin-point

and small, and 48 hours incubation was required to

enable accurate counting of the number of mutants

produced. In the presence of glucose-6-phosphate (25

mg/1) fewer mutant colonies were found, and these

were mostly of pin-point size. These observations

can be explained as follows. For staphylococci in

the absence of glucose-6-phosphate (G6P) prevention

of fosfomycin uptake by the alpha-glycerophosphate

system is only required, whereas in the presence

of G6P prevention of uptake by the hexosephosphate

system is necessary as well. Inhibition of one

pathway will allow some growth, however inhibition

of both will enable growth to proceed only very

slowly. According to Woodruff et al (1977) such

fosfomycin-resistant mutants are of reduced

virulence, and in vivo do not appear to present

the number of problems that would be expected on

the basis of in vitro data.

Fosfomycin has been used extensively in Japan

and Spain, however it is not available for

clinical use in countries such as Australia, U.K

and the U.S.A. Published reports of the clinical

efficacy of fosfomycin in the treatment of

staphylococcal infections are sparse. However, Lau

e t a l . (1986) have used fosfomycin successfully to148

treat three patients with MRSA septicaemia.

Fosfomycin appears to be a promising

antibiotic, but more information on its clinical

efficacy is required together with guidelines

suggesting the most appropriate antibiotics with

which to combine it.

i i . Antistaphylococcal Activity of Fusidic A c i d .

Using an inoculum of 10^ cfu, 90% of the 100

strains of MGRSA studied (Table Via) were inhibited

by 0.7 mg/1 of fusidic acid. Reports of similar

good activity against MRSA have been made by

Guenther and Wenzel (1984) and Verbist (1990). No

substantial increase in MIC was found on

increasing the inoculum size from 10^ to 10^ cfu

(Table VIb) , however there was a two-fold increase

in MIC on increasing the inoculum from 10^ to

10^ cfu. Hilson (1962) reported fusidic acid to

produce a striking inoculum effect with large

inocula (10^ cfu) which he believed was due to an

alkaline reaction in the nutrient medium resulting

in fusidic acid degradation.

Various claims have been made concerning the

bactericidal activity displayed by fusidic acid.

Initially, fusidic acid was shown to be

bactericidal (Chabbert 1953), however the cellophane

transfer method he used to obtain these results

149

was unreliable because of a high degree of

antibiotic carry-over (Barber & Waterworth, 1962).

Chokkavelu et a l . (1984) have also reported fusidic

acid to be bactericidal against MRSA, however many

workers consider fusidic acid to be bacteriostatic

(Hilson, 1962; Verbist, 1990).

In our replica-plating experiments, fusidic acid

was not bactericidal, although at 8.0 mg/1 a

reduction in bacterial numbers was apparent. In

the time-kill studies (figure 3b) fusidic acid at

10 mg/1 reduced the initial inoculum 10 to 100 -

fold (see figure 3b) after 8 hours, but by 24

hours regrowth by resistant mutants occurred.

Godtfredsen et al. (1962) performed time-kill

experiments with an inoculum of 10^ cfu/ml and

found a similar initial rate of killing to ours

with no subsequent overgrowth, however with larger

inocula overgrowth by resistant mutants took place.

Mutation to fusidic acid resistance readily

occurs in vitro (Table VII), but according to

Shanson (1990) such variants are not as significant

a cause of clinical resistance as would be

expected. Fusidic acid has been used with success

to treat MRSA infections although to prevent the

development of resistance its use in appropriate

combination is recommended (Jensen, 1968).

150

iii. Antistaphylococcal Activity of Nitrofurantoin.

In the 1940s, the nitrofurans were first

recognised as potentially useful antimicrobial

agents, and since this time several thousand

compounds belonging to this class have been

synthesized (Chamberlain, 1976). Despite the large

number of nitrofuran compounds which have been

evaluated as antimicrobial agents, only three -

furazolidone, nitrofurantoin and nitrofurazone have

seen widespread clinical use. Nitrofurazone is a

topical agent (see section 2.3i), furazolidone is

only used for the treatment of gastrointestinal

infections, and nitrofurantoin only achieves

therapeutically useful levels in urine and bile.

Nitrofurantoin has good antistaphylococcal

activity (Table Via). Like us, Chamberlain (1976) has

found S. aureus to be inhibited by 6.0- 12 mg/1

nitrofurantoin. No inoculum effect was detected for

nitrofurantoin. Nitrofurantoin was bactericidal in

both replica-plating (MBCq q , 20 mg/1) and time-kill

studies (figure 3c). Herlich and Schweiger (1976) have

reported nitrofurantoin to exert a bacteriostatic

effect by blocking the initiation of translation,

and also to be bactericidal by inducing non­

r e p a y a b l e DNA lesions. In our time-kill studies

(figure 3c) one strain (SA 1) overgrew in the

presence of 32 mg/1 nitrofurantoin. This strain

had developed reduced susceptibility to

151

nitrofurantoin (MIC = 160 mg/1). Overgrowth bystrain SA1 did not occur in broth containing 64

mg/1 nitrofurantoin.

An attractive property of nitrofurantoin is the

lack of development of bacterial resistance in the

clinical situation despite 40 years of use. For

example, Schneierson (1960) in surveys conducted

during the 1950s found little resistance to

nitrofurantoin in S. au r e u s . No nitrofurantoin

resistance was found in the MGRSA we studied.

Peacock et a l . (1980) reported nitrofurantoin to be

active against more than 90% of MGRSA tested,

however, on the basis of disc susceptibility

testing 7.5% of MGRSA were resistant. McOsker e_t

al (1990) have suggested that the lack of bacterial

resistance is because nitrofurantoin binds non-

specifically to almost every ribosomal protein thus

having multiple sites of action.

i v . Antistaphylococcal Activity of Novobiocin.

Novobiocin was uniformly active against the

MGRSA studied (Table Via). Walsh et a l . (1985)

reported a novobiocin MIC q q of 0.25 mg/1 against

MRSA, we obtained a MIC^ q of 0.23 mg/1 against

our strains. No inoculum effect was seen for

novobiocin (Table V I b ) . In replica-plating studies

novobiocin was mainly bacteriostatic, although at

higher concentrations (eg. 8.0 mg/1) some bactericidal

152

activity was observed. In time-kill studies (figure

3d), novobiocin initially slowly killed bacteria,

however this was then masked by overgrowth of

resistant mutants. Johnston et a l . (1987) have also

reported overgrowth by resistant mutants in time-

kill studies.

A spontaneous frequency of mutation to

novobiocin resistance of 2.5 x 10“ ® - 7.6 x 10“9 was

detected for low concentrations of novobiocin (4 mg/1),

but no resistant mutants grew at higher novobiocin

concentrations (20 mg/1). Previous experience with

novobiocin monotherapy has shown that staphylococcal

resistance to novobiocin can readily develop (Kirby

et a l . . 1956; Ward et a l .. 1981). Use of novobiocin

in appropriate combinations is recommended. Arathorn

et a l . (1990) have reported that short courses of

oral novobiocin-rifampicin are effective in

eradicating MRSA carriage.

v . Antistaphylococcal activity of Pristinamycin.

Pristinaraycin belongs to a group of antibiotics

formerly known as the "Virginiamycins", but nowadays

more often referred to as the "Streptogramins" or

"Synergistins” .

Few reports of the properties of pristinamycin

are available in English literature. This is

perhaps because pristinamycin has only been used

in France and a number of other continental

153

European countries. Pristinamycin had good activity

against MGRSA (Table V i a ) , and both erythromycin-

sensitive and erythromycin-resistant strains were

susceptible. Our findings agree with those of

Maskell et a l . (1988) who reported pristinamycin

M I C q q S of 0.4 mg/1 against erythromycin-sensitive

and erythromycin-resistant MRSA. No inoculum effect

was found for pristinamycin (Table V I b ) , and this

agrees with the findings of Barber and Waterworth

(1964) .

Pristinamycin was bactericidal against some

strains in both replica-plating and kill-curve

studies. This was investigated further to determine

whether there was a correlation with macrolide and

lincosamide sensitivity status. In replica-plating

studies pristinamycin was bactericidal against

erythromycin-sensitive and inducibly (MLSg) resistant

strains. Pristinamycin was not bactericidal against

strains possessing constitutive MLSg resistance.

Similar trends were found in our time-kill studies

(figure 3e) with pristinamycin at a concentration

of 4.0 mg/1.

Pristinamycin resistance was not found in the

MGRSA we surveyed, and no resistant mutants were

obtained in our spontaneous mutation rate

experiments (Table VII). Clinically, resistance to

pristinamycin does not appear to be a problem as

there have only been a few, sporadic reports of

resistant clinical isolates (Petit et a l . , 1983;

154

Duval, 1985). Pristinamycin is a safe, well-tolerated

antibiotic useful in the treatment of

staphylococcal infections (Bastin et a l ., 1982).

v i . Antistaphylococcal activity of Rifamp icin.

Like us (Table V i a ) , numerous workers have found

rifampicin to be extremely active against MRSA.

Rifampicin is bactericidal at near MIC

concentrations, however in time-kill studies (figure

3f) an initial reduction of inoculum was followed

by overgrowth of resistant mutants even at

concentrations substantially greater than MIC.

A spontaneous frequency of mutation to

resistance of c. 10“® was found in our experiments

(Table VII). Moorman and Mandell (1981) have

detected similar mutation rates to ours.

Unfortunately, development of staphylococcal

resistance to rifampicin in the clinical

situation (Binda et a l ., 1971) has necessitated that

rifampicin be used in appropriate combination

(Kapusnik et a l . t 1984; Eng et a l . , 1985). Despite

its good antistaphylococcal activity, rifampicin was

initially regarded as primarily an anti-tuberculous

agent (Garrod et a l . , 1981), and even today its

potential efficacy in treating staphylococcal

infections is not fully recognized (eg. Chambers,

1988). Other workers (e.g. Acar et a l ., 1983; Clumeck

et a l . , 1984) have shown rifampicin to be a most155

useful component of successful combined chemotherapy

against MRSA.

v i i . Antistaphylococcal activity of Teicoplanin and

Vancom ycin.

Vancomycin is currently the antibiotic of choice

for treating systemic infections caused by

methicillin-resistant staphylococci (Watanakunakorn,

1982; Kucers, 1984; Cafferkey et a l . , 1985b). When

vancomycin was first introduced into clinical use

a significant number of side effects were reported

(e.g. thrombophlebitis, flushing and rashes were

common). Additionally, vancomycin was also shown

to be nephrotoxic and ototoxic, and there had been

a number of reports of histamine-release reactions

-"red- man syndrome" (Farber, 1984). Initially,

vancomycin preparations were of poor purity, the

term "Mississippi Mud" was used to describe some of

the earliest. Nowadays with advances in production

and purification methods vancomycin is available as

a white powder. Slow intravenous vancomycin

infusion over a period of one hour along with

monitoring of antibiotic levels has resulted in a

major reduction of antibiotic-associated side

effects.

Teicoplanin, like vancomycin, is a glycopeptide

antibiotic, however teicoplanin can be injected (once

daily). There is some debate as to the clinical

156

efficacy of teicoplanin (Calain & Waldvogel, 1990).

Some workers (eg. Glupczynski et a l . t 1986) have

reported an unacceptable number of treatment

failures in serious infections which have been

mostly attributed to inadequate serum levels.

We found MIC q q S for teicoplanin and vancomycin

of 0.9 and 1.7 mg/1 respectively (Table Via). Many

other workers have reported similar results for S .

aureus (Watanakunakorn, 1981; Greenwood, 1988).

Vancomycin was slightly less active than

teicoplanin, and for both antibiotics a negligible

inoculum effect was found (Table VIb).

In our replica-plating studies teicoplanin and

vancomycin had variable bactericidal activity (Maple

et a l . , 1989a). The degree of bactericidal activity

exhibited by these antibiotics depended upon the

time at which replica-plating was performed (see

Table VIII). For example, teicoplanin and vancomycin

had MBC q q S of 19.0 and 13 mg/1 respectively at

24 hours incubation, however on replica-plating

after 26 hours incubation MBCq q S of 4.0 mg/1 and

2.5 mg/1 respectively were found.

Time-kill studies (figure 3g) also showed a

variable bactericidal effect depending upon

glycopeptide concentration, and the time of exposure

to glycopeptide. Vancomycin produced a greater rate

of killing than teicoplanin, and for both

antibiotics the time required to achieve a 99.9%

kill depended upon the concentration used (figure 3g).

157

Variable MBCs of vancomycin against MGRSA have

also been reported by Foldes et a l . (1983) and

Traub et a l . (1984). Lagast et a l . (1986) have also

shown vancomycin to be more rapidly bactericidal

than teicoplanin. On the basis of our 22 hour

readings for teicoplanin and vancomycin (Table VIII)

some of our strains could be described as

"tolerant" (a ratio of MBC to MIC of greater than

32). The finding of "tolerance” has been implicated

as a cause of sub-optimal clinical response with

glycopeptide therapy (Sorell et a l . , 1982). Kaye

(1980) has questioned the clinical significance of

"tolerance". Some of our strains could be

described as "tolerant" at 22 hours but not after

26 hours exposure to vancomycin. Like others

(Pelletier, 1984), we feel that the laboratory

aspects of "tolerance" need further clarification.

Vancomycin is the mainstay of chemotherapy of

serious infections due to MRSA, and also of

empirical therapy in the absence of susceptibility

data. It is of major importance to microbiologists

and clinicians alike that vancomycin resistance

does not develop in clinical isolates of S .

a ureus. In vitro vancomycin resistance is very

difficult to produce (Griffith, 1984; Watanakunakorn,

1988). A number of workers (Watanakunakorn, 1990)

have found it easier to produce in vitro

resistance to teicoplanin. Furthermore, of great

concern is a recent report by Kaatz et a l .(1990a)

158

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of the clinical development of teicoplanin

resistance in S. a ureus. Sporadic resistance to

teicoplanin and vancomycin has been reported in

coagulase-negative staphylococci (Johnson et a l . ,

1990). Also of great concern to microbiologists

are recent reports of outbreaks of infection with

enterococci possessing transferable vancomycin

resistance (Johnson et a l .t 1990).

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developing resistance to a wide range of

antimicrobial compounds. The emergence of vancomycin

resistance in these organisms would seem to be

inevitable.

160

Log

cFu/ml

F I G U R E 3

Graph A

Killing curves for Fosfomycin at 25 mg/l

______________-A

Strain3-

A C o n t r o l S O X- n o a n t i b i o t i c a d d e d

1 .

T --------1---------- 7 ---------1 -6 8 24

Time (hours)

Log1

0 cF

u/m

lF I G U R E 3

Graph BKilling curves for Fusidic acid at 10mg/l

4.

2 .

Strain-■-- RMI

A U S 1 0-A— sox- A C o n t r o l S O X

- n o a n t i b i o t i c a d d e d

1 .

~ l --------------1-------------------1--------------T "

2 4 6 8 24

Time (hours)

Log1

0 cF

u/m

l

F I G U R E 3

Graph CKilling curves for Nitrofurantoin at 32 mg/l

1 0-,

5-

StrainR M IS A IS O X

^ C o n t r o l S O X- n o a n t i b i o t i c a d d e d

,_ _ K illing c u r v e o f 6 4 m g / l n i t r o f u r a n t i o na g a i n s t s t r a i n S A I

24Time (hours)

Log1

0 cF

u/m

lF I O U T R E 3

Graph DKilling curves for Novobiocin at 1 mg/l

10

2 -

1 .

Strain- ■ - - R M I- • - - S A I- A — S O X

A C o n t r o l S O X- n o a n t b i o t i c a d d e d

Time (hours)

— r~24

Log.

cFu/

ml

F I G U R E 3

Graph EKilling curves for Pristinamycin at 4 mg/l

10

Strain □ - - - H K 2 ( e r y t h S ) ® - - - S A I ( e r y t h R , d i n d S ) ■ - - R M I ( e r y t h R , c l i n d R ) ▲ - - S O X— - A C o n t r o l S O X

- n o a n t i b i o t i c a d d e d

4.

24Time (hours)

Log

cFu/

ml

FIGURE 3

Graph F

Killing curves for Rifampicin at 5 mg/l

Strain ■ - - R M I • - - S A I ▲ - - S O X A C o n t r o l S O X

- n o a n t i b i o t i c a d d e d

24Time (hours)

166

Log1

0 cF

u/m

l

F I G U R E 3

Graph G

Killing curve for strain SAI exposed to various concentrations of Teicoplanin and Vancomycin

- # - - T e i c o p l a n i n 4 m g / l- • — T e i c o p l a n i n 1 6 m g / l- • T e i c o p l a n i n 6 4 m g / l- O V a n c o m y c i n 4 m g / l- O — V a n c o m y c i n 1 6 m g / lO - - V a n c o m y c i n 6 4 m g / l- n C o n t r o l S A I - n o A n t i b i o t i c a d d e d

Time (hours)

167

2.2 New Antibiotics active against MGRSA.

During recent years a number of new antibiotic

moieties have been discovered which have shown

good activity against MRSA. Most notable of these

are the daptomycins (daptomycin), lipoglycodepsipeptides

(ramoplanin), oxazolidinones (DuP 105 and DuP 721) and

paulomycins (paldimycin). The activity of these

antibiotics against MGRSA is shown in Table IX.

There have also been attempts to modify the

structure of existing antibiotics to produce

compounds possessing greater antistaphylococcal

activity, or more amenable pharmacokinetics for the

treatment of staphylococcal infections. Examples of

these are the chemical synthesis of a water

soluble pristinamycin (RP 59500), and the development

of new 14, 15 or 16-membered macrolides.

i . Antistaphylococcal Activity of Daptomycin.

The antibacterial activity of daptomycin (an

acidic lipopeptide compound) is primarily due to an

interaction with cellular lipoteichoic acid (Canepari

e t a l ., 1990). Daptomycin is active against both

aerobic and anaerobic Gram-positive bacteria, however

it has little activity against Gram-negative

organisms. The in vitro activity of daptomycin is

enhanced by supplementing susceptibility testing

media with calcium chloride (Eliopoulos et a l . ,

168

1986). Andrew et a l . (1987) have reported that the

enhancement of activity is specifically produced by

calcium ions. Hence, daptomycin MICs vary with the

calcium ion content of the susceptibility testing

medium used, and these can vary greatly. For

example, IsoSensitest agar contains only 1.55 mg

Ca++ /1 compared with 26.4 Ca++ in Mueller Hinton

agar, furthermore these amounts may show

considerable batch to batch variation.

In our studies (Table IX) daptomycin was

uniformly active against MGRSA of diverse origins.

We obtained a MIC^ q of 1.4 mg/l and MIC^ q of

2.6 mg/l in IsoSensitest agar supplemented with

10 mg/l Ca + + (as recommended by Andrew et a l . ,

1987) compared to MICs of greater than 8 mg/l in

non-supplemented media. Our results are similar to

those obtained by Andrew et al (1987) against MRSA.

In replica-plating studies (Table I X ) , daptomycin was

bactericidal at MIC or near MIC concentrations.

Van der Auwera (1989) has reported daptomycin to

be rapidly bactericidal against MRSA.

Preliminary human pharmacokinetic data indicate

that daptomycin achieves plasma levels of 8.5 mg/l

one hour following intravenous dosing of lmg/kg

(Black et a l . , 1986). In an animal model of

methicillin-resistant S. aureus pneumonia, treatment

with daptomycin was of similar efficacy to

treatment with vancomycin (Kephart & Esposito, 1988).

Initial clinical trials with daptomycin were

169

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170

Table IX

prematurely suspended in June 1988 because of

unexplained treatment failures in patients with

endocarditis or bacteraemias caused by Gram-positive

cocci. It is believed that these failures were

due to a profound diminution of bactericidal

activity in the presence of albumin (Garrison ejt

a l . , 1990). Currently, clinical trials are underway

in which a more aggressive dosing strategy has

been adopted, however problems with toxicity at

these increased doses have been encountered.

i i . Antistaphylococcal Activity of DuP 721 and DuP 10 5 .

DuP 721 and DuP 105 are members of a new

series of synthetic antimicrobial agents - the

oxazolidinones. These agents are principally active

against Gram-positive cocci (Slee et a l ., 1987).

DuP 721(p-acetylphenyloxooxazolidinylmethylacetamide)

has consistently been shown to be more active

than DuP 105 which is a methy1-sulfinyl derivative

(Slee et a l . , 1987; Neu et a l . , 1988). Like Daly

et a l . (1988) we found DuP 721 and DuP 105 to be

bacteriostatic (Table IX).

Both DuP 721 and DuP 105 showed uniform

activity against MGRSA of diverse geographical

origins which possessed resistance to many classes

of antimicrobial agents. The absence of cross­

resistance may be because the functional

oxazolidinone group of the oxazolidinones is only

171

rarely found in pharmaceuticals (Brumfitt & Hamilton-

Miller, 1988). Little human pharmacokinetic data is

available for DuP 721 and DuP 105, and there have

been no reports of clinical trials conducted with

these agents.

iii. Antistaphylococcal Activity of Paldimycin.

Paldimycin is a paulomycin-related antibiotic

composed of two closely related substances (U 67963

and U 67964) derived from paulomycins A and B

(Argoudelis et a l . , 1987a). Paldimycin, like the

corresponding paulomycins, is sensitive to heat and

acidic or alkaline environments (Argoudelis et a l .,

1987b); furthermore, the activity of paldimycin is

also medium dependent (Rolston et a l . , 1987).

Paldimycin showed greatest activity in Antibiotic

Medium No. 2 (pH 6.8), in which we determined a

MIC^ q of 0.27 mg/l and MIC^ q of 0.75 mg/l

following incubation at 37°C for 18 h (Table IX).

In replica-plating studies, paldimycin was

bactericidal against most strains tested at near

MIC concentrations. Using nutrient broth (pH 6.8)

Chandrasekar and Sluchak (1989) have also reported

similar paldimycin activity against MRSA. Very

little pharmacokinetic or clinical data is

available on paldimycin, and it seems that the

commercial development of this agent for

clinical use will not be progressed.

172

i v . Antistaphylococcal Activity of Ramoplanin.

Ramoplanin (formerly called A 16686 and MDL 62198)

is a complex of three closely-related polypeptides

produced by Actinoplanes sp. ATCC 33076 (Cavalleri

et a l . , 1984). The manufacturers of ramoplanin

recommend that it be called a

"lipoglycodepsipeptide" . Ramoplanin specifically

inhibits bacterial cell wall synthesis (Cavalleri e_t

a l . , 1984), and its activity is primarily restricted

to Gram-positive bacteria (Pallanza et a l . t 1984).

We have found ramoplanin to be highly, and

uniformly active against MGRSA (Table IX). In

replica-plating studies ramoplanin was bactericidal

at near MIC concentrations (Table IX), and time-

kill studies (figure IVe) showed it to produce

99.9% killing in 4 hours or less. A number of

workers (Neu & Neu, 1986; Jones & Barry, 1989) have

reported similar findings.

Cross-resistance of ramoplanin with currently

used antibiotics has not been found. Little data

is available regarding the pharmacokinetics and

clinical efficacy of ramoplanin, although it has

been suggested by O'Hare et a l . (1990) that

ramoplanin will probably be used as a topical

ag e n t .

173

v • Ant istaphylococcal Activity of new 14, 15 and16-membered macro lides.

Recently, a number of new macrolides have been

developed which can be differentiated by the

number of carbon atoms constituting the macrocyclic

lactone ring. Erythromycin is a 14-membered

macrolide, and although alterations have been made

to its formulation it is believed that new

compounds with improved activity (eg. against H .

influenzae) and pharmacological properties (eg. longer

serum half-life) can be developed to replace it.

A number of new 14-membered macrolides such as

clarithromycin, dirithromycin and roxithromycin have

been found to possess improved pharmacokinetics

relative to erythromycin. None of these compounds

are markedly more active than erythromycin against

S . a ureus, and none are active against erythromycin

resistant strains. Similarly, azithromycin (a 15-

membered macrolide) showed little activity against

MGRSA (Table X).

The 16-raembered macrolides such as miocamycin,

rokitamycin and spiramycin are active against MRSA

possessing inducible MLSg-type resistance. Our

findings on the activity of these compounds

against MGRSA are shown in Table X. MGRSA are

most susceptible to rokitamycin followed by

miocamycin and then spiramycin. Table X shows the

importance of determining the erythromycin

174

sensitivity status of the test panel of strains.If the majority of strains used are constitutively

resistant then neither agent would appear to have

useful activity against MRSA. Hardy et a l . (1988)

reported miocamycin, rokitamycin and spiramycin to

have little activity against MRSA, because most of

the strains tested were constitutively resistant.

Spiramycin has been succesfully used to treat

staphylococcal infections (Macfarlane et a l . , 1968).

Spiramycin is not a new antibiotic, however it is

only available in certain countries (eg. France). We

have found (Table V) constitutive MLSg resistance in

47% of MGRSA from worldwide sources, and in view

of this there is only limited scope for use of

16-membered macrolides in the treatment of MGRSA

infections.

v i . Ant istaphylococcal Activity of RP 59500 - an

injectable streptogramin.

Streptogramins (eg. pristinamycin) can only be

administered by the oral route, which limits their

use in severe infections. We have previously

observed that pristinamycin has good activity

against MGRSA (Table Via) and that staphylococcal

resistance does not appear to be a problem (Table

V). To utilise these attributes of pristinamycin,

Rhone Poulenc Sante are in the process of

developing a water soluble "pristinamycin"- RP 59500.

175

to ►a CO to 3E > M > o CD 53 >►d 3 >d o H* N 3 z o d »d oH' H- *- o H- *< H 3 pi-cn co d H- o pi­ rt t—t CO pi­ cn H*CD c+ p c+ p s' 3* w c+ s' CD <Ol H- B p B 3 d M H* d cn H*o 3 B *< 0 0 o pi- o o pi-o p o *< o B B H 3 3 o <3 H* o H- *< *< M t+ '<Vj 3 H- 3 o 0 o H* oo 3 H- H' < H* p 0H- 3 3 CD 3 R Hj3 M p

CO 3 I-4o o 4- o o o o 3 CD CO 4»-• • • • • • • w d 3 c+ »h-4 I—4 CO I—4 CD CO CO o CD CO00 00 cn CO cn CO CO H* H-*o 3 co CD H- pi- p cnc+ 3 CO H* 3II d CO e+ <!p H* p A pI-4 H* c+ 3 H* 3►—4 3 H* Pi­ 3 o*3 0) < CO pi-o o -0 o I—4 o o w CD c+ CD• • • • • • • o co d d I-4to to o 4*. ~o 4- 4* (£> pi- p 3 osCO CO Oi CD © d H- p 1p 3 c+H- CO H* 33 • 0 (DCO 3 B3 • CO p o'3 CD H* o I-4 CDV V M CO 3 do o 4*. o I-4 o H- a CD• • • • • CO CO cn co 3 H* o aH-4 I—4 00 I—4 o CO to o r+ o 3 o00 cn CD 3 p H- 0-> 1—13 cr 3 M 3II r+ I-1 o a> pCD H- o oCO o' c+ dCO CO -—- I-4 H* o3 c+ a: < o I-*V V w d t-4 • 3 H*o o -o o H4 o p CO- a• • • • • CO CO CO H- w d CDto to to £» 00 CO CO o 3 >_- CD o CO4- to CO CO Hj •H*CO dc* 3p o H*3 53 COc+ CO C+3 > H-3 CD 3o o V V V V V M CO o CO p• • o H« o pi- o B4>> CO CO CO CO CO CO cn CO 3 d o <00 CD to to to to CO o c+ co p B o3 p pi- H* d H*3 H* 3 d 3II r+ c+ CO H*3 COCO c+ H* pcn CO H- p 3 33 r+ < 3 r ao o V V V V V M 3 CD a• • o p00 CD CO CO CO CO CO CO H- CO h-4CO to to to CO CO o 3 cn CO

176

Table

RP 59500 has been produced from semi-synthetic

modifications of the two major components of

pristinamycin, and the selection and combination of

two promising derivatives RP 57669 and RP 54476 to

yield RP 59500 methanesulfonate (Barriere et a l . ,

1990).

RP 59500 showed comparable activity to

pristinamycin against MGRSA possessing inducible and

constitutive MLS resistance (Table X). Like us,

other workers have reported RP 59500 to have good

activity against MRSA (Mitsuhashi et a l .,1990).

v i i . The lack of new agents available for use in

the treatment of infections due to M G R S A .

Although daptomycin, the oxazolidinones, paldimycin,

ramoplanin and the new macrolides showed good

activity against MGRSA, none of these agents

currently appears to offer the clinician a future

alternative choice to vancomycin. Daptomycin has

shown disappointing efficacy in clinical trials, and

there is concern over toxicity associated with

higher doses. The development of the oxazolidinones

and paldimycin does not appear to have been

progressed by their respective manufacturers.

Ramoplanin is a promising compound, but will most

likely be only available for topical use. The new

14- and 15-raembered macrolides, although having

improved pharmacological properties are no more

177

active against MGRSA than erythromycin. The 16-

membered macrolides (josamycin, miocamycin and

rokitamycin were more active against MGRSA, however

approximately 50% of the strains were still

resistant. Finally, RP 59500 may be an alternative

agent to vancomycin in the future, but its

efficacy still has to be determined by clinical

trials, at least in countries where it is still

not available.

178

2.3 Topical Antibiotics for the Treatment of MRSAC a r r i a g e .

The control of outbreaks of MRSA

infection/colonisation is expensive both in terms

of the logistical requirements necessary for

preventing cross-infection (Mehtar et a l , 1989), and

the increased costs in terms of prolonged length

of stay and more expensive antibiotic treatments

associated with such outbreaks (Cheng & French, 1988).

Guidelines (Working Party, 1986; Working Party, 1990)

issued by a combined working party of the

Hospital Infection Society and British Society for

Antimicrobial Chemotherapy recommend that patients

whether infected or colonised with MRSA should be

immediately isolated, and that every attempt should

be made to eradicate carriage by use of topical

antibiotics and/or antiseptics. Furthermore, staff

may be carriers of MRSA, and it is imperative in

order to control the dissemination of infection

and prevent their prolonged removal from duty, that

effective eradication can be achieved.

There have been conflicting reports of the

efficacy of antiseptics in eradicating MRSA

carriage. Bartzokas et a l . (1984) and Brady et a l .

(1990) have reported triclosan to be effective in

clearing MRSA carriage, whereas Pearraan et a l .

(1985) and Cookson and Phillips (1989) have found

various antiseptic regimens ineffective in clearing

179

MRSA. The use of topical antibiotic preparations

such as bacitracin, tetracycline and neomycin,

together or in combination with antiseptics is of

limited efficacy in clearing carriage (Casewell &

Hill, 1986; Chow & Yu, 1989). Topical therapy with

fusidic acid (White et a l . , 1989) or systemic

therapy with rifampicin (Yu et a l ., 1986) has been

found to be effective in clearing staphylococcal

carriage, however the emergence of resistance to

these antibiotics following such use is a cause

for concern. Recently, the new topical agent

mupirocin (pseudomonic acid) has been introduced into

clinical use, and reports of its efficacy in

clearing MRSA carriage have been most encouraging

(Casewell & Hill, 1987). Unfortunately, following the

clinical use of mupirocin the emergence of

bacterial resistance has been reported (Rahman e_t

a l . , 1987; Baird & Coia, 1987). In this section, the

antistaphylococcal activity of mupirocin is compared

to that of potential alternative topical agents.

180

i . Antistaphylococcal Activity of Azelaic Acid,Nitrofurazone and Silver Sulphadiazine compared toMupirocin.

The in vitro activity in terms of M I C ^ q , M I C q q ,

M B C50 , MBC90 of azelaic acid, nitrofurazone and

silver sulphadiazine compared to mupirocin against

80 strains of MGRSA is shown in Table XI.

Azelaic acid is a saturated dicarboxylic acid.

Topical azelaic acid has a beneficial effect in

the treatment of acne, where it has been shown

to produce on average, a 224-fold reduction in

micrococcaceae and 30-fold reduction in

Propionobacterium spp. (Bladon et a l . , 1986). Azelaic

acid is commonly used as a 15-20% cream with no

deleterious effect, furthermore, oral administration

of up to 20 grams a day is tolerated (Breathnach

et a l ., 1984). There have been few reports of the

antibacterial activity of azelaic acid, and we have

found none which show its activity against MGRSA.

We have found azelaic acid to be uniformly

active against MGRSA from many different

geographical sources (Table XI), and it is

bactericidal both in replica-plating studies and

time-kill studies (figure 4a). The efficacy of

azelaic acid in clearing MRSA carriage would seem

well worth investigating.

Nitrofurazone ("Furacin") is a nitrofuran

derivative possessing broad-spectrum activity which

181

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p N 03 Oi—1O O•p 3 H-cr fD CO03P-H-03NH*PfD

2o 00 M• 00 i—*On OI—* On vO O OnOn O

tO00

to

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3OQ

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182

has been used in various clinical situations since

its introduction in 1944. Currently, nitrofurazone

is recommended primarily for topical use in

wounds, second and third degree burns, and as

prophylaxis in skin grafting. Nitrofurazone solution

has been used as a bladder, eye and mouth

irrigant, and nitrofurazone cream has been used to

treat bacterial vaginitis and cervicitis. We found

nitrofurazone to be uniformly active against all

strains tested. Replica-plating studies (Table XI)

and time-kill studies (figure 4b) showed

nitrofurazone to be bactericidal at near MIC

concentrations. A considerable number of clinical

reports have shown nitrofurazone to be effective

against infections caused by Gram-positive and

Gram-negative bacteria, and despite over 30 years

of use clinical resistance is still not a problem

(Hooper & Covarrubias, 1983). Should mupirocin be

ineffective in clearing MRSA wound carriage,

nitrofurazone would appear to be an appropriate

alternative, however we are not aware of the

results of any clinical trials using nitrofurazone

in this indication.

Silver sulphadiazine ("Flamazine") was uniformly

active against the MGRSA studied (Table XI) despite

many of the strains being sulphonamide-resistant.

Silver sulphadiazine was less active than

nitrofurazone, however like nitrofurazone , replica-

plating and time-kill studies (Figure 4c) showed

183

silver sulphadiazine to be bactericidal at near

MIC concentrations. No difference in the rate of

killing was observed between sulphonamide-sensitive

and sulphonamide-resistant strains. Silver

sulphadiazine is a broad-spectrum agent which has

been shown to be beneficial in the treatment and

prevention of burn infections (Carr et a l . t 1973).

In the U.K, "Flamazine" is recommended (Lowbury e£

a l ., 1982) as first-line treatment for the

prevention of burn wound infection, however,

following its prophylactic use a preponderant flora

of sulphonamide-resistant Gram-negative bacilli can

emerge. There is little reported data regarding

the antibacterial activity of silver sulphadiazine

despite the widespread clinical use of this agent.

Rode et al (1989) have reported that a combination

of 1% silver sulphadiazine and 0.2% chlorhexidene

as topical therapy in a burns unit failed to

control MRSA wound colonisation/infection which was

subsequently brought under control on replacement

by mupirocin.

Mupirocin (Table XI) is highly active against

MGRSA, although concentrations substantially greater

than MIC are required for a bactericidal effect.

The consistent success of mupirocin in clearing

staphylococcal carriage has led to its

recommendation (Working Party, 1986; Working Party

1990) for the treatment of MRSA colonisation. No

mupirocin resistance was found in the MGRSA we

184

tested. High-level (greater than 1000 mg/1)

resistance to mupirocin in MRSA is increasingly

been found, and has been shown to be plasmid-

mediated (Rahman et a l ., 1990).

185

FIGURE A-Graph A

Killing curves for Azelaic acid at 2.5 gram/l (0.25%)

10 _

Control SOX - no antibiotic addad

Eao

RMISAI

SOX

24T i m e ( h o u r s )

Graph B

Killing curves for Nitrofurazone at 60 mg/l

10

— -A Control SOX - no antibiotic addad

9

8

7

6

5

4 SAI

iSOX3

2RM

1

0T i m e ( h o u r s )

FIGURE AGraph C

Killing curves for silver sulphadiazine at 250 mg/l

10

9 Control SOX ■ no antibiotic addad

8

7

6

5

4SAI

3RMI

2

1

SOX02 4

T i m e ( h o u r s )

Graph D

Killing curves for Mupirocin at 4.0 mg/l

10

-■A Control SOX - no antibiotic addad

9

8

7

6SOX

SAI5o

RMI

4

3

2

1

02 4

T i m e ( h o u r s )

Log

cFu

/ml

FIGURE ^Graph E

Killing curves for Ramoplanin at 1 mg/l and 2.0 mg/l

10 _Control SOX

A - no antibiotic added A S O X ^

Ramoplanin 1mg /ISAI

RM I

RM ISO XSAI

Ramoplanin20mg/l

T im e (hours)

188

3. Do fluoroquinolones have a useful therapeutic role against MGRSA?

Ciprofloxacin was the first fluoroquinolone to

be developed for general systemic use, and became

clinically available during the mid-1980s.

Ciprofloxacin possessed uniform inhibitory activity

against MRSA resistant to multiple antibiotics eg.

aminoglycosides and macrolides. Additionally,

ciprofloxacin was rapidly bactericidal against these

organisms (Smith & Eng, 1985).

The novel mode of action of the

fluoroquinolones, whose primary site of action is

subunit A of DNA gyrase means that cross­

resistance with other antibiotics was not found.

Furth ermore,it was believed that bacterial

resistance to these agents would not be a

clinical problem (Smith, 1984). In general,

fluoroquinolones are non-toxic, can be given orally

or by injection, and achieve good tissue levels.

In view of the problems of resistance and

toxicity encountered with other available

antistaphylococcal agents there was initial

enthusiasm for using fluoroquinolones against MRSA.

This early optimism has been tempered by recent

reports of widespread fluoroquinolone resistance in

MRSA. In this section the antistaphylococcal

properties of selected fluoroquinolones are

described, followed by an assessment of the problem

189

of development of resistance, and finally, promising

new fluoroquinolones are discussed.

3.1 Activity of the present clinically used

fluoroquinolones against M G R S A .

i . Inhibitory and bactericidal activity of

ciprofloxacin, enoxacin, norfloxacin, ofloxacin and

pefloxacin against M G R S A .

Currently, five fluoroquinolones, ciprofloxacin,

enoxacin, norfloxacin, ofloxacin and pefloxacin are

available for general clinical use. The inhibitory

and bactericidal activity of these agents compared

to nalidixic acid is shown in Table XII.

Ciprofloxacin, ofloxacin and pefloxacin are similarly

active against MGRSA, and enoxacin and norfloxacin

are less active. The non-fluorinated quinolone,

nalidixic acid is inactive against staphylococci.

By replica-plating, the fluoroquinolones were shown

to be bactericidal at near MIC concentrations (Table

XII). Our findings agree with those reported by

many other workers (eg. Smith, 1986; Fass & Helsel,

1987) .

The relative activity of fluoroquinolones in

vivo depends upon their pharmacokinetic properties.

These are summarised in Table XIII. Norfloxacin has

the lowest ratio of in vitro activity relative to

achievable serum levels (i.e therapeutic ratio), and

190

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HjPOH* oa M

H*3

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ch-» WOS fl>o a

.191

Table XII

These values

can be

increased with

multiple dosing

to different

extents.

* P I r t X O td O I-* "I n<j 03 X I-1 (0

03 XCL Ow cn

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192

is not used for treating systemic infections.Ciprofloxacin, ofloxacin and pefloxacin have higher

serum concentrations and greater in vitro activity,

compared to norfloxacin. In Table XIII the peak

serum concentrations of these agents are shown.

Multiple oral dosing with ofloxacin or pefloxacin

can produce higher peak serum levels (c. 8.0 mg/l).

For many Gram-negative bacteria these blood levels

greatly exceed MIC, however for Gram-positive

bacteria the therapeutic ratio is much less.

i i . Time-kill curves for ciprofloxacin, enoxacin,

ofloxacin and pefloxacin against M G R S A .

The killing kinetics against MGRSA of

ciprofloxacin, enoxacin, ofloxacin and pefloxacin have

been studied using various concentrations of agent.

Results shown in figures V ai, bi, ci and di were

with the fluoroquinolones at MIC (determined by

broth dilution studies) concentrations. Here killing

was observed for up to 8 hours following

inoculation, however, by 24 hours all the strains

had overgrown producing turbid broths. In figure

V aii, bii, cii and dii, fluoroquinolones at 4 x MIC

concentrations were used. At these concentrations

ciprofloxacin and j ofloxacin were usually

bactericidal, while enoxacin and pefloxacin showed

an initial bactericidal effect followed by

overgrowth by all strains after 24 hours.

193

Figure o

10

9.

0 8 12 244Tim* (hour*)

I ■ at 0.25 mg/l against MGRSA.

10,

8 12 240 4Tim* (hours)

• ■ ■

I I I . at 3 mg/l against MGRSA.

A1ft

ef

3.

0 8 124 24Tim. (hours)

■ •

I I - at 1 mg/l against MGRSA.

Strain- RMI

- SAI

' SOX- Control SOX

- no antibiotic added

Killing curve of Ciprofloxacin

t-igure o

B10-.

40 8 12 24Tim* (hours)

I . at 1 mg/l against MGRSA.

10-i

24o 4 8 12Tim* (hour*)

I I I . at 10 mg/l against MGRSA.

10

e

f

o 4 8 12 24Tim* (hours)

■ mI I . at 4 mg/l against MGRSA.

StrainRMI

SAI

SOX

Control SOX - no antibiotic added

Killing curve of Enoxacin. 195

r i g u r e o

10 _

12 24eo 4Tim* (hours)

m

I . at 0.5 mg/l against MGRSA.

10,

o

f

o 4 6 12 24Tim* (hours)

i l l . at 8 mg/l against MGRSA.

10,

o 4 8 24

I I . at 2 mg/l against MGRSA.

Strain■m— rm i

+ ------ SAI

A — soxControl SOX - no antibiotic added

Killing curve of Ofloxacin. 196

figure o

D10 -

of

o s 12 244

I.

Tims (houn)

at 0.5 mg/l against MGRSA.

10.

3.

12 244 80Tim* (hours)

j j j . at 8 mg/l against MGRSA.

10-19 .

241280 4

II.

Tims (hours)

at 2 mg/l against MGRSA.

StrainRMI

• ------ SAI

± — SOX

Control SOX - no antibiotic added

Killing curve of Pefloxacin. 197

Overgrowth by strain RMI sometimes occurred with

ciprofloxacin and ofloxacin.

The highest fluoroquinolone concentrations used

in the time-kill studies were ciprofloxacin (3.0

mg/l), enoxacin (10 mg/l), ofloxacin (8.0 mg/l) and

pefloxacin (8.0 mg/l). For ciprofloxacin, ofloxacin

and pefloxacin these concentrations were similar to

the respective peak serum levels following oral

dosing. The corresponding figure for enoxacin is

c. 4.0 mg/l, but at this concentration (i.e 4 x MIC)

overgrowth by all strains had been found in the

previous experiments. In order to establish whether

overgrowth could be prevented by a higher

concentration, an arbitrary concentration of 10 mg/l

enoxacin was used in the final time-kill

experiments. With these higher concentrations of

ciprofloxacin, enoxacin, ofloxacin and pefloxacin a

99.9% reduction in the initial inoculum was

usually maintained for 24 hours incubation (figure

V aiii, biii, ciii and diii) and broth turbidity

was not apparent even after 48 hours incubation.

On repeating certain time-kill experiments we

found considerable variation in the viable counts

obtained after 24 hours incubation. We have used

median data values (compiled from three separate

experiments) for 24-hour data points in figure V.

The production of broth turbidity was also a

variable phenomenon. Sometimes broths appeared

turbid at 24 hours although on viable counting

198

only 10^ - 1C)6 cfu/ml were present. Normally, broth

turbidity does not become apparent until a viable

count of c. 10® cfu/ml is achieved. Smith et a l .

(1986) who also observed this phenomenon, attributed

it to cell swelling and the production of multi-

cellular forms. When we have quoted the finding

of broth turbidity, viable counting has always been

used to confirm that the turbidity was due to a

viable count of 10® - 10^ cfu/ml.

To assess whether overgrowth was due to

fluoroquinolone instability in IsoSensitest broth

following prolonged incubation at 37°C (in the

dark) fluoroquinolone levels were assayed in

uninoculated broth before and after incubation. No

significant difference (t-test, p = 0.9) in

fluoroquinolone activity was found in pre-incubation

broth compared to post-incubation broth. The

development of resistance during the time-kill

studies is discussed in the next section (3.2i).

There have been differing reports of the

activity of ciprofloxacin in time-kill studies. For

example, with ciprofloxacin at 4 x MIC, Smith and Eng

(1985) reported initial killing with no overgrowth,

whereas Foster e t a l . (1986) found overgrowth

following initial killing. Antibiotic carry-over

resulting in inhibition of growth on viable

counting plates could significantly influence

experimental findings. We investigated the

significance of antibiotic carry-over in our

199

experiments. If 0.1 ml of 4 x MIC, or higher

concentrations, of fluoroquinolones was dropped onto

a plate seeded with S. aureus NCTC 6571 (ie. strain

SOX), after 24 hours incubation inhibition and

clearing of growth could be seen where the drop

fell. However, if 0.1 ml aliquots of the same

fluoroquinolone concentrations were thoroughly spread

on plates, no significant difference (t-test, p = 0.9)

in viable counts on these treated plates compared

to non-treated plates was found.

i i i . Spontaneous plate mutation rates to resistance

for ciprofloxacin, enoxacin, norfloxacin, ofloxacin

and pefloxacin compared to nalidixic a c i d .

For strains RM1 , SA1 and SOX spontaneous

mutation rates were determined by plating out c.

lO^ cfu onto agar containing ciprofloxacin at 5

mg/1 and 25 mg/1, enoxacin at 10 mg/1 and 50

mg/1, norfloxacin at 10 mg/l and 50 mg/1, ofloxacin

at 5 mg/l and 25 mg/l and pefloxacin at 5 and

25 mg/l. Resistant mutants were only obtained at

frequencies of 3.0 x 10“ 9 to 5.0 x 10“ 9 on plates

containing 10 mg/l norfloxacin. In no case were

resistant mutants found to the other

fluoroquinolones. For the older non-fluorinated

quinolone, nalidixic acid at 256 mg/l, frequencies

of mutation to resistance of 10“ - 10“ ® were

observed. Numerous in vitro studies (Wolfson &

200

Hooper, 1985) have reported much lower mutation

rates to fluoroquinolone resistance compared to

those found for the older quinolones (eg. nalidixic

acid ) .

According to Smith (1990) frequencies of mutation

to resistance of 10“ ^ - 10“® occur for S. aureus in

the presence of 5 x MIC concentrations of enoxacin

and norfloxacin, and 10~9 _ 10“ 10 in the presence

of 5 x MIC concentrations of ciprofloxacin and

ofloxacin. At 10 x MIC concentrations of

ciprofloxacin, enoxacin, norfloxacin and ofloxacin,

resistant mutants could only be obtained with

norfloxacin (frequency 10” ^). We have made similar

findings. Kayser and Novak (1987) and French et al

(1988) showed that high-level fluoroquinolone

resistance can easily be produced by passaging

strains in the presence of two-fold increasing

drug concentrations.

201

3 . 2 Fluoroquinolone____resistance____ in_____MGRSA -

development of resistance in vitro, and properties

of resistant strains derived in vitro and from

clinical sources.

i . Development of fluoroquinolone resistance in

vitro during time-kill experiments.

As an extension of our findings of regrowth

in the time-kill experiments (figure V), four

strains (RFH 10, AUS 10, SA 1 and SOX) were grown for

48 hours in the presence of the same

concentrations of ciprofloxacin, enoxacin, ofloxacin

and pefloxacin as previously used. Strains RFH 10,

AUS 10 and SA 1 were chosen because they possessed

a range of susceptibilities to ciprofloxacin.

Strain RFH 10 was moderately susceptible/resistant

to ciprofloxacin (MIC 2.0 mg/l) and strains AUS 10

and SA 1 were susceptible to ciprofloxacin (MIC 0.5

mg/l and 0.25 mg/l respectively). Strain SOX

(ciprofloxacin MIC, 0.25 mg/l) was used as a

control in these experiments.

Viable counts were performed on the time-kill

broths at 24 and 48 hours incubation, and the

susceptibilities to fluoroquinolones of any

organisms isolated were determined. Our results

are shown in Table XIV (a, b, c and d).

For ciprofloxacin at 0.25 rag/1, there was

initial killing followed by regrowth, and by 24

202

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2 0 6

hours incubation

were found. The

the overgrowing

and SA 1) was

NCTC 6571) a

susceptibility

ciprofloxacin,

turbid broths,

only 2.6 x 10^

incubation, and

hours incubation,

isolated at the end of

2-16 fold decrease

susceptibility. MICs

unchanged. At 3.0

RFH 10 overgrew

strain possessed

fluoroquinolone

only 102 - 105

10, SA 1 and

fluoroquinolone

were unchanged. Strain

48 hours incubation,

ciprofloxacin (data

produced mutants

susceptibilities.

Similar

have been

XIV b

108 - 109 cfu/ml

susceptibility of

RFH 10, AUS 10

SOX (S. aureus

fluoroquinolone

.0 mg/l

and SOX produced

a viable count of

found after 24 hours

not appear until 36

AUS 10, SA 1 and SOX

the experiment showed a

fluoroquinolone

RFH 10 were

only strain

broth. This

decrease in

counts of

strains AUS

incubation. The

these organisms

isolated after

of 5.0 mg/l

strain RFH 10

fluoroquinolone

slight variations

pefloxacin (Table

mutants with

2 0 7

turbid broths of

fluoroquinolone

strains of MGRSA (ie.

unchanged. For strain

8-16 fold decrease in

occurred (Table X l V a ) . With 1

strains RFH 10, SA 1

For strain AUS 10

cfu/ml was

turbidity did

Strains

in

for strain

mg/l ciprofloxacin

to produce a turbid

a 2-32 fold

susceptibility. Viable

cfu/ml were found for

SOX after 48 hours

susceptibilities of

SOX was not

In the presence

not shown) only

with decreased

trends, although with

found for enoxacin and

and d). Overgrowth by

decreased fluoroquinolone susceptibilities occurred

at 0.5 and 2.0 mg/l pefloxacin, and 1.0 and 4.0

mg/l enoxacin. In the presence of 10 mg/l

enoxacin and 8.0 mg/l pefloxacin, only strain RFH

10 could produce mutants and overgrow. A different

trend was observed for ofloxacin, strains AUS 10, SA 1

and SOX only overgrew in broth containing 0.5

mg/l. At 2,0 mg/l ofloxacin only strain RFH 10

overgrew, and at 8.0 mg/l even strain RFH 10 could

not produce mutants.

These findings show that fluoroquinolone

resistance can be easily selected in the presence

of therapeutic concentrations of ciprofloxacin,

enoxacin, ofloxacin and pefloxacin. However, the

propensity of strains to become resistant may be

different for individual fluoroquinolones. Thus,

selection of resistance was least with ofloxacin,

which may be due to the fact that ofloxacin

possesses an extra mechanism of killing (Lewin &

Smith, 1988) against staphylococci not found for

ciprofloxacin. This extra mechanism of killing of

ofloxacin may also account for the observation

that this antibiotic was the most active of the

agents studied against the fluoroquinolone-resistant

mutants.

Irrespective of the particular fluoroquinolone

or concentration used, resistant mutants were found

to possess similar reductions in susceptibility,

suggesting that only a one-step mutation could

2 0 8

take place. Furthermore, decreases in

fluoroquinolone susceptibility were proportional to

the initial susceptibilities of strains. For

example, with ciprofloxacin strain SOX could only

mutate to a ciprofloxacin susceptibility of 2.0

mg/l (a 8-fold decrease in susceptibility) and

strain RFH 10 could only mutate to a

ciprofloxacin susceptibility of 16.0 mg/l (again a

8-fold decrease in susceptibility). Generally,

mutation to resistance in the presence of one

fluoroquinolone resulted in cross-resistance to the

others. The degree of reduction in susceptibility

differred between the fluoroquinolones. The least

reduction in susceptibility was found for ofloxacin

followed by ciprofloxacin. The greatest reduction

in susceptibility was found for pefloxacin.

Fluoroquinolone resistance appears to develop in

a stepwise manner. For example, following exposure

to 1.0 mg/l ciprofloxacin, strains initially

sensitive to ciprofloxacin (MIC 0.25 - 0.5 mg/l) may

develop borderline resistance (i.e ciprofloxacin MICs

2.0-4.0 mg/l). In the clinical situation such

strains exist (eg. RFH 10), and these can then

mutate to clinical resistance (ie. ciprofloxacin MIC

= 16 mg/l) following further exposure to

fluoroquinolones. Laboratories may well fail to

detect those strains possessing subclinical

resistance using current disc testing techniques.

As a consequence of failing to detect borderline

2 0 9

resistance a permanent reservoir of organisms with

the potential to rapidly develop clinical

resistance may build up following widespread

fluoroquinolone usage.

Ofloxacin appears to be the most appropriate

fluoroquinolone to use against MGRSA, at least with

a view to minimising the risk of emergence of

resistance. Furthermore, depending upon the initial

fluoroquinolone MICs of strains which mutate to

resistance different levels of fluoroquinolone

resistance might be expected in a diverse

collection of resistant strains. In order to

determine whether the phenomena discovered in our

time-kill experiments are relevant to the clinical

situation, we have investigated the properties of

an internationally diverse collection of

fluoroquinolone-resistant MGRSA.

i i . Quinolone susceptibility patterns of clinical

isolates of ciprofloxacin-resistant M G R S A .

Our in vitro studies show that fluoroquinolone

resistance can emerge in the presence of

therapeutic concentrations of fluoroquinolones.

Irrespective of the fluoroquinolones used, the same

patterns of resistance are obtained, and the level

of this resistance depends upon the initial

susceptibility of the strains. Thus, in v i v o , it

might be expected that clinical isolates of

2 1 0

fluoroquinolone-resistant MGRSA would possess similar

susceptibility patterns. To investigate this

possibility, we have collected MGRSA from 7 centres

worldwide (two centres in France and Israel,and one

centre in Germany, Italy and the USA) and have

determined their susceptibility to a range of

fluoroquinolone agents. Our findings are shown in

Table XV.

From the MICs in Table XV different levels

and patterns of fluoroquinolone resistance are

apparent. Even strains from the same centres

showed different patterns and levels of resistance

eg. strain IS1 vs. strain IS2, strain TEX1 vs

strain TEX2. Other workers (Kojima e t a l ., 1990;

Yamamoto, 1990) have also reported different

fluoroquinolone susceptibility patterns in clinical

isolates. Kojima et al (1990) have proposed that

different mechanisms of resistance may occur (i.e

mutation of DNA gyrase, or reduced permeability due

to an alteration in the cytoplasmic membrane). In

v i t r o , French et a l . (1988) and Limb et a l . (1987)

have shown that passage in subinhibitory

concentrations of fluoroquinolones can result in a

variety of levels and patterns of fluoroquinolone

resistance. Thus, it would appear from our results

with the clinical isolates of ciprofloxacin-

resistant MGRSA that a number of different

mechanisms of fluoroquinolone resistance exist, or

that fluoroquinolone resistance has evolved along a

211

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2 1 2

TABLE XV.

Patterns of

quinolone resistance

in an

international

Abbreviations N.A

- nalidixic

acid, Acr

- acrosoxacin,

Cip -

ciprofloxacin Enx

- enoxacin,

Nfx -

norfloxacin, Ofx

- ofloxacin

and Pfx

- pefloxacin.

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213

number of separate evolutionary pathways, or a

combination of both. We have been unable to

determine the fluoroquinolone resistance mechanisms

in the strains studied because of the practical

difficulties encountered in investigating them in S .

aureus (Ubukata et a l . 1989).

Against the ciprofloxacin-resistant MGRSA,

ciprofloxacin and ofloxacin were found to be the

most active (median MIC = 16 mg/l) of the

fluoroquinolones tested, followed by pefloxacin

(median MIC = 32 mg/l) and enoxacin and norfloxacin

(median MICs = 64 mg/l). The older, non-fluorinated

quinolone - acrosoxacin was also tested against the

ciprofloxacin-resistant strains, and contrary to our

expectations some strains were found to be

sensitive (eg. RFH 1, F3, IT6). The phenomenon of

"reversed incomplete cross-resistance" between the

fluoroquinolones and older non-fluorinated quinolones

has been seldom reported. Van Caekenberghe and

Pattyn (1987) have reported "reversed incomplete

cross-resistance" with acrosoxacin for a strain of

Flavobacterium multivorans. Fluoroquinolone-resistant

mutants derived in vitro showed little acrosoxacin

resistance. Our results for strain RFH 10 are

shown in Table XVI.

There have been few reports which have

monitored the clinical emergence of fluoroquinolone

resistance during treatment. Milne and Faiers (1988)

reported the rapid development of ciprofloxacin

2 1 4

Abbreviations: Acr

- acrosoxacin,

Cip - ciprofloxacin,

Enx -

enoxacin,Nfx

- norfloxacin,

N.A - nalidixic

acid, Ofx

- ofloxacin,

Pfx - pefloxacin.

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2 1 5

resistance in MRSA following use of ciprofloxacin

to treat MRSA septicaemia. Initially, this isolate

had a ciprofloxacin MIC of 0.5 mg/l which

increased to 4.0 mg/l, and finally 16 mg/l, before

the patient died. The serum ciprofloxacin level 2

hours after a 100 mg intravenous dose was only

0.7 mg/l. This dosage appeared inadequate, and it

may be appropriate to monitor ciprofloxacin levels

in blood to ensure that satisfactory concentrations

are maintained when dealing with staphylococcal

infections.

i i i . Are the reports of high incidences of

fluoroquinolone resistance in hospitals due to the

propensity of MGRSA to develop fluoroquinolone

resistance or are they due to epidemic spread of

resistant strains?

According to Shalit et a l . (1989) during a

widespread epidemic of ciprofloxacin-resistant MRSA

in a general hospital in no instance was a

quinolone-resistant organism isolated from a patient

following fluoroquinolone treatment. Raviglione et

a l . (1990) found exactly the opposite: ciprofloxacin-

resistant MRSA being isolated only from patients

in whom there had been prior use of

ciprofloxacin. Daum et a l . (1990) and Smith et a l .

(1990) found initial cases of colonization or

2 1 6

infection with ciprofloxacin-resistant MRSA followed

use of ciprofloxacin. Subsequent cases were not

related to ciprofloxacin therapy rather hospital

transmission of existing strains. We have made our

own assesment of whether the problem of

ciprofloxacin resistance is primarily one of cross­

infection or is due to development of resistance

in separate strains.

We have examined ciprofloxacin-sensitive and

ciprofloxacin-resistant MGRSA from one of the first

reported outbreaks of ciprofloxacin-resistant MRSA

(Isaacs et a l . , 1988) which occurred in a Texas

hospital. Ciprofloxacin-resistant MGRSA from two

centres in Israel which have been suggested to be

cross-infecting strains have also been studied.

The results of phage-typing are shown in Table

XVII. Many of the strains from Texas gave a

strong reaction with phage 75 at 100 x R T D ,

although at RTD this reaction was weak or non­

existent. All the ciprofloxacin-sensitive MGRSA from

Texas showed the same phage-type, however a number

of different patterns were observed for the

ciprofloxacin-resistant strains. Strains TEX 2, TEX 10

and TEX 16 reacted with a variety of phages from

lytic groups I, II and III of the International

Set at RTD, although slight differences in patterns

were observed between these strains. Four strains

from Israel (IS 1, IS 2, IS 3, IS 4) were non-typable

with the International Set even at RTD x 100.

2 1 7

However, 3 of these 4 did react with supplementary

or Nuan phages (Richardson et a l . , 1988) . The

remaining 4 Israeli strains showed different typing

patterns.

In order to type non phage-typable strains, and

possibly differentiate strains of the same phage

type, plasmid analysis, antibiotic susceptibility

typing and biotyping was performed on the strains.

The results of antibiotic susceptibility typing, and

biotyping are shown in Table XVIII. The non phage-

typable Texas strains (ie. strains 13 and 20) were

different from each other in terms of

ciprofloxacin MIC, plasmid content, antibiotic

susceptibility profile and biotype. A 36 md

plasmid was found in all Texas strains, however in

strain 13, a 2.5 md plasmid was also present.

Strain 13 was resistant to tetracycline and also

rifampicin. The ciprofloxacin-resistant, phage-type 75

strains (i.e 1,3,7,11,17 and 5 and 14) could be

split into 3 groups on the basis of antibiotic

susceptibility profiles and biotyping. Strains

1,7,11 and 17 had similar ciprofloxacin MICs (16 or

32 mg/l) and produced sheep blood haemolysis but

not egg yolk lipase. Strain 3 had a similar

biotype to strains 1,7,11 and 17, however a

ciprofloxacin MIC of only 4.0 mg/l was found.

Strains 5 and 14 differed from 1,7,11 and 17 because

they did not produce sheep blood haemolysis.

Strains 2, 10 and 16 were widely susceptible to

2 1 8

ciprofloxacin sensitive

strains

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ciprofloxacin sensitive

strains.

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220

ciprofloxacin sensitive

strains

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ciprofloxacin sensitive

strains

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222

a variety of phages, and had different biotypes.

The ciprofloxacin- sensitive MGRSA from Texas

showed much less variation in typing properties

compared to the ciprofloxacin-resistant strains.

They were negative for egg-yolk lipase, had a 36

md plasmid, and possessed the same antibiotic

sensitivities. Strains 6 and 18 differed from the

other ciprofloxacin sensitive strains because they

produced haemolysis on sheep blood agar.

All the strains from Israel had identical

biotypes except for the non phage-typable strain

IS 2 which was the only Israeli strain to produce

a negative egg yolk lipase reaction. Despite

identical biotypes and plasmid-types (a cryptic

plasmid was found in all strains) strains 6 and 7

differed from the others in terms of antibiotic

sensitivity profiles. Combining our phage typing,

biotyping and antibiotic sensitivity profile data

of the Israeli strains, no two strains were found

to be identical.

The results of these studies suggest that the

problem of ciprofloxacin resistance in the

hospitals we have studied is due more to the

development of resistance in separate strains

rather than cross-infection due to a single strain.

Daum et a l . (1990) have shown that ciprofloxacin-

resistant MGRSA causing outbreaks of hospital

infection in the USA can be of a number of

different phage types. Hadorn et a l . (1990) have

223

used ribotyping (probing of DNA coding for

ribosomal RNA) to confirm that their ciprofloxacin-

resistant Israeli strains are all the same. The

results for our Israeli strains suggest that

independent evolutionary events have occurred. Some

workers question the discriminatory power of

ribotyping (Judith Richardson, PHLS Colindale, personal

communication), and our typing methods may

differentiate strains of the same ribotype.

In our typing studies we have found that

methods such as biotyping, phage typing with the

International Set (even at RTD x 100), determination

of antibiotic susceptibility profiles or plasmid-

typing, used on their own sometimes fail to

provide sufficient strain differentiation. However,

when a variety of methods are combined the

prospects of strain differentiation are much

improved. Thus, we agree with Cookson et a l .

(1986) in that a combination of strategies is

required to distinguish between strains so as to

confirm known or suspected routes of acquisition.

Finally, exposure to fluoroquinolones and/or

mutation to fluoroquinolone resistance has been

reported to alter the phenotypes of strains.

Crumplin (1990) has suggested that mutation to

fluoroquinolone resistance can result in pleiotropic

effects. This is because DNA gyrase has a

significant role in sustaining normal bacterial

function, any alteration in DNA gyrase activity

224

would disrupt "normal" function. We have found

similar physiological profiles (in terms of APISTAPH

profile) for ciprofloxacin- sensitive and

ciprofloxacin-resistant MGRSA. Smith (1990) has

reported loss of coagulase activity in S. aureus

following mutation to resistance in v i t r o , however

both our in vitro derived mutants, and clinically

resistant strains produced coagulase. Should loss

of coagulase production occur in the clinical

situation, S. aureus may be misidentif ied as S .

epidermidis. We have also found that the phage-

types of laboratory derived fluoroquinolone-resistant

mutants were no different from those of parent

strains .

3.3 New fluoroquinolones with improved activity

against M G R S A .

Since the introduction of ciprofloxacin, and

other related fluoroquinolones in the mid-1980s,

considerable effort has been expended by many

pharmaceutical companies in developing new

fluoroquinolones. Initially, many of these new

compounds, such as fleroxacin (Leigh et a l ., 1988),

possessed only improved pharmacokinetics compared to

ciprofloxacin.

Recently, newer compounds have been developed

with much improved antistaphylococcal activity eg.

tosufloxacin (Barry & Jones, 1989) and sparfloxacin

2 2 5

(Kojima et a l . , 1989). The activities of these

agents against ciprofloxacin-sensitive and

ciprofloxacin-resistant MGRSA are shown in Table

XIX. Compared to ciprofloxacin, sparfloxacin and

tosufloxacin showed approximately a ten-fold

improvement in activity against ciprofloxacin-

sensitive MGRSA. Against ciprofloxacin-resistant

MGRSA, sparfloxacin and tosufloxacin were active

against strains with low-level (MIC 2-8 mg/1)

resistance, but not against those showing higher

levels of resistance. Our results for sparfloxacin,

obtained against isolates of ciprofloxacin-resistant

MGRSA are similar to those reported by Chaudhry

et a l . (1990). Tosufloxacin only attains peak serum

levels of c. 0.25 mg/1 (personal communication,

Abbott Laboratories, Illinois). Hence, despite its

excellent in vitro activity tosufloxacin has a

similar therapeutic ratio to ciprofloxacin. According

to Montay et a l . (1990), sparfloxacin attains peak

serum levels of 1.0-2.0 mg/1 depending on the

dose given. Thus, sparfloxacin may be more active

in vivo than ciprofloxacin or ofloxacin. Another

new fluoroquinolone with excellent in vitro (MIC^q

0.004 mg/1) activity against MGRSA is WIN 57273

(Kaatz & Seo. 1990b), however, we have been unable to

obtain supplies of this compound for laboratory

testing .

In the future there may be new fluoroquinolones

available with greatly enhanced antistaphylococcal

226

♦MIC for

50% and

90% of

isolates tested,

respectively.

4*O

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2 2 7

Table XIX.

Susceptibilities of

160 ciprofloxacin-sensitive

MGRSA and

activities compared to ciprofloxacin and ofloxacin.

If these new agents can attain satisfactory serum

levels so that their increased activity is

reproduced in vivo (i.e they have an improved

therapeutic ratio) their use may result in reduced

development of fluoroquinolone resistance. This is

because, as we have shown, if fluoroquinolone

concentrations are high enough sensitive strains

cannot mutate to resistance. Additionally, strains

with decreased fluoroquinolone susceptibilities (eg.

RFH 10) may prove susceptible.

228

4.0 GENERAL DISCUSSION AND CONCLUSIONS

4.1 "Ba r b e r ’s Law - the spread of resistant

staphylococci can be controlled either by not

giving antibiotics or by preventing the

transmission of the resistant organisms between

persons". Parker et a l ., 1974.

The antibiotic susceptibility profiles (Table IV)

of 100 strains of MGRSA from 32 centres in 23

countries have been determined. This is the first

survey to document the international problem of

multiple antibiotic resistance in MGRSA. Many

differing susceptibility profiles were found, some

strains being sensitive to a range of currently

available antistaphylococcal agents, others resistant

to many of these agents.

During the late 1960s and continuing into the

1970s multiple antibiotic resistance in

Staphylococcus aureus declined (Parker et a l . , 1974).

Initially, it was thought that a decrease in

tetracycline usage (although not an overall

reduction in use of antibiotics) was responsible

for the decline in staphylococcal multiple

antibiotic resistance. This explanation was rejected

by Ayliffe and his colleagues (Ayliffe et a l . ,

1979) who also found that use of one antibiotic

(in their case- erythromycin) selected an outbreak

strain resistant to tetracycline, erythromycin and

229

novobiocin. While rigid antibiotic policies

significantly reduced multiple antibiotic resistance

in S . au r e u s , Ridley ^t al. (1970) reported

isolations of MRSA to increase. It would appear

that the spread of MRSA is not directly

influenced by antibiotic usage, which sharply

contrasts with the role of penicillin in promoting

the spread of penicillin-resistant staphylococci.

The reasons why methicillin and gentamicin

resistant S. aureus so rapidly appeared worldwide

following the initial discovery of these organisms

during the mid-1970s are not clear. Only the

phage type 80/81 S . aureus of the 1950s has shown

a similar capacity for rapid, worldwide spread

(Shanson, 1981). More than 50% of our MGRSA were

non phage-typable with the "International Set" of

phages even at RTD x 100. 23 of the 42 typable

strains were lysed by phage 85 alone, or phage 85

and other Group III phages as well (Table I). The

remaining typable strains had mixed Group I + III

lysis pat terns.

The results of typing with supplementary

phages are shown in Table XX. Four reserve, non­

specific phages (88A, 90, 83C and 932) were used

together with a new set of experimental ("Nuan")

phages (616, 617, 618, 620, 622, 623, 625, 626, 629 and

630) developed by Richardson e_t al. (1988).

According to these workers all of the "Nuan" phages

were lytic group-III related except phage 625

230

Table XX.Supplementary phage-types of MGRSA used in antibiotic resistance studies.

Country Strain SupplementaryCode phage-type

"Nuan"phage-type

AUSTRIA

AUSTRALIA

BELGIUM

BRAZIL

CHILE

ENGLAND

V 8V 15

AS 1 AS 2

AS 3

AS 6 AS 10

BL 1 BL 3

BL 4

BZ 1 BZ 2 BZ 4 BZ 1 2 BZ15 BZ16 BZ17 BZ18

CH 3 CH 4

C 1 UK16 UK1 7

F.R.G.

RFH RFH RFH RFH RFH RFH10 RFH11

G 1 G 2 G 3

NTNT

NT88A/932

90/932

NTNT

90/932 88A/90/83C/932

90/932

NTNTNT

90/932932NT

932NT

90/932NT

932NTNT932932

88A/93288A/932

93288A/90/932

83C

932932932

NTNT

618/630616/617/620/622/626/630

616/617/618/620/622/625/626/630

NTNT

618/620/623616/617/618/620/622/623617/620/622/623/629/630

NTNT

629 618/629

NTNT

617/618/620/622/630618

630 NT

617/620/622/626NT

625/629617/620/622/626617/620/622/626616/617/622/626

617/620/622/626/630616/617/622/630

616/617/620/622/626/630618

617/618/620/622/626618/620618/620

C o n t d .

231

Table XX. contd .

Supplementary phage-types of MGRSA used in antibiotic resistance studies.

Country Strain Supplementary "Nuan"Code phage-type phage-type

F.R.G. G 4 932 617/618/620/622/626/630contd . G 5 NT 618/620

G 6 NT 618/620

FRANCE F 1 NT 623F 2 NT NTF 3 NT 623F 4 NT 623F 5 NT NTF 11 NT NTF 1 2 90/932 NTF 13 NT NTF14 NT NTF25 932 NT

GDR EG 6 NT NTEG 1 2 NT NT

GREECE AT 1 NT 620AT 3 NT NT

HONG KONG HK 1 90/932 617/618/622/629HK 2 90/932 617/618/622/626HK 3 90/932 617/618/622

ITALY IT 6 NT 629IT 7 NT NTIT 8 NT NTIT11 932 618/620/623/625/629IT1 2 88A/90 NT

JAPAN JP 1 NT NTJP 2 NT NT

KUWAIT KW 4 NT NTKW 5 NT 616/617/618/620/622/630KW 7 NT 617/618/620/622/626

POLAND PL 1 88A/932 617/620/622/626PL 2 88A 623/625PL 3 88A/932 616/617/620/622/626/630PL 4 88A/932 617/620/622/626

C o n t d .

232

Table XX. contd.Supplementary phage- types of MGRSA used in antibioticresistance stud ies.

Country Strain Supplementary "Nuan"Code phage-type phage-type

PORTUGAL P 1 NT 616/617/622/623P 2 NT NTP 3 NT NT

REPUBLIC IR 1 NT 617/622OF IR 2 NT 617/622IRELAND IR 4 NT 617/620/622/626

IR14 NT 617/620/622/626IR 15 NT 617/620/622/626IR 17 NT NTIR18 NT 617/620/622

SOUTH SA 1 932 NTAFRICA SA 2 932 618

SA 4 NT 618/629SPAIN SP 1 88A/932 NT

SP 2 NT NTSP 3 90/932 NT

SWITZERLAND SW 1 NT NTSW 2 90/932 617/622/623/626

TURKEY T 6 NT 625T 7 NT 617/620/626T 9 NT NTT10 NT 625

USA US 7 NT NTUS 12 88A/83C/932 616/617/618/620/622/

623/625/626/630US 13 88A/83C/932 616/617/618/620/622/

623/625/626/630RM 1 NT 618/623/629RM 2 NT 623/629RM 3 NT 618/623/629RM 4 NT 617/620/622/623RM 5 NT 617/620/622/623SC10 NT NTSC11 83C 623/629

U.S.S.R RS 2 NT 616/617/620/622/625/626/630

233

which was Group-I related. Many of the previously

non-typable MGRSA could be typed using these

phages, and by inference many of these strains

would appear to be Group Ill-related.

A questionnaire survey conducted in the UK and

Ireland between 1982 and 1983 established a high

incidence of MGRSA in hospitals in South-East

England (Cooke et.______ a_l. , 1986). Subsequent

bacteriological examination of these strains by

Marples et a l . (1986) showed that a single epidemic

isolate, EMRSA-1 was primarily responsible for the

high incidence seen. A very similar strain was

also causing an epidemic of infections in Eastern

Australian hospitals (Townsend et a l . , 1987). The

properties of EMRSA-1 have been studied in depth

by Cookson and Phillips (1988) who stated EMRSA-

1 to have chromosomally-mediated resistance to

tetracycline, minocycline, erythromycin (MIC greater

than 1000 mg/1), clindamycin (MIC 250 mg/1) and

streptomycin (MIC 60 mg/1). EMRSA-1 is usually

sensitive to fusidic acid, neomycin and rifampicin.

Aminoglycoside resistance (due to production of APH

(2M )/AAC(6’) is often encoded on a 15-23 Md plasmid

commonly found in EMRSA-1.

Richardson et al (1988) have shown strains of

EMRSA-1 to have characteristic phage-types. Some

strains react with phage 85 (usually weakly). These

Mtype-85 strains have a Nuan phage profile of

616/617/622/626/630, and may react with phages

234

88A/932. Other EMRSA may react with phages 84 and

85, sometimes with the supplementary phages 88A/932,

and these 84/85 strains show a wider pattern of

Nuan phage lysis ie. 616/617/618/620/622/626/630.

Unfortunately, Richardson and her colleagues (1988)

also observed that similar profiles with the

"Nuan" phages could be obtained in apparently

unrelated strains. The variety of phage-types

(Tables I and XX) and antibiotic resistance

patterns (Table IV) found in the strains we studied

suggests that the spread of MGRSA is probably not

due to the widespread dissemination of a single

clone .

We have found that 44% of MGRSA produce the

bifunctional aminoglycoside-modifying enzyme APH

(2")/AAC ( 6 1), while 56% produce this enzyme along

with APH(3')-IV as well. These mechanisms were

inferred from the results of disc sensitivity test

data to six aminoglycoside antibiotics (Van de

Klundert et a l ., 1984). Attempts were also made to

detect the genes responsible for aminoglycoside

resistance using specific probes currently under

development by Dr G. Miller of Schering-Plough

Research, Bloomfield, New Jersey, USA. These studies

are still under way, however preliminary results

show agreement with the mechanisms inferred using

Van de Klun dert’s method. Ounissi et a l . (1990)

reported virtually a 100% correlation between the

presence of enzymes APH (3f) and APH (2")/AAC (6*) in

235

S. aureus as deduced from susceptibility data toaminoglycoside agents and the detection of these

enzymes by specific probes. For streptomycin the

correlation is not so good, as streptomycin

resistance may be due to a number of enzymes or

ribosomal mutation.

In our strains the presence of APH (2” )/AAC (6’)

positively correlated with sensitivity to neomycin

and resistance to gentamicin (sisomicin), kanamycin

and tobramycin. The presence of APH ( 3 f) and APH

(2")/AAC ( 6 ’) positively correlated with resistance

to gentamicin (sisomicin), kanamycin and neomycin.

The finding that the same enzyme - APH (2")/AAC ( 6 ’)

was responsible for gentamicin, kanamycin and

tobramycin resistance in MGRSA worldwide suggests

that the genes for this resistance have

disseminated worldwide. This might at first seem

unlikely because gentamicin resistance genes have

been found to be chr omosomally located in some

strains (Storrs et a l . , 1988), while in others they

may be carried on completely different plasmids.

For example, gentamicin resistance in Australian

strains is commonly carried on non-conjugative Inc 1

plasmids, whereas in strains from the USA the

resistance determinant is usually found on large

conjugative plasmids (Lyon & Skurray, 1987). These

differences have been explained by the

identification of a transposon (Tn 4 0 0 1 ) carrying

the genetic determinants of gentamicin resistance

236

in the aforementioned Australian and U.S. strains

(Byrne et a l ., 1990).

Our results, together with the genetic and

molecular findings of Byrne et a l . (1990) suggest

that gentamicin resistance in MRSA has been

produced by the worldwide dissemination of a

transposon (Tn 4001) into MRSA, and not the spread

of a resistant clone. The factors responsible for

such widespread dissemination are not clear. Thus,

B a r b e r ’s law does not seem to apply to the spread

of gentamicin, kanamycin and tobramycin resistance

in MRSA.

Parker et a l . (1974) believed that multiple

antibiotic-resistant hospital strains of S. aureus

declined during the 1960s because of reduced

transmission by improved infection control measures,

rather than decreased antibiotic usage. Such

measures may have been effective against the

multi-resistant S . au r e u s , but they were not

effective against MRSA, which increased in numbers

as the 1960s progressed (Parker & Hewitt, 1970). With

this in mind we totally agree with the following

statement made by Parker and his colleagues (Parker

et a l . , 1974).

’’The days are past when the microbiologistcould attribute resistance problems solely to the misuse of antibiotics by clinicians, and then retire to his laboratory. We must accept that a great deal of antibiotic treatment will be given in hospitals, and see that this is done with as little ill effect as possible. The first element in our policy must be to have an efficient infection control organisation. In planning the use of individual agents, it is insufficient to ensure

237

that an effective agent is used for the treatment of all clinical infections, because this ignores the action of the agent on organisms at carrier sites; it is the total exposure of the hospital population to the agent that matters. The use of antibiotics in combination has so often failed to prevent the accumulation of resistant strains,notably in staphylococci with neomycin and bacitracin, and more recently with cotrimoxazole, that it cannot be relied upon in the future. On the other hand, we have several effective and unrelated agents for the treatment of staphylococcal infection, and resistance to these is at present infrequent or irregular in distribution. This situation offers some hope that an intelligent and flexible policy of diversification in the use of antibiotics might be a useful means of slowing up the rate at which resistances accumulate."

In this study, we have shown multiple

antibiotic resistance to be a particular problem

in MGRSA, for example more than 60% of strains

examined were resistant to 10 or more antibiotics.

With this in mind do we still (like Parker e_t

a l . , 1974) have "several effective and unrelated

agents" available to treat infections due to MGRSA?

238

4.2 Antibiotic options for the treatment of MRSAinfections and/or colonisation.

i . Options available for treatment of systemic

infections.

Table V shows that over 50% of MGRSA tested

were resistant to tobramycin, netilmicin, amikacin,

neomycin, streptomycin, tetracycline, minocycline,

erythromycin, clindamycin (including inducible

resistant strains) and trimethoprim. 41% of strains

were resistant to chloramphenicol, 28% were

resistant to rifampicin, 15% were resistant to

fusidic acid, and one strain was resistant to

novobiocin. No resistance was found to

nitrofurantoin, pristinamycin, teicoplanin and

vancomycin. Use of chloramphenicol often results in

rapid emergence of resistance, and this agent is

nowadays considered as sub-optimal therapy for

treatment of staphylococcal infections. Although

little resistance was found to novobiocin, its use

has often been associated with the rapid

development of resistance. Novobiocin has been

shown to be hepatotoxic, and its manufacturers

(Upjohn) seem most unwilling to encourage its use.

Currently, fusidic acid and rifampicin are

regarded as acceptable alternative agents to use

in place of vancomycin. From our in vitro studies

these agents are highly active against MGRSA.

239

However, because of the propensity of MGRSA todevelop resistance following exposure to either of

these agents, their use in appropriate combination

is recommended. 28% of MGRSA were resistant to

rifampicin, and for many of these strains the only

available recognised alternative chemotherapy is

vancomyc i n .

Because of the current lack of resistance to

vancomycin, and its proven efficacy in the

treatment of MRSA infections, vancomycin is the

first choice antibiotic to use against MGRSA.

Clinically, vancomycin use does have its problems,

because of its ototoxicity and nephrotoxicity

levels have to be monitored, and it has to be

slowly infused so as to avoid thrombophlebitis.

However, of greater concern to us is the

increasing reliance placed on vancomycin for

treating many Gram-positive infections. The resulting

greater bacterial exposure to vancomycin in the

hospital environment increases the likelihood of

development of resistance. Already increasing

problems with vancomycin resistance are being

encountered. There have recently been reports not

only of outbreaks of infection with enterococci

possessing plasmid-mediated vancomycin resistance, but

also of the isolation of vancomycin-resistant

coagulase-negative staphylococci (Johnson et a l . 1990) .

If P a r k e r ’s predictions are correct it only seems

to be a matter of time before vancomycin

240

resistance occurs in MRSA. With this in mind wehave attempted to identify agents which might be

used in place of vancomycin.

Fosfomycin, nitrofurantoin and pristinamycin had

good activity against MGRSA. Resistance to

fosfomycin was found in 21% of MGRSA studied,

however no resistance was found to nitrofurantoin

and pris tinamycin. Nitrofurantoin use is limited to

urinary tract infections, although the related

nitrofuran- furazolidone could be used to treat

MGRSA bowel carriage or enterocolitis. Intravenous

sodium nitrofurantoin has also been used in the

past. Fosfomycin and pristinamycin have been used

in some countries (eg. Spain, France), although they

are not available in others (eg. U.K) and

comparatively little is known of their

antistaphylococcal activity. We have shown

pristinamycin to be uniformly active against all

strains and are most encouraged by these findings.

Clinical resistance to fosfomycin (MIC greater than

64 mg/1) was only found in 14 strains of MGRSA.

Development of fosfomycin resistance in vitro was

found to readily occur, however the relative lack

of clinically isolated strains suggests that in

vitro findings may not necessarily be relevant to

the in vivo situation. Fosfomycin has been used

with success in the treatment of MRSA septicaemia

(Lau et a l . , 1986) and it could become a useful

alternative agent to vancomycin.

The activity of antimicrobial agents currently

under development eg. daptomycin, the oxazolidinones

(DuP 105 and DuP 721), paldimycin, ramoplanin, new 14,

15- and 16- membered macrolides and an injectable

streptogramin (RP 59500) have been assessed against

MGRSA. We have found daptomycin, the oxazolidinones,

paldimycin, ramoplanin and RP 59500 to be uniformly

active against MGRSA. However, only daptomycin and

RP 59500 (an injectable pristinamycin) are currently

being progressed as far as clinical trials.

Preliminary clinical trials with daptomycin were

disappointing and re-evaluation of this compound is

currently being undertaken. Rokitamycin was the

most active of the macrolides tested, however it

will only be of limited use because even though

it had good activity against erythromycin-sensitive

and inducible resistant MGRSA it was still

inactive against clindamycin-resistant strains.

From these studies it appears that few agents

are presently under development which could be

used as alternatives to vancomycin for systemic

therapy. Hence, P a r k e r ’s aim of "diversification in

the use of antibiotics" does not appear to be a

current reality.

242

i i . Options for treatment of MGRSA carriage.

Much consideration has been given to infection

control measures directed towards the elimination

of MRSA from the hospital environment (Working

Party, 1986 & 1990). Infection control measures can

range from treatment of colonised individuals with

antiseptics and/or topical antibiotics in an

attempt to rid them of carriage to closure of

whole wards and removal of colonised patients to

specialist isolation facilities.

Until recently, no single agent has consistently

eradicated MRSA from carriage sites, and there has

been considerable debate as to the efficacy of

antiseptics or topical antibiotics in such

indications (Casewell & Hill, 1986; Brumfitt & Hamilton-

Miller, 1989). Now that mupirocin is available (both

in skin and nasal preparations) we have an agent

of consistent and proven efficacy in clearing

nasal and skin carriage (Casewell & Hill, 1987).

Initial screening studies of staphylococci failed

to detect resistance to mupirocin (Casewell & Hill,

1985), and in our own survey of 100 MGRSA no

resistance was found. However, staphylococcal

resistance to mupirocin may become an increasing

problem, and naturally occurring resistant strains

have even been found (Rahman et a l . , 1990). Because

of emerging bacterial resistance, and the potential

toxicity (due to the polyethylene glycol base in

243

skin formulations) of mupirocin when applied to

extensive burns (Rode et a l . , 1989) other alternative

topical agents have been looked for. We have

found azelaic acid, nitrofurazone, ramoplanin, and

silver sulphadiazine to be uniformly active against

the MGRSA tested. We were particularly interested

by azelaic acid, a naturally occurring compound

reported to possess a considerable number of

probiotic effects (Breathnach et al . , 1984).

Antiseptics and topical antibiotics may hinder

wound healing (Leaper & Simpson, 1986), and it would

be interesting to learn of the influence of

azelaic acid on such processes.

4.3 Role of fluoroquinolones against M G R S A .

Ciprofloxacin was the first fluoroquinolone

possessing broad-spectrum activity and

therapeutically useful blood-levels to be developed

and enter clinical use. There was some initial

enthusiasm that ciprofloxacin might be efficacious

against MRSA in view of its novel mode of

action, uniform activity and rapid killing against

these organisms (Smith & Eng, 1985). Other

fluoroquinolones- enoxacin, ofloxacin and pefloxacin-

soon followed ciprofloxacin into clinical use, and

were similarly regarded as potentially useful

antistaphylococcal agents. Early in v i t r o , in vivo

244

and clinical studies were fairly optimistic with

regard to the efficacy of ciprofloxacin in

resolving S. aureus skin structure infections and

osteomyelitis (Neu, 1987). Specifically, ciprofloxacin

was shown to successfully eradicate MRSA

colonisation (Mulligan et a l . , 1987) and it was

thought to be a major advance in the treatment

of MRSA osteomyelitis (Neu, 1987).

Staphylococcal resistance to ciprofloxacin did

not appear to be a problem until 1989. Isaacs e_t

a l . (1988) are usually credited as the first

workers to report an outbreak of infection with

ciprofloxacin-resistant MRSA. However, in French

hospitals pefloxacin-resistant MRSA had posed

serious problems since 1986 (Acar & Buu-Hoi, 1988;

Jean-Pierre et a l . , 1988). In 1989, we showed

ciprofloxacin resistance in MGRSA to be an

international problem (Maple et a l . , 1989c), and

warned of the dangers of inappropriate

ciprofloxacin usage (Maple et a l . , 1989b). Since

this time there have been numerous reports of

ciprofloxacin resistance in MRSA.

Our studies outlined in Section 3 determined

which, if any, fluoroquinolone was most appropriate

for treating MGRSA infections, and sought to

explain the high incidences of ciprofloxacin

resistance reported for MRSA. Ciprofloxacin,

ofloxacin and pefloxacin were all highly active

against MGRSA, however because the peak serum levels

245

of these agents differ (Table XIII), ofloxacin and

pefloxacin appear to have a greater therapeutic

index than ciprofloxacin. The fluoroquinolones were

rapidly bactericidal, and only low mutation rates

to resistance were found.

Fluoroquinolone-resistant strains emerged during

our time-kill experiments, and this resistance could

readily develop following exposure to therapeutic

concentrations of ciprofloxacin, enoxacin and

pefloxacin. Resistance did not readily emerge in

the presence of therapeutic concentrations of

ofloxacin. Our results using strains of MGRSA with

different initial fluoroquinolone susceptibilities

showed that development of fluoroquinolone

resistance occurred in a stepwise manner.

The ability of a strain to become resistant

depended upon its initial fluoroquinolone

susceptibility and the concentration of

fluoroquinolone to which it was exposed. In this

manner strains with initial ciprofloxacin MICs of

0.25 mg/1 and 2.0 mg/1 could increase their

resistance to 2.0 mg/1 and 16.0 mg/1 respectively.

If this was repeated in v i v o , clinical isolates of

ciprofloxacin-resistant MRSA showing different levels

of resistance would be expected. Of more concern

is that any strain can develop fluoroquinolone

resistance in this way, and presumably the extent

of such resistance will depend on the amount of

fluoroquinolone usage. Perhaps these observations

246

might explain Schaefler’s (1989) observations with

ciprofloxacin-resistant MRSA in New York.

Ciprofloxacin-resistant MRSA isolated in 1987 were

characteristically susceptible to 2.0-4.0 mg/1

ciprofloxacin, whereas strains isolated after January

1988 were often more resistant (ciprofloxacin MICs

of 12.5-25 mg/1). Ciprofloxacin-resistant strains

appeared simultaneously in many different hospitals

and phage-typing and antibiotic resistance profiles

suggested they had independently evolved.

The same pattern of fluoroquinolone resistance

was selected in vitro by ciprofloxacin, enoxacin,

ofloxacin and pefloxacin. On the other hand,

different levels and patterns of fluoroquinolone

resistance were found in the clinical isolates of

MGRSA studied. The latter may be a result of

different mechanisms of fluoroquinolone resistance.

Of particular interest was the observation of

reversed incomplete resistance to acrosoxacin in

some of our isolates. Kojima e t a l . (1990) have

suggested that fluoroquinolone resistance can be

due to altered DNA gyrase or permeability/efflux

mechanisms.

There is some controversy at present as to

whether the high incidence of ciprofloxacin-

resistant MRSA is due to independent evolution of

strains or cross-infection by a few resistant

strains. Central to this debate is our current

ability to differentiate MGRSA. We have shown

247

(Table I) that a considerable number of MGRSA are

non-typable by the International Set of phages, and

new typing methods are required to differentiate

such strains. We have typed many of our strains

by using supplementary phages, antibiotic

susceptibility profiles, plasmid-content and a small

number of biological tests (sheep blood haemolysis

and egg yolk lipase production). Using this system

we have differentiated between many ciprofloxacin-

resistant isolates showing that the spread of

ciprofloxacin resistance is due to development of

resistance in individual strains as well as cross­

infection by resistant strains.

Due to the ease with which fluoroquinolone

resistance apparently develops in MGRSA, we are

pessimistic over the future therapeutic usefulness

of these agents against MGRSA. If fluoroquinolone

use is considered necessary for treatment then we

would suggest that ofloxacin is the most

appropriate agent to use. In the future new

fluoroquinolones may become available (eg.

sparfloxacin) which have a better therapeutic ratio

than ofloxacin, and such agents may prove more

effective in preventing the development of

resistant strains.

248

4.4 The future problem of antibiotic resistance inM G R S A .

In this work we have shown that multiple

antibiotic resistance in MGRSA presents a

significant therapeutic problem. Of great concern

is our observation that there are very few

currently available alternative agents for the

treatment of MGRSA carriage or infection. The

significance of these findings depends upon the

environment in which MGRSA is being isolated. In

countries where health care budgets are sufficient

for hospitals to devote time and expenditure to

control of MGRSA by implementation of rigorous

infection control measures the problems posed be

even highly multiple resistant strains of MGRSA

can be limited. However, in countries where such

measures cannot be financed although new

generations of increasingly potent antibiotics can

be purchased, the conditions are ripe for the

development and spread of highly multiple resistant

strains. Should vancomycin resistance emerge the

consequences for treatment of MGRSA infections

could be disastrous.

We have also seen that certain types of

antibiotic resistance can rapidly spread worldwide,

as in the case of gentamicin resistance in MRSA.

The rapid spread of this resistance is most

probably due to dissemination of the resistance

genes on a transposable genetic element. As such,

249

spread of resistance cannot be directly controlled

by antibiotic policy or even cross-infection

control measures. The location of resistance genes

on transposable genetic elements poses serious

potential resistance problems (Lyon & Skurray, 1987).

For S. aureus there is already considerable

evidence that such elements can alternate between

chromosomal or plasmid sites, be rapidly

disseminated between strains, and can be tranferred

to or acquired from different species (eg. S .

epidermidi s ) and even different genera (eg.

Enterococcus faecalis) .

It has been suggested (Koch, 1981; Foster, 1983)

that the genes responsible for antibiotic

resistance have evolved over many thousands of

years amongst soil organisms (particularly antibiotic

producers). Thus, by developing totally synthetic

antimicrobial agents with novel mechanisms of

action eg. the fluoroquinolones, it was believed

that bacterial resistance would not be a problem.

Our experience with ciprofloxacin resistance in

MGRSA shows that, as with trimethoprim and the

s ulphonamides, this is not the case.

Since the introduction of penicillin into

clinical use during the 1940s and the subsequent

development of other antibiotics there has been a

continual conflict with hospital staphylococci which

have shown a fecundity for developing resistance

to each new antibiotic. The impact of S. aureus

250

on the hospital environment has waxed and waned

over the years, as have the types of strain

responsible for such problems.

In the late 1940s extensive problems were

caused by Group I type (eg. type 52A) penicillinase

producing S. aureus which had a prediliction for

maternity and neonatal units. During the 1950s

these and type 80/81 strains were a cause of

major concern. Late in the 1950s multiple

antibiotic-resistant Group III strains became a

significant problem. In the 1960s Group I strains

ceased to appear in the hospital environment, and

following the introduction of methicillin and its

congeners most multiple resistant Group III strains

could be effectively dealt with. During the late

1960s and into the 1970s the influence of S .

aureus on hospital practice declined, however in

the late 1970s and continuing into the 1980s

major hospital problems due to MGRSA emerged.

These continue, for example, recently a small

outbreak of infections (one fatality) occurred in

our hospital due to a highly multiple-resistant

MGRSA. This MGRSA was resistant to many

antibiotics including ciprofloxacin, fusidic acid and

mupirocin, and could only be treated with vancomycin.

If vancomycin resistance does appear in MGRSA

our assessment of the current therapeutic options

available suggests that we are ill equipped for

this eventuality.

251

REFERENCES

ABRAHAM, E. P., CHAIN, E., FLETCHER, C. M . , GARDNER, A. D., HEATLEY, N. G., JENNINGS, M. A. (1941). Further observations on Penicillin. Lancet ii: 177-189.

ACAR, J. F., COURVALIN, P., CHABBERT, Y. A. (1970). Methicillin-resistant staphylococcaemia: bacteriological failure of treatment with cephalosporins, pp. 280- 285. Antimicrobial Agents and Chemotherapy (1971).

ACAR, J. F., GOLDSTEIN, F. W., DUVAL, J. (1983). Use of rifampicin for the treatment of serious staphylococcal ond Gram- negative bacillaryinfections. Reviews of Infectious Diseases 5 (S u p p l . 3), 502- 506.

ACAR, J. F., B U U - H O I , A. Y. (1988). Resistance patterns of important Gram- positive pathogens. Journal of Antimicrobial Chemotherapy 21: (S u p p l . C ), 41- 47.

ACAR, J. F., CLUZEL, R., COURVALIN, P., DUVAL, J., FLEURETTE, J., MEGRAUD, F., MEYRAND, M . , SOUSSY, C. J., THABAUT, A. (1990). RP 59500: a collaborative study of a semi-synthetic streptogramin in seven general hospitals in France. Abstracts of the 30th International Conference of Antimicrobial Agents and Chemotherapy (Atlanta), Abstract no. 780.

ALLEN, J. D., EVANS ROBERTS, C., KIRBY, W. M. M. (1962). Staphylococcal septicaemia treated with methicillin. Report of twenty-two cases. New England Journal of Medicine 266: 111-116.

ALY, R., SHINEFIELD, H. I., STRAUSS, W. G., M A I B A C H , H.I. (1977). Bacterial adherence to nasal mucosal cells. Infection and Immunity 17: 546-549.

252

A M Y E S , S. G. B., TAIT, S. (1990). Trimethoprimresistance in staphylococci. Journal of Medical Microbiology 31: 4-7.

ANDREW, J. H., WALE, M. C. J., WALE, L. J., GREENWOOD, D.(1987). The effect of cultural conditions on the activity of LY 146032 against staphylococci and streptococci. Journal of Antimicrobial Chemotherapy 20: 213- 221.

ANDREWS, J. M., B A Q U E R O , F., BELTRAN, J. M., CANTON, E., CROKAERT, F., G O B E R N A D O , M., GOMEZ- L U S , R., LOZA, E., NAVARRO, M., OLAY, T., RODRIGUEZ, A., VICENTE, M. V., WISE, R., YOURASSOWSKY, E. (1983). Internationalcollaborative study on standardization of bacterial sensitivity to fosfomycin. Journal of Antimicrobial Chemotherapy 12: 357- 361.

ANNEAR, D. I. (1968). The effect of temperature on resistance of Staphylococcus aureus to methicillin and some other antibiotics. Medical Journal ofAustralia 1: 444-446.

ARATH00N, E. G., HAMILTON, J. R., HENCH, C. E., STEVENS, D. A. (1990). Efficacy of short courses of oral novobiocin-rifampin in eradicating carrier state of methicillin-resistant Staphylococcus aureus and in vitro killing studies of clinical isolates. Antimicrobial Agents and Chemotherapy 34: 1655-1659.

ARGOUDELIS, A. D., BACZYNSKYJ, L., B U E J E , J. A.,MARSHALL, V. P., MIZSAK, S. A., WILEY, P. F. (1987a). Paulomycin-related antibiotics: paldimycins andantibiotics 273a2» Isolation and characterization. Journal of Antibiotics 40: 408- 418.

253

ARGOUDELIS, A. D., BACZYNSKYJ, L., MIZSAK, S. A., SHILLIDAY, F. B., SPINELLI, P. A., DEZWAAN, J. (1987b). Paldimycins A and B and antibiotics 273a2a and 273aab» Synthesis and characterization. Journal of Antibiotics 40: 419- 436.

ARTHUR, M., BRISSON-NOEL, A., COURVALIN, P. (1987). Origin and evolution of genes specifying resistance to macrolide, lincosamide and streptograminantibiotics: data and hypotheses. Journal ofAntimicrobial Chemotherapy 20: 783- 802.

ASHESHOV, E. H. (1975). The genetics of tetracyclineresistance in Staphylococcus a ureus. Journal ofMedical Microbiology 88: 132- 140.

A YLIFFE, G. A. G., GREEN, W., LIVINGSTON, R., L O W B U R Y , E. J. L. (1977). Antibiotic- resistant Staphylococcus aureus in dermatology and burn wounds. Journal ofClinical Pathology 30: 40-44.

AYLIFFE, G. A. G., LILLY, H. A., LOWBURY, E. J. L. (1979). Decline of the hospital staphylococcus?Incidence of multi-resistant S . aureus in threeBirmingham hospitals. Lancet i: 538-541.

BAIRD, D., C0IA, J. (1987). Mupirocin-resistant Staphylococcus au r e u s . Lancet ii: 387-388.

BAIRD-PARKER, A. C. (1963). A Classification ofMicrococci and Staphylococci based on Physiologicaland Biochemical tests. Journal oj GeneralMicrobiology 30: 409-427.

BAIRD-PARKER A. C. (1972). Classification andIdentification of Staphylococci and their Resistanceto Physical Agents ^In: COHEN, J. 0. (ed). TheStaphylococci. New York, Wiley-Interscience Press, p. 1- 2 0 .

254

BAQUERO, F., LOPEZ-BREA, M., V A L L S , A., CANEDO, T.(1977). Fosfomycin and plasmidic resistance. Chemotherapy 23 (Suppl. 1) t 133-140.

BARBER, M. (1947). Staphylococcal infection due to penicillin-resistant strains. British Medical Journal 2: 863-865.

BARBER, M. (1960). Drug-resistant staphylococcal infection. British Journal of Obstetrics and Gynaecology 67: 727-732.

BARBER, M. (1964). Naturally occurring methicillin-resistant staphylococci. Journal o_f GeneralMicrobiology 35: 183-190.

BARBER, M., R0ZWAD0WSKA-D0WZENK0, M. (1948). Infection by penicillin-resistant staphylococci. Lancet ii: 641-644.

BARBER, M., B U R S T O N , J. (1955). Antibiotic-resistant staphylococcal infection. A study of antibiotic sensitivity in relation to bacteriophage types. Lancet ii: 578-583.

BARBER, M., WATERWORTH, P. M. (1962). Antibacterialactivity in vitro of fucidin. Lancet i: 931- 932.

BARBER, M., WATERWORTH, P. M. (1964). Antibacterialactivity of lincomycin and pristinamycin: acomparison with erythromycin. British MedicalJournal 2: 603- 606.

BARBER, M., WATERWORTH, P. M. (1966). Activity ofgentamicin against pseudomonas and hospital staphylococci. British Medical Journal 1: 203-205.

255

BARRETT, F. F., McGEHEE, R. F., FINLAND, M. (1968). Methicillin-resistant Staphylococcus aureus at Boston City Hospital. Bacteriologic and epidemiologic observations. New England Journal of Medicine 279: 441-448.

BA R R I E R E , J. C., BOUANCHAUD, D. H., HARRIS, N. V., PARIS, J. M., ROLIN, 0., SMITH, C. (1990). The design, synthesis and properties of RP 59500 and related semi-synthetic streptogramin antibiotics. Abstracts of the 30th International Congress on Antimicrobial Agents and Chemotherapy (Atlanta), Abstract No. 768.

BARRY, A. L., JONES, R. N. (1989). In-vitro activities of temafloxacin, tosufloxacin (A-61827) and five other fluoroquinolone agents. Journal of Antimicrobial Chemotherapy 23:527-536.

BARTZOKAS, C. A., PAT0N, J. H., GIBSON, M. F., GRAHAM,R., McLOUGHLIN, G. A., CROTON, R. S. (1984). Control and eradication of methicillin-resistantStaphylococcus aureus on a surgical unit. NewEngland Journal of Medicine 311: 1422-1425.

BASTIN, R., RAPIN, M., KERNBAUM, S. (1982). Controlled study of pristinamycin versus oxacillin in staphylococcal infections. Pathologie et Biologie(Paris) 6: 473- 475.

BEAVEN, D. W., BURRY, A. F. (1956). Staphylococcalpneumonia in the newborn. An epidemic with 8 fatalcases. Lancet ii: 211-215.

BENNER, E. J., KAYSER, F. H. (1968). Growing clinical significance of methicillin-resistant Staphylococcus aureus. Lancet ii: 741-744.

256

B I N D A , G., DOMENICHINI, E., GOTTARDI, A., ORLA N D I , B., ORTELLI, E., PACINI, B., FOWST, G. (1971). Rifampicin, a general review. Arzneimittelforschung 12a: p.1962.

BISMUTH, R., ZILHAO, R., SAKAMOTO, H., GUESDON, J - L . , COURVALIN, P. (1990). Gene heterogeneity for tetracycline resistance in Staphylococcus spp. Antimicrobial Agents and Chemotherapy 34: 1611—1614.

BLACK, H. R., BRIER, G. L., W O L N Y , J. D., N Y H A R T , E. H. (1986). Preliminary pharmacology and pharmacokinetics of LY 146032, a new peptolide antibiotic. Abstracts of the 26th Interscience Conference onAntimicrobial Agents and Chemotherapy, New Orleans, Abstract 894.

BLADON, P. T., BURKE, B. M., CUNLIFFE, W. J., FORSTER, R. A., HOLLAND, K. T., KING, K. (1986). Topical azelaic acid and the treatment of acne: a clinical andlaboratory comparison with oral tetracycline.British Journal of Dermatology 114: 493-499.

BLAKE, S. C., CHAPMAN, P. C., EATON, N, MERRI K I N , D. J. (1990). BRL 44154 - A novel penicillin activeagainst methicillin- resistant staphylococci - in vitro and in vivo properties.Abstracts of the 7th Mediterranean Congress ofChemotherapy, Barcelona, Spain, 20- 25th May, 1990.

BLUM, A. (1982). Culturing an epidemic of staphylococci in the mass media. Medical Journal of Australia 1: 475- 479.

BONDI A., Jr., DIETZ, C. C. (1945). Penicillin resistant staphylococci. Proceeedings of the Society for Experimental Biology and Medicine 60: 55-58.

257

BOROWSKI, J., JAKUBICZ, P., ZAREMBA, M. et al. (1988).Nationwide studies on occurrence of methicillin resistant Staphylococcus aureus and Staphylococcuscoagulase-negative strains in Polish hospitals. Some characteristics of MRSA strains. Abstract P2 / 12. 1st International Conference of the Hospital Infection Soc i e t y , 1988.

BRADY, L. M., THOMSON, M., PALMER, M. A., HARKNESS, J. L. (1990). Succesful control of endemic MRSA in a cardiothoracic surgical unit. Medical Journal of Australia 152: 240-245.

BREATHNACH, A. S., NAZZAR0-P0RR0, M. , PASSI, S. (1984). Azelaic acid. British Journal of Dermatology 111: 115-120.

BREED, R.S., MURRAY, E.G.D., SMITH, N.R. (1957). Bergey's Mannual of Determinative Bacteriology.Seventh Edition, London, E. & S. Livingstone.

BRIDGES, R. A., B E R E N D E S , H., GOOD, R. A. (1957).Serious reactions to novobiocin. Journal___ ofPediatrics 50: 579- 585.

BRITISH SOCIETY FOR ANTIMICROBIAL CHEMOTHERAPY(1988). Break-points in in____ vitro antibioticsensitivity testing. Journal_____of AntimicrobialChemotherapy 21: 701-710.

BROWN, D. F. J., REYNOLDS, P. E. (1980). Intrinsic resistance to B-lactam antibiotics in Staphylococcus au r e u s . FEBS Letters 122: 275-278.

BRUMFITT, W., HA MILTON-MILLER, J. M. T. (1988). In vitro microbiological activities of DuP 105 andDuP 721, novel synthetic oxazolidinones. Journal of Antimicrobial Chemotherapy 21: 711- 720.

258

BRUMFITT, W., MAPLE, P. A. C., HAMILTON-MILLER, J. M. T.(1989). Antibiotic sensitivity patterns of methicillin-resistant Staphylococcus aureus and their use in biotyping. Abstract 778/PP40, p. 325, Abstracts of 4th European Congress of Clinical Micro biology, Nice, France.

BRUMFITT, W., HAMILTON-MILLER, J. M. T. (1989).Methicillin-resistant Staphylococcus a u r e u s . NewEngland Journal of Medicine 320: 1188-1196.

BULGER, R. J., SHERRIS, J. C. (1968). Decreasedincidence of antibiotic resistance amongStaphylococcus au r e u s . A study in a university hospital over a 9-year period. Annals of Internal Medicine 69: 1099-1107.

BULLOCH, W. (1938). The History of Bacteriology.First Edition, London, Oxford University Press.

BURDESKA, A., THEN, R. L. (1990). Clinical importance of trimethoprim resistance in staphylococci isolated in Europe. Journal of Medical Microbiology 31: 11-14.

BYRNE, M. E., GILLESPIE, M. T., SKURRAY, R. A. (1990). Molecular analysis of a gentamicin resistancetransposonlike element on plasmids isolated fromNorth American Staphylococcus aureus strains.Antimicrobial Agents and Chemotherapy 34: 2106-2113.

CAFFERKEY, M. T., HONE, R., FALKI N E R , F. R., KEANE, C. T., POMEROY, H. (1983). Gentamicin and methicillin- resistant Staphylococcus aureus in Dublin hospitals: clinical and laboratory studies. Journal of Medical Microbiology 16: 117-127.

259

CAFFERKEY, M. T., HONE, R., COLEMAN, D . , POMEROY, H.,McGRATH, B., RUDDY, R., KEANE, C. T. (1985a).Methicillin-resistant Staphylococcus aureus in Dublin1971-1984. Lancet ii: 705-708.

CAFFERKEY, M. T., HONE, R., KEANE, C. T. (1985). Antimicrobial chemotherapy of septicaemia due tomethicillin-resistant Staphylococcus_______ aureus.Antimicrobial Agents and Chemotherapy 28: 819- 823.

CALAIN, P., WALDVOGEL, F. (1990). Clinical efficacy ofteicoplanin. European_____Journal_____ of_____ClinicalMicrobiology and Infectious Disease 9: 127- 129.

CANEPARI, P., BOARETTI, M., DEL MAR LLEO, M., SATTA, G.(1990). Lipoteichoic acid as a new target foractivity of antibiotics: mode of action ofdaptomycin (LY 146032). Antimicrobial Agents and Chemotherapy 34: 1220- 1226.

CARR, H. S., WL0DK0WSKI, T. J., ROSENKRANZ, H. S. (1973). Silver sulfadiazine: in vitro antibacterial activity. Antimicrobial Agents and Chemotherapy 4: 585-587.

CARROLL, J. D., POMEROY, H. M., RUSSELL, R. J.,ARBUTHN0TT, J.P., KEANE, C. T., McCORMICK, 0. M. ,COLEMAN, D. C. (1989). A new methicillin- andgentamicin- resistant Staphylococcus____ aureus inDublin: molecular genetic analysis. Journal___ ofMedical Microbiology 28: 15-23.

CASEWELL, M. W., HILL, R. L. R. (1985). In-vitroactivity of mupirocin ("pseudomonic acid") againstclinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 15: 523-531.

2 6 0

CASEWELL, M. W. (1986). Epidemiology and control of the ’modern’ methicillin-resistant Staphylococcusaureus. Journal of Hospital Infection 7: (Suppl. A)t 1- 1 1 .

CASEWELL, M. W., HILL, R. L. R. (1986). The Carrier State: methicillin-resistant Staphylococcus aureus.Journal of Antimicrobial Chemotherapy 18: (Suppl. A), 1-12.

CASEWELL, M. W. , HILL, R. L. R. (1987). Mupirocin (”pseudomonic acid”)- a promising new topicalantimicrobial agent. Journal of AntimicrobialChemotherapy 19: 1-5.

CAVALLERI, B., PAGANI, H., VOLPE, G., SELVA, E., PARENTI,F. (1984). A-16686, a new antibiotic fromActinoplanes. I. Fermentation, isolation andpreliminary physico-chemical characteristics. Journal of Antibiotics 37: 309-317.

CHABBERT, Y., TERRIAL, G. (1952). Evolution actuelledes types de resistance aux antibiotiques chez lesstaphylocoques pathogenes. Annales De L f InstitutPasteur 83: 499-505.

CHAMBERLAIN, R. E. (1976). Chemotherapeutic properties of prominent nitrofurans. Journal of Antimicrobial Chemotherapy 2: 325-336.

CHAMBERS, H. F. (1988). Methicillin-resistantstaphylococci. Clinical Microbiology Reviews 1: 173-186.

CHAMBERS, H. K., SACHDEVA, M. (1990). Binding of B- lactam antibiotics to penicillin-binding proteins in methicillin-resistant Staphylococcus aureus. Journal of Infectious Diseases 161: 1170-1176.

261

CHANDRASEKAR, P. H., SLUCHAK, J. A. (1989). Susceptibility of staphylococci to paldimycin and emergence of resistance in vitro. Journal of Antimicrobial Chemotherapy 24: 821-824.

CHAUDHRY, A. Z., KNAPP, C. C., SIERRA-MADERO, J.,WASHINGTON, J. A. (1990). Antistaphylococcal activities of sparfloxacin (CI-978; AT-4140), ofloxacin andciprofloxacin. Antimicrobial Agents and Chemotherapy 34: 1843-1845.

CHENG, A. F., FRENCH, G. (1988). Methicillin-resistant Staphylococcus aureus bacteraemia in Hong Kong. Journal of Hospital Infection 12: 91-101.

CHOKKAVELU, V., CHANDRASEKAR, P., R0LST0N, K., LeFROCK, J. L., SCHELL, R. F. (1984). Activity of eleven antimicrobial agents against methicillin-,methicillin- and rifampicin- resistant Staphylococcus aureus . Chemotherapy 30: 97-101.

CHOPRA, I. (1976). Mechanisms of resistance to fusidic acid in Staphylococcus aureus. Journal of General Microbiology 96: 229-238.

CHOW, J. W., YU, V. L. (1989). Staphylococcus aureus nasal carriage in haemodialysis patients, its role in infection and approaches to prophylaxis. Archives of Internal Medicine 149: 1258-1262.

CLARKE, H. T., JOHNSON, J. R., ROBINSON, R. (1949). The chemistry of Penicillin. Princeton University Press, New Jersey.

CLUMECK, N., MARCELIS, L., AMRI-LAMRASKI, M. H., GORDTS,B. (1984). Treatment of severe staphylococcal infections with a rifampicin-minocycline association. Journal of Antimicrobial Chemotherapy 13 (Suppl. C), 17- 22.

262

COCITO, C. (1979). Antibiotics of the virginiamycin family, inhibitors which contain synergisticcomponents. Microbiological Reviews 43: 145- 198.

COLEBROOK, L., KENNY, M. (1936). Treatment of human puerperal infections, and experimental infections in mice, with prontosil. Lancet i: 1279-1286.

COLEMAN, D. C., POMEROY, H., ESTRIDGE, J. K., KEANE, C. T., CAFFERKEY, M. T., HONE, R., FOSTER, T. J. (1985). Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin- resistant Staphylococcus aureus from Dublin hospitals. Journal of Medical Microbiology 20: 157- 167.

COMMUNICABLE DISEASE REPORT (1986). Two- region survey of methicillin-resistant Staphylococcus aureus (MRSA). Communicable Disease Report 86/10.

COOKE, E. M., CASEWELL, M. W., EMMERSON, A. M., GASTON,M., de SAXE, M., MAYON-WHITE, R. T., GALBRAITH, N. S.(1986). Methicillin-resistant Staphylococcus aureus in the U.K. and Ireland. A questionaire survey. Journal of Hospital Infection 8: 143-148.

C00KS0N, B., TALSANIA, H., NAIDOO, J., PHILLIPS, I.(1986). Strategies for typing and properties of epidemic methicillin- resistant Staphylococcus aureus. European Journal of Clinical Microbiology 5: 702-709.

C00KS0N, B. D., PHILLIPS, I. (1988). Epidemicmethicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 21, (Suppl. C), 57-65.

C00KS0N, B. D., PHILLIPS, I. (1989). Letter to the editor. New England Journal of Medicine 321: 1344.

263

COOVADIA, Y. M.f BHANA, R. H., JOHNSON, A. P., HAFFEJEE,I., MARPLES, R. R. (1989). A laboratory- confirmed outbreak of rifampicin- methicillin resistant Staphylococcus aureus (RMRSA) in a newborn nursery. Journal of Hospital Infection 14: 303- 312.

COPPENS, L., HANSON, B., KLASTERSKY, J. (1983). Therapy of staphylococcal infections with cefamandole orvancomycin alone or with a combination ofcefamandole and tobramycin. Antimicrobial Agents and Chemotherapy 23: 36-41.

COURTIEU, A. L., DRUGEON, H., BILLAUDEL, S. (1977).Susceptibility to fosfomycin of hospital strainsisolated in Nantes (France). Frequency of mutation to resistance. Chemotherapy 23 (Suppl. 1), 25- 36.

COURVALIN, P., DAVIES, J. (1977). Plasmid- mediated aminoglycoside phosphotransferase of broad substrate range that phosphorylates amikacin. Antimicrobial Agents and Chemotherapy 11: 619- 624.

COWAN, S. T., STEEL, K. J. (1974). Identification of Medical Bacteria. 2nd edition, Cambridge University Press (Cambridge).

CRAVEN, D. E., REED, C., KOLLISCH, N., DeMARIA, A.,LICHTENBERG, D., SHEN, K., McCABE, W. R. (1981). A large outbreak of infections caused by a strain of Staphylococcus aureus resistant to oxacillin and aminoglycosides. American Journal of Medicine 71: 53-58.

CRAVEN, D. E., RIXINGER, A. I., GOULARTE, T. A., McCABE, W. R. (1986). Methicillin- resistant Staphylococcus aureus bacteraemia linked to intravenous drug abusers using a "shooting gallery". American Journal of Medicine 80: 770-776.

2 6 4

CROSSLEY, K., LOESCH, D., LANDESMAN, B., MEAD, K., CHERN, M. , STRATE, R. (1979). An outbreak of infectionscaused by strains of Staphylococcus aureusresistant to methicillin and aminoglycosides. 1.Clinical studies. Journal of Infectious Diseases139: 273-279.

CRUICKSHANK, R., DUGUID, J. P., MARMION, B. P., SWAIN, R. H. A. (1975). Medical Microbiology (The Practice ofMedical Microbiology). 12th edition, ChurchillLivingstone (Edinburgh).

CRUMPLIN, G. C. (1990). Mechanisms of resistance to the 4-quinolone antibacterial agents. Journal of Antimicrobial Chemotherapy 26 (Suppl. F), 131-144.

CULLMANN, W., STIEGLITZ, M., BAARS, B., OPFERKUCH, W.(1985). Comparative evaluation of recently developed fluoroquinolone compounds - with a note on thefrequency of resistant mutants. Chemotherapy 31:19-28.

CUNDLIFFE, E. (1972). The mode of action of fusidicacid. Biochemical and Biophysical ResearchCommunications 46: 1794-1801.

DALY, J. S., ELI0P0UL0S, G. M., REISZNER, E., MOELLERING Jr., R. C. (1988). Activity and mechanism of actionof DuP 105 and DuP 721, new oxazolidinonecompounds. Journal of Antimicrobial Chemotherapy 21: 721-730.

DAUM, T. E., SCHABERG, D. R., TERPENNING, M. S., SOTTILE, W. S., KAUFFMAN, C. A. (1990). Increasing resistanceof Staphylococcus aureus to ciprofloxacin.Antimicrobial Agents and Chemotherapy 34: 1862-1863.

265

DEVAUD, M., KAYSER, F. H., HUBER, R. (1977). Resistance of bacteria to the newer aminoglycoside antibiotics: an epidemiological and enzymatic study. Journal of Antibiotics XXX: 655- 664.

DORNBUSCH, K., MILLER, G. H., HARE, R. S., SHAW, K. J. and the ESGAR Study Group (1990). Resistance to aminoglycoside antibiotics in Gram- negative bacilliand staphylococci isolated from blood. Report froma European collaborative study. Journal ofAntimicrobial Chemotherapy 26: 131- 144.

DOUTHWAITE, A. H., TRAFFORD, J. A. P. (1960). A new penicillin. British Medical Journal 2: 687-690.

DOWDING, J. E. (1977). Mechanisms of gentamicin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 11: 47- 50.

DUGUID, J. P. (1951). The demonstration of bacterial capsules and slime. Journal of Pathology andBacteriology 63: 673-685.

DUNKLE, L. M., NAQVI, S. H., McCALLUM, R., LOFGREN, J. P.(1981). Eradication of epidemic methicillin- gentamicin resistant Staphylococcus aureus in anintensive care nursery. American Journal ofMedicine 70: 455- 458.

DUVAL, J. (1985). Evolution and epidemiology of MLSresistance. Journal of Antimicrobial Chemotherapy 16: (Suppl. A), 137- 149.

DYKE, K. G. H. (1969). Penicillinase production andintrinsic resistance to penicillins in methicillin-resistant cultures of Staphylococcus aureus. Journalof Medical Microbiology 2: 261-278.

266

EDITORIAL (1977). Topical Antibiotics. British Medical Journal 1: 1494.

EDITORIAL BOARD (1958). Conservation of the generic name Staphylococcus Rosenbach, designation of thename Staphylococcus aureus Rosenbach as thenoraenclatural type of the genus StaphylococcusRosenbach, and designation of a neotype culture ofStaphylococcus____ aureus Rosenbach. Opinion 17.International_____ Bulletin_______ of______ BacteriologicalNomenclature and Taxonomy 8: 138-153.

ELEK, S. D., HILSON, G. R. F. (1954). Combined agardiffusion and replica plating techniques in the study of antibacterial substances. Journal ofClinical Pathology 7: 37-44.

ELIOPOULOS, G. M., WILLEY, S., REISZNER, E., SPITZER, P.G., CAPUTO, G., MOELLERING Jr., R. C. (1986). In vitroand in vivo activity of LY 146032, a new cycliclipopeptide antibiotic. Antimicrobial Agents andChemotherapy 30: 532-535.

ELWELL, L. P., WILSON, H. R., KNICK, V. B.,KEITH, B. R.(1986). In vitro and in vivo efficacy of the combination trimethoprim-sulfamethoxazole againstclinical isolates of methicillin-resistantStaphylococcus aureus. Antimicrobial Agents andChemotherapy 29: 1092-1094.

ENG, R. K., SMITH, S. M., TILLEM, M., CHERUBIN, C.(1985). Rifampicin resistance: development ofresistance during the therapy of methicillin-resistant Staphylococcus aureus infection. Archives of Internal Medicine 145: 146-148.

ERIKSEN, K. R., ERICHSEN, I. (1963). Resistance to the newer penicillins. British Medical Journal 1: 746.

2 6 7

EYKYN, S. J. (1988). Staphylococcal sepsis. The changing pattern of disease and therapy. Lancet i: 100-104.

FARBER, B. B. (1984). Vancomycin: renewed interest inan old drug. European Journal____ of___ClinicalMicrobiology 3: 1-3.

FASS, R. J., HELSEL, V. L. (1987). In vitro antistaphylococcal activity of pefloxacin alone andin combination with other antistaphylococcal drugs.Antimicrobial Agents and Chemotherapy 31: 1457-1460.

FINKELSTEIN, R., MARKEL, A., REINHERZ, G., HASHMAN, N., MERZBACH, D. (1989). The emergence of methicillin- resistant Staphylococcus aureus infections in an Israeli hospital. Journal of Hospital Infection 14: 55-61.

FINLAND, M., HAIGHT, T. H. (1953). Antibioticresistance of pathogenic staphylococci. Archives of Internal Medicine 91: 143-158.

FINLAND, M. (1955). Emergence of antibiotic-resistant bacteria. New England Journal of Medicine 253: 909-922.

FINLAND, M. (1974). An Historical Introduction I_n JACKSON, G. G., ESCARZAGA, E., GARR0D, L. P., NEU, H., UEDA, Y. (eds). Gentamicin: review and commentary on selected world literature. New York, Council forInterdisciplinary Communication in Medicine Ltd.,pp. iii- viii .

FLEMING, A. (1929). On the antibacterial action of cultures of a Penicillium, with special referenceto their use in the isolation of B. influenzae. British Journal of Experimental Pathology 10: 226-236.

268

FOLDES, M . , MUNRO, R., SORRELL, T. C., SHANKER, S., TOOHEY, M. (1983). In vitro effects of vancomycin, rifampicin, and fusidic acid, alone and in combination, against methicillin- resistantStaphylococcus aureus. Journal of AntimicrobialChemotherapy 11: 21-26.

FOSTER, J. K., LENTINO, J. R., STRODTMAN, R., DiVINCENZO, C. (1986). Comparison of in vitro activity of quinolone antibiotics and vancomycin against gentamicin- and methicillin-resistant Staphylococcus aureus by time-kill studies. Antimicrobial Agents and Chemotherapy 30: 823-827.

FOSTER, T. J. (1983). Plasmid-determined resistance to antimicrobial drugs and toxic metal ions inbacteria. Microbiological Reviews 47: 361-409.

FRENCH, G. L., LING, J., LING, T., HUI, Y. W. (1988). Susceptibility of Hong Kong isolates ofmethicillin- resistant Staphylococcus aureus toantimicrobial agents. Journal____ of AntimicrobialChemotherapy 21: 581-588.

FRIMODT-MOLLER, N., THAMDRUP ROSDAHL, V., SORENSEN, G., HARTVIG HARTZEN, S., WEIS BENTZON, M. (1986).Relationship between penicillinase production andthe in-vitro activity of methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, and cephalothin against strains of Staphylococcus aureus of different phage patterns and penicillinase activity. Journal of Antimicrobial Chemotherapy 18: 27-33.

269

FRONGILLO, R. F., DONATI, L., FEDERICO, G., MARTINO, P., MORONI, M., ORTONA, L., PALUMBO, M. , PASTICCI, B. M. , PIZZIGALLO, E., PRIVITERA, G., SERRA, P., SIGNORINI, M. , VENDITTI, M., PAULUZZI, S. (1986). Clinical comparative study on the activity of cefamandole in the treatment of serious staphylococcal infections caused by methicillin-susceptible and methicillin-resistant strains. Antimicrobial Agents andChemotherapy 29: 789-796.

GARGAN, R. A., BRUMFITT, W., HAMILTON-MILLER, J. M. T.(1982). A concise biotyping system differentiating strains of Escherichia coli. Journal of ClinicalPathology 35: 1366-1369.

GARRISON, M. W., VANCE-BRYAN, K., LARSON, T. A., TOSCANO, J. P., ROTSCHAFER, J. C. (1990). Assesment of effects of protein binding on daptomycin and vancomycinkilling of Staphylococcus aureus by using an invitro pharmacodynamic model. Antimicrobial Agentsand Chemotherapy 34: 1925-1931.

GARROD, L. P. (1944). The laboratory control ofPenicillin treatment. British Medical Journal 1:528-530.

GARROD, L. P. (1968). Methicillin-resistantstaphylococci. Lancet ii: 871.

GARROD, L. P., LAMBERT, H. P., 0 fGRADY, F. (1981).Antibiotic and Chemotherapy. 5th edition, Edinburgh, Churchill Livingstone, pp. 73-77.

GEDEBOU, M., KRONVALL, G., HABTE-GABR, E., RINGERTZ, S.(1987). The bacteriology of nosocomial infections at Tikur Anbessa teaching hospital, Addis Ababa.Acta Pathologica, Micr obiologica , et ImmunologicaScandinavica, Section B 95: 331- 336.

270

GELLERT, M. , O ’ DEA, M. H., ITOH, T., TOMIZAWA, J. (1976). Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proceedings of the National Academy of Sciences, USA. 73: 4474- 4478.

GIAMARELLOU, H., PAPAPETROPOULOU, M., DAIKOS, G. K.(1981). ’Methicillin resistant’ Staphylococcus aureus infections during 1978- 1979: clinical andbacteriological observations. Journal_______ofAntimicrobial Chemotherapy 7: 649-655.

GILLESPIE, W. A., ALDER, V. G. (1952). Production of opacity in egg-yolk media by coagulase-positive staphylococci. Journal of Pathology and Bacteriology 64: 187-199.

GILLESPIE, W. A., SIMPSON, K. (1958). Staphylococcal infection in a maternity hospital, epidemiology and control. Lancet ii: 1075-1080.

GLUPCZYNSKI, Y., LAGAST, H., VAN DER AUWERA, P., THYS, J. P., CROKAERT, F., YOURASSOWSKY, E., MEUNIER-CARPENTIER, F., KLASTERSKY, J., KAINS, J. P., LEGRAND, J. C. (1986). Clinical evaluation of teicoplanin for therapy of severe infections caused by Gram-positive bacteria. Antimicrobial Agents and Chemotherapy 29: 52-57.

GODTFREDSEN, W., R0H0LT, K., TYBRING, L. (1962).Fucidin, a new orally active antibiotic. Lancet i: 928-931.

GOTO, S. (1977). Fosfomycin, antibacterial activity in vitro and in vivo. Chemotherapy 23 (Suppl. 1), 63-74.

GRAHAM, D. R., CORREA- VILLASENOR, A., ANDERSON, R. L.,VOLLMAN, J. H., BAINE, W. B. (1980). Epidemic neonatalgentamicin- methicillin- resistant Staphylococcusaureus infection associated with nonspecific topical

271

use of gentamicin. Journal of Pediatrics 97: 972-978.

GRANINGER, W. , LEITHA, T., HAVEL, M., GEORGOPOULOS, A.(1984). In vitro activity of fosfomycin against methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Infection 12: 293- 295.

GREENWOOD, D. (1988). Microbiological properties ofteicoplanin. Journal of Antimicrobial Chemotherapy 21 (Suppl. A), 1- 13.

GRIFFITH, R. S. (1984). Vancomycin use - an historical review. Journal of Antimicrobial Chemotherapy 14(Suppl. D), 1- 5.

GRUBB, W. B., TOWNSEND, D.E., ASHDOWN, N., TJIA, T., McGLASHAN, C., LENG, T. (1986). Genetic analysis ofmethicillin-resistant Staphylococcus aureus fromSingapore Hospitals. European Journal of ClinicalMicrobiology 5: 728-730.

GUENTHNER, S. H., WENZEL, R. P. (1984). In vitroactivities of teichomycin, fusidic acid,flucloxacillin, fosfomycin, and vancomycin againstmethicillin- resistant Staphylococcus______aureus .Antimicrobial Agents and Chemotherapy 26: 268- 269.

GUPTA, S. P., CHAKRAVATI, R. N. (1954). Determination of bacterial sensitivity to common antibiotics.Indian Journal of Medical Research 42: 159-164.

HACKBARTH, C. J., CHAMBERS, H. F. (1989a). Methicillin-resistant staphylococci: detection methods andtreatment of infections. Antimicrobial Agents andChemotherapy 33: 995-999.

272

HACKBARTH, C. J., CHAMBERS, H. F. (1989b). Methicillin- resistant staphylococci: genetics and mechanisms ofresistance. Antimicrobial Agents and Chemotherapy33: 991-994.

HADORN, K., LENZ, W. , KAYSER, F. H., SHALIT, I., KRASEMANN, C. (1990). Use of a ribosomal RNA gene probe for the epidemiological study of methicillin and ciprofloxacin resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 9: 649-653.

HAJEK, V., MARSALEK, E. (1971). The Differentiation of Pathogenic Staphylococci and a Suggestion for theirTaxonomic Classification. Zentralblatt furBakteriologie Mikrobiologie und Hygiene, I. Abt. Originale (Series) A 217: 176-182.

HAJEK, V. (1976). Staphylococcus intermedius, a New Species isolated from Animals. International Journal of Systematic Bacteriology 26: 401-408.

HALEY, R. W., HIGHTOWER, A. W., KHABBAZ, R. F., THORNSBERRY, C., MARTONE, W. J., ALLEN, J. R., HUGHES, J. M. (1982). The emergence of methicillin- resistant Staphylococcus aureus infections in United Stateshospitals. Possible role of the house staff-patient transfer circuit. Annals___ of InternalMedicine 97: 297-308.

HALLMAN, F.A. (1937). Pathogenic staphylococci in the anterior nares: their incidence and diffentiation.Proceedings of____ the Society___ for ExperimentalMedicine and Biology 36: 789-794.

HAMILTON-MILLER, J. M. T. (1982). B- lactamases andtheir clinical significance. Journal_____ ofAntimicrobial Chemotherapy 9: (Suppl. B) , 11-19.

273

HAMILTON-MILLER, J. M. T., RAMSAY, J. (1967). Stability of cephaloridine and cephalothin to staphylococcalpenicillinase. Journal of General Microbiology 49: 491-501.

HANSEN, S. L., FREEDY, P. K. (1984). Variation in the abilities of automated, commercial, and referencemethods to detect methicillin-resistant(heteroresistant) Staphylococcus aureus.Journal of Clinical Microbiology 20: 494-499.

HARDY, D. J., SWANSON, R. N., HENSEY, D. M., RAMER, N.R., BOWER, R. R., HANSON, C. W., CHU, D. T. W., FERNANDES, P. B. (1987). Comparative antibacterialactivities of temafloxacin hydrochloride (A-62254) and two reference fluoroquinolones. Antimicrobial Agents and Chemotherapy 31: 1768-1774.

HARDY, D. W., HENSEY, D. M., BEYER, J. M. , VO JTKO, C., McDo n a l d , E. J., FERNANDES, P. V. (1988). Comparative in vitro activities of new 14, 15 and 16- memberedmacrolides. Antimicrobial Agents and Chemotherapy 32: 1710-1719.

HENNESSEY, R. S. F., MILES, R. A. (1958). Staphylococcus aureus type 80 and human infections in Uganda. British Medical Journal 2: 893-895.

HERRLICH, P., SCHWEIGER, M. (1976). Nitrofurans, agroup of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNAclasses. Proceedings of the National Academy of Sciences, USA 73: 3386-3390.

HILS0N, G. R. F. (1962). In-vitro studies of a new antibiotic (fucidin). Lancet i: 932-933.

274

HOEPRICH, P. D. (1969). Gentamicin versusStaphylococcus aureus . Journal o_f InfectiousDiseases 119: 391-392.

HOOPER, G., COVARRUBIAS, J. (1983). Clinical use and efficacy of furacin: a historical perspective.Journal of International Medical Research 11: 289-293.

HUCKER, G. J. (1948). In BREED, R.S., MURRAY, E.G.D.,HITCHENS, A.P. (eds.), Ber gey1 s______Mannual______ofDeterminative Bacteriology. Sixth Edition, London, Balliere, Tindall & Cox, p 235.

IGARI, J., SHIMOJI, K., UEZU, N., NAKASONE, I, TAIRA, K., KAKUZU, R. (1988). In vitro susceptibilities of clinical isolates of Staphylococcus aureus. Japan Journal of Antibiotics 41: 1205-1211.

IPSEN, T., GAHRN- HANSEN, B. (1988). Occurrence of methicillin- resistant Staphylococcus aureus in a department of orthopedic surgery 1970- 1986.European Journal of Clinical Microbiology and Infectious Diseases 7: 400-403.

ISAACS, R. D., KUNKE, P. J., COHEN, R. L., SMITH, J. W.(1988). Ciprofloxacin resistance in epidemic methicillin-resistant Staphylococcus aureus. Lancet ii: 843.

JACKSON, G. G. (1969). Introduction. Journal of Infectious Diseases 119: 341.

JAWETZ, E. (1961). Polymixin, colistin and bacitracin. Pediatric Clinics of North America 8: 1067-1071.

JEAN-PIERRE, H., BOYER, G., JEAN, A., DARBAS, H. (1988).Evolution de la resistance des Staphylococcusaureus a la pefloxacine. Etude portant sur 782souches isolees en 1985 et 1986. Pathologie et

275

Biologie 36: 956-958.

JEFFERSON, J., McKNIGHT, A. G. (1969). Topical Gentamicin. British Journal of Clinical Practice 23: 133-138.

JELJASZEWICZ ( 1972). Toxins (Hemolysins) In: COHEN J.0. (ed) The Staphylococci. New York, WileyInterscience Press, p 249-280.

JELJASZEWICZ, J. (1983). Infections Caused by Staphylococci. Infection 11 (Suppl. 2), 109-111.

JENSEN, K. (1968). Methicillin-resistant staphylococci. Lancet ii: 1078.

JENSEN, K., LASSEN, H. C. A. (1969). Combined treatment with antibacterial chemotherapeutical agents in staphylococcal infections. Quarterly Journal of Medicine, New Series 38: 91-106.

JEPSEN, 0. B. (1986). The demise of the "old”methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection 7: (Suppl. A), 13-17.

JEV0NS, M. P. (1961). nCelbeninM-resistantstaphylococci. British Medical Journal i: 124-125.

JEV0NS, M. P., COE, A. W., PARKER, M. T. (1963). Methicillin resistance in staphylococci. Lancet i: 904-907.

JOHNSON, A. P., UTTLEY, A. H., WOODFORD, N., GEORGE, R. C. (1990). Resistance to vancomycin and teicoplanin: an emerging clinical problem. Clinical Microbiology Reviews 3: 280-291.

276

JOHNSTON, B. L., KWOK, R. Y. Y., MULLIGAN, M. E. (1987). In vitro activity of novobiocin and rifampin alone and in combination against oxacillin-resistant Staphylococcus aureus. Diagnostic Microbiology and Infectious Diseases 8: 137-147.

JOLLY, J., GOLDBERG, M. (1989). Methicillin resistance in staphylococci: an evaluation of conditions for detection. Medical Laboratory Sciences 46: 2-5.

JONES, R. N., BARRY, A. L. (1989). In vitro evaluation of ramoplanin (A 16686 or MDL 62198). A new depsipeptide complex for potential topical use. Diagnostic Microbiology and Infectious Diseases 12:279-282.

JORDENS, J. Z., DUCKWORTH, G. J., WILLIAMS, R. J. (1989). Production of "virulence factors" by "epidemic" methicillin-resistant Staphylococcus aureus in vitro. Journal of Medical Microbiology 30: 245-252.

KAATZ, G. W., SEO, S. M., DORMAN, N. J., LERNER, S. A. (1990a). Emergence of teicoplanin resistance duringtherapy of Staphylococcus aureus endocarditis.Journal of Infectious Diseases 162: 103- 108.

KAATZ, G. W., SEO, S. M. (1990b). WIN 57273, a new fluoroquinolone with enhanced in vitro activity versus Gram-positive pathogens. Antimicrobial Agents and Chemotherapy 34: 1376-1380.

KAHAN, F. M., KAHAN, J. S., CASSIDY, P. J., KR0PP, H. (1974). The mechanism of action of fosfomycin (Phosphonomycin) . Annals of the New York Academy of Sciences 235: 364- 385.

KAPLAN, M. P., TENENBAUM, M. J. (1982). Staphylococcus aureus: Cellular Biology and Clinical Application. American Journal of Medicine 72: 243-258.

277

KAPUSNIK, J. E., PARENTI, F., SANDE, M. A. (1984). The use of rifarapicin in staphylococcal infections- a review. Journal of Antimicrobial Chemotherapy 13 (Suppl. C), 61-66.

KAYE, D. (1980). The clinical significance oftolerance of Staphylococcus aureus. Annals of Internal Medicine 93: 924-926.

KAYSER, F. H. (1975). Methicillin-resistantstaphylococci 1965-1975. Lancet ii: 650-653.

KAYSER, F., NOVAK, J. (1987). In vitro activity of ciprofloxacin against Gram-positive bacteria.American Journal of Medicine 82 (Suppl. 4A), 33-39.

KEPHART, P. A., ESPOSITO, A. L. (1988). Comparison of the investigational drug, LY 146032, with vancomycin in experimental pneumonia due to methicillin-resistant Staphylococcus aureus . Journal ofAntimicrobial Chemotherapy 21: 33-39.

KERR, S., KERR, G. E., MACKINTOSH, C. A., MARPLES, R. R. (1990). A survey of methicillin- resistant Staphylococcus aureus affecting patients in England and Wales. Journal of Hospital Infection 16: 35-48.

KHALIFA, K. I., HEIBA, A. A., HANCOCK, G. (1989). Nontypable bacteriophage patterns of methicillin- resistant Staphylococcus aureus involved in ahospital outbreak. Journal of Clinical Microbiology 27: 2249-2251.

KIDSON, A., LILLY, H. A., LOWBURY, E. J. L. (1979). Flucloxacillin treatment of methicillin-,,resistant" and sensitive staphylococcal infection. Journal of Antimicrobial Chemotherapy 5: 359-364.

278

KING, K., BRADY, L. M., HARKNESS, J. L. (1981). Gentamicin-resistant staphylococci. Lancet ii: 698-699.

KIRBY, W. M. M. (1944). Extraction of highly potent penicillin-inactivator from penicillin-resistantstaphylococci. Science 99: 452-3.

KIRBY, W. M. M., AHERN, J. J. (1953). Changing pattern of resistance of staphylococci to antibiotics. Antibiotics and Chemotherapy I I I : 831-835.

KIRBY, W. M. M., HUDSON, D. G., NOYES, W. D. (1956). Clinical and laboratory studies of novobiocin, a new antibiotic. Archives of Internal Medicine 98: 1-7.

KIRMANI, N., TUAZON, C. U., MURRAY, H. W. , PARRISH, A.E., SHEAGREN, J. N. (1978). Staphylococcus aureus carriage rate of patients receiving long-term haemodialysis. Archives of Internal Medicine 138: 1657-1659.

KLASTERSKY, J., HENSGENS, C., DANEAU, D. (1975). Therapy of staphylococcal infections: a comparative study of cephaloridine and gentamicin. American Journal ofthe Medical Sciences 269: 201-207.

KLASTERSKY, J., VAN DER AUWERA, P. (1986).Cephalosporins, vancomycin, aminoglycosides and other drugs, especially in combination, for the treatment of methicillin-resistant staphylococcal infections. Journal of Antimicrobial Chemotherapy 17: (Suppl. A) 19-24.

KNOX, R. (1960). A new penicillin (BRL 1241) active against penicillin-resistant staphylococci. British Medical Journal 2: 690-693.

KOCH, A. L. (1981). Evolution of antibiotic resistancegene function. Microbiological Reviews 45: 355-378.

279

KOJIMA, T., INOUE, M. , MITSUHASHI, S. (1989). In vitro activity of AT-4140 against clinical bacterial isolates. Antimicrobial Agents and Chemotherapy 34: 1123-1127.

KOJIMA, T., INOUE, M., MITSUHASHI, S. (1990). In vitro activity of AT-4140 against quinolone- andmethicillin- resistant Staphylococcus______aureus.Antimicrobial Agents and Chemotherapy 34: 1123-1127.

KOSMIDIS, J. (1988). Staphylococcal infections in hospital: the Greek experience. Abstract S4/ 2. 1st International Conference of the Hospital Infection Society, 1988.

KRESKEN, M., WIEDEMANN, B. (1986). Development ofresistance in the past decade in central Europe. Journal of Antimicrobial Chemotherapy 18: (Suppl. C), 235-242.

KUCERS , A. (1972). The Use of Antibiotics, aComprehensive Review with Clinical Emphasis. London, William Heinemann Books Ltd.

KUCERS, A. (1984). Vancomycin. Journal______ofAntimicrobial Chemotherapy 14: 564-567.

LACEY, R. W. (1975). Antibiotic resistance plasmidsof Staphylococcus aureus and their clinicalimportance. Bacteriological Reviews 39: 1-32.

LACEY, R. W. (1984). Antibiotic resistance inStaphylococcus aureus and streptococci. British Medical Bulletin 40: 77-83.

LACEY, R. W. (1987). Multi- resistant Staphylococcus aureus - a suitable case for inactivity? Journal of Hospital Infection 9: 103-105.

280

LACEY, R. W., MITCHELL, A. A. B. (1969). Gentamicin- resistant Staphylococcus aureus. Lancet ii: 1425-1426.

LACEY, R. W., ALDER, V. G., GILLESPIE, W. A. (1970). The survival of Staphylococcus aureus on human skin. An investigation using mixed cultures. British Journal of Experimental Pathology 51: 305-313.

LACEY, R. W., GRINSTEAD, J. (1973). Genetic analysis of methicillin- resistant strains of Staphylococcus aureus; evidence for their evolution from a single clone. Journal of Medical Microbiology 6: 511-526.

LACEY, R. W., STOKES, A. (1977). Susceptibility of the 'penicillinase- resistant' penicillins andcephalosporins to penicillinase of Staphylococcus aureus. Journal of Clinical Pathology 30: 35-39.

LACEY, R. W., STOKES, A. (1979). Studies on recently isolated cultures of methicillin-resistantStaphylococcus_____aureus . Journal_____ of_____GeneralMicrobiology 114: 329-339.

LACEY, R. W., LORD, V. L., H0WS0N, G. L. (1984). In vitro evaluation of miokamycin: bactericidal activityagainst streptococci. Journal of AntimicrobialChemotherapy 13: 5-13.

LAGAST, H., DODIAN, P., KLASTERSKY, J. (1986).Comparison of pharmacokinetics and bactericidal activity of teicoplanin and vancomycin. Journal of Antimicrobial Chemotherapy 18: 513-520.

LAU, W. Y., TEOH-CHAN, C. H., FAN, S. T., LAU, K. F.(1986). In vitro and in vivo study of fosfomycinin methicillin-resistant Staphylococcus____ aureussepticaemia. Journal of Hygiene, Cambridge 96: 419- 423.

281

LAURELL, G., WALLMARK, G. (1953). Studies on Staphylococcus aureus pyogenes in a children's hospital. Acta Pathologica et MicrobiologicaScandinavia 32: 438-447.

LEADING ARTICLE (1964). New Penicillins. BritishMedical Journal 2: 323-324.

LEADING ARTICLE (1965). Staphylococci resistant toneomycin and bacitracin. Lancet ii: 421-422.

LEADING ARTICLE (1968). Methicillin-resistantstaphylococci. Lancet ii: 759.

LEADING ARTICLE (1981). Of gentamicin and staphylococci. Lancet ii: 127-128.

LEAPER, D. J., SIMPSON, R. A. (1986). The effect of antiseptics and topical antimicrobials on wound healing. Journal of Antimicrobial Chemotherapy 17: 135-137.

LECLERCQ, R., NANTAS, L., SOUSSY, C. J., DUVAL, J.(1990). Activity of RP 59500 (RP), a new parenteral semisynthetic streptogramin (Sg) againstStaphylococcus aureus strains according tomechanisms of resistance to macrolide-lincosamide- streptogramin antibiotics (MLS). Abstracts of the 30th International Conference on Antimicrobial Agents and Chemotherapy (Atlanta), Abstract no. 786.

LE GOFFIC, F., CAPMAU, M. L., BONNET, D., CERCEAU, C.,SOUSSY, C., DUBLANCHET, A., DUVAL, J. (1977). Plasmid-mediated pristinamycin resistance PAC IIA: a newenzyme which modifies Pristinamycin IIA. Journal ofAntibiotics XXX: 665-669.

282

LEIGH, D., BINT, A., GOULD, I. (Eds). Fleroxacin, a long acting fluoroquinolone with broad spectrum activity. Journal of Antimicrobial Chemotherapy 22 (Supplement D ), 1988.

LEPPER, M. H., MOULTON, B., DOWLING, H. F., JACKSON, G.C, KOFMAN, S. (1954). Epidemiology of erythromycin- resistant staphylococci in a hospital population -effect on therapeutic activity of erythromycin.Antibiotics Annual 1953-1954, 308-313 (1954).

LEWIN, C., SMITH, J. T. (1988). Bactericidal mechanisms of ofloxacin. Journal of Antimicrobial Chemotherapy22 (Suppl. C ), 1-8.

LIMB, D. I., DABBS, D. J. W., SPENCER, R. C. (1987). In- vitro selection of bacteria resistant to the 4-quinolone agents. Journal_____of_____AntimicrobialChemotherapy 19: 65-71.

LINNEMANN Jr. C. C., MASON, M., MOORE, P., KORFHAGEN, T. R., STANECK, J. L. (1982). Methicillin- resistantStaphylococcus aureus: experience in a generalhospital over four years. American Journal ofEpidemiology 115: 941- 950.

LOCKSLEY, R. M., COHEN, M. L., QUINN, T. C., TOMPKINS, L.S., COYLE, M. B., KIRIHARA, J. M., COUNTS, G. W. (1982). Multiply antibiotic-resistant Staphylococcus aureus: Introduction, transmission, and evolution ofnosocomial infection. Annals of Internal Medicine 97: 317-324.

LOWBURY, E. J. L., CASON, J. S., MacG. JACKSON, D., MILLER, R. W. S. (1962). Fucidin for staphylococcal infection of burns. Lancet ii: 478-480.

283

LOWBURY, E. J. L., BABB, J. R., BROWN, V. I., COLLINS, B. J. (1964). Neomycin-resistant Staphylococcus aureus in a burns unit. Journal of Hygiene, Cambridge 62: 221-228.

LOWBURY, E. J. L., LILLY, H. A., KIDSON, A. (1977)."Methicillin- resistant" Staphylococcus______ aureus:reassesment by controlled trial in burns unit. British Medical Journal 1: 1054-1056.

LOWBURY, E. J. L., AYLIFFE, G. A. J., GEDDES, A. M.,WILLIAMS, J. D. (1982). Control of hospitalinfection, a practical handbook. 2nd edition, Chapman & Hall Ltd. (London).

LYON, B. R., GILLESPIE, M. T., BYRNE, M. E., MAY, J. W., SKURRAY, R. A. (1987). Plasmid- mediated resistance to gentamicin in Staphylococcus aureus: the involvement of a transposon. Journal of Medical Microbiology 23: 101-110.

LYON, B. R., SKURRAY, R. (1987). Antimicrobial resistance of Staphylococcus aureus: genetic basis.Microbiological Reviews 51: 88-134.

MACFARLANE, J. A., MITCHELL, A. A. B., WALSH, J. M. , ROBERTSON, J. J. (1968). Spiramycin in the prevention of postoperative staphylococcal infection. Lancet i: 1-4.

MADIRAJU, M. V. V. S., BRUNNER, D. P., WILKINSON, B. J.(1987). Effects of temperature, NaCl, and methicillin on penicillin-binding proteins, growth, peptidoglycan synthesis, and autolysis in methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 31: 1727-1733.

284

MAPLE, P. A. C., HAMILTON-MILLER, J. M. T., BRUMFITT, W. (1989a). Comparative in vitro activity of vancomycin, teicoplanin, ramoplanin (formerly A 16686), paldimycin, DuP 721 and DuP 105 against methicillinand gentamicin resistant Staphylococcus aureus.Journal of Antimicrobial Chemotherapy 23: 517-525.

MAPLE, P. A. C., HAMILTON-MILLER, J. M. T., BRUMFITT, W. (1989b). World-wide antibiotic resistance in methicillin- resistant Staphylococcus aureus. Lancet i: 537-540.

MAPLE, P. A. C., HAMILTON-MILLER, J. M. T., BRUMFITT, W. (1989c). Ciprofloxacin resistance in methicillin- and gentamicin-resistant Staphylococcus aureus. European Journal of Clinical Microbiology and InfectiousDiseases 8: 622-623.

MAPLE, P., BRUMFITT, W., HAMILTON-MILLER, J. M. T.(1990d). A review of the antimicrobial activity ofthe fluoroquinolones. Journal of Chemotherapy 2:280-294.

MARANDON, J. L., OEDING, P. (1966). Investigations onanimal Staphylococcus aureus strains. 1. Biochemical characteristics and phage typing. Acta Pathologica et Microbiologica Scandinavia 67: 149-156.

MARANDON, J. L., OEDING, P. (1967). Investigations onanimal Staphylococcus aureus strains. 2. Antigens.Acta Pathologica et Microbiologica Scandinavia 70: 300-304.

MARKOWITZ, N., P0HL0D, D. J., SARAVOLATZ, L. D., QUINN, E. L. (1983). In vitro susceptibility patterns of methicillin- resistant and - susceptible Staphylococcus aureus strains in a population of parenteral drug abusers from 1972- 1981. Antimicrobial Agents and Chemotherapy 23: 450-457.

285

MARPLES, R. R., RICHARDSON, J. F., de SAXE, M. (1986). Bacteriological characters of strains ofStaphylococcus aureus submitted to a referencelaboratory related to methicillin resistance. Journal of Hygiene, Cambridge 96: 217-223.

MARQUES, A. R., PETRILLO, V., HOEFEL, H. (1989).Methicillin- resistant Staphylococcus aureus in ageneral hospital in Brazil. Journal of HospitalInfection 14: 380-381.

MASKELL, J. P., SEFTON, A. M. , YONG, J., CHI, S. J., WILLIAMS, J. D. (1988). Comparative in vitro activityof erythromycin, vancomycin and pristinamycin.Infection 16: 365-370.

MATHEWS, P. R., STEWART, P. R. (1984). Resistanceheterogeneity in methicillin-resistant Staphylococcus aureus. FEMS Microbiology Letters 22: 161-166.

MAYON-WHITE, R.T., DUCEL, G., KERESELIDZE, T., TIKOMIROV, E. (1988). An international survey of the prevalence of hospital-acquired infection. Journalof Hospital Infection 11 (Suppl. A): 43-48.

MCDONALD, M., HURSE, A., SIM, K. N. (1981). Methicillin- resistant Staphylococcus aureus bacteraemia. Medical Journal of Australia 2: 191-194.

McDOUGAL, L. K., THORNSBERRY, C. (1984). Newrecommendations for disk diffusion antimicrobial susceptibility tests for methicillin-resistant(heteroresistant) staphylococci. Journal of Clinical Microbiology 19: 482-488.

McGOWAN Jr., J. E. (1988). Gram-positive bacteria: spread and antimicrobial resistance in university and community hospitals in the USA. Journal of Antimicrobial Chemotherapy 21: (Suppl. C) , 49-55. 286

McOSKER, C. C., POLLACK, J. R., ANDERSEN, J. A. (1990). Inhibition of bacterial protein synthesis bynitrofurantoin macrocrystals: an explanation for thecontinued efficacy of nitrofurantoin Iii HARRISON, L.H. (ed.), Management of Urinary Tract Infections.Royal Society of Medicine Services International Congress and Symposium Series No. 154.

MEDICAL JOURNAL OF AUSTRALIA (1982). Methicillin- resistant Staphylococcus aureus. Medical Journal ofAustralia 1: 445-480.

MEHTAR, S., DRABU, Y. J., MAYET, F. (1989). Expenses incurred during a 5 week epidemic methicillin- resistant Staphylococcus aureus outbreak. Journal ofHospital Infection 13: 199-200.

MELO CRISTINO, J. A. G., TORRES PEREIRA, A., AFONSO, F.(1985). Infection with methicillin- gentamicin- resistant Staphylococcus aureus strains in apaediatric surgery unit in Lisbon. Journal ofHospital Infection 6: 426-428.

MILATOVIC, D. (1986). Vancomycin for treatment ofinfections with methicillin- resistant Staphylococcus aureus: are there alternatives? European Journal of Clinical Microbiology 5: 689-692.

MILLER, G. H., SABATELLI, F. J., HARE, R. S., WAITZ, J. A. (1980). Survey of aminoglycoside resistance patterns. Developments in Industrial Microbiology 21: 90-104.

MILNE, L. M., FAIERS, M. C. (1988). Letter to the editor. Lancet ii: 843.

287

MINUTH, J. N., HOLMES, T.M., MUSHER, D. M. (1974). Activity of tetracycline, doxycycline, and minocycline against methicillin- susceptible and -resistant staphylococci. Antimicrobial Agents and Chemotherapy 6: 411-414.

MITCHELL, A. A. B. (1964). New epidemic strain of Staphylococcus aureus. Emergence and spread in a General Hospital. Lancet i: 859-862.

MITSUHASHI, S., INOUE, M., INOUE, K. (1990). In vitro evaluation of RP 59500 (a streptogramin family). Abstracts of the 30th International Conference on Antimicrobial Agents and Chemotherapy (Atlanta), Abstract no. 772.

MOLLBY, R., WADSTROM, T. (1973). Purification of staphlyococcal beta-, gamma- and delta-hemolysins In: JELJASZEWICZ, J., HRYNIEWICZ, W. (eds) Staphylococci and Staphylococcal Infections. Warsaw, Polish Medical Publishers, 298-313.

MOLLBY, R. (1983). Isolation and properties of membrane damaging toxins Ijas EASMON C. S. F., ADLAM,C. (eds) Staphylococci and Staphylococcal Infections. The organism in vivo and in vitro. Academic Press (London), 619-669.

MONTAY, G., BRUNO, R., THEBAULT, J. J., VERGINOL, J. C., CHASSARD, D . , EBMEIER, M., GAILLOT, J. (1990). Dose- dependent pharmacokinetic study of sparfloxacin (SPX) in healthy young volunteers. Abstracts of the 30th International Congress on Antimicrobial Agents and Chemotherapy, Atlanta, Abstract no. 1248.

288

MONTEFIORE, D., ROTIMI, V. 0., ADEYEMI-DORO, F. A. B.(1989). The problem of bacterial resistance to antibiotics among strains isolated from hospital patients in Lagos and Ibadan, Nigeria. Journal of Antimicrobial Chemotherapy 23: 641-651.

MONTIEL, F., KALTWASSER, G., VALDIVIESO, C., LAM, M.(1988). In vitro susceptibility to 10 antibiotics of methicillin- resistant (MRSA) Staphylococcus aureus in Chile. Diagnostic Microbiology and Infectious Diseases 10: 145- 148.

MOORMAN, D. R., MANDELL, G. L. (1981). Characteristics of rifampin-resistant variants obtained fromclinical isolates of Staphylococcus____ aureus.Antimicrobial Agents and Chemotherapy 20: 709- 713.

MORGAN, M. G., HARTE- BARRY, M. J. (1989). Methicillin- resistant Staphylococcus aureus: a ten- year survey in a Dublin hospital. Journal of Hospital Infection 14: 357- 362.

M0UT0N, R. P., MULDERS, S. L. T., de KNIFF, J., HERMANS, J. (1989). Comparison of test systems for recognition of methicillin resistance inStaphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 8: 968-973.

MULLIGAN, M. E., RUANE, P. J., JOHNSTON, L., WONG, P., WHEEL0CK, J. P., MacDONALD, K., REINHARDT, J. F., JOHNSON, C. C., STATNER, B., BLOMQUIST, I., McCARTHY, J., O ’BRIEN, W., GARDNER, S., CITRON, D.M.(1987). Ciprofloxacin for eradication ofmethicillin-resistant Staphylococcus________aureuscolonization. American Journal of Medicine 82,(Suppl. 4A), 215-219.

289

MURAKAMI, K., TOMASZ, A. (1989). Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus. Journal of Bacteriology 171: 874-879.

NEU, H. C. (1974). Gentamicin, an overview In: JACKSON, G. G., ESCARZAGA, E., GARROD, L. P., NEU, H., UEDA, Y. (eds). Gentamicin; review and commentary on selected world literature. New York, Council for Interdisciplinary Communication in Medicine, pp 179-191.

NEU, H. C. (1987). Ciprofloxacin: an overview andprospective appraisal. American Journal of Medicine 82, (Suppl. 4A), 395-404.

NEU, H. C., NEU, N. M. (1986). In vitro activity of A 16686, a new glycopeptide. Chemotherapy 32: 453-457.

NEU, H. C., NOVELLI, A., SAHA, G., CHIN, N-X. (1988). In vitro activities of two oxazolidinone antimicrobial agents, DuP 721 and DuP 105. Antimicrobial Agents and Chemotherapy 32: 580-583.

NOBLE, W. C., WILLIAMS, R.E.O., JEVONS, M. P., SHOOTER, R. A. (1964). Some aspects of nasal carriage of staphylococci. Journal of Clinical Pathology 17: 79-83.

NOBLE, W. C., VALKENBURG, H. A., WOLTERS, C. H. L. (1967). Carriage of Staphylococcus aureus in random samples of a normal population. Journal of Hygiene, Cambridge 65: 567-573.

0GST0N, A. (1882). Micrococcus Poisoning. Journal of Anatomy (London) 17: 24-58.

O ’HARE, M. D., GHOSH, G., FELMINGHAM, D., GRUNEBERG, R.N. (1990). In vitro studies with ramoplanin (MDL62198); a novel lipoglycopeptide antimicrobial.Journal of Antimicrobial Chemotherapy 25: 217-220.

290

OUNISSI, H., DERLOT, E., CARLIER, C., COURVALIN, P.(1990). Gene homogeneity for aminoglycoside-modifying enzymes in Gram-positive cocci. Antimicrobial Agents and Chemotherapy 34: 2164-2168.

PALLANZA, R., BERTI, M., SCOTTI, R., RANDISI, E., ARIOLI, V. (1984). A-16686, a new antibiotic fromActinoplanes. II. Biological properties. Journal of Antibiotics 37: 318-324.

PARKER, M. T., HEWITT, J. H. (1970). Methicillin resistance in Staphylococcus aureus. Lancet i: 800-804.

PARKER, M. T., ASHESHOV, E. H., HEWITT, J. H., NAKHLA, L.S., BROCK, B. M. (1974). Endemic staphylococcal infections in hospitals. Annals of the New YorkAcademy of Sciences 236: 466-484.

PARKER, M. T. (1983). Staphylococcus and Micrococcus; the anaerobic gram-positive cocci JLn: WILSON, G. S., MILES, A. A., PARKER, M. T. (ed.) Topley and Wilson’s principles of bacteriology, virology and immunity. London, Edward Arnold, 218-245.

PATTIS0N, J. R., MANSELL, P. E. (1973). Fucidin-resistant staphylococci in current hospitalpractice. Journal of Medical Microbiology 6: 235-244.

PAUL, J. (1833). Case of congenital fungus haematodes in which amputation of the thigh was performed in the tenth week of the child’s life.Lancet i: 439-445.

PAVILLARD, R., HARVEY, K., DOUGLAS, D., HEWST0NE, A.,ANDREW, J., COLLOPY, B., ASCHE, V., CARSON, P., DAVIDSON,A., GILBERT, G., SPICER, J., T0S0LINI, F. (1982).Epidemic of hospital-acquired infection due tomethicillin-resistant Staphylococcus aureus in majorVictorian hospitals. Medical Journal of Australia 1:

291

451-454.

PEACOCK, J. E., MARSIK, F. J., WENZEL, R. P. (1980).Methicillin-resistant Staphylococcus_______ aureus:Introduction and spread within a hospital. Annals of Internal Medicine 93: 526-532.

PEARMAN, J. W., CHRISTIANSEN, K. J., ANNEAR, D. I., GOODWIN, C. S., METCALF, C., DONOVAN, F. P., MACEY, K. L., BASSETTE, L. D., POWELL, I. M., GREEN, J. M., HARPER, W. E., McKELVIE, M. S. (1985). Control of methicillin-resistant Staphylococcus aureus (MRSA) in anAustralian metropolitan teaching hospital complex. Medical Journal of Australia 142: 103-108.

PELLETIER, L. L. (1984). Lack of reproducibility ofmacrodilution MBCs for Staphylococcus aureus.Antimicrobial Agents and Chemotherapy 26: 815-818.

PETIT, J-C, COHEN, F., COUCHOUD, S., DAGUET, G. L.(1983). Staphylococcus aureus oxacilline etgentamicine-resistants: survenue et evolution sur une periode de 6 mois. Biomedicine & Pharmacotherapy 37: 429-433.

PIPER, J., HADFIELD, T., McCLESKEY, F., EVANS, M.,FRIEDSTROM, S., LAUDERDALE, P., WINN, R. (1988). Efficacies of Rapid Agglutination Tests for Identification of Methicillin-Resistant StaphylococcalStrains as Staphylococcus aureus. Journal ofClinical Microbiology 26: 1907-1909.

P0CHEE, E., CREWE-BROWN, H. H. (1988). Methicillinresistant Staphylococcus aureus at Ga-Rankuwahospital. Abstract 09/ 4. 1st____ InternationalConference of the Hospital Infection Society, 1988.

292

POHLOD, D. J., SARAVOLATZ, L. D., SOMERVILLE, M. M.(1987). In vitro susceptibility of Gram-positive cocci to LY 146032, teicoplanin, sodium fusidate, vancomycin and rifampicin. Journal of Antimicrobial Chemotherapy 20: 197-202.

P0RTH0USE, A., BROWN, D. F. J., GRAEME SMITH, R., ROGERS, T. (1976). Gentamicin resistance in Staphylococcus aureus. Lancet i: 20-21.

PUTLAND, R. A., GUINNESS, M. D. (1985). Autobac susceptibility testing of methicillin-resistant Staphylococcus aureus isolated in an Australian hospital. Journal of Clinical Microbiology 22: 822-827.

QUIE, P. G., HILL, H. R., TODD DAVIS, A. (1974). The Defective Phagocytosis of Staphylococci. Annals of the New York Academy of Sciences 236: 233-243.

RAHMAN, M., NOBLE, W. C., C00KS0N, B. (1987). Mupirocin- resistant Staphylococcus aureus. Lancet ii: 387.

RAHMAN, M., CONNOLLY, S., NOBLE, W. C., C00KS0N, B., PHILLIPS, I. (1990). Diversity of staphylococci exhibiting high-level resistance to mupirocin. Journal of Medical Microbiology 33: 97-100.

RAMMELKAMP, C. H., MAX0N, T. (1942). Resistance of Staphylococcus aureus to the action of Penicillin. Proceedings of the Society for Experimental Biology and Medicine 51: 386-389.

RAVIGLIONE, M. C., BOYLE, J. F., MARIUZ, P., PABL0S- MENDEZ, A.,CORTES, H., MERL0, A. (1990). Ciprofloxacin- resistant methicillin-resistant Staphylococcus aureus in an acute-care hospital. Antimicrobial Agents and Chemotherapy 34: 2050-2054.

293

REPORT (1986). Guidelines for the control ofepidemic methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection 7: 193-201.

RICHARDS, F., McCALL, C., COX, C. (1971). Gentamicin treatment of staphylococcal infections. Journal of the American Medical Association 215: 1297-1300.

RICHARDSON, J. F., CHITTASOBHON, N., MARPLES, R. R.(1988). Supplementary phages for the investigationof methicillin-resistant Staphylococcus aureus.Journal of Medical Microbiology 25: 67-74.

RIDLEY, M., BARRIE, D., LYNN, R., STEAD, K. C. (1970).Antibiotic-resistant Staphylococcus_____ aureus andhospital antibiotic policies. Lancet i: 230-233.

RIMLAND, D. (1987). Nosocomial infections withmethicillin and tobramycin resistant Staphylococcusaureus: implications of physiotherapy in hospital-wide dissemination. American Journal of the MedicalSciences 290: 91-97.

RODE, H., HANSL0, D., DE WET, P. M., MILLAR, A. J. W., CYWES, S. (1989). Efficacy of mupirocin inmethicillin-resistant Staphylococcus_____ aureus burnwound infection. Antimicrobial______ Agents____ andChemotherapy 33: 1358-1361.

RODRIGUEZ, A., OLAY, T., VICENTE, M. V. (1986). Invitro activity of fosfomycin alone and combinedagainst methicillin- resistant staphylococci In.: Fosfomycin, Proceedings of the internationalsymposium. Instituto de Farmacologia Espaniola (CEPA), Madrid, 1987.

R0G0LSKY, M. (1979). Nonenteric Toxins ofStaphylococcus aureus. Microbiological Reviews 43: 320-360.

294

ROLINSON, G. N. (1961). "Celbenin"-resistantstaphylococci. British Medical Journal 2: 125-126.

ROLSTON, K. V. I., LeBLANC, B., HO, D. H., BODEY, G. P. (1987). In vitro activity of paldimycin (U- 70138F) against Gram-positive bacteria isolated from patients with cancer. Antimicrobial Agents and Chemotherapy 31: 650-652.

ROSENDAL, K., BULOW, P., BENTZON, M. W., ERIKSEN, K. R. (1976). Staphylococcus aureus strains isolated in Danish hospitals from January 1st, 1966, to December31st, 1974. Acta_____ Pathologica MicrobiologicaScandinavica Section B 84: 359-368.

ROUNTREE, P. M., THOMSON, E. F. (1949). Penicillin- resistant and streptomycin-resistant staphylococci in a hospital. Lancet ii: 501-504.

ROUNTREE, P. M., BARBOUR, R. G. H., THOMSON, E. F. (1951). Incidence of penicillin-resistant and streptomycin-resistant staphylococci in a hospital. Lancet i: 435-436.

ROUNTREE, P. M., FREEMAN, B. M. (1955).Infections caused by a particular phage type of Staphylococcus aureus. Medical Journal of Australia 2: 157-161.

ROUNTREE, P. M., FREEMAN, B. M., JOHNSTON, K. G. (1956). Nasal carriage of Staphylococcus aureus by variousdomestic and laboratory animals. Journal ofPathology and Bacteriology 72: 319-321.

ROUNTREE, P. M., BEARD, M. A. (1965). The spread of neomycin-resistant staphylococci in a hospital. Medical Journal of Australia i: 498-502.

295

SARAVOLATZ, L. D., MARKOWITZ, N., ARKING, L. M., POHLOD,D., FISHER, E. (1982). Methicillin-resistantStaphylococcus aureus. Epidemiologic observationsduring a community acquired outbreak. Annals of Internal Medicine 96: 11-16.

SAROGLOU, G., CROMER, M., BISNO, A. L. (1980).Methicillin- resistant Staphylococcus______aureus:interstate spread of nosocomial infections withemergence of gentamicin- methicillin- resistantstrains. Infection Control 1: 81-89.

SCHAEFLER, S. (1982). Bacteriophage-mediatedacquisition of antibiotic resistance byStaphylococcus aureus type 88. Antimicrobial Agents and Chemotherapy 21: 460-467.

SCHAEFLER, S. (1989). Methicillin-resistant strains ofStaphylococcus aureus resistant to quinolones.Journal of Clinical Microbiology 27: 335-336.

SCHAEFLER, S., JONES, D., PERRY, W., RUVINSKAYA, P. L., BARADET, T., MAYR, E., WILSON, M. E. (1981). Emergence of gentamicin- and methicillin -resistantStaphylococcus aureus strains in New York Cityhospitals. Journal of Clinical Microbiology 13:754-759.

SCHAEFLER, S., JONES, D., PERRY, W., BARADET, T., MAYR,E., RAMPERSAD, C. (1984). Methicillin- resistant Staphylococcus aureus strains in New York Cityhospitals: inter- hospital spread of resistantstrains of type 88. Journal of ClinicalMicrobiology 20: 536-538.

SCHITO, G. C., VARALDO, P. E. (1988). Trends in theepidemiology and antibiotic resistance of clinicalStaphylococcus strains in Italy. Journal__ofAntimicrobial Chemotherapy 21: (Suppl. C), 67- 78.

296

SCHNEIERSON, S. S. (1960). 1959 bacterialsusceptibility to nitrofurantoin as compared to 1953 to 1955. Antibiotics and Chemotherapy 10: 747-752.

SCHLEIFER, K. H. (1986). In BUTLER, J.P. (ed.), Bergey's Mannual of Systematic Bacteriology. Baltimore,Williams & Wilkins, p 1003.

SHALIT, I., BERGER, S. A., GOREA, A., PRIMERNMAN, H.(1989). Widespread quinolone resistance amongmethicillin-resistant Staphylococcus aureus isolates in a general hospital. Antimicrobial Agents andChemotherapy 33: 593-594.

SHANSON, D. C. (1981). Antibiotic- resistantStaphylococcus aureus. Journal of Hospital Infection2: 11-36.

SHANSON, D. C. (1990). Clinical relevance ofresistance to fusidic acid in Staphylococcusaureus. Journal of Antimicrobial Chemotherapy 25 (Suppl. B), 15-21.

SHANSON, D. C. KENSIT, J. G., DUKE, R. (1976). Outbreak of hospital infection with a strain of Staphylococcus aureus resistant to gentamicin andmethicillin. Lancet ii: 1347-1348.

SHAW, C., STITT, J.M., COWAN, S.T. (1951). Staphylococci and their Classification. Journal of General Microbiology 5: 1010-1023.

SHAW, W. V. (1984). Bacterial resistance to chloramphenicol. British Medical Bulletin 40: 36-41.

SLEE, A. M., WU0N0LA, M. A., McRIPLEY, R. J., ZAJAC, I.,ZAWADA, M. J., BARTHOLOMEW, P. T., GREGORY, W. A.,FORBES, M. (1987). Oxazolidinones, a new class of

297

synthetic antibacterial agents: in vitro and invivo activities of DuP 105 and DuP 721. Antimicrobial Agents and Chemotherapy 31: 1791- 1797.

SKURRAY, R. A., ROUCH, D. A., LYON, B. R., GILLESPIE, M. T., TENNENT, J. M., BYRNE, M. E., MESSEROTTI, L. J., MAY, J. W. (1988). Multi- resistant Staphylococcus aureus: genetics and evolution of Australian strains. Journal of Antimicrobial Chemotherapy 21: (Suppl. C) , 19-38.

SMITH, J. T. (1984). Awakening the slumbering potential of the 4-quinolone antibacterials. The Pharmaceutical Journal 233: 299-305.

SMITH, J. T. (1990). Mutation rates to 4-quinolone resistance. Arzneimittel-Forschung/Drug Research 40: 1- 1 1 .

SMITH, S. M. (1986). In vitro comparison of A- 56619, A-56620, amifloxacin, ciprofloxacin, enoxacin,norfloxacin, and ofloxacin against methicillin- resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 29: 325-326.

SMITH, S. M., ENG, R. H. K. (1985). Activity of ciprofloxacin against methicillin-resistantStaphylococcus aureus. Antimicrobial Agents and Chemotherapy 27: 688-691.

SMITH, S. M., ENG, R. H. K., BERMAN, E. (1986). The effect of ciprofloxacin on methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 17: 287-295.

SMITH, S. M., MANGIA, A., ENG, R. H. K., RUGGERI, P., CYTRYN, A., TECSON-TUMANG, F. (1988). Clindamycin for colonization and infection by methicillin-resistant Staphylococcus aureus. Infection 16: 95-97.

298

SMITH, S. M., ENG, R. H. K., BAIS, P., FAN-HAVARD, P., TECSON-TUMANG, F. (1990). Epidemiology ofciprofloxacin-resistance among patients withmethicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 26: 567-572.

SOMPOLINSKY, D., SAMRA, Z., KARAKAWA, W. W., VANN, W. F., SCHNEERSON, R., MALIK, Z. (1985). Encapsulation and Capsular Types in Isolates of Staphylococcus aureus from different Sources and Relationship to Phage Types. Journal of Clinical Microbiology 22: 828-834.

SORRELL, T. C., PACKHAM, D. R., SHANKER, S., FOLDES, M., MUNRO, R. (1982). Vancomycin therapy for methicillin- resistant Staphylococcus aureus. Annals of Internal Medicine 97: 344-350.

SOUSSY, C. J., BOUANCHAUD, D. H., FOUACE, J., DUBLANCHET, A., DUVAL, J. (1975). A gentamycin resistance plasmid in Staphylococcus aureus. Annales de Microbiologie 126 B: 91-94.

SOUSSY, C. J., DUBLANCHET, A., CORMIER, M., BISMUTH, R., MIZON, F., CHARDON, H., DUVAL, J., FABIANI, G. (1976). Nouvelles resistances plasmidiques de Staphylococcus aureus aux aminosides (gentamicine, tobramycine, amikacine). Nouvelle Presse Medicale 5: 2599-602.

SPELLER, D. C. E., RAGHUNATH, D., STEPHENS, M., VIANT, A.C., REEVES, D. S., WILKINSON, P. J., BROUGHALL, J. M. , HOLT, H. A. (1976). Epidemic infection by a gentamicin-resistant Staphylococcus aureus in three hospitals. Lancet i: 464-466.

SPINK, W. W. (1954). Staphylococcal infections and the problem of antibiotic-resistant staphylococci. Archives of Internal Medicine 94: 167-196.

299

SPINK, W. W., HALL, W. H. (1945). Penicillin therapy at the University Hospitals. Annals of Internal Medicine 22: 510-525.

SPRATT, B. G. (1978). Escherichia coli resistance to B-lactam antibiotics through a decrease in the affinity of a target for lethality. Nature 274:713-715.

STEWART, G. T., NIXON, H. H., COLES, H. M. T. (1960). Report on clinical use of BRL 1241 in children with staphylococcal and streptococcal infections. British Medical Journal 2: 703-706.

STEWART, G. T., HOLT, R. J. (1963). Evolution of natural resistance to the newer penicillins. British Medical Journal 1: 308-311.

ST0RRS, M. J., C0URVALIN, P., FOSTER, T. J. (1988). Genetic analysis of gentamicin resistance in methicillin- and gentamicin- resistant strains of Staphylococcus aureus isolated in Dublin hospitals. Antimicrobial Agents and Chemotherapy 32: 1174-1181 .

SUBCOMMITTEE (1965). Subcommittee on Taxonomy of Staphylococci and Micrococci - Minutes of First Meeting. International Bulletin of Bacteriological Nomenclature and Taxonomy 15: 107-108.

SUTHERLAND, R., R0LINS0N, G. N. (1964). Characteristics of methicillin-resistant staphylococci. Journal of Bacteriology 87: 887-899.

SYKES, R. B., MATTHEW, M. (1976). The B-lactamases of Gram-negative bacteria and their role in resistance to B-lactam antibiotics. Journal of Antimicrobial Chemotherapy 2: 115-157.

300

TENNENT, J. M., LYON, B. R., GILLESPIE, M. T., MAY, J. W., SKURRAY, R. A. (1985). Cloning and expression of Staphylococcus aureus plasmid-mediated quaternaryammonium resistance in Escherichia_____ coli.Antimicrobial Agents and Chemotherapy 27: 79-83.

TENNENT, J. M., MAY, J. W., SKURRAY, R. A. (1986). Characterisation of chloramphenicol resistance plasmids of Staphylococcus aureus and S. epidermidis by restriction enzyme mapping techniques. Journal of Medical Microbiology 22: 79-84.

THELESTAM, M. (1983). Modes of membrane damagingaction of staphylococcal toxins I_n: EASMON, C. S.F., ADLAM, C. (eds) Staphylococci and staphylococcal infections. London, Academic Press, 2: 705-744.

THOMPSON R. E. M., HARDING, J. W. , SIMON, R. D. (1960).Sensitivity of Staphylococcus_____pyogenes tobenzyl penicillin and BRL 1241. British MedicalJournal 2: 708-709.

TIMBURY, M. C., WILSON, T. S., HUTCHINSON, J. G. P., GOVAN, A. D. T. (1958). A staphylococcus type-80 epidemic in a maternity hospital illustrating some special features. Lancet ii: 1081-1084

T0MASZ, A., DRUGE0N, H. B., deLENCASTRE, H. M., JABES,D., McDOUGAL, L., BILLE, J. (1989). New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrobial Agents and Chemotherapy 33: 1869-1874.

T0PLEY, W. W. C., WILSON, G. S. (1936). The principles of bacteriology and immunology. Second Edition, London, Edward Arnold & Co.

301

TOSCANO, W. A., STORM, D. R. (1982). Bacitracin. Pharmacology and Therapeutics 16: 199-210.

TOWNSEND, D. E., ASHDOWN, N., BRADLEY, J. M., PEARMAN, J. W., GRUBB, W. B. (1984). "Australian” methicillin-resistant Staphylococcus aureus in a Londonhospital? Medical Journal of Australia 141: 339-340.

TOWNSEND, D. E., ASHDOWN, N., BOLTON, S., BRADLEY, J., DUCKWORTH, G., M00RH0USE, E. C., GRUBB, W. B. (1987). The International spread of methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection 9: 60-71.

TRALLERO, E. P., ARENZANA, J. M. G., EGUILUZ, G. C., CANCER, R. C. (1988). Prevalence of methicillin-resistant Staphylococcus aureus in a Spanishhospital. Reviews of Infectious Diseases 10: 627-628.

TRAUB, W., SPOHR, M., BAUER, D. (1984). Gentamicin and methicillin resistant, clinical isolates ofStaphylococcus aureus: comparative in vitro and in vivo efficacy of alternative antimicrobial drugs. Chemotherapy 30: 102-112.

TUAZ0N, C. U., PEREZ, A., KISHABA, T., SHEAGREN, J. N. (1975). Staphylococcus aureus among insulin-injecting diabetic patients: an increased carrier rate.Journal of the American Medical Association 231: 1272.

TURNIDGE, J., LAWSON, P., MUNRO, R., BENN, R. (1989). A national survey of antimicrobial resistance inStaphylococcus aureus in Australian teachinghospitals. Medical Journal of Australia 150: 65-72.

UBUKATA, K., ITOH-YAMASHITA, N., KONNO, M. (1989).Cloning and expression of the nor A gene forfluoroquinolone resistance in Staphylococcus aureus.Antimicrobial Agents and Chemotherapy 33: 1535-1539.

302

VAN DER AUWERA, P. (1989). Ex vivo study of serum bactericidal titers and killing rates of daptomycin (LY 146032) combined or not combined with amikacin compared with those of vancomycin. Antimicrobial Agents and Chemotherapy 33: 1783-1790.

VAN CAEKENBERGHE, D. L., PATTYN, S. R. (1987). Reversed incomplete cross-resistance among the older andnewer quinolone antibiotics. Journal______ ofAntimicrobial Chemotherapy 19: 404.

VAN DE KLUNDERT, J. M., VIEGENTHART, J. S., VAN D00RN,E., BONGAERTS, G. P. A., MOLENDIJK, L., M0UT0N, R. P.(1984). A simple method for the identification ofaminoglycoside- modifying enzymes. Journal____ ofAntimicrobial Chemotherapy 14: 339-348.

VARALDO, P. E., CIPRIANI, P., FOCA, A., GERACI, C.,GIORDANO, A., MADEDDU, M. A., ORSI, A., POMPEI, R.,PRENNA, M., REPETTO, A. et al. (1984). Identification, clinical distribution, and susceptibility tomethicillin and 18 additional antibiotics ofclinical Staphylococcus isolates: nationwideinvestigation in Italy. Journal of ClinicalMicrobiology 19: 838-843.

VERBIST, L. (1990). The antimicrobial activity of fusidic acid. Journal of Antimicrobial Chemotherapy 25 (Suppl. B), 1-5.

VERHOEF, J., VERBRUGH, H. A. (1981). Host Determinants in Staphylococcal Disease. Annual Reviews of Medicine 32: 107-122.

WADSTR0M, T. (1983). Biological effects damaging toxins JEn: EASM0N, C. S. F.,Staphylococci and staphylococcal infections Academic Press, 2: 671-704.

303

of cell ADLAM, C.

London,

WAITZ, J. A., WEINSTEIN, M. J. (1969). Recent microbiological studies with gentamicin. Journal of Infectious Diseases 119: 355-360.

WAKEFIELD, D. S., PFALLER, M., MASSANARI, M. , HAMMONS, G. T. (1987). Variation in methicillin- resistantStaphylococcus aureus occurrence by geographiclocation and hospital characteristics. Infection Control 8: 151-157.

WALSH, T. J., HANSEN, S. L., TATEM, B. A., AUGER, F., STANDIFORD, H. C. (1985). Activity of novobiocin against methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 15: 435-440.

WARD, T. T., WINN, R. E., HARTSTEIN, A. I., SEWELL, D. L. (1981). Observations relating to an inter- hospital outbreak of methicillin- resistant Staphylococcus aureus: role of antimicrobial therapy in infectioncontrol. Infection Control 2: 435-439.

WATANAKUNAKORN, C. (1981). The antibacterial actionof vancomycin. Reviews of Infectious Diseases 3 (Suppl.), 210-215.

WATANAKUNAKORN, C. (1982). Treatment of infections due to methicillin-resistant Staphylococcus aureus. Annals of Internal Medicine 97: 370-378.

WATANAKUNAKORN, C. (1988). In vitro induction ofresistance in coagulase-negative staphylococci to vancomycin and teicoplanin. Journal of Antimicrobial Chemotherapy 22: 321-324.

WATANAKUNAKORN, C. (1990). In vitro selection ofresistance of Staphylococcus aureus to teicoplaninand vancomycin. Journal_____ of_____ AntimicrobialChemotherapy 25: 69-72.

304

WEHRLI, W. (1983). Rifampicin: mechanisms of action and resistance. Journal of Infectious Diseases 5: 407-411.

WEISBLUM, B. (1984). Inducible erythromycin resistance in bacteria. British Medical Bulletin 40: 47-53.

WHITE, A. (1964). The use of gentamicin as a nasal ointment. American Journal of the Medical Sciences 248: 52-55.

WHITE, D. G., COLLINS, P. 0., BOSWELL, R. B. (1989).Topical antibiotics in the treatment of superficial skin infections in general practice - a comparison of mupirocin with sodium fusidate. Journal of Infection 18: 221-229.

WIEDEMANN, B., KRESKEN, M. (1984). The incidence anddevelopment of resistance in Staphylococcus aureusfrom three European countries. Journal ofAntimicrobial Chemotherapy 14: (Suppl. D), 27-34.

WILLIAMS, J. D., WALTHO, C. A., AYLIFFE, G. A. J.,LOWBURY, E. J. L. (1967). Trials of fiveantibacterial creams in the control of nasal carriage of Staphylococcus aureus. Lancet ii: 390-392.

WILLIAMS, R. E. 0. (1946). Skin and Nose Carriage of bacteriophage types of Staph, aureus. Journal of Pathology and Bacteriology 58: 259-268.

WILLIAMS, R. E. 0. (1959). Epidemic Staphylococci.Lancet i: 190-195.

WILLIAMS, R. E. 0. (1963). Healthy carriage ofStaphylococcus aureus: its prevalence and importance. Bacteriological Reviews 27: 56-71.

305

WILLIAMS, R. E. 0., HARPER, G. J. (1946). Determination of coagulase and alpha-haemolysin production bystaphylococci. British Journal of ExperimentalPathology 27: 72-81.

WINSLOW, C.-E. A., ROGERS, A. F. (1906). A Statistical Study of Generic Characters in the Coccaceae. Journal of Infectious Diseases 3: 485-546.

WINSLOW, C.-E. A., ROTHBERG, W., PARSONS, E.I. (1920). Notes on the Classification of the White and Orange Staphylococci. Journal of Bacteriology 5: 145-168.

WISE, R. (1990). Macrolide progress. Journal ofAntimicrobial Chemotherapy 26: 5-6.

W0LFS0N, J. S., HOOPER, D. C. (1985). Thefluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrobial Agents and Chemotherapy 28: 581-586.

WOODRUFF, H. B., MATA, J. M. , HERNANDEZ, S., MOCHALES,S., RODRIGUEZ, A., STAPLEY, E. 0., WALLICK, H., MILLER,A. K., HENDLIN, D. (1977). Fosfomycin: laboratorystudies. Chemotherapy 23 (Suppl. 1) , 1-22.

WORKING PARTY (1986). Guidelines for the control of epidemic methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection 7: 193-201.

WORKING PARTY (1990). Revised guidelines for thecontrol of epidemic methicillin-resistantStaphylococcus aureus. Journal of Hospital Infection 16: 351-357.

WYATT, T. D., FERGUSON, W. P., WILSON, T. S., McCORMICK,E. (1977). Gentamicin resistant Staphylococcus aureusasssociated with the use of topical gentamicin.Journal of Antimicrobial Chemotherapy 3: 213-217.

306

YAMAMOTO, T., TAKUBO, S., FUJITA, K., OGURI, T., YOKOTA, T. (1990). Cloning and restriction analysis of DNA conferring new quinolone antimicrobial agent resistance from Staphylococcus aureus and other coagulase-negative Staphylococcus species. FEMSMicrobiology Letters 68: 335-340.

YOUNG, H. K., SKURRAY, R. A., AMYES, S. G. B. (1987).Plasmid-mediated trimethoprim resistance inStaphylococcus aureus. Characterisation of the firstGram-positive plasmid dihydrofolate reductase (typeSI). Biochemical Journal 243: 309-312.

YU, V. L., GOETZ, A., WAGENER, M., SMITH, P. B., RUES, J.D., HANCHETT, J., ZURAVLEFF, J. J. (1986).Staphylococcus aureus nasal carriage and infectionin patients on haemodialysis. New England Journal of Medicine 315: 91-96.

307

Appendices

Further Information on Methods Used.

A . Influence____ o_f__ antibiotic____ carry-over___ in

fluoroquinolone MBC and time-kill experiments.

Some workers (eg. Kaatz & Seo, 1990b; Hardy et al.,

1987) have reported that antibiotic carry-over

influenced their results in time-kill experiments.

One of the advantages of the replica-plating

method of Elek and Hilson was that it avoided

the broth antibiotic carry-over associated with

viable counting from fluoroquinolone-containing

broth. Broth carry-over could not be avoided in

the time-killing experiments. Some workers have

found that they can only eliminate the influence

of fluoroquinolone carry-over by diluting broth

100-fold in water. However, this approach has

limitations when counting small numbers of

organisms. We observed no fluoroquinolone inhibitory

effects (ie. areas of reduced or no growth on

counting plates) in our viable counting from

fluoroquinolone-containing broths. However, to

determine the influence of fluoroquinolone carry­

over on our viable counts the following experiment

was performed.

0.1 ml samples of IsoSensitest broth and

IsoSensitest broth containing the highest

308

concentrations of ciprofloxacin, enoxacin, ofloxacin

and pefloxacin used were spread in triplicate onto

IsoSensitest agar- plates. Immediately after, the

plates were spread with 0.1 ml of a bacterial

suspension containing approximately 500 cfu/ml of

S. aureus NCTC 6571. After 24 hours incubation at

37°C the plates were counted. On comparison of

the viable counts on antibiotic spread plates to

those on non-antibiotic spread plates no difference

(Student’s T-test, p = 0.9) in viable count was

found.

B . Development of a biotyping system for MGRSA.

i . Use of API STAPH profiles.

’’API STAPH” is a biochemical test system

designed to identify staphylococci and micrococci.

While it is designed to facilitate speciation

rather than sub-speciation (the latter being the

aim of biotyping), our previous experience (Gargan

et al. , 1982) with Gram-negative species is that

this type of system can be of some use in

biotyping. We obtained 11 different biotypes (ie.

API codes) for the MGRSA we identified. However

55% of strains fell into two biotypes (6736153 and

6736113). A variety of other profiles (eg. 6776153,

6736115, 6736112, 6734113 etc.) were found for small

numbers of strains.

309

i i. Use of antibiotic susceptibility profiles.

In a previous study (Brumfitt et al. , 1989) we

found that of 23 antibiotics tested, sensitivity to

netilmicin, amikacin, neomycin, tetracycline,

clindamycin, chloramphenicol, trimethoprim, rifampicin

and ciprofloxacin provided most discrimination (ie.

between 15 and 85% of strains were sensitive).

Using this system 59 antibiotypes were obtained,

and the greatest number of strains of any one

type was 4.

iii . Determination of____ haemolysin___ profiles by

microtitre method.

Staphylococci produce a variety of haemolysins

(alpha, beta, gamma and delta), each of which is

capable of lysing erythrocytes of different species

origins to varying degrees (Mollby, 1983). To

identify and quantify production of these

haemolysins we used the micro-titration method

reported by Jordens et al. (1989).

Three known specific haemolysin producing

organisms, S . aureus NCTC 7121, S . aureus NCTC 5664

and S . aureus NCTC 10345 were used as controls in

the assay. S. aureus NCTC 7121 (Wood 46) only

produces alpha haemolysin and in our assay had a

titre of 128 against rabbit erythrocytes in the

presence of fibrinogen. S. aureus NCTC 5664 (Y2,

310

Sweden) produced beta and gamma haemolysins. Broth

supernatant from this strain was active against

human, rabbit and sheep erythrocytes, and this

activity was reduced in the presence of heparin.

Following incubation at 0°C an increase in titre

was found against all erythrocytes, but particularly

with sheep erythrocytes. S. aureus NCTC 10345

produced delta haemolysin and broth supernatants of

this organism had little activity against rabbit

or sheep erythrocytes (titre 0-2), and not much

more against human erythrocytes (titre 2-8). Little

reduction in titre ( 1 or 2 wells ) occurred in the

presence of fibrinogen. On cooling, an increase in

titre was found similar to that seen with S .

aureus NCTC 5664.

Using this method we tested strains from Israel

and Texas. Titres of 4-32 were obtained with

rabbit blood + fibrinogen, however little activity

(maximum titre of 4) was found with human or

sheep erythrocytes, and this was not changed in

the presence of heparin or fibrinogen. Because we

only reliably detected alpha haemolysin in these

strains using this method, we felt that because of

its expense and lack of discrimination it was not

worthwhile to continue. Also from previous

biotyping studies we had found that strains could

be differentiated on the basis of sizes of zones

of haemolysis on sheep blood agar. There was a

correlation between sizes of zones of haemolysis

311

on sheep blood agar and titre of alpha

haemolysin. Strains producing large zones, had

greater titres than those producing small zones.

Elek and Levy (1954) reported that discrepancies

can occur between the haemolysins detected using

culture filtrates compared to those determined by

growth on agar. According to Mollby (1983) agar

plate tests are more reliable than broth methods

for detecting mixtures of haemolysins: for example

beta-haemolysin production may readily be lost by

growth in liquid media.

iv. Sheep blood haemolysis.

Williams and Harper (1946) found 93% of S. aureus

of human origin produced alpha-haemolysin as

detected by antitoxin neutralization on sheep blood

agar. These workers also found other haemolysins

responsible for sheep blood lysis and suggested

that other toxins may be produced. We are of the

same opinion; however, using the method of Jordens

et al. (1989) we have failed to identify them.

v . Egg-yolk reaction.

The observations that most strains of coagulase-

positive staphylococci produced opacity when grown

in media containing egg yolk prompted Gillespie

and Alder (1952) to study this reaction in detail.

312

They found that it was probably due to production

of a lipase, and that isolates of S. aureus from

hospital inpatients were often egg yolk negative

as were most penicillin-resistant staphylococci. On

the contrary, most S. aureus from outpatients were

egg-yolk positive.

Approximately 60% of MGRSA tested were egg-yolk

positive, which contrasts with Gillespie and

Alder’s findings because these strains were

predominantly in-patient isolates resistant to many

antibiotics including penicillin. We found the egg-

yolk reaction most useful in differentiating

strains.

vi. TWEEN 80 reaction.

The results for Tween 80 hydrolysis mirrored

those obtained for the egg-yolk reaction.

vii. Pigmentation on Milk Agar.

The interpretation of colour varies between

workers, for example Lacey and Stokes (1979)

classified strains as orange or yellow whereas

Putland and Guinness (1985) classified strains as

gold, cream or white. Lacey and Stokes (1979) found

most (c. 90%) MRSA produced orange pigment and

Putland and Guinness (1985) observed that most (67%)

of their strains produced gold pigment. Most (c.

313

70%) of our MGRSA produced orange/gold pigment,

however this figure was highly subjective as

considerably different results were obtained when

different people read the plates. We are of the

opinion that this method is too subjective, and

the results we obtained were not used to

differentiate strains in our fluoroquinolone

resistance studies. Interestingly, two of the Irish

MGRSA produced a lime yellow pigment.

viii. Plasmid-typing.

The Takahashi and Nagano (1984) method was

inconsistent in reliably detecting plasmids.

Although small and large plasmids were isolated,

there was considerable variability in results on

repeating experiments. Large plasmids (30 mD or

larger) were sometimes observed, yet on other

occassions they were not. A further problem was

the low plasmid yield (ie. production of only faint

bands) obtained. The PHLS (Johnson) method

consistently detected large and small plasmids.

The plasmid contents and sizes of 45 strains of

MGRSA used in the resistance survey (Table IV) were

studied, and Table XXI shows our findings for 33 of

those strains. It is evident that the MGRSA

314

Selection of MGRSA of Worldwide Origins

Origin Susceptibility to Plasmid ContentCountry Code MLS Chlor Tet and sizes (mD)Australia AS2 I R R 20, 2.0AS6 C S R 30

AS10 c S R 30Chile CH3 c R S 5.0Belgium BEL1 I R R 20, 4.0Brazil BZ1 I R R 2.0

BZ12 I S S —

England RFH1 I R R 22, 4.0RFH2 I R R 22, 4.0RFH4 c S R 20, 1.4RFH11 c R R 20, 3.0, 1.4Cl I R R 30, 4.8UK16 I R R 20, 4.8, 2.0

E .Germany EG 6 I S R 4.6, 3.5France Fll c S R 26

F12 c R R 20, 4.8F13 c S R —

Hong Kong HK1 s S R 25, 3.5Ireland IR1 I s R —

IR15 I s R 26Japan JAP 2 c s S 22Poland PL1 c R R 24, 2.0Portugal PI c S R 1.6

P2 c S R 1.6Russia RUS2 s R R to » o

S. Africa SA1 I R R 00 > 3.0Spain SP1 I S R 2.0SP3 I S R 30Switzerland SW2 I R R o•CM

Turkey T6 c R R 20, 2.0USA RM1 c S S 18

SC10 c S S 43US12 c S R 34C = constitutive resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics, I = inducibleresistance. Criteria for S and R are same as used for Table IV.

315

studied contained a variety of plasmids of

different sizes. For the 45 strains studied, only

three strains (BZ12, F13 and IR1) were found to

contain no plasmids, all the other strains were

found to contain from 1-3 plasmids. In Table XXI

the susceptibility to macrolide, lincosamide and

streptogramin (MLS) antibiotics, chloramphenicol and

tetracycline of strains is compared to plasmid

content. Chloramphenicol resistance was found to be

always plasmid-mediated, whilst tetracycline resistance

or resistance to MLS antibiotics could have been

plasmid or chromosomally mediated in different

strains .

Although this work was interesting it was felt

that further plasmid analysis studies were

inappropriate because of the difficulties in

transferring or curing single plasmids from strains

containing multiple plasmids which is requisite for

assigning the location of resistance factors.

Furthermore, following Lyon and Skurray's excellent

review of the genetic basis of antimicrobial

resistance in S. aureus (Lyon & Skurray, 1987) we came

to the opinion that further studies of the genetic

basis of resistance in our strains would be of

limited significance to the project as a whole.

316

C . Detection and Isoelectric focusing of type SI

enzyme.

Detection and isoelectric focusing of type SI

enzyme was carried out at the Bacteriology

Research Laboratory, Old Medical School, Edinburgh

University. The method used was being developed by

S. Tait, and formed a major part of his PhD

thesis. Because of the complexity of the method,

the fact that it is as yet unpublished, and

because the results obtained were of limited

significance to this study, the methodology shall

only be briefly outlined.

Cultures were initially grown in 10 ml

IsoSensitest broth containing 2.0 mg/1 trimethoprim

for 12 hours at 37°C. These starter cultures were

then aseptically added to 2.0 litre flasks

containing 1.0 litre IsoSensitest broth. After

shaking overnight (80 rpm) at 37°C, bacteria were

harvested by centrifugation (6000 rpm for 10

minutes). They were washed in saline and

resuspended in 10 ml phosphate-mercaptoethanol

buffer. The bacteria were lysed with lysostaphin

and then sonicated. The extracts were centrifuged

at 16,000 rpm for 1 hour, and the supernatant

collected. Nucleic acids in these crude

preparations were precipitated by addition of

streptomycin sulphate, they were then centrifuged

(10,000 rpm for 30 minutes at 4°C) and the

I 317

supernatant collected. Ammonium sulphate was added

to 50% saturation (to remove protein contaminants)

and the supernatant decanted. This was then

saturated to 80% with ammonium sulphate and the

precipitate (containing the proteins we do want) was

collected by centrifugation at 4°C for 30 minutes

at 10,000 rpm. The pellet was resuspended and

dialysed in phosphate-mercaptoethanol buffer. It was

then ready for isoelectric focusing. Polyacrylamide

gels were pre-run for 20 minutes (1500 volts,15

watts), and samples (and controls containing purified

type SI) were streaked over the surface at the

anode. When the standards had focussed the gel

was sequentially stained with NADPH solution

followed by dihydrofolate and then mercaptoethanol

solution.

318

D . Suppliers of Strains of MGRSA

We are indebted to the following who supplied

the strains of MGRSA used for this work: J.F.

Acar, 0. Ang, G.F. Ara j , C.A. Bartzokas, E. Bergogne-

Berezin, J. Borowski, M.T. Cafferkey, P.Y. Chau, R.L.

Cohen, J. Cooper, M. Dan, S. Dixson, A.A. Forder, A.

Georgopoulos, H. Giamarellou, M. Gobernado, F.W.

Goldstein, W. Graninger, G.G. Grassi, D. Hanslo, C. Hohne,

J.F. John Jr, F.H. Kayser, C.T. Keane, C.C. Linnemann, D.

Merzbach, R.C. Moellering Jr, E.C. Moorhouse, H.C. Neu,

C.E.O. Pires de Campos, V. Prado, G.W. Smith, K.H. Spitzy,

J.L. Staneck, A, Torres Pereira, E.P. Trallero, W.H.

Traub, K. Ubukata, C. Watanakunakorn, E. Yourassowsky.

319

Summary of Conclusions and Suggestions for futurew o r k .

Although a substantial amount of work has been

published in recent years concerning the genetics

and mechanisms of antibiotic resistance possessed

by MGRSA, few workers have attempted to answer the

question as to whether antibiotic resistance in

MGRSA poses a significant clinical problem. In

endeavoring to answer this we discovered a number

of new facts regarding the emergence of antibiotic

resistance in MGRSA, and specifically the development

of fluoroquinolone resistance.

i . New facts emerging.

From the international survey of antibiotic

resistance in MGRSA we showed that these organisms

possessed differing degrees of multiple antibiotic

resistance. Some strains were highly multiple

antibiotic resistant whilst others were considerably

less so. Most of the highly multiple resistant

strains emerged from Brazil, France and Turkey,

countries which have a reputation for failing to

control antibiotic usage. Strains showing least

multiple resistance originated from Chile, East

Germany, Hong Kong, Italy, Russia, U.K and the USA.

In these countries antibiotic usage is either

320

controlled or newer classes of antibiotic are

simply not available.

In terms of MIC a considerable diversity of

resistance phenotypes was seen, both with reference

to the variety of different antibiotic resistance

patterns, and the levels (low or high) of resistance

to individual antibiotics such as ciprofloxacin,

fusidic acid, gentamicin, minocycline and streptomycin.

Resistance to macrolide, lincosamide and streptogramin

(MLS-type) antibiotics was closely associated with

methicillin and gentamicin resistance. Gentamicin

resistance in strains of worldwide origin was

associated with the same enzyme - APH(2")/A A C (6f) .

Phage typing with the International Set and

additional experimental phages revealed most strains

to belong to Group III or be Group III-related.

A comprehensive assesment of the antebacterial

activity of currently available antistaphylococcal

agents was carried out by studying MIC, MBC and

killing curves. Fosfomycin and pristinamycin appeared

as potentially useful alternatives to vancomycin.

Further evidence was obtained that the phenomenon

of tolerance to vancomycin and teicoplanin was due

to methodological variables. New data were obtained

on the activity against MGRSA of azelaic acid,

nitrofurazone and silver sulphadiazine.

We were among the first workers to find

resistance to fluoroquinolones in MGRSA, and showed

it to be present in strains from a number of

countries. Ciprofloxacin-resistant MGRSA from clinical

sources had different levels and patterns of

quinolone resistance. The phenomenon of "reversed

incomplete cross-resistance" between fluoroquinolones

and older non-fluorinated quinolones was seen in a

number of strains. In broth time-kill experiments

fluoroquinolone resistance readily emerged in the

presence of peak serum levels of ciprofloxacin,

enoxacin and pefloxacin. Resistance did not readily

emerge in the presence of peak serum concentrations

of ofloxacin. No change in phage-type or

susceptibility to agents other than quinolones was

detected following mutation to fluoroquinolone

resistance. Fluoroquinolone resistance developed in

a stepwise manner, and irrespective of the

particular concentration or fluoroquinolone used,

resistant mutants possessed similar reductions in

susceptibility indicating that only an initial one-

step mutation could occur. Typing of ciprofloxacin-

sensitive and resistant MGRSA from one of the

first outbreaks to be reported showed that

resistance had evolved in a number of different

strains.

322

i i . New thoughts on Antibiotic Resistance in MGRSA.

It has previously been stated that a single

clone of MRSA has spread worldwide. Our work

showed MGRSA from several sources to be different

and we suggest that antibiotic resistance has

evolved in separate strains. The aminoglycoside

modifying enzyme A P H (2")/AAC( 6 ?) was present in all

MGRSA which indicates that the gene for this

enzyme has become disseminated worldwide. Genes

coding for resistance to aminoglycosides and MLS

antibiotics have been found in antibiotic producing

Streptomyces spp., and also other Gram-positive

bacteria such as streptococci and coagulase negative

staphylococci. These organisms may have acted as a

reservoir for gene transfer to MGRSA. The

unrestricted use of antibiotics provides the

selective pressure for multiple resistance to appear

in MRSA, and that in the case of aminoglycoside

resistance and resistance to MLS antibiotics this

resistance may readily be acquired through

interstrain, interspecies or intergeneric transfer.

iii. Future work to be d o n e .

The data from the survey of antibiotic

resistance in MGRSA enabled us to qualitatively

assess the problem of multiple antibiotic resistance

323

in MGRSA. What is needed is a survey to

quantitatively determine the international problem of

antibiotic resistance in MRSA and relate this to

patterns of antibiotic usage and clinical origin of

strains. Currently, the European Society for Clinical

Microbiology and Infectious Diseases is attempting

to survey the incidence of MRSA in different

countries, however antibiotic usage patterns are not

being ascertained.

Recently, much work has been published on the

genetic control of resistance in MGRSA and it

appears that a great deal of antibiotic resistance

is transposon-mediated. Certain chromosomal "hot

spots" for the integration of transposons have been

identified and a further understanding of these

would be most useful. Determination of the location

(chromosome or plasmid) of resistances in the

strains we studied would be useful as a means of

further understanding how resistance has evolved and

spread.

The most important aim for the future is to

attain an international concensus regarding the

appropriate use of antibiotics with a view to

limiting the opportunities for emergence of multiple

resistance .

WORLD-WIDE ANTIBIOTIC RESISTANCE IN METfflCIIXIN-RESISTANT

STAPHYLOCOCCUS AUREUS

P. A. C. M a p l e J. M. T. H a m i l t o n - M i l l e r W . B r u m f i t t

Departtnent of Medical Microbiology, Royal Free Hospital School of Medicine, London NW3 2QG

Summary Antibiotic resistance patterns were determined for 106 strains of methicillin-

resistant Staphylococcus aureus (MRSA) from 21 countries. Resistance to gentamicin, tobramycin, netilmicin, amikacin, streptomycin, or erthromycin was recorded in more than 90% of strains. Resistance to the other compounds tested was as follows: tetracycline 86%, minocycline 76%, trimethoprim 69%, clindamycin 66%, neomycin 59%, chloramphenicol 39%, rifampicin 26%, fosfomycin 22%, ciprofloxacin 17%, fusidic acid 12%, bacitracin 2%, and novobiocin 1%. All the stains were sensitive to mupirocin, pristinamycin, ramoplanin, teicoplanin, and vancomycin. There were geographical patterns of resistance: MRSA from the UK and Australia were predominantly resistant to trimethoprim, whereas many strains from centres in Europe and the USA were sensitive. MRSA that were resistant to ciprofloxacin were of French and German origin. 15 strains, 12 of which came from France, Turkey, or Brazil, were resistant either to thirteen or to fourteen agents.

INTRODUCTION

O u t b r e a k s of infection attributable to methicillin- resistant Staphylococcus aureus (MRSA) have been reported world wide, and, since 1976, aminoglycoside-resistant strains have become increasingly prevalent.1,2 Many strains of MRSA are multiresistant, and vancomycin is at present the antibiotic of choice for systemic infection.3 Resistance to vancomycin would be extremely worrying: a report of plasmid mediated resistance in enterococci4 has fuelled this concern.

During the past three years, we have collected strains of MRSA from 28 centres in 21 countries. We have looked at the chemotherapeutic options and we have determined the incidence and degree of multiresistance in different geographical regions.

MATERIALS AND METHODS

Bacterial StrainsW e studied 106 strains of M R S A from the following 21 countries

(if more than one centre, number in parentheses): Australia, Austria, Belgium, Brazil, England (3), Federal Republic of

Germany, France (2), German Democratic Republic, Greece, Hong Kong, Ireland (2), Italy, Japan, Kuwait, Poland, Portugal, South Africa (2), Spain, Switzerland, Turkey, and the U S A (3).

MicrobiologyMinimum inhibitory concentrations (MIC) of the 23 antibiotics

listed in table I were determined by the agar dilution method. Briefly, doubling dilutions of antibiotic were incorporated into ‘IsoSensitest Agar’ (Oxoid, UK). A Denley Multipoint Inoculator (Denley Instruments, UK) was used to inoculate the agar plates with 10s organisms from peptone water broths grown at 37°C for 24 h. The inoculated plates were incubated for 24 h at 37°C before recording MICs. MICs of fosfomydn were done in the presence of glucose-6-phosphate (25 mg/1).

Methicillin resistance was confirmed by incubation of strains on nutrient agar against 25 jig methicillin strips (Mast Laboratories, U K ) for 40 h at 30°C.

RESULTS

The MICs of 23 antimicrobial agents against 106 strains of MRSA are shown in tables II and ill. We classified strains as sensitive, moderately resistant, or resistant to each agent according to the MICs (see table i). Compounds could be

TABLE I—MICS USED TO CLASSIFY STRAINS AS SENSITIVE, MODERATELY RESISTANT, OR RESISTANT

Antibiotic

MIC (mg/1)

Sensitive Moderate Resistant

Gentamicin* <1 N/A 2*8Tobramycin* <1 N/A ^ 16Netilmicin* <1 2,4 2*8Amikacin* <4 8,16 ^32Streptomycin* <16 N/A ^64Neomycin* <4 N/A ^ 16Bacitradnt ^8 N/A ^32Tetracycline <4 N/A 2*32Minocycline <4 N/A ^8Erythromycin <1 N/A 2? 8Chloramphenicol < 16 N/A 2*32Fosfomycint <16 32,64 2*128Clindamycin <0-25 N/A ^16Trimethoprim <0-25 1,2 2*4Rifampirin <012 1 ^16Fusidic acid <0-5 4 2*16Novobiocin <0-5 2 N/APristmamycin <2 N/A N/ACiprofloxacin <1 2,4 2*16Mupirocin <0-5 N/A N/ARamoplanin <1 N/A N/AVancomycin <2 N/A N/ATeicoplanin <1 N/A N/A

MIC = minimum inhibitory concentration.N/A = not applicable. tIU.Definitions of sensitivity as suggested by *a working party of the British Society of Antimicrobial Chemotherapy5 and by tan international study group.6

grouped in relation to the distribution of recorded MICs: for mupirocin, pristinamycin, ramoplanin, teicoplanin, and vancomycin, there was a single peak in the distribution—ie, all the strains were sensitive; for streptomycin, neomycin, bacitracin, tetracycline, minocycline, erythromycin, chloramphenicol, clindamycin, and novobiocin there were two peaks (ie, one group of strains sensitive, the other resistant); and for trimethoprim, rifampicin, fusidie acid, and ciprofloxacin there were three groups of MRSA— namely, sensitive, moderately resistant, and resistant. For the remaining compounds (gentamicin, tobramycin, netilmicin, amikacin, and fosfomycin) there was no obvious pattern of distribution.

Table IV summarises the prevalence and diversity of resistance of the strains to 23 antibiotics. Resistance/ moderate resistance to the aminoglycosides (gentamicin, tobramycin, netilmicin, amikacin, neomycin, and

TABLE II—DISTRIBUTION OF MICS OF VARIOUS ANTIBIOTICS FOR 106 STRAINS OF MRSA

No of strains

MIC (mg/1)

Antibiotic <1 2 4 8 16 32 64 3*128

Gentamicin 9 0 0 1 1 1 3 91Tobramycin 7 0 0 0 3 11 19 66Netilmicin 8 5 8 32 22 20 4 7Amikacin 3 0 5 11 22 30 19 16Streptomycin 0 0 6 2 1 0 4 93Neomycin 27 14 2 0 2 6 16 39Bacitracin* 68 23 9 4 0 1 1 0Tetracycline 11 2 2 0 0 1 15 75Minocycline 18 4 3 5 26 46 4 0Erythromycin 9 0 0 0 0 0 0 97Chloramphenicol 0 0 2 56 7 4 12 25Fosfomycin 2 3 24 31 23 6 2 15

*IU.

TABLE III—DISTRIBUTION OF MICS IF VARIOUS ANTIBIOTICS FOR 106 STRAINS OF MRSA

No of strains

MIC (mg/1)

Antibiotic <012 0-25 05 1 2 4 8 3*16

Clindamycin 35 1 0 0 0 0 0 70Trimethoprim* 0 39 0 23 4 1 6 33Rifampicin 78 0 0 3 0 0 0 25Fusidie acid 50 40 3 0 0 7 0 6Novobiocin 21 82 2 0 1 0 0 0Pristinamycin 14 46 36 9 1 0 0 0Ciprofloxacin* 1 8 53 26 4 9 0 5Mupirocin 0 84 22 0 0 0 0 0Ramoplanin 0 25 74 7 0 0 0 0Vancomycin 0 1 4 90 11 0 0 0Teicoplanin 0 6 32 68 0 0 0 0

*As trimethoprim and ciprofloxacin are synthetic they should be called “antimicrobials” but we have included them as antibiotics for convenience.

4TABLE IV—INCIDENCE AND DISTRIBUTION OF ANTIBIOTIC

RESISTANCE IN 106 STRAINS OF MRSA

Antibiotic

Resistant strainsModerately

resistant strains Sensitive strains

No of isolates

No of centres

(countries)No of isolates

No of centres

(countries)No of isolates

No of centres

(countries)

Gentamicin 97 28(21) 0 0 9 3(3)Tobramycin 99 28 (21) 0 0 7 2(2)Netilmicin 85 27(21) 13 8(8) 8 4(4)Amikacin 65 23(19) 33 19 (14) 8 2(2)Streptomycin 97 28(21) 0 0 9 4(4)Neomycin 63 23 (18) 0 0 43 16(14)Bacitracin 2 2(2) 0 0 104 28(21)Tetracycline 91 28(21) 0 0 15 7(7)Minocycline 81 26 (19) 0 0 25 12(12)Erythromycin 97 26 (19) 0 0 9 5(5)Chloramphenicol 41 17(12) 0 0 65 22 (18)Ramoplanin 0 0 0 0 106 28 (21)Fosfomycin 15 6(5) 8 7(6) 83 25 (20)Clindamycin 70 22 (18) 0 0 36 18(13)Trimethoprim 40 14(11) 27 15(13) 39 15(14)Rifampicin 25 10(10) 3 1(1) 78 23(19)Fusidie acid 6 5(4) 7 4(4) 93 21 (19)Novobiocin 0 0 1 1(1) 105 28 (21)Pristinamycin 0 0 0 0 106 28 (21)Ciprofloxacin 5 2(2) 13 7(6) 88 27 (21)Mupirocin 0 0 0 0 106 28(21)Vancomycin 0 0 0 0 106 28 (21)Teicoplanin 0 0 0 0 106 28 (21)

5 6 7 8 9 10 11 12 13 14Total of antibiotics tested to which strains are resistant or moderately resistant

Degree of multiresistance in MRSA strains.

streptomycin), and resistance to clindamycin, erythromycin, tetracycline, and minocycline was widespread. By contrast, all MRSA were sensitive to mupirocin, pristinamycin, ramoplanin, teicoplanin, and vancomycin. The strains that were resistant to ciprofloxacin (MIC 16 mg/1) were of French and German origin whereas those that were moderately resistant (MIC=2 or 4 mg/1) originated from various centres in Europe, USA, and Asia. MRSA that were resistant/moderately resistant to fosfomycin came from France, Germany, Italy, Turkey, and Brazil. 2 strains (1 from Switzerland, 1 from Brazil) were resistant to bacitracin. For 1 strain (from Ireland) the MIC of novobiocin was high (2 mg/1).

12 of the 15 strains resistant/moderately resistant either to thirteen or to fourteen antibiotics (figure) came from France, Turkey, and Brazil. The other 3 strains were from the USA, Kuwait, and Switzerland. 2 of the 15 strains were sensitive to rifampicin, 3 to fosfomycin, and 11 to fusidie acid and ciprofloxacin.

DISCUSSION

Gentamicin resistance in MRSA is encountered world wide; resistance can be chromosomally or plasmid mediated.7 Various patterns of aminoglycoside resistance have been recorded; for example, Rimland8 has reported tobramycin-resistant, gentamicin-sensitive strains, and Schaefler et al9 observed MRSA that were resistant to gentamicin, tobramycin, and amikacin. Even when the organisms are sensitive, the clinical use of aminoglycosides, either alone10 or with P-lactams,11 has proved to be less than satisfactory for the treatment of staphylococcal infections.

Resistance to antibiotics of the “macrolide, lincosamide, and streptogramin” (MLS) group—represented in the present study by erythromycin, clindamycin, and pristinamycin—is due either to methylation of ribosomal RNA, or to enzymic modification of the antibiotic. The first type of MLS resistance may be either constitutive (strains are resistant to erythromycin and clindamycin, but remain sensitive to pristinamycin), or inducible (strains are resistant to erythromycin only, but clindamycin resistance can develop in the presence of subinhibitory concentrations of erythromycin). Many of the MRSA that were sensitive to clindamycin showed inducible clindamycin resistance. None of the strains in the present study was resistant to pristinamycin, although resistance has been reported as a rare event. Our findings concerning MLS resistance are similar to those of Duval.12

Where the MRSA strains were trimethoprim-sensitive— commonly in the USA13—co-trimoxazole (trimethoprim/ sulphamethoxazole) has been used successfully.14 MRSA from the UK and Australia were resistant to trimethoprim, whereas strains from many European centres were sensitive.

Resistance to rifampicin and to fusidie acid is due to chromosomal mutations followed by selection. Selection is less likely if these agents are used in appropriate combinations.1516 We are concerned that as many as 28 of the 106 strains studied were not sensitive to rifampicin, particularly those from France, Turkey, and Brazil.

There are few clinical reports on the use of novobiocin, fosfomycin, and ciprofloxacin for treatment of infections caused by MRSA. We have found widespread resistance to ciprofloxacin: this is disconcerting because the antibiotic has a novel structure and has only recently entered clinical use. Our concern is heightened by reports from the USA of ciprofloxacin resistance in MRSA after treatment.1718 There have, however, been encouraging reports about the use of fosfomycin for MRSA infections.19 Development of resistance during treatment can occur, but does not seem to arise if fosfomycin is not used alone.20

Only 1 of our strains was resistant to novobiocin: in vitro, there is promising activity with this compound and the related compound, coumermycin.2U2 None of the strains in the present study was resistant to vancomycin or to the structurally similar teicoplanin. S aureus has remained sensitive to vancomycin, although reports of resistance by 5 epidermidis,23 and, more recently, of plasmid mediated resistance by enterococci4 warn against complacency. Teicoplanin, which is currently under clinical evaluation,24 is less toxic and is easier to administer than vancomycin.

Opinions vary on the efficacy of topical antibiotics for eradicating MRSA carriage. Like many others25 we believe that certain topical agents should only be regarded as an adjunct to rigorous measures of hospital control of infection: Undoubtedly, scrupulous handwashing and access to isolation facilities are much more effective in controlling the spread of MRSA. Bacitracin is ineffective,26 and many strains are resistant to neomycin and tetracycline. There are encouraging reports about the efficacy of mupirocin,27 but even with this drug, resistance has been encountered.28 The novel lipoglycodepsipeptide antibiotic, ramoplanin29 (formerly called A-16686), may have an important future role as a topical agent.

In the present study, numerous patterns of resistance to antibiotics were recorded. Although the severity of multiresistance ranged from 5 to 14 antibiotics, the choice of effective recognised chemotherapy in many instances was limited to rifampicin, to fusidie acid, or to vancomycin. For the highly multiresistant strains—ie, those resistant to 13 or 14 antibiotics—vancomycin was often the only antibiotic so far approved by the regulatory bodies to which the strains were sensitive. A thorough in-vitro and in-vivo assessment of fosfomycin, novobiocin (or the related compound coumermycin), and pristinamycin, in terms of efficacy and development of resistance, is essential if we are to increase therapeutic options. In view of the prevalence of

ciprofloxacin resistance found in the present study and by others17 we feel that the use of fluoroquinolones against M RSA should be re-evaluated.

12 of the 15 highly multiresistant strains came from France, Turkey, and Brazil: we have yet to determine whether this multiresistance is a consequence o f strains with enhanced genetic adaptability, or of selection caused by favourable environmental factors. Additional resistance can be prevented: first, it is essential that antibiotics that can be used systemically should not also be used topically; and, second, for antibiotics where m utation to resistance is likely (eg, ciprofloxacin, fosfomycin, fusidie acid, and rifampicin), use of appropriate combinations will minimise development of resistance.

Finally, because of the possibility of spread o f resistant strains from one country to another, close international cooperation is the key to limiting occurrence and spread.

This study has been aided by the Special Trustees o f the Royal Free Hospital. A fellowship was made available to J. M . T . H -M . by th e - Leverhulme Trust.

W e are indebted to the following, who supplied the strains used in this study: J. F. Acar, O. Ang, G. F. Araj, C. A. Bartzokas, E. Bergogne-Berezin, J. Borowski, M . T . Cafferkey, P. Y. Chau, J. Cooper, S. D ixson, A. A. Forder, A. Georgopoulos, H . Giamarellou, F. W . Goldstein, W. Graninger, G. G. Grassi, D . Hanslo, C. H ohne, F. H . Kayser, C. T . Keane, C. C. Linnemann, R. C. Moellering, E. C. M oorhouse, H . C. N eu , C. E. O. Pires de Campos, G. W . Smith, K. H. Spitzy, J. L. Staneck, A. Torres-Pereira, E. P. Trallero, W. H . Traub, K. Ubukata, C. Watanakunakom, R. P. Wenzel, E. Yourassowsky.

Correspondence should be addressed to W . B.

REFERENCES

1. Shanson D C, Kensit JG , Duke R. Outbreak of hospital infection with a strain ofStaphylococcus aureus resistant to gentamicin and methicillin. Lancet 1976; ii: 1347—48.

2. Soussy CJ, Dublanchet A, Cormier M , et al. Nouvelles resistances piasmidiques deStaphylococcus aureus aux aminosides (gentamicine, tobramycine, amikacine). Nouv Presse Med 1976; 5: 2599-602.

3. Milatovic D. Vancomycin for treatment of infections with methicillin-resistantStaphylococcus aureus: are there alternatives? E urJ Clin Microbiol 1986; 5:689-92.

4. LeClercq R, Derlot E, Duval J, Courvalin P. Plasmid mediated resistance tovancomycin and teicoplanin in Enterococcus faecium. N Engl J M ed 1988; 319: 157-61.

5. British Society for Antimicrobial Chemotherapy. Break-points in in-vitro antibioticsensitivity testing. J Antimicrob Chemoiher 1988; 21: 701-10.

6. Andrews JM , Baquero F, Beltran JM , et al. International collaborative study onstandardization of bacterial sensitivity to fosfomycin. J Antimicrob Chemoiher 1983; 12: 357-61.

7. Lyon BR, Skurray R. Antimicrobial resistance of Staphylococcus aureus: genetic basis.Microbiol Rev 1987; 51: 88-134.

8. Rimland D. Nosocomial infections with methicillin and tobramycin resistantStaphylococcus aureus: Implications of physiotherapy in hospital-widedissemination. Am J M ed Sci 1987; 290: 91-97.

9. Schaefler S, Jones D, Perry W, et al. Emergence of gentamicin and methicillin resistantStaphylococcus aureus strains in New York City hospitals. J Clin Microbiol 1981; 13: 754-59.

10. Cafferkey M T , Hone R, Keane CT. Antimicrobial chemotherapy of septicaemia dueto methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 1985; 28: 819-23.

11. Acar JF , Courvalin F, Chabbert YA. Methicillin-resistant staphylococcaemia:bacteriological failure of treatment with cephalosporins. Antimicrob Agents Chemoiher!1970. American Society for Microbiology, 1971: 280-85.

8

12. Duval J. Evolution and epidemiology of MLS resistance. J Antimicrob Chemother1985; 16 (suppl A): 137-49.

13. Elwell LP, Wilson RH, Knick VB, Keith BR. In vitro and in vivo efficacy of thecombination trimethoprim-sulfamethoxazole against clinical isolates of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 1986; 29: 1092-94.

14. Markowitz N, Saravolatz L, Pohlod D, et al. Comparative efficacy and toxicity oftrimethoprim-sulfamethoxazole versus vancomycin in the therapy of seriousS. aureus infections. In: 23rd Interscience Conference Antimicrobial Agents Chemotherapy 1983; Abs No 903:201. Washington DC: American Society for Microbiology, 1983.

15. Kapusnik JE, Parenti F, Sande MA. The use of rifampicin in staphylococcalinfections—a review. J Antimicrob Chemother 1984; 13 (suppl C): 61-66.

16. Jensen K. Methicillin-resistant staphylococci. Lancet 1968; ii: 1078.17. Piercy EA, Barbara D, Luby JP, Mackowiak PA. Ciprofloxacin for methicillin-

resistant Staphylococcm aureus infections. Antimicrob Agents Chemother 1989; 33: 128-30.

18. Isaacs RD, Kunke PJ, Cohen RL, Smith JW. Ciprofloxacin resistance in epidemicmethicillin-resistant Staphylococcia aureus. Lancet 1988; ii 843.

19. Lau WY, Teoh-Chan CH, Fan ST, Lau KF. In vitro and in vivo study of fosfomycinin methicillin resistant Staphylococcus aureus. J Hyg t Camb) 1986; 96:419-23.

20. Baron D, Drugeon H, Courtieu AL, Nicolas F. Septicemies et infections graves agermes multiresistants. Resultats du traitement par la fosfomycine. Med Malad Infect 1981; 11:255-61.

21. Walsh TC, Hansen SL, Tatem BA, Auger F, Standiford HC. Activity of novobiocinagainst methicillin resistant Staphylococcus aureus. J Antimicrob Chemoiher 1985; 15:435-40.

22. Unowsky J, Chandrasekar PH, De Lorenzo W, Levine DP. In vitro and in vivo activityof coumermycin and other antibacterial agents against methicillin resistant strains of Staphylococcus aureus. Chemotherapy 1986; 32:499-505.

23. Schwalbe RS, Stapleton JT, Gilligan PH. Emergence of vancomycin-resistance incoagulase negative staphylococci. N EnglJ Med 1987; 316:927-31.

24. Stille E, Sietzen W, Dieterich HA, Fell JJ. Clinical efficacy and safety of teicoplanin.J Antimcrob Chemother 1988; 21 (suppl A): 69-79.

25. Editorial. Staphylococci resistant to neomycin and bacitracin. Lancet 1965; ii: 421-22.26. McNally T, Lewis MR, Brown DR. Effect of rifampicin and bacitracin on nasal

carriers of Staphylococcus aureus. Antimicrob Agents Chemother 1984; 25:422-26.27. Casewell MW, Hill RLR. Mupirocin (“pseudomonic acid”)—a promising new topical

antimicrobial agent. J Antimicrob Chemother 1987; 19:1-5.28. Baird D, Coia J. Mupirocin-resistant Staphylococcta aureus. Lancet 1987; ii: 387-88.29. Pallanza R, Scotti R, Beretta G, Cavalleri B, Arioli V. In vitro activity of A-16686, a

potential antiplaque agent. Antimicrob Agents Chemother 1984; 26:462-65.

Printed in Great Britain © 1989 The Lancet, by Robquest Ashford Kent 46 Bedford Square London W C 1 B 3SL

Journal oj Antimicrobial Chemotherapy (1989) 23, 517-525

Comparative in-vitro activity of vancomycin, teicoplanin, ramoplanin (formerly A16686), paldimycin, DuP 721 and DuP 105 against methicillin and gentamicin resistant Staphylococcus aureus

P. A. C. Maple, J. M. T. Hamilton-Miller* and W. Brumfitt

Department o f Medical Microbiology, Royal Free Hospital School o f Medicine, PondStreet, Hampstead, London NW3 2QG, UK

The in-vitro activities of five anti-staphylococcal agents, teicoplanin, ramoplanin, paldimycin, D u P 721 and D u P 105 have been compared to vancomycin. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBGs) have been determined for a collection of methicillin and gentamicin resistant Staphylococcus aureus (MGRSA), comprising 75 strains obtained from 22 centres.

In terms of geometric mean MICs (inoculum size 105 cfu) paldimycin was the most active agent (0-4 mg/1) followed by ramoplanin (0-75 mg/1), teicoplanin (1*0 mg/1),D u P 721 and vancomycin (2-0 mg/1) and D u P 105 (6-8 mg/1).

Ramoplanin was bactericidal within six hours to all strains at a concentration of 1 0 mg/1. The M B C 90s for vancomycin and teicoplanin were > 32 mg/1 after 22 h exposure to antibiotic and 2-5 and 4 0 mg/1 respectively after 26 h exposure. Paldimycin was bactericidal against only some strains, while D u P 721 and D u P 105 were not bactericidal.

Ramoplanin is the most interesting of the new antibiotics, on account of its rapid and consistent bactericidal activity.

Introduction

Before 1975, strains of Staphylococcus aureus resistant to gentamicin were rarely isolated (Soussy et al., 1975; Porthouse et al., 1976). Outbreaks of hospital infection with strains of S. aureus resistant to methicillin and gentamicin (MGRSA) were first reported in 1976 (Shanson, Kensit & Duke, 1976; Soussy et al., 1976). Subsequently, such strains have shown increasing prevalence worldwide (Leading article, 1981; Thompson & Wenzel, 1982; Townsend et al., 1987).

Vancomycin is currently the antibiotic of choice for treating systemic infections caused by methicillin resistant staphylococci (Watanakunakorn, 1982; Kucers, 1984). Concern that the now widespread use of vancomycin will lead to the emergence of resistance has prompted the search for new agents. The toxicity of vancomycin, its inconvenient administration and its cost, are reasons for contemplating alternative antimicrobial therapy.

Recently, a number of new compounds with activity predominantly against Gram- positive species have been reported. We have investigated five such compounds with characteristics which make them potential alternatives to vancomycin.

♦Corresponding author.

5170305-7453/89/040517 + 09 $02.00/0 © 1989 The British Society for Antimicrobial Chemotherapy

1988). Ramoplanin (formerly called A 16686 and M D L 62198) and paldimycin also have good anti-staphylococcal activity (Neu & Neu, 1986; Pfaller, Bale & Barrett, 1987). The latter two compounds are structurally novel, paldimycin being derived from the paulomycins (Argoudelis et al., 1987a) while ramoplanin is a lipoglycodepsipep- tide, resembling but distinct from daptomycin. D uP 721 and DuP 105 are entirely new chemical entities, substituted oxazolidinones (Slee et al., 1987).

In this study, we report the activity o f these antimicrobial agents against a collection o f M GRSA comprising 75 strains from 22 centres in 17 countries distributed over five continents.

Materials and methods

Media

Iso-Sensitest Agar (ISA), Nutrient Agar (NA) and Antibiotic Medium No. 2 (AM) were from Oxoid Ltd. Basingstoke.

Bacterial strains

The 75 strains o f M GRSA were kindly supplied from Liverpool, UK; Crewe, UK; London, UK; Dublin, Eire; Paris, France; Brussels, Belgium; Hamburg, FGR; Vienna, Austria; Zurich, Switzerland; Lisbon, Portugal; Athens, Greece; Pavia, Italy; Istanbul, Turkey; Hong-Kong; Melbourne, Australia; Tokyo, Japan; Ohio, USA; Botucatu, Brazil; Cape Town, South Africa.

Twenty strains o f methicillin sensitive S. aureus (MSSA) from the routine microbiology laboratory at The Royal Free Hospital were used for additional studies on the bactericidal activity o f vancomycin and teicoplanin.

The antimicrobial agents used were: vancomycin hydrochloride (Sigma Chemicals, St Louis, Missouri, USA), gentamicin sulphate (Roussel Laboratories, London, UK), teicoplanin and ramoplanin (Merrell Dow, Lepetit Research Centre, Milan), paldimycin (Upjohn Co., Kalamazoo, Michigan, USA), D uP 721 and DuP 105 (Du Pont de Nemours, Geneva, Switzerland).

Microbiological methods

The strains of S. aureus were confirmed as tube coagulase-positive, catalase-positive, Gram-positive cocci.

To test for methicillin resistance, strains and appropriate controls were streaked on to NA, and then paper strips containing 25 jug methicillin (M ast Laboratories, Merseyside) were laid on the agar at right angles to the inocula. Plates were read after incubation for 40 h at 30°C.

Minimum inhibitory concentrations (MICs) were determined by incorporating doubling dilutions o f antimicrobial agents into agar. For paldimycin, because of its better stability at pH 6*5, AM was used, while ISA was used for all the other agents. Using a multi-point inoculator (Denley Instruments, Billingshurst, Sussex) all plates

Activity of five new antibiotics against MGRSA 519

were inoculated within 2 h of pouring. The MIC was the lowest concentration of antimicrobial agent inhibiting visible growth after 18 h incubation at 37°C.

Minimum bactericidal concentrations (MBCs) were measured by inoculating plates with 105 cfu, incubating these for periods ranging from 6 to 40 h at 37°C, and replicating on to ISA using velvet pads (Elek & Hilson, 1954). MBCs were the minimum concentration of antimicrobial agent killing at least 99*9% of the original inoculum.

S. aureus NCTC 6571 was used as a control throughout. Each determination was reproducible on at least two separate occasions.

Timed kill curves were performed on a selection of strains. The organisms were incubated overnight in Iso-Sensitest Broth (Oxoid) to produce a growth of 108-109 cfu/ml. Erlenmeyer flasks containing 100 ml sterile Iso-Sensitest Broth were inoculated with 1 ml of overnight culture and antibiotic was added to a concentration of 4, 16 or 64 mg/1. The flasks were placed in an orbital incubator (Gallenkamp) and shaken at 120 rpm for 36 h at 37°C. A flask free of all antibiotics was used to produce a control growth curve. Quantitative counts at 0, 3, 6, 20, 22, 24, 26 and 36 h of exposure to antibiotic were performed on ISA plates which were incubated for 24 h at 37°C prior to counting.

Results

All the strains were resistant to gentamicin (MICs > 1 6 mg/1) and methicillin.The MICs of teicoplanin, ramoplanin, DuP 721 and DuP 105 are compared with

vancomycin in Table I. All the agents had narrow ranges of activity, except paldimycin where MICs ranged from 0*12-2*0 mg/1. Both paldimycin and ramoplanin were highly active, with geometric mean MICs of 0*4 and 0*75 mg/1 respectively. Teicoplanin was twice as active as vancomycin and DuP 721. Comparing the geometric means for the agents no more than a doubling of MIC was seen on increasing the inoculum 103 to 105 cfu.

Table II shows the MBCs of the agents tested against all the strains when replica- plating was performed after 22 h exposure to antibiotic. Ramoplanin was rapidly and uniformly bactericidal at a concentration of 1*0 mg/1. Vancomycin and paldimycin displayed irregular bactericidal activity. Their MBCs for more than half the strains were

Table I. Inhibitory activity of vancomycin, teicoplanin, ramoplanin, paldimycin, D u P 721 andD u P 105 against M G R S A

M I C (mg/1)Inoculum 103 cfu Inoculum 105 cfu

geometric geometricAntimicrobial agent range m i c 50 m i c 90 mean range m i c 50 MIC90 mean

Vancomycin 0*5-20 0*8 1*5 1*2 1 0-4*0 1*5 2*2 2*0Teicoplanin 0*25-1*0 0*44 0*85 0*56 0*5-40 0*8 1*2 1*0Ramoplanin 0*12-1*0 0*3 0*46 0*38 0*5-20 0*5 1*0 0*75Paldimycin 0*06-2*0 0-16 0*3 0*24 0*12-2*0 0*17 0*5 0*4D u P 721 0*25-1*0 0*7 10 093 1 *0-4*0 1*6 3*0 2*0D u P 105 2*0-80 3*0 6*0 4*6 2*0-8 *0 5*2 7*4 6*8

DZu jr. a. l. iviapie ei at.

Table II. Bactericidal activity of vancomycin, teicoplanin, ramoplanin, paldimycin, D u P 721( and D u P 105 against

M G R S A

Antimicrobial agent rangemg/l

m b c 50 m b c 90Vancomycin < 4 - > 3 2 <4 >32Teicoplanin < 2- > 32 12 >32Ramoplanin 10 10 10Paldimycin < 2 - > 6 4 <2 >6 4D u P 721 >128 >128 >128D u p 105 >128 >128 >128

4 and 2 mg/1 respectively, while some strains were not killed at concentrations of >32 mg/1. Teicoplanin under these conditions was less bactericidal than either vancomycin or paldimycin. These unexpected observations led us to investigate further the bactericidal activity of these antibiotics. Twenty of the previously tested organisms were randomly selected and MBCs were determined after 20, 22, 24, 26 and 40 h exposure to antibiotic. The results are summarised in Table III. Vancomycin and teicoplanin showed MBC90s of 13 and 19 mg/1 at 24 h but 2*5 and 4 mg/1 at 26 h. For 20 strains of MSSA, at 24 h, MBC90s of 2-0 and 16 mg/1 were obtained for vancomycin and teicoplanin respectively. The killing curves (Figure 1) also show that vancomycin has a slightly more rapid bactericidal effect than teicoplanin against MGRSA and MSSA. For paldimycin, MBCs were virtually the same at 6, 24 and 36 h when AM was used. In ISA, or NA, paldimycin was not bactericidal to any of the strains. The influence on the activity observed for paldimycin of medium, temperature and length of incubation was investigated (Table IV). When AM (pH 6*5) was used, the same MIC was observed irrespective of temperature or length of incubation. Much higher MICs were observed when paldimycin was incorporated into NA (pH 7*4) and ISA (pH 7-4). MICs and MBCs in NA and ISA were greatly effected by the temperature and length of incubation.

Table III. MICs and M B C s of vancomycin and teicoplanin at different times against 20 strainsof M G R S A

Time of exposure to M I C (mg/1) M B C (mg/1)antimicrobial agent Antimicrobial Number of strains indicated in appropriate row

(h) agent 0-5 1 0 2 0 4 0 0-5 1 0 2 0 4 0 80 16 32 64 128 >128

20 vancomycinteicoplanin 13

157

5 1 113 3

9 8 3

22 vancomycin 15 5 4 5 4 1 1 1 4teicoplanin 13 7 2 1 1 3 10 3

24 vancomycin 15 5 13 1 6teicoplanin 13 7 10 2 2 3 3

26 vancomycin 15 5 2 15 3teicoplanin 13 7 ' 7 3 3 5 2

40 vancomycinteicoplanin 13

157

510

109

101

Logi

c c

fu/m

lActivity of five new antibiotics against MGRSA 521

Es3UoO'o_ l

4020 22 24 26

T i m e ( h )

Figure 1. Typical killing curves of vancomycin (V) and teicoplanin (T) against MGRSA (------------------ ) andMSSA (-----------). The concentrations of antibiotic used were 4 mg/1 (a), 16 mg/1 (b) and 64 mg/1 (c).Control growth curves (no antibiotic added): O, MSSA; X, MGRSA.

522 P. A. C. Maple et al.

Table IV. Influence of medium, temperature and length of incubation on the activity ofpaldimycin against 20 strains of M G R S A

M e d i u mTemperature of incubation (°C)

Time of incubation (h)

Inoculum 105 cfu (mg/1) Range M I C 50 M I C 90

A M 2a 37°C 18 0-12-2*0 0*32 1*0N A b 37°C 18 2*0-32*0 11-0 22*0A M 2 37°C 36 0*12-2*0 0*32 1*0N A 37°C 36 >64*0 >64*0 >64*0A M 2 30°C 18 0*12-4*0 0*35 1*0N A 30°C 18 0*5-20 0*7 1*5A M 2 30°C 36 0*12-4*0 0*35 1*0N A 30°C 36 1*0-64*0 10*0 27*0

“Oxoid antibiotic medium N o. 2, pH 6*5. bOxoid nutrient agar, pH 7-4.

Discussion

The MICs reported here (Table I) for vancomycin and teicoplanin resemble those found by previous workers for S. aureus (W atanakunakorn, 1984; Greenwood, 1988). Vancomycin was slightly less active than teicoplanin, and for both antibiotics an insignificant inoculum effect was found. Vancomycin and teicoplanin are held to be bactericidal against the various types of staphylococci (Geraci, 1977; Williams & Gruneberg, 1984).

Our initial findings (Table II) for vancomycin and teicoplanin of variable bactericidal activity after 22 h exposure to antibiotics, but virtually uniform bactericidal activity after 26 h, led us to investigate the effect of time of replica-plating on the MBCs observed (Table III). We also timed bactericidal activity in broth (Figure 1), and investigated the bactericidal activity of the antibiotics on MSSA by both methods. Replica-plating and broth time-kill studies showed vancomycin and teicoplanin to be bactericidal to MSSA more rapidly than the MGRSA, a phenomenon also seen by Neu & Labthavikul (1983) with teicoplanin. Against both MGRSA and MSSA, vancomycin was more rapidly bactericidal than teicoplanin, which mirrors results obtained in serum (Lagast, Dodian & Klastersky, 1986).

Foldes et al. (1983) and Traub, Spohr & Bauer (1984) have reported variable MBCs of vancomycin for MGRSA. The strains used by the latter were also used in this study. Similarly, W atanakunakorn (1978) and Norden & Keleti (1981) have reported high MBCs of vancomycin for MSSA. With our 22 h readings some of the MGRSA could have been considered ‘tolerant’ (M BC: MIC ^ 32) a finding which may predict sub- optimal clinical response in some infections (Sorell et al., 1982), although this is disputed (Kaye, 1980). However, tolerance was not found at 26 h and this calls into question the common practice of determining MBCs after ‘overnight’ incubation, or after 24 h. We feel that recognized laboratory guidelines, such as those proposed in the USA (NCCLS, 1987) would prove useful in this area of work.

Ramoplanin was rapidly bactericidal, uniformly killing all strains at a concentration of 1 *0 mg/1, within 6 h. This agent should be further studied as a systemic agent, especially for endocarditis, or, if its pharmacological properties prevent such use, topically for eradication of skin and nasal carriage of MGRSA.

Paldimycins, like the corresponding paulomycins, are sensitive to heat and acidic or alkaline environments (Argoudelis et al., 19876). Rolston et a l, (1987) also found the activity of paldimycin to be medium dependent. There was significant loss of activity when paldimycin was incorporated in NA (pH 7-4), influenced both by the nature of the medium, and temperature. We have found paldimycin to be stable in AM (pH 6-8) over 36 h, irrespective of temperature (Table IV).

DuP 721 and DuP 105 are synthetic agents containing a novel oxazolidinone functional grouping. Like other workers, we havfc found DuP 721 to be more active than DuP 105 against S. aureus (Neu et al., 1988), and that both compounds are bacteriostatic (Daly et al., 1988). 1

In conclusion, all the antimicrobial agents except DuP 105, showed quantitatively superior or similar in-vitro activity to vancomycin in terms of MIC. Further work is required with ramoplanin, DuP 721 and paldimycin to establish their pharmacokinetic profiles and in-vivo efficacy. Paldimycin augments serum killing of S. aureus (Cialdella, Ulrich & Marshall, 1988). However, until detailed published studies correlating its activity in vitro with that in vivo are available, a state of confusion over conditions for susceptibility testing will exist.

We believe ramoplanin to be a most promising antibiotic, on account of its rapid, uniform bactericidal activity against MGRSA, and also because of its reported good activity against streptococci, especially faecal types (Pallanza et al., 1984). Teicoplanin is undergoing clinical trials now, and should it have similar clinical efficacy to vancomycin, offers the prospect of more easily administered and better tolerated treatment.

Finally, our MBC studies showed teicoplanin to be less rapidly bactericidal than vancomycin for MGRSA and MSSA. Whether this observation is clinically relevant is debatable, especially when considering the influence of technical variableson the observation of increased MBC (Pelletier, 1984).

Acknowledgement

This work was sponsored entirely by grants from The Special Trustees of The Royal Free Hospital and from The Leverhulme Trust. We are grateful to the following for supplying strains of S. aureus: C. A. Bartzokas, J. Cooper, G. W. Smith, E. C.Moorhouse, C. T. Keane, J. F. Acar, E. Yourassowsky, W. H. Traub, K. H. Spitzy,F. H. Kayser, A. Torres Pereira, H. Giamarellou, G. G. Grassi, O. Ang, P. Y. Chau, S. Dixson, K. Ubukata, C. W atanakunakorn, C. Linnemann, C. E. O. Pires de Campos, A. A. Forder and D. Hanslo.

References

Argoudelis, A. D ., Baczynskyj, L., Buege, J. A., Marshall, V. P., Mizak, S. A. & Wiley, P. F. (1987a). Paulomycin — related antibiotics: paldimycins and antibiotics 273a2. Isolation and characterisation. Journal o f Antibiotics 40, 408-18.

Argoudelis, A. D., Baczynskyj, L., Mizak, S. A., Shilliday, F. B., Spinelli, P. A. & DeZwaan, J. (19876). Paldimycins A and B and antibiotics 273a2a and 273a2/!. Synthesis and characterisation. Journal o f Antibiotics 40, 419-36.

Cialdella, J. I., Ulrich, R. G. & Marshall, V. P. (1988). Augmentation o f serum bactericidal activity by paldimycin. Journal o f Antibiotics 41, 660-66.

Daly, S. J., Eliopoulos, G. M., Reiszner, E. & Moellering, R. C. (1988). Activity and mechanism

524 P. A. C. Maple et al.

of action of D u P 105 and D u P 721, new oxazolidinone compounds. Journal of Antimicrobial Chemotherapy 21, 721-30.

Elek, S. D. & Hilson, G. R. F. (1954). Combined agar diffusion and replica plating techniques in the study of antibacterial substances. Journal o f Clinical Pathology 7, 37-44.

Foldes, M., Munro, R., Sorrell, T. C., Shanker, S. & Toohey, M. (1983). In-vitro effects of vancomycin, rifampicin, and fusidie acid, alone and in combination, against methicillin- resistant Staphylococcus aureus. Journal o f Antimicrobial Chemotherapy 11, 21-6.

Geraci, J. E. (1977). Vancomycin. Mayo Clinic Proceedings 52, 631-64.Greenwood, D. (1988). Microbiological properties of teicoplanin. Journal o f Antimicrobial

Chemotherapy 21, Suppl. A, 1-13.Kaye, D. (1980). The clinical significance of tolerance of Staphylococcus aureus. Annals of

Internal Medicine 93, 924-96.Kucers, A. (1984). Vancomycin. Journal o f Antimicrobial Chemotherapy 14, 564— 7.Lagast H., Dodian, P. & Klastersky, J. (1986). Comparison of pharmacokinetics and

bactericidal activity of teicoplanin and vancomycin. Journal o f Antimicrobial Chemotherapy 18, 513-20.

Leading article. (1981). Of gentamicin and staphylococci. Lancet ii, 127-8.National Committee for Clinical Laboratory Standards (1987). Methods for determining

bactericidal activity of antimicrobial agents. Proposed Guideline M26-P. NCCLS, Villanova, PA.

Neu, H. C. & Labthavikul, P. (1983). In vitro activity of teichomycin compared with those of other antibiotics. Antimicrobial Agents and Chemotherapy 24, 425-8.

Neu, H. C. & Neu, N. M. (1986). In vitro activity of A16686, a new glycopeptide. Chemotherapy 32, 453-457.

Neu, H. C, Novelli, A., Saha, G. & Chin, N.-X. (1988). In vitro activities of two oxazolidinone antimicrobial agents, D u P 721 and D u P 105. Antimicrobial Agents and Chemotherapy 32, 580-3.

Norden, C. W., & Keleti, E. (1981). Antibiotic tolerance in strains of Staphylococcus aureus. Journal o f Antimicrobial Chemotherapy 7, 599-605.

Pallanza, R., Berti, M., Scotti, R., Randisi, E. & Arioli, V. (1984). A-16686, a new antibiotic from Actinoplanes. II Biological properties. Journal o f Antibiotics 37, 318-24.

Pelletier, L. L. (1984). Lack of reproducibility of macrodilution M B C ’s for Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 26, 815-8.

Pfaller, M. A., Bale, M. & Barrett, M. (1987). In-vitro activity of paldimycin against methicillin- resistant and susceptible isolates of Staphylococcus aureus and S. epidermidis. Journal o f Antimicrobial Chemotherapy 20, 286-8.

Porthouse, A., Brown, D. F. J., Graeme Smith, R. & Rogers, T. (1976). Gentamicin resistance in Staphylococcus aureus. Lancet i, 20-1.

Rolston, K. V. I., LeBlanc, B., Ho, D. H. & Bodey, G. P. (1987). In vitro activity of paldimycin (U-70138F) against Gram-positive bacteria isolated from patients with cancer. Antimicrobial Agents and Chemotherapy 31, 650-2.

Shanson, D. C., Kensit, J. G. & Duke, R (1976). Outbreak of hospital infection with a strain of Staphylococcus aureus resistant to gentamicin and methicillin. Lancet ii, 1347-8.

Slee, A. M., Wuonola, M. A., McRipley, R. J., Zajac, I., Zawada, M. J., Bartholomew, P. T. et al. (1987). OxazPlidinones, a new class of synthetic antibacterial agents: in vitro and in vivo activities of D u P 105 and D u P 721. Antimicrobial Agents and Chemotherapy 31, 1791-7.

Sorrell, T. C., Packham, D. R., Shanker, S., Foldes, M. & Munro, R. (1982). Vancomycin therapy for methicillin-resistant Staphylococcus aureus. Annals o f Internal Medicine 97, 344-50.

Soussy, C. J., Bouanchaud, D. H., Fouace, J., Dublanchet, A. & Duval, J. (1975). A gentamicin resistance plasmid in Staphylococcus aureus. Annales de Microbiologie 126B, 91-4.

Soussy, C. J., Dublanchet, A., Cormier, M., Bismuth, R., Nizon, F., Chardon, F. et al. (1976). Nouvelles resistances plasmidiques de Staphylococcus aureus aux aminosides (gentamicine, tobramycine, amikacine). Nouvelle Presse Medicale 5, 2599-602.

Stille, W., Sietzen, W., Dieterich, H.-A. & Fell, J. J. (1988). Clinical efficacy and safety of teicoplanin. Journal o f Antimicrobial Chemotherapy 21, Suppl. A, 69-79.

Activity ot live new antibiotics against MGK5A 525

Thompson, R. L. & Wenzel, R. P. (1982). International recognition of methicillin-resistant strains of Staphylococcus aureus. Annals o f Internal Medicine 97, 925-6.

Townsend, D. E., Ashdown, N., Bolton, S., Bradley, J., Duckworth, G., Moorhouse, E. C. et al. (1987). The international spread of methicillin resistant Staphylococcus aureus. Journal o f Hospital Infection 9, 60-71.

Traub, W. H., Spohr, M. & Bauer, D. (1984). Gentamicin and methicillin resistant, clinical isolates of Staphylococcus aureus: comparative in vitro and in vivo efficacy of alternative antimicrobial drugs. Chemotherapy 30, 102-12.

Watanakunakorn, C. (1978). Antibiotic-tolerant Staphylococcus aureus. Journal o f Antimicrobial Chemotherapy 4, 561-8.

Watanakunakorn, C. (1982). Treatment of infections due to methicillin-resistant Staphylococcus aureus. Annals o f Internal Medicine 97, 370-8.

Watanakunakorn, C. (1984). Mode of action and in-vitro activity of vancomycin. Journal o f Antimicrobial Chemotherapy 14, Suppl. D, 7-18.

Williams, A. H. & Gruneberg, R. N. (1984). Teicoplanin. Journal o f Antimicrobial Chemotherapy 14, 441-8.

{Received 11 August 1988; revised version accepted 20 December 1988)

622 Eur. J. Clin. Microbiol. Infect. Dis.

more, for detection o f catheter-related bacteremia, this combination had 100% sensitivity and a positive predictive value similar to the actual rate o f catheter- related bacteremia.

References

1. Vanherweghem, J. L., Cabolet, P., Dhaene, M., Gold­man, M., Stolear, J. C., Sabot, J. P., Waterlot, Y., Marchal,M.: Complications related to subclavian catheters for hemodialysis. American Journal o f Nephrology 1986, 6: 3 39 -345 .

2. Qeri, J., Corrado, M. L., Seligman, S. J.: Quantitative culture o f intravenous catheters and other intravascular inserts. Journal of Infectious Diseases 1980, 141: 781— 786.

3. Maki, D. G., Weise, C. E., Sarafin, H. W.: A semiquan- titative culture method for identifying intravenous- catheter related infection. New England Journal of Medicine 1977, 296: 1305-1309 .

4. Liffares, J., Sitges-Serra, A., Garau, J., Perez, J. L., Martin, R.: Pathogenesis of catheter sepsis: a prospec­tive study with quantitative and semiquantitative cultures of catheter hub and segments. Journal of Clinical Microbiology 1985, 21: 357—360.

5. Mausner, J. S., Bahn, A. K.: Epidemiology. Saunders, Philadelphia, 1974, p. 243—252.

6. Norwood, M. A. G., Civetta, J. M.: Evaluating sepsis in critically ill patients. Chest 1987, 92: 137—144.

7. Collignon, P. J., Soni, P., Pearson, I. Y., Woods, W. P., Munro, R., Sorrell, T. C.: Is semiquantitative culture of central vein catheter tips useful in the diagnosis of catheter-associated bacteremia? Journal o f Clinical Microbiology 1986, 24: 5 3 2 -5 3 5 .

8. Bozzetti, F., Terno, G., Camerini, E., Baticci, F., Scarpa, D., Pupa, A.: Pathogenesis and predictability of central venous catheter sepsis. Surgery 1982, 91: 3 8 3 -3 8 9 .

9. Moyer, M. A., Edwards, L. D., Farley, L.: Comparative culture methods on 101 intravenous catheters: routine, semiquantitative and blood cultures. Archives of Internal Medicine 1983, 143: 6 6 -6 9 .

10. Sitges-Serra, A., Puig, P., Jaurrieta, E., Garau, J., Alastrue, A., Sitges-Creus, A.: Catheter sepsis due to Staphylo­coccus epidermidis during parenteral nutrition. Surge­ry, Gynecology and Obstetrics 1980, 151: 481—483.

Ciprofloxacin Resistance in Methicillin- and Gentamicin-Resistant Staphylococcus aureus

P. Maple*, J. Hamilton-Miller, W. Brumfitt

A total of 112 Staphylococcus aureus strains resistant to methicillin and gentamicin were collected from 31 centres in 22 countries worldwide. Many strains

Department o f Medical Microbiology, The Royal Free Hospital School o f Medicine, Pond Street, Hampstead, London NW3 2QG, UK.

were multi-resistant. In tests to determine the sus­ceptibility of the organisms to ciprofloxacin 16 strains (14.3 %), originating from France, the FRG, Israel and Italy, were shown to be resistant to this agent. To limit ciprofloxacin resistance, a reappraisal is necessary of fluoroquinolone usage against methi­cillin- and gentamicin-resistant Staphylococcus aureus.

In recent years, there have been a growing number o f reports o f infection or colonization due to methicillin - and gentamicin-resistant Staphylococcus aureus (MGRSA). As many o f these strains are also resistant to numerous other^ antibiotics besides the beta- lactams and gentamicin (1), in certain instances the choice o f effective antistaphylococcal chemotherapy may be severely restricted. Ciprofloxacin, on account o f its structural novelty and reported in vitro activity against MRSA (2), has found favour with some clinicians for treatment o f infection or colonization with these organisms.

The first specific report o f ciprofloxacin resistant MRSA appeared in October 1988 (3). Subsequent accounts show that resistance to fluoroquinolones in these organisms can become common (4 , 5). In order to determine how widespread this phenomenon is, we tested a total o f 112 strains o f MGRSA from 31 centres in 22 countries for susceptibility to cipro­floxacin.

Materials and M ethods. Strains o f MGRSA have been collected from 31 centres worldwide over the last three years. For this study, a total o f 112 strains were used. No more than four and no fewer than two strains from any one centre were represented in the study collection. For each centre, only strains with different phage-types or biotypes were used. The sources o f the strains were: Australia (S. Dixson), Austria (K. H. Spitzy), Belgium (E. Yourassowsky), Brazil (C. E. O. Pires de Campos), UK (J. Cooper, W. Brumfitt, C. A. Bartzokas), Federal Republic o f Germany (W. H. Traub), France (J. F. Acar, E. Bergogne Berezin), German Democratic Republic (C. Hohne), Greece (H. Giamarellou), Hong Kong (P. Y. Chau), Republic o f Ireland (C. T. Keane, E. C. Moorhouse), Israel (D. Merzbach), Italy (G .G . Grassi), Japan (K. Ubukata), Kuwait (G. F.Araj), Poland (J. Borowski), Portugal (A. Torres Pereira), South Africa (A. A. Forder, D. Hanslo), Spain (E. P. Trallero), Switzerland (F. H. Kayser), Turkey (O. Ang), USA (R. C. Moellering, R. P. Wenzel, C.C. Linnemann, H. C. Neu, C. Watanakunakorn).

All the strains were coagulase-positive (tube test), catalase-positive, gram-positive cocci which grew up to the edge o f paper strips containing 25 p% o f methicillin (Mast laboratories, UK) after incubation for 40 h at 30 °C on nutrient agar. All strains were gentamicin-resistant (MIC > 1 6 mg/1).

because the colonization was secondary to a hema­togenous seeding. Catheters remained in situ for a mean of 19.7 days (range 5 to 69 days). Twenty- six of the 50 catheters were significantly colonized and in eight cases (31%) the patient developed a catheter-related bacteremia. The distribution of the microorganisms can be seen in Table 1. The skin smear at the catheter entry site and hub cultures were positive in 31 and 14 cases respectively.The sensitivity, specificity and predictive values of the quantitative and semiquantitative cultures for the individual catheter segments and both segments together can be seen in Table 2. The best results were obtained with the combination of semiquantitative and quantitative cultures of the intradermal catheter segment.Norwood et al. (6) concluded that semiquantitative culture of the intradermal portion of the catheter is the most appropriate method for detection of catheter colonization or catheter-related bacteremia whether

Table 1: Distribution o f microorganisms isolated in cases of catheter colonization and catheter-related bacteremia.

MicroorganismCatheter

colonization® (n = 26)

Catheter-related bacteremia

(n = 8)

Coagulase-negativestaphylococci

22 (6) 3

Staphylococcus aureus 4 (2 ) 3Corynebacterium spp. 4 (4 ) 0Pseudomonas aeruginosa 3 (2 ) 0Streptococcus faecalis 2 (2 ) 1Proteus vulgaris 1 (1 ) 1Alcaligenes sp. 1 (1 ) 0

aNumber o f organisms isolated in polymicrobial cultures in parenthesis.

for investigatory or clinical purposes. Other authors (7, 8) believe that routine cultures of the intra­dermal segment may be unnecessary, maintaining that the catheter tip (the intravascular segment) is the most important segment which should be cultured in order to diagnose catheter-related bacteremia. Maki et al. (3) found that in long catheters, the intra­dermal segment showed heavier growth than the catheter tip and recommended semiquantitative culture of both catheter segments. However, in another study (9) using a larger number of catheters no significant differences could be found between the semiquantitative cultures of the intradermal and intravascular catheter segments.Culture of the external surface of catheters is not useful for detecting colonising microorganisms when the source is not the skin at the catheter entry site but the catheter hubs (10). Consequently, Linares et al. (4) suggest semiquantitative culture of the external surface together with quantitative culture of the intraluminal surface of the catheter tip (or the intravascular catheter segment) as the best method for detecting catheter colonization and catheter- related bacteremia.Theoretically, at least, the best method should combine high sensitivity and specificity in detec­tion of catheter colonization and high sensitivity in detection of catheter-related bacteremia. Further­more, the positive predictive value for catheter- related bacteremia should be similar to the rate of cases of catheter colonization which in fact develop catheter-related bacteremia.In summary, we found that in jugular or subclavian catheters inserted for hemodialysis, a combination of semiquantitative culture of the external surface and quantitative culture of the intraluminal surface of the intradermal catheter segment provided the best means of detecting catheter colonization. Further-

Table 2: Results o f culture techniques for detection o f catheter colonization and catheter-related bacteremia in 50 hemo­dialysis catheters.

Sensitivity (%) Specificity (%) Positive predictive Negative predictiveCulture technique Colonization Bacteremia Colonization Bacteremia ™lue «*) for value (%) for

bacteremia bacteremia

SemiquantitativeIntravascular segment 78 75 96 71 33 93Intradermal segment 78 80 94 63 26 95Both catheter parts 94 80 93 50 22 93

QuantitativeIntravascular segment 43 62 96 88 50 97Intradermal segment 46 80 100 88 54 95Both catheter parts 65 86 50 78 40 97

Both techniquesIntravascular segment 83 87 93 64 31 96Intradermal segment 89 100 94 60 29 100

MICs of gentamicin and ciprofloxacin were deter­mined by the agar dilution method. Doubling dilutions of antibiotics were incorporated into molten Iso-Sensitest agar (Oxoid, UK) held at 50 °C. Inocula of approximately 5 X 105 C F U from 24 h-old peptone water cultures grown at 37 °C were delivered onto the plates using a multi-point inocula- tor (Denley Instruments, UK). MICs were read after incubation for 24 h at 37 °C. MBCs were determined after exposure for 24 h to antibiotic at 37 °C by replica-plating onto fresh Iso-Sensitest agar plates which were incubated for 24 h at 37 °C. MBCs were defined as the minimum concentration of antibiotic killing at least 99.9 % of the original inoculum.Phage typing was performed with the International Set at routine test dilution (RTD) and 100 x RTD. Biotyping was performed by a modification of the method of Putland and Guinness (6), which detects haemolysis (sheep blood agar), lipolysis (egg yolk agar) and pigmentation (milk agar). Antibiotic sensitivity profiles were also determined by means of the disc method using DST Agar (Oxoid, UK) supple­mented with 7 % lysed horse blood. The discs con­tained as follows (content in parenthesis): netilmicin (10 jtrg), amikacin (10 Mg), neomycin (10 Mg), tetra­cycline (10 Mg), clindamycin (2 Mg), chloramphenicol (10 Mg), trimethoprim (1.25 Mg) and rifampicin (5 Mg)- An inoculum of approximately 5 X 10s C F U per plate was used. Sensitivity results were read after incubation for 20 h at 37 °C. Strains were classed as resistant if the diameter of the inhibition zone (total diameter minus disc diameter) was < 6 mm, and sensitive if the diameter was > 6 mm.

Results and Discussion. The following MIC break­points of ciprofloxacin were adopted after con­sultation with Bayer, UK: sensitivity < 1 mg/1, inter­mediate sensitivity > 1 mg/1 but < 4 mg/1, and resistance > 4 mg/1. Applying these criteria 94 strains were classified as sensitive, two as intermediate and 16 as resistant (Table 1). The ciprofloxacin-resistant strains originated from the FRG. France, Israel and Italy. Ciprofloxacin MICs of > 16 mg/1 and MBCs of > 32 mg/1 were found for M G R S A from France and Israel.Until now, fluoroquinolone (ciprofloxacin or pefloxa- cin) resistant strains of M R S A have been reported from the U S A (3,5), France (4) and the U K (7).

All these strains were found in hospitals where either ciprofloxacin or pefloxacin had been used to treat infections or colonization. As we had made no attempt to collect M G R S A specifically resistant to ciprofloxacin for this study, the high incidence of ciprofloxacin resistance is cause for concern.Ciprofloxacin has been reported to have good in vitro activity against MRSA, and has been suggested as an important new alternative for treatment of M R S A colonization (8). In common with other investigators (9), we found ciprofloxacin to be bactericidal: many of the sensitive strains tested were killed by a concentration of 1 mg/1 (Table 1).The finding of an incidence of 14.3 % ciprofloxacin resistance in this collection of organisms contradicts earlier claims that such resistance would rarely be encountered (10). Our ciprofloxacin-resistant M G R S A strains were also resistant to enoxacin, nor­floxacin, ofloxacin and pefloxacin. Thus we would urge caution before fluoroquinolones are used for the management of patients with MGRSA. It would be unfortunate if ciprofloxacin-resistant strains were to emerge due to inappropriate use of fluoroquino­lones, especially as the antimicrobial agents available for treatment of infections caused by M G R S A are already so limited. We advocate that ciprofloxacin (or any other fluoroquinolone) should not be used for the eradication of carriage. Furthermore, if quinolones are required for treatment, then their use in appropriate combination should be seriously considered in order to reduce the chance of selecting for resistance.

References

1. Maple, P. A . C., Hamilton-Miller, J. M. T., Brumfitt, W.:World-wide antibiotic resistance in methicillin-resistant Staphylococcus aureus. Lancet 1989, i: 53 7 —540.

2. Smith, S. M., Eng, R. H. K.: Activity o f ciprofloxacin against methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 1985, 27: 6 8 8 -6 9 1 .

3. Isaacs, R. D ., Kunke, P. J., Cohen, R. L., Smith, J. W.:Ciprofloxacin resistance in epidemic methicillin-resistant Staphylococcus aureus. Lancet 1988, ii: 843.

4. Jean-Pierre, H., Boyer, G., Darbas, H.: Evolution de la resistance des Staphylococcus aureus a la pefloxacin. Etude portant sur 782 souches isole'es en 1985 et 1986. Pathologie Biologie 1988, 36: 9 5 6 -9 5 8 .

Table 1: MICs and MBCs o f ciprofloxacin for 112 strains o f methicillin- and gentamicin-resistant Staphylococcus aureus col­lected worldwide.

Concentration o f ciprofloxacin (mg/1)

< 0 .2 5 0.5 1 2 4 8 16 32 > 3 2

No. o f strains inhibited 10 52 32 2 9 0 3 4 0No. of strains killed 2 5 70 15 10 3 0 0 7

5. Schaefler, S.: Methicillin-resistant strains o f Staphylo­coccus aureus resistant to quinolones. Antimicrobial Agents and Chemotherapy 1989, 27: 335—336.

6. Putland, R. A ., Guinness, M. D. G.: Autobac susceptibi­lity testing o f methicillin-resistant Staphylococcus aureus isolated in an Australian hospital. Journal o f Clinical Microbiology 1985, 22: 8 2 2 -8 2 7 .

7. Milne, F. M., Faiers, M. C.: Ciprofloxacin resistance in epidemic methicillin-resistant Staphylococcus aureus. Lancet 1988, ii: 843.

8. Mulligan, M. E., Ruane, P. J., Johnston, L., Wong, P., Wheelock, J. P., MacDonald, K., Reinhardt, J. F., John­son, C. C., Statner, B. B., Blomquist, I., McCarthy, J., O’Brien, W., Gardner, S., Hammer, L., Citron, D. M.: Ciprofloxacin for eradication o f methicillin-resistant Staphylococcus aureus colonization. American Journal o f Medicine 1987, 82, Supplement 4A: 215—219.

9. Smith, S. M., Eng, R. H. K., Berman, E.: The effect of ciprofloxacin on methicillin-resistant Staphylococcus aureus. Journal o f Antimicrobial Chemotherapy 1986, 17: 2 8 7 -2 9 5 .

10. Smith, J. T.: Mode o f action o f the 4-quinolone anti­bacterial agents. In: Ciprofloxacin product monograph. ADIS Press, Auckland, New Zealand, 1986, p. 1 9 -3 1 .

In Vitro Activity of Cefoperazone- Sulbactam Combinations against Cefoperazone-Resistant Clinical Bacterial Isolates

G. M. Eliopoulos1,3*, K. Klimm1,M. J. Ferraro2,3, R.C. Moellering, Jr.1,3*

From July 1987 to January 1988,452 cefoperazone- resistant bacterial isolates were identified among strains subjected to routine susceptibility testing in a clinical microbiology laboratory. The 452 isolates were tested for susceptibility to cefoperazone, sulbac­tam, and a 2:1 combination of these drugs by agar dilution techniques. The greatest benefit of the cefo- perazone-sulbactam combination was noted against Bacteroides spp. and Acinetobacter spp. The com­bination demonstrated clinically significant synergism against approximately 20% of strains of Pseudomonas aeruginosa.

Department o f Medicine, New England Deaconess Hospital, 185 Pilgrim Road, Boston, Massachusetts 02215, USA. Departments o f Medicine and Microbiology, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114, USA.Harvard Medical School, 25 Shattuck Street, Boston, Mas­sachusetts 02115, USA.

The beta-lactamase inhibitors sulbactam, clavulanic acid and tazobactam effectively inhibit a number of commonly encountered beta-lactamases of either plasmid or chromosomal origin (1). Although ex- panded-spectrum cephalosporins such as cefopera­zone are relatively resistant to hydrolysis by many of these enzymes, occasional bacterial isolates de­monstrate substantially greater susceptibility to cefo- perazone-sulbactam combinations than to the cephem alone (2). The purpose of this study was to examine the activity of cefoperazone-sulbactam combinations against the cefoperazone-resistant bacteria encoun­tered in a busy clinical microbiology laboratory over a period of seven consecutive months.

Materials and Methods. During the period of July 1987 through January 1988, all routine clinical iso­lates subjected to antimicrobial susceptibility test­ing at the Massachusetts General Hospital, Boston, Massachusetts, were examined for susceptibility to cefoperazone. Aerobic and facultative bacteria re­sistant to cefoperazone as tested by the disk diffu­sion method (zone diameter < 15 m m ) and anaerobic isolates resistant as tested by the disk elution method (growth in 30Mg/ml) were referred for subsequent testing by agar dilution reference methods (3). Sus­ceptibility to cefoperazone and sulbactam (Pfizer, USA), alone and in a 2:1 combination, was determined (2). Antibiotics were incorporated into Mueller- Hinton agar (BBL Microbiology Systems, USA) for testing aerobic organisms. Inocula of approximately 104 CFU/spot were applied to plates using a multi­prong inoculating device. Anaerobes were tested on Wilkins-Chalgren agar (Oxoid, UK) using inocula of about 105/spot. Plates were incubated in air at room temperature for 18h or in an anaerobic atmosphere (GasPak, BBL) for 48h at 35 °C.

Results and Discussion. During the study period, 452 organisms were referred for further testing based on resistance to cefoperazone as determined by aforementioned criteria (Table 1). Cefoperazone- resistant organisms accounted for 6 % of routine clinical isolates of gram-negative bacteria screened during a representative three-month interval. Rates of resistance to cefoperazone varied widely between species, ranging from 0.2% of Escherichia coli to 42% of Acinetobacter anitratus. Resistance to cefo­perazone based on disk diffusion testing predicted resistance by dilution testing (MIC > 64Mg/ml) (2) in 97.6% of aerobic isolates. Of the 369 facultative and aerobic organisms determined to be cefopera­zone-resistant by disk diffusion, only eight were in­hibited by a cefoperazone concentration of 32Mg/ml, while one was inhibited at 16 pglmt. Among the Bacteroides spp., 18 of 83 (21.7%) judged to be re­sistant by the elution method demonstrated inter­mediate levels of susceptibility (MIC = 32 /Ltg/ml) by