FEMS Microbiology Letters 234 (2004) 275–280

www.fems-microbiology.org

Identification and genetic characterisation of orthopaedicStaphylococcus isolates collected in Italy by

automated EcoRI ribotyping

Agnese Andollina a, Alessandra De Cesare b, Giorgia Bertoni a, Liliana Modelli a,Gerardo Manfreda b,*

a Research Laboratory of Clinical Pathology and Microbiology, Rizzoli Orthopaedic Institute, Bologna, Italyb Department of Food Science, Alma Mater Studiorum, University of Bologna, Via S. Giacomo 9, 40126 Bologna, Italy

Received 19 January 2004; received in revised form 9 March 2004; accepted 22 March 2004

First published online 9 April 2004

Abstract

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis,

both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from

surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph� system for 96% of the isolates.

All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson’s index of dis-

crimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among

isolates with the identical ribotyping profile.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Staphylococcus; Automated ribotyping; Identification; Characterisation

1. Introduction

Staphylococcus aureus and the coagulase-negative

staphylococci (CNS) represent the 31.5% and 30.2% ofthe isolates collected from surgical injuries at the

Rizzoli Orthopaedic Institutes (Bologna, Italy) during

the year 2000. In the hospital routine work the staph-

ylococci identification is often not completed by their

genetic characterisation to strain level. In fact, many

characterisation techniques are very laborious and

produce unrepeatable results. However, the isolates

characterisation might be very useful, from an epide-miological point of view, to differentiate sporadic and

clonal strains, to trace their sources and to limit their

diffusion.

* Corresponding author. Tel.: +39-051-2096765/2096766; fax: +39-

051-2096764.

E-mail address: gmanfreda@disa.unibo.it (G. Manfreda).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.03.044

Among the earliest schemes for classification of

staphylococci and micrococci there was that of Baird

Parker, which remained themethod of choice until that of

Kloos and Schleifer, with the identification of several newStaphylococcus species [1]. More recently, several molec-

ular approaches have been proposed for the identification

of Staphylococcal taxa. These approaches fall into two

categories: those based on the detection of species-specific

sequences, that are not present in the chromosome of all

staphylococci, and those based on the detection of se-

quence variations in ubiquitous elements, such as rRNA

and tRNA operons or chaperonin-encoding genes [2].Staphylococcus aureus and CNS epidemiological

studies have been traditionally performed by bacterio-

phage typing [3,4], plasmid pattern analysis [5,6], whole-

cell protein profile analysis [7], immunoblotting profile

analysis [8]. More recently, many genotyping methods

have been applied, such as pulsed-field gel electropho-

resis (PFGE) [9], fluorescent amplified fragment length

. Published by Elsevier B.V. All rights reserved.

276 A. Andollina et al. / FEMS Microbiology Letters 234 (2004) 275–280

polymorphism (FAFLP) [10], polymerase chain reaction

restriction fragment length polymorphism (PCR-RFLP)

[11] and multilocus sequence typing [12]. The major

limitations of most typing methods include a lack of

standardisation, a need for highly skilled technical staff,and significant hands-on time required for performance.

Due to its facility of use and speed of analysis, an au-

tomated genotyping technique, such as that represented

by automated ribotyping, may allow clinical laborato-

ries to apply molecular typing more broadly. Automated

ribotyping, which is possible using the RiboPrinter�

system (DuPont, Qualicon Inc.), is based on the analysis

of the genomic fragments generated by restriction di-gestion of the rRNA operons, highly conserved and

present in several copies on the bacterial chromosome

[13]. The most conserved regions of the operons allow

identification of a bacterium to genus and species level,

whereas variable and flanking regions permit discrimi-

nation between strains [14]. Manual ribotyping has been

described as a useful molecular technique for typing of

staphylococci [15–18]. In this study automated EcoRIribotyping was used to confirm the identification of 102

Staphylococcus isolates previously submitted to API

Staph�. Moreover, the discriminatory power of auto-

mated ribotyping as typing method was calculated and

the genetic relationships among strains were quantified

using cluster analysis.

2. Materials and methods

2.1. Bacterial isolates

A total of 102 Staphylococcus isolates, collected from

surgical injuries during three months (October–Decem-

ber) of the year 2000 at the Rizzoli Orthopaedic Insti-

Table 1

Identification and characterization results

Species Ribotyping

N. isolates N. RGs N. unique

RGs

N. comm

RGs

S. aureus 38 23 19 4

S. epidermidis 28 12 7 5

S. haemolyticus 9 5 4 1

S. hominis 9 3 1 2

S. warneri 5 2 1 1

S. lugdunensis 4 3 2 1

S. capitis 3 3 3 –

S. sciuri 2 2 2 –

S. xylosus 2 2 2 –

S. simulans 1 1 1 –

S. cohnii 1 1 1 –

Total 102 57 43 14

aOne S. haemolyticus identified by ribotyping was biochemical ide S. lugdbOne S. hominis and one S. capitis identified by ribotyping was biochemiccOne S. warneri identified by ribotyping was biochemical identified as S.

tutes (Bologna, Italy), were analysed. All isolates were

collected from patients hosted in ten different ortho-

paedic units (i.e. hospital wards, ambulatories, intensive

care room, transplant room) and were previously iden-

tified by using the API Staph� system (BioMerieuxItalia S.p.a, Rome, Italy) (Table 1). In order to be

subjected to ribotyping, a pure culture of each isolate

was grown on Brain Heart Infusion agar (BD, Becton

Dickinson and Company) at 37 �C for 24 h.

2.2. Automated ribotyping

The pure cultures obtained on BHI agar plates weresubmitted to identification and characterisation on the

RiboPrinter� as recommended in the manufacturer’s

instructions for the Qualicon RiboPrinter� Microbial

Characterisation System (DuPont Qualicon, Wilming-

ton, DE). The DNA was cut with the restriction en-

zyme EcoRI (Qualicon, Inc.) and the fragments were

electrophoretically separated and transferred to a

membrane. A sulfonated DNA probe was then hybri-dised to the genomic DNA on the membrane. The ge-

netic fingerprint was visualised and captured using a

chemioluminescent detection system and a charge-cou-

pled device camera. The computer workstation analy-

sed the images and generated a ribotyping pattern for

each Staphylococcus isolates using proprietary algo-

rithms. The ribotyping patterns were defined by the

position and intensity of the DNA fragments that theyrepresented. The identification of each isolate was ob-

tained when the corresponding pattern matched one of

the pattern of the DuPont Identification library of the

RiboPrinter� with a similarity >0.86. The characteri-

sation consisted of combining profiles within a simi-

larity range equal or larger than 0.93 to form a

dynamic ribogroup (RG) that reflected the genetic re-

API STAPH

on N. isolates N. biotypes N. unique

biotypes

N. common

biotypes

38 6 1 5

30 12 6 6

8a 4 2 2

8b 5 3 2

4c 1 – 1

5 3 2 1

2b 1 – 1

2 1 – 1

2 1 – 1

2 1 – 1

1 1 1 –

102 36 15 21

unensis.

al identified as S. epidermidis.

simulans.

A. Andollina et al. / FEMS Microbiology Letters 234 (2004) 275–280 277

latedness of the isolates [14]. The profiles collected

during this research were labelled with alphanumeric

codes (i.e. 153-42S4), which defined the ribogroup of

the isolate examined.

2.3. Simpson’s index of discrimination (SID)

The suitability of EcoRI ribotyping and API Staph�

for differentiation of Staphylococcus isolates was deter-

mined using the Simpson’s numerical index, as described

by Hunter and Gaston [19]. This index represents the

probability that two randomly chosen isolates would be

distinguished by the technique used. As the numericalindex approaches the maximum value of one (repre-

senting 100% discriminatory ability) the higher the

probability that the technique will be able to discrimi-

nate between two unrelated isolates.

2.4. Cluster analysis

Automated EcoRI ribotyping profiles were importedinto the program named BioNumerics (version 2.0;

Applied Maths) and dendrograms were generated based

on the banding pattern similarities. Unweighted pair

group method with arithmetic averages (UPGMA) and

Pearson correlation coefficient were used.

Fig. 1. Staphylococcus aureus cluster analysis results.

3. Results

All isolates of this study set were successfully ribo-

typed. The ribotyping identification results confirmed

those obtained biochemically for all isolates but four

(Table 1). In particular, all S. aureus, S. epidermidis,

S. lugdunensis, S. sciuri, S. xylosus, S. simulans and

S. cohnii identified by ribotyping were biochemically

confirmed (Table 1). Moreover, one of nine isolatesidentified as S. haemolyticus by ribotyping was bio-

chemically classified as S. lugdunensis, biotype 6716152;

one of nine isolates identified as S. hominis was bio-

chemically classified as S. epidermidis, biotype 6306152;

one of three isolates identified as S. capitis was bio-

chemically classified as S. epidermidis, biotype 6306111;

one of five isolates identified as S. warneri was bio-

chemically classified as S. simulans, biotype 6430113.Among the 57 ribogroups identified, 75% were asso-

ciated with single isolates and labelled as unique,

whereas 25% were shared between two to ten isolates

and labelled as common (Table 1). Both common, as

well as unique ribotyping profiles, were characterised by

8–13 well defined non-overlapping bands, with a mo-

lecular weight ranging from less than one to more than

48 kbp. Among the 36 biotypes identified, 42% wereassociated with single isolates and 58% to two to six

isolates. The SID calculated for S. aureus was 0.94,

whereas the SID for CNS, referring for a group of iso-

lates which comprise several different species, was 0.96.

TheS. aureus ribotyping profileswere clustered in three

main groups (Fig. 1), containing 17, 12 and 9 strains, re-

spectively, with a similarity ranging from 52% to 98%. Asexpected, using the BioNumerics software the majority of

the isolates classified within the same ribogroup clustered

with a similarity equal or larger than 93%. However,

strains A-SR 06 and A-SR 89, as well as A-SR 42 and A-

SR 97, clustering with similarities of 93%, were assigned

to different ribogroups because they have a different

number of bands, resulting in different genetic profiles.

Otherwise, strain A-SR07, with ribogroup 153-42S4,clustered with a similarity of 90%with strainsA-SR71,A-

SR90, A-SR94, A-SR77, A-SR92, A-SR15, A-SR05 be-

longing to the same 153-42S4 ribogroup.

The CNS ribotyping profiles belonging to the same

species generally clustered together (Fig. 2). In particu-

lar, all S. haemolyticus strains were grouped with a

similarity ranging from 68% to 98%. The only exception

was represented by isolate H-SR 60 clustering along

278 A. Andollina et al. / FEMS Microbiology Letters 234 (2004) 275–280

with the S. hominis strains with a similarity ranging from

85% to 90%. The S. lugdunensis isolates were grouped

along with the S. cohnii labeled as CO-SR91 with a

Fig. 2. Coagulase-negative staphylococci cluster analysis results.

similarity ranging from 68% to 95%. The S. xylosus,

capitis and sciuri isolates clustered together, as well as

the S. epidermidis strains. However the isolates belong-

ing to this last species were grouped in two clusters with

a similarity lower than 30%. Finally the S. simulans

isolate clustered alone and did not showed any signifi-

cant similarity to the isolates of the other species. From

the biochemical point of view, strain H-SR60 showed

identical biotype to strains H-SR03, H-SR17 and H-

SR43 showing different ribogroups. Moreover, strains

E-SR04, E-SR112, E-SR18 and E-SR24 shared their

biotypes with strains of the same species classified in a

different cluster.

4. Discussion

The correct identification of Staphylococcus isolates,

as well as their characterisation to strain level, is crucial

to develop efficient epidemiological surveillance pro-

grams and to manage an outbreak investigation, inwhich a cluster of infections due to organisms of the

same species is identified. In particular, the strain typing

provides evidence of biological and genetic relatedness

among isolates, and allows to determine the presence of

clonal spread and sources of infection [20–22].

One of the aims of this research was to evaluate the

possibility to use the RiboPrinter� to confirm the

identification results of 102 Staphylococcus isolates ob-tained using the rapid biochemical test named API

Staph�. The ribotyping identification results confirmed

those obtained biochemically for all isolates but four. In

particular, two isolates biochemically identified as

S. epidermidis were identified as S. hominis and S. capitis

by ribotyping. Moreover, two isolates biochemically

identified as S. lugdunensis and S. simulans, respectively,

were classified as S. haemolyticus and S. warneri usingribotyping. The RiboPrinter� identification process is

based on the analysis of rRNA restriction fragments,

highly conserved within genus and species, whereas

biochemical tests are based on the expression of proteins

and enzymes environmentally conditioned. This differ-

ence might explain the lack of correspondence between

some identification results obtained using the two

methods. Otherwise, it seems significant that the groupof species identified by the two techniques was the same.

For the evaluation of genotyping techniques for bac-

teria characterisation, Struelens [23] proposed strict cri-

teria, such as high typeability, reproducibility, stability

and discriminatory power. All the isolates of this study

were successfully ribotyped (100% typeability) and the

ribotyping discriminatory power, calculated using the

SID, was very high for both S. aureus (0.94) and CNS(0.96). These results might be due to the presence of six

rRNAoperons [24] inStaphylococcus genome. In fact, the

ribotyping discriminatory power generally increases with

A. Andollina et al. / FEMS Microbiology Letters 234 (2004) 275–280 279

the number of rRNA operons, that varies from one, in

Rickettsia prowazekii and Mycoplasma pneumoniae to a

maximum of 15 in Clostridium paradoxum [25].

Due to its complete automation and standardisation,

the stability and reproducibility of the ribotyping patternsis very high [26]. In fact, the pattern of one isolate is always

the same also after one year period. Staphylococcus ri-

botyping usingHidIII and EcoRI, as restriction enzymes,

has been described from other authors [16,27]. They

found that EcoRI produces more bands and ribotypes

than HidIII. The results of this research confirm that

EcoRI is a good enzyme for the discrimination of different

genetic types, within the sameStaphylococcus species, andfor Staphylococcus identification. In fact, the ribogroup

designation obtained for all strains tested was always

linked to the Staphylococcus species.

The similarity among the ribotyping profiles collected

in this research was quantified by cluster analysis using

the BioNumerics software. The reason why some S.

aureus isolates (i.e. A-SR06 and A-SR89, A-SR42 and

A-SR97) showing similarities of 93% were classifiedwithin different ribogroups and isolates with the same

ribogroup (i.e. A-SR07 and A-SR05) clustered with

similarity <93% is related to the different algorithms

used in the RiboPrinter� and BioNumerics software to

perform the similarity analysis.

The S. aureus strains with ribogroup 153-56S4 shared

the same biotype (Table 2), whereas isolates with ribo-

groups 153-42S4, 153-47S3 and 153-51S4 were charac-terised by two biotypes that differed for only one

biochemical test (i.e. b-naphtyl-acid phosphate, N-ace-

tyl-glucosamine and potassium nitrate). The four

S. warneri strains in ribogroup 153-51S6 shared the

same biotype, as did the two S. epidermidis strains in

ribogroup 153-46S8 (Table 2). In contrast, all other

CNS isolates showed different biotypes for strains with

Table 2

Biotypes identified among isolates with the same ribogroup

Species Common RGs (N. isolates) B

S. aureus 153-42S4(8) 6

153-47S3(5) 6

153-51S4(3) 6

153-56S4(3) 6

S. epidermidis 153-46S6(10) 6

153-42S1(3) 6

153-56S6(4) 6

153-89S5(2) 6

153-46S8(2) 6

S. haemolyticus 153-46S7(5) 6

S. hominis 153-46S1(4) 6

153-51S1(4) 6

S. warneri 153-51S6(4) 6

S. lugdunensis 153-47S8(2) 6

a Biotypes 6712151 and 6306152 correspond to Staph lugdunensis and Stap

the same ribogroup. In particular, the biotypes of the

S. haemolyticus strains in ribogroup 153-46S7, the

S. hominis strains in ribogroups 153-46S1 and 153-51S1

as well as the S. lugdunensis strains in ribogroup 153-

47S8 differed by two or more biochemical reactions. Incontract, the differences in the biochemical profiles of

the S. epidermidis strains in ribogroups 153-46S6, 153-

42S1, 153-56S6 and 153-89S5 were limited to single re-

actions, such as production of the enzymes urease or

arginine dihydrolase (ADH) or production of a-naphtyl-acid phosphate (PAL). Finally, the same biotypes (i.e.,

S. aureus 6736153; S. epidermidis 6706112, 6706113,

6702112; S. hominis 6216153, 6212132) were identifiedfor isolates with different ribogroups.

If further studies will confirm that automated EcoRI

ribotyping is suitable to obtain both the identification as

well as the genetic characterisation of Staphylococcus

isolates during the same analysis, clinical laboratories

having to deal with large number of isolates might select

the RiboPrinter� as first identification and typing

method. The automated ribotyping analysis is per-formed in 8 h and the RiboPrinter� can process 32

isolates per day. API Staph� test, PCR-RFLP, PFGE,

AFLP, and DNA sequencing time to results are 1, 1, 3, 2

and 2 days, respectively [28]. The cost of the analysis of

each sample by automated ribotyping (i.e. 51 Euro) is

comparable with that obtained adding the cost of the

manual API Staph� test (i.e. 6.5 Euro for each sample)

to that of mostly used typing methods. It has been es-timated that the PCR-RFLP, PFGE, AFLP, and DNA

sequencing costs are about 11, 38, 20 and 40 Euro per

sample [28]. Moreover the automated ribotyping cost

decrease over the years in relation to the number of

isolates tested resulting lower than that of manual ri-

botyping and PFGE in laboratories analysing more than

2000 isolates per year [29].

iotypes for each ribotype (N. isolates)

732153(2); 6736153(6)

736113(2); 6736153(3)

736152(2); 6734152(1)

736151(3)

706112(5); 6706113(2); 6702112(1); 6702113(1); 6704113(1)

706110(1); 6706113(1); 6706132(1)

702112(2); 6706112(2)

706111(1); 6706113(1)

706113(2)

232150(3); 6610171(1); 6712151(1)a

216153(3); 6212132(1)

306152(1)a; 6212132(1); 6216152(1); 6216153(1)

332113(4)

712110(1); 6716152(1)

h epidermidis, respectively.

280 A. Andollina et al. / FEMS Microbiology Letters 234 (2004) 275–280

Acknowledgements

The authors would like to thank Dr. Ludwig Mo-

roder (APPA, Bolzano, Italy) for assistance in the

cluster analysis.

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