A STUDY ON CURRENT ANTIMICROBIAL SUSCEPTIBILITY

PATTERN OF TYPHOIDAL SALMONELLAE CAUSING ENTERIC

FEVER IN SCHOOL GOING CHILDREN AND YOUNG ADULTS

Dissertation submitted for

M.D. MICROBIOLOGY BRANCH – 1V

DEGREE EXAMINATION

THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY

CHENNAI – 600 032

TAMILNADU

MAY 2018

CERTIFICATE

This is to certify that this dissertation titled “A STUDY ON CURRENT

ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF TYPHOIDAL

SALMONELLAE CAUSING ENTERIC FEVER IN SCHOOL GOING

CHILDREN AND YOUNG ADULTS ” is a bonafide record of work done

by Dr. ANITHA.M, during the period of April 2016 to March 2017 under the

guidance of Prof.Dr.MANGALA ADISESH, M.D., Professor of Microbiology,

Institute of Microbiology , Madras Medical College and Rajiv Gandhi

Government General Hospital, Chennai - 600003, in partial fulfillment of the

requirement of M.D. MICROBIOLOGY Degree Examination of The Tamilnadu

Dr.M.G.R. Medical University to be held in May 2018.

Dr.R.NARAYANA BABU, MD.,DCH Dr.ROSY VENNILA., M.D., Dean, Director, Madras Medical College & Institute of Microbiology, Rajiv Gandhi Government Madras Medical College & General Hospital, Rajiv Gandhi Government Chennai – 600003 General Hospital, Chennai – 600003

DECLARATION

I, Dr.ANITHA.M, Post Graduate , Institute of Microbiology, Madras

Medical College, solemnly declare that the dissertation titled “A STUDY ON

CURRENT ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

TYPHOIDAL SALMONELLAE CAUSING ENTERIC FEVER IN

SCHOOL GOING CHILDREN AND YOUNG ADULTS ”is the bonafide

work done by me at Institute of Microbiology, Madras Medical College under the

expert guidance and supervision of Prof.Dr. MANGALA ADISESH M.D.,

Professor, Institute of Microbiology, Madras Medical College. The dissertation is

submitted to the Tamil Nadu Dr.M.G.R Medical University towards partial

fulfillment of requirement for the award of M.D., Degree (Branch IV) in

Microbiology.

Place: Chennai

Date: Dr. ANITHA.M

Signature of the Guide

Prof. Dr.MANGALA ADISESH, MD., Professor,

Institute of Microbiology Madras Medical College, Chennai-600 003.

ACKNOWLEDGEMENT

I wish to express my sincere thanks to the Honourable Dean

Dr.R.NARAYANA BABU, MD, D.Ch ., Madras Medical College & RGGGH,

Chennai for permitting me to use the resources of this Institution for my study.

I express my sincere thanks to Dr. ROSY VENNILA , M.D., Director,

Institute of Microbiology, Madras Medical College & RGGGH, Chennai.

I feel fortunate and indebted to be under the guidance of Prof.Dr.

MANGALA ADISESH , M.D., Institute of Microbiology, for suggesting the

topic for my dissertation and for her valuable advice, guidance in preparing and

compilation of my work, throughout my study period. She is a source of

inspiration in my endeavours.

I specially thank to our Prof.Dr.U.UMADEVI M.D., for her valuable

guidance ,and co-ordinating me in doing Molecular workup for my study at

Christian Medical College, Vellore.

I extend my sincere thanks to our Professors Dr. S.THASNEEM BANU

M.D., Dr.R.VANAJA M.D., Dr.C.P.RAMANI M.D., for their support,

guidance and valuable advice.

I extend my sincere thanks to Prof. Dr.N.DEVASENA M.D., Department

of Microbiology, Institute of Child Health and Hospital for Children for her

support and guidance .

I extend my whole hearted gratitude and special thanks to my co-guide

Dr.R.DEEPA., M.D., Senior Assistant Professor for her expert guidance and

support in doing my study.

I express my sincere thanks to our Assistant Professors

Dr.N.RATHNAPRIYA M.D., Dr.K.USHAKRISHNAN M.D.,

Dr.K.G.VENKATESH M.D., Dr.C.S.SRIPRIYA M.D.,

Dr.N.LAKSHMIPRIYA,M.D.D.Ch., Dr. DAVID AGATHA M.D., and

Dr.B.NATESAN M.D., for their support and guidance in my study.

My sincere thanks to Prof.Dr.T.RAVICHANDRAN.,M.D.,D.Ch.,

Director and Professor , Institute of Child Health and Hospital for Children for his

support and guidance. My sincere thanks to Prof.Dr.K.SRINIVASAGALU

M.D., former Director and Professor., Institute of Internal Medicine for his

guidance during my study period .

I thank Dr. Evangeline Mary, M.D.,M.B.A.,Department of Community

Medicine , Govt. Stanley Medical College, Chennai, for guiding me in analyzing

the results statistically .

I would like to thank all my colleagues, and all our technicians and staffs

in Institute of Microbiology, Madras Medical College for their support and

cooperation.

I would like to thank the Institutional Ethics Committee for approving my

study.

I feel indebted to my parents who had been an everlasting support and

encouragement and for their heartful blessings.

I thank my husband Dr. K. Chandrasekaran M.S, for taking great care,

support and encouragement without which this work would not have been

possible.

I thank all my patients without whom this study would not have been

completed.

Above all I thank the Almighty for his blessings and grace by giving me

this opportunity to acquire knowledge .

TABLE OF CONTENTS

SI. NO TITLE PAGE

No.

1 INTRODUCTION 1

2 AIMS & OBJECTIVES 4

3 REVIEW OF LITERATURE 5

4 MATERIALS & METHODS 50

5 RESULTS 68

6 DISCUSSION 79

7 LIMITATIONS OF THE STUDY 90

7 SUMMARY 91

8 CONCLUSION 93

9 BIBLIOGRAPHY

ANNEXURE-I ABBREVATIONS

ANNEXURE-II CERTIFICATE OF APPROVAL

ANNEXURE-III PROFORMA

ANNEXURE-IV CONSENT FORM

ANNEXURE-V MASTER CHART

LIST OF TABLES

S. NO TITLE PAGE

NO.

1 DISTRIBUTION OF PATIENTS IN THE STUDY POPULATION 68

2 AGE GROUP AND SEX DISTRIBUTION IN THE STUDY POPULATION 69

3 DISTRIBUTION OF PATIENTS BASED ON HOSPITAL ADMISSION 70

4 CARDINAL SYMPTOMS AMONG STUDY POPULATION 70

5 CARDINAL SIGNS AMONG STUDY POPULATION 71

6 DISTRIBUTION BASED ON FOOD HABITS 71

7 DISTRIBUTION OF BACTERIAL GROWTH IN BLOOD CULTURE 72

8 DISTRIBUTION OF PATHOGENS ISOLATED FROM BLOOD CULTURE 72

9 MONTHWISE DISTRIBUTION OF SALMONELLA ISOLATES 73

10 DISTRIBUTION BASED ON FOOD HABITS AND TYPHOID FEVER 74

11 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF S.TYPHI AND S.PARATYPHI A BY DISC DIFFUSION METHOD 74

12 DISTRIBUTION OF MIC OF CIPROFLOXACIN BY MICROBROTH DILUTION METHOD –CLSI AND EUCAST INTERPRETATIVE CRITERIA

76

13 ANTIMICROBIAL SUSCEPTIBILITY TESTING PROFILE OF TYPHOIDAL SALMONELLA ISOLATES , MOLECULAR CHARACTERISATION & CLSI AND EUCAST GUIDELINES

77

14 RESULTS OF SEQUENCE ANALYSIS OF SALMONELLA ISOLATES 78

LIST OF FIGURES

S. NO TITLE PAGE

NO

1 DISTRIBUTION OF PATIENTS IN THE STUDY POPULATION 68

2 AGE GROUP AND SEX DISTRIBUTION IN THE STUDY POPULATION 69

3 SEASONAL DISTRIBUTION OF ENTERIC FEVER 73

4 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF S.TYPHI AND S.PARATYPHI A BY DISC DIFFUSION METHOD

75

CERTIFICATE – II

This is to certify that this dissertation work titled “A STUDY ON

CURRENT ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

TYPHOIDAL SALMONELLAE CAUSING ENTERIC FEVER IN

SCHOOL GOING CHILDREN AND YOUNG ADULTS” of the

candidate DR.ANITHA .M with Registration Number 201514002 for the award

of M.D. in the branch of MICROBIOLOGY. I personally verified the

urkund.com website for the purpose of plagiarism Check. I found that the

uploaded thesis file contains from introduction to conclusion pages and result

shows 3 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with Seal.

Introduction

1

INTRODUCTION

Enteric fever is a systemic infection caused by the human adapted

athogens Salmonella enterica serovar Typhi (S.Typhi). A similar but often

less severe disease is caused by S.Paratyphi A, B, and sometimes

S.Paratyphi C. These organisms are the important cause of febrile illness in

crowded and impoverished populations with poor sanitation that are

exposed to unsafe water and food [1].

Advances in public health and hygiene have led to the virtual

disappearance of enteric fever from developed countries , but the disease

remains endemic in many developing countries and become a major public

health problem [1,2] .The global annual incidence of enteric fever was

estimated between 11.9 million and 26.9 million cases , in the year 2010

[3,4] . Case fatality rate remains 1% ranging between 1,29,000 and 1,61,000

typhoid deaths annually.[4]

The incidence of typhoid and paratyphoid varies geographically , with

South-Central and South-East Asia having the highest incidence-typically

exceeding 100 cases per 1,00,000 persons-years for typhoid and with lower,

variable rates for paratyphoid fever.

Infants ,children and adolescents experience the greatest burden of

illness[1]. Chronic carriage occurs occurs following primary infection in

2

approximately 2-5% of cases in the absence of appropriate antimicrobial

therapy and is strongly dependant on age and sex. [5]

Enteric fever carries a mortality rate of 30 % , if not treated properly,

whilst appropriate antimicrobial therapy reduces the mortality rate to as low

as 0.5% .[6] Hence, timely treatment with appropriate antimicrobial agents is

important for reducing the mortality and morbidity. But the resistance to

antimicrobial agents and its changing trends becomes a major challenge in

the management of both S.Typhi and S.Paratyphi .

After the first reported outbreak of chloramphenicol resistant S. Typhi

in 1972, there has been a steady increase in the number of multidrug

resistant (MDR) strains of S.Typhi-resistance to Ampicillin,

Chloramphenicol, Trimethoprim-Sulphamethoxazole - over the next two

decades [7,8,9]. But the rate of MDR strains was at a lower range among

S.Paratyphi A.[9]

Ciprofloxacin and Ceftriaxone (III generation cephalosporin)

become the drug of choice for treating MDR strains. With the increasing

use of Fluroquinolones since 1990s , there was a gradual decrease in MDR

strains with emergence of Nalidixic acid resistant strains (NARST).

Meanwhile, this switch to Ciprofloxacin and selective pressure exerted by

the irrational use of ciprofloxacin in human and veterinary therapeutics

3

leads to emergence of resistance , resulting in clinical failure and delayed

treatment response .

The regular revisions of Clinical Laboratory Standards Institute

(CLSI) guidelines in the interpretive criteria in 2011 and addition of new

fluroquinolones in 2015 and 2016 indicate the urgency and need to revise

breakpoints to optimise the dose of fluroquinolones and use of this drug

effectively in susceptible clinical isolates[10,11,12]

The most common cause of resistance to Nalidixic acid and

decreased susceptibility to fluoroquinolones in serovar Typhi is

chromosomal mutation in the quinolone resistance determining

region(QRDR) of the DNA gyrase subunit gyrA.[13,14 ]

With the changing patterns in antibiogram, it is necessary to

continually monitor the antibiotic resistance pattern and understand the

mechanism involved. Hence, this study was undertaken to characterise the

prevalent serotypes and their resistance patterns and analyse the molecular

mechanisms involved, so that appropriate strategies can be adopted in the

management of enteric fever.

Aims & Objectives

4

AIMS AND OBJECTIVES

AIM :

To determine the current trend of Antimicrobial Susceptibility Pattern

of Typhoidal Salmonellae in School going children & Young adults.

OBJECTIVES :

To isolate and identify the Salmonella species causing Enteric fever

through blood cultures.

To perform the Antimicrobial susceptibility testing for the isolates.

To do Molecular characterisation of isolates with reduced

susceptibility to fluroquinolone.

Review of Literature

5

REVIEW OF LITERATURE

HISTORY

Before the 19th century, typhus and typhoid fever thought to be same

disease. Many clinical distinctions were proposed,but none reliably

distinguished these syndromes. In 1829 in Paris, P. Ch. A. Louis

distinguished typhoid fever from other fevers on the basis of intestinal

lymph node and spleen pathology [15]. He described the clinical phenomena

of rose spots, intestinal perforation, and hemorrhage.

William Jenner in 1850 settled the dispute between typhus and

typhoid fever [16]. He differentiated typhoid fever based on the pathologic

evidence of enlargement of the Peyer’s patches and mesenteric lymph

nodes. He also noted that prior attacks of typhoid protected against

subsequent attacks, this was not the case in fever due to typhus. In 1869,

Wilson proposed the term “Enteric fever”, given the anatomic site of

infection[17].

In 1873,William Budd demonstrated that food, water, and fomites

could be the reason for transmitting typhoid fever [18]. Karl Joseph Eberth

(1879) first observed the typhoid bacillus in mesenteric lymph nodes and

spleen ; Gaffkey(1884) in Germany isolated the bacillus [19]. Hence,typhoid

bacillus was then called Eberth-Gaffky bacillus or Eberthella typhi.

6

The genus “Salmonella” was named after Daniel Elmer Salmon, a

veterinary pathologist, following the isolation of American-hog-cholera

bacillus (S.choleraesuis) in 1885.

In 1896, Pfeiffer and Kalle made the first typhoid vaccine using heat

killed organisms [20]. In the same year, Georges Fernand Isidore Widal , a

French physician and others demonstrated that convalescent sera from

typhoid patients caused the organisms to “stick together in large balls and

lose their motility”. He coined the term ‘agglutinin’ to describe this

observation .This technique provided a clinical tool for the identification of

Salmonella and was used extensively by Kauffman and White during the

1920s to1930s , for classification of over 2000 serotypes. [21].

In 1948, Theodore Woodward et al, reported the successful treatment

of Malaysian typhoid patients with Chloromycetin, and thus the modern

age of antimicrobial therapy for typhoid fever began.

A small animal model for typhoid fever was developed in the early

1950s, when animal experiments illustrated the susceptibility of in bred

mouse strains to infection by Salmonella Typhimurium. In1952, Zinder and

Lederberg , discovered genetic transduction, the transfer of genetic

information from one cell to another by a virus particle (bacteriophage P22)

using S.Typhimurium strains [22]. The combination of a convenient animal

7

model and powerful genetic techniques available for the study of

S.Typhimurium resulted in wide spread study of this model system of

Salmonella pathogenesis.

The healthy carrier state was realised with the typhoid Mary episode.

Typhoid Mary, a New York city cook, born as Mary Mallon, worked as a

cook for a number of establishment in the United States. Several outbreaks

of typhoid fever were attributed to her from 1900-1907 , leads to 47 cases

and 3 deaths[23]. Incidence rate was decreased in parallel with improved

sanitation, safety measures in food and water supplies, identification and

treatment of chronic carriers and use of Typhoid vaccines that reduced the

susceptibility of hosts to infection.

8

ENTERIC FEVER

Based on the clinical patterns in Human Salmonellosis , Salmonella

serptypes can be grouped into Typhoidal Salmonella and Non-typhoidal

Salmonella (NTS) . In human infections , the four different clinical

manifestations are Enteric fever, Gastroenteritis , Bacteraemia and other

Extra-intestinal complications and Chronic carrier state.

Enteric fever is a severe systemic illness characterized by fever and

abdominal symptoms that is caused by dissemination of Salmonella Typhi

and Salmonella Paratyphi. In endemic areas, most patients presenting to

hospitals with enteric fever are between 5 and 25 years of age. Those

younger than 4 years of age are more likely to have a nonspecific febrile

illness not recognized as typhoid. When children younger than 1 year of age

acquire typhoid, the disease is often more severe and is associated with a

higher rate of complications. The incubation period averages 10-14 days but

ranges from 5-21 days depending on the inoculum ingested and the immune

status of the person. In addition, patients with immunosuppression, biliary

and urinary tract abnormalities, and reticuloedothelial system defects, such

as hemoglobinopathies, malaria, schistosomiasis, bartonellosis, and

histoplasmosis,are at increased risk of severe disease.[24,25]

9

Epidemiology of Enteric Fever

In contrast to other Salmonella serotypes,the etiologic agents of

Enteric fever -Salmonella Typhi and Salmonella Paratyphi A,B, and C

serotypes –have no known hosts other than humans [26].

Burden of illness :

Enteric fever continues to be a major public health problem, with very

high social and economic impact because of hospitalization of patients with

acute disease and the complications and loss of income attributable to the

duration of clinical illness [25] .

Global Scenario :

In 2000, it was estimated that nearly 21.7 million illnesses caused

by S.Typhi and 5.5 million illnesses caused by S.Paratyphi A, B,and C

annually and an incidence ranging from 25-1000 cases per 1,00,000

population in endemic regions. The crude and laboratory adjusted estimate

was 10.8 and 21.7 million cases of typhoid fever. [1] .

Regions with high incidence of typhoid fever (>100/1,00,000

cases/year) include South-Central Asia and South-East Asia.

Infants,children and adolescents experience the greatestburden of illness in

these regions. Regions of medium incidence (10-100/1,00,000 cases/year)

include the rest of Asia, Africa, Latin America and the Caribbean and

10

Oceania, except for Australia and New Zealand and low in the other parts of

the world (10 cases/10,00,000) [1] .

The recent analysis of global typhoid fever morbidity , by Buckle et

al in 2010 reported , crude and adjusted estimate counting for low sensitivity

of blood culture for isolation of bacteria was of 13.5 and 26.9 million cases.

Indian Scenario :

The estimated prevalence of laboratory confirmed typhoid and

paratyphoid was 9.7% and 0.9% respectively. Pooled estimates of incidence

were 377 (170-801) and 105 (74-148) per 1,00,000 person years

respectively. Typhoid fever showed a significant decline in prevalence over

a period of time and was detected among clinically suspected febrile cases

or during outbreaks. But Paratyphoid fever did not show any trend over time

and there was no clear association with the risk factors involved.Children in

the age group of 2-4 years old had the high incidence rate [5] .

Typhoid and Paratyphoid fevers were included in Global Burden of

Disease 2010 (GBD 2010) project, when they estimated to account for 12.2

million disability-adjusted life years [27] .

11

Case definition : [25]

Confirmed case of typhoid fever

A patient with fever (38°C and above) that has lasted for at least three

days, with a laboratory-confirmed positive culture (blood, bone marrow,

bowel fluid) of S. typhi.

Probable case of typhoid fever

A patient with fever (38°C and above) that has lasted for at least three

days, with a positive serodiagnosis or antigen detection test but without S.

typhi isolation.

Chronic carrier

Excretion of S. typhi in stools or urine (or repeated positive bile or

duodenal string cultures) for longer than one year after the onset of acute

typhoid fever. Short-term carriers also exist but their epidemiological role is

not as important as that of chronic carriers. Some patients excreting S. typhi

have no history of typhoid fever.

Mode of transmission : [25,26]

Humans are the only natural host and reservoir. The infection is

transmitted by ingestion of food or water contaminated with faeces. Sexual

transmission has also been reported. Established risk factors are

contaminated water supply, ice cream, flavoured iced drinks or food from

12

street vendors. Shellfish taken from contaminated water, and raw fruit and

vegetables washed with sewage contaminated water , have been sources of

past outbreaks .The highest incidence occurs where water supplies serving

large populations are contaminated with faeces.

Epidemiological data suggest that waterborne transmission of S. typhi

usually involves small inocula, whereas foodborne transmission is

associated with large inocula and high attack rates over short periods. The

inoculum size (103 to 106 ) and the type of vehicle in which the organisms

are ingested greatly influence both the attack rate and the incubation period

(highly variable 1- 6 weeks) .

Small number of patients (1-5%) with acute infection develop a

chronic carrier state,which has allowed the disease to persist during inter-

epidemic periods. Levine et al. (1982) studied the role of chronic carriers as

a reservoir of infection in Santiago, Chile, where the crude rate of 694

carriers per 1,00,000 inhabitants was found.

Risk factors

Environmental factors

Housing in close proximity to open sewers and highly contaminated

water bodies , residence in low elevation areas , and rainy season are the

important environmental risk factors for transmission of typhoid fever [18,25]

13

Host factors

Typhoidal Salmonella are able to survive at low gastric pH 1.5.

Conditions that decrease stomach acidity like antacids, histamine-2 receptor

antagonists (H2 blockers), proton pump inhibitors, past infection with

Helicobacter pylori, gastrectomy, and achlorhydria facilitate infection [26] .

Genetic polymorphisms in regulatory region of PARK2 and

PACGR, protein aggregate that is essential for breaking down the bacterial

signalling molecules that dampen the macrophage response , are found

disproportionately in persons infected with S.Typhi, and Mycobacterium

lepra e [22].

Role of HLA- linked genes in susceptibility or resistance to this

infection has been studied. HLADRB1*0301/6/8, HLA-DQB180201-3, and

Tumour Necrosis Factor-α (TNFα*2-308) are belived to be associated with

susceptibility to typhoid fever. HLA-DRB1*12 is associated with protection

against complicated typhoid fever [28 ] .

14

CLASSIFICATION AND TAXONOMY

Salmonella is a genus of family of Enterobacteriaceae. Existence of

multiple Salmonella speices was taxonomically accepted before

1983.Currently, the genus Salmonella is divided into two species:

Salmonella enterica and Salmonella bongori, as a result of experiments

indicating a high degree of DNA similarity. Salmonella enterica which

contains six species (I,II, IIIa,IIIb,IV and VI) and Salmonella bongori,

which was formerly subspecies V. Almost all the serotypes of S.enterica

subspecies I are pathogenic for humans, except for rare human infections

with subspecies IIIa and IIIb,that were previously designated as genus

Arizonae.

Members of the seven Salmonella subspecies can be serotyped into

more than 2500 serotypes (serovars) based on antigenically diverse surface

structures : somatic(O) antigens, the carbohydrate component of

lipopolysaccharide, the surface Vi antigen (restricted to S.Typhi and

S.ParatyphiC), and flagellar (H) antigen [29,30,31].The name usually refers to

the location where the Salmonella serotype was first isolated.

According to the current Salmonella nomenclature system in use at

the U.S. Centre for Disease Control and Prevention and World Health

Organisation laboratories the full taxonomic designation Salmonella

enterica subspecies enterica serotype Typhi can be shortened to Salmonella

serotype Typhi or Salmonella Typhi.29

15

Classification of Salmonella species and subspecies [32]

Subspecies No of serotypes within subspecies

Salmonella enterica subspecies enterica(I) 1531

Salmonella enterica subspecies salmae(II) 505

Salmonella enterica subspecies arizonae(IIIa) 99

Salmonella enterica subspecies diarizonae(IIIB) 336

Salmonella enterica subspecies houtenae(IV) 73

Salmonella enterica subspecies indica(VI) 13

Salmonella bongori(Formerly subgenera V) 22

Total (genus salmonella) 2579

Biochemical differentiation of subspecies of Salmonella [30]

Subspecies Enterica Salamae Arizonae Diarizonae Houtenae Indica Dulcitol + + - - - d ONPG (2h) - - + + - d Malonate - + + + - - Gelatinase - + + + + + Sorbitol + + + + + - d-Tartarate + - - - - - Galacturonate - + - + + + Mucate + + + - (70%) - + Salicin - - - - + - Growth in KCN - - - - + - Beta glucoronidase d d - + - d

Lysed by phage O1 + + - + - +

+,>90 % strains of positive ; - ,>90% of strains negative ; d,some strains

positive ,others negative.

16

In 2003, a total of 2555 serovars were identified in Kaufmann-White

scheme. In 2007, since L.Le Minor described most of the presently known

serovars, “Kauffmann-White scheme” was redesignated as “White –

Kauffmann-Le minor” scheme.[32]

HABITAT

Salmonellae are primarily intestinal pathogens of human and animals

including wild birds, domestic pets, and rodents. They are found frequently

in sewage, rivers, and in soil in which they do not multiply significantly.

Under suitable environmental conditions, they may survive for weeks in

waters and for years in soil.

In contrast to other Salmonella serotypes, the etiologic agents of

Enteric fever- S.Typhi and S.Paratyphi A,B, and C – have no known host

other than humans [30] .

PHENOTYPE

Morphology

Salmonellae are gram negative, non -spore forming, facultative

anaerobic bacilli that measures about 2 to 3 by 0.4 to 0.6 micrometer in

size. Like other Enterobacteriaceae, they produce acid on glucose

fermentation , reduce nitrates, and do not produce cytochrome oxidase. All

organisms are motile as a result of peritrichous flagella, except

17

S.Gallinarum- Pullorum which is non-motile. Most serotypes do not ferment

lactose, but nearly 1% of organisms are able to ferment lactose. This

property of differential metabolism of sugars can be used to distinguish

many Salmonella serotypes; Salmonella Typhi is the only organism that

does not produce gas on sugar fermentation.[30]

Culture Characters and Growth Requirements [30,31]

Salmonella grow over a wide temperature range from 15–45o C,

optimally at 37o C at pH 4-8. Under special conditions they may proliferate

at <48 o C and withstand pH<4. In aerobic and anaerobic conditions they

grow readily on ordinary media. Most are prototropic,i.e capable of growing

on a glucose-ammonium minimal medium , but some strains are

auxotrophic and require enrichment with one or more amino acids or

vitamins, e.g. cysteine or nicotinamide ; most Typhi strains require

tryptophan.

After 24 hours of incubation at 370C,most strains of S.Typhi produce

moderately large (2- 3 mm in diameter), grey white, moist, circular colonies

, with smooth convex surface and entire edge, resembling the colonies of

other enterobacteria in Nutrient agar plate. Non-haemolytic grey white

colonies in Blood agar plate. Lactose non fermenting colonies in Mac

Conkey agar plate.

18

Paratyphi A produce relatively small size colonies. ‘Rough’, non-

virulent strains (S →R variation) form opaque granular colonies with an

irregular surface and indented edge.

Paratyphi B produce large mucoid colonies,or colonies surrounded

by thick mucoid ‘slime wall’ made up of ‘M’antigen of Kauffmann ,best

developed at low temperature, low humidity and high osmolarity (Anderson

and Rogers 1963). The “mucoid wall test” is positive with most strains of

Paratyphi B (Kauffmann 1966) [31] .

Most of the strains grow abundantly and give uniform turbidity in

liquid medium like peptone water and nutrient broth.On prolonged

incubation thin surface pellicle will be seen. ‘ Rough’ (R) variants tends to

autoagglutinate , producing granular deposits and sometimes a thick pellicle

formation [30].

Various Differential and Selective media are available for the

isolation of salmonellae from faeces and other samples that are heavily

contaminated with other bacteria [26] .

Low- selective media, such as MacConkey agar and Leifson’s

deoxycholate citrate agar (DCA) , and intermediate –selective media, such

as Taylor’s xylose lysine deoxycholate agar (XLD), Salmonella-Shigella

agar, or Hektoen enteric agar are widely used for screening .

19

Selective chromogenic medium , such as CHROMagar are more

specific than other selective medium. Tetrathionate and Selenite based

enrichment broths are often used to facilitate the recovery of low numbers

of organisms.

Highly –selective media, such as selenite with brilliant green ,

reserved in outbreak situations for carrier detection .Wilson & Blair’s

brilliant- green bismuth sulphite agar (BBSA) is a valuable isolation

medium for S.Typhi.

BIOCHEMICAL ACTIVITIES [30]

Salmonella enterica subspecies enterica can be phenotypically

identified by the following biochemical reactions include

1. Fermentation of glucose, maltose, mannitol and sorbitol with the

production of acid and gas.(S.Typhi, Gallinarium-anaerogenic).

2. Absence of fermentation of sucrose, lactose, salicin, and adonitol.

3. Failure to produce indole, hydrolyse urea, deaminate phenylalanine.

4. Positive methyl red reaction and a negative Voges-Proskauer reaction

20

There a few exceptions to these.

Biochemical Reactions Of Salmonella enterica subspecies enterica[31 ,33]

BIOCHEMICAL REACTIONS

INTERPRETATION

Cytochrome oxidase Negative Catalase Positive Nitrate reduction Reduces nitrates to nitrites Phenylalanine deaminase test Fails to deaminate phenylalanine

Hugh Leifsons OF media Shows both oxidative and fermentative pattern Fermentation of glucose Produces acid only or acid and gas KCN Sensitive Indole Not produced Methyl red Positive Vogue proskaeur Acetoin not produced Simmons citrate Utilized (except S.Typhi and S.Paratyphi A) Urease Not produced

Triple sugar iron agar(TSI)

Alkaline/acid with speck of H2S - S.Typhi Alkaline/acid with gas and no H2S - S.Paratyphi A Alkaline/acid with plenty of H2S - S.Paratyphi B

Biochemical differences between S.Typhi and S.Paratyphi A [31,33]

Biochemical Test S.Typhi S.Paratyphi A Glucose fermentation Production of acid only Acid with gas Xylose fermentation ± - Arabinose fermentation - + Mucate fermentation ± Does not ferment Dulcitol fermentation - + Rhamnose fermentation - + d-tartarate Acid only Does not ferment Lysine + - Arginine + + Ornithine - +

21

ANTIGENIC STRUCTURE [30]

The antigens used to define the serological types of Salmonella include:

1. The O antigens, heat stable polysaccharides that form part of cell wall

polysaccharide (LPS).

2. The H antigen, heat labile proteins of the flagella with diphasic

variation

3. Surface Vi antigen, surface polysaccharide that inhibits agglutinabilty

of organism by homologous ‘O’ antisera of which Vi antigen of

Typhi is most important.

Vi ANTIGEN

Daniels et al (1989) demonstrated that Vi antigen is a capsular

polysaccharide of α-(1→4) linked N-acetyl-D-galactosaminouronic acid

variably acetylated at C2/C3positions. It prevents immune serum mediated

killing, is antiphagocytic and increases resistance to peroxide. Properties of

Vi are determined by structural (via B) and functional (via A) elements at

distinct chromosomal sites (Makela & Stocker 1969).

Felix and Pitt (1934) demonstrated that Typhi strains cultured from

the blood of typhoid fever patients were inagglutinable in O9 serum

showed greater virulence in mice than O-agglutinable strains and proposed

the name ‘Vi’ (virulence) antigen for the surface structure.

22

Vi is produced by 3 strains of Salmonella serotypes Typhi, Paratyphi

C and Dublin, that are genetically distant. The presence of ‘via’ genes in all

but a few (1%) strains of Typhi and Paratyphi C suggest Vi is an

established property of these serotypes.

Continued laboratory culture of Vi+ strains may lead to loss of Vi

production. Daniels et al , in 1989, demonstrated that isolates of Paratyphi

C produce less Vi per cell than Typhi , release it more rapidly in the

medium and show higher frequency of reversion from Vi+ to Vi- than

Typhi.

Vi vaccine gives excellent protection in controlled trials in areas with

high attack rates of Typhoid fever. (Felix & Pitt 1934; Robbins and Robbins

1984; Tacker et al.1986; Klugman et al.1987; Daniels et al. 1989) [34,35,36] .

PATHOGENESIS

Salmonella infections begin with ingestion of bacteria in

contaminated food or water. Waterborne transmission involves the ingestion

of fewer bacilli and has a long incubation period and lower attack rate

compared with food borne transmission [37,38].

Median infectious dose to produce disease is approximately 106

bacilli. Gastric acidity represents the initial barrier to salmonella

colonization. On exposure to acid in vitro, Salmonella display an adaptive

23

tolerance response that probably facilitates bacterial survival in the stomach

and passage to the small intestine [39] .

Interactions with intestinal epithelium and induction of enteritis

Salmonella must evade host antimicrobial factors secreted into the

intestinal lumen,including antimicrobial peptides, bile salts, IgA, and

traverse a protective mucous barrier before encountering intestinal epithelial

cells [40,41] .

Salmonella express an array of distinct fimbriae that contribute to

tight adherence to intestinal epithelial cells. Salmonella preferentially adhere

to and enter the specialized microfold cells (M cells) that overlie lymphoid

tissue within payer’s patches. The bacteria remain in an endocytic vacuole,

where they replicate or be transported across the cytoplasm through

“bacteria-mediated endocytosis” and released in to the blood or lymphatic

circulation.

Salmonella pathogenicity island I (SPI I) encodes salmonella-

secreted invasion proteins (ssps) and a type III secretion system(T3SS).

T3SS is required for bacterial mediated endocytosis and intestinal epithelial

invasion. Salmonella mutants laking a functional SPI-I, T3SS cannot invade

epithelial cells as observed in tissue culture and animal models SPI-1 codes

for two protein SipC and SipA, induce membrane ruffling and

macropinocytosis [43].

24

Interactions with Macrophages & Systemic infection

Salmonella sense the acidic environment of the Salmonella-

containing vacuole (SCV) and activate various regulatory proteins PhoP

and PhoQ, required for adaptation in intracellular environment of host cells.

These proteins regulates transcription of over 200 genes which is required

for survival within macrophages. PhoQ is the sensor protein for the

phagosome environment by sensing acidic pH and antimicrobial peptides to

activate gene expression [44-46]

Encoded on SPI-2 is an additional T3SS , which directly delivers the

bacterial proteins in to the macrophage cytoplasm favours the survival and

promotes virulence.

Host response and immunity

The innate immune system senses invasive Salmonella infections by

recognition of lipopolysaccharide by toll-like receptor 4 (Tlr-4), bacterial

lipoproteins by Tlr-2 , and flagellin by Tlr-5 by a signalling system Ipaf and

peptidoglycan by Nod1,Nod2 [ 47] . Activation of these receptors leads to

synthesis of cytokines that orchestrate the inflammatory response and

instruct the subsequent antigen-specific immune response.

Macrophage activation and efficient killing of salmonella is

associated with production of specific antibody by B cells [48] .The infected

25

macrophages carry the bacteria to the mesenteric lymph nodes, multiply

there and reach blood stream via thoracic duct resulting in primary

bacteraemia. Primary bacteraemia being transient , seeds the liver, spleen,

lymph node and bone marrow where the bacteria continue to multiply.

Following multiplication in large numbers, the bacteria are released into the

bloodstream resulting in secondary bacteraemia and leads to the onset of

clinical disease.

CLINICAL MANIFESTATION [26]

Salmonella serotypes most often produce characteristic clinical

manifestations that have been given the syndrome designations such as

gastroenteritis, enteric fever, bacteremia and vascular infection, localized

infections and chronic carrier state.

ENTERIC FEVER

Enteric fever is classically described as an acute illness with fever

and abdominal tenderness. The symptoms are nonspecific and may be

insidious in onset.The differential diagnosis of gradual onset of fever and

abdominal pain with hepatosplenomegaly also includes malaria, amoebic

liver abscess, visceral leishmaniasis and viral syndromes such as dengue

fever.

26

The incubation period is typically 10 -14 days, but ranges fron 5 - 21

days based on the inoculum ingested and the health and immune status of

the person. Following ingestion of the organism, persons may develop

enterocolitis with diarrhoeal illness lasting for several days; these

symptoms usually resolve before the onset of fever.

Diarrhoea is common among children under 1 year of age .

Constipation is present in 10% to 38% of patients. Fever and abdominal

pain are the cardinal manifestations, but only 75% of patients presented with

fever and only 30% to 40% of patients will have abdominal pain at

presentation [49,50] . Nonspecific symptoms , such as dull frontal headache,

chills, diaphoresis, anoexia, cough, weakness, sore throat, muscle pain and

dizziness are frequent before fever onset [ 51] .

Initially fever is low grade and rises by the second week of illness to

39º to 40º. Patients with typhoid fever usually appears acutely ill. Relative

bradycardia is neither a sensitive nor a specific sign of typhoid

fever,occurring in less than 50% of patients. Approximately 30% of

patients will have rose spots on the trunk at the end of first week [52].

Organisms can be cultured from punch biopsies of these lesions and the

pathology is characterized by a perivascular mononuclear infiltrate.

27

Approximately 20-50% of patients have hepatosplenomegaly, 3% of

adults develop necrotizing cholecystitis with localized right upper quadrant

pain. Sometimes patient may present as pancreatitis .

2-40 % patients presents with neurologic manifestations, which

includes meningitis, neuritis, Guillain-Barre syndrome, and 5% to 10%

patients presents as neuropsychiatric illness including apathy, psychosis,

and confusion.This so called typhoid state has been described as ‘coma

vigil ’, picking at the bed clothes and muscle twitching are characteristic.

Nearly 10%-15% of patients develop severe disease , which depends

on host immune status, strain virulence, inoculums and choice of

antimicrobial therapy.Most common complications include Gastrointestinal

bleeding (10-20%) and intestinal perforation (1- 3%) occur in third and

fourth weeks of illness and results from hyperplasia, ulceration and necrosis

of the Peyer’s patches.

Rare complications include endocarditis, pericarditis, orchitis, and

focal abscesses and granuloma,arthritis,osteomyelitis,pancreatitis .

Hematologic abnormalities associated with typhoid include include

leukopenia, anemia, and disseminated intravascular coagulation.

Upto 10%of patients develop mild relapse, usually within 2 to 3

weeks of fever resolution and associated with the same strain type and

susceptibility profile. Reinfection may be distinguished from relapse using

molecular typing.

28

CHRONIC CARRIER STATE

0.2% to 0.6% of patients with non typhoidal salmonellosis

develop chronic carrier state . Up to 10% of untreated patients excrete

S.Typhi in feces upto 3 months and 1% to 4% develop chronic carriage [53]

. The frequency of chronic carriage is higher in infants, women, in persons

with biliary abnormalities or concurrent bladder infection with Schistosoma

haematobium, and in infants [54,55].

Chronic carriage of S.typhi and S.Paratyphi A has been associated

with an increased incidence of carcinoma of the gallbladder and of other

gastrointestinal malignancies [56] .

LABORATORY DIAGNOSIS OF ENTERIC FEVER

Clinical diagnosis of typhoid fever is difficult because of lack of

specific clinical signs and also due to altered clinical course of the disease

due to empirical treatment.

BACTERIAL CULTURE

Isolation of S.Typhi or S.Paratyphi by culturing blood, bone

marrow, another sterile sites ,stool or other intestinal secretions and rose

spots becomes the definitive diagnosis. Culture confirms the diagnosis and

provides an isolate to perform antimicrobial susceptibility testing ,

epidemiological typing and molecular characterisation [2,57] .

29

BLOOD CULTURE :

Blood culture processing done by conventional methods or in the

recent years automatic blood culture systems are also available.

Conventional blood culture makes use of BHI broth or bile broth or

sometimes biphasic media for optimal recovery of salmonella. For the

optimum yield of the organism the volume of blood to culture broth in

traditional systems should be 1:10 or even more. This dilutes antibacterial

substances present in the blood. Commercial blood culture system contains

resins , which allows higher volume of blood to be tested in a lesser volume

of broth [ 25] .

Conventional blood culture are incubated at 37ºC aerobically. The

bottles are examined visually for evidence of growth (hemolysis, turbidity)

during 6 -18 hrs. Blind subcultures from BHI broth has to be done on

Blood and Mac Conkey agar plates on day 1, even if doesn’t show any

signs of growth, there after serial subculturing done on alternative days till

7 days. The growth if any is identified by standard biochemical reactions

and confirmed. On day 7, all the blood culture bottles are subcultured before

discarded as negative.

Sensitivity of blood culture is upto 80% in patients who was not on

prior antibiotics, but sensitivity drops down to as low as 40 % in areas of

30

endemicity, where antimicrobials are taken very often before correct

evaluation [ 57,58 ] .

Culture positivity rate is high in first and second week of illness, but

in the absence of antimicrobial exposure cultures will still remain positive in

third week [ 57,60] .

Sensitivity is further reduced if only small quantities of S.Typhi (<10

organisms/ml ) are present in patients’s blood, frequently it is less than one

or less . Hence, volume of blood sample collected for culture enhances the

recovery of organism [ 59-63 ]. Increase of yield by 3.2% for each 1 ml of

blood sampled [ 64] .

Due to the higher levels of bacteremia in children compared to that in

adults, at least 10-15 ml of blood from schoolchildren and adults, and 2-4 ml

from toddlers and preschool children should be taken to achieve optimal

isolation rates [25] .

It is unsafe to collect large blood samples from children, especially in

infants. Baron and colleagues have determined the recommendations for

volumes of blood to be collected from infants and children [ 64] .

31

SUGGESTED BLOOD VOLUMES FOR CULTURES FROM

INFANTS AND CHILDREN [64]

Weight of patient(kg)

Total Blood

Volume (ml)

Blood Volume(ml) (Culture 1)

Blood Volume(ml) (Culture 2)

Total volume for

culture (ml)

% of total blood

volume

≤1 50-99 2 2 4

1.1-2 100-200 2 2 4 4

2.1-12.7 >200 4 2 6 ³

12.8-36.3 >800 10 10 20 2.5

>36.3 >2200 20-30 20-30 40-60 1.8-2.7

LYSIS CENTRIFUGATION SYSTEM [33,64]

Is an isolator system that has a special tube which contains saponin

which lyses both RBC & WBS. The system also contains polypropelene

glycol which decreases foaming, SPS as anticoagulant, EDTA to chelate

ions and inhibit complement cascade and coagulation, inert flurochemicals

to cushion and concentrate the organism during centrifugation. The tube is

centrifuged at 3000 rpm for 15 min and sediment is subcultured onto

appropriate media. This method increases the rapid recovery of intracellular

microorganisms and allow for quantification.

32

BONE MARROW CULTURE :

Higher colony counts are present in bone marrow compared to blood

and counts not reduced even with 5 days of prior antimicrobial therapy [64,65].

The sencitivity of bone marrow culture is variable 55-90% and specificity

upto 30% . Inspite of greater sensitivity, bone marrow culture is of less

clinical value because of invasive procedure, pain and expensive when

compared to blood culture.

Because amost all S.Typhi and S.Paratyphi are associated with the

mononuclear cell- platelet fraction , blood clot culture, centrifuge of blood

and culture of the buffy coat fraction, or the lysis direct plating –lysis

centrifugation method can substantially reduce the time o isolation of the

organism and variably improve sensitivity [66,67] .

SEROLOGICAL TESTS:

ANTIBODY DETECTION

1.WIDAL TEST

Widal test measures agglutinating antibodies against LPS (O),

flagellar (H) antigens of S.Typhi, and flagellar (AH, BH) antigens of

S.Paratyphi A & B respectively in sera of individuals with suspected

Enteric fever. It is simple and inexpensive procedure but lacks sensitivity

(47-77 %) and specificity (50-92 %). Acute and convalescent –phase serum

33

samples taken approximately 10 days apart is required to perform the test ;

positive result is determined by a 4-fold increase in antibody titre.

However, in infected patients antibody titres often rise before the

clinical onset,making it difficult to determine the 4-fold rise in antibody

titre.False negative and false positive results are common when single acute

phase serum is used for detection. Knowledge of the background levels of

antibodies in local population may aid in better interpretation of the test

when performed among patients with high prior probability of infection[67-70]

2.ELISA

Enzyme –linked immunosorbent assays (ELISA) have been use dto

study the normal antibody response to LPS, flagella, Vi capsular

polysaccharide, or outer membrane protein antigens. Anti-LPS antibodies

and Antiflagellum antibodies are more sensitive than Widal “O” and “H”

antigen based test [71] .

3. SDS-PAGE

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis ( SDS-

PAGE) immunoblotting used to detect antibodies against LPS and flagellar

antigens of S.Typhi and S.Paratyphi [72].

34

4. RAPID SEROLOGICAL TEST [73].

There are a number of commercially available point-of-care rapid

serologic tests for enteric fever .

TUBEX TF TEST :

Detects antibody against S.Typhi LPS with an inhibition assay format

and a visual result readout, with 56-100% sensitivity and 58-100 %

specificity .

TYPHIDOT :

Measures IgM and IgG antibodies against a 50-kDa outer membrane

protein of S.Typhi in a immunodot test format, with 67-98 % sensitivity and

58-100 % specificity.

TYPHIDOT M :

Measures IgM antibodies , after removal of IgG antibodies, against a

50-kDa outer membrane protein oh S.Typhi in a dot blot format, with

slightly higher of sensitivity 47-98% and specificity of 65-93 %.

ANTIGEN DETECTION

Rapid latex agglutination test (LAT ) detects specific antigens in

culture supernatants. With a sensitivity of 100%, specificity of 97.6%, and

positive and negative predictive values of 90.9% and 100%, respectively.

LAT can be used for the presumptive diagnosis of enteric fever in remote

35

health centers . LAT could detect the antigen in 100% of the sera of patients

with negative blood culture and positive Widal, indicating better sensitivity

as compared to blood culture.

COAGGLUTINATION TEST:

It is a slide agglutination method that uses killed staphylococci

(Cowan I strain) bearing protein A which binds with Fc fragment of IgG

specific against somatic O antigen of S.enteritidis.The test will be positive

in first week of fever with sensitivity of 86.67% and specificity of 88.83 %.

It will become negative after the first week of illness [ 76 ] .

MOLECULAR METHODS

Nuclei acid amplification test, including conventional and real - time

PCR, have been developed for the detection of both S.Typhi and

S.Paratyphi A mainly in blood sample. Targets include Hd flagellin fliC-d ,

the Vi capsular gene viaB , the tyvelose epimerase gene ( tyv), the paratose

synthase gene (prt), the 16sRNA gene , hilA ( a regulatory gene in

Salmonella pathogenicity island [SPI-1] , and the gene encoding 50 kDa

outer membrane protein ST50 .

OTHER LABORATORY TESTS

Hematologic abnormalities associated with typhoid include

leukopenia, anaemia, and subclinical disseminated intravascular

36

coagulopathy and elevated creatitine kinase and liver function tests (

aspartate transaminase and alanine transaminase ) . Liver biopsies

demonstrated focal Kupffer cell hyperplasia and mononuclear cell

infiltration of the portal space [26] .

DIAGNOSIS OF TYPHOID CARRIERS

Detection of carrier is important public health measure. It is useful

for screening food handlers and cooks to detect carrier state. Carrier state

can be determined by isolating the organism in stool, bile or urine. The

frequency and intensity of bacillary shedding vary widely and it is essential,

therefore, to test repeated samples. For the detection of urinary carriers,

repeated urine cultures should be carried out.

STOOL CULTURE

The sensitivity of stool culture increases from about 10 % in a single

sample to about 30 % by testing multiple samples during 3 rd – 5 th week of

illness . Faeces can be cultured on selective media, both directly and after

preliminary culture in a liquid enrichment medium. The common media

used are Selenite F broth, DCA,XLD agar. Wilson and Blair’s medium is a

good selective medium.

37

BILE CULTURE

Culture of bile obtained from a overnight duodenal string capsule

provides a sensitivity similar to that of blood culture and helpful in isolating

the infectious agent both from patients and carriers.

DETECTION OF Vi ANTIGEN :

Demonstration of Vi antigens has been used as a screening test for the

carrier state.The test was found to be 70% sensitive and it still increase with

multiple number of samples.

Carrier tracing in cities can be done by ‘sewer swab technique’.

TABLE : DETECTION METHODS AND POSITIVITY RATE

DURING DIFFERENT STAGES OF ILLNESS

STAGE OF ILLNESS METHODS RESULT

(% POSITIVITY )

1 st week Blood culture 95

2 nd week Blood culture 40-50

Widal Test Low antibody titre

3 rd week Blood culture 15-20

Stool and Urine culture 80

Widal Test 100

4 th week Blood culture 5-10

Stool and Urine culture 90

Widal Test 100

38

VARIOUS TYPING METHODS :

BACTERIOPHAGE TYPING [30]

The underlying principle of phage typing is the host specificity of the

bacteriophages. Several phage-typing schemes have been developed for

serotypes of clinical and epidemiological importance.

Typhi phage types

In 1938, Craigie and Yen , developed the first phage typing scheme

based on the principle of phage adaptation for differentiation of Typhi .

Progressive adaptations in this scheme were made of Vi phage II, which is

specific for Vi (capsular) antigen of Typhi ( Felix and Pitt 1934), is highly

adaptable and shows high degree of specificity. The adaptation is due in

part to the selection of spontaneously occurring host-range phage mutants

by the bacterium and in part to a non mutational phenotypic modification of

the phage by the host strain.

In 1947, Craige and Felix, standardized the method of phage typing

and with further adaptations the internationally recognized total number of

phage type is 106. The scheme is now used in specialised WHO approved

reference centers world wide.

The types most widespread around the world are E1 and A, followed

by B2, C1, D1 and F1. A serious limitation of the usefulness of Vi phage

39

typing is that A or E1 may be so common in a country as to limit the

epidemiological information. These can now be overcome by further

discrimination using battery of biochemical test and more advanced

molecular typing methods.

BIOTYPING [30]

Subdividing common salmonella serotypes according to their

biochemical characters is of value in epidemiological investigations.

Anderson et al., in 1978 and Barker et al. ,in 1980 explained about the

usefulness of combined phage type-biotype studies.

Combined phage type-biotype studies help:

to determine with greater confidence the fine relationships among

strains

to characterize variants that arise from a strain in the course of its

epidemic spread and

to indicate likely phage type interconversions.

MOLECULAR TYPING METHODS [30]

A range of typing methods based on characterization of the genotype

of the organism by analysis of plasmid and chromosomal DNA have now

been developed. Typing methods based on characterisation of Plasmid

40

DNA include Plasmid profile typing, Plasmid fingerprinting and

Identification of plasmid mediated virulence genes.

Chromosomally based techniques include Ribotyping, Random

cloned chromosomal sequence (RCCS) , Insertion sequence (IS) 200,

Pulsed field gel electrophoresis and PCR based methods such as Random

amplified polymorphic DNA typing (RAPD), Repetitive extragenic

palindromic element typing (REP-PCR), Variable number of tandem repeats

finger printing (VNTR) The methods most extensively used for

epidemiological investigations are plasmid typing, RCCS typing, IS200

fingerprinting. PFGE and AFLP are more recently used methods.

ANTIMICROBIAL RESISTANCE

DEVELOPMENT OF ANTIMICROBIAL RESISTANCE AMONG

TYPHOIDAL SALMOELLA STRAINS :

CHLORAMPHENICOL RESISTANCE :

Chloramphenicol, the first successful therapeutic drug of typhoid

fever since 1948. Chloromphenicol binds to the 50S subunit of bacterial

ribosomes, which inhibit peptide chain elongation.

Anderson et al., in 1950 first reported resistance to chloromphenicol

in Salmonella Typhi isolates from England. The first epidemic caused by

Chloromphenicol-resistant strain was that which occurred in Mexico in

41

1972 [77]. At about the same time there was a first substantial outbreak in

Calicut Kerala, India in which 7 of 13 strains were resistant to

chloromphenicol and invitro transmissibility to E.coli was demonstrated by

Paniker and Vimala 1972, and in both outbreaks mortality was high [78]. In

the succeeding 5 years, outbreaks occurred in several other countries,

notably,Vietnam, Indonesia, Korea, Chile, Bangladesh.

Resistance mechanism to chloramphenicol include enzyme

inactivation by acetylation of the drugs through chloramphenicol acetyl

transferases (CATs).. Resistance to Chloramphenicol was mediated by self

transmissible plasmids of the HII incompatibility type (IncHI) during the

outbreak . In addition to chloramphenicol resistance,these plasmids often

carried genes conferring resistance to other drugs, such as streptomycin,

sulphonamides and tetracyclines [79] .Other mechanisms include inactivation

by phosphotransferases efflux systems and mutation at target sites and loss

of OMP.

MULTI DRUG RESISTANCE

With greater number of reports of chloromphenicol resistant S.Typhi

and S.Paratyphi A emerging worldwide, Ampicillin, Cotrimoxazole, and

Tetracycline became the drug of choice. There was a good response to these

drugs until resistance developed.

42

By the late 1980s, Multiple- drug resistance (MDR), defined as

resistance to ampicillin, chloramphenicol, and trimethoprim-

sulfamethoxazole, was reported from multiple countries [ 80-82] .

Resistance to Ampicillin is mostly by production of β-lactamase

enzymes that hydrolyse the β-lactam ring and sometimes by impaired

penetration of drug to target PBP( penicillin binding protein) in the bacteria.

Resistance to Co-trimoxazole also emerged after few years of wide

spread use. Low level resistance to trimethoprim is due to drug resistant

variants of the chromosomal folA gene encoding the bacterial DHFR . High

level resistance is achieved by a bypass mechanism through genes that are

plasmid mediated. Some of these genes are dfr1, dfrA 3, dfrA10, dfrB6.

Sulphonamide resistance is by mutations in the gene folP that encodes for

DHPS. Acquired resistance is by plasmid mediated genes such as sul1 and

sul2.

MDR- THE GLOBAL PICTURE

One of the first report of MDR was in 1988, when there was an

outbreak of typhoid fever in the Kashmir valley involving 230 cases, 11

isolates of S.Typhi were multidrug-resistant(MDR); resistance to all first

line drugs simultaneously . In Shangai 22/142(15%) S.Typhi isolated

between 1988 and 1989 were MDR and this was shown to be carried on a

43

98MDa plamid which could be transferred to E.coli (Zhang,1991). In recent

reports during 2002 and 2003 isolates of S.Typhi from Malaysia and

Indonesia were found to be susceptible to all antibiotics, including Nalidixic

acid.

MDR - IN INDIA

After the 1988 outbreak in Kashmir valley including 230 cases, a

number of reports of MDR salmonella were reported from different parts of

the country. The maximum number of MDR isolates was seen in Central

India(71%) whereas it was least in the South(55%) [83].

In 1989 MDR S.typhi was present in Eastern India associated with a

120 MDa plasmid [84]. In 1996, Harish et al reported 33% MDR isolates of

S.Typhi, in Pondicherry. Madhulika et al., in 2003 reported 38.8% MDRST

from Pondicherry. Padma Krishnan et al., in Chennai, reported 12% of

S.Typhi isolates were found to be MDR out of a total of 50 isolates showing

a significant decline [85]. .In contrast a study in Kerala by Ayana et al in

2007, showed no isolates of MDRST [86] .

FLUROQUINOLONES [87]

The answer to MDR came with the discovery of Nalidixic acid, the

prototype of the quinolones in 1962.The second generation quionolones

44

with the addition of a fluoride atom solved the problem with a broadened

spectrum of activity including Gram positives and a good systemic action.

Mechanism of action

The important targets of fluoroquinolones are bacterial enzymes

DNA gyrase and DNA topoisomerase IV with 2 pairs of subunit. DNA

gyrase is a tetramer with 2 subunit, gyr A and gyrB (A2B2) encoded by gyr

A and gyr B genes respectively. DNA gyrase is responsible for introducing

negative supercoils into DNA and for relieving topological stress during

replication. The active site of the enzyme is located at the 122nd aminoacid,

which is usually tyrosine. Subunits of topoisomerase IV and parC and par E

(A2B2), encoded by parC and pare genes respectively, associated with

decatenating the daughter replions.

Quinolones bind to DNA gyrase or topoisomerase IV and induce a

conformational change in the enzyme causing it to break the DNA and

prevent relegation by forming a quinolone-DNA-enzyme complex.

Fluoroquinolones are generally bactericidal but at higher concentrations,

they are bacteriostatic attributed to a number of factors, including reduced

RNA synthesis. At concentrations below 0.5 µg/ml in vivo, Ciprofloxacin

leaves a residual population of Ciprofloxacin susceptible cells whose growth

is inhibited by the drug are not killed. Adaptive mutations may occur in vivo

under conditions of reduced ciprofloxacin concentrations, which are likely

45

to occur following cessation of treatment, when the drug is eliminated

slowly over a few weeks.

Mechanism of resistance

Resistance to FQs is by two mechanisms, target and non target.

Alteration in the target enzymes, DNA gyrase and topoisomerase IV is an

important mechanism of resistance. Other non target mechanisms include

decreased accumulation either by efflux or by decrease uptake of drug and

plasmid mediated quinolone resistance.

Quinolone resistance in Salmonella is usually associated with

mutation in DNA gyrase mostly in the QRDR region of the A subunit.

Plasmid mediated resistance genes of qnr (qnr A, qnr B, qnr S, qnr D) and

qnr S1 have been described. Point mutations in QRDR region spanning

amino acids 67 to 106 confers resistance to Nalidixic acid and reduced

susceptibility to FQs. Most of the mutation occur at the position 83 where

the aminoacid is usually serine designated Ser 83, through threonine maybe

found in some; Thr83. Some mutation also occur at position 87. Substitution

of the aminoacids would alter the affinity and therefore the susceptibility to

quinolones. Substitutions to Tyr, Phe or Ala have been identified at codon

83, and to Asp, Gly, TYR, or Lys at codon 87. These show different levels

of decreased susceptibility to quinolones.

46

Topoisomerase IV is considered a secondary target for quinolones in

salmonella. Quinolone resistance mutations in parC occur at codon Ser 80

or less frequently at codon Glu84 and are invariably associated with gyrA

mutation that give phenotypic expression for parC mutation [88] .

In 2003, parC mutations were reported in Salmonella isolates which

were both sensitive or had reduced susceptibility to ciprofloxacin. Therefore

all parC mutations do not appear to play a role in resistance. Isolates with a

single gyrA mutation were less resistant to fluroquinolones than those with

an additional par C mutation (Tyr57 Ser or Ser80 Arg), while those with

double gyrA mutations were more resistant [ 89] .

Efflux pump decreases the accumulation of drug inside the bacterial

cell due to increased efflux , caused by mutation leading to over-expression

of AcrAB-TolC efflux pump.

NARST AND CIPROFLOXACIN

A few years after the introduction of fluoroquinolones as therapy for

typhoid fever, treatment failure was reported with ciprofloxacin. In

1992,from UK, Rowe et al reported that decreased susceptibility to

fluoroquinolones , often associated with Nalidixic acid resistant ( NAR

).Isolates fully susceptible to ciprofloxacin by disc testing typically have a

ciprofloxacin MIC 0.03 µg/ml and are susceptible to nalidixic acid. It was

47

soon observed that a number of isolates with a MIC of 0.125-1.0µg/ml that

were susceptible to ciprofloxacin by disc diffusion were associated clinical

failure i.e no remittance after 7 days of treatment. These were resistant to

nalidixic acid both by disc diffusion and MIC (32µg/ml). they were termed

NAR or NARST [7,8] .

NAR IN INDIA

Kadhiravan et al (2005) did a study on 60 blood culture –proven

typhoid fever patients. All the 60 isolates were sensitive to ciprofloxacin by

disc diffusion testing and had MIC of 0.016 to 2 mcg/ml. 37% of isolates

were MDRST and 78% showd resistance to NA. Study showed Nalidixic

acid resistance was 82% sensitive and 100% specific for identifying isolates

low level resistance to ciprofloxacin. Authors concluded that fluroquinolone

breakpoints of NCCLS guidelines need to be redefined and quinolones

cannot be used as a first line drugs against S.Typhi infection.

Harish et al (2006) in a prospective study obtained 51 Salmonella

isolates from blood samples of 629 clinically suspected enteric fever cases

between 2004-2005. Of the 51 salmonella, 27(53%) were S.typhi and 24

(47%) were S.paratyphi A. Among 27 isolates of S.typhi only 2were

sensitive to Nalidixic acid and rest were resistant to nalidixic acid. All the

NARST strains had MIC of ≤1µg/ml for ciprofloxacin, ofloxacin and

gatifloxacin. Among the 24 NAR S.paratyphi A serotypes, 20 had MIC of

48

≤1µg/ml for all the three quinolones,while 4 had ciprofloxacin MIC

between 8-32µg/ml. Authors concluded because of increased resistance to

fluoroquinolones and high prevalence S.Paratyphi may emerge as the main

cause of enteric fever in India.

PEFLOXACIN - AS A SURROGATE MARKER FOR QUINOLONE

RESISTANCE

Earlier in 2012 the interpretative breakpoints for ciprofloxacin had

been revised, where the susceptibility cut off using disc diffusion was raised

from 21 to 31 mm and the MIC value was lowered from 1 to 0.06 µg/ml. In

2013, the disc diffusion interpretative criterion of levofloxacin and

ofloxacin for S.Typhi was removed . Meanwhile , the MIC interpretative

criteria for levofloxacin and ofloxacin have been lowered to ≤ 0.12 µg/ml

susceptible, 0.25-1 µg/ml intermediate and ≥ 2 µg/ml resistant.

Various studies globally reported false negative results of

ciprofloxacin susceptibity while using Nalidixic acid as a surrogate

marker[90,91]. Nalidixic acid does not detect plasmid mediated reistance of

fluoroquinolones.

In 2015, CLSI and The European Committee on Antimicrobial

Susceptibility Testing (EUCAST) have recommended the use of 5 µg

Pefloxacin disc diffusion test as reliable surrogate marker to identify

49

fluroquinolone susceptibility to S.Typhi [ 92,93] . Pefloxacin is understood to

identify chromosomal (gyrA, gyrB, parC and pare ) ; plasmid ( qnrA, qnrB,

qnrS) mediated fluoroquinolone resistance better than Nalidixic acid and

Ciprofloxacin by disc diffusion. In addition , using pefloxacin can avoid

the testing of Ciprofloxacin by disc diffusion and MIC distribution of

levofloxacin , ofloxacin and ciprofloxacin [92].

Materials & Methods

50

MATERIALS AND METHODS

This study was conducted at the Institute of Microbiology and

Institute of Child Health and Hospital for Children , Egmore, Chennai- in

association with Institute of Internal Medicine, Madras Medical College &

Rajiv Gandhi Government General Hospital . Molecular characterisation

was done at Department of Clinical Microbiology, Christian Medical

College ,Vellore .

Study Design : Cross-sectional study.

Study Duration : One year (June 2016-May 2017).

Ethical Consideration :

All patients satisfying the inclusion criteria will be documented , and

taken up for the study after obtaining informed written consent in both

regional language and English. This study was reviewed and approved by

Institutional Ethics Committee .

Sample size : 257 patients.

Sample size ( n) calculated using the formula,

n = Z2 x P(1-P ) / d2

where, Z = statistics for a level of confidence [ 1.96 ], P = expected

prevalence [ 9 %], d=absolute precision [ 3.5].

51

Statistical Analysis : Descriptive statistics , SPSS version 21.

INCLUSION CRITERIA :

Febrile children and young adults in the age group of 5-24 years

admitted as inpatients and outpatients with one or more of the following

criteria will be included.

Fever more than 3 days duration

Symptoms of Enteric fever such as abdominal pain, altered bowel

habits, nausea, vomiting, and signs such as pyrexia,toxic look, coated

tongue with sparing of margins, splenomegaly, hepatomegaly,

hepatosplenomegaly.

EXCLUSION CRITERIA:

Patients who were on antibiotics for the past one week .

Patients with fever accompanied by other localising signs

&symptoms.

SAMPLE COLLECTION AND TRANSPORT :

Venous blood was collected under aseptic precautions from patients

and clinically evaluated for Enteric fever.10 ml of blood sample collected

from adult patients and in children blood sample collected based on their

body weight in Brain Heart Infusion broth ( BHI ) transported safely and

rapidly to the laboratory for conventional blood culture [ 25,64] .

52

Preparation of site :

1. Peripheral vein to be drawn was chosen and disinfected using 70%

alcohol.

2. Skin over the venipuncture site was cleansed with 70 % alcohol in a

circular fashion, approximately 5 cm in diameter , rubbed vigorously

and allowed to air-dry.

3. Starting in the centre of the circle , 2% tincture of iodine was applied

in ever-widening circles until the entire circle has been saturated with

iodine, it was then allowed to dry for 1minute.

4. Sterile needle was inserted into the vein and blood was drawn ,

transferred to 50 ml of BHI broth, making 1: 5 or 1:10 dilution of

blood in broth.

5. After the sample was collected , the site should be cleansed again

with 70% alcohol.

Time of collection : [24]

Blood was collected from patients during febrile episodes, and before

the antibiotic therapy was initiated.

53

SAMPLE PROCESSING :

Incubation Conditions :

Blood culture bottles was incubated aerobically at 370 C for 18-24

hours. All the blood culture bottles were examined for evidence of growth

(hemolysis , turbidity ) during 6 - 18 hours of incubation. Blind subcultures

were done on Nutrient agar plate, Mac Conkey agar plate,5% Sheep Blood

agar plate, after 24 hours of incubation .Then further subcultures were

done after 48 hours, 72 hours and 1 week of incubation .

IDENTIFICATION OF SALMONELLA : [31,33]

COLONY MORPHOLOGY :

On Nutrient agar plate colonies of Salmonella species were large

2- 3 mm , moist, translucent, low convex, discrete colonies with smooth

surface with entire edges.

On Blood agar, Salmonella species form moist greyish non

haemolytic colonies and on MacConkey agar plate produce lactose non

fermenting colonies.

Colonies morphologically resembling Salmonella species were

subjected to preliminary test- Gram stain , Motility by Hanging drop

method, Catalase and Oxidase test.

54

GRAM STAIN :

Salmonella was gram negative bacilli measuring approximately 2-4 x

0.6 µm, uniformly stained with parallel sides and rounded ends, non-

capsulated, non-sporing.

Presumptive identification of the isolates were done using standard

biochemical tests such as Hugh Leifson’s oxidative fermentative test,

Nitrate reduction test, Indole production, Methyl red and Voges Proskaeur

reaction, Citrate utilization ,Urease production, Phenylpyruvic acid test, 1 %

carbohydrate fermentation test for Glucose, Lactose, Xylose, Arabinose and

Moeller’s Decarboxylation test [Table 3,4].

SLIDE AGGLUTINATION TEST : [31]

Confirmation of the isolates were done by Slide agglutination test

using specific antisera- Polyvalent ‘O’ antisera, and Salmonella Typhi ‘H’ ,

Salmonella Paratyphi ‘AH’ and ‘BH’ ( Institute of Preventive Medicine,

Guindy ).

Procedure :

1) A sterile, grease free glass slide was taken and an identification

linewas drawn in the upper surface of the slide, and two circles

measuring 2 x 1 cm were drawn underneath, and labelled as Control

and Test.

55

2) Using a sterile inoculating loop portion of pure growth from a fresh

subculture in a non-selective medium (Nutrient agar plate) was taken

and emulsified in a drop of physiological saline (20 µL) and mixed

thoroughly in a ‘Control’ ring.

3) Rocked the slide back and forth and observed for any

autoagglutination under a bright light and over a black background .

4) The saline suspension was carefully examined to ensure that it is

even and does not show any clumping . If still autoagglutination

occurs ,the culture is cannot be serotyped.

5) Test was further proceed if there is no autoagglutination .

6) Emulsified a portion of pure growth in a drop of physiological saline

in ‘Test’ ring and a drop of (equal volume ) of Polyvalent ‘O’

antisera was added and mixed well.

7) Tilt the slide back and forth and observed for agglutination

.Clumping was seen within 30 seconds to 1 minute, if the reaction

was positive, .

8) Similarly the test was proceeded for flagellar antigens , using

polyvalent ‘H’ antisera for S.Typhi isolates, ‘AH’ for S.Paratyphi A

and ‘BH’ for S.Paratyphi B.

56

ANTIBIOTIC SUSCEPTIBILITY TESTING : [94]

KIRBY-BAUER DISC DIFFUSION METHOD :

Antibiotic susceptibility testing of the isolates was done by Kirby-

Bauer disc diffusion method according to Clinical Laboratory Standards

Institute (CLSI -2016) guidelines for the following drugs - Ampicillin

(10 µg), Chloramphenicol (30µg), Cotrimoxazole (1.25/23.75 µg).

Ciprofloxacin (5µg), Nalidixic acid(30 µg),Pefloxacin(5 µg ),Cefotaxime

(30 µg),Ceftriaxone (30 µg), Azithromycin(15 µg).

3-4 well isolated , morphologically similar colonies were taken with

a sterile loop and inoculated into peptone water and incubated at 370 C for 2

hours. Turbidity was adjusted to 0.5 McFarland standards and a lawn

culture was made on Muller-Hinton agar and appropriate antibiotic discs

were placed. Plates were incubated at 370 C for 16-18 hours.The zones of

inhibition were measured and interpreted according to CLSI 2016

guidelines-M100- S26 document. Quality control was done using ATCC

E.coli 25922 strain.

57

TABLE : KIRBY-BAUER DISC DIFFUSION METHOD

Antibiotic

Disc content (in

µg )

Diameter of Zone of inhibition (in mm)

Susceptible Intermediate Resistant

Ampicillin 10 17 14-16 13

Chloramphenicol 30 18 13-17 12

Cotrimoxazole 1.25/23.75 16 11-15 10

Nalidixic acid 30 19 14-18 13

Ciprofloxacin 5 31 21-30 20

Pefloxacin 5 24 - 23

Cefotaxime 30 26 23-25 22

Ceftriaxone 30 23 20-22 19

Azithromycin 15 13 - 12

MINIMUM INHIBITORY CONCENTRATION ( MIC ) : [ 94,95,96]

Determination of Ciprofloxacin minimum inhibitory concentration

(MIC) by using broth-micro dilution method (BMD) :

Broth- microbroth dilution (BMD) is a technique in which a bacterial

suspension at a predetermined concentration is tested against various

concentrations of antimicrobial agent in a liquid medium with a

predetermined formulation as per Clinical Laboratory Standards Institute

(CLSI) guidelines –M07-A10 document. MIC of the Salmonella isolates

for the drug Ciprofloxacin monohydrate(HiMedia) by Broth Micro Dilution

method.

58

WEIGHING ANTIMICROBIAL POWDER :

1000 X Volume (ml) X Concentration (µg/ml) Weight (mg) = ------------------------------------------------------------ Potency (µg/mg) P = Potency of the antibiotic base, 980 µg/mg

V = Volume of distilled water required, 10 ml

C = Final concentration of the solution, 1024 µg/ml

W = Weight of the antibiotic substance to be dissolved in distilled

water,10.44 mg.

PREPARATION OF ANTIBIOTIC STOCK SOLUTIONS :

A stock solution containing 1024 ug/ml of the antibiotic to be tested

was prepared i.e., 10.44 mg of antibiotic powder was weighed and dissolved

in 10 ml of distilled water. The required dilutions of the antibiotics were

made by doubling dilutions from 1024 µg /ml to 128 µg/ml . Range of

concentration of antibiotics was decided by breakpoints determined by CLSI

2016 guidelines .

QUALITY CONTROL STRAINS :

Positive control-.ATCC E.coli 25922 ( QC range 0.25- 1 µg/ml)

59

INOCULUM PREPARATION :

1. Inoculate 2- 3morphologically similar colonies of 24 hours pure

growth from nutrient agar plate into 1.5 ml nutrient broth. Incubate at

370 C for 2 hours and then adjust to 0.5 McFarland standards.

2. 100 µl of 0.5 McFarland adjusted inoculum is transferred to 2 ml of

physiological saline and used as final inoculums for performance of

MIC testing.

PROCEDURE :

1. 96 well microtitre plate was chosen and labelled for Quality control

strain ( Positive control- E.coli ATCC), Test isolates and Sterility

control.

2. 100 µl of Cation Adjusted Muller Hinton Broth (CAMHB ) was

added in all the wells .

3. First column in microtitre plate was the Growth control for the

isolates containing 100 µl of sterile drug free CAMHB, and 10 µl of

corresponding inoculum suspension.

4. Last row was the sterility control of medium containing 100 µl of

CAMHB.

5. 100 µl of 128 µg/ml of stock solution was added in second column

containing 100 µl of CAMHB, and serially diluting to further wells

60

from higher to lower concentrations (labeled as 128 µg/ml t0 0.06

µg/ml).

6. 10 µl of the final inoculum was added to the well of microtitre plate.

7. 10 µl of final inoculums was streaked on nutrient agar plates for

purity check.

8. Nutrient agar plate and sealed microtitre plates were incubated at 37 0

c for 16-18 hours

INTERPRETATION OF RESULTS :

1. Purity of inoculum suspension in Nutrient agar plate was checked .

2. The growth in positive control well was checked .

3. The growth ( turbidity ) in Growth control well of the corresponding

isolates were confirmed.

4. Minimum inhibitory concentration (MIC) was read as the lowest

concentration of the antibiotic without visible growth / turbidity.

MOLECULAR ANALYSIS OF CIPROFLOXACIN RESISTANCE

GENES: [ 97,98 ]

Mutational analysis in Quinolone –resistance –determining regions

(QRDR) of the DNA gyrase subunit II genes gyrA,gyrB and parC gene of

DNA topoisomerase subunit IV from Salmonellae isolates with decreased

susceptibility or resistant to ciprofloxacin were studied by performing

Polymerase chain reaction (PCR) and DNA sequencing.

61

Genomic DNA Extraction :

1. Pure growth from 24 hours culture from nutrient agar plate was

emulsified in 200 µl of physiological saline and vortexed .

2. DNA extracted using Automatic DNA extracter-QIA symphony SP

(Qiagen-Boston).

UNIPLEX PCR DETECTION OF gyrA , gyrB and parC GENE:

Uniplex PCR was performed with the Applied Biosystems VeritiTM

96-well Thermal cycler (Thermo Fisher Scientific ). For each of the DNA

sample a reaction mix was prepared with the following components.

REACTION MIX COMPOSITION :

S.No COMPONENTS REACTION VOLUME FOR EACH CYCLE

1 Master mix 10µl

2 MilliQ water 4µl

³ Q solution 2µl

4 Primer mix 2µl

5 DNA Template 2µl

62

CYCLING CONDITIONS :

Cycling conditions for 30 cycles run- gyrA,gyrB,and parC -

1 Initial Denaturation 920 C 5 min

2 Denaturation 920 C 1min

3 Annealing 620 C 1min

4 Extension 740 C 2min

5 Final Extension 740 C 1min

PRIMERS : [99]

Antimicrobial Resistance gene sequence for Salmonella species.

S.No Oligo Name 5 < -----------Sequence ----------- >3 Base Pair

1 gyrA F TGTCCGAGATGGCCTGAAGC 347 bp

2 gyrA R TACCGTCATAGTTATCCACG

3 gyrB F CAAACTGGCGGACTGTCAGG 345 bp

4 gyrB F TTCCGGCATCTGACGATAGA

5 parC F CTATGCGATGTCAGAGCTGG 270 bp

6 parC R TAACAGCAGCTCGGCGTATT

AGAROSE GEL ELECTROPHORESIS :

PCR products were analysed using gel electrophoresis. 1% (w/v)

Agarose gel was prepared using 1 x TAE buffer (Sigma-Aldrich Pvt.

Ltd.,India) and 6µL of Ethidium bromide was added. The molten agarose

gel was cast in the electrophoresis tray with combs in place.

63

After solidifying, the cast was placed in the electrophoresis tank with

the buffer. 5µl DNA molecular weight marker (100 bp DNA ladder) was

loaded in one slot, and positive control ( Salmonella Typhi ATCC® 19430

TM ) in another slot . 5µl of PCR products were loaded in the respective

slots. Electrodes were connected and set a voltage of 100 V ; 400 mA for 1

hour.

On Gel documentation in (Bio-Rad,Hercules,California,USA)

presence of bands were observed at 347 bp, 345 bp, and 270 bp indicates

the presence of gyrA, gyrB and parC genes respectively in all the isolates

and in positive control strain .

DNA SEQUENCING :

Principle:

Sequencing of the amplicons was carried out by the

dideoxynucleotide chain-termination method, using an automated DNA

sequencer ABI PRISM 3500 Genetic Analyser (Applied Biosystems, USA ).

Pre-Clean Up Procedure :

Pre-clean up of the PCR products were done using QIA Quick PCR

Purification kit.

1. Add 50 µl of Buffer PB to 10 µl of PCR product and mix in a

QIA quick column.

64

2. Centrifuge – 30,000 rpm for 1 minute and discard the supernatant .

3. Wash with 750 µl Buffer PE and then centrifuge -30,000 rpm for 1

minute and discard the supernatant.

4. To elute the DNA add 30 µl of Elution buffer, let the column stands

for 1 minute and then centrifuge.

DNA TEMPLATE QUALITY:

Poor template quality was the most common cause of sequencing

problem.

Examining DNA Quality :

1. Spectrophotometric method :

Checked DNA template concentration and purity using Nano drop

method . This method reveals any protein contamination.1 µl of PCR

product was loaded in Thermo Scientific µDropTM plate and analysed

using SkanIT- software tool ).16 samples can be analysed at a time. If the

concentration was more than 15 µl, it was diluted with equal volumes of

MilliQ water accordingly.

2. Agarose gel electrophoresis and documentation :

Agarose gel electrophoresis reveals contaminating DNAs and

RNAs. DNA templates were subjected Agarose gel electrophoresis and

documentation along with a positive control ( Salmonella enterica

65

subspecies enterica serovar Typhi ATCC® 19430 TM ) and a molecular

ladder (100 bp ) which showed single band at 347 bp, 345 bp, and 270 bp

indicates the presence of gyrA, gyrB and parC genes respectively in all the

isolates and in positive control strain .

SEQUENCE PCR :

SEQUENCING REACTION DILUTIONS :

S.No COMPONENTS REACTION

VOLUME FOR FULL CYCLE

REACTION VOLUME FOR

16th CYCLE

1 Ready Reaction Mix 8 µl 0.5 µl

2 Dilution Buffer - 1.75 µl 3 Template(200 ng/µl) 1 µl 1 µl 4 Primer(2 pmol/µl) 8 µl 0.5 µl

5 MilliQ water 7 µl 4.75 µl

6 Total 20 µl 10 µl

CYCLING CONDITIONS :

1 Initial Denaturation 96 22 sec

2 Denaturation 96 10 sec

3 Annealing 50 5 sec

4 Extension 60 4 min

5 Final Extension 4 4 min

Amplicons obtained were subjected to Post-clean up procedure .

66

POST-CLEAN UP PROCEDURE ( TUBE METHOD ) :

Using Big dye terminator Vs 3.1 clean up –Tube method post clean

up were done .

The reaction product were transferred to 1.5 ml tube, 12 µl of Master

mix I was added to 10 µl of amplicons per reaction. Then 52 µl of Master

mix II was added and the contents were mixed well and incubated at room

temperature for 15 minutes. Spin at 12000 rpm for 20 minutes at room

temperature. The supernatant were decanted .

Then 250 µl of 70 % freshly prepared ethanol was added and spin at

12000 g for 10 minutes at room temperature. Supernatent discarded and 12

µl of elution buffer ( Hi –Di formamide ) was added . The sample tubes

were covered with septa , denatured , snap chilled and proceeded with

Capillary electrophoresis.

After the completion of the procedure, electrophorogram pattern

obtained and analysed with the reference sequence .

Reference sequences of S.Typhi for all QRDR genes (gyrA,gyrB and

parC) were obtained from http://www.ncbi.nlm.nih.gov/gene ., with

following accession numbers : gyrA- CAD07504, gyrB-NC _003198,parC-

NC_003198.1.

67

Sequence chromatograph files were analysed using software tool.

Forward and reverse DNA sequences were assembled using EMBOSS

merger software, nucleotide ambiguities were resolved by NUCLEOTIDE

massager tool and alignment of sequences were done by CLUSTAL

OMEGA software tool. Nucleotides were translated to proteins using

ExPASY Protein software and analysed for mutation by comparing with the

references.

Results

68

RESULTS

TABLE 1 : DISTRIBUTION OF PATIENTS IN THE STUDY

POPULATION (n=257)

STUDY SITE No.of Patients Percentage (%)

RGGGH 157 61.1

ICH 100 38.9

Total 257 100.0

61.1% were young adults and 38.9% were children

FIGURE: 1 DISTRIBUTION OF PATIENTS IN THE STUDY

POPULATION ( n=257)

RGGGH61%

ICH39%

Study Site Distribution

69

TABLE 2 : AGE GROUP AND SEX DISTRIBUTION

IN THE STUDY POPULATION

STUDY POPULATION

AGE GROUP (n=257)

GENDER TOTAL

MALE FEMALE

CHILDREN 5-7 22 29 51 8-10 18 18 36

11-12 11 4 15

YOUNG ADULTS

13-17 16 14 30 18-24 73 52 125

TOTAL 140 117 257

Mean age group is 15.27 , Standard deviation is 6.66.

Both genders were equally affected among children.

Male preponderance was observed among young adult population.

FIGURE: 2 AGE GROUP AND SEX DISTRIBUTION IN THE

STUDY POPULATION

2218

1116

73

29

18

4

14

52

5 - 7years 8 - 10years 11 -12years 13 - 17years 18- 24years

Age Group and Gender DistributionMale Female

70

TABLE 3 : DISTRIBUTION OF PATIENTS BASED ON HOSPITAL

ADMISSION

INPATIENTS/ OUTPATIENTS No.of patients Percentage (%)

INPATIENTS 177 68.9

OUTPATIENTS 80 31.1

Total 257 100.0

68.9% were admitted and treated as inpatients.

TABLE 4 : CARDINAL SYMPTOMS AMONG STUDY

POPULATION

SYMPTOMS Young adults (n = 157)

Percentage (%)

Children ( n=100)

Percentage (%)

Fever with abdominal pain 27 17.2 7 7

Fever with abdominal pain and Diarrhoea 52 33.1 68 68

Fever with abdominal pain, Diarrhoea,

Nausea and vomiting 49 31.2 25 25

Fever with constipation 13 8.3 0 0

Fever with nonspecific symptoms 16 10.2 0 0

The predominant symptom among Enteric fever suspected febrile

patients were fever, abdominal pain and diarrhoea .

71

TABLE 5 : CARDINAL SIGNS AMONG STUDY POPULATION

CARDINAL SIGNS Young Adults

Percentage (%) Children Percentage

(%)

Pyrexia,toxic look , pallor 61 38.9 47 47

Pyrexia, toxic look, coated tongue 74 47 44 44

Pyrexia, toxic look, coated tongue,hepatomegaly 0 0 2 2

Pyrexia, toxic look, coated tongue,Splenomegaly 5 3.2 2 2

Pyrexia, toxic look, coated tongue,hepatosplenomegaly. 3 1.9 3 3

Pyrexia,pallor 14 8.9 2 2

The predominant sign among the study population were pyrexia,

toxic look and coated tongue.

TABLE 6 : DISTRIBUTION BASED ON FOOD HABITS ( n=257)

H/O EATING FOOD

OUTSIDE Young adults (n=157) Children (n= 100)

YES 76 (48.4%) 25 ( 25 %)

NO 81 (51.6% 75 ( 75%)

72

TABLE 7 : DISTRIBUTION OF BACTERIAL GROWTH IN

BLOOD CULTURE

BACTERIAL GROWTH

YOUNG ADULTS (n=157)

CHILDREN (n=100)

TOTAL (n=257)

Growth 10 5 15 (5.9 %)

No growth 147 95 242 (94.1 %)

The isolation rate by blood culture method was 5.9%

TABLE 8 : DISTRIBUTION OF PATHOGENS ISOLATED FROM

BLOOD CULTURE

PATHOGENS ISOLATED

YOUNG ADULTS

(n =157) n(%)

CHILDREN ( n=100) n(%)

TOTAL (n=257) n(%)

S.Typhi 6 (3.82 %) 5 (5%) 11 (4.3%)

S.Paratyphi 1 (0.64%) - 1 (0.4%)

E.coli 2 (1.27%) - 2 (0.77%)

Klebsiella pneumonia 1 (0.64%) - 1 (0.4%)

Salmonella species isolated from conventional blood culture in 12

cases (4.7%). Among this ,7 (4.45 %) isolated from young adult population

and 5 isolates (5 % ) from children.

73

TABLE 9 : MONTHWISE DISTRIBUTION OF SALMONELLA

ISOLATES ( n=12)

MONTH No. Of isolates Percentage (%)

June 3 25.0

July 3 25.0

August 3 25.0

September 1 8.3

October 1 8.3

April 1 8.3

Maximum isolation of was observed from June to August.

FIGURE : 3 SEASONAL DISTRIBUTION OF ENTERIC FEVER

3 33

1 11

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5 11

Month

74

TABLE 10 : DISTRIBUTION BASED ON FOOD HABITS AND

TYPHOID FEVER

STUDY SITE Outside food habits S.Typhi S.Paratyphi

A TOTAL

RGGGH Yes 6 0 6

No 0 1 1

ICH Yes 1 0 1

No 4 0 4

TOTAL 11 1 12

Among Young adults, eating outside food was significantly

associated with Typhoid fever due to S.Typhi and S.Paratyphi A with ‘ p’

value 0.021 (p < 0.05- significant ) by Fisher’s Exact Test . But this

association was not statistically significant among children.

TABLE 11 : ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

S.TYPHI AND S.PARATYPHI A BY DISC DIFFUSION METHOD

( % of Susceptibility )

SALMONELLA SPECIES

AMPI 10 µg

COT 25 µg

CK 30 µg

CTX 30 µg

CFT 30 µg

AZIT 15 µg

NA 30 µg

PEF 5 µg

CIP 5 µg

S.TYPHI ( n= 11) 100 100 100 100 100 100 0 0 0

S.PARATYPHI A (n=1 ) 100 100 100 100 100 100 0 0 0

75

FIGURE 4 : ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

S.TYPHI (n=11) AND S.PARATYPHI A (n=1) BY DISC DIFFUSION

METHOD

Abbrevation: AMPI – ampicillin, COT- cotrimoxazole, CK-

chloramphenicol , CTX-cefotaxime , CFT-ceftriaxone , AZIT-

azithromycin, NA- nalidixic acid, PEF- pefloxacin , CIP-ciprofloxacin.

Antimicrobial susceptibity pattern of both S.Typhi and S.Paratyphi A

showed similar pattern .

All the 12 isolates were resistant to Nalidixic acid ( NAR) and

Pefloxacin. None of the isolates were MDR - based on their susceptibility

to Ampicillin , Cotrimoxazole , Chloramphenicol. All the isolates were

susceptible to Third generation Cephalosporins and Azithromycin.

12 12 12 12 12 12

8

12 12

4

0

2

4

6

8

10

12

14

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

S

I

R

76

TABLE 12 : DISTRIBUTION OF MIC OF CIPROFLOXACIN BY

MICROBROTH DILUTION METHOD

MIC CLSI interpretative criteria EUCAST interpretative criteria

CIPROFLOXACIN SUSCEP TIBLE

≤0.06 µg/ml

INTER MEDIATE 0.125-0.5

µg/ml

RESIS TANT

≥ 1 µg/ml

SUSCEP TIBLE

≤0.06 µg/ml

RESIS TANT

≥0.06 µg/ml

S.Typhi (n=11) 0 3 8 0 11

S.Paratyphi A ( n=1) 0 1 0 0 1

TOTAL (n=12) 0 4 (33.3%)

8 (66.7%) 0 12

(100%)

All the 12 Pefloxacin resistant isolates ( Surrogate marker of

Ciprofloxacin resistance ) were subjected to Ciprofloxacin MIC testing

by Brothmicro dilution method and was found to be 66.7 % resistant and

33.3 % intermediate using CLSI interpretative criteria , while all the 12

isolates were found to be resistant by interpretative criteria of

EUCAST .

77

TABLE 13 : RESULTS OF MOLECULAR CHARACTERISATION

OF TYPHOIDAL SALMONELLAE AND COMPARISON WITH

ANTIMICROBIAL SUCEPTIBILTY PATTERN (n=12)

Isolate ID

RESISTANT PHENOTYPE

[ NAL-PEF-CIP ] DISC

DIFFUSION

CIPRO MIC

(µg/ml)

MUTATION ANALYSIS OF QRDR GENES

Ciprofloxacin MIC

Interpretation

gyrA gyrB parC CLSI EUCAST

ST 1 NAL-PEF 0.125 + - - I R

ST 2 NAL-PEF 2 + - - R R

ST 3 NAL-PEF 4 + - + R R

ST 4 NAL-PEF 2 + - - R R

ST 5 NAL-PEF 2 + - + R R

ST 6 NAL-PEF 1 + - - R R

ST 7 NAL-PEF-CIP 0.125 + - - I R

ST 8 NAL-PEF-CIP 4 + - + R R

ST 9 NAL-PEF 0.125 + - - I R

ST 10 NAL-PEF 2 + - + R R

ST 11 NAL-PEF-CIP 1 + - - R R

ST 12 NAL-PEF 0.125 + - - I R

All the Pefloxacin resistant Salmonella isolates were found to be

Ciprofloxacin resistant by MIC according to EUCAST interpretative

criteria, which was further confirmed by molecular characterisation of

QRDR gene mutation.

78

TABLE 14 : RESULTS OF SEQUENCE ANALYSIS OF QRDR

GENE AMONG SALMONELLA ISOLATES ( n=12)

MOLECULAR CHARACTERISATION OF QRDR GENES BY DNA SEQUENCING

Sample ID GyrA gyrB ParC

ST1 Serine (83) – Phenylalanine No mutation No mutation

ST2 Serine (83) – Tyrosine No mutation No mutation

ST 3

Serine (83) – Phenylalanine,

Aspartic acid (87) – Asparagine

No mutation Serine (80) – Isoleucine

ST4 Aspartic acid (87) – Asparagine No mutation No mutation

ST5 Serine (83) – Phenylalanine No mutation Serine (80) –

Isoleucine

ST³ Aspartic acid (87) Asparagine No mutation No mutation

ST7 Serine (83) – Phenylalanine No mutation No mutation

ST8

Serine (83) – Phenylalanine,

Aspartic acid (87) – Asparagine

No mutation Serine (80) – Isoleucine

ST9 Serine (83) – Phenylalanine No mutation No mutation

ST10

Serine (83) – Phenylalanine,

Aspartic acid (87) – Asparagine

No mutation Serine (80) – Isoleucine

ST11

Serine (83) – Phenylalanine, Glycine (133) – Glutamic acid

No mutation No mutation

ST12 Serine (83) – Phenylalanine No mutation No mutation

Most common mutation observed was Ser 83-Phe followed by Asp87-Asn

in gyrA gene.

Discussion

79

DISCUSSION

This study was performed on 257 Enteric fever suspected febrile

patients with an objective to isolate and determine the antimicrobial

susceptibility pattern of Typhoidal Salmonellae among school aged

children and young adults. Among 257 patients , 157 (61%) young adults

and 100 (39%) children were included in the study [ Table 1] .

Among those febrile patients 177 ( 68.9%) were admitted and treated

as inpatients and the remaining 80 patients ( 31.1%) were treated as

outpatients.[ Table 3].

The predominant symptoms observed were fever, abdominal pain

and diarrhoea in children (68 %) and adult population (33 %) [Table 4].

The predominant signs were pyrexia, toxic look and coated tongue in

children (44%) and adults (47%) [59,60] [Table 5] .

Authors from various parts of the world have done similar studies

among enteric fever patients . Stuart et al., found that school aged children

or young adults between 5-25 years of age were the most commonly

affected age group in the areas of endemicity [ 101,102,103] . He also observed

that the predominant symptom was fever (75%) followed by abdominal

pain ( 40%). Scragg et al., observed that diarrhoea was the predominant

symptom in children .

80

Crump et al.,observed that poor sanitation and lack of access to safe

water and food is the major risk factor for transmission of Typhoidal

Salmonella [ 1 ] . Oslen et al., observed asymptomatic carriage infood

handlers plays a major role in transmission of typhoid bacillus [106] .

In this study 48.4 % of adults and 25 % of children with febrile

illness had a history of eating outside foods such as ice cream , fruit salads ,

soft drinks , flavoured milks , sweets and food from local street vendors in

unsanitary conditions using water of questionable quality [Table 7 ]. None

of the patients included in the study were vaccinated against Typhoid fever

Conventional blood culture was done for 257 patients suspected to

patients to isolate and perform antimicrobial susceptibility testing .

Among these, eleven Salmonella Typhi ( 4.3 % ) and one Salmonella

Paratyphi A ( 0.4 % ) were isolated [ Table 9 ]. In India, varying prevalence

of typhoid fever has been reported over a period of time and geographical

distribution. The systematic review and meta analysis study done in 2016

reported the prevalence of Salmonella Typhi to be about 9.7 % and that of

Salmonella Paratyphi A was 0.9 %. [5] .

The World Health Organisation , in 2008 , reported the prevalence of

typhoid fever as 28.1 per 1000 febrile episodes in India.

81

Blood culture positivity is usually high during the first week of illness

but can also be isolated from subsequent weeks also if there is no prior

antibiotic therapy [2,25] . The low isolation rate of Salmonella species from

blood culture could be attributed to many factors- prior antibiotic therapy,

low bacterial count ( sometimes as low as one ) ,poor selection of cases,

volume of blood sampled [25] .

In this study ,the isolation rate in blood culture of Salmonella species

was 4.7 % ( n=12), with isolation of S.Typhi about 4.3 % (n=11)and that

of S.Paratyphi A (n= 1) being 0.4 % .

Among the 12 Salmonella isolates , 4.45 % ( n=7) were isolated from

young adults and 5 % ( n= 5) were isolated from children [Table 8 ].

Typhoid fever affects all age group and both the sexes are equally

affected.In the areas of endemicity the predominant age group affected are

school-aged children or young adults, within the age group of 5-25 years of

age.

Among children 5-7 (19.9%) years of age group were most

commonly affected with female preponderance ; in adult population studied

18-24 (48.6% )years of age were most commonly affected with male

preponderance, with mean age group was 15.27 [Table 2]. Mohanty et al,

observed that the most common age group affected were 5-19 years [9].

82

Typhoid fever is known to show seasonal variation and is mainly

associated with hot summer months. Many studies worldwide and Indian

studies showed peak incidence around the end of dry season .Mohanty et

al,observed peak incidence of enteric fever occurs between April and June

followed by July- September . In this study the peak isolation was between

June-August ( 75%) and there after decline in frequency to 8.3% for each

month, in September, October and March [9] .

The food habits of the patients with enteric fever was studied and

found that 58.3% of laboratory confirmed typhoid fever patients had the

history of having food items from outside street vendors. Eating food

prepared in unhygienic conditions and with untreated water is the major risk

factor for transmission. Also Levine et al, studied the role of chronic carriers

as a reservoir of infection in Santiago,where a crude rate of 694 carriers per

1,00,000 inhabitants was found [1,25] .

Similar to these observations , this study showed significant

association ( p <0.05 ) between H/O eating food outside and Salmonella

isolation among adult population.

MDR –defined as resistance to ampicillin , chloramphenicol and

trimethoprim-sulphamethoxazole., was reported from many countries

.Studies globally have found that 80% of S.Typhi isolates ( 1993-1996)

83

were MDR [107,108]. Currently in India, the MDRST varies from

7-55%[110,111] .MDR in Central India declined from 91% in 1991 to 22% in

2002[107,108].

Multicentric study in India, conducted by Dahiya.S et al,in 2014-

2015, observed the overall MDR prevalence was less than 3%. However ,in

each tertiary care hospital included in this study the MDR frequency were

found to be 6 % [109].

In this present study, all the 12 isolates were susceptible to

Ampicillin (100%), Chloramphenicol (100%)and Trimethoprim-

sulphamethoxazole (100%), and none of the isolates were Multidrug

resistant [ Table 12].

Overall there is a general trend of decline in MDR strains . The

reason could be due to discontinuation of these drugs in the treatment of

typhoid fever, thus relieving the selection pressure. Another reason could be

the loss of plasmids such as the R-plasmid that confer multi drug resitance ,

or emergence of de novo susceptible strains may also contribute for this

decline.

Ciprofloxacin has remained the drug of choice of enteric fever for the

last many years. However, isolates of S.Typhi and S.Paratyphi A with

reduced susceptibity to fluoroquinolones have now appeared in the

84

subcontinent , Vietnam and Tajikistan , and the treatment failures with

fluoroquinolones has also been noted.

In vivo adaptive mutations have been reported to occur on continuous

sub-inhibitory exposure to ciprofloxacin [ 112] . Also, the frequent use of

Ciprofloxacin for trivial infections is likely to promote resistance to these

agents.

In this present study,75% (9 /12) isolates were in the intermediate

category, 25% (3/12) isolates were resistant, and none of the isolates were

susceptible to ciprofloxacin by Kirby-Bauer’s Disc Diffusion Method

[Table 12 ]

When the isolates were subjected to MIC determination by Broth

micro dilution method , 33.3% of isolates (4/12) were in the intermediate

category, and 66.7% (8/12) became resistant. None of the isolates were in

the susceptible range of MIC based on CLSI interpretation [ Table 13 ].

This decreased fluroquinolone susceptibility was detected by using

Nalidixic acid in earlier days, since it was considered to be the surrogate

marker of fluroquinolone resistance. But, according to recent changes in

CLSI and EUCAST guidelines, Pefloxacin (5µg) disc has been suggested as

the best surrogate marker of fluoroquinolone susceptibility rather than

Nalidixic acid and Ciprofloxacin disc diffusion itself . Nalidixic acid disc

85

diffusion method does not detect all the possible mechanisms of

fluroquinolone resistance. But , Pefloxacin can detect all the possible

mechanisms of resistance except the one mediated by the plasmid aac(6´)-

lb-cr.

Fang et al., studied the fluoroquinolone resistance in Salmonella and

the utility of Pefloxacin disc diffusion in 2015, reported that Pefloxacin

disc diffusion provides better separation of ciprofloxacin susceptible and

non-susceptible strains than other disk diffusion, including ciprofloxacin

itself [121]. In this study all isolates were resistant to Nalidixic acid and

Pefloxacin , both showed 100% resistant pattern to all the 12 isolates

[Table 13].

Nine isolates ( 75% ) which were intermediate susceptibility to

ciprofloxacin were found to be resistant by Pefloxacin disc diffusion. This

was further confirmed by molecular characterisation of QRDR genes

(gyrA,gyrB and parC) [ Table 13 and14 ] [122] .

According to CLSI breakpoints, 33.3% (4/12) of isolates showed

intermediate susceptibilityto ciprofloxacin by MIC which were resistant by

Pefloxacin disc diffusion. This observation correlates well with

Ciprofloxacin MIC breakpoints of EUCAST guidelines .It is simple and

86

easy to perform Pefloxacin disc diffusion over MIC of fluroquinolones for

detecting its susceptibility [ 91,92,93 ] .

Quinolone resistance is mediated by chromosomal mutations in

QRDR genes- gyrA , gyrB and parC , parE. All the 12 isolates S.Typhi ( n

=11) and S.Paratyphi A ( n= 1) isolates which were Pefloxacin resistant

were subjected to PCR followed by Sangers sequencing of QRDR genes-

gyrA, gyrB and parC [ 113,114] .

Mutation in gyrA gene was identified in all the 12 isolates (100%) .

Four isolates (33%) harboured mutation in parC gene. None of the isolates

showed mutation in gyrB gene [83] .

Menezes et al, reported in a study conducted from 2005-2009 at

Pondicherry, out of 11 isolates tested with ciprofloxacin MIC ranging from

≥1 to ≤ 4 µg/ml , showed three mutations, two mutations within the QRDR

of gyrA (at codon 83 and 87) and a single point mutation at parC at codon

80 .No mutations were observed in gyrB and par E of any of the isolates

tested [11] .

Similarly ,in the present study, most common mutations observed

were at codon 83 where a C →T transition led to the substitution of

phenylalanine for serine .This single point mutation leads to low level

fluoroquinolone resistance , and didn’t cause considerable rise of MIC, and

87

the isolates were in the intermediate category (MIC -0.125µg/ml). While

treating this patient with higher dose of ciprofloxacin it might translate into

possible/probable clinical failure [114].

Renuka et al.,in 2004 reported that double or triple mutation in gyrA

or parC genes ,or even double mutations in gyrA cause high level

ciprofloxacin resistance in a study conducted in North India [113] .

Another mutation observed at codon 83 was substitution of Tyrosine

for Serine where C→A transition occured. But, this mutation caused

significant rise in MIC value and the isolate was resistant .

Five out of twelve isolates ( 41.7%) in this study had amino acid

substitution , Asparagine to Aspartic acid where G →A transition

occurs.This single point mutation cause significant elevation of MIC upto

1-2 µg/ml .When an isolate harbours double mutation, MIC values further

rise from 4 - >32 µg/ml .This observation is consistent with the reported in

studies of Balaji et al [114] .

Balaji et al, in 2015,studied 33 isolates with Ciprofloxacin

intermediate susceptibility range by doing molecular characterisation and

observed that both double and single amino acid substitutions were seen at

positions 83 and 87 in all the isolates for gyrA .No mutations were detected

for gyrB , while parC mutations were observed at 80 and 84 positions.

88

Double or triple mutation in gyrA cause significant increase in MIC value

rather than single point mutation .

Mutation at codon 133, has been rarely reported in literature.In this

study, single isolate of S.Typhi had a mutation at codon 133 ,where

Glutamic acid substitution for Glycine occured and was associated with

resistant range of MIC. Hamidian et al reported this mutation for the first

time ,in a study conducted at Iran in 2010 [115].

No mutation was observed in gyrB in all the 12 isolates ,consistent

with previous studies conducted in Pondicherry from 2005-2009 by Harish

et al.

Song et al ,in 2010 , repoted mutations in gyrB gene , mutation at

Phe-464, Leu-465, causing rise in MIC value [116] .

Mutation at codon 80 of parC gene was observed in 33% (4) of

isolates, where isoleucine substitution for serine occured. When this

mutation occurs along with another single point mutation in gyrA will lead

on to higher range of ciprofloxacin MIC value.

All the 12 isolates were subjected to Ceftriaxone and Cefotaxime

disc diffusion, showed 100% susceptibitity pattern, making as an effective

alternative for treating fluroquinolone resistance Salmonella isolates.

89

All the 12 culture proven Enteric fever cases were treated with

parenteral ceftriaxone according to their standard dosing regime and fever

defervescence was observed on day 3 or 4 following antimicrobial

therapy. Patient followed up and complete recovery was reported.

This is in contrary to other studies, where high level resistance to

ceftriaxone was observed due to the presence of CTX-M-15 and SHV-12

extended spectrum beta lactamases ( ESBLs) [11] . Recent reports of clinical

failure ,relapse to ceftriaxone and ESBL detection limits its use . None of

the isolates in the present study were ESBL producers.

Azithromycin shows excellent tissue penetration and achieves

intracellular concentration well. Hence it is equivalent or superior to other

alternative drugs in the management of uncomplicated typhoid fever with

prompt resolution of symptoms and low prevalence of relapse and carrier

state [117,118] .

All the 12 (100 %) isolates showed susceptibility to Azithromycin by

disc diffusion method . Several studies have reported Azithromycin MIC

distributions for typhoidal and non-typhoidal Salmonella strains [119].

Clinical breakpoints for disc diffusion and MIC testing have been adopted

by CLSI for Salmonella Typhi but not yet for Salmonella Paratyphi A .

Some sporadic cases of treatment failure with Azithromycin was reported

in literature [ 120]. Hence judicious use and continuous monitoring of creep

in MIC for azithromycin is essential to combat resistance.

Limitations of the study

90

LIMITATIONS OF THE STUDY

1. Our study had a limitation in the number of isolates studied.

Molecular analysis of large number of isolates for various resistance

mechanisms and its correlation with fluoroquinolone resistance might

help in devising methodology for detection of Ciprofloxacin

resistance in Salmonella isolates which is imperative for the

management of Typhoid fever.

2. Serotyping and Phage typing of the isolates were not done, that could

have thrown light on the pattern of strains in the population of

patients attending this tertiary care centre.

Summary

91

SUMMARY

One Hundred children (39 %) and one fifty seven (61 %) young

adults patients with clinical suspicion of Enteric fever were subjected to

conventional blood culture.

The isolation rate of Salmonella species was 4.7% . Among children the

isolation rate was 5% and 4.45% in young adults.

Predominant isolate among the Salmonella species was S.Typhi (91.6%)

followed by S.Paratyphi A (8.3%).

Predominant age group affected 5-7 years (19.9%) with female

preponderance, mean age 7.2 years.

Among young adult patients, 18-24 years old were most commonly

affected with male preponderance, mean age 19.9 years.

Peak incidence of Typhoid fever was observed from June-August. (75%-

isolation )

58.3% affected patients had H/O eating food ouside home with

significant association between Salmonella species isolation among adult

patients.

None of the individuals had Typhoid vaccination.

Analysis of Antimicrobial susceptibility pattern in this study revealed

re-emergence of susceptibility to all the first line antimicrobials-

ampicillin, chloramphenicol and co-trimoxazole in all the 12 isolates. No

MDR Salmonella species have been isolated.

92

100% resistance to Nalidixic acid & Pefloxacin by disc diffusion

method was observed.

Nine ( 75%) isolates were intermediate and three (25% ) isolates were

resistant to Ciprofloxacin by Disc diffusion method.

All 12 (100% ) isolates were susceptible to Ceftriaxone , Cefotaxime,and

Azithromycin by disc diffusion method. Enteric fever cases in this study

were treated with injection ceftriaxone according to standard dosing

regime and complete recovery was observed , with fever defervescence

by day 4 following antimicrobial therapy .

Four ( 33.3%) isolates were intermediate (0.125µg/ml) and eight

(66.7%) isolates were resistant (1-4µg/ml) to Ciprofloxacin by

Brothmicro dilution method of MIC determination.

All the Pefloxacin resistant isolates harboured point mutation in genes

encoding QRDR region when subjected to PCR and Sequencing.

Mutation analysis revealed that , all the 12 (100%) isolates showed

mutation in gyrA , with most common mutation observed were

Ser83→Phe ,followed by Asp87 →Asn ; no mutation observed in gyrB

gene ; Ser80→Isoleucine mutation in parC gene were observed among 4

( 33.3%) isolates.

Interpretative criteria of EUCAST guidelines for Ciprofloxacin MIC

correlates well with Pefloxacin resistance in all the 12 isolates , which

were confirmed by molecular characterisation .

Conclusion

93

CONCLUSION

This study demonstrates that children and young adults were the

predominant population affected with typhoid fever and had a strong

association between eating food outside and isolation of Salmonella

species.The predominant isolation was S.Typhi, and followed by

S.Paratyphi A.

Antimicrobial susceptibility pattern was similar in both the species,

with re-emergence of susceptibility to ampicillin, chloramphenicol and co-

trimoxazole. Both the species showed high resistance to Nalidixic acid and

Pefloxacin.The use of Pefloxacin as a surrogate marker for fluoroquinolone

susceptibility is promising , since it detects all the mechanisms for

fluoroquinolone resistance in this study , and clearly separates ciprofloxacin

susceptible and resistant strains .

Pefloxacin disc diffusion was found to be the best in detecting

fluoroquinolone resistance in centres where routine MIC testing and

molecular characterisation was not possible .

Interpretative criteria for Ciprofloxacin MIC by Microbroth dilution

method in EUCAST guidelines have a better correlation with molecular

characterisation , with gyrA mutation at codon 83 was predominant

followed by gyrA mutation at codon 87.

Ceftriaxone and Azithromycin seem to be the effective therapeutic

option for fluroquinolone resistant Salmonella isolates.

COLOUR PLATES

Gram stain of Salmonella species showing gram negative bacilli

Mac Conkey – Lactose non fermenting colonies

Blood Agar- Non hemolytic grey white colonies

BIOCHEMICAL REACTIONS OF SALMONELLA TYPHI

BIOCHEMICAL REACTIONS OF SALMONELLA PARATYPHI A

SLIDE AGGLUTINATION TEST – SALMONELLA TYPHI

SLIDE AGGLUTINATION TEST – SALMONELLA PARATYPHI A

ANTIMICROBIAL SUSCEPTIBILITY PATTERN - DISC DIFFUSION METHOD

CIPROFLOXACIN MIC-BROTH MICRODILUTION METHOD

g/ml

MOLECULAR CHARACTERIZATION OF QRDR GENE

gyr A

LANE 1-12 - Salmonella Isolates showing bands at 347 bp

LANE 13 - NC Negative control

LANE 14 - DNA Ladder 100 bp

LANE 15 - PC Positive control ( ATCC Salmonella Typhi 19430 )

MOLECULAR CHARACTERIZATION OF QRDR GENE

gyr B

LANE 1-12 - Salmonella Isolates showing bands at 345 bp

LANE 13 - NC Negative control

LANE 14 - DNA Ladder 100 bp

LANE 15 - PC Positive control ( ATCC Salmonella Typhi 19430 )

MOLECULAR CHARACTERIZATION OF QRDR GENE

parC

LANE 1-12 - Salmonella Isolates showing bands at 270 bp

LANE 13 - NC Negative control

LANE 14 - DNA Ladder 100 bp

LANE 15 - PC Positive control ( ATCC Salmonella Typhi 19430 )

SEQUENCE ANALYSIS

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ANNEXURE-I

ABBREVATIONS

Arg - Arginine

Asn - Asparagine

Asp - Aspartic acid

AST - Antimicrobial susceptibility testing

AMPI - Ampicillin

AZIT - Azithromycin

BMD - Broth microdilution

Bla - β lactamase

bp - base pair

CTX - Cefotaxime

CFT - Ceftriaxone

Ck - Chloramphenicol

CIP - Ciprofloxacin

COTRI - Cotrimoxazole

CFU - Colony Forming Units

CMI - Cell mediated immunity

CLSI - Clinical Laboratory Standards Institute

DNA - Deoxynucleotide

ESBL - Extended spectrum beta lactamase

EUCAST - The European Committee on Antimicrobial Susceptibility

Testing

Gly - Glycine

Glu - Glutamic acid

Gyr - Gyrase

H - Flagella

IFN γ - Interferon gamma

Ile - Isoleucine

Inc - Incompatibility group

LAT - Latex agglutination test

µg - Microgram

µL - Microlitre

MDR - Multi-drug resistant

MHA - Muller-Hinton agar

MHC - Major Histo Compatibility

MIC - Minimum inhibitory concentration

NA - Nalidixic acid

NAR - Nalidixic acid resistant

Par - Topoisomerase

PF - Pefloacin

PCR - Polymerase Chain Reaction

Phe - Phenylalanine

QRDR - Quinolone Resistant Determining Region

SCV - Salmonellae containing vacuole

Ser - Serine

SPI-1 - Salmonella pathogenicity island-1

SPI-2 - Salmonella pathogenicity island-2

Tyr - Tyrosine

TTSS - Type III secretion system

ANNEXURE-II

ANNEXURE-III

PROFORMA

Name : IP NO: OP NO : Age: Ward: UNIT : Sex: Address & Contact No :

School area details :

Parents’ details :

H/O Fever in family members :

Presenting Symptoms :

H/O Vaccination :

Clinical Evaluation & Provisional Diagnosis :

MICROBIOLOGICAL ANALYSIS

Sample collection ( Date & Time ) : Details of Cultre :

Final Culture Report:

Antimicrobial Susceptibility Pattern : (Disc Diffusion ) :

Ciprofloxacin MIC :

Molecular characterization of QRDR genes:

ANNEXURE-IV

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

1 1 1 1 Archana 20 2 1 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 02 1 1 1 Balu 19 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3 1 2 1 Kishore 5 1 1 6 1 2Salmonella

TyphiS S S S S S R R I 2 0.125 1 7 7

4 1 2 1 Udhaya kumar 6 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 05 1 2 1 Janani 5 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 06 1 1 1 Jeevanandam 19 1 5 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 07 1 1 1 Chandru 20 1 2 4 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0

8 1 1 1 Amudha 23 2 4 2 1 2Salmonella

TyphiS S S S S S R R I 2 2 2 7 7

9 1 1 1 Dhanalakshmi 19 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 010 1 1 1 Chandrasekar 17 1 2 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 011 1 1 1 Sumathi 24 2 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 012 1 1 1 Ellammal 23 2 1 1 2 2 Micrococci 0 0 0 0 0 0 0 0 0 0 0 0 0 013 1 1 1 Krishnan 20 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 014 1 1 1 Ponnii 18 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 015 1 2 1 Aparna 10 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 016 1 2 1 Irfan 9 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 017 1 2 1 Archana Bai 10 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 018 1 2 1 Shanthini 8 2 2 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 019 1 1 2 Moorthy 22 1 5 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 020 1 1 2 Tulasiram 17 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 021 1 1 2 Nirmal Kumar 20 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 022 1 1 2 Vinoth Kumar 24 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 023 1 1 1 Srinivasan 23 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 024 1 1 2 Balan 19 1 3 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 025 1 1 1 Ramesh 24 1 4 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 026 1 1 2 Yuvaraj 20 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 027 1 2 1 Akshaya 6 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 028 1 2 1 Sowndarya 11 2 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 029 1 2 1 Deepak 11 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

30 1 2 1 Yithish 5 1 2 6 2 2Salmonella

TyphiS S S S S S R R I 2 4 6 7 4

31 1 2 1 Kavin 10 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 032 1 1 1 Jebin 24 2 4 4 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 033 1 1 1 Sharmila 15 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 034 1 2 2 Absal 10 1 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 035 1 2 1 Pooja 8 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 036 2 1 2 Nandakumar 14 1 2 1 2 2 Micrococci 0 0 0 0 0 0 0 0 0 0 0 0 0 037 2 1 2 Gopinath 20 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

38 2 1 1Jerlin Roopavathy

13 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

39 2 1 2 Dilip kumar 18 1 3 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 040 2 1 2 Roshan 19 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

41 2 1 2 Sandeep kumar 22 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ANNEXURE V - MASTER CHART

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

42 2 2 2 Archana 7 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

43 2 1 1 Raheema 13 2 2 2 1 2Salmonella

TyphiS S S S S S R R I 2 2 3 7 7

44 2 2 1 Shanthi 9 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 045 2 2 1 Roshan 6 1 2 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 046 2 2 1 Manisha 8 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 047 2 2 1 Sanjitha 10 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 048 2 2 1 Mahesh 7 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 049 2 2 1 Thenmozhi 12 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 050 2 1 2 Vinoth 23 1 5 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 051 2 1 2 Ashwin 24 1 4 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 052 2 1 2 Bharathy 22 2 2 4 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 053 2 1 2 Tamilarasan 16 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 054 2 1 1 Mahadevan 18 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 055 2 1 1 Pavithra 18 1 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 056 2 1 1 Zaheela 14 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 057 2 2 1 Ilavarasi 7 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

58 2 1 1 Ibrahim Kareem 22 1 3 6 1 2Salmonella

TyphiS S S S S S R R I 2 2 1 7 4

59 2 2 1 Tamil 7 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 060 2 2 1 Sanjay 6 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 061 2 2 1 Varshini 5 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 062 2 2 1 Ashwin kumar 5 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 063 2 2 1 Hasika 9 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 064 2 2 1 Ajay 8 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 065 2 2 1 Praveen 8 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 066 2 2 1 Kaviya 10 2 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 067 2 2 1 Madan 6 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 068 2 2 1 Karthika 7 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 069 2 2 1 Varshini 8 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 070 2 1 2 Jayabalan 19 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 071 2 2 1 Madhushree 8 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 072 2 1 2 Sridhar 20 1 5 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 073 2 1 2 Manjula 17 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 074 2 2 1 Karthika 10 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 075 2 2 1 Kavin 6 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 076 2 2 1 Raman 7 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 077 2 2 1 Senu 11 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 078 2 2 1 Deva 5 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 079 2 2 1 Karthick 9 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 080 2 2 1 Vetrivel 6 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 081 2 2 1 Logith Prasath 9 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

82 2 1 1 Abinesh 17 1 3 6 1 2Salmonella

TyphiS S S S S S R R I 2 1 3 7 7

83 2 2 1 Shyam 10 1 2 1 1 2 Micrococci 0 0 0 0 0 0 0 0 0 0 0 0 0 084 3 2 1 Prasad 12 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

85 3 2 1Shabeena Begam

8 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

86 3 2 1 Malar 10 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 087 3 2 1 Nithish 11 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

88 3 2 1Pradeep Chandran

9 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

89 3 2 1 Tharun 8 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 090 3 2 1 Saranya 6 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 091 3 2 1 Yamini 12 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 092 3 2 1 Tajudheen 6 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 093 3 2 1 Gokulraj 7 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 094 3 2 1 Nithish Kumar 8 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 095 3 2 1 Manoj 12 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 096 3 1 1 Rasiyam 19 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

97 3 1 1 Praveen Kumar 23 1 3 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

98 3 1 2 Arul Kumar 22 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

99 3 1 2 Mahesh Kumar 20 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

100 3 1 2 Gunithi Gomal 22 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

101 3 1 2 Rajkumar 21 1 4 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0102 3 2 1 Varsha 7 2 2 3 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0103 3 2 1 Rohini 5 2 2 4 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0104 3 2 1 Adhira 6 2 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0105 3 1 2 Shanmugam 19 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0106 3 1 2 Masthan 24 1 1 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0107 3 1 2 Gayathri 16 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0108 3 1 2 Arul Kumar 21 1 1 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0109 3 2 1 Vinay 9 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0110 3 2 1 Dhakshayini 6 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0111 3 1 2 Kuppusamy 18 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

112 3 2 1 Jansi Rani 5 2 2 4 2 2Salmonella

TyphiS S S S S S R R R 2 0.125 1 7 7

113 3 2 2 Rakshitha 8 2 1 3 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0114 3 2 2 Bargavi 11 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0115 3 1 2 Jyothi 17 2 5 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0116 3 1 2 Indumathi 16 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0117 3 2 1 Roshan 6 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0118 3 2 1 Ellan Jesika 5 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0119 3 2 1 Yaseem 6 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

120 3 2 1 Thaman 6 1 3 1 2 2Salmonella

TyphiS S S S S S R R R 2 4 6 7 4

121 3 2 1 Anifa Banu 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0122 3 2 1 Charan 7 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0123 3 2 1 Sharmila 6 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0124 3 2 1 Nandhakumar 12 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0125 3 2 1 Subashree 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0126 3 2 1 Yuvaraj 12 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0127 3 2 1 Janani 10 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

128 3 2 1 Munna 6 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0129 3 2 1 Vijaya kumar 12 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0130 3 2 1 Sana Farhath 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0131 3 2 1 Saidharshan 5 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0132 3 2 1 Udhayakumar 8 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0133 3 2 1 Meganathan 5 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0134 3 1 1 Goutham 20 1 1 2 2 2 S.Paratyphi A S S S S S S R R I 2 0.125 1 7 7135 4 2 1 Muheeb 9 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0136 4 2 1 Harini 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0137 4 2 1 Niveditha 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0138 4 2 1 Sachin 6 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0139 4 2 1 Nida Fathima 7 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0140 4 2 1 Kowsikar 8 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

141 4 2 1 Kishore kumar 6 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

142 4 2 1 Krithika 6 2 2 6 2 2Salmonella

TyphiS S S S S S R R I 2 2 6 7 4

143 4 2 1 Kishore 11 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0144 4 2 1 Udaya kumar 8 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0145 4 2 1 Niveditha 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0146 4 2 1 Umalakshmi 6 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0147 5 2 1 Santhosh 10 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0148 5 2 1 Porkodi 7 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0149 5 2 1 Archana 7 2 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0150 5 2 1 Vanashri 6 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0151 5 2 1 Akshaya 5 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0152 5 2 1 Rani 8 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0153 5 2 1 Pradeep 9 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0154 5 1 1 Chandiran 23 1 1 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0155 5 1 1 Dhanalakshmi 22 2 5 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0156 5 1 1 Sandhiya 19 2 5 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0157 5 1 1 Beijili 24 2 4 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

158 5 1 1Ushman Hameed

23 1 3 6 1 2Salmonella

TyphiS S S S S S R R R 2 1 5 7 7

159 5 1 1 Palani 22 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0160 5 1 1 Tamilarasi 23 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0161 6 1 1 Asha 19 2 2 1 2 2 E.coli 0 0 0 0 0 0 0 0 0 0 0 0 0 0162 6 1 1 Vignesh 16 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0163 6 1 1 Arul 24 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0164 6 1 1 Krishnan 19 1 2 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0165 6 1 1 Ponni 22 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0166 6 1 1 Jothy 19 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0167 6 1 2 Abdul kareem 16 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0168 6 1 2 Rajeshwari 23 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0169 6 1 2 Hariharan 22 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

170 6 1 2 Mohana 24 2 4 2 1 2 K.pneumoniae 0 0 0 0 0 0 0 0 0 0 0 0 0 0

171 6 1 2 Vijaya 24 2 5 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

172 6 1 1 Kokila 20 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0173 7 1 1 Vishnupriya 18 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0174 7 1 1 Deepa 18 2 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0175 7 1 2 Kaviyarasan 22 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0176 7 1 1 Raveendiran 24 1 2 7 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0177 7 1 1 Muthukumar 23 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0178 7 1 1 Indumathy 20 2 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0179 7 1 1 Dinesh 17 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0180 7 1 1 Muthupriya 19 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0181 7 1 1 Malar 18 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0182 7 1 1 Gopi 24 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0183 7 1 1 Sangeetha 21 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0184 8 1 1 Jayakumar 18 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0185 8 1 1 Jithan 15 1 3 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0186 8 1 1 Sheela 24 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0187 8 1 2 Tharun 18 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0188 8 1 2 Rathika 24 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0189 8 1 2 Stephen Raj 18 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0190 8 1 1 Srinivasan 23 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0191 8 1 1 Dhanamani 23 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0192 8 1 1 Sivaraman 11 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

193 8 1 2 Naveen kumar 16 1 4 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

194 8 1 2 Fathima 21 2 5 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0195 8 1 2 Rajammmal 23 2 5 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0196 9 1 2 Nithish 19 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

197 9 1 2 Suriya Prakash 20 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

198 9 1 2 Suresh 23 1 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0199 9 1 2 Sangeetha 20 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0200 9 1 2 David 19 1 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0201 9 1 2 Priya 23 2 3 2 2 2 E.coli 0 0 0 0 0 0 0 0 0 0 0 0 0 0202 9 1 2 Ajay kumar 24 1 4 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0203 9 1 2 Rajasekar 19 1 5 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0204 9 1 2 Jothi 24 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0205 9 1 2 Ramesh 21 1 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0206 9 1 2 Elbin Jacob 22 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0207 9 1 2 Monika 20 2 2 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0208 10 1 2 Brijesh Kumar 20 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0209 10 1 1 Kumudha 20 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0210 10 1 1 Ranjani 17 2 4 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0211 10 1 1 Vijayan 20 1 5 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0212 10 1 1 Monish 13 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0213 10 1 1 Mohana 14 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0214 10 1 1 Preety 18 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0215 10 1 1 Ganthi 19 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0216 10 1 1 Yasmin 18 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0217 10 1 1 Ahmed 13 1 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SAMPLE NO

MONTHSTUDY

SITEIP/OP NAME AGE SEX

CARDINAL SYMPTOMS

CARDINAL SIGNS

OUTSIDE FOOD

HABITS

TYPHOID VACCINATI

ON

ORGANISM ISOLATED

AMPI COT CK CTX CFT AZIT NA PEF CIPRO

MDR Multidrug resistant

Salmonella species

CIPRO- MICRO BROTH

DILUTION

gyrA gyrB parC

218 10 1 2 Sakthivel 15 1 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0219 10 1 1 Dinesh 23 1 4 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0220 10 1 1 Ruby Stella 20 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0221 10 1 1 Priyanka 16 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0222 10 1 2 Vijayalakshmi 20 2 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0223 10 1 2 Shanmugaraj 22 1 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0224 10 1 2 Suja 22 2 4 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0225 11 1 2 Femina 24 2 5 7 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0226 11 1 2 Devaraj 19 1 5 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0227 11 1 2 Vidhya 24 2 5 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0228 11 1 1 Parthiban 23 1 1 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0229 11 1 1 Ramadoss 21 1 1 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0230 11 1 1 Ilavarasan 21 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0231 11 1 1 Divya 22 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0232 11 1 1 Saravanan 20 1 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0233 11 1 1 Sharmila 17 2 2 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0234 11 1 1 Ajithkumar 20 1 4 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

235 11 1 1 Mani 23 1 2 4 1 2Salmonella

TyphiS S S S S S R R I 2 0.125 1 7 7

236 11 1 2 Raja 24 1 5 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0237 11 1 2 Hariharan 15 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0238 11 1 1 Sanjay 23 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0239 11 1 1 Manjula 14 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0240 11 1 1 Subbulakshmi 23 2 3 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0241 11 1 2 Naseer 24 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0242 11 2 1 Harikrishnan 11 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0243 11 1 2 Sujeetha 10 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0244 12 1 1 Vijay 23 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0245 12 1 1 Charles 24 1 1 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0246 12 1 2 Venkatesan 24 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0247 12 1 2 Diwakar 22 1 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0248 12 1 2 Devi 24 2 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0249 12 1 2 Nithish 13 1 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0250 12 1 1 Yuvaraj 15 1 2 1 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0251 12 1 2 Dhavania 23 2 1 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0252 12 1 2 Gnanodhaya 15 2 2 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0253 12 1 1 Sakthivel 23 1 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0254 12 1 2 Vinothkumar 22 1 3 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0255 12 1 2 Ranjitham 20 2 3 2 1 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0256 12 1 2 Inayathula 19 1 1 1 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0257 12 1 2 Jamuna 21 2 3 2 2 2 No growth 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Month codeStudy Site

Code IP/OP Code Sex CodeOutside

food habits

Code ASTPattern CodeSymptoms

CodeCode Signs Code MDR Code

Antibiotics

AbbrevationCipro-MIC

gyrA CODE gyrB CODE parC CODE

June 1 RGGGH 1 IP 1 Male 1 Yes 1 Suceptibile SFever,Abdomi

nal pain1

Pyrexia,Toxic look,Pallor

1MDR

isolate1AMPI ampicillin ≤0.06 Sensitive

ser(83) Phenylala

nine1

No mutation

7ser (80)-isoleucin

e4

July 2 ICH 2 OP 2 Female 2 No 2 Intermediate I

Fever, Abdominal

pain, Diarrhoea

2Pyrexia,Toxic look,Coated

tongue2

Susceptible isolate

2 COT cotrimoxazole 0.125-0.5Intermed

iateser(83)

Tyrosine2

August 3 Resistant R

Fever, Abdominal

pain, Diarrhoea, Vomitting

3

Pyrexia, Toxic look, Coated

tongue, Hepatomegaly

3 CKchloramphenic

ol≥1 Resistant

Asp(87)-Asn

3

September 4 Not tested 0Fever,

Constipation4

Pyrexia, Toxic look, Coated

tongue, Splenomagaly

4 CFT ceftriaxone

ser(83) Phe,Gly(1

33)-Glutamic

acid

5

October 5Fever,Non

specific symptoms

5Pyrexia,Rose spots,Coated

tongue5 AZIT azithromycin

ser(83)Phe,Asp(87)

Asn6

November 6

Pyrexia,Toxic look,Coated

tongue,Hepatosplenomegaly

6 NA nalidixic acid

December 7 Pyrexia,Pallor 7 PEF pefloxacin

January 8 CIPRO ciprofloxacin

February 9 CTX cefotaxime

March 10

April 11

May 12