Study on Molecular Characterisation of Anti-Microbial Resistance in Salmonella Enterica Serovar typhi and paratyphi from blood culture isolates in a tertiary care hospital

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A Study on Molecular Characterisation of Anti-microbial Resistance in Salmonella enterica serovar typhi and paratyphi from

Blood Culture Isolates in a Tertiary Care Hospital.

Dissertation submitted in

Partial fulfillment of the Regulations required for the award of M.D. DEGREE

In

MICROBIOLOGY – BRANCH IV The Tamil Nadu

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CERTIFICATE

This is to certify that the enclosed work “A Study on Molecular Characterisation of Anti-microbial Resistance In Salmonella enterica serovar typhi and paratyphi from Blood Culture Isolates In a Tertiary Care Hospital” submitted by Dr. S. VIJI to The Tamilnadu Dr. MGR Medical University is based on bonafide cases studied and analysed by the candidate in the Department of Microbiology, Coimbatore Medical College and Hospital , Coimbatore during the period from July

2016 to June 2017 under the guidance and supervision of Dr. N.Mythily, MD., Professor and Head of Department , Department of

Microbiology and the conclusion reached in this study are her own.

Guide

Dr. N.MYTHILY, M.D.,

Professor & HOD

Department of Microbiology, Coimbatore Medical College, Coimbatore.

Dr.B.ASOKAN, M.S., M.Ch., Dr. N.MYTHILY , M.D.,

Dean, Professor & HOD,

Coimbatore Medical College and Hospital, Department of Microbiology,

Coimbatore – 14. Coimbatore Medical College,

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DECLARATION

I, Dr. S. VIJI , solemnly declare that the dissertation entitled “A Study on Molecular Characterisation of Anti-microbial Resistance In Salmonella enterica serovar typhi and paratyphi from Blood Culture Isolates In A Tertiary Care Hospital” was done by me at Coimbatore Medical College Hospital, Coimbatore during the period from July 2016 to June 2017 under the guidance and supervision of Dr. N. Mythily, M.D., Professor & HOD, Department of Microbiology, Coimbatore Medical College, Coimbatore.

This dissertation is submitted to The Tamilnadu Dr. MGR. Medical University towards the partial fulfilment of the requirement for the award of M.D. Degree (Branch – IV) in Microbiology.

I have not submitted this dissertation on my previous occasion to any University for the award of any degree.

Place: Coimbatore Date :

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ACKNOWLEDGEMENT

I express my deep debt of gratitude to our respectful Dean, Dr.B. Asokan M.S., Mch., for permitting me to do this study.

I thank

Dr.A.Arjunan, M.D.,

Vice Principal, Coimbatore

Medical College, Coimbatore for her encouragement and suggestions

in completing the study.

I wish to place my deep sense of gratitude and sincere thanks to Dr. N. Mythily MD., Professor and Head of the Department of Microbiology, for the constant encouragement and timely advice given to me during the course of my post-graduation.

I express my deep sense of gratitude and indebtedness to Professor Dr.N. Mythily MD., as a guide , for her constant guidance, valuable advice and inspiration throughout my study.

I sincerely place my thanks to Associate Professor Dr.P.Sankar,M.D., for his support and encouragement.

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My special thanks to my post graduate colleagues Dr.K.Sivaram, Dr.N.Vandarkuzhali and Dr. P.Santhanalakshmi and other post graduates in the department of Microbiology for their co-operation in completing my study.

I take this opportunity to thank all the technical staffs in the Department of Microbiology who gave me their kind co-operation throughout my study.

I affectionately thank my family members who are giving their constant support throughout my entire post-graduation course without which this work would not have been successful.

I am thankful to God, who have been with me all throughout my way to reach the destination.

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CERTIFICATE - II

This is to certify that this dissertation work titled “A Study on Molecular Characterisation of Anti-microbial Resistance In Salmonella enterica serovar typhi and paratyphi from Blood Culture Isolates in a Tertiary Care Hospital.” of the candidate Dr. S. Viji with registration Number 201514254 for the award of Doctor of Medicine 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 2% percentage of plagiarism in the dissertation.

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CONTENTS

S.NO CONTENTS PAGE NO

1. INTRODUCTION 01

2. AIMS AND OBJECTIVES 07

3. REVIEW OF LITERATURE 08

4. MATERIALS AND METHODS 37

5. RESULTS 65

6. DISCUSSION 69

7. SUMMARY 77

8. CONCLUSION 79

9. BIBLIOGRAPHY

10. ANNEXURE

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LIST OF TABLES

S.No TABLES

1

PERCENTAGE OF ENTERIC CULTURE POSITIVES

2

DISTRIBUTION OF SALMONELLA SPECIES

3

GENDER DISTRIBUTION OF ENTERIC FEVER CASES

4

AGE WISE DISTRIBUTION OF SALMONELLA SPECIES

5 CORRELATION BETWEEN NEUTROPHIL COUNT AND BLOOD

CULTURE POSITIVE ENTERIC FEVER

6 CORRELATION BETWEEN HEPATOSPLENOMEGALY AND

BLOOD CULTURE POSITIVE ENTERIC FEVER

7 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

Samonella typhi

8 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

Salmonella paratyhi A

9

QUINOLONE RESISTANCE AMONG SALMONELLA SPECIES 10

ANALYSIS OF RESISTANT GENES BY MOLECULAR METHODS 11

MUTATION ANALYSIS OF gyrA , gyrB and parC GENES.

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LIST OF CHARTS

S.No CHART

1 PERCENTAGE OF ENTERIC CULTURE POSITIVES

2 DISTRIBUTION OF SALMONELLA SPECIES

3 GENDER DISTRIBUTION OF CULTURE POSITIVES

4 AGE WISE DISTRIBUTION OF SALMONELLA SPECIES

5 CORRELATION BETWEEN NEUTROPHIL COUNT AND CULTURE

POSITIVES

6 CORRELATION BETWEEN HEPATOSPLENOMEGALY

AND CULTURE POSITIVES

7 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

Salmonella typhi

8 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

Salmonella paratyphi A

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LIST OF COLOUR PLATES

S.NO COLOUR PLATES

1 Nutrient Agar plate : Salmonella typhi colonies

2 MacConkey Agar plate: Non - lactose Fermenting colonies of Salmonella typhi

3 Blood Agar plate : Grey white colonies of Salmonella typhi

4 DCA plate : Salmonella typhi with black head colonies

5 DCA plate : Salmonella paratyphi A with translucent colonies

6 Gram’s Stain – Gram Negative bacilli

7 Biochemical Reactions of Salmonella typhi

8 Biochemical Reactions of Salmonella paratyphi A

9 Sugar Fermentation by Salmonella typhi

10 LAO – Lysine Decarboxylated in Salmonella typhi

11 LAO – Ornithine Decarboxylated in Salmonella paratyphi A

12 Mueller Hinton Agar – AST by Kirby – Bauer Method Salmonella typhi

Sensitive to Ciprofloxacin, Pefloxacin & Nalidixic acid

13 Mueller Hinton Agar – AST by Kirby – Bauer Method Salmonella typhi

Resistant to Ciprofloxacin, Pefloxacin & Nalidixic acid

14 Mueller Hinton Agar – AST by Kirby – Bauer Method Salmonella typhi

Sensitive to Chloramphenicol, Cotrimoxazole, Ceftriaxone & Azithromycin

15 MIC – E Test for Ciprofloxacin

16 Multiplex PCR for Plasmid Mediated Quinolone Resitance genes

( qnrA, qnrB, qnrS )

17 Multiplex PCR for Chromosomal Mediated Quinolone Resistance genes

(gyrA, gyrB, parC)

18 Gene Sequencing – gyrA, gene at quinolone resistance determining region

18 (a) DNA Sequence alignment Mutation Analysis : gyrA

19 Gene Sequencing – parC, gene at quinolone resistance determining region

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LIST OF ABBREVIATIONS

S. typhi Salmonella typhi

S.paratyphiA Salmonella paratyphi A

CLSI Clinical Laboratory Standards Institute

PCR Polymerase chain Reaction

MDR Multi – drug resistant

DNA Deoxyribonucleic Acid

MIC Minimum Inhibitory Concentration

MHA Mueller Hinton Agar

µg Microgram

Mac MacConkey Agar

BAP Blood Agar Plate

DCA Deoxycholate Citrate Agar

XLD Xylose Lysine Deoxycholate

H2S Hydrogen sulphide

K/A Alkaline / Acid

ONPG O-nitrophenyl beta D-galactopyranoside

HCl Hydrochloric acid

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AIDS Acquired Immuno Deficiency Syndrome

WHO World Health Organisation

ELISA Enzyme Linked Immunosorbent Assay

EUCAST European Committee on Antimicrobial Susceptibility Testing

ESBL Extended Spectrum Beta Lactamases

TSI Triple Sugar Iron

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INTRODUCTION

Enteric fever is a world health problem, which occurs predominantly in the developing countries like India. The term enteric fever comprises of both typhoid fever and paratyphoid fever. Typhoid fever is caused by Salmonella enterica subspecies enterica serovar typhi.

The organism that causes Paratyphoid fever can be either of the three serovars Salmonella paratyphi A, Salmonella paratyphi B (known as scholtmuelleri) or Salmonella paratyphi C (also called hirschfeldii).15

Salmonella paratyphi A is the common organism that occurs world wide, paratyphi B is more prevalent in Europe and paratyphi C is a very rare pathogen. The ratio of enteric fever caused by Salmonella typhi to that caused by Salmonella paratyphi is about 10 to 1.1.15

World Health Organisation has estimated, that the occurrence of typhoid fever being 21.7 million cases and 2,50,000 mortality occurring annually.11,12 Among that , the 80% of cases and deaths occur in Asia, the remaining occurs mainly in Africa and latin America.4,12,13 In

developing countries like India, typhoid fever occurs with an incidence of 102 to 2,219 cases, per 1,00,000 population.12

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facilitate the transmission of the disease are poor sanitation, unprotected drinking water supply, lack of hand washing and toilet access.

Enteric fever is an acute systemic illness characterized by step ladder pattern type of remittent fever, rose spots, coated tongue, hepatosplenomegaly and relative bradycardia. The complications of enteric fever are gastrointestinal bleeding, intestinal perforation and the neurological manifestations that occurs rarely like meningitis, cerebellar ataxia and neuropsychiatric symptoms.

The 10% of the enteric fever cases who are not treated properly will become carriers and they excrete the bacilli in feces or urine. 22 The multiplication of typhoid bacilli occurs in the gall bladder and are excreted in feces. Faecal carriers are more common than the urinary carriers. This carrier state contributes for the occurrence of epidemics and endemics.

Antibiotics are the mainstay for treatment of enteric fever. Prompt treatment of the disease with appropriate anti-microbials in appropriate time is a major criteria in reducing the mortality from 30% to 0.5%.10

Irrational use of antibiotics has led to the emergence of resistance in Salmonella species. The emergence of antimicrobial resistance is a problem in the management of enteric fever, that becomes a challenge for both clinicians and the microbiologists.

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Ampicillin and Cotrimoxazole. 11,12 Multi drug resistant strains are those

which are resistant to Chloramphenicol, Ampicillin and Cotrimoxazole.4

Due to the emergence of multi-drug resistant ( MDR ) Salmonella typhi strains, there occurred many epidemics of typhoid fever globally, especially in Southeast Asia. 4, 13

Resistance to Chloramphenicol in S.typhi are plasmid mediated, that occurs due to the acquisition of the R plasmid that encodes for an enzyme acetyl transferase, which inactivates chloramphenicol. In S.typhi, the resistance to Cotrimoxazole are due to the mutations in the chromosomal gene that encodes, the enzyme dihydrofolate reductase type VII. The development of multi-drug resistance is due to single plasmid that belongs to the incompatibility group, H I1 and this transmissible.4

The emergence of multi-drug resistant Salmonella strains, made the fluoroquinolones like Ciprofloxacin being the drug of choice in the treatment of typhoid fever. But indiscriminate use of fluoroquinolones led to the development of resistance against these agents also. This condition is further complicated by the emergence of quinolone resistant strain and there are studies that show, a steady increase in Minimum Inhibitory Concentration (MIC) of Ciprofloxacin. 4,13

Since then several studies showed that, though the isolates are fully susceptible to Ciprofloxacin by disc diffusion tests, there occurs constant treatment failure. 4,13. A high level of Ciprofloxacin resistant strains of

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Nalidixic acid being a reliable indicator for decreased ciprofloxacin susceptibility, it is considered as a surrogate marker for resistance to fluoroquinolones. Whereas Clinical and Laboratory Standards Institute (CLSI 2015) recommends Pefloxacin as a surrogate marker for identification of fluoroquinolone resistance in a dose of 5µg by disc diffusion.18

Fluoroquinolones target DNA gyrase and topoisomerase IV, which are bacterial enzymes that are a part of a complex which uncoils and recoils the bacterial DNA for transcription.4 Salmonella typhi commonly develops fluoroquinolone resistance through specific mutations in gyr A and par C, that codes for binding region of DNA gyrase and topoisomerase IV, respectively.

A single point mutation in gyrA gene confers a partial resistance, if a second gyrA point mutation is added the resistance increases. However a mutation in parC gene when added to a single gyrA mutation confers full invitro resistance to first generation fluoroquinolones.4 The other mechanisms of antimicrobial resistance in Salmonella isolates are plasmid mediated resistance, where plasmids of incompatibility group are important vectors of antibiotic resistance and rapid efflux of the drug.

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resistance to Ceftriaxone and recently AmpC beta-lactamase producing Salmonella typhi has been identified.4

Azithromycin can be used for treating Ceftriaxone, Ofloxacin and Chloramphenicol resistant Salmonella isolates.4 Azithromycin is very effective in the eradication of intra-cellular Salmonella and gastro intestinal carriage is eliminated and it is also a potential drug in paediatric population where quinolones are contraindicated.4

Development of resistance to azithromycin has also been detected in Salmonella typhi and S. paratyphi A isolates.13 Salmonella species develop resistance to antimicrobial agents either due to chromosomal changes or the exchange of genetic material via plasmids or transposons.2

The risk factors for the emergence of antibiotic resistance in Salmonella typhi are overuse, misuse and inappropriate prescription. Difficulties in the diagnosis of S.typhi by culture methods are the factors behind irrational prescriptions of antibiotics. 12

The occurrence of relapse is common in the partially and fully resistant strains than the fully susceptible strains. As there is a steady increase in drug resistance among Salmonella species, it is necessary to continuously monitor the antimicrobial susceptibility patterns of Salmonella enterica isolates to update the therapeutic guidelines.13

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on the understanding of local patterns of the antimicrobial resistance and on the results of the antimicrobial susceptibility testing of the Salmonella isolates from the individual patients.

Since serological methods of demonstration of antibodies in patients serum is possible only at second or third week of illness which may cause delay in the diagnosis of enteric fever, this study is targeted at isolation of Salmonella typhi and S.paratyphi from blood culture at an earlier stage.

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AIMS AND OBJECTIVES

AIM OF THE STUDY :

The aim of the study is to isolate, identify the Salmonella typhi and Salmonella paratyphi from blood culture and to detect its anti-microbial resistance pattern, to study the molecular characterization of resistance genes.

OBJECTIVES:

1) To isolate Salmonella typhi and Salmonella paratyphi from blood culture using conventional methods.

2) To confirm the isolated organisms by slide agglutination test using High Titre Sera.

3) To evaluate the anti-microbial susceptibility pattern of the organisms isolated, by Kirby-Bauer disc diffusion method.

4) To determine the antibiotic resistance pattern and to detect the Minimum Inhibitory Concentration of resistance drugs by E-Strip methods.

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REVIEW OF LITERATURE

Historical Review of Salmonella :

In 1826, Bretonneau, detected an enteric lesion of the disease and Louis named it as typhoid in 1829. In 1856 Budd described the disease to be transmitted by faeces of the patients.20 In 1880, Eberth identified

typhoid bacillus in the mesenteric nodes and in the spleen of fatal cases of typhoid fever and Gaffky in 1884 isolated the bacillus. Since then the organism was called as Eberth-Gaffky bacillus or Eberthella typhi.22

Salmon and Smith, in 1885, identified a bacillus that caused hog cholera and named it as Salmonella cholera-suis. It was the first bacillus to be isolated in the genus Salmonella. Then onwards it was realized that, typhoid bacillus also belonged to this genus, with some differences in biochemical reactions. Eberth-Gaffky bacillus was renamed as Salmonella typhi and the genus Eberthella had been no longer used.20

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Taxonomy of Salmonella typhi :

Domain : Bacteria

Phylum : Proteobacteria

Class : Gammaproteobacteria

Order : Enterobacteriales

Family : Enterobacteriaceae

Genus : Salmonella

Species : Salmonella enterica

Subspecies : Salmonella enterica enterica

Serovar : Salmonella enterica serovar Typhi

Morphology :

Salmonella typhi, Salmonella paratyphi A,B,C all belong to the genus Salmonella that comes under the family Enterobacteriaceae. Salmonella are gram negative bacilli, about 2-4 x 0.6 micrometer, non-capsulated, non-sporing, non acid-fast most serotypes are motile with peritrichous flagella, except S.gallinarum and S.pullorum, which are non-motile.21

Classification :

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1) Clinical classification :

This classification is the oldest one, but still used nowadays and also termed as User friendly classification. Salmonellae is classified into two group,

i) Typhoidal Salmonella :

This comprises of both serovars Salmonella typhi and Salmonella paratyphi. These are the causative agents of enteric fever in human beings and they are defined to the human host alone.

ii) Non-typhoidal Salmonella :

The rest of the serotypes that inhabit the intestine, of a wide range of animals causing food- borne gastroenteritis and septicemia in human beings comes under non-typhoidal Salmonella

2) Kauffmann-White Scheme Antigenic Classification:

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i) Serogroups :

The genus Salmonellae are classified into serogroups based on the presence of somatic O antigen.

ii) Serotypes :

Every serogroup is further classified into serotypes, based on the presence of flagellar antigens.

3) Molecular classification :

The genus Salmonella are classified into two species – Salmonella enterica and Salmonella bongori, based on DNA Hybridization studies The species of Salmonella enterica, further have six subspecies, namely enterica, salamae, arizonae, diarizonae, houtenae and indica. Then the subspecies are further classified into serotypes as described in Kauffmann-White scheme.

Kauffmann-White Antigenic Classification for Salmonella 22

Serogroup

Serotype name

O Ag * Vi Ag

H Ag

New Old Phase 1 Phase 2

2 A S.paratyphi A 1,2,12 _ A (1,5)

4 B S.paratyphi B 1,4,5,12 _ B 1,2

7 C1 S.paratyphi C 6,7 + C 1,5

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Cultural Characteristics :

Salmonellae are aerobic and facultative anaerobes, readily grows on simple media at pH of 6-8 and at optimum temperature of 37ᵒC. On Nutrient agar, after 24 hours of incubation at 37ᵒC the colonies are large 2-3 mm in diameter, circular, low convex, surface smooth, translucent,with entire edges and, easily emulsifiable. 20

On MacConkey agar, Salmonella colonies appear as large, circular, 1-3 mm in diameter, low convex, non-lactose fermenting, colonies. On blood agar, the colonies appear as large, circular, moist, 1-3 mm in diameter, greyish white, low convex colonies with entire edges.

One of the selective media for Salmonella is Deoxycholate citrate (DCA) media that contains high concentration of bile salts. S.typhi grow as pale, nearly colourless, smooth and shiny, translucent colonies with black centers due to the production of H2S. S.paratyphiA do not produce

H2S, so the colony morphology on DCA will be pale, nearly colourless,

smooth, shiny and translucent colonies.

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S.typhi, which decarboxylate lysine, initially produce yellow colonies from the utilization of xylose and later on the colonies become red due to the decarboxylation of lysine with black head centers due to production of hydrogen sulphide.20, whereas S.paratyphi A colonies are red colour in XLD as they do not produce H2S.

On Wilson and Blair bismuth sulphite medium, H2S producing

S.typhi and S.paratyphi B cause reduction of sulphite to sulphide, thereby producing jet black colonies with a metallic sheen. Salmonella paratyphi A do not form H2S, so they produce green coloured colonies in Wilson and

Blair bismuth sulphite media. 22

In Salmonella –Shigella agar, another selective media, the colonies of S.typhi and S.paratyphi B appear colourless with black centers, owing to the production of H2S, whereas S. paratyphi A produce colourless

colony without black centers.24

Hektoen enteric agar, one of the recently formulated media, S.typhi produces blue-green colonies, with black centers from H2S production,

whereas S.paratyphi A produce blue green colonies without black centers.24

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The commonly employed enrichment media in isolation of Salmonella species from faeces are Selenite F broth, tetrathionate broth and gram negative broth. Whereas isolation of Salmonella from blood samples require either Bile broth or BHI broth.

Biochemical reactions :

Salmonellae are catalase positive and oxidase negative. They reduce nitrates to nitrites. Indole is not formed, citrate is utilized except by S.typhi, S.paratyphi A, urease is not hydrolysed. In triple sugar iron, Salmonella typhi produce [K/A] alkaline/acid slant with speck of H2S,

Salmonella paratyphi A produce K/A with gas, whereas Salmonella paratyphi B produce K/A with abundant H2S. 24

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Biochemical reactions of Salmonella species 22,24

Species Indole Citrate Urease TSI Gas/H2S Lysine Arginine Ornithine

S.typhi _ _ _ K/A

-/+ (speck)

+ - -

S.paratyphi A _ _ _ K/A +/ - - - +

S.paratyphi B _ + _ K/A + / ++ + + +

Pathogenesis :

The infective dose of Salmonella typhi is 103 – 106 bacilli to initiate the infection in humans.

Mode of transmission :

Enteric fever is transmitted commonly by feco-oral route, through contaminated food and water. The carriers who are asymptomatic, or the patients those who have recently recovered from the illness, excretes enormous S.typhi in the stool which causes contamination of food and water.

The following are the modes of transmission,

i) handling food directly without hand washing

ii) Flies act as vector in transmission of bacilli

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Roughly about 10% of those who have recovered from enteric fever excretes the bacilli in the faeces for about 12 weeks and 2-3% became carriers in future and hence become the source of infection.

Predisposing factors for transmission :

The transmission of Salmonella enterica species are promoted by the following factors like

i) antacid ingestion

ii) achlorhydria

iii) prior Helicobacter pylori infection

iv) inflammatory bowel disease

v) prior GIT surgery

vi) suppression of the intestinal flora by antibiotics

The organisms enter the intestine via M cells which are epithelial cells that lines the mucosa of the intestine. The bacilli forms the membrane ruffles on the cell membrane of M cells. These ruffles enclose the adherent bacteria inside the large vesicles. This process of uptake is called bacteria mediated endocytosis. 22

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necessary for the bacterial uptake. Following entry, the bacilli remain inside the vacuoles in the cytoplasm. 22

Salmonellae present in the vacuoles cross the epithelial layer to reach submucosa, where the macrophages phagocytose the bacilli. Salmonella typhi cause alterations on the surface, hence the organism are resistant to the lysosomal enzymes of macrophages. This process is regulated by the organisms regulatory systems such as PhoP/PhoQ system, that renders the expression of outer membrane proteins, thus facilitating the alterations in lipopolysaccharides. 22

The organisms that are present in the macrophages spread through the lymphatics to enter the blood stream, thus leading to transient primary bacteremia. The Salmonellae then disseminates throughout the reticulo endothelial tissues like liver, spleen, lymph nodes, bone marrow and the other organs like gall bladder, kidneys and lungs where further multiplication takes place. Secondary bacteremia occurs from the seeded organs, that leads to the onset of clinical illness.

Antigenic Structure and Virulence Factors :

Salmonellae have the following antigens on their cell wall. They are

i) Somatic antigen (O)

ii) Flagellar antigen (H)

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The non-specific antigens like fimbriae are present in some strains of Salmonellae.

Somatic antigen (O) :

The somatic antigen O is a phospholipid-protein-polysaccharide complex and it is an integral part of the cell wall lipopolysaccharide. It is similar to endotoxin. Somatic (O) antigen is less immunogenic.

The antibody to O antigen develops early and disappears early, so its presence indicates recent infection. When mixed with antisera, O antigen suspension agglutinates and forms compact, granular,chalky clumps. But agglutination occurs slowly and the optimum temperature for agglutination is 55ᵒ C.22 The antibody to O antigen is cross reactive, so

H antigen is a more reliable indicator. 20

Flagellar antigen H :

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Vi antigen:

It is a surface polysaccharide envelope or capsular antigen that envelopes the O antigen. Vi antigen is related to its virulence. It is present in only few serotypes like S.typhi, S.paratyphi C and S.dublin. The bacilli are inagglutinable to the O antiserum, when it contains Vi antigen. By heating at 100ᵒC for one hour, the strain becomes agglutinable with O antisera, when Vi antigen is removed and exposes the O antigen. Vi antigen is destroyed by 1N HCl and 0.5 N NaOH but not by alcohol or formaldehyde.

Vi antigen is not used in the diagnostic purpose as it is poorly immunogenic and the antibody titres are low. Hence Vi antigen is not employed in the Widal test. However it is believed that the complete absence of Vi antibody in a case of typhoid fever indicates poor prognosis. The Vi antibody disappears early in convalescence, so its presence indicates the development of carrier state. 20

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M antigen :

It is a loose extracellular polysaccharide slime which contains colanic acid. These antigens are non-specific, as they are present in serotypes of Salmonella as well as in many strains of E.coli. These antigens resemble Vi antigens by masking agglutination with O antibodies. When heated to 100ᵒ C, M antigen gets removed thereby rendering the bacilli agglutinable to O antibody. 21

Fimbrial antigens :

These antigens are non–specific and present among various members of the enterobacteriaceae. These antigens are shared between many serotypes of Salmonellae, hence cross reactions occur in agglutination tests, if bacteria is in fimbrial phase and the sera contains fimbrial antibodies. 21

Antigenic variations :

Salmonellae that possess various antigens undergo many types of phenotypic and genotypic variations.

Variations of O Antigen :

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i) S -- R variation :

Mutation causes Smooth to Rough variation and this variation is associated with the loss of O antigen and of virulence of the organism.

ii) Lysogenic conversion :

Salmonella when gets infected with a bacteriophage, they either loose or gain O antigen, this is called as lysogenic conversion.

Variations in H antigen :

i) OH -- O variation :

In this variation the motile strains of Salmonellae may loose their flagella and they become non-motile. But the inhibition of flagella is only temporary.

ii) Phase variation :

The flagellar antigens exist in two phases, each phase comprising of distinct set of flagellar antigens. Phase 1 antigens are serotype specific and designated as a,b,c,, etc. Phase 2 antigens are nonspecific or group antigens, they are few in number that are designated as 1,2,3,etc.

Variation in Vi antigen : ( V-W Variation )

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is lost completely and such strains W forms are agglutinable by O antisera and not by Vi antisera. 22

Clinical manifestations of Enteric fever :

The incubation period of enteric fever is about 10-14 days. With every spike, the fever peaks to higher level then gradually falls down (step ladder pattern type of remittent fever). The other symptoms like headache, chills, cough, sweating, myalgia and arthralgia occurs. During the episode of fever, 25% of the patients develop rose spots, that are faint, salmon coloured, blanching, maculopapular rash occurring over trunk and abdomen.6

Rajesh Upadhyay et al in 2015 from New Delhi explained abdominal pain, nausea, vomiting, constipation in adults, diarrhea in children and anorexia are the early intestinal manifestations that may occur. The important clinical signs that are elicited are relative bradycardia, hepatospleenomegaly, and epistaxis.15

About 10% of the cases develop complications like gastrointestinal bleeding, intestinal perforation and peritonitis in the third or fourth week of illness. In enteric fever the high case fatality rate may be seen in those patients with altered sensorium.

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The other rare complications that may occur are neurological manifestations like meningitis, encephalomyelitis, Gullain-Barre syndrome, cranial or peripheral neuritis, cerebellar ataxia and neuro-psychiatric symptoms like hysteria, delirium, paranoid pyschosis and aggressive behaviour.

The major serious complications that are documented with typhoid fever are haemorrhage that may cause sudden death in some patients. Hepatitis, myocarditis, pneumonia, disseminated intravascular coagulation, thrombocytopenia and haemolytic uremic syndrome are the other rare complications that may occur. About 15% of enteric fever cases died due to prolonged fever in the pre-antibiotic era. Few others develop genito-urinary manifestations or develop relapse or carrier state.

The factors that influence the severity and outcome of the infection are; duration of illness before the initiation of treatment, the choice of antimicrobials, age, vaccination history, the virulence of the bacterial strain, host factors (e.g. AIDS or other causes of immune-suppression)

and whether the individual was taking other medications such as H2 blockers or antacids to diminish gastric acid.

Carriers:

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i) Convalescent carriers : Those who shed the bacilli in faeces for three weeks to three months after recovery of illness are called convalescent carriers.

ii) Temporary carriers : Those who shed the bacilli in faeces for more than three months but less than a year are called temporary carriers. iii) Chronic carriers: Those who shed the bacilli in faeces for more

than a year are called chronic carriers, which occurs in about 1-4% of enteric fever cases.

Detection of carriers :

The stool culture and bile culture methods are used to detect the faecal carriers and urine culture detects the urinary carriers. The demonstration of Vi antibodies is also used to detect the carriers. Isolation of Salmonella from sewage is carried out either by Sewer –swab technique or by filtration method to trace the carriers in the communities.

Classification of typhoid fever cases ( WHO Guidelines )

Confirmed case The patients with fever for more than 3 days and culture positive for S.typhi (blood, urine, stool culture) Probable case The patients who have fever for more than 3 days

with a positive serological test but S.typhi is not isolated in culture.

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Laboratory Diagnosis :

The laboratory diagnosis of enteric fever begins with the basic investigations like complete blood counts with differential leukocyte count. The clinical diagnosis of enteric fever is confirmed either by isolation of bacilli by culture methods or by the demonstration of antibodies in the serum.

The gold standard test for the diagnosis of enteric fever is isolation of organism by blood culture, so it is mandatory to perform it in all clinically suspected enteric fever cases. Blood culture is the ideal method for diagnosis in the first week of illness, which becomes positive in about 90% of cases.

Bone marrow culture can be done, when blood culture is negative especially when the patient is on antibiotics. Duodenal aspirate culture can be done if both blood and bone marrow culture are negative. Stool and urine culture are useful for the isolation of Salmonella in the third and fourth week of illness.

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Typing of Salmonellae :

This is done mainly for the purpose of surveillance and for determining the source of infection. There are several typing methods like

1. Phenotypic methods that includes Phage typing, Bacteriocin typing, Biotypnig and Antibiogram typing

2. Genotypic methods : The most commonly used methods are the following,

i) Plasmid typing : This is done on the basis of the numbers and molecular weight of plasmids that are present in Salmonella species.

ii) Chromosomal based : This is done by Pulse field gel electrophoresis, Ribotyping and Restriction fragment length polymorphism. DNA sequencing can be done to detect mutations in resistant genes.

Treatment of Enteric fever :

WHO guidelines states that, for fully sensitive isolates either 15 mg/kg of ciprofloxacin or 15 mg/kg of Ofloxacin for a duration of 10-14 days can be given. In case of multi-drug resistant strains either 15mg/kg of Ciprofloxacin or Cefixime of 15-20 mg/kg for 10-14 days should be prescribed. For quinolone resistant isolates, Ceftriaxone 75

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Treatment of Carriers :

Carriers should be treated either by Amoxycillin with probenicid of 30 mg/kg, orally tid for 6-12 weeks or Cotrimoxazole 4-20 mg/kg, oral bid for 6-12 weeks. Ciprofloxacin in a dose of 1500 mg oral bid for 4 weeks can also be used for treating carriers.

In 1948 chloramphenicol has been introduced as the drug of choice for treatment of enteric fever. This drug inhibits the bacterial synthesis and act as bacteriostatic agent. In the past, as Chloramphenicol is orally active and also a broad spectrum antibiotic, it was irrationally used resulting in the development of resistance. Moreover because of its adverse effect and its contraindication during pregnancy and in neonates, Ampicillin and Cotrimoxazole were used as an alternative source in treatment of enteric fever.

Enteric fever responded to ampicillin, amoxicillin and cotrimoxazole. Later on due to emergence of multidrug resistant Salmonella typhi, fluoroquinolones like ciprofloxacin, ofloxacin and pefloxacin are used for the treatment. These drugs act by inhibiting bacterial enzymes DNA gyrase and topoisomerase IV, which play a major role in division, coiling and supercoiling of bacterial DNA during multiplication. 4,18

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considered to be a surrogate marker for fluoroquinolone resistance. Since Nalidixic acid do not determine the low level resistance in Salmonella species, CLSI 2015 and EUCAST suggested Pefloxacin as the reliable surrogate marker of fluoroquinolone resistance. 18

Due to the development of resistance to fluoroquinolones by Salmonella species, the Cephalosporins like Ceftriaxone, Cefixime, Cefipime and Cefpodoxime are widely used for the treatment of enteric fever. Recently third generation cephalosporins like ceftriaxone, cefotaxime and cefoperazone are used effectively in treatment. The mechanism of

action of Cephalosporins are inhibition of bacterial cell wall synthesis. 8

Jaspal Kaur on 2013 explained Azithromycin, a broad spectrum azilide, can be the drug of choice over fluoroquinolones, cephalosporins and chloramphenicol because of the following reasons.4

i) negligible relapse rate,

ii) its defervescence is rapid,

iii) effective in removing intracellular Salmonella,

iv) gastrointestinal carriage is eradicated,

v) potential drug in paediatrics where quinolones are contraindicated.

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protein synthesis. The carbapenems are a class of beta lactam antibiotics with broad spectrum activities and they are stable to the hydrolysis of ESBL producing isolates. The carbapenems and tigecycline are the potent drugs in case of ceftriaxone resistant isolates.4

History of Antimicrobial resistance :

In 1950, the Chloramphenicol resistant S.typhi first emerged in UK and subsequently this resistance was observed in Greece and Israel. There were sporadic reports of Chloramphenicol resistance world wide, including India. The plasmid encoded Chloramphenicol resistance was first observed in early 1970יs followed by epidemics in Central America. However in 1972, chloramphenicol resistant S.typhi strains became a major problem in causing outbreaks worldwide.4,8

In 1980 multi-drug resistant S.typhi strains emerged in south east Asia. A single plasmid is responsible for multi-drug resistance and this is highly transmissible. This plasmid belongs to incompatibility group, H I1.In 1987 multi-drug resistant strains of S.typhi were first reported in Pakisthan. In India MDR strains of S.typhi were first reported in 1990 and an outbreak called Dombivali fever reported in Mumbai. There were sporadic reports of multi drug resistant S.typhi strains all over India. 4,12

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gradually developed resistance to fluoroquinolones. Saha et al and Kumar et al in 2007 declared third generation cephalosporins also developed resistant to S.typhi strains.4,12

Mechanism of Antimicrobial resistance :

Typhoidal Salmonella exhibits antimicrobial resistance by two factors

i) Acquisition of foreign genes via plasmids

ii) Mutation on chromosomes.

Resistance can be achieved by horizontal acquisition of resistant genes, mobilized via insertion sequences, transposons, and conjugative plasmids. The antimicrobial resistance is also exhibited by recombination of foreign DNA into the chromosome or by mutations in different chromosome loci. 8

Plasmid mediated resistance :

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The mechanism of drug resistance mediated by acquisition of R plasmids are due to

i) Inactivation of the drug

ii) reduced membrane permeability

iii) modification of drug site

iv) rapid efflux of antibiotic

i) Inactivation of drug :

This is the common cause of resistance that inactivates antimicrobials. The enzyme beta lactamase present in various bacteria is best known example for inactivation of the drug. Enzyme beta lactamase cause hydrolysis of beta lactam ring of penicillin and cephalosporins. The initial strains of antibiotic resistant S.typhi carried chloramphenicol acetyl transferase type I, which encodes an enzyme that inactivates chloramphenicol via acetylation.4,8

Datta et al reported that S.typhi has acquired R plasmid in the intestine of human beings from other enteric bacteria. Chloramphenicol resistant S.typhi have emerged due to acquisition of R plasmid which encodes the enzyme acetyl transferase that inactivates chloramphenicol. 4,8

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resistance and this plasmid belongs to the incompatibility group, H I1 and this is highly transmissible.

ii) Reduced membrane permeability :

The pathogens by preventing the entrance of the drug they become resistant. The new genetic information changes the nature of proteins in the membrane, which leads to the alteration in the membrane permeability. Such an alteration cause a change in the membrane transport system pores, and hence the antibiotics can no longer cross that membrane. Salmonella typhi, exhibited resistance to tetracycline, quinolones and some aminoglycosides by this mechanism.

iii) Modification of target site :

The mechanism of drug resistance in S.typhi occurs when the target enzyme or the cellular structure of the pathogen gets modified, so that it is no longer susceptible to the drug.

iv) Rapid extrusion or efflux of the antibiotic :

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The genes like plasmid mediated beta lactamases, tetracycline –resistance genes, and aminoglycoside modifying enzymes are organized on transposons.

Chromosome mediated resistance :

Chromosomal resistance is due to the mutation in the gene that codes for either the target of the drug or the transport system in the membrane that controls the uptake of the drug. The irrational use of the antibiotic has led to the emergence of chromosomal mediated drug resistance phenomenon against fluoroquinolones. This has been attributed to a single point mutation in the quinolone resistance determining region of topoisomerase gene gyrA, which encodes DNA gyrase. 8

The fluoroquinolones target DNA gyrase and topoisomerase IV. These are bacterial enzymes that are responsible for the uncoiling and recoiling of bacterial DNA for transcription. Salmonella typhi most commonly develops fluoroquinolone resistance through specific mutations in gyrA and par C which codes for DNA gyrase and topoisomerase IV, respectively.

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The quinolones that are used in the treatment of enteric fever are ciprofloxacin, levofloxacin, ofloxacin and gatifloxacin. In gatifloxacin and moxifloxacin the primary target is gyrA gene, and for ciprofloxacin and levofloxacin it is the parC gene.This explains the varied pattern of susceptibilities, so all the fluoroquinolones should be tested individually.4

Resistance to trimethoprim is due to mutations in the chromosomal gene that encodes the enzyme dihydrofolate reductase. The resistance to sulfonamides is mediated by a chromosomal mutation in the gene encoding for the target enzyme dihydropteroate synthetase, that reduces the binding affinity of the drug.

Extensive use of Cephalosporins leads to the development of resistance by producing Extended Spectrum Beta Lactamases. S.typhi produces a variety of ESBL types like TEM, SHV, CTXM enzymes. TEM types of ESBL was first discovered in a patient called Temonieria and hence named as TEM. SHV was named so because they are sulfhydryl variable.CTX-M are ESBL s that have the tendency to hydrolyse Cefotaxime.

Prophylaxis for Enteric fever :

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The early diagnosis of illness and treatment with appropriate antibiotics prevents the development of carriers, who play a major role in the transmission of the disease. The prompt detection of carriers by stool / urine culture or by demonstration of Vi antibodies, aids in treating the carriers early.

Vaccines :

Vaccination of individuals protects only for short duration and it is indicated in following conditions,

i) those who are travelling to endemic areas

ii) household contacts

iii) school children and hostellers

The three types of vaccines available for typhoid fever are,

i) parenteral TAB vaccine

ii) parenteral Vi polysaccharide vaccine

iii) typhoral vaccine

i) Parenteral TAB vaccine :

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ii) Parenteral Vi polysaccharide vaccine :

This vaccine contains purified Vi polysaccharide antigen obtained from S.typhi strain Ty2. It is advised after two years of age, given as a single dose of 25 µg of Vi antigen intramuscular or subcutaneous. It confers protection for 2 years.

iii) Typhoral vaccine :

It is an oral, live, attenuated S.typhi Ty2 1 a vaccine given after six years of age, orally before food on 1,3,5 and /or 7th day i.e totally

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MATERIALS AND METHODS :

Place of study :

This Study on Molecular Characterisation of Anti-Microbial Resistance in Salmonella Enterica Serovar typhi and paratyphi From Blood Culture Isolates in a Tertiary Care Hospital was carried out in fever OP and in Medicine / Paediatric Department in Coimbatore Medical College and Hospital, Coimbatore.

Duration of study :

The study was conducted over a period of one year from July 2016 to June 2017

Type of study : Prospective Study

Approval :

Ethical clearance was obtained from Institutional Ethics Committee and informed consent was obtained from the patients and also the parents or guardians of the accompanying children.

Sample size :

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Inclusion Criteria :

Patients with signs and symptoms of enteric fever like ( step ladder pattern type of remittent fever, relative bradycardia, hepatosplenomegaly and rose spots) of all age groups attending on an outpatient or inpatient basis were included in this study.

Exclusion criteria :

Those patients who had taken antibiotics within a week were excluded from this study.

Evaluation :

A detailed information about this study was informed to the patients, parents / guardian and informed consent obtained before the study. A detailed history regarding the patients name, age, address, duration and history of presenting illness, treatment history about antibiotics taken were obtained.

Sample Collection :

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Under aseptic precautions, 5 ml and 10 ml of venous blood was collected from children and adults respectively in patients of acute febrile illness. Blood samples collected were inoculated aseptically into blood culture bottle containing 25 - 50 ml of Brain heart infusion broth and incubated aerobically at 37°C.

The broth was examined regularly for bacterial growth like turbidity and subculture was done on Nutrient agar, Mac Conkey agar and on Blood Agar plate. The plates were incubated for 24 hours at 37°C. Any growth was further processed for identification as per the standard procedure. The organisms were identified by their colony morphology, Gram staining methods, motility test and following biochemical reactions with suitable controls.

 Catalase  Oxidase  Indole test  Citrate test  Urease test

 Triple sugar iron test  Nitrate reduction test

 Methyl red and voges-Proskauer test  Sugar fermentation tests

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GRAM STAIN :

A smear was prepared from 18-24 hours old colony from the agar plate. After air drying and heat fixation, Gram staining was done using 1% methyl violet as primary stain, grams iodine as mordant, acetone as decolouriser and dilute carbol fushcin as counterstain. Gram reaction, shape and arrangement of the bacteria were observed. Salmonella species appear as gram negative rods.

MOTILITY :

Hanging drop procedure was done using, the saline suspension of the colony and the edge of the drop was focused under high power microscope to observe for the motility of the bacteria. Salmonella species are motile except for S.gallinarum and S. pullorum.

CATALASE TEST:

This test is done to determine the ability of the organism to produce catalase which splits hydrogen peroxide into oxygen and water. The release of oxygen produces brisk effervescence.

Procedure

 One or two colonies to be tested were picked up by a sterile glass

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 Production of brisk effervescence indicates catalase production.

Salmonella species were catalase positive. OXIDASE TEST :

It is done to determine the presence of bacterial cytochrome oxidase that takes part in aerobic respiration by transferring electrons to oxygen forming water.

Procedure

Freshly prepared oxidase reagent (tetramethyl p-phenylene diamine dihydrochloride) was poured on a filter paper placed in a petridish and the colony to be tested was smeared on the filter paper and development of a deep purple colour in 10 seconds shows the organism was oxidase positive, otherwise negative. Salmonella species were oxidase negative.

INDOLE TEST :

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CITRATE UTILIZATION TEST :

This test is done to determine the ability of organism to utilize citrate as a sole source of carbon and inorganic ammonium salt as a sole source of nitrogen. The organisms was streaked on the surface of a slant of Simmon’s citrate medium and incubated at 37ºC for 18-24 hours. Development of deep blue colour of the medium / presence of growth was taken as positive. Salmonella typhi and Salmonella paratyphi A do not utilize citrate where as Salmonella paratyphi B utilizes citrate.

UREASE HYDROLYSIS TEST :

The organisms ability to produce urease which hydrolyses urea into ammonia and carbon dioxide is determined by this test. The organisms was streaked on the slope of Christensen’s urease medium and incubated at 37ºC for 18-24 hours. Those organisms which hydrolyse urea will produce pink colour change. Salmonella species do not hydrolyse urea.

TRIPLE SUGAR IRON (TSI) TEST :

This test is done to determine the ability of organisms to ferment a specific carbohydrate incorporated in a basal growth medium with or without production of gas and H2S production.

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18-24 hours at 37ºC, and examined for the presence of growth and fermentation. Salmonella species produces alkaline slant and acid butt reaction ( K/A ) because of the fermentation of glucose alone which occurs in minimal concentration. Salmonella typhi produces K/A with speck of H2S present at the junction of slant and butt. Salmonella

paratyphi A produce K/A with gas but no H2S whereas Salmonella

paratyphi B produces K/A with gas and abundant H2S.

NITRATE REDUCTION TEST :

This was tested after growing the bacterium for 2-3 days at 37°c in nitrate broth. Those organisms which reduce nitrates to nitrites was detected by the addition of α-napthylamine (reagent A) and sulfanilic acid (reagent B), which forms a red diazonium dye, p-sulfobenzene –azo –α –naphthylamine. A red colour developing within a few minutes denotes a

positive reaction, while absence of colour indicates negative reaction. Salmonella species belonging to Enterobacteriaceae reduce nitrates to nitrites.

METHYL RED TEST ( MR ) :

This test is used to identify those organisms that produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the medium.

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was added to the broth. Appearance of red colour at the surface of the medium is considered positive. Salmonella species are Methyl red positive.

VOGES PROSKAUER (VP ) test :

VP test is used to identify those organisms which produces neutral acid end products acetoin from glucose fermentation. The isolates to be tested were inoculated into MR/VP broth and incubated at 37°C for 48 to 72 hours. First reagent A 0.6 ml of 5 % α- napthol in absolute alcohol was added, followed by 0.2 ml of reagent B ( 40% KOH ). The tube was shaken gently without plugging, so that the medium gets exposed to atmospheric oxygen. The tubes were allowed to stand undisturbed for 10-15 minutes. A positive test is represented by the development of a red colour, indicating the presence of diacetyl, the oxidative product of acetoin. Salmonella species are VP negative.

CARBOHYDRATE FERMENTATION TEST :

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DECARBOXYLASE & DIHYDROLASES TEST(Lysine –Arginine-Ornithine):

This test is done to determine the ability of the organism to decarboxylate an amino acid to form an amine with resulting alkalinity. Moellers decarboxylase medium is the base most commonly used for determining the decarboxylase capabilities of Enterobacteriaceae. The amino acid to be tested is added to the decarboxylase base before inoculation of the test organism. A control tube consisting only the base without the amino acid was also set up in parallel.

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Differentiation of serotypes within the Salmonella enterica.

TESTS S.typhi S.paratyphi A S.paratyphi B

Triple Sugar Iron

K/A with speck of H2S

K/A with gas & no H2S

K/A with abundant H2S,

gas Citrate Not utilized Not utilized Utilized Lysine

decarboxylation

Decarboxylated Not

decarboxylated

Not

decarboxylated Ornithine

decarboxylation

Not

decarboxylated

decarboxylated Decarboxylated

Arginine

decarboxylation

Not

decarboxylated

Not

decarboxylated

Decarboxylated

Glucose + / ( no gas ) + / ( gas + ) + / ( gas + )

Xylose _ _ +

Arabinose _ + +

Slide Agglutination Test :

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The isolates were therefore confirmed by slide agglutination test using the High titre sera of Salmonella typhi O, H, Salmonella paratyphi AH and Salmonella paratyphi BH procured from King Institute of Preventive Medicine and Research, Chennai. In a sterile clean glass slide the colony to be tested was emulsified in normal saline to form a uniform smooth milky white suspension. To this suspension 1-2 drops of High titre sera were added and the slide was rotated thoroughly for few seconds.

A positive result is indicated by visible clumping with clearing of suspension, whereas if the milky white suspension remains unchanged it indicates a negative result. Salmonella typhi agglutinates with S.typhi O, H antisera, S. paratyphi A agglutinates with S.paratyphi AH antisera and S.paratyphi B agglutinates with S.paratyphi BH antisera.

ANTIBIOTIC SUSCEPTIBILITY TESTING:

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Mueller-Hinton agar was prepared according to the manufacturer’s instructions and poured into petri dishes of 9 cm diameter to a depth of 4mm.

Preparation of 0.5 McFarland standards

1. Prepare 1% solution of sulphuric acid by adding 1ml of concentrated sulphuric acid to 99 ml of distilled water. Mix well.

2. Prepare 1% solution of barium chloride by dissolving 0.5g of dihydrate barium chloride in 50ml of distilled water.

3. Add 0.6 ml of BaCl² solution to 99.4 ml of H2SO4 solution

to make upto 100 ml.

This can be stored at room temperature for up to six months. The 0.5 McFarland standard provides an optical density equivalent to the density of 1.5 × 108 colony forming units/ml.

List of Antibiotics:

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Procedure :

A sterile swab was immersed in the prepared bacterial suspension and lawn culture was done on Mueller Hinton Agar plate. The surface of MHA plate was swabbed in three directions ensuring even and complete distribution of the inoculum over the entire plate. Within 15 minutes of inoculation the antimicrobial discs were applied using a sterile forceps.

The antimicrobial discs used were procured from Himedia. Each disc was pressed down to ensure complete contact with the surface of MHA. The plates were inverted for incubation to prevent accumulation of moisture on the agar surface which would interfere with the interpretation of the test.

The plates were incubated at 37 °C for 18-24 hours. The zone of inhibition was measured using the zone measuring scale and interpreted according to the CLSI standards. However, Azithromycin interpretation was based on the disc manufacturers recommendations. Minimum inhibitory concentrations (MIC) were determined for ciprofloxacin for all Salmonella isolates which showed resistance by Kirby –Bauer disc diffusion methods using Epsilon test strips.

MIC Determination by E – strips :

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a predefined quantitative gradient which is used to determine the Minimum Inhibitory Concentration (MIC) in mcg/ml of different antimicrobial agents against microorganisms as tested on MHA, following overnight incubation. MIC of ciprofloxacin was determined by using Ciprofloxacin Ezy MICTM Strip (CIP) of concentration 0.002 - 32 mcg/ml procured from HIMEDIA.

Preparation of Inoculum :

The 4-5 similar colonies to be tested was transferred with a loop to peptone water and incubated at 35-37°C for 2-8 hours until turbidity develops. Compare the inoculum turbidity with that of 0.5 McFarland standard.

Test Procedure :

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Interpretation:

The plate was read after the determined time of incubation. The MIC of the isolate was interpreted where the zone of inhibition intersects with the strip.

Molecular characterization of Antimicrobial Resistance Genes : The Quinolone resistant Salmonella enterica species were subjected for the plasmid mediated quinolone resistance genes qnrA, qnrB and qnrS, as well as chromosomal mediated resistance genes gyrA, gyrB and parC by Multiplex PCR.

Requirements:

 Micro Pipettes of variable volume 0.5-10 µl, 10-100 µl, and 100-1000 µl

 Pipette tips with aerosol barrier

 Spin columns

 Collection tubes

 Vortex mixer

 Water bath

 Centrifuge (Refrigerated) with rotor for 1.5ml reaction tubes

 1.5ml / 2ml microcentrifuge tubes

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DNA Extraction:

The DNA extraction procedure yields purified DNA of more than 30kb in size obtained after the lysis of the cell. This kit utilizes the silica based membrane technology in the form of a spin column. The isolated DNA can be used directly for the PCR amplification.

Components of extraction

 Phosphate buffered saline  Lysozyme

 Digestion buffer  Binding buffer

 Proteinase K ( protease )  Internal control template  Isopropanol

 70% Ethanol  Elution buffer

Storage and stability:

 The bacterial genomic DNA extraction kit was stored at room

temperature.

 The proteinase K and Lysozyme were stored at -20 0 C.

Bacterial pellet preparation:

About 4-6 colonies of the test strains were inoculated in peptone water and incubated at 370C overnight. 1-1.5 ml of bacterial culture was

Figure

FIG 2: MacConkey Agar – Non-lactose fermenting colonies of                                             Salmonella typhi

FIG 2:

MacConkey Agar – Non-lactose fermenting colonies of Salmonella typhi p.85
FIG 1: NUTRIENT AGAR PLATE – Salmonella typhi colonies

FIG 1:

NUTRIENT AGAR PLATE – Salmonella typhi colonies p.85
FIG 3: BLOOD AGAR PLATE : Salmonella typhi – greyish white colonies

FIG 3:

BLOOD AGAR PLATE : Salmonella typhi – greyish white colonies p.86
FIG 4: DEOXYCHOLATE CITRATE AGAR –

FIG 4:

DEOXYCHOLATE CITRATE AGAR – p.86
FIG 5: DEOXYCHOLATE CITRATE AGAR – Salmonella paratyphi A

FIG 5:

DEOXYCHOLATE CITRATE AGAR – Salmonella paratyphi A p.87
FIG 6: Gram’s Stain – Gram Negative bacilli

FIG 6:

Gram’s Stain – Gram Negative bacilli p.87
FIG 8: Biochemical Reactions  of  Salmonella  paratyphi A

FIG 8:

Biochemical Reactions of Salmonella paratyphi A p.88
FIG 7: Biochemical Reactions of Salmonella typhi

FIG 7:

Biochemical Reactions of Salmonella typhi p.88
FIG 10: LAO – Lysine Decarboxylated in Salmonella typhi

FIG 10:

LAO – Lysine Decarboxylated in Salmonella typhi p.89
FIG 9: Sugar Fermentation by Salmonella typhi

FIG 9:

Sugar Fermentation by Salmonella typhi p.89
FIG 11: LAO – Ornithine Decarboxylated in Salmonella paratyphi A

FIG 11:

LAO – Ornithine Decarboxylated in Salmonella paratyphi A p.90
FIG 12: Mueller Hinton Agar – AST by Kirby – Bauer Method

FIG 12:

Mueller Hinton Agar – AST by Kirby – Bauer Method p.91
FIG 13: Mueller Hinton Agar – AST by Kirby – Bauer Method

FIG 13:

Mueller Hinton Agar – AST by Kirby – Bauer Method p.91
FIG 15: MIC – E Test for  Ciprofloxacin

FIG 15:

MIC – E Test for Ciprofloxacin p.92
FIG 14: Mueller Hinton Agar – AST by Kirby – Bauer Method

FIG 14:

Mueller Hinton Agar – AST by Kirby – Bauer Method p.92
FIG 17:  Multiplex PCR for Chromosomal Mediated Quinolone Resistance genes (gyrA, gyrB, parC)

FIG 17:

Multiplex PCR for Chromosomal Mediated Quinolone Resistance genes (gyrA, gyrB, parC) p.93
FIG 16:  Multiplex PCR for Plasmid Mediated Quinolone Resistance genes

FIG 16:

Multiplex PCR for Plasmid Mediated Quinolone Resistance genes p.93
FIG 18: Gene Sequencing – gyrA, gene at quinolone resistance determining region

FIG 18:

Gene Sequencing – gyrA, gene at quinolone resistance determining region p.94
FIG  19:  Gene Sequencing – parC, gene at quinolone resistance determining region

FIG 19:

Gene Sequencing – parC, gene at quinolone resistance determining region p.96
TABLE 1: PERCENTAGE  OF  ENTERIC  CULTURE  POSITIVES

TABLE 1:

PERCENTAGE OF ENTERIC CULTURE POSITIVES p.103
TABLE 3:  GENDER  DISTRIBUTION  OF  ENTERIC FEVER CASES

TABLE 3:

GENDER DISTRIBUTION OF ENTERIC FEVER CASES p.103
TABLE 2:  DISTRIBUTION   OF SALMONELLA SPECIES (n=28)

TABLE 2:

DISTRIBUTION OF SALMONELLA SPECIES (n=28) p.103
TABLE 4:  AGE  WISE   DISTRIBUTION OF  SALMONELLA SPECIES (n=28)

TABLE 4:

AGE WISE DISTRIBUTION OF SALMONELLA SPECIES (n=28) p.104
TABLE 5: CORRELATION  BETWEEN   NEUTROPHIL COUNT

TABLE 5:

CORRELATION BETWEEN NEUTROPHIL COUNT p.105
TABLE  6: CORRELATION BETWEEN HEPATOSPLENOMEGALY

TABLE 6:

CORRELATION BETWEEN HEPATOSPLENOMEGALY p.105
TABLE 7: ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

TABLE 7:

ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF p.106
TABLE  8:  ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF

TABLE 8:

ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF p.107
TABLE  9:  QUINOLONE RESISTANCE AMONG

TABLE 9:

QUINOLONE RESISTANCE AMONG p.108
TABLE  10:  ANALYSIS OF RESISTANT GENES BY

TABLE 10:

ANALYSIS OF RESISTANT GENES BY p.108
TABLE  11: MUTATION  ANALYSIS  OF

TABLE 11:

MUTATION ANALYSIS OF p.109

References

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