studies on virulence, antimicrobial resistance and genetic diversity of salmonella typhimurium ...

145 Pages·2016·18.85 MB·English

STUDIES ON VIRULENCE, ANTIMICROBIAL
RESISTANCE AND GENETIC DIVERSITY OF
SALMONELLA TYPHIMURIUM ISOLATES

FROM NORTH INDIA

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Submitted to the

G.B. Pant University of Agriculture & Technology

Pantnagar - 263 145, Uttarakhand, India

By

Prasanna Kumar V
M.V.Sc. (Veterinary Public Health)

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF

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(Veterinary Public Health & Epidemiology)

August, 2016
ACKNOWLEDGEMENT

First of all I bow my head towards the Almighty, the one above all of us, God, for
answering all my prayers, for giving me energy, endurance and showered many blessings to
accomplish my endeavors.
………
With Regardful memories
It is a matter of pleasure to glance back and recall the path one traverses during the
days of hard work and pre-perseverance. It is still great at this juncture to recall all the faces
and spirit in the form of teachers, friends, near and dear ones. I would consider this work
nothing more than incomplete without attending to the task of acknowledging the
overwhelming help I received during this endeavor of mine.
I avail this opportunity to express my deep sense of gratitude and sincere regards to
Dr. S.P. Singh, Dean Student Welfare, Professor and Head, Department of Veterinary
Public Health and Epidemiology and Chairman of my Advisory Committee for his scholastic
guidance, diligent help, continuous inspiration, critical appreciation and all the trust he had
in me during the entire course of this study. I consider myself fortunate to have worked under
his competent guidance.
I am very thankful to members of my advisory committee, Dr. A.K. Upadhyay,
Professor, Department of Veterinary Public Health and Epidemiology, Dr. A.H. Ahmad,
Professor, Department of Veterinary Pharmacology and Toxicology and Dr. Mumtesh Kumar
Saxena, Associate Professor, Animal Biotechnology Centre, Department of Physiology and
Biochemistry for their generous help, cooperation, suggestions and support during the entire
course of my research work. I shall ever stay indebted to them.
I express my appreciation to Dr. Maansi, Assistant Professor, Department of
Veterinary Public Health and Epidemiology for her assistance whenever required. I am very
thankful to Dr. Deepak Kumar, Assistant Professor, Department of Veterinary Public Health
and Epidemiology for his valuable suggestions, generous help and constant inspiration. I also
owe my sincere gratitude to Dr. Stuti Vatsya, Professor and Head, Veterinary Parasitology
for her kind help and support during the course of study.
I extend my gratitude and sincere regards to Dean, College of Veterinary and Animal
Sciences and Dean, PGS for providing necessary amenities during the course of investigation.
“
I am sincerely thankful to ICAR funded project Outreach Programme on Zoonotic
” “
Diseases and WHO funded project Monitoring of antimicrobial drug resistance in food-
”
borne pathogens for the financial support of my research activities.
I am highly privileged to be a part of Department of Veterinary Public Health and
Epidemiology, C. V & A. Sc, GBPUAT, Pantanagar.
I am highly thankful to Director, VTCC, NRCE, Hissar; Dr. S.K. Kotwal, Professor
and Head, Div. of VPH&E, SKUAST, Jammu and Dr. Purushottam Kaushik, Asst.
Professor, VPH, BVC, Patna for providing their isolates without which this work would not
have seen the light of the day.
I extend my deep sense of gratitude to Dr. Nitin Kurkure, Asso. Prof. and Head,
Dept. of Veterinary Pathalogy, Nagpur Veterinary College, Nagpur; Dr. Sandeep
Chaudhari, Asso. Prof. and Head, Dept. of VPE, Nagpur Veterinary College, Nagpur for
providing facilities to perform PFGE at their laboratory and Satyajit Kale (S.R.F) for
helping me in learning the method.
I wish to express my regard to the technical and supporting staff of our department. I
thank Mr. Sajid Hussain, Mr. Ajay (S.R.F), Mr. Nazurul Hussain, Mr. Pawan, Mr.
Indrajeet, Mr. Chandrabushan, Mr. Shankar and Mr. Fakira for providing a pleasant
working environment in the department and assisted in completing this work successfully and
timely. I am also thankful to security personnel for their kind cooperation during my research
work in off hours and holidays.
I render my special gratitude to Dr(s). Srinivas (D.D), Nagaraj. S (A.D),
Subramanya, Someshekar, Prabhu, Vasanthi, Ravindra, Madhu, Sridar, Praveen and
Manjunath S.P for their support and encouragement in both field (Dept. of A.H & V.S) and
academics. I extend the same to Dr. Madhusudhan, Dr. Prem and Dr. Vishwanath.
I am sincerely thankful to my senior Dr. Bhaskar Ganguly, for his scholastic
guidance, valuable suggestions and for always being there, whenever I hit a roadblock; Dr.
Nirbhay Kumar for his relentless help and Dr. Amit Prasad, for his moral support. My
sincere gratitude to Dr(s). Suman Kumar, Ramees, Aman Kamboj, Shiva and Prutvi2 for
their assistance.
’
I am thankful to my juniors Abhinav, Monika, Kiran, V.P. Singh, Vimal,
Rajashree, Neelam, Rahul, Shweta, Geetika and Aman for their help.
I will always cherish the uninterrupted cooperation and the funfilled days with my
friends Pratima, Sonia, Manali, S.K. Sheetal, Mahesh and others during my stay at
Pantnagar. A special mention to my friend Dr. Mehtab Parmar for his help which finds no
words to express.
“ ”
I take this opportunity to thank my seniors of Pantnagar Kannada Balaga : Dr(s)
Anoop, Erayya, Kalmesh, Jaba, Praveen, Basavaraj, Sajeesh, and juniors Shiva, Jairam,
Rudragouda, Srinivasu and others for their help, support and wonderful company during my
stay here. I also admit my incapability to express my sense of gratitude to Guru sir, Santosh sir
and Raghavan sir for many things during my stay at Pantnagar.
“ ”
I thank seniors and juniors of IVRI Kannada Balaga . A special heartfelt mention
to my friends Chethan and Rakesh and for their hospitality during my stay in holidays and
weekends.
I also deeply acknowledge my friends, Aswartha, Chandregowda, Sharath, Suresh,
and the entire 2005 batch (ZEUS Series) for joyful, unforgettable days in my student life
and I extend the same to my school friends, Yogi, Prasanna, Rahul, Prashanth and many.
There are no words to express my deep sense of gratitude and profound regards to my eternal
friends Shylu and Vasu for their boundless love and caring in every aspect of my life. I am
immensely thankful to my sister Lucky for her love, care, unremitting babble, joyous time and
much more.
Finally and foremost, mere any words from the vastness of literature fail to express
my deepest feeling of indebtedness and respect to my beloved parents and brother Pradeep,
whose tender care, love and endless prayers brought me to this stage of life. I am fortunate to
have extremely supportive family friend Munirathnamma madam for her care and prayers. A
special word of appreciation to Mama, Akka and all my relatives for their love and support.
I also thank all my beloved teachers from my school days to now, whose directions helped in
many assessment. Heartfelt thanks to my Guru G.S.R sir for his invaluable teachings in all
aspects of my life. I devote this thesis to my beloved Mother Smt. Parvathamma and my
teachers.
I thank all those whose name could not find a separate place but helped and
encouraged me directly or indirectly.

Pantnagar (Prasanna Kumar V)
Aug, 2016 Author
CERTIFICATE

This is to certify that the thesis entitled “Studies on virulence,
antimicrobial resistance and genetic diversity of Salmonella Typhimurium
isolates from North India” submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy with major in Veterinary Public Health
and Epidemiology and minor in Animal Biotechnology of the College of Post
Graduate Studies, G. B. Pant University of Agriculture and Technology, Pantnagar,
is a record of bona fide research carried out by Mr. Prasanna Kumar V,
Id. No. 41187 under my supervision and no part of the thesis has been submitted
for any other degree or diploma.
The assistance and help received during the course of this
investigation have been duly acknowledged.

Pantnagar (S.P. Singh)
August, 2016 Chairman
Advisory Committee
CERTIFICATE

We, the undersigned, members of the Advisory Committee of
Mr. Prasanna Kumar V, Id. No. 41187 a candidate for the degree of Doctor of
Philosophy with major in Veterinary Public Health and Epidemiology and
minor in Animal Biotechnology agree that the thesis entitled “Studies on
virulence, antimicrobial resistance and genetic diversity of Salmonella
Typhimurium isolates from North India” may be submitted in partial
fulfillment of the requirements for the degree.
CONTENTS

S. NO. CHAPTERS PAGE
1. INTRODUCTION
2. REVIEW OF LITERATURE
3. MATERIALS AND METHODS
4. RESULTS AND DISCUSSION
6. SUMMARY AND CONCLUSION
LITERATURE CITED
APPENDICES
VITA
ABSTRACT
LIST OF TABLES

Table No. Title Page
1 Details of Salmonella Typhimurium isolates used in the study
2 State-wise distribution of S. Typhimurium isolates
3 Primers used for multiplex PCR targeting ompC and typh genes
4 Primer profile used for detection of virulent genes
5 Primer profile used for detection of anti-microbial resistance gene
and class 1 integron
6 PCR conditions employed for different virulence genes
7 PCR conditions employed for different resistance gene and class 1
integron
8 Distribution of different virulence genes among S. Typhimurium
isolates
9 Prevalence of virulence gene among S. Typhimurium isolates
10 Antimicrobial resistance pattern of S. Typhimurium isolates
11 Antibiotic resistance profile of S. Typhimurium isolate
12 Multiple drug resistant (MDR) pattern of S. Typhimurium isolates
13 Resistance pattern and multiple antibiotic resistance index of S.
Typhimurium isolates as per source
14 Resistance pattern and multiple antibiotic resistance index of S.
Typhimurium isolates as per place of isolation
15 Distribution of multidrug resistant S. Typhimurium among different
locations
16 Distribution of antimicrobial genes in resistant and intermediate S.
Typhimurium
17 Details of Class 1 integron in MDR S. Typhimurium
18 Genotypic characteristics of antimicrobial resistant S. Typhimurium
LIST OF FIGURES

Fig. No. Title Page
1 General overview of the present classification of Salmonella
enterica
2 Latex Agglutination Test
3 PCR product of ompC and typh genes
4 PCR product of invA gene
5 PCR product of sipA gene
6 PCR product of sopE1 gene
7 PCR product of fliC gene
8 PCR product of mgtC gene
9 PCR products of spvC gene
10 PCR product of stn and sopB genes
11 PCR product of gipA gene
12 Distribution of Virulence genes in S. Typhimurium from Uttar
Pradesh
13 Distribution of Virulence genes in S. Typhimurium from
Uttarakhnad
14 Distribution of Virulence genes in S. Typhimurium from Bihar
15 Distribution of Virulence genes in S. Typhimurium from Assam
16 Distribution of Virulence genes in S. Typhimurium from
Jammu and Kashmir
17a Escherichia coli ATCC25922 (quality control) isolate on MHA
plate showing zones of inhibition for different antibiotics
17b Salmonella Typhimurium (S23) isolate on MHA plate
showing zones of inhibition for different antibiotics
18a Distrubution of MDR isolates as per location
18b Distrubution of MDR isolates as per source
19 Fluorescence of MDR Salmonella Typhimurium isolates on
agar plates containing increasing concentrations of Ethidium
bromide
20 PCR product of bla gene
TEM
21 PCR product of aadA1 gene
22 PCR product of strA gene
23 PCR product of strB gene
24 PCR product of sul1 gene
25 PCR product of sul2 gene
26 PCR product of tet(A) gene
27 PCR products of class 1 integron
28 Representative ERIC-PCR profile of different Salmonella
Typhimurium isolates
29 Dendrogram showing genotypic similarity among Salmonella
Typhimurium isolates based on ERIC-PCR fingerprints
30 PFGE profile of different S. Typhimurium isolates
31 Dendrogram showing genotypic similarity among Salmonella
Typhimurium isolates based on PGFE fingerprints
32 Dendrogram showing genotypic similarity among Salmonella
Typhimurium isolates based on ERIC-PCR fingerprints