Status of Antimicrobial Resistance in Bacteria Isolated From Kenyan Chickens

106 Pages·2017·1.38 MB·English

STATUS OF ANTIMICROBIAL RESISTANCE IN BACTERIA ISOLATED FROM
KENYAN CHICKENS

TINO AYUL DENG AJAK (BSc. University of Juba)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR
THE MASTER OF SCIENCE DEGREE OF UNIVERSITY OF NAIROBI IN APPLIED
MICROBIOLOGY (BACTERIOLOGY OPTION)

DEPARTMENT OF VETERINARY PATHOLOGY, MICROBIOLOGY AND
PARASITOLOGY, FACULTY OF VETERINARY MEDICINE
UNIVERSITY OF NAIROBI

2017
i
DECLARATION
ii
DEDICATION
I dedicate this work to the soul of my late father Dr. Ayul Deng Ajak
My mother Nyabage Chol Deng
My brothers Oyenj Ayul, Banian Ayul, Amooj Ayul and Kimo Ayul
Thanks for the prayers, patience and Love

iii
ACKNOWLEDGEMENTS
I would like to give thanks to God for His goodness and mercy endures forever; without The
Almighty God, I wouldn‘t have been able to accomplish my studies.
Many thanks to my supervisors: Prof. Lilly C. Bebora, Prof. Samuel M. Kariuki, Prof. Peter K.
Gathumbi and Dr. Gerald M. Muchemi for their valuable comments, guidance and support
through this study. Special thanks to my lead supervisor Prof. Lilly C. Bebora for everything she
has done to make sure this research becomes a success.
Thanks to Dr. Mahacla O. Odongo for his guidance and support. I appreciate all staff in the
Department of Veterinary Pathology, Microbiology and Parasitology, University of Nairobi for
supporting me during the studies. I thank Mr. Ezekiel H. Weda, Mrs. Mary N. Mutune, Mrs.
Lydia N. Maina, Mrs. Ann K. Munene and Mrs. Charity Gathenya for their assistance with the
laboratory work.
Special thanks to my mother for her endless love, encouragement and inspiration throughout my
studies. Much appreciation to my auntie Dr. Sarah O. Dak for her love and support. I would like
to thanks my friends, George Otieno, Jackson Mushiri and Jasinta Mamur for their moral and
technical support.
I would also like to thank the German Academic Exchange Service (DAAD) for awarding me a
scholarship, without which this research would not have been possible. Many thanks to Prof.
Samuel M. Kariuki, Mr. John N. Kiiru, Mr. Ronald Ngetich and Mr. Samuel Njoroge of the
Center for Microbiology Research, Kenya Medical Research Institute (KEMRI) for technical
support in molecular analysis.
May God bless you all.
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TABLE OF CONTENTS
DECLARATION ...................................................................................................................... ii
DEDICATION ........................................................................................................................ iii
ACKNOWLEDGEMENTS .................................................................................................... iv
TABLE OF CONTENTS ..........................................................................................................v
LIST OF TABLES .................................................................................................................. ix
LIST OF FIGURES ..................................................................................................................x
LIST OF APPENDICES ......................................................................................................... xi
LIST OF ABBREVIATIONS ................................................................................................ xii
ABSTRACT ............................................................................................................................ xv
CHAPTER 1: GENERAL INTRODUCTION .........................................................................1
1.1 Background information .................................................................................................1
1.2 Objectives ..........................................................................................................................2
1.2.1 Overall objective .........................................................................................................2
1.2.2 Specific objectives .......................................................................................................2
1.3 Hypothesis .........................................................................................................................3
1.4 Justification of study ..........................................................................................................3
CHAPTER 2: LITERATURE REVIEW .................................................................................4
2.1 Antimicrobials and antimicrobial resistance .......................................................................4
2.2 Global approaches/concerns to antimicrobial resistance .....................................................5
2.3 Status of antimicrobial resistance in Kenya ........................................................................8
2.4 Scope of antimicrobial resistance in bacteria ............................................................... 10
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2.5 Modes of action of antimicrobials .................................................................................... 11
2.6 Mechanisms of antimicrobial resistance in bacteria .......................................................... 11
2.7 Predisposing factors for development of antimicrobial resistance ..................................... 12
2.8 Detection of antimicrobial resistance/susceptibility in bacteria ......................................... 12
2.8.1 Phenotypic methods .................................................................................................. 12
2.8.1.1 Diffusion technique ............................................................................................. 13
2.8.1.2 Minimum Inhibitory Concentration (MIC) .......................................................... 13
2.8.2 Genotypic methods .................................................................................................... 14
2.8.2.1 Definition and Examples ..................................................................................... 14
2.8.2.2 Polymerase Chain Reaction (PCR) ...................................................................... 15
2.9 Extended – spectrum beta-lactamases .............................................................................. 16
2.10 Bacteria that inhabit the gastrointestinal tract (GI) of chickens ....................................... 17
2.11 Usage of chicken as the study animal ............................................................................. 18
CHAPTER 3: DETERMINATION OF ANTIMICROBIAL RESISTANCE PROFILES OF
BACTERIA ISOLATED FROM CHICKEN ........................................................................ 19
3.1 INTRODUCTION ........................................................................................................... 19
3.2 MATERIALS AND METHODS ..................................................................................... 19
3.2.1 Study Area and study animals ................................................................................ 19
3.2.2 Study design .............................................................................................................. 20
3.2.3 Sample Size Calculation ............................................................................................ 20
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3.2.4 Sampling method and data collection ........................................................................ 21
3.2.5 Sample collection, handling and processing ............................................................... 21
3.2.6 Disposal of carcasses ................................................................................................. 21
3.2.7 Isolation and identification of bacteria ....................................................................... 21
3.2.8 Phenotypic antimicrobial sensitivity testing of the bacterial isolates .......................... 23
3.2.9 Statistical analysis ..................................................................................................... 24
3.3 RESULTS ....................................................................................................................... 24
3.3.1 Aerobic bacteria isolated from the study chickens ..................................................... 24
3.3.2 Antibiotic resistance test results of the bacterial isolates ............................................ 26
3.3.3 Multidrug resistance demonstrated by the bacterial isolates ....................................... 29
3.4 DISCUSSION ................................................................................................................. 35
CHAPTER 4: DETECTION OF ANTIMICROBIAL RESISTANT GENES IN E. COLI
ISOLATES USING POLYMERASE CHAIN REACTION ................................................. 42
4.1 INTRODUCTION ........................................................................................................... 42
4.2 MATERIALS AND METHODS ..................................................................................... 42
4.2.1 Study organisms ........................................................................................................ 42
4.2.2 Isolation of DNA by boiling method .......................................................................... 43
4.2.3 Polymerase Chain Reaction procedure ....................................................................... 43
4.2.4 Gel preparation, Electrophoresis and photography ..................................................... 44
4.2.4.1 Gel preparation ................................................................................................... 44
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4.2.4.2 Electrophoresis and photography ........................................................................ 44
4.3 RESULTS ....................................................................................................................... 45
4.4 DISCUSSION ................................................................................................................. 49
CHAPTER 5: OVERALL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
................................................................................................................................................. 53
5.1 OVERALL DISCUSSION ............................................................................................... 53
5.2 CONCLUSIONS ............................................................................................................. 57
5.3 RECOMMENDATIONS ................................................................................................. 58
REFERENCES ........................................................................................................................ 59
APPENDICES ......................................................................................................................... 82
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LIST OF TABLES
Table 3.2: Overall antimicrobial resistance patterns demonstrated by the isolated bacteria ........ 28
Table 3.3: Multidrug resistance patterns demonstrated by isolates from chickens in the three
study groups ....................................................................................................... 31
Table 3.4: Antimicrobial inclusion rates in the multi-drug resistance blocks: overall and per study
group ................................................................................................................. 34
Table 4.1: Primers and annealing temperatures used in the PCR ............................................... 44
Table 4.2: Proportion of isolated genes in E. coli strains ........................................................... 45
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LIST OF FIGURES

Figure 3.1: Researcher working in the Bacteriology laboratory.................................................. 22
Figure 3.2: Overall proportion of bacteria isolated from chickens .............................................. 25
Figure 3.3: Antimicrobial susceptibility profiles (clear zones) for one E. coli isolates to various
antimicrobial discs on Mueller Hinton agar ........................................................... 29
Figure 4.1: PCR amplification reaction for E. coli bla gene – negative reaction ................... 46
TEM
Figure 4.2: PCR amplification reaction for E. coli bla gene – negative reaction ................. 47
CTX-M
Figure 4.3: PCR amplification reaction for E. coli dfrA1 gene – positive reaction for 3 samples 48
x