Contamination levels and transferability of antimicrobial resistance by Escherichia coli isolated

120 Pages·2015·1.57 MB·English

by  Joyce Arua

Contamination levels and transferability of antimicrobial resistance by
Escherichia coli isolated from raw retail chicken meats in Nairobi,
Kenya.

Joyce Arua Odwar

A thesis submitted in partial fulfilment for the degree of Master of
Science in Medical Microbiology in the Jomo Kenyatta University of
Agriculture and Technology.

2015
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
University.
Signature: _____________________ Date: ___________________
Joyce Arua Odwar
This thesis has been submitted for examination with our approval as University
supervisors.
Signature: _____________________ Date: ___________________
Prof. Samuel Kariuki
KEMRI, Kenya.
Signature: _____________________ Date: ___________________
Dr. Gideon Kikuvi
JKUAT, Kenya.

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DEDICATION
I would like to dedicate this thesis to my husband, my dearest parents Mr. and Mrs.
George Odwar and my two siblings who mean the world to me. Their unconditional
love, encouragement, patience and support motivated me to complete my project
through difficult and challenging times.

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ACKNOWLEDGMENTS
While it is not possible to allocate to each one the credit that is due to them, the
following deserve special attention for making it possible for me to carry out the project
smoothly and complete it successfully; Above all I acknowledge and give glory to the
Almighty God, the giver of life, health, intellect, wisdom and strength. Special gratitude
goes to my supervisors Prof. Samuel Kariuki and Dr. Gideon Kikuvi for their words of
advice, encouragement and dedicating their time to direct me compile my thesis. To Dr.
John Kiiru who assisted by providing his expertise in the field. To all laboratory staff
who tirelessly assisted me with my work both directly and indirectly. To Mr. Kariuki
Ngummo who sacrificed his time and resources in assisting me in my data analysis.

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TABLE OF CONTENTS
DECLARATION ........................................................................................................ II
DEDICATION .......................................................................................................... III
ACKNOWLEDGMENTS ........................................................................................ IV
LIST OF TABLES .................................................................................................... IX
LIST OF FIGURES .................................................................................................... X
LIST OF PLATES .................................................................................................... XI
LIST OF APPENDICES .......................................................................................... XII
LIST OF ABBREVIATIONS .................................................................................XIII
ABSTRACT ............................................................................................................ XVI
CHAPTER ONE .......................................................................................................... 1
1.0 INTRODUCTION ................................................................................................ 1
1.1 Background information ..................................................................................... 1
1.2 Statement of the problem .................................................................................... 3
1.3 Justification of the study ..................................................................................... 3
1.4 Research questions ............................................................................................. 4
1.5 Objectives........................................................................................................... 5
1.5.1 General objective ......................................................................................... 5
1.5.2 Specific objectives ....................................................................................... 5
CHAPTER TWO ......................................................................................................... 6
2.0 LITERATURE REVIEW .................................................................................... 6
2.1 Poultry production ............................................................................................... 6
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2.1.1 Global poultry production ............................................................................ 6
2.1.2 Poultry production and consumption in Kenya ............................................. 6
2.2 Biology of E. coli ............................................................................................... 7
2.2.1 Role of E. coli in causing disease ................................................................. 8
2.2.2 Sources of contamination by E. coli in chicken meat .................................... 9
2.2.3 Laboratory diagnosis and treatment of E. coli infection .............................. 10
2.2.4 Prevention of E. coli contamination in food ................................................ 11
2.3 Antimicrobial consumption in food producing animals ..................................... 12
2.3.1 Global scenario of antimicrobial consumption in food producing animals .. 12
2.3.2 The effects of antibiotics use in food animals on human health ................... 13
2.4 Antimicrobial resistance ................................................................................... 15
2.4.1 Food animals as a source of Antimicrobial resistance ................................. 15
2.4.2 Antimicrobial resistance in E. coli isolated from poultry............................. 16
2.4.3 Transfer of Antibiotic resistance ................................................................. 18
CHAPTER THREE ................................................................................................... 20
3.0 MATERIALS AND METHODS ....................................................................... 20
3.1 Study design ...................................................................................................... 20
3.2 Study site .......................................................................................................... 20
3.3 Study samples ................................................................................................... 22
3.4 Sample size calculation ..................................................................................... 22
3.5 Sampling procedure .......................................................................................... 22
3.6 Laboratory Procedures ...................................................................................... 23
3.6.1 Enumeration of E. coli and total coliform bacteria from retail chicken meat 23
3.6.2 Isolation and presumptive identification of E. coli ...................................... 24
3.6.3 Confirmative identification of E. coli ......................................................... 24
3.6.4 Antimicrobial Susceptibility testing ............................................................ 25
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3.6.5 Template DNA preparation ........................................................................ 26
3.6.6 Primers and PCR amplification .................................................................. 27
3.6.7 Agarose gel electrophoresis ........................................................................ 28
3.6.8 Bacterial in-vitro conjugation tests ............................................................. 29
3.6.9 Plasmid DNA extraction............................................................................. 30
3.7 Data management and analysis ......................................................................... 31
3.8 Ethical considerations ....................................................................................... 31
CHAPTER FOUR...................................................................................................... 32
4.0 RESULTS ........................................................................................................... 32
4.1 Contamination rate and microbial count determined from the raw retail chicken 32
4.2 Prevalence of antimicrobial resistance among E. coli isolates............................ 35
4.3 Antibiotic resistance profiles among E. coli isolates. ......................................... 35
4.4 Distribution of antimicrobial resistance in E. coli from chicken meat from the 5
classes of retail outlets .............................................................................................. 36
4.5 Prevalence of E. coli isolates with virulence genes ............................................ 37
4.6 Prevalence of antimicrobial resistance among virulent and non-virulent E. coli. 40
4.7 Conjugative transfer of resistance from resistant E. coli to non-resistant recipient
................................................................................................................................. 42
4.8 Plasmid DNA profiles in Donors and Trans-conjugants .................................... 43
CHAPTER FIVE ....................................................................................................... 45
5.0 DISCUSSION ..................................................................................................... 45
5.1 Contamination levels in raw retail chicken by E. coli and E. coli/coliform count45
5.2 Prevalence of antimicrobial resistant E. coli isolated from raw retail chicken .... 51
5.4 Association between antimicrobial resistance and retail outlet classification ..... 54
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5.5 E. coli isolates harbouring virulence markers .................................................... 55
5.6 Association between antibiotic resistance and virulence genes in E. coli isolated
................................................................................................................................. 56
5.7 Co-transfer of antimicrobial resistance .............................................................. 59
5.8 CONCLUSIONS .............................................................................................. 62
5.9 RECOMMENDATIONS ................................................................................. 62
5.10 LIMITATIONS OF THE STUDY ................................................................... 64
REFERENCES .......................................................................................................... 65

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LIST OF TABLES
Table 3-1: Control strains of diarrheagenic E. coli .................................................. 27
Table 4-1: Comparison of mean microbial counts among the 5 groups of retail
outlets ................................................................................................... 35
Table 4-2: Antibiotic susceptibility profiles among E. coli isolates in retail chicken
meats........................................................................................................36
Table 4-3: Relationship between retail outlet classification and antibiotic resistance
............................................................................................................. 37
Table 4-4: Classification of pathogenic E. coli isolated from raw retail chicken meats
............................................................................................................. 38
Table 4-5: Relationship between virulence of E. coli isolated and antimicrobial
resistance .............................................................................................. 40
Table 4-6: Antibiotic resistance profiles exhibited by E. coli donors and trans-
conjugants ............................................................................................ 43

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LIST OF FIGURES
Figure 3-1: Map of Nairobi showing the administrative divisions and locations .. 21
Figure 4-1: Box plot representing distribution of E. coli count among the 5
classifications of retail outlets ........................................................ 33
Figure 4-2: Box plot representing distribution of coliform count among the 5
classifications of retail outlets........................................................... 34
Figure 4-3: Drug resistance phenotypes identified in pathogenic and non-
pathogenic E. coli isolated from raw retail chicken ........................... 41

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