i Antibiotic Susceptibility Patterns and Detection of Genes Responsible for Resistance of Klebsiella

125 Pages·2013·1.6 MB·English

Antibiotic Susceptibility Patterns and Detection of Genes Responsible for
Resistance of Klebsiella species and Escherichia coli Isolated from
Environmental Sources around Nairobi, Kenya

Pamela Muringo Njugu

A Thesis Submitted in Partial Fulfillment for the Degree of Masters of Science
in Botany (Microbiology) in the Jomo Kenyatta University of Agriculture and
Technology

2011
i
DECLARATION

This thesis is my original work and has not been presented for a degree in any other
university.

Signature:………………………………… Date:…………………
Pamela Muringo Njugu

This thesis has been submitted for examination with our approval as university
supervisors.

Signature:……………………………….. Date:………………….
Dr. Viviene Matiru
JKUAT, Kenya

Signature:………………………………… Date:………………….
Dr. Samuel M. Kariuki
KEMRI, Kenya

ii
DEDICATION

To Lucy Wangari Njugu, Rose Wanjiku Njugu, Calvin Mwihuri Njugu and George
Ngondi Michuki for enabling and facilitating me to achieve all I have.

iii
ACKNOWLEDGEMENT

There are a number of people without whom this thesis might not have been written,
and to whom I am greatly indebted.
First and foremost I would like to thank God for the gift of knowledge and
intelligence.
I extend my sincere gratitude to my supervisors, Dr. Samuel Kariuki and Dr. Viviene
Matiru for their great assistance, valuable advice, encouragement and support
throughout the study. I thank Mr. John Kiiru for his guidance and assistance in
providing laboratory materials and procedures. I am greatly indebted to Mr George
Michuki who has been a great source of encouragement and has given me a lot of
moral support throughout. I appreciate his assistance in statistical analysis and
provision of some laboratory materials.
I express my gratitudes to Agnes Munyalo, Jane Muyodi, Job Ongechi, Lawrence
Thiong’o, Ronald Ngetich and all other staff of the Centre for Microbiology
Research, Kenya Medical Research Institute, for their support and encouragement in
the laboratory work. I also thank my fellow classmates and lecturers for their
encouragement and moral support. More thanks go to the administration of Kenya
Medical Research Institute and Jomo Kenyatta University of Agriculture and
Technology for their general support in my MSc course.
Finally to my loving family who have been my pillar, and a very great source of
encouragement and inspiration throughout this study, and also my life. A very
iv
special thank you for your practical, emotional and financial support that made this
MSc course a success. I love you all.

MAY GOD BLESS YOU ALL!
v
TABLE OF CONTENTS
DECLARATION ................................................................................................... ii
DEDICATION ...................................................................................................... iii
ACKNOWLEDGEMENT .................................................................................... iv
TABLE OF CONTENTS ...................................................................................... vi
LIST OF TABLES ..................................................................................................x
LIST OF FIGURES .............................................................................................. xi
LIST OF APPENDICES ...................................................................................... xii
LIST OF ABBREVIATIONS AND ACRONYMS ............................................ xiii
ABSTRACT .......................................................................................................... xv
CHAPTER ONE .....................................................................................................1
1.0 INTRODUCTION .............................................................................................1
1.1 Statement of the problem ....................................................................................4
1.2 Justification .........................................................................................................4
1.3 Hypotheses..........................................................................................................5
1.4 Objectives ...........................................................................................................5
1.4.1 General Objective .........................................................................................5
1.4.2 Specific Objectives .......................................................................................6

vi
CHAPTER TWO ....................................................................................................7
2.0 LITERATURE REVIEW .................................................................................7
2.1 Enterobacteriaceae Infections ..........................................................................7
2.1.1 Escherichia coli (E. coli) characteristics ....................................................7
2.1.2 Escherichia coli Infections .......................................................................9
2.1.3 Klebsiella Species Characteristics ........................................................... 12
2.1.4 Klebsiella species Infections.................................................................... 14
2.2 Antibiotics resistance ..................................................................................... 17
2.2.1 Environmental damage mediated by antibiotics in water environments.... 20
2.3 Mechanisms of Antibiotics Resistance ........................................................... 21
2.3.1 Plasmids .................................................................................................. 23
2.3.2 Integrons ................................................................................................. 24
2.3.2.1 Integrons as a source of resistance genes .............................................. 26
2.3.2.2 Prevalence of Integrons ........................................................................ 27
2.3.3 Extended Spectrum β-Lactamases (ESBLs) Production ........................... 28
2.3.4 Ambler Class C β-Lactamases Production ............................................... 33
2.3.5 Aminoglycoside Modifying Enzymes ...................................................... 35
2.4 Clinical importance of antibiotic resistant pathogens ..................................... 39
CHAPTER THREE .............................................................................................. 40
3.0 MATERIALS AND METHODS .................................................................... 40
3.1 Study site and Specimen source......................................................................... 40
vii
3.2 Sampling and study design ................................................................................ 40
3.3 Sample Size ...................................................................................................... 41
3.4 Laboratory Procedures ...................................................................................... 42
3.4.1 Isolation of bacteria .................................................................................... 42
3.4.2 Identification of bacteria ............................................................................. 42
3.4.3 Antibiotic susceptibility testing ................................................................... 43
3.4.4 Determination of minimum inhibitory concentrations ................................. 44
3.4.5 Identification of Extended Spectrum β-Lactamases (ESBLs) ...................... 45
3.4.6 Isolation of DNA by boiling method ........................................................... 46
3.4.7 Conjugation experiments ............................................................................ 46
3.4.8 Polymerse chain reaction (PCR) amplification of resistance genes .............. 47
3.4.8.1 Detection of aminoglycosides modifying enzymes ............................... 47
3.4.8.2 Detection of Integrons class 1 and 2 ..................................................... 48
3.4.8.3 Electrophoresis ..................................................................................... 48
3.4.9 Plasmid studies ........................................................................................... 49
3.4.9.1 Harvesting and Extraction. ................................................................... 49
3.5 Data management .............................................................................................. 50
3.6 Ethical considerations ....................................................................................... 50
CHAPTER FOUR................................................................................................. 52
4.0 RESULTS ........................................................................................................ 52
viii
4.1 Samples ............................................................................................................ 52
4.2 Isolation and Identification of bacteria............................................................... 52
4.3 Identification of isolates by biochemical typing ................................................. 53
4.4 Antibiotics susceptibility testing ........................................................................ 56
4.5 Minimum inhibitory concentrations (MIC) ........................................................ 65
4.6 Detection of resistance genes............................................................................. 65
4.6.1 Detection of Int 1 and Int 2 genes ............................................................... 65
4.6.2 Detection of AME genes ............................................................................. 67
4.7 Plasmid studies ................................................................................................. 68
4.8 Conjugation experiments ................................................................................... 68
CHAPTER FIVE .................................................................................................. 70
5.0 DISCUSSION .................................................................................................. 70
CHAPTER SIX ..................................................................................................... 78
6.0 CONCLUSIONS AND RECOMMENDATIONS .......................................... 78
6.1 Conclusions ................................................................................................... 78
6.2 Recommendations ......................................................................................... 79
REFERENCES ..................................................................................................... 80
APPENDICES ....................................................................................................... 97

ix
LIST OF TABLES

Table 1. Mechanisms of Antimicrobial Resistance among Enteric Gram-
negative Bacilli................................................................................... 23
Table 2. Sources of water samples and locations collected ............................... 40
Table 3. Isolates identified by biochemical typing ........................................... 55
Table 4. Isolates distribution by source ............................................................ 55
Table 5. Representative zones of inhibition diameters and their interpretations
for beta lactam antibiotics ................................................................... 57
Table 6. Representative zones of inhibition diameters and their interpretations
for aminoglycosides antibiotics .......................................................... 58
Table 7. Representative zones of inhibition diameters and their interpretations for
nucleic acid inhibitor antibiotics ......................................................... 59
Table 8. Representative zones of inhibition diameters and their interpretations for
fluoroquinolones antibiotics................................................................ 60
Table 9. MICs of multidrug resistant Klebsiella species and Escherichia coli
isolates ............................................................................................... 65

x