Molecular characterisation of extended spectrum
beta-lactamases producing Enterobacteriaceae
Montso Kotsoana Peter
21261660
Bachelor of Science (BSc) Honours in Microbiology
(2013)
Bachelor of Science in Animal Health (2012)
Dissertation submitted in fulfilment of the requirements for the degree
Master
of Science in Biology (Molecular Microbiology) at the Mafikeng
Campus of the North-West University
Supervisor
: Professor CN ATEBA
Co-supervisor
: Professor NP SITHEBE
i
DECLARATION
I, Montso Kotsoana Peter, declare that the dissertation entitled “Molecular
characterisation of extended spectrum beta-lactamases producing
Enterobacteriaceae”, hereby submitted for the degree of Master of Science in Biology (Molecular Microbiology), has not previously been submitted by me for a degree at this or any other university. I further declare that this is my work in design and execution and that all materials contained herein have been duly acknowledged.
ii
DEDICATION
I dedicate this study to my father, Mr Elia Mokhosi Montso (RIP) and my mother, Mrs Mary Mamojabeng Montso for nurturing and taking care of me.
iii
ACKNOWLEDGEMENTS
I wish to thank the National Research Foundation - DST (NRF-DST) for the financial support. It is thanks to this support that I was able to complete this study.
I wish to express my sincere gratitude to my supervisor, Professor Collins Njie Ateba for his valuable guidance, support and commitment throughout this journey. The relationship was one of ‘father and son’. I learned a lot from you, and I thank you for dedicating your time to supervise this study. Thanks for the care, concern and support, especially during the period before I receiving the scholarship. I am also grateful to my co-supervisor, Professor Patricia Nomathamsanqa Sithebe. Her words of encouragement and motherly advice kept me focused throughout this study.
Special thanks go to my mother, Mrs Montso Mary Mamojabeng, my brother and sisters for their spiritual and moral supporting. I am greatly indebted to my aunts, Sister Francina Montso and Ms Anadleda Montso.
My sincere thanks also to Mr Motumi Lesitsi and Mrs Motumi Alice (affectionately known as ‘Miss’), for their enormous love and support throughout my studies. Despite the challenges they had, they were always supporting me. I am where I am today because of your advice and prayers. I wish to also thank Mr Gwangwa Jerry and Mrs Gwangwa Mosele for their financial support, care and love. Indeed, your support inspired me to fulfil my lifelong dream. Words alone cannot express the depth of gratitude I owe you.
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I am also grateful to my best friend, Khomotso Herminah Mongadi for her advices, caring, sweet love and being the bond that holds us together.
I thank the North West University, Faculty of Agriculture, Science and Technology and the Department of Biological Sciences for allowing me to undertake my studies at this University. I also wish to thank the North West University Postgraduate bursary for funding this study.
Members of Molecular Microbiology Group are not be left out of this list. They deserve special thanks for their enormous contributions throughout the study. Your guidance, ideas and criticisms are highly appreciated. I also acknowledge the support of Ms Madira Manganyi and Mr Morapedi Johannes towards the completion of this study.
Above all, my sincere appreciation and gratitude go to God Almighty for His blessings and for giving me the strength to complete this study.
v
ABSTRACT
Extended Spectrum beta-lactamase producing Enterobacteriaceae have been responsible for numerous outbreaks of infections worldwide. Escherichia coli and
Klebsiella pneumoniae species are the main producers of ESBLs, causing hospital and
community acquired infections. The aim of this study was to isolate and identify ESBL producing Enterobacteriaceae from cattle faeces and raw beef samples and determine the presence of ESBL determinants using specific PCR assays. A total of 151 samples were analysed and 259 presumptive isolates screened for characteristics of
Enterobacteriaceae using biochemical tests and 16S rRNA universal gene. A total of
196 isolates were confirmed as E. coli and K. pneumoniae through amplification of uidA,
uspA and ntrA genes fragments respectively. Antimicrobial susceptibility test was
performed to determine antibiotic resistance profiles of the isolates. Large proportions (66.7-100%) of isolates were resistant to Amoxicillin, Aztreonam, Ceftazidime, Cefotaxime and Piperacillin.
Correlations between antibiotic resistant isolates from the various sources was determined using the Pearson's product of moment and scored as significant if P≤0.05. Phenotypic characterisation by cluster analysis of antibiotic inhibition zone diameter data used to determine the commonness of isolates and resolve the differences of isolates from various sources and/or locations revealed a close association between isolates derived from cattle faeces and beef samples. This implies that the isolates may have originated from the common progeny.
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A total of 196 isolates were screened for the presence of ESBL determinants. Among all the isolates analysed, the prevalence of ESBL was 53.1%. The blaTEM, blaSHV and
blaCTX-M genes were detected in 85.5%, 69.6% and 58% of the isolates respectively and
this was most prevalent among E. coli isolates. Similarly, the blaCTX-M gene was the
most frequently detected in K. pneumoniae isolates. On the contrary, blaOXA gene was
highly prevalent in K. pneumoniae. The genetic relatedness of the isolates was determined using ERIC PCR technique. The DNA fragments showed great similarities in the banding patterns among E. coli and K. pneumoniae isolates. From the findings of this study, there is a need for continuous monitoring of ESBL producing strains in food products and food producing animals.
vii TABLE OF CONTENTS TITLE Page DECLARATION……… i DEDICATION……… ii ACKNOWLEDGEMENTS………... iii ABSTRACT………... v
TABLE OF CONTENTS……….. vii
LIST OF TABLES………. xiv
LIST OF FIGURES……….. xv
LIST OF APPENDICES………... xviii
LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS………… xix
DEFINITION OF CONCEPTS……….. xxi
CHAPTER ONE………... 1
INTRODUCTION AND PROBLEM STATEMENT……….. 2
1.1 GENERAL INTRODUCTION………. 2
1.2 PROBLEM STATEMENT……… 4
1.3 AIM AND OBJECTIVES OF THE STUDY………... 6
1.3.1 Aim of the study……… 6
1.3.2 Objectives of the study………... 6
CHAPTER TWO……….. 7
LITERATURE REVIEW……….. 8
2.1 ENTEROBACTERIACEAE………. 8
2.2 MECHANISMS OF ATIMICROBIAL AGENT……….. 9
viii
2.4 RESISTANCE TO BETA-LACTAM ANTIBIOTICS………. 12
2.5 EXTENDED SPECTRUM BETA-LACTAMSES IN ENTEROBACTERIACEAE………. 13
2.5.1 Beta-lactamases……….. 13
2.5.2 Extended beta-lactamases (ESBLs)………. 14
2.5.3 Types of ESBLs……… 15
2.5.3.1 TEM type beta-lactamase……….. 15
2.5.3.2 SHV type beta-lactamase……….. 16
2.5.3.3 CTX-M type beta-lactamase……….. 16
2.5.3.4 OXA type beta-lactamase……….. 17
2.6 TRANSFER OF RESISTANT DETERMINANTS……… 18
2.6.1 Transformation………. 18
2.6.2 Transduction………. 19
2.6.3 Conjugation……….. 19
2.7 ANIMAL SPECIES AS RESERVOIRS OF ESBL-PRODUCING ENTEROBACTERIACEAE………. 20
2.8 CONTAMINATION OF MEAT WITH ESBL-PRODUCING ENTEOBACTERIACEAE……… 22
2.9 CLINICAL IMPLICATIONS OF ESBL-PRODUCING ENTEROBCATERIACEAE IN HUMANS………. 23
2.10 TREATMENT OPTIONS FOR ESBL INFECTIONS……….. 24
2.11 DETECTING ESBL-PRODUCING ENTEROBACTERIACEAE……... 26
ix
2.12.1.1 Double disk synergy test………. 26
2.12.1.2 Three dimensional test... 27
2.12.1.3 E-test for ESBL………. 27
2.12.1.4 VITEK 2 ESBL test (BioMériux, France)……….. 28
2.12.2 Genotypic detection of ESBL-producing Enterobacteriaceae……….. 28
2.12.2.1 Molecular detection of ESBLs……… 28
2.12.2.2 Genotypic typing of ESBLs………. 30
2.15 CONCLUSION……….. 31
CHAPTER THREE……….. 33
MATERIALS AND METHODS……….. 34
3.1 Materials……… 34
3.1.1 Control strains……….. 34
3.2 METHODS……… 34
3.2.1 Study area and sample size ………... 34
3.2.2 Ethical clearance………. 35
3.2.3 Collection of samples……….. 35
3.3 BACTERIAL ISOLATION……… 35
3.3.1 Beef samples……… 35
3.3.2 Cattle faeces samples………. 36
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3.4.1 Cellular morphology………. 38
3.4.2 Oxidase test……….. 38
3.4.3 Triple Sugar Iron (TSI) test………. 39
3.4.4 Simmons’ citrate test………... 39
3.5 CONFIRMATORY TESTS……….. 40
3.5.1 Analytical Profile Index (API) 20E………. 40
3.5.2 Screening of Enterobacteriaceae for ESBL activity using the Brilliance ESBL Agar (BESBLA)……… 41
3.6 BACTERIAL STORAGE………. 41
3.7 MOLECULAR CHARACTERISATION OF ENTEROBACTERIACEAE………. 42
3.7.1 DNA Extraction………. 42
3.7.2 Determination of DNA quality and quantity……….. 43
3.8 Amplification of 16S rRNA universal gene fragments……… 43
3.9 Genus specific PCR……… 44
3.9.1 Confirmation of identities of potential E. coli isolates through PCR amplification of uidA and uspA gene fragments ……… 44
3.9.2 Confirmation of identities of potential Klebsiella pneumoniae isolates through PCR amplification of ntrA gene fragment ………. 45
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3.11 ANTIMICROBIAL SUSCEPTIBILITY TEST……… 48
3.12.0 GENOTYPIC CHARACTERISATION OF ESBL-PRODUCING ISOLATES………. 50
3.12. Detection of ESBL (blaCTX-M, blaOXA, blaSHV, blaTEM and blaCMY group)
genes by Multiplex PCR analysis ………... 50
3.13 GENOTYPIC TYPING OF E. COLI AND K. PNEUMONIAE ISOLATES USING ERIC-PCR METHOD ………... 52
3.14 AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS…….. 54
3.15 STATISTICAL ANALYSIS……….. 54
CHAPTER FOUR………. 56
RESULTS……….. 57
4.1 Detection of Enterobacteriaceae isolates from cattle faeces and beef samples…... 57
4.2 Identification of isolates based on preliminary and confirmatory biochemical tests………... 57
4.3 Distribution of ESBL-producing isolates in cattle faeces and beef samples………... 59
