screening for antibiotic resistance genes in escherichia coli and salmonella enterica isolated from

251 Pages·2017·1.96 MB·English

SCREENING FOR ANTIBIOTIC RESISTANCE GENES
IN ESCHERICHIA COLI AND SALMONELLA ENTERICA
ISOLATED FROM DRINKING WATER FROM PARTS OF
KADUNA STATE, NIGERIA

BY

OLAJOKE MOFOLUKE OLUKOSI

DEPARTMENT OF MICROBIOLOGY,
FACULTY OF LIFE SCIENCES,
AHMADU BELLO UNIVERSITY,
ZARIA, NIGERIA.

FEBRUARY, 2017

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SCREENING FOR ANTIBIOTIC RESISTANCE GENES IN
ESCHERICHIA COLI AND SALMONELLA ENTERICA ISOLATED
FROM DRINKING WATER FROM PARTS OF KADUNA STATE,
NIGERIA.

BY

Olajoke Mofoluke OLUKOSI
B.Sc (A.B.U. 2004), M.Sc (A.B.U. 2008)
Ph.D/SCIE/6689/10-11

A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES,
AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILLMENT FOR
THE AWARD OF A DOCTOR OF PHILOSOPHY DEGREE IN MICROBIOLOGY

DEPARTMENT OF MICROBIOLOGY,
FACULTY OF LIFE SCIENCES,
AHMADU BELLO UNIVERSITY,
ZARIA, NIGERIA.

FEBRUARY, 2017
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DECLARATION
I declare that the work in this thesis entitled “SCREENING FOR ANTIBIOTIC
RESISTANCE GENES IN ESCHERICHIA COLI AND SALMONELLA ENTERICA
ISOLATED FROM DRINKING WATER FROM PARTS OF KADUNA STATE,
NIGERIA” was performed by me in the Department of Microbiology, Ahmadu Bello
University, Zaria, under the supervision of Professors J.B. Ameh, I.O. Abdullahi and
C.M.Z. Whong.
The information derived from the literature has been duly acknowledged in the text and a
list of references provided. No part of this thesis was previously presented for another
degree or diploma at this or any other institution.

OLUKOSI, Olajoke M.
____________________ ______________________ _____________________
Name of Student Signature Date

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CERTIFICATION
This thesis entitled SCREENING FOR ANTIBIOTIC RESISTANCE GENES IN
ESCHERICHIA COLI AND SALMONELLA ENTERICA ISOLATED FROM DRINKING
WATER FROM PARTS OF KADUNA STATE, NIGERIA by Olajoke Mofoluke
OLUKOSI meets the regulations governing the award of the degree of Doctor of
Philosophy of the Ahmadu Bello University, and is approved for its‟ contribution to
knowledge and literary presentation.

Prof. J.B. Ameh
______________________ _____________________ _________________
Chairman, Supervisory Committee Signature Date

Prof. I.O. Abdullahi
___________________________ _____________________ _________________
Member, Supervisory Committee Signature Date

Prof. C.M.Z. Whong
___________________________ _____________________ _________________
Member, Supervisory Committee Signature Date

Prof I.O. Abdullahi
__________________________ _____________________ _________________
Head of Department Signature Date

Prof. K. Bala
___________________________ _____________________ _________________
Dean, School of Postgraduate Studies Signature Date

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ACKNOWLEDGEMENTS
I give all the glory to God Almighty, the giver of life and every good thing. He that is able
to bring to completion what He started, I‟m forever grateful to Him.
I acknowledge the scholarly and professional contributions of my supervisors, Prof. J.B.
Ameh, Prof. I.O. Abdullahi and Prof C.M.Z. Whong. Their concern and input had an
immense impact on this work, and I‟m deeply thankful for their constant availability,
support and constructive criticisms. Thank you sirs, may the good Lord bless you all, amen.
I wish to thank the staff of Microbiology Department, A.B.U. Zaria for their support. I wish
to acknowledge the support of Prof. S.E. Yakubu, Dr. E.E. Ella, Mrs T.E. Addai, Mrs D.O.
Toryilla, Mal. Shittu, Mr. Alex, Mal. Shuaibu Garba, Dr. H. Raji, Dr. S.L. Afegbua, Dr.
M.S. Aliyu and Dr. A.B. Suleiman. They contributed in diverse ways to the success of this
study and I‟m very grateful. I also wish to thank the staff of the Sanitary Microbiology
Laboratory of the Department of Water Resources and Environmental Engineering, A.B.U.
Zaria, especially Mr. Paul Alika and Mallam Yahaya for their assistance in some aspects of
the work. I thank the staff of the Multiuser Laboratory, Department of Chemistry, A.B.U.
Zaria, for their assistance.
I wish to appreciate the staff of the Bioscience Unit, IITA, Ibadan for their assistance on the
molecular aspect of the work. Thanks for your support and consideration. I also thank Mr.
T. Fadason for his input and support in analyzing some of my data. I thank Mr. and Mrs.
Nathaniel Ikilama and Mal. Idris for their support in the statistical analyses of the data. I
appreciate Mal. Isa who chauffeured me to my various study sites.
I am forever indebted to my amazing parents, Prof. And Mrs J.O. Olukosi. They stood
behind me throughout the duration of this study and beyond. I pray that their labour of love
will never be in vain. I am grateful for their support morally, financially, spiritually and
otherwise, thanks for being exemplary parents, God bless you. I also thank all my siblings
and their spouses especially Mr Dayo Olukosi for his constant support in diverse ways over
the course of this study. I appreciate my in-law, Dr. A.R. Akintunde and all my extended
family members for their encouragement. I wish to appreciate my parents-in-law, Pastor
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and Mrs. E.A. Alalade and the entire family for their support and encouragement, God bless
you all.
I appreciate the contributions of past and current post-graduate students and co-workers in
the laboratory. They include among others: Mrs Marie Yomi, Dr. I. Mzungu, Mal. Umar,
Dr. Ngozi Okpo, Dr. Tafena Amapu, Dr. Peter Shiaka, Mr. Matthew, Mr. Rufus, Dr. Grace
Abakpa and Dr. Adenike Ogunnaike. I wish to thank my friends Joke Lawal, Ozozoma
Abu, Titilayo Anjorin, Dr. S.L. Afegbua, Amaka Ndugbu, Adenike Onyemachi and Sharon
Balogun for their concern and moral support. God bless you all.
Last but not the least; I‟m immensely grateful to my wonderful husband Dr. Olusegun
Alalade for his moral, financial, and spiritual support and his diverse contributions to this
work. I say thank you for your patience and understanding over the period of this study. I
pray God blesses you and enlarge your coast. To my wonderful children Oluwatoni and
Toluwanimi, I say thank you for your patience and endurance. God bless and uplift you in
Jesus name. Amen.

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TABLE OF CONTENTS

Page
Title page.................................................................................................................................i
Declaration..............................................................................................................................ii
Certification...........................................................................................................................iii
Acknowledgements................................................................................................................iv
Table of contents....................................................................................................................vi
List of Figures......................................................................................................................xiii
List of Tables........................................................................................................................xiv
List of Plates........................................................................................................................xvii
List of Appendices.............................................................................................................xviii
Abbreviations.......................................................................................................................xix
Abstract.................................................................................................................................xx
CHAPTER ONE
INTRODUCTION.................................................................................................................1
1.1General Overview.............................................................................................................1
1.2Statement of the Research Problem..............................................................................4
1.3 Justification of the Study...............................................................................................5
1.4 Aim of Study ..................................................................................................................5
1.5 Specific Objectives...........................................................................................................6
CHAPTER TWO
LITERATURE REVIEW..............................................................................................................7
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2.1 Water Pollution...............................................................................................................7
2.2 Challenges of Unhygienic Environments......................................................................9
2.3 Drinking Water Availability .......................................................................................10
2.4 Drinking Water Standards..........................................................................................14
2.4.1 Physical and chemical indicators of drinking water quality........................................16
2.4.2 Heavy metals in drinking water...................................................................................21
2.5 Bacteriological Indicators of Water Pollution.................................................................25
2.5.1 Feacal coliforms...........................................................................................................27
2.5.2 Escherichia coli............................................................................................................28
2.6 Escherichia coli O157: H7 (E. coli O157: H7) ............................................................34
2.6.1 Epidemiology of E. coli O157:H7................................................................................35
2.6.2 Infections Caused By E. coli O157:H7........................................................................37
2.6.3 Mode of Spread............................................................................................................38
2.6.4 Virulence Factors of E. coli O157:H7..........................................................................39
2.6.5 Diagnosis of Infections Caused by E. coli O157:H7 ...............................................41
2.6.6 Treatment of E. coli O157:H7 infections.....................................................................42
2.6.7 Vaccine.........................................................................................................................42
2.6.8 Survival of E. coli O157:H7 in the environment.........................................................42
2.7 Salmonella species.........................................................................................................43
2.7.1 Epidemiology of Salmonella enterica..........................................................................46
2.7.2 Infections caused by Salmonella enterica....................................................................47
2.7.3 Mode of Spread of Salmonella enterica Infections......................................................48
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2.7.4 Virulence factors of Salmonella enterica.....................................................................49
2.7.5 Diagnosis of salmonellosis...........................................................................................50
2.7.6 Treatment of Salmonella enterica infections..............................................................50
2.8 Antibiotics......................................................................................................................51
2.8.1 Antibiotics that inhibit synthesis of cell wall...............................................................52
2.8.2 Inhibition of protein synthesis......................................................................................53
2.8.3 Nucleic acid synthesis inhibition..................................................................................55
2.8.4 Cell membrane disruption ...........................................................................................56
2.8.5 Metabolic antagonism..................................................................................................57
2.9 Antibiotic Resistance....................................................................................................58
2.10 Mechanisms of Drug Resistance in Bacteria.............................................................60
2.10.1 Drug resistance by target site modification................................................................60
2.10.2 Inactivation of antibiotics ..........................................................................................63
2.10.3 Ribosome protection .................................................................................................65
2.10.4 Reduced membrane permeability ..............................................................................65
2.10.5 Active drug efflux ....................................................................................................66
2.11 Multidrug Resistance (MDR) ....................................................................................68
CHAPTER THREE
MATERIALS AND METHODS........................................................................................70
3.1 Study Area.....................................................................................................................70
3.2 Study Design..................................................................................................................70
3.3 Sampling Methodology.................................................................................................72
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3.3.1 Sample size..................................................................................................................72
3.3.2 Sampling plan……………………….………………………………………………..72
3.3.3 Collection of samples…………………………………………….……………..…....73
3.4 Sample Analysis............................................................................................................76
3.4.1 Physicochemical and heavy metal analysis of water samples………..………………76
3.4.2 Bacteriological analyses…………………………...…………………………………78
3.5 Gram Staining of Isolates…………………………………...………………….…….80
3.6 Biochemical Characterization of the Isolates………………...…………..…………81
3.6.1 Indole test…………………………………………...………………………………..81
3.6.2 Methyl-red Voges-Proskaeur (MRVP) test…………………………………………..81
3.6.3 Citrate utilization test……………………………………………...…………………82
3.6.4 Motility test……………………………………………………………...………...…82
3.6.5UreaseTest……………………….................................................................................82
3.6.6 Triple Sugar Iron test (TSI)..........................................................................................82
3.6.7 Identification of organisms using Microgen Enterobacteriaceae Gram Negative-A
(GN-A) ID kit………….....…………………..…………………………………….83
3.7 Identification of E. coli O157:H7………………………………………………….....84
3.8 Identification of Salmonella enterica…………………………………………...…....84
3.9 Antibiotic susceptibility testing of the isolates………………………………………84
3.10 Molecular Analyses……………………………………...………………………......85
3.10.1 Extraction of genomic DNA …………………………………………...….........….85
3.10.2 Primer design………………………………………....……………………………..86
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