ISOLATION AND MOLECULAR CHARACTERIZATION OF

SHIGA TOXIN PRODUCING Escherichia coli (STEC)

ASSOCIATED WITH READY-TO-EAT GAME MEAT IN SOUTH
WEST, NIGERIA

BY

ADEYA, CHIDINMA CELINE

18010101026

A RESEARCH SUBMITTED TO THE DEPARTMENT OF

BIOLOGICAL SCIENCES, COLLEGE OF BASIC AND APPLIED
SCIENCES, MOUNTAIN TOP UNIVERSITY, MAKOGI, IBAFO,
OGUN STATE, NIGERIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF THE BACHELOR OF SCIENCE (B. Sc.) IN
MICROBIOLOGY

SEPTEMBER, 2022

i
 DECLARATION

I hereby declare that this project written under the supervision of Dr. O. E. Fayemi is
a product of my own laboratory analysis and literature search. Information derived
from various sources have been duly acknowledged in the text and a list of references
provided. This Project research has not been presented anywhere for the award of any
degree or certificate.

ii
 CERTIFICATION

This is to certify that this Project research titled “ISOLATION AND

MOLECULAR CHARACTERIZATION OF SHIGA TOXIN PRODUCING
Escherichia coli (STEC) ASSOCIATED WITH READY-TO-EAT GAME
MEAT IN SOUTH WEST, NIGERIA” was carried out by ADEYA, CHIDINMA
CELINE, with matriculation number 18010101026. This Project meets the
requirements governing the award of the Bachelor of Science (B. Sc.) degree in
Microbiology department of biological sciences of Mountain Top University, Ogun
State, Nigeria and is approved for its contribution to knowledge.

_________________________ _________________________
Dr. O. E. FAYEMI Date
(Project Supervisor)

_________________________ _________________________
Dr. (Mrs.) C. I. AYOLABI Date
(Ag. Head of Department)

iii
 DEDICATION

My utmost gratitude goes to God for making this research, and my stay throughout
school a success, for unending divine provision, insight and help, I am forever
grateful.

iv
 ACKNOWLEDGEMENTS

I dedicate this work to the Almighty God for his grace, health and everything needed
to make this work a success.

My profound gratitude goes to the school, Mountain Top University for providing a
world class superior, fully equipped laboratory for the students of Biological Sciences.

I would like to appreciate the efforts of my supervisor, Dr. O. E. Fayemi for

supervision, guidance and contributions, for the completion of this work.
I would also like to appreciate the Head of Department (H. O. D), Dr. Mrs. C. I.
Ayolabi for her efforts and contributions geared towards the completion of this project.
I also wish to express my sincere gratitude to my Mother Mrs. Roberta Adeya, my
father Mr. Francis Adeya and my siblings; Adekemi, Adebayo and Christabell for
their unfailing support in every way from the onset to the completion of this project.
I would like to appreciate Dr. G. B. Akanni, Mrs. O. T Adegbala, Miss J. Anyasi, Mr.
F. Okunbi and the entire Department of Biological Sciences for their time, efforts,
guidance and impacting knowledge.
Finally, my appreciation goes to my friends and colleagues: Blessing A., Divine M.,
Comfort O., Doyin, Enoch, Joshua, Ayomide Opawande for being available and
always helping out in time of need.

v
 TABLE OF CONTENTS

Title Page........................................................................................................................i
Declaration .....................................................................................................................ii
Certification ................................................................................................................. iii
Dedication ..................................................................................................................... iv
Acknowledgements ........................................................................................................ v
Table of Contents .......................................................................................................... vi
List of Tables ................................................................................................................ ix
List of Figures ................................................................................................................ x
List of Plates ................................................................................................................. xi
Abbreviations ...............................................................................................................xii
Abstract ...................................................................................................................... xiii
CHAPTER ONE: INTRODUCTION ........................................................................ 1
1.0 Introduction .............................................................................................................. 1
1.1 Background of Study ............................................................................................... 1
1.2 Statement of Problem ............................................................................................... 2
1.3 Justification of Study ............................................................................................... 2
1.4 Aim and Objectives.................................................................................................. 2
CHAPTER TWO: LITERATURE REVIEW ........................................................... 3
2.0 Literature Review..................................................................................................... 3
2.1 Game Meat ............................................................................................................... 3
2.2 Implication of Game Meat in Foodborne Diseases.................................................. 3
2.3 Escherichia coli ....................................................................................................... 4
2.3.1 Brief Hstory of Escherichia coli .................................................................... 4
2.3.2 Characteristics of Escherichia coli ................................................................ 4
2.3.3 Pathotypes of Escherichia coli....................................................................... 7
2.3.3.Enterotoxigenic Escherichia coli (ETEC) ..................................................... 9
2.3.3.2 Enyeropathogenic Escherichia coli (EPEC) ............................................... 9
2.3.3.3 Enteroaggregative Escherichia coli (EAggEC) .......................................... 9
2.3.3.4 Diffusely adherent Escherichia coli (DAEC) ........................................... 10
2.3.3.5 Enteroinvasive Escherichia coli (EIEC) ................................................... 10
2.3.3.6 Shiga toxin Producing Escherichia coli (STEC) ...................................... 10
vi
 2.3.4 Epidemiology of Escherichia coli ............................................................... 11
2.3.5 Pathogenesis of Escherichia coli ................................................................. 11
2.3.6 Transmission of Escherichia coli................................................................. 12
2.3.7 Antimicrobial Resistance and Susceptibility of Escherichia coli ................ 12
CHAPTER THREE: METHODOLOGY ............................................................... 13
3.0 METHODOLOGY ................................................................................................ 13
3.1 Study Area ............................................................................................................. 13
3.2 Sample Collection .................................................................................................. 15
3.3 Sterilization ............................................................................................................ 15
3.4 Materials and Equipments Used ............................................................................ 15
3.5 Preparation of Culture Media................................................................................. 15
3.6 Sample Preparation ................................................................................................ 16
3.7 Serial Dilution ........................................................................................................ 17
3.8 Plating .................................................................................................................... 17
3.9 Sub-culturing.......................................................................................................... 17
3.10 Biochemical Tests ................................................................................................ 18
3.10.1 Gram Staining ............................................................................................ 18
3.10.2 Catalase test ............................................................................................... 19
3.10.3 Oxidase test ................................................................................................ 19
3.10.4 Motility test ................................................................................................ 19
3.10.5 Oxidative Fermentative test ....................................................................... 19
3.10.6 Preservation of Samples ............................................................................. 20
3.11 DNA Extraction ................................................................................................... 20
3.11.1 Materials Used ........................................................................................... 20
3.11.2 Activation of Isolates ................................................................................. 20
3.11.3 Pre- washing................................................................................................ 21
3.11.4 Boiling......................................................................................................... 21
3.12 Polymerase Chain Reaction (PCR) ...................................................................... 21
3.13 Gel Electrophoresis .............................................................................................. 22
CHAPTER FOUR: RESULTS ................................................................................. 24
4.0 RESULTS .............................................................................................................. 24
4.1 Results .................................................................................................................... 24
4.1.1 TVC Result .................................................................................................. 24
4.1.2 Biochemical tests of E. coli isolates ............................................................ 24
vii
 4.1.3 Prevalence of Shiga toxin E. coliI in game meat ........................................ 27
CHAPTER FIVE: DISCUSSION............................................................................. 30
5.0 DISCUSSION ........................................................................................................ 30
5.1 DISCUSSION ........................................................................................................ 30
CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS ..................... 32
6.1 CONCLUSIONS AND RECOMMENDATIONS ................................................ 32
REFERENCES ............................................................................................................ 33

viii
 LIST OF TABLES

TABLE TITLE PAGE

Table 3.1 Table showing the Sample obtained and location 14
Table 3.2 Table showing the target genes, virulence factors and primer 23
sequences (Persson et al., 2007)
Table 3.3 Protocol for Thermocycler 24
Table 3.4 Multiplex PCR Components for PCR Treatment 24
Table 4.1 Total Viable Count (TVC) of E. coli in the game meat 25
(n=55) from various locations in South western Nigeria.
Table 4.2 The results of biochemical tests for presumptive 26
identification of E. coli in all isolates.
Table 4.3 Table Showing the Number of Positive samples and their 30
location

ix
 LIST OF FIGURES

FIGURE TITLE PAGE

Figure 2.1 Detailed Structure of Escherichia coli 6
Figure 2.2 Classification of Escherichia coli pathotypes 8
Figure 4.1 A representation showing game meat species and positive 28
samples

x
LIST OF PLATES

PLATE TITLE PAGE

Plate 4.1 Agarose gel electrophoresis image of
Multiplex-PCR products (Human estA,
Porcine estA, vtx1, vtx2, ipaH, eae, elta). 29
Lane L: marker (100-bp ladder), lane 30 and
31: E. coli isolate (Porcine estA)
Plate 4.2 Agarose gel electrophoresis image of
Multiplex-PCR products (Human estA,
Porcine estA, vtx1, vtx2, ipaH, eae, elta). 30
Lane L: marker (100bp-ladder). Lane 28, 33,
34, 38 (Human estA), Lane 30, 32, 35, 45
(Vtx1)

xi
 ABBREVIATIONS

BHI – Brain Heart Infusion broth

BPW – Buffered Peptone Water
CFU/ml – Colony Forming Units/ml
DAEC – Diffusely Adherent Escherichia coli
DEC – Diarrheagenic Escherichia coli
DPEC – Diarrheagenic Pathogenic Escherichia coli
ExPEC – Extra-intestinal Pathogenic Escherichia coli
EAggEC – Enteroaggregative Escherichia coli
EHEC – Enterohemorrhagic Escherichia coli
EIEC – Enteroinvasive Escherichia coli
EPEC – Enteropathogenic Escherichia coli
ETEC – Enterotoxigenic Escherichia coli
HUS – Hemolytic Uremic Syndrome
MAC – Mac Conkey agar
NA – Nutrient agar
NMEC – Neonatal Meningitis Escherichia coli
PCR- Polymerase Chain Reaction
SMAC – Sorbitol Mac Conkey agar
STEC – Shiga toxin producing Escherichia coli
TVC – Total Viable Count
UPEC – Uropathogenic Escherichia coli
WHO -World Health Organization

xii
 ABSTRACT

Game meat is a term that includes all animals derived from wildlife. It is indigenous
to China and Africa. In Nigeria, a variety of game meat is consumed, Grasscutter and
Antelope being the dominant species consumed. The presence of pathogenic
microorganisms in the game meat that is commonly consumed may spell doom to the
public health, and conversely the society at large. Therefore, this research was carried
out to determine the presence of pathogenic Escherichia coli, specifically Shiga toxin
producing E. coli in various game meat species sourced from Lagos, Ogun, Oyo,
Ondo and Osun State in the South West region of Nigeria. A total of 55 game meat
samples were aseptically obtained at designated sales points and E. coli was isolated
using Sorbitol Mac Conkey agar (SMAC) and Mac Conkey agar (MAC).
Morphological and biochemical tests were performed on the isolates. The suspected
isolates were then genotypically characterized using Multiplex PCR. Among the game
meat sampled (n=55), 25.4% of the game meat samples were confirmed positive for
Shiga toxin producing E. coli genes, out of which 12.7% and 3.6% possessed the
Enterotoxigenic Human and Porcine estA gene 9.1%, positive with the Vtx1 gene that
produces Shiga toxin. This means that few of the game meat samples were Shiga
Toxin producing E. coli (STEC). The presence of pathogenic E. coli is a risk to the
public health as it could cause foodborne illnesses and diseases including diarrhea and
Hemolytic Uremic Syndrome (HUS).

Keywords: Escherichia coli, Game meat, PCR, Shiga toxin producing Escherichia
coli, South west.

xiii
 CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF STUDY

Game meat refers to any animal gotten from wildlife. Game meat is used as a source
of food for man, and in recent times, as a profitable source of income. In global
distribution, game meat is indigenous to Africa and Asia (Ariyo et al., 2018). Game
meat is most commonly referred to as ‘bush meat’ in Nigeria (Okeke et al., 2013).
The consumption of game meat is on the rise, and this might pose a threat to the
general public health as Game meat is said to be a zoonotic agent (Gomes-Neves et al.,
2021). The presence of pathogenic microorganisms in game meat consumed is of
great concern as this poses risk to the health of the consumers which may lead to the
outbreak of several foodborne diseases (Fayemi et al., 2021). The microorganisms
that have been associated with game meat includes Toxoplasma gondii (Almeria et al.,
2021), Trichinella nativa (McIntyre et al., 2007), Enterohemorrhagic Escherichia coli,
Clostridium botulinum, Clostridium perfringes, Campylobacter spp, Salmonella spp,
Shigella spp (Todd, 2014).
Escherichia coli is one of the most important abundant microorganisms found
distributed in nature owing to its ubiquitous nature (Kolenda et al., 2015).
Diarrheagenic pathogenic Escherichia coli (DPEC) strains are among the most
important microorganisms known for causing foodborne diseases, and has also been
implicated as the most common causative agent of diarrhea (Mohamed et al., 2018).
Diarrhea as defined by the World health Organization (WHO), is the excessive,
frequent passing of watery stools for a duration of 3 -7 days. Diarrheal diseases
however preventable and treatable, is the second leading cause of death in children
under 5 years globally, with an annual mortality rate of 520, 000 [World Health
Organization, 2017]. In Africa, diarrheal diseases are ranked among the main causes
of morbidity and mortality affecting children under 5 (Adjuik et al, 2006). The Shiga
toxin Producing Escherichia coli (STEC), a diarrheagenic pathotype is known to
cause diarrhea and serious potentially life-threatening diseases such as the Hemolytic
Uremic Syndrome (Ayoade et al., 2021).

1
1.2 STATEMENT OF PROBLEM

The ready-to-eat game meat sold by different vendors from various locations in
Nigeria are not always prepared under standard conditions, leaving a high probability
of being contaminated with pathogenic microorganisms. This is a call for concern on
the food safety in the consumption of these game meat as it could lead to an
unpredictable outbreak of food borne diseases such as diarrhea or in worst case even
zoonotic endemic or epidemic diseases as game meat is a notable source of zoonotic
diseases.

1.3 JUSTIFICATION OF STUDY

The consumption of game meat in Nigeria is experiencing an increase. The presence

of pathogenic E. coli, particularly the Pathotype Shiga Toxin Producing Escherichia
coli (STEC) will be investigated in the Game meat to ascertain the safety of
consuming game meat. Therefore, this study is necessary for the investigation of the
microbiological quality and safety in consuming game meat.

1.4AIM AND OBJECTIVES

The aim and objectives of this study are as follows.

⚫ To isolate Escherichia coli in ready-to-eat game meat from various locations
within South western Nigeria.
⚫ To determine the virulence genes present in the isolated Shiga Toxin Producing
Escherichia coli (STEC).

2
 CHAPTER TWO

LITERATURE REVIEW

2.1 GAME MEAT

Game meat refers to any animal gotten or sourced from wildlife. Game meat was
formerly the main source of meat that was available for consumption before the
domestication of some species (Olaniyan et al., 2016). In Past times it was a food
source for personal consumption by people indigenous to traditional settings such as
villages. Recently, this is not the case, game meat consumption is becoming more
accepted as it is not only limited to the hunters and traditional indigenes in remote
areas but now more appreciated due to its nutritional value (Gomes-Neves et al.,
2021). The consumer interest of game meat is on the rise due to the rapidly growing
population of wild game species (Branciari et al., 2022). Game meat is high in Protein,
mineral content and low fat and cholesterol levels (Lizana et al., 2022). A quantitative
based survey showed that people consume game meat based on rational motives such
as preference of taste, nutritional value and availability (Niewiadomska et al., 2020).
Commercial hunting has allowed and encouraged the sales of different species of bush
meat at pricey rates in specific cities, towns known as ‘hotspots’, which does not only
provide the availability of bush meat for consumption but also serve as a lucrative
source of income (Okeke et al., 2013). In Africa, Antelope is the most prevalent game
meat consumed (Faraz and Waheed, 2018).

2.2 IMPLICATION OF GAME MEAT IN FOOD BORNE

DISEASES

Given that there are no hygienic standards or norms in the wild, the origin of game
meat is a major cause for concern. Due to the hunting procedure, the hygiene
throughout the post-hunting process, processing, and transportation, the safety of
game meat is frequently questioned (Gomes-Neves et al., 2021). To reduce the
potential risk to the consumer, it is crucial to ensure the sanitation and safety
standards of game meat. It has been shown that 43% of the foodborne pathogens
3
linked to recent outbreaks of infectious diseases around the world are descended from
wildlife species (Hedman et al., 2020). Shiga Toxin producing Escherichia coli is a
zoonotic agent associated with game meat and its products, which could compromise
the safety, hence, leading to the development of foodborne diseases such as diarrhea,
hemorrhagic colitis, etc. (Naseer et al., 2017).
According to reports, game meat sold in the local market in Wuhan, China, was a
source of the SARS-CoV-2 virus outbreak, which was recognized as the first
respiratory foodborne illnesses to spread globally (Jalava, 2020). A further important
factor in ensuring the safety of consuming game meat is the hygiene involved, which
relates to the processes, protocols, and procedures for promoting health.

2.3 ESCHERICHIA COLI

2.3.1 BRIEF HISTORY OF ESCHERICHIA COLI

Escherichia coli was first discovered and described by the German pedriatrist and
microbiologist Theodor Escherich. In 1884, Theodor Escherich focused his research
on the bacterial flora of infantile diarrhea (Kaper et al., 2005). Theodor described
Escherichia coli as normal inhabitants of the intestine and also proposed that they
were responsible for some infectious diseases of the intestine and the urinary tract
(Friedmann, 2014). He isolated 19 different bacteria from the stool samples of
children he observed, carried out Gram staining on the organisms and described in
detail the organism Bacterium coli commune, now known as Escherichia coli.
(Shulman et al., 2017).

2.3.2 CHARACTERISTICS OF ESCHERICHIA COLI

Escherichia coli is a Gram-negative bacilli of length 1µm and width of 0.35µm

(Malik et al., 2022). Escherichia coli are non-sporulating Facultative anaerobes and is
a member of the Enterobacteriaceae family, hence, constitutes a part of the normal
micro flora of the gut (Saba et al., 2015). E. coli is ubiquitous in nature; as it is found
in soil, water, food due to contamination from faeces or from animals (Kolendaet al.,
2015). E. coli becomes pathogenic as a result of acquisition of virulent genes by
mobile elements such as transposons, plasmids bacteriophages and even through
4
pathogenicity islands (Campilongo et al., 2014; Razzaq et al., 2016). Certain food
products like meat, dairy products, vegetables have been implicated in food borne
disease outbreak caused by Escherichia coli.(Saad et al., 2021). It is regarded as the
most studied bacterial specie because it can easily be propagated and easy to be
genetically manipulated (Abdelrahman et al., 2022).

5
Figure 2.1: Detailed Structure of Escherichia coli Adapted from Terlizzi et al (2017).

6
2.3.3 PATHOTYPES OF ESCHERICHIA COLI

Several Escherichia coli strains have been discovered and have been grouped as either
Extra-intestinal pathogenic Escherichia coli (ExPEC) or Intestinal pathogenic
Escherichia coli. The intestinal pathogenic Escherichia coli otherwiseknown as
Diarrheagenic Escherichia coli (DEC) comprises of 6 pathotypes, they include
Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic Escherichia coli (EPEC),
Diffusely Adherent Escherichia coli (DAEC), Enteroinvasive Escherichia coli (EIEC),
Enteroaggregative Escherichia coli (EAggEC) and the Shiga toxin producing
Escherichia coli (STEC), (Sikorski et al., 2021). Among the Extra-intestinal
pathogenic E. coli, there are two major pathotypes; Uropathogenic Escherichia coli
(UPEC) and Neonatal Meningitis Escherichia coli (NMEC).

7
Figure 2.2: Classification of Escherichia coli pathotypes Adapted from Wakeham
(2013).

8
2.3.3.1 ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

Enterotoxigenic Escherichia coli is the leading cause of diarrhea in developing

countries. As the name implies, Enterotoxigenic E. coli possess two enterotoxins used
for causing diarrhea namely; heat labile and heat stable enterotoxins respectively
(Gomez-Aldapa et al., 2013). The major virulence factor possessed by ETEC is the
fimbriae, that facilitate attachment to the lumen, followed by the secretion of
enterotoxins to induce fluid efflux from the intestine (Curtis et al., 2017).

2.3.3.2 ENTEROPATHOGENIC ESCHERICHIA COLI (EPEC)

Enteropathogenic Escherichia coli (EPEC) is the most implicated organism in

infantile diarrhea (Al-Charrakh et al., 2012). The mechanism of pathogenicity
involves the localized adhesion to the intestinal mucosa and effacement (tight binding)
to the intestinal mucosa.

2.3.3.3 ENTEROAGGREGATIVE ESCHERICHIA COLI (EAggEC)

The Enteroaggregative Escherichia coli (EAggEC), is an E. coli pathotype that has

been linked and associated with acute and persistent diarrhea in children, and
traveler’s diarrhea (Mahindroo et al., 2021). The mechanism of causing diarrhea is
said to be a complex process, however, detailed research has proven that there are 3
necessary EAggEC to be implicated as the causative organism of diarrhea (Mavarro-
Garcia and Elias, 2011). The first stage, is the colonization of the intestinal mucosa by
the abundant adherence of aggregates of E. coli to the intestinal mucosa. The second
stage involves the release of enterotoxins and cytotoxins to the colonized intestinal
mucosa. The final stage is for the bacteria to induce inflammation of the intestinal
mucosa, hence, stimulating diarrhea [Mavarro-Garcia and Elias, 2011]. This
pathotype is characterized by the clinical manifestation of watery diarrhea (Savavino
et al., 1996).

9
2.3.3.4 DIFFUSELY ADHERENT ESCHERICHIA COLI (DAEC)

Diffusely adherent pathotype of E. coli is characterized by the presence of a diffusely

adherence pattern on HeLa and HEp-2 epithelial cells and associated with Persistent
and acute diarrhea in children (Javadi et al., 2020). The Diffusely Adherent E. coli is
detected by distinguishable diffusely adherence to the tissue of the intestinal lining
without possessing Hemagglutinin (Yamamoto et al., 1996). About 75% of Diffusely
Adherent E. coli possess adhesins from the Afa/Dr family responsible for the
presentation of adhesion (Mansan-Almeida et al., 2013)

2.3.3.5 ENTEROINVASIVE ESCHERICHIA COLI (EIEC)

The Enteroinvasive Escherichia coli (EIEC) was first found in 1971, and was
implicated for causing diarrhea in supposedly healthy people (Abbasi et al., 2015).
Enteroinvasive E. coli possess a mechanism of pathogenicity resembling Shigella
dystenteriae (Pasqua et al., 2017). As the name implies, Enteroinvasive E. coli
invades the lining of the epithelium and facilitates spreading of the bacteria to
colonize the surface with its enterocytes. The serotype O96:H19 is capable of also
producing biofilms to colonize the epithelium of the intestine (Gomez-Duarte et al,
2018).

2.3.3.6 SHIGA TOXIN PRODUCING ESCHERICHIA COLI (STEC)

As the name implies, Shiga toxin producing E. coli is a pathotype of E. coli that
produces Shiga toxin. This Shiga toxin produced is similar to the toxin produced by
the bacteria Shigella dysenteriae. It is otherwise known as Enterohaemorrhagic
Escherichia coli (EHEC) or Verotoxigenic E. coli Shiga toxin Producing E. coli is a
foodborne zoonotic agent that has been associated with global outbreaks that was
responsible for serious public health implications (Nguyen & Sperandio, 2012). Shiga
toxin Producing Escherichia coli (STEC) results in several foodborne illnesses from
mild diarrhea to severe cases like Hemolytic Uremic Syndrome (HUS) which
damages the kidney (Ayoade et al., 2021). The genesis of most infections caused by
STEC are due to the common serotype O157:H7, which has been linked to the
consumption of contaminated food and water containing the faeces of infected
animals (Saba et al., 2015). In 1982, the Shiga toxin producing E. coli (STEC)
10
serotype O157:H7 was discovered as a pathogen (Omer et al., 2018). STEC is further
divided into two serogroups; O157 and non-O157 groups (Babolhavaeji et al., 2021).
STEC colonizes the intestines and produces Shiga toxins that stimulate inflammatory
response, hence, characterized by the clinical manifestation of bloody diarrhea.

2.3.4 EPIDEMIOLOGY OF ESCHERICHIA COLI

An estimated 265,000 STEC infections occur in the United States of America yearly
ranging from mild to potentially life-threatening infections i.e Hemolytic Uremic
Syndrome (Tack et al., 2021). Outbreaks of E. coli O157:H7 and similar organisms
known for producing the Shiga toxin have occurred in many countries including
Australia, Japan, the UK, and many other European countries (Todd, 2014;
Pennington et al., 2014). The epidemiology of diarrheal diseases in Africa caused by
the Diarrheagenic Escherichia coli (DEC) is rather poorly understood as a result of
lack and/or inadequate facilities needed to study and characterize them (Cissé et al,
2017). However, countries in Africa which have reported data comprise of Kenya,
Nigeria, Côte d’Ivoire, and the Central African Republic, the O157 STEC have been
isolated from sporadic cases of diarrhea and HUS, and have also been associated with
some diarrheal disease outbreaks, especially in southern Africa. In Nigeria, Non-O157
STEC have also been linked to sporadic cases and outbreaks of diarrhea (Fairbrother
& Nadeau, 2006).

2.3.5 PATHOGENESISOF ESCHERICHIA COLI

Escherichia coli infections usually develop as a result of ingestion of Contaminated

meat or meat product containing E. coli, usually STEC (Tack et al, 2021).
The consumed pathogenic Escherichia coli then replicates within the gut and
colonizes the lining of the intestinal mucosa. The eaeA gene in STEC is responsible
for encoding the protein, intimin, which facilitates the adhesion and strong effacement
to the lining of the intestinal mucosa (Khalifa et al, 2019). The STEC then secretes
either one or both Shiga toxins; Stx1 and Stx2 that corrodes the lining of the intestine,
leading to the loss of electrolytes, often presented as watery stool (diarrhea) which
may/ may not be bloody (Konate et al., 2017).

11
2.3.6 TRANSMISSION OF ESCHERICHIA COLI

Escherichia coli could be transmitted directly from the consumption of animals such
as dogs, cats, livestock; Cattle, sheep, game birds or wild game reservoirs such as
wild dog, rabbits, deers, wild cats etc ( Lutwick, 2014). Escherichia coli could also be
transmitted from person-to-person by fecal-oral route (Fairbrother & Nadeau, 2006).

2.3.7 ANTIMICROBIAL RESISTANCE AND SUSCEPTIBILITY

OF ESCHERICHIA COLI

Resistance to antibiotics is a major problem that has paralleled the discovery of

antibiotics. The misuse and abuse of antibiotics at sub-inhibitory concentrations has
led to the development of adaptive resistance (Ghosh et al., 2019). Evidence from
surveillance data shows that resistance in Escherichia coli is highest for antibiotics
and antimicrobial agents that have been in use for the longest period of time in both
human and animal medicine (Issa et al., 2020). E. coli of animal origin have been
reported to often show resistances to relatively older antimicrobial agents, including
Tetracyclines, Phenicols, Sulfonamides, Trimethoprim, and Phosphomycin (Poirelet
al., 2018).In recent times, the increasing development of multi-drug resistance to
antibiotics displayed by E. coli isolates, including Shiga Toxin Producing Escherichia
coli is alarming (Davies and Davies, 2010). There is therefore the need for antibiotics
susceptibility testing for effective anti-biotherapy of detected STEC isolates
(Aguilera-Alonso et al., 2022). However, despite being resistant to different classes of
antibiotics, STEC is also susceptible to antibiotics when used in the correct
concentrations.

12
 CHAPTER THREE

METHODOLOGY

3.1 STUDY AREA

The sampling locations for this research study were Lagos, Ogun, Oyo, Ondo and
Osun States in Nigeria. These states fall under the South-Western Geo-political zone
of Nigeria. These states were chosen due to their high population status in Nigeria.
There is high availability of affordable, ready-to-eat game meat that are usually sold
in several designated ‘hotspots’ in the sampling locations. The ‘hotspots’ of game
meat are areas that are known specifically for the sales of different types of game
meat. Most of these hotspots for the sales of these game meat are not hygienic and
have questionable or poor sanitary etiquette, hence, the reason why Lagos, Ogun, Oyo,
Ondo and Osun State were chosen as the area of study for this research.

13
Table 3.1: Table showing the Sample obtained and location
LOCATION NAME OF ISOLATE ID TOTAL
ISOLATE
LAGOS STATE
Pangolin PA1LI, PA2L1 2
Festac Sparrow SPL1 1
Bird BIL1 1

Oluwo Market Deer D2L1 1 25

Bush dog B2L1 1
Grasscutter G1L1 1
Etu ETL1 1
Wild Cat WCL1 1
Atika ATL1 1
Agbonrin AGL1 2

Epe Fish Market Antelope A 1 L1, A 2 L1, A 3 L1, 3

Monkey M1L1, M2L1 2
Rabbit R1L1, R2L1 2
Bush dog B1L1 1
Grasscutter G2L1, G3L1, G 4L1 3
Porcupine PL1 1
Deer DL1 1
OGUN STATE
Antelope AN 1S1, AN 2S1 2
Sango-Ota Grasscutter GRS1 1 12
Rabbit RAS1 1
Bush rat BUS1 1
Igala IGS1 2

Abeokuta Antelope ANA1 1

Rabbit RAA1 1
Hedgehog HEA1 1
Guinea fowl GUA1 1
Alligator ALA1 1
ONDO STATE
Oja-Oba Market Civet Cat CC1O1 1
Rabbit RAO1 1 9
Antelope AN1O1, AN2O1, AN3O1 3
Grasscutter GR1O1 1

Ore GR2O1 1
Grasscutter GUO1 1
Guinea Fowl CC2O1 1
Civet Cat
OSUN STATE
Sabo Hare HS1 1 5
Sese SS1 1
Antelope A 4 S1, A5S1, A6S1 3

OYO STATE
Ibadan Aparo API1 1
Eta ETI1 1 4
Esii Tuku ESI1 1
Guinea Fowl GFI1 1

TOTAL 55

14
3.2 SAMPLE COLLECTION

Ready-to-eat game meat samples were purchased from the vendors at designated
hotspots and collected in sterile zip-lock bags and transported to the laboratory. The
game meats were mainly dried cured meat. A total of 55 samples were gotten from
different bush meat hotspots across the sampling locations; Lagos, Ogun, Oyo, Ondo
and Osun state.

3.3 STERILIZATION

Sterilization of micro-pipette tips, Eppendorf tubes at 121°C in the autoclave and petri
dishes, glassware (conical flasks, beakers) at 160°C in the oven was carried out at
each phase of work to ensure septic condition and to prevent cross-contamination.
The work bench/area was regularly disinfected with 70% alcohol. A flame, provided
by the Bunsen burner was always kept on, ensuring the sterility of the air while
working.

3.4 MATERIALS AND EQUIPMENTS USED

Materials used: Micro pipette, Micro-pipette tips, Beakers, conical flask, Petri dishes,
Eppendorf tubes, Cryovial tubes, Test tubes, test tube racks, filter paper, spatula,
inoculating loop, hockey stick.
Equipment used: Autoclave, oven, weighing balance, Stomacher, Incubator
(Memmert) Vortex mixer, distiller, water bath.

3.5 PREPARATION OF CULTURE MEDIA

For the isolation, enumeration and identification of Escherichia coli, selective and
differential media were employed. A Selective media is a media that favours the
growth of a specific organism, while inhibiting the growth of other microorganisms
that are not the target organism of interest. On the other hand, differential media refers

15
to a media that allows the growth of more than one microorganism of interest but with
morphologically distinguishable colonies.
Mac Conkey agar was used, as the differential agar to differentiate the lactose
fermenting bacteria from the non-lactose fermenting bacteria. It produces pink
coloured colony of the suspected Escherichia coli isolate.
The Sorbitol Mac Conkey agar (SMAC) was used for the presumptive isolation and of
the STEC serovar O157:H7. It was prepared by dissolving 50.03g of the powder in
1000ml of water and autoclaved at 121°C for 15 minutes.
Nutrient agar was used for the identification of total viable count. It was prepared
according to the manufacturer’s instruction; 28g of Nutrient agar powder in 1000ml of
water and autoclaved at 121°C for 15 minutes.
The Brain Heart Infusion broth (BHI) is a general-purpose media that was used to
cultivate and maintain the isolates. It was prepared by dissolving 37g in 1L of water at
121°C.
Buffered Peptone Water (BPW) was used for the primary enrichment of the samples.
It contains Peptone at low concentration which provides nutrients for survival of
microorganisms. Buffered Peptone water is also very important for the resuscitation
of cells that have been injured by processes of food preservation. 0.1% Buffered
Peptone Water (BPW) was used for the serial dilution. 0.1% Buffered Peptone water
(BPW) was obtained by dissolving 20g of the Peptone Powder in 1000ml of water,
then it was autoclaved at 121°C for 15 minutes.

3.6 SAMPLE PREPARATION

25g of meat was measured aseptically for each bush meat sample using the weighing
balance. The 25g of each sample was finely chopped using a sterile knife and then put
into a conical flask containing 225ml of 0.1% Buffered Peptone Water. The samples
were homogenized in the stomacher for 2 minutes. This is referred to as the primary
enrichment.

16
3.7 SERIAL DILUTION

1000µl (1ml) was pipetted from the primary enrichment of each sample and
introduced into the test tube containing 9ml of 0.1 % BPW. To obtain the diluents,
1ml was pipetted from the first dilution factor (100 ) and into the second dilution
factor. This process was repeated till the dilution factor of 10−6 was attained.

3.8 PLATING

The spread plate technique was used for plating into the prepared Sorbitol Mac
Conkey Agar (SMAC) and Mac Conkey Agar (MAC) plates. 100µl (0.1ml) of the
10−1 and 10−3 diluent factors were inoculated into the Mac Conkey Agar (MAC) and
Sorbitol Mac Conkey Agar (SMAC) plates and spread using 100% ethanol. This was
done by dipping the hockey stick into the 100% alcohol, flaming with the Bunsen
burner and letting to cool off, then spreading the plate to ensure even distribution of
the innoculum. Replicates were made for each sample. The plates were then inverted
and incubated at 37°C for 18-24 hours.

3.9 SUB-CULTURING

The incubated plates were checked after a duration of about 24 hours for growth. The
morphological characteristics such as the shape, colour, colony morphology, elevation,
edge and other characteristics were also recorded in detail. A sub-culture is done to
purify the isolated bacterial colonies from a mixed bacterial culture. Hence, sub-
culturing is done to obtain pure isolates or culture. The Nutrient Agar is used for sub-
culturing from the Sorbitol Mac Conkey Agar (SMAC). The Nutrient agar plates are
divided into four quadrants and labeled E1 to E4. The process of sub-culturing
involves the selection of distinct colonies from the plates for each sample. For
Escherichia coli, the white and pink colonies are suspected to be Escherichia coli;
O157 and non- O157 respectively. Two distinct white colonies and pink colonies are
selected from each sample plate’s diluent factors. The E1 and E2 quadrants is for the
white colonies and the E3 and E4 quadrants are for the pink colonies. The sub-

17
culturing involves streaking a loop full of the selected colonies into each of the
designated quadrants using the inoculating loop.

3.10 BIOCHEMICAL TESTS

For the identification of the presumptive isolates, after the morphological

characterization further tests were carried out to confirm if the isolates were truly the
organism of interest. Below are the tests carried out for the confirmation of the
suspected E. coli isolates.

3.10.1 GRAM STAINING

Gram staining test is a test done to differentiate bacteria on the basis of their cell wall.
This was done to differentiate the bacterial isolates based on the staining property of
their cell wall, into either Gram positive or negative, with the use of dyes to enhance
the visibility of their cell wall. This was done by making a smear of the selected
isolate on a glass slide, it was then heat fixed by passing it over the Bunsen burner
flame. The slides were then flooded with Crystal Violet which served as the primary
stain for 1 minute and washed off with water. Next, the slides were flooded with
Iodine (mordant) and allowed to sit for 1 minute, then washed off with water. The
slides were decolorized with 70% alcohol for 20 seconds and rinsed with water. The
final step was counterstaining the slides with Safranin for 30 seconds and then rinsed
off. On the basis of the Gram Staining, the isolates that retained the colour of the
Primary stain (Crystal Violet) after being treated with the 70% alcohol are Gram
Positive while the isolates that were decolorized after treatment with alcohol and
retained the colour of the Counterstain (Safranin) ranging from pink to red are Gram
Negative. The stained slides are dried with blotting paper and immersion oil is applied
to the slides and observed under the microscope using the oil immersion objective
lens (x100). The observations were documented.

18
3.10.2 CATALASE TEST

Catalase test is can be used in the identification of Enterobacteriaceae. Therefore, this

test was carried out to check the isolate that possess the enzyme catalase, that breaks
down Hydrogen Peroxide (𝐻2 𝑂2) into oxygen and water. On a clean microscopic slide,
a loop full of distinct colony from the pure culture plate was picked using the
inoculating loop and smeared onto the slide. Few drops of 3% Hydrogen Peroxide
was dispensed on the fixed slide and if it produced bubbles which is Oxygen gas, then
it means the organism is catalase positive. If there was no production of bubbles then
it means the organism is unable to produce the enzyme, hence, Catalase negative.

3.10.3 OXIDASE TEST

This test was carried out to check if the isolates could produce the enzyme oxidase. A
filter paper was flooded with the oxidase reagent. A loop full of the isolate was
inoculated on the filter paper. A purple coloration after 1 minute indicated that the test
was oxidase positive, and colourless (no colour change) indicates oxidase negative.

3.10.4 MOTILITY TEST

The Motility test as the name implies, was carried out to determine if the suspected E.
coli isolates were capable of moving independently using metabolic energy. A sterile
inoculating needle was used to pick the suspected colony and was stabbed into the
center of the semi-solid agar in the test tube. A diffuse zone of growth that extends
from the line of inoculation indicates that the tested organism is motile.

3.10.5 OXIDATIVE FERMENTATIVE TEST

The Oxidative Fermentative test was carried out to determine if the suspected E. coli
isolates could metabolize glucose by fermentation or aerobic respiration (oxidatively).
10ml of the oxidative fermentative medium was prepared and autoclaved at 121°C for
15 minutes. 10ml of Glucose was dispensed into Durrham tubes containing 100ml of
distilled water. Thereafter, 1ml of the solution containing glucose was dispensed in

19
the Oxidative fermentative medium, followed by the inoculation of the suspected
isolates. A colour change from Green to Orange was observed.

3.10.6 PRESERVATION OF SAMPLES

A loop full of each isolate was inoculated into test tubes containing 5ml Brain Heart
Infusion broth (BHI) for 18-24 hours. After this, the solution was homogenized and
750µl was taken from the tube and dispensed into Eppendorf tubes. 750µl of 20%
glycerol was added to the Eppendorf tubes, homogenized and stored at -80°C in an
ultra-freezer.

3.11 DNA EXTRACTION

DNA extraction is a method used to extract or separate pure DNA from biological
samples (Game meat). It separates the pure DNA from cellular membrane, proteins
and other cellular components. Hence, DNA extraction was carried out to extract the
DNA from microbial cells of the game meat samples, using the Boiling Technique.

3.11.1 MATERIALS USED

The following materials were used in the DNA extraction process. They include:
Microbial cells (Preserved Game meat samples), Sterilized distilled water,
Micropipette tips, Micropipette, Centrifuge, Heating block, Eppendorf tubes,
Autoclave, Brain Heart Infusion Broth (BHI), Vortex mixer, Inoculating loop, Ice.

3.11.2 ACTIVATION OF ISOLATES

⚫ 1ml of pure Brain Heart Infusion broth (BHI) was prepared, and dispensed
into 2ml Eppendorf tubes. It was sterilized in the autoclave at 121ºC for 15
minutes.
⚫ After setting it down to cool, 100µl of each of the thawed preserved stock
cultures into the various Eppendorf tubes containing 1ml BHI.
⚫ The isolates were incubated at 37ºC for 2 days (48 hours).

20
3.11.3 PRE-WASHING

⚫ Each of the activated isolates were centrifuged at 5000g for 3 minutes.

⚫ The BHI supernatant was decanted into the waste container, leaving the
supernatant at the bottom of the Eppendorf tube.
⚫ 1.5ml of the sterilized distilled water was added into the tubes, they were
mixed and centrifuged at 5000g for 3 minutes.
⚫ The supernatant was discarded. 200µl of distilled water was added to the tubes
mixed using the Vortex mixer.

3.11.4 BOILING

⚫ The Heating block was turned on and allowed to heat up to boiling

temperature (100ºC).
⚫ The Eppendorf tubes containing the pre- washed isolates were carefully sealed
and placed in the heating block, then covered with the lid to prevent them
from popping out.
⚫ It was allowed to boil for 15 minutes.
⚫ The boiled DNA was placed into ice to cool for 5 minutes.
⚫ It was centrifuged at 7000g for 6 minutes.
⚫ 150µl of the DNA supernatant was carefully transferred into the properly
coded fresh Eppendorf tubes.

3.12 POLYMERASE CHAIN REACTION (PCR)

Polymerase Chain reaction (PCR) is a commonly used technique to make billions of

copies of DNA from a small sample. It was created by the American Biochemist and
Doctor of Chemistry Kary Muler in the year 1985. The extracted DNA samples from
each Game meat sample was amplified using the Multiplex Polymerase Chain (PCR)
technique. The cocktail mixture was prepared and loaded into the Thermocycler. The
Multiplex PCR technique was used to amplify multiple DNA sequences together
simultaneously and was carried out in two treatments. The initial denaturation at 95°C
for 5 minutes; 35 cycles of 95°C for 2 minutes; 42°C for 30 seconds; and 72°C for 4
minutes, thereafter followed by a final elongation step at 72°C for 10 minutes
included in Table 3.2.
21
 3.13 GEL ELECTROPHORESIS

Gel Electrophoresis is a method used to separate and analyze the DNA molecules on
the basis of their size and charge. Each DNA was loaded into the perforated agarose
gel. The agarose gel was placed in the buffer and current was passed through via the
negative terminal. The positive samples migrated towards the positive terminal and
was viewed under Ultra Violet light with the aid of gel documentation system.

Table 3.2 Table showing the target genes, virulence factors and primer sequences
adapted from Persson (2007).
TARGET VIRULENCE SEQUENCE (5΄-) FINAL
GENE FACTOR CONCENTRATION(µm)
Human estA STFh TTTCGCTCAGGATGCTAAACCAG 0.4
CAGGATTACAACACAATTCACAGCAGTA
Porcine estA STIp CTTTCCCCTCTTTTAGTCAGTCAACTG 0.4
CAGGATTACAACAAAGTTCACAGCAG
vtx1 VT1 GTTTGCAGTTGATGTCAGAGGGA 0.25
CAACGAATGGCGATTTATCTGC
vtx2 VT2 GCCTGTCGCCAGTTATCTGACA 0.5
GGAATGCAAATCAGTCGTCACTC
Eae Intimin GGYCAGCGTTTTTTCCTTCCTG 0.15
TCGTCACCARAGGAATCGGAG
eltA LTI AAACCGGCTTTGTCAGATATGATGA 0.45
TGTGCTCAGATTCTGGGTCTCCT
ipaH IPaH TTGACCGCCTTTCCGATACC 0.1
ATCCGCATCACCGCTCAGAC
16SrDNA 16SrDNA GGAGGCAGCAGTGGGGAATA 0.25
TGACGGGCGGTGTGTACAAG

22
Table 3.3: Protocol for Thermocycler
ANALYSIS STEP TEMPERATURE(°C) TIME
1x Initial denaturation 95 15 min
35x Denaturation 94 50 sec
Annealing 57 40 sec
Polymerization 72 50 sec
1x Final Polymerization 72 3 min
1x Hold 4 ∞

Table 3.4: Multiplex PCR Components for the PCR Treatment

S/N REAGENT INITIAL FINAL V/R(µl)
CONCENTRATION CONCENTRATTION
(µm) (µm)
1 Master mix 5x 1x 2
2 StFp 20 0.4 0.2
3 StRp 20 0.4 0.2
4 Vtx1Tf 20 0.25 0.125
5 Vtx1tR 20 0.25 0.125
6 Vtx2tF 20 0.5 0.25
7 Vtx2tR 20 0.5 0.25
8 ipaHF 20 0.1 0.05
9 ipaHR 20 0.1 0.05
10 MgCl2 25 1.5 0.6
11 dH2O 4.15
12 DNA

23
 CHAPTER FOUR

RESULTS

4.1 RESULTS

4.1.1 TVC RESULT

The microbial analysis of the isolates for Total viable count (TVC) of Shiga Toxin
Producing E. coli (STEC) and general E. coli gotten from culture on SMAC are
shown in Table 4.1. The TVC for the game meat samples were relatively high,
starting from 4.3 x 106 Cfu/ml which was the Eta to the Monkey with the highest
count of 9.5 x 106 Cfu/ml, followed by antelope with a TVC ranging from 8.3-8.6
x106 Cfu/ml.

4.1.2 BIOCHEMICAL TESTS OF E. COLI ISOLATES

Table 4.2 summarizes the biochemical tests carried out on all presumptive E. coli
isolates. All the samples were negative to Gram staining, Catalase positive, Oxidase
negative, Motility positive and fermentative, which are all characteristics of E. coli.

24
Table 4.1: Total Viable Count (TVC) of E. coli in the game meat (n=55) from various
locations in South western Nigeria.
LOCATION GAME MEAT NUMBER OF TOTAL VIABLE
SAMPLES COUNT (Cfu/mL)
Lagos Pangolin 8.6 x 106
Quail 4.1x 106
Deer 8.1 x 106
Bush dog 6.4 x 106
Grasscutter 8.5 x 106
Etu 5.5 x 106
Wild cat 25 7.3 x 106
Atika 6.3 x 106
Agbonrin 4.5 x 106
Antelope 8.7 x 106
Monkey 9.5 x 106
Rabbit 7.5 x 106
Porcupine 8.3 x 106
Ogun Antelope 8.6 x 106
Grasscutter 8.4 x 106
Rabbit 7.8 x 106
Bush rat 12 6.2 x 106
Igala 6.7 x 106
Hedgehog 5.2 x 106
Guinea fowl 4.8 x 106
Alligator 7.3 x 106
Ondo Civet cat 7.2 x 106
Rabbit 7.4 x 106
Antelope 9 8.8 x 106
Grasscutter 8.3 x 106
Guinea fowl 4.4 x 106
Osun Hare 4.8 x 106
Sese 5 6.8 x 106
Antelope 8.6 x 106
Oyo Aparo 5.5 x 106
Eta 4.3 x 106
Esii Tuku 4 7.1 x 106
Guinea fowl 5.0 x 106
TOTAL 55

25
 Table 4.2:The results of biochemical tests for presumptive identification of E. coli in
all isolates.
Sample ID Gram Catalase Oxidase Motility Oxidative/Fermentative Suspected
Staining Organism
PA1LI - + - + F E. coli
PA2L1 - + - + F E. coli
SPL1 - + - + F E. coli
BIL1 - + - + F E. coli
D2L1 - + - + F E. coli
B2L1 - + - + F E. coli
G1L1 - + - + F E. coli
ETL1 - + - + F E. coli
G2L1 - + - + F E. coli
BUS1 - + - + F E. coli
RAA1 - + - + F E. coli
HEA1 - + - + F E. coli
GUO1 - + - + F E. coli
SS1 - + - + F E. coli
ETI1 - + - + F E. coli

Note: ‘+’ indicates positive result, ‘-’ indicates negative result and ‘F’ indicates
Fermentative.

26
4.1.3 PREVALENCE OF SHIGA TOXIN E. COLI IN GAME MEAT

The prevalence of the game meat samples (n=55) show that Antelope and Grasscutter
are the most prevalent (100%).

Number of bushmeat Positve sample

14

12

10

Figure 4.1: The prevalence of presumptive Shiga toxin producing E. coli in game
meat (n=55).

27
Plate 4.1: Agarose gel electrophoresis image of Multiplex-PCR products (Human
estA, Porcine estA, vtx1, vtx2,ipaH, eae, elta). Lane L: marker (100-bp ladder), lane
30 and 31: E. coli isolate (Porcine estA).

28
 TREATMENT 1 L 28 29 30 31 32 33 34 35 36

Vtx1(260)bp
Human estA(151)bp

+ve samples
28,30,31,33,34,35,3
8,45
L 37 38 39 40 41 42 43 44 45

Vtx1(260)bp
Human estA(151)bp

TREATMENT 1: Human estA (StFh and StRh

primers) – 151bp TREATMENT 2: Porcine estA (StFp and StRp
vtx1 – 260bp primers) – 160bp
vtx2 – 420bp eae – 377bp
ipaH – 647bp eltA – 479bp

Plate 4.2: Agarose gel electrophoresis image of Multiplex-PCR products (Human

estA, Porcine estA, vtx1, vtx2, ipaH, eae, elta). Lane L: marker (100bp-ladder). Lane
28, 33, 34, 38 (Human estA), Lane 30, 32, 35, 45 (Vtx1).

4.1.4 PREVALENCE OF POSITIVE SAMPLES ACCORDING TO

LOCATION
All the game meat samples were positive for E. coli, however, the Table 4.3 shows
the number of Positive Samples of the E. coli gene of interests (Vtx1, Human and
Porcine estA). Ogun state has the highest number of positive samples (12.7%), Ondo
and Osun States with the lowest number of positive samples (1.8%).

Table 4.3: Number of Positive samples and their location

LOCATION LAGOS OGUN OYO ONDO OSUN

NO. OF 3 7 2 1 1
POSITIVE
SAMPLES

29
 CHAPTER FIVE

DISCUSSION

5.1 DISCUSSION

Escherichia coli is ranked as the most studied bacterial pathogen due to the impact on
public health (Pokharel et al., 2020).The existence of several game meat species and
availability at relatively affordable rates further encourages the sales, hence,
consumption of game species in Nigeria. Shiga Toxin Producing E. coli (STEC) has
majorly been implicated in cattle and Sheep (Colleloet al., 2015).
This research aimed at isolating and detecting E. coli from ready-to-eat game meat
samples sold at various game meat hotspots in the South West region of Nigeria.
The game meat samples were investigated to assess the safety in consuming game
meat due to the insufficient knowledge on the microbiological quality of game species
in Nigeria.
In all the game meat samples, E. coli was suspected, E. coli is used as an indicator of
fecal matter, meaning that the contamination of the game species is probably due to
fecal contamination, poor sanitary conditions, improper handling or even storage
lapses.
The Total Variable Count (TVC) was also used an indicator to assess the microbial
safety of the game meat samples. The TVC of the game meat samples ranged from
4.3 to 9.5 x 106 log10Cfu/ml shows that they all possessed relatively high microbial
load but still tolerable as it does not exceed the Food and Organization (FAO) limit of
105 Cfu/ml (FAO, 2005). Despite the fact that the TVC does not assess the microbial
quality of the food, it is used as an indicator of processing, handling, storage of the
sample.
The PCR results of the presumptive E. coli (n=55) show that all the samples for E.
coli are positive, however, the target E. coli genes of interest identified was the
Human estA, Porcine estA, and Vtx1; 12.7%, 3.6% and 9.1% respectively. Antelope
was the most implicated game meat specie. A recent study on beef products in Lagos
state, also confirmed the presence of STEC in ready-to-eat beef products using
Multiplex PCR (Fayemiet al., 2021).

30
Shiga Toxin Producing Escherichia coli (STEC) has been known for causing
unpredictable and deadly outbreaks of infectious diarrhea, Hemorrhagic Uremic
Syndrome and hemorrhagic colitis (Vallance et al., 2002). Therefore, the presence of
STEC in game meat that is now being commonly consumed is a health risk which
could potentially be a hazard.

31
 CHAPTER SIX

CONCLUSIONS AND RECOMMENDATIONS

6.1 CONCLUSIONS AND RECOMMENDATIONS

Based on this research finding, the presence of pathogenic E. coli and Shiga toxin
producing E. coli (STEC) was suspected and detected in few of the game meat species.
The frequency of the contaminated samples albeit low, still indicates that there are
sanitary lapses in the processing of these ready-to-eat game meat. These game meat
species are casually sold, and the presence of STEC in them is an indication that the
microbiological safety is compromised, hence, a risk to the public health. There
should be strict enforcement of Hazard and/or risk-based food safety protocols to
prevent or mitigate the occurrence and incidence of pathogenic microorganisms in
game meat by appropriate food safety and Public health authorities.
Periodical quality audits to monitor the Processes used in handling and processing
game meat and its products. Generally, the game meat must be properly and
thoroughly cooked to eliminate microorganisms. Surveillance systems should be put
in place to track and the monitor occurrence of foodborne illnesses and/or diseases as
a result of consuming game meat.

32
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