BACTERIOLOGICAL QUALITY OF FRESH CUT FRUITS

SOLD IN IHIAGWA AND EZIOBODO MARKET IN OWERRI

WEST LOCAL GOVERNMENT AREA OF IMO STATE

BY
ANAEME, NETOCHUKWU FRANCIS

REG. NO: 20181085695

DEPARTMENT OF MICROBIOLOGY
SCHOOL OF BIOLOGICAL SCIENCES

SUPERVISED BY
Prof. (Mrs.) J. C. Orji

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE
AWARD OF BACHELOR OF TECHNOLOGY (B.TECH) IN
MICROBIOLOGY

June 2024
 APPROVAL PAGE
This project titled Bacteriological quality of fresh cut fruits sold in Ihiagwa and
Eziobodo market in Owerri West Imo State was written by Anaeme,
Netochukwu Francis with registration number (20181085695) and has been read
and approved as meeting the requirements of the school of Biological Sciences,
Federal University of Technology Owerri for the award of Bachelor of
Technology (B.Tech) Degree in Microbiology.

………………………. ……………………….
Prof. (Mrs.) J. C. Orji Date
Supervisor

………………………. ……………………….
Prof. Ifechukwu Enyinna Adieze Date

Head of Department

………………………. ……………………….
Prof. Chinwe S. Alisi Date
Dean, school of Biological Sciences

………………………. ……………………….
...................................
External Examiner Date

i
 CERTIFICATION
I certify that this project ‘Bacteriological quality of fresh cut fruits sold in
Ihiagwa and Eziobodo market in Owerri West Imo State’ is an original work by
Anaeme, Netochukwu F. with registration number (20181085695) of the school
of biological sciences, Federal University Of Technology Owerri in accordance
with the requirements of the school of biological sciences.

…………………….. ………………………
Anaeme, Netochukwu. F Date

ii
 DEDICATION
This study is dedicated to the Anaeme families.

iii
 ACKNOWLEDGEMENTS

Firstly the researcher thanks the Almighty creator who is the source of His
living and inspirations to him alone be the glory for he gives understanding,
wisdom and ideas which led in the writing of this work and its successful
completion in the midst of obvious challenges.
My gratitude goes to Prof. (Mrs.) J. C. Orji my project Supervisor who in her
busy schedule brought out time to go through his work, he sincerely thank Prof.
Wesley Braide for his assistance and support throughout the experimental
procedures, The researcher expresses his profound gratitude to Prof. Ifechukwu
Enyinna Adieze (Head of Department) Department of Microbiology in the
faculty of biological sciences, Federal University of Technology Owerri and all
the staffs in the department.
Words will not be enough to express his gratitude to his loving parents Prof. and
Dr. Mrs. F .O Anaeme and my loving sister Chisom and the rest of family
members for standing beside him all these time. To my fellow course mates and
friends Golden, Nze Ozioma jesus, Divinelove oluebube, Nnewdimma, Dr.
Coins, Ebuka, Ifeanyi, Jane, Faustina, Marvis chimezie, Walter, Success and so
many others who contributed to the work, May God Almighty Bless You All.

iv
 TABLE OF CONTENTS

COVER PAGE
TITLE PAGE
APPROVAL PAGE i
CERTIFICATION PAGE ii
DEDICATION iii
ACKNOLEDGEMENTS iv
TABLE OF CONTENTS v
LIST OF TABLES vii
ABSTRACT vii
CHAPTER ONE: INTRODUCTION 1
1.1 Background to the Study 1
1.2 Statement of the Problem 3

1.3 Aim and Objective 4

1.4 Scope of the Study 4

CHAPTER TWO: REVIEW OF LITERATURE

2.1 Major Concepts of the study 5
2.1.1 Effects of polyethylene bags for packaging of sliced fruits by street
vendors in Nigeria. 5
2.1.2 Microbiological quality of pre-cut fruits sold in retail outlets in Nigeria
2.1.3 Bacterial pathogens associated with fruit contamination 6
2.1.3.1 Pathogenic Escherichia coli 7
2.1.3.3 Salmonella spp 11
2.1.3.4 Shigella spp 12
2.1.3.5 Staphylococcus aureus 13
2.1.3.6 Listeria monocytogenes 14
2.1.3.7 Vibrio 14
2.1.3.8 Spore Formers 15
2.2 Theoritical Framework 17
2.3 Empirical Studies 20
2.4 Summary of Related Literature 21

v
CHAPTER THREE: METHODOLOGY

3.1 Collection of samples 23

3.2 Preparation of media and diluents 23

3.3 Characterization and Identification of Microbial Isolates 25

3.4 Biochemical characterization of bacterial isolates 27

CHAPTER FOUR: RESULTS 30

CHAPTER FIVE: DISCUSSION OF FINDINGS AND SUMMARY OF

THE STUDY

5.1 Discussion 43

5.2 Conclusion 44

REFERENCES 45

Appendix 53

vi
 LIST OF TABLES

Table 1: Total Counts and Colonial Characteristics of Bacterial Isolates on

Nutrient Agar 30
Table 2: Total Counts and Colonial Characteristics of Fungal Isolates on Potato

Dextrose Agar 32

Table 3: Total Counts and Colonial Characteristics of Bacterial Isolates on

Mannitol Salt Agar. 34

Table 4: Total Counts and Colonial Characteristics of Bacterial Isolates on

Cetrimide Agar 35

Table 5: Total Counts and Colonial Characteristics of Bacterial Isolates on

Salmonella Shigella Agar 36

Table 6: Total Counts and Colonial Characteristics of Bacterial Isolates on

Eosin Methylene Blue Agar 37
Table7: Microscopic and Biochemical Characteristics of Bacteria isolated from
Samples 39

Table8: Antibiotic Sensitivity Test of Gram Positive Bacterial Isolates 40

Table9: Antibiotic Sensitivity Test of Gram Negative Bacterial Isolate 41
Table 10: Percentage Occurrence of Bacteria and Fungi in Samples 42

vii
 ABSTRACT

In Nigeria the consumption of fresh cut fruits has become increasingly popular
particularly in urban areas like Owerri Metropolis. However the bacteriological
quality of these fruits is a concern as they may be contaminated with harmful
bacteria. The risk of contamination is higher in Nigeria due to inadequate food
safety regulations, poor handling practices and insufficient storage facilities.
The aim of this study is to investigate the prevalence of harmful bacteria in
fresh cut fruits sold in Ihiagwa and Eziobodo markets and to evaluate the
bacteriological quality of different types of fresh cut fruits (e.g., watermelon,
pineapple, pawpaw) in Ihiagwa and Eziobodo market. Microbiological studies
was carried out with reference to standard methods. Microbial isolates where
characterized based on cultural (colonial) and microscopic and few biochemical
methods. Microorganisms that were not identified by the colonial and
microscopic characteristics were further subjected to a few biochemical tests.
Identification was done with reference to standard manuals. Antimicrobial
sensitivity test was carried out to determine sensitivity and resistance of specific
pathogens to a wide range of antimicrobial agents. In the result of this research
the percentage distribution of bacterial and fungal isolates where as follows; for
bacterial isolates Staphylococcus sp (11.9%), Pseudomonas aeruginosa
(23.8%), Bacillus sp (19.4%), Enterococcus faecalis (14.9%), Enterobacter sp
(14.9%), Shigella sp (7.5%) and Salmonella sp (7.5%) while for fungal isolates
only Saccharomyces cerevisiae was present. However the study showed that
harmful bacteria and fungi where found in the fresh cut fruit samples. The
medical significance of some of the isolates had been reported. Contaminants
might have come in contact with these fruits due to poor hygiene. Proper care
should be taken in the processing of these products to guarantee consumers’
confidence and satisfaction.

viii
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

A popular distinction between vegetables and fruits is difficult to uphold. In

general, those plants or plant parts that are usually consumed with the main course
of a meal are popularly regarded as vegetables, while those mainly used as desserts
are considered as fruits thus cucumber and tomato are fruits botanically, since they
are the portion of the plant containing seeds, but are commonly regarded as
vegetables. Vegetables are parts of plants that are consumed by humans or other
animals as food. The original meaning is still commonly used and is applied to
plants collectively to refer to all edible plant matter including the flowers, fruits,
stems, leaves, roots, and seeds. An alternate definition of the term is applied
somewhat arbitrarily often by culinary and cultural tradition. It may exclude foods
derived from some plants that are fruits, flowers, nuts, and cereal grains, but include
savory fruits such as tomatoes and courgettes, flowers such as broccoli, and seeds
such as pulses.
Fruits and vegetables (F&V) are considered in dietary guidance because of their
high concentrations of dietary fiber, vitamins, minerals, especially electrolytes and
more recently phyto-chemicals especially antioxidants (Slavin and Lloyd, 2012).
Various reviews have associated low intake of fruits and vegetables with chronic
diseases such as cardiovascular diseases, blood pressure, hypercholesterolemia,
osteoporosis, many cancers, chronic obstructive pulmonary diseases, respiratory
problems as well as mental health (Adebawo et al., 2006). Despite an increasing
focus on the health benefits of fruits and vegetables their consumption is below the
recommended intake among adults (Schneider et al, 2007). Therefore considering
how nutritional related health problems have risen drastically globally, it seems
critical that formal nutrition education aiming to increase knowledge of fruits and
vegetables intake be given priority in health education programs. Sufficient intake
of fruit and vegetables (F&V) has been related epidemiologically with reduced risk
of many non-communicable diseases. Currently much interest are focused on the
vital role of antioxidants which impart bright colour to F&V and act as scavengers
cleaning up free radicals before they cause detrimental health effects (Kaur and
Kapoor, 2001). Moreover fibers found in F&V have been shown to reduce intestinal
passage rates by forming a bulk, leading to a more gradual nutrient absorption
(Anderson et al, 2010) hence preventing constipation. They can be fermented in the
colon increasing the concentration of short chain fatty acids having anti
carcinogenic properties and maintaining gut health (Lattimer and Haub, 2010).
Several studies have highlighted the CVD risk-reducing potential of F&V whereby
their intake were strongly associated with lower cardiovascular risk factors such as
lower blood pressure (BP), cholesterol and triacylglycerol thus preventing
premature cardiovascular disorders (Adebawo et al., 2006). Recently Habauzit et al.
(2013) reported that fruits containing a high amount of anthocyanins, flavonols and
1
procyanidins such as berries, grapes and pomegranate are effective at decreasing
cardiovascular risk while citrus fruits and apples had a moderate effect on BP and
blood lipid level. An increased consumption of carotenoid rich F&V maintains the
cholesterol level in blood since they reduce oxidative damage and cause an increase
in LDL oxidation resistance (Williamson, 1996). An increased consumption of
cruciferous vegetables was also reported to cause a decrease in the risk of intestinal,
bowel, thyroid, pancreatic and lung cancer (Park, 2011). F&V have also been
suggested to prevent osteoporosis in adults mainly for their rich sources of calcium
and other vitamins which are vital in bone health (Southon, 2000). The high fiber
content of F&V may play a role in calcium absorption and reduce the acid load of
the diet (New, 2001), enhancing bone formation and suppressing bone resorption
which consequently result in greater bone strength (Shen, 2012).
Fresh cut fruits are a popular and convenient option for consumers seeking a
healthy and refreshing snack. However, the processing and handling of fresh cut
fruits can increase the risk of contamination by harmful microorganisms (Balogun
et al., 2020). The World Health Organization (WHO) estimates that approximately
600 million people fall ill annually due to food borne diseases resulting in 420,000
deaths worldwide (WHO, 2020).
In Nigeria the consumption of fresh cut fruits has become increasingly popular
particularly in urban areas like Owerri Metropolis. However the bacteriological
quality of these fruits is a concern as they may be contaminated with harmful
bacteria like Salmonella, Escherichia coli and Shigella (Akinjogunla et al., 2020).
The risk of contamination is higher in Nigeria due to inadequate food safety
regulations, poor handling practices, and insufficient storage facilities (Oyedeji et
al., 2017). Several studies have investigated the bacteriological quality of fresh cut
fruits in various parts of Nigeria. For example, a study conducted in Lagos found
that 71.4% of fresh cut fruits sampled were contaminated with bacteria including
Staphylococcus aureus and E. coli (Adeyemi et al., 2017). Another study in Abuja
found that 63.6% of fresh cut fruits sampled were contaminated with fungi
including Aspergillus and Penicillium (Olorunfemi et al., 2020).
Despite these findings, there is a lack of research on the bacteriological quality of
fresh cut fruits sold in Owerri Metropolis. This knowledge gap necessitates a study
to investigate the bacteriological quality of fresh cut fruits sold in this region. This
study aims to determine the prevalence of bacterial contaminants in fresh cut fruits
sold in Owerri Metropolis and assess the potential health risks associated with their
consumption.
Fresh cut fruits are a nutrient dense snack that provides essential vitamins, minerals,
and antioxidants. However, the processing and handling of fresh cut fruits can
increase the risk of contamination by harmful microorganisms (Balogun et al.,
2020). The Centers for Disease Control and Prevention (CDC) estimates that
approximately 48 million people get sick from foodborne illnesses annually in the
United States, resulting in 128,000 hospitalizations and 3,000 deaths (CDC, 2020).

2
In addition to the risk of foodborne illness, fresh cut fruits may also be
contaminated with pesticide residues, heavy metals, and other harmful substances
(Adeyemi et al., 2017). The use of pesticides and other agricultural chemicals in
fruit production can lead to residues on the fruits, which can pose health risks to
consumers (Adeyemi et al., 2017).
The processing and handling of fresh cut fruits also pose a risk of contamination.
Fresh cut fruits are typically washed, cut, and packaged in facilities that may not
adhere to proper sanitation and hygiene practices (Balogun et al., 2020). This can
lead to cross-contamination of fruits with harmful microorganisms, increasing the
risk of foodborne illness (Balogun et al., 2020).
1.2 Problem Statement
The bacteriological quality of fresh cut fruits sold in Owerri metropolis is a
significant public health concern as the fruits may be contaminated with harmful
bacteria posing a risk of food borne illnesses to consumers. This statement outlines
the core issue which can be broken down into several aspects:
- Fresh cut fruits: A growing trend in convenience foods, fresh cut fruits are more
susceptible to contamination due to handling and processing.
- Owerri metropolis: The specific geographic location, which may have unique
factors contributing to the problem, such as inadequate regulations or poor fruit
handling practices.
- Bacteriological quality: The presence of harmful bacteria, like Salmonella, E. coli,
and Listeria, which can cause severe foodborne illnesses.
- Public health concern: The potential impact on consumers, particularly vulnerable
populations like the elderly, pregnant women, and young children.
- Food borne illnesses: A range of symptoms, from mild to severe, including
gastrointestinal issues, fever, and even life-threatening complications.
This problem statement sets the stage for a comprehensive investigation which
including sampling and testing fresh cut fruits for bacterial contamination,
Identifying risk factors and sources of contamination, evaluating existing
regulations and fruit handling practices, developing strategies for improvement such
as education campaigns, improved storage and handling practices, and enhanced
regulation enforcement and running anti microbiological susceptibility test to know
the exact drugs that can help cure infected patients. By addressing this problem, the
research aims to contribute to ensuring the safety and quality of fresh cut fruits
consumed in Owerri metropolis, ultimately protecting public health.

3
1.3 Aim and Objective

This study is aimed at determining the “Bacteriological quality of fresh cut fruits
sold in Ihiagwa and Eziobodo markets”. The objectives include:
1. To investigate the prevalence of harmful bacteria in fresh cut fruits sold in
Ihiagwa and Eziobodo market.
2. To evaluate the bacteriological quality of different types of fresh cut fruits
(e.g., watermelon, pineapple, pawpaw) in Ihiagwa and Eziobodo market.
3. To compare the bacteriological quality of fresh cut fruits from different
sources (e.g., markets and street vendors) in Ihiagwa and Eziobodo market.
4. To identify the most common bacterial contaminants in fresh cut fruits sold
in Ihiagwa and Eziobodo market.
5. To determine the antibiotic resistance patterns of bacterial isolates from fresh
cut fruits in Ihiagwa and Eziobodo market.
6. To provide recommendations for improving the bacteriological quality of
fresh cut fruits sold in Ihiagwa and Eziobodo market based on the study's
findings.
1.4 Scope of the study
This research was carried out in Ihiagwa and Eziobodo market located in Owerri
West Local Government Area of Imo State where some sliced fruits like
watermelon and pineapple where purchased from random sellers from different
selling points. The samples where macerated in a stomacher blender and
homogenously in sterile distilled water, Using serial dilution method the samples
where inoculated into a growth media to check for most occurring colonies and an
antimicrobial sensitivity test was carried out to check for both Gram positive and
Gran negative Bacterial isolates.

4
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 MAJOR CONCEPTS OF THE STUDY

2.1.1 EFFECTS OF POLYETHYLENE BAGS FOR PACKAGING OF

SLICED FRUITS BY STREET VENDORS IN NIGERIA.

Polyethylene bags can insulate fruits, potentially retaining heat and causing
temperatures to rise which can affect fruit quality and ripening, Polyethylene bags
can trap moisture leading to a humid microenvironment that fosters bacterial growth
and fruit decay (Adelowo and Adelowo, 2013). Trapped ethylene gas can stimulate
fruit ripening and senescence, potentially causing fruits to become overripe or
spoiled more quickly (Beuchat, 2000). Reduced gas exchange can lead to a buildup
of CO2 and a reduction in O2, potentially affecting fruit respiration and ripening
(Buck et al., 2003). Polyethylene bags may also leach chemicals such as phthalates
or BPA, into the fruit potentially affecting flavor, texture, and safety (Omorodion
and Nwiyege, 2023).

In aspect of physical damage fruits packaged in polyethylene bags may be more

susceptible to physical damage, such as bruising or punctures due to the bag's
material properties (Afolabi and Adesanya. 2015). The effects of polyethylene bags
on fruits can vary depending on factors like; Bag thickness and material properties,
Fruit type and ripeness, Storage conditions (temperature, humidity, etc.), handling
and transportation practices

Street vending is a common practice in Nigeria, with many vendors selling sliced
fruits to consumers. However the microbiological quality of these fruits is a concern
as they may be contaminated with harmful microorganisms. Previous studies have
shown that street-vended fruits are often contaminated with microorganism
including bacteria, mold, and yeast (Adelowo and Adelowo, 2013) and (Beuchat,
2000). Polyethylene packaging may not be effective in preventing microbial
contamination as it can permeate moisture and allow bacterial growth (Bhattacharya
and Mukherjee, 2015). In Nigeria, street vendors often lack proper training on food
handling and hygiene practices, which can contribute to microbial contamination
(Federal Ministry of Health, Nigeria, 2014).

2.1.2 MICROBIOLOGICAL QUALITY OF PRE-CUT FRUITS SOLD IN

RETAIL OUTLETS IN NIGERIA
Consumption of fruits and vegetables has increased significantly in many countries
during the past decade. Vegetables and fruits have been associated with outbreaks
of food-borne disease in many countries. Organisms involved include bacteria,
5
fungi, viruses and parasites (Jay, 1996; De Roever, 1998). Pre-cut fruits (pineapple,
strawberries, cantaloupes, watermelon and grapes) suspected to be contaminated
with Norovirus has been reported. Kaplan and Campbell (1982) implicated
Norovirus in fruit salad. Outbreaks of salmonellosis have been associated with the
consumption of cut watermelon and cantaloupe (CDC, 1979; Reis et al., 1990;
CDC, 1991; Blostein, 1993; CDC, 2009) in the United States of America. Fruits
may be fresh, canned and may be whole, pre directly after peeling, cut or pureed.
Fruits may be eaten raw, pre-cut or sliced into pieces. Pre-cut fruits refer to fruits
that have been cut open, sliced into pieces but remain in the fresh state and are
stored or displayed for sale or for serving in retail outlets (fresh fruit packs in
supermarkets, cut fruits in buffets) assorted fruits offered by restaurants and
vendors (Kaplan and Campbell, 1982; Lund, 1992; De Roever, 1998).
Epidemiological data have shown that food cross-contamination during preparation
contributes remarkably to the occurrence of food-borne diseases (Gilling et al.,
2001; Kusumaningrum et al., 2004; CDC, 2009). Commercial pre-cut fruit vending
in Nigeria consists of a small number of operations in most cities. However, this
sector has not been considered by agencies responsible for food services as having a
role in the introduction of potential food borne bacteria or parasites. Fruits are
known to carry natural non-pathogenic microflora but contamination with
pathogens from human, animal and environmental sources can sporadically occur at
various stages of preparation before consumption. Furthermore the risk of
foodborne illnesses caused by pathogens could be increased in fresh cut fruit
products because they could be contaminated with pathogens during processing
(Gombas et al., 2017). Additionally, the growth of pathogens in fresh-cut fruit
products can be enhanced by nutrients in the fruit cells exposed during the peeling
and cutting processes (Qadri et al., 2015). Contamination of food borne pathogens
in fresh cut fruit products has been recently reported; for example, L.
monocytogenes in cut apples and melons in Canada (Zhang et al., 2020)
and Bacillus cereus in cut apples in Korea (Tango et al., 2018). Nevertheless,
studies on the microbial contamination of fresh-cut fruit products are limited.
Information on microbial contamination assessment of related products should be
provided to ensure the microbial safety of fresh-cut fruit products distributed in
retail stores.

2.1.3 BACTERIAL PATHOGENS ASSOCIATED WITH FRUIT

CONTAMINATION
2.1.3.1 PATHOGENIC ESCHERICHIA COLI
Escherichia coli typically colonize the gastrointestinal tract of human infants within
a few hours after birth. Usually, E. coli and its human host coexist in good health
and with mutual benefit for decades. These commensal E. coli strains rarely cause
disease except in immune compromised hosts or where the normal gastrointestinal
barriers are breached as in peritonitis, for example. The niche of commensal E.
6
coli is the mucous layer of the mammalian colon. The bacterium is a highly
successful competitor at this crowded site, comprising the most abundant facultative
anaerobe of the human intestinal microflora. Despite the enormous body of
literature on the genetics and physiology of this species, the mechanisms
whereby E. coli assures this auspicious symbiosis in the colon are poorly
characterized. One interesting hypothesis suggests that E. coli might exploit its
ability to utilize gluconate in the colon more efficiently than other resident species,
thereby allowing it to occupy a highly specific metabolic niche (Sweeney et al.,
1996).
However, there are several highly adapted E. coli clones that have acquired specific
virulence attributes, which confers an increased ability to adapt to new niches and
allows them to cause a broad spectrum of disease. These virulence attributes are
frequently encoded on genetic elements that can be mobilized into different strains
to create novel combinations of virulence factors, or on genetic elements that might
once have been mobile but have now evolved to become 'locked' into the genome.
Only the most successful combinations of virulence factors have persisted to
become specific 'PATHOTYPES' of E. coli that are capable of causing disease in
healthy individuals. Three general clinical syndromes can result from infection with
one of these pathotypes: enteric/diarrhoeal disease, urinary tract infections (UTIs)
and sepsis/meningitis. Among the intestinal pathogens there are six well-described
categories: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC),
enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC),
enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC)( Nataro and
Kaper, 1998). UTIs are the most common extraintestinal E. coli infections and are
caused by uropathogenic E. coli (UPEC). An increasingly common cause of
extraintestinal infections is the pathotype responsible for meningitis and sepsis
meningitis associated E. coli (MNEC). The E. coli pathotypes implicated in
extraintestinal infections have recently been called ExPEC(Russo and Johnson,
2000). EPEC, EHEC and ETEC can also cause disease in animals using many of
the same virulence factors that are present in human strains and unique colonization
factors that are not found in human strains. An additional animal pathotype known
as avian pathogenic E. coli (APEC) causes extraintestinal infections primarily
respiratory infections, pericarditis, and septicaemia of poultry. The various
pathotypes of E. coli tend to be clonal groups that are characterized by shared O
(lipopolysaccharide, LPS) and H (flagellar) antigens that define SEROGROUPS (O
antigen only) or SEROTYPES (O and H antigens)( Nataro and Kaper, 1998;
Whittam, 1996).Pathogenic E. coli strains use a multi-step scheme of pathogenesis
that is similar to that used by other mucosal pathogens, which consists of
colonization of a mucosal site, evasion of host defences, multiplication and host
damage. Most of the pathogenic E. coli strains remain extracellular, but EIEC is a
true intracellular pathogen that is capable of invading and replicating within
epithelial cells and macrophages. Other E. coli strains might be internalized by
epithelial cells at low levels, but do not seem to replicate intracellularly.

7
Pathogenic E. coli strains possess specific adherence factors that allow them to
colonize sites that E. coli does not normally inhabit, such as the small intestine and
the urethra. Most frequently these adhesins form distinct morphological structures
called fimbriae (also called pili) or fibrillae, which can belong to one of several
different classes. Fimbriae are rod-like structures of 5–10 nm diameter that are
distinct from flagella. Fibrillae are 2–4 nm in diameter, and are either long and wiry
or curly and flexible (Cassels and Wolf, 1995). The Afa adhesins that are produced
by many diarrhoeagenic and uropathogenic E. coli are described as afimbrial
adhesins, but in fact seem to have a fine fibrillar structure that is difficult to
visualize (Keller et al., 2002). Adhesins of pathogenic E. coli can also include
outer-membrane proteins, such as intimin of UPEC and EHEC, or other non-
fimbrial proteins. Some surface structures trigger signal transduction pathways or
cytoskeletal rearrangements that can lead to disease. For example, the members of
the Dr family of adhesins that are expressed by DAEC and UPEC bind to
the DECAY-ACCELERATING FACTOR (DAF, also known as CD55), which
results in activation of phosphatidylinositol 3-kinase (PI-3-kinase) and cell-surface
expression of the major histocompatibility complex (MHC) class I-related
molecule MICA(Tieng et al., 2002). The IcsA protein of EIEC nucleates actin
filaments at one pole of the bacterium, which allows it to move within the
cytoplasm and into adjacent epithelial cells on a 'tail' of polymerized actin
(Goldberg and Theriot, 1995). Even surface structures that are present on
commensal E. coli strains can induce signalling cascades if the organism encounters
the appropriate receptor. The LPS of E. coli and other Gram-negative bacteria binds
to Toll-like receptor 4 (TLR4), triggering a potent cytokine cascade that can lead to
septic shock and death (Tapping et al, 2000). Flagellin, the main component of
flagella, can bind to TLR5, thereby activating interleukin (IL)-8 expression and an
inflammatory response (Hayashi et al., 2001).

2.1.3.2 CAMPYLOBACTER

Campylobacter infections are among the most common bacterial infections in

humans. They produce both diarrheal and systemic illnesses. In industrialized
regions, enteric Campylobacter infections produce an inflammatory sometimes
bloody diarrhea or dysentery syndrome (WHO, 2022). Campylobacter
jejuni usually is the most common cause of community acquired inflammatory
enteritis in developing regions diarrhea may be watery (WHO 2022). Infections
with Campylobacter like organisms can produce an enterocolitis/proctocolitis
syndrome in homosexual males who are at increased risk for Helicobacter
cinaedi and Helicobacter fennelliae infections (Totten et al., 1985). Bacteremia also
can occur with H fennelliae (WHO 2022). C jejuni infections also may produce
serious bacteremic conditions in individuals with AIDS. Most reported bacteremias
have been due to Campylobacter fetus infection. Campylobacter lari, which is
found in healthy seagulls also has been reported to produce mild recurrent diarrhea
in children. Campylobacter upsaliensis may cause diarrhea or bacteremia
whereas Campylobacter hyointestinalis which has biochemical characteristics
8
similar to those of C fetus, causes occasional bacteremia in immunocompromised
individuals. Campylobacter organisms also may be an important cause of traveler's
diarrhea especially in Thailand and surrounding areas of Southeast Asia. In a study
of American military personnel deployed in Thailand more than half of those with
diarrhea were found to be infected with Campylobacter species.

Campylobacter is said to be prevalent in food animals such as poultry, cattle, pigs,

sheep, and ostriches, as well as pets, including cats and dogs. The known routes
of Campylobacter transmission include fecal-oral, person-to-person sexual contact,
unpasteurized raw milk and poultry ingestion, and waterborne (through
contaminated water supplies), exposure to sick pets, especially puppies also has
been associated with Campylobacter outbreaks (WHO, 2022). Transmission
of Campylobacter organisms to humans usually occurs via infected animals and
their food products. Most human infections result from the consumption of
improperly cooked or contaminated foodstuffs. Chickens may account for 50% to
70% of human Campylobacter infections. Most colonized animals develop a
lifelong carrier state.Campylobacter has been found in shellfish (Rincé et al., 2018;
Wilson et al., 1996). The infectious dose is 1000-10,000
bacteria. Campylobacter infection has occurred after ingestion of 500 organisms by
a volunteer; however, a dose of fewer than 10,000 organisms is not a common
cause of illness. Campylobacter species are sensitive to hydrochloric acid in the
stomach. Conditions in which acid secretion is blocked, for example, by antacid
treatment or disease, predispose patients to Campylobacter infections.

Symptoms of Campylobacter infection begin after an incubation period of up to a

week. The sites of tissue injury include the jejunum, the ileum, and can extend to
involve the colon and rectum. C jejuni appears to invade and destroy epithelial
cells. C jejuni is attracted to mucus and fucose in bile, and the flagella may be
important in both chemotaxis and adherence to epithelial cells or mucus. Adherence
may also involve lipopolysaccharides or other outer membrane components. Such
adherence would promote gut colonization. PEB 1 is a superficial antigen that
appears to be a major adhesin and is conserved among C jejuni strains. Some strains
of C jejuni produce a heat-labile, cholera-like enterotoxin, which is important in
watery diarrhea observed in infections. Infection with the organism produces
diffuse, bloody, edematous, and exudative enteritis. The inflammatory infiltrate
consists of neutrophils, mononuclear cells, and eosinophils. Crypt abscesses
develop in the epithelial glands, and ulceration of the mucosal epithelium occurs.
Cytotoxin production has been reported in Campylobacter strains from patients
with bloody diarrhea. In a small number of cases, the infection is associated with
the hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura through
9
a poorly understood mechanism. Endothelial cell injury, mediated by endotoxins or
immune complexes, is followed by intravascular coagulation and thrombotic
microangiopathy in the glomerulus and the gastrointestinal mucosa.
Campylobacter species produce the bacterial toxin cytolethal distending toxin
(CDT), which produces a cell block at the G2 stage preceding mitosis. CDT inhibits
cellular and humoral immunity via the destruction of immune response cells and
necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions.
This leads to slow healing and results in disease symptoms (Smith and Bayles,
2006). In patients with HIV infection, Campylobacter infections may be more
common, may cause prolonged or recurrent diarrhea, and may be more commonly
associated with bacteremia and antibiotic resistance. C fetus is covered with a
surface S-layer protein that functions like a capsule and disrupts c3b binding to the
organisms, resulting in both serum and phagocytosis resistance. C jejuni infections
also show recurrence in children and adults with immunoglobulin deficiencies.
Acute C jejuni infection confers short-term immunity. Patients develop specific
immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA)
antibodies in serum; IgA antibodies also develop in intestinal secretions. The
severity and persistence of C jejuni infections in individuals with
AIDSand hypogammaglobulinemia indicate that both cell-mediated and humoral
immunity are important in preventing and terminating infection.The oral cavity
contains numerous Campylobacter species such as Campylobacter concisus, that
have been associated with a subtype of inflammatory bowel disease(Zhang, 2015;
Kaakoush et al., 2014). Campylobacter gracilis is associated with periodontal
disease (Zhang et al., 2021). pleuropulmonary disease and bacteremia.

Campylobacter infections are usually self-limited and rarely cause mortality. Exact
figures are unavailable, but occasional deaths have been attributed
to Campylobacter infections, typically in elderly or immunocompromised persons
and secondary to volume depletion in young, previously healthy individuals.
Campylobacter infections have no clear racial predilection they organisms are
isolated more frequently from males than females. Homosexual men appear to be at
increased risk for infection with atypical Campylobacter species such
as Helicobacter cinaedi and H fennelliae.

Campylobacter infections can occur in all age groups studies show a peak incidence
in children younger than 1 year and in persons aged 15 to 29 years. The age-specific
attack rate is highest in young children. In the United States, the highest incidence
of Campylobacter infection in 2010 was in children younger than 5 years and was
24.4 cases per 100,000 population (MEDLINE Link, 2011)However, the rate of
fecal cultures positive for Campylobacter species is greatest in adults and older
10
children. Asymptomatic Campylobacter infection is uncommon in adults. In
developing countries Campylobacter infection is very common in the first 2 years
of life. Asymptomatic infection is also more common (Das et al., 2021).

2.1.3.3 SALMONELLA SPP

Salmonellosis ranges clinically from the common Salmonella gastroenteritis
(diarrhea, abdominal cramps, and fever) to enteric fevers (including typhoid fever)
which are life-threatening febrile systemic illness requiring prompt antibiotic
therapy. Focal infections and an asymptomatic carrier state occur. The most
common form of salmonellosis is a self-limited, uncomplicated gastroenteritis.
Salmonellae are Gram-negative, flagellated, facultatively anaerobic bacilli
possessing three major antigens: H or flagellar antigen; O or somatic antigen; and
Vi antigen (possessed by only a few serovars). H antigen may occur in either or
both of two forms, called phase 1 and phase 2. The organisms tend to change from
one phase to the other. O antigens occur on the surface of the outer membrane and
are determined by specific sugar sequences on the cell surface. Vi antigen is a
superficial antigen overlying the O antigen; it is present in a few serovars, the most
important being S typhi.
Antigenic analysis of salmonellae by using specific antisera offers clinical and
epidemiological advantages. Determination of antigenic structure permits one to
identify the organisms clinically and assign them to one of nine serogroups (A-I),
each containing many serovars. H antigen also provides a useful epidemiologic tool
with which to determine the source of infection and its mode of spread.
Contaminated food is the major mode of transmission for non-typhoidal
salmonellae because salmonellosis is a zoonosis and has an enormous animal
reservoir. The most common animal reservoirs are chickens, turkeys, pigs, and
cows; dozens of other domestic and wild animals also harbor these organisms.
Because of the ability of salmonellae to survive in meats and animal products that
are not thoroughly cooked, animal products are the main vehicle of transmission.
The magnitude of the problem is demonstrated by the following recent yields of
salmonellae: 41% of turkeys examined in California, 50% of chickens cultured in
Massachusetts, and 21% of commercial frozen egg whites examined in Spokane,
WA.
The epidemiology of typhoid fever and other enteric fevers primarily involves
person-to-person spread because these organisms lack a significant animal
reservoir. Contamination with human feces is the major mode of spread, and the
usual vehicle is contaminated water. Occasionally, contaminated food (usually
handled by an individual who harbors S typhi) may be the vehicle. Plasmid DNA
fingerprinting and bacteria phage lysotyping of Salmonella isolates are powerful
epidemiologic tools for studying outbreaks of salmonellosis and tracing the spread
of the organisms in the environment.
11
In typhoid fever and non-typhoidal salmonellosis, two other factors have
epidemiologic significance. First, an asymptomatic human carrier state exists for
the agents of either form of the disease. Approximately 3% of persons infected
with S typhi and 0.1% of those infected with non-typhoidal salmonellae become
chronic carriers. The carrier state may last from many weeks to years. Thus, human
as well as animal reservoirs exist. Interestingly, children rarely become chronic
typhoid carriers. Second, use of antibiotics in animal feeds and indiscriminant use
of antibiotics in humans increase antibiotic resistance in salmonellae by promoting
transfer of R factors.
Salmonellosis is a major public health problem because of its large and varied
animal reservoir, the existence of human and animal carrier states, and the lack of a
concerted nationwide program to control salmonellae.
2.1.3.4 SHIGELLA SPP

Gram-negative, facultative anaerobes of the genus Shigella are the principal agents
of bacillary dysentery. This disease differs from profuse watery diarrhea, as is
commonly seen in choleraic diarrhea or in enterotoxigenic Escherichia
coli diarrhea, in that the dysenteric stool is scant and contains blood, mucus, and
inflammatory cells. In some individuals suffering from shigellosis, moderate
volume diarrhea is a prodrome or the sole manifestation of the infection. Bacillary
dysentery constitutes a significant proportion of acute intestinal disease in the
children of developing countries, and this infection is a major contributor to stunted
growth of these children. Shigellosis also presents a significant risk to travelers
from developed countries when visiting in endemic areas, and sporadic food or
water-borne outbreaks occur in developed countries (Bennish et al., 1992).

The pathogenic mechanism of shigellosis is complex involving a possible

enterotoxic and/or cytotoxic diarrheal prodrome, cytokine-mediated inflammation
of the colon, and necrosis of the colonic epithelium (Butler et al., 1986). The
underlying physiological insult that initiates this inflammatory cascade is the
invasion of Shigella into the colonic epithelium and the lamina propria. The
resulting colitis and ulceration of the mucosa result in bloody, mucoid stools, and/or
febrile diarrhea. Humans are the primary reservoir of Shigella species, with captive
subhuman primates as accidental hosts. In developing countries with prevailing
conditions of inadequate sanitation and overcrowded housing, the infection is
transmitted most often by the excreta of infected individuals via direct fecal-oral
contamination. Flies may contribute to spread from feces to food. The most
common species, S dysenteriae and S flexneri, are also the most virulent. In
developed countries, sporadic common source outbreaks, predominantly
involving S sonnei, are transmitted by uncooked food or contaminated water. The
latter outbreaks usually involve semipublic water systems such as those found in
camps, trailer parks, and Indian reservations. Direct fecal-oral spread can also occur
in institutional environments such as child day-care centers. Mental hospitals, and
nursing homes. Homosexual men are also at increased risk for direct transmission
12
of Shigella flexneri infections, and chronic, recrudescent illness complicating HIV
infection has been reported (Keusch and Bennish, 1991).
2.1.3.5 STAPHYLOCOCCUS
Bacteria in the genus Staphylococcus are pathogens of man and other mammals.
Traditionally they were divided into two groups on the basis of their ability to clot
blood plasma (the coagulase reaction). The coagulase-positive staphylococci
constitute the most pathogenic species S aureus. The coagulase-negative
staphylococci (CNS) are now known to comprise over 30 other species. The CNS
are common commensals of skin, although some species can cause infections. It is
now obvious that the division of staphylococci into coagulase positive and negative
is artificial and indeed, misleading in some cases. Coagulase is a marker for S
aureus but there is no direct evidence that it is a virulence factor. Also, some natural
isolates of S aureus are defective in coagulase. Nevertheless, the term is still in
widespread use among clinical microbiologists. Because S aureus is a major cause
of nosocomial and community-acquired infections, it is necessary to determine the
relatedness of isolates collected during the investigation of an outbreak. Typing
systems must be reproducible, discriminatory, and easy to interpret and to use. The
traditional method for typing S aureus is phage-typing. This method is based on a
phenotypic marker with poor reproducibility. Also, it does not type many isolates
(20% in a recent survey at the Center for Disease Control and Prevention), and it
requires maintenance of a large number of phage stocks and propagating strains and
consequently can be performed only by specialist reference laboratories.
Many molecular typing methods have been applied to the epidemiological analysis
of S aureus, in particular, of methicillin-resistant strains (MRSA). Plasmid analysis
has been used extensively with success, but suffers the disadvantage that plasmids
can easily be lost and acquired and are thus inherently unreliable. Methods designed
to recognize restriction fragment length polymorphisms (RFLP) using a variety of
gene probes, including rRNA genes (ribotyping), have had limited success in the
epidemiology of MRSA. In this technique the choice of restriction enzyme used to
cleave the genomic DNA, as well as the probes, is crucial. Random primer PCR
offers potential for discriminating between strains but a suitable primer has yet to be
identified for S aureus. The method currently regarded as the most reliable is pulsed
field gel electrophoresis, where genomic DNA is cut with a restriction enzyme that
generates large fragments of 50-700 kb.
2.1.3.6 LISTERIA MONOCYTOGENES
The gram-positive bacterium Listeria monocytogenes is a ubiquitous, intracellular
pathogen which has been implicated within the past decade as the causative
organism in several outbreaks of foodborne disease. Listeriosis, with a mortality
rate of about 24%, is found mainly among pregnant women, their fetuses, and
immunocompromised persons, with symptoms of abortion, neonatal death,
septicemia, and meningitis. Epidemiological investigations can make use of strain-
typing procedures such as DNA restriction enzyme analysis or electrophoretic
13
enzyme typing. The organism has a multifactorial virulence system, with the thiol-
activated hemolysin, listeriolysin O, being identified as playing a crucial role in the
organism's ability to multiply within host phagocytic cells and to spread from cell to
cell. The organism occurs widely in food, with the highest incidences being found
in meat, poultry, and seafood products. Improved methods for detecting and
enumerating the organism in foodstuffs are now available, including those based on
the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction.
As knowledge of the molecular and applied biology of L. monocytogenes increases,
progress can be made in the prevention and control of human infection.
2.1.3.7 VIBRIO
Vibrio is a genus of ubiquitous bacteria found in a wide variety of aquatic and
marine habitats; of the >100 described Vibrio spp., ~12 cause infections in humans.
Vibrio cholerae can cause cholera, a severe diarrhoeal disease that can be quickly
fatal if untreated and is typically transmitted via contaminated water and person-to-
person contact. Non-cholera Vibrio spp. (for example, Vibrio parahaemolyticus,
Vibrio alginolyticus and Vibrio vulnificus) cause vibriosis - infections normally
acquired through exposure to sea water or through consumption of raw or
undercooked contaminated seafood. Non-cholera bacteria can lead to several
clinical manifestations, most commonly mild, self-limiting gastroenteritis, with the
exception of V. vulnificus, an opportunistic pathogen with a high mortality that
causes wound infections that can rapidly lead to septicaemia. Treatment for Vibrio
spp. infection largely depends on the causative pathogen: for example, rehydration
therapy for V. cholerae infection and debridement of infected tissues for V.
vulnificus-associated wound infections, with antibiotic therapy for severe cholera
and systemic infections. Although cholera is preventable and effective oral cholera
vaccines are available, outbreaks can be triggered by natural or man-made events
that contaminate drinking water or compromise access to safe water and sanitation.
The incidence of vibriosis is rising, perhaps owing in part to the spread of Vibrio
spp. favoured by climate change and rising sea water temperature.

2.1.3.8 SPORE FORMERS

Bacterial species have different coping mechanisms with selective harsh
environmental conditions. One of the most common coping mechanisms for
bacteria is forming spores to protect themselves against ecological degrading
agents. Bacterial spores are the most dormant form of bacteria since they exhibit
minimal metabolism and respiration as well as reduced enzyme production.
Typically, Gram-positive bacteria are best known for producing intracellular spores
called endospores as a survival mechanism. Endospores are highly retractile and
thick-walled structures formed inside the bacterial cells. It is most common
for Bacillus species as well as Clostridium species to create endospores. (Hariram
14
and Labbé, 2015). B. cereus is a member of the Bacillus species and is well known
for its ability to cause food borne illness as a result of its spores surviving various
temperatures. Similarly, C. perfringens spores are acid-soluble proteins that show
high resistance to chemicals and heat.(Paredes-Sabja., 2008)
Endospores can resist inactivation from ethanol treatment. (Palombo, 2020) They
also can survive high temperatures for up to 150°C, making specific Gram-positive
species heat resistant. Further, bacterial spores can show typical viability signs at
temperatures near the absolute zero. Endospores are resistant to the chemical
agents, e.g., triphenylmethane dyes, and can even protect the bacterial cells against
ultraviolet radiation, extreme pH gradients, drought, and nutrition depletion.
Endospores germinate back into vegetative cells (an active bacterial cell that
undergoes metabolism) when surrounding environmental conditions favor bacterial
growth and reproduction. Several stimulants revert bacterial cells to their active
vegetative cells, such as optimal close-to-body temperature and diffusion of
nutrients and water through bacterial cell walls through alteration of their surface
tension.
The process of spore formation is a multistep process. It starts from replication of
the bacterial DNA, followed by the formation of the forespore, which is, by
definition, pinching of the cellular plasma membrane between the replicated
chromosome. Then, a cortex forms between the inner and outer membrane by
extending the second cellular membrane to enclose the forespore with calcium and
dipicolinic acid. Finally, the external spore coat surrounds the endospore before its
release (Koser and McClelland, 1917).
Microscopic examination to delineate the morphology of endospores involves
differential staining processes such as malachite green and fluorescence staining
techniques. Staining dormant bacterial samples with malachite green as the primary
stain and safranine as the counteract stain results in the appearance of green oval
endospores enclosed inside pink vegetative bacterial cells (D'Incecco et al.,
2018). There are different locations of the endospores inside the bacterial cell. For
instance, central endospores are located in the middle of the bacterial cell, while the
terminal endospore appears at the end. There is also a subterminal type of
endospores that appears between the middle and the end of the cell (de Andrade
Cavalcante et al., 2019).
Despite their sturdy and resistant nature to environmental threats, endospores can
get affected by certain eradication factors. During the 17th century, John Tyndall, a
famous European physicist, discovered Tyndallization. The latter is the process of
heating liquids and objects at a temperature of 80 to 100°C for 30 minutes; then, the
sample is incubated. The procedure is repeated for three consecutive days. The
principle behind successive heating for three days is that heating endospores for the
first-time results in reverting them into vegetative cells killed through repetitive
heat in the second and third days (Gould, 2006).

15
2.1.4 ANTI MICROBIAL SENSITIVITY TEST
The majority of infectious diseases are bacterial in origin. With the discovery of
laboratory methods to grow these microorganisms using an appropriate growth
medium known as “culture” determining the sensitivity and resistance of specific
pathogens to a wide range of antimicrobial agents becomes necessary so that
healthcare providers can immediately institute proper treatment regimens to their
patients (Lagier et al., 2015).
Antimicrobial susceptibility testing (AST) is a laboratory procedure performed by
medical technologists (clinical laboratory scientists) to identify which antimicrobial
regimen is specifically effective for individual patients. On a larger scale, it aids in
the evaluation of treatment services provided by hospitals, clinics, and national
programs for the control and prevention of infectious diseases. Recently,
researchers have had to implement continuous surveillance activities for resistance
patterns due to the mutations in bacterial DNA (Sawatzky et al., 2015; Graham et
al., 1985).
Clinical laboratories currently employ several methods depending on the laboratory
test menu that they provide. These approaches include the disk diffusion and
minimum inhibitory concentration (MIC) methods. Commercial systems also
became available across health centers and hospital facilities, utilizing both
phenotypic and genotypic characterization of bacterial resistance. While routine
antimicrobial susceptibility testing for gram-positive (e.g., Staphylococcus aureus)
and gram-negative bacteria (e.g., Pseudomonas aeruginosa) are commonly
available in peripheral laboratories, drug susceptibility testing (DST)
for Mycobacterium tuberculosis are usually carried out within more complex
facilities like reference laboratories. Despite the differences in the techniques for
susceptibility tests, all laboratories must be critical on each step of the sampling and
testing process so that test results are obtainable with consistently high levels of
accuracy and reliability.

2.1.5 SOURCES OF CONTAMINATION

Fruits can become contaminated with harmful bacteria, viruses, and parasites at
various stages, from farm to table. Some common sources of contamination include:

a. Animal feces: Animal waste can contain harmful pathogens like E. coli,
Salmonella, and Listeria which can contaminate fruits during farming,
processing, or transportation.

16
 b. Human handling: Poor hygiene practices by farm workers, packers, or
handlers can transfer pathogens from their hands to fruits.

c. Contaminated water: Irrigation water, washing water, or ice used for

cooling can contain harmful pathogens, which can spread to fruits.

d. Soil and environment: Fruits can become contaminated with pathogens

present in the soil, decaying matter, or environmental sources.

e. Wildlife and pests: Animals like birds, deer, or rodents, as well as pests like
insects or worms, can carry pathogens that can contaminate fruits.

f. Farm equipment and tools: Unclean or unsanitized equipment can transfer

pathogens to fruits during harvesting, pruning, or processing.

g. Packaging materials: Contaminated packaging materials, like boxes or bags,

can transfer pathogens to fruits during storage or transportation.

h. Transportation vehicles: Uncleaned or unsanitized vehicles can spread

pathogens to fruits during transportation.

i. Storage facilities: Unclean or unsanitized storage facilities can allow

pathogens to spread to fruits during storage.

j. Cross-contamination: Fruits can become contaminated by coming into

contact with other contaminated fruits, surfaces, or objects.

2.2 THEORITICAL FRAMEWORK

This study relies on the following theories for its notional underpinning:

2.2.1 Storage Conditions Theory

The Storage Conditions Theory states that the microbial quality of fresh-cut fruits
is significantly impacted by various factors during storage, including temperature,
humidity, atmosphere, time, and handling and sanitation practices. This theory has
been extensively researched and described by scientists such as Dr. Maria Rivera-
Lopez and Dr. Jeffrey K. Brecht.

Temperature is a critical factor in maintaining the microbial quality of fresh-cut

fruits. Refrigeration temperatures between 32°F and 50°F (0°C and 10°C) slow
down microbial growth, while temperatures above 50°F (10°C) accelerate growth
17
(Rivera-Lopez, 2019). For example, a study on fresh-cut apples found that storage
at 39°F (4°C) resulted in significantly lower microbial counts compared to storage
at 50°F (10°C) (Brecht, 2017).

Humidity also plays a crucial role in microbial growth on fresh-cut fruits. High
humidity (above 80%) fosters microbial growth, while low humidity (below 60%)
inhibits growth (Avena-Bustillos et al., 2017). Modified atmosphere packaging
(MAP) with low oxygen and high carbon dioxide levels can also inhibit microbial
growth, as demonstrated in a study on fresh-cut strawberries (Zhang et al., 2020).

Time is another essential factor in maintaining microbial quality. Longer storage

times increase the risk of microbial contamination and growth (Rivera-Lopez,
2019). Proper handling and sanitation practices are also critical in preventing
microbial contamination, as demonstrated in a study on fresh-cut melons (Parnell et
al., 2017).

In conclusion, the Storage Conditions Theory emphasizes the importance of optimal

storage conditions, including temperature, humidity, atmosphere, time, and
handling and sanitation practices, in maintaining the microbial quality of fresh-cut
fruits. By understanding and controlling these factors, the risk of microbial
contamination and growth can be minimized, ensuring the safety and quality of
fresh-cut fruits for consumption.

2.2.2 Handling Practices Theory: The Handling Practices Theory, as researched

and described by Lund (2011) and Saba (2017), emphasizes the importance of
proper handling techniques to prevent microbial contamination and growth on
fresh-cut fruits. Dr. Lund's study found that poor handling practices can lead to a
significant increase in microbial loads on fresh-cut fruits (Lund, 2011). Dr. Saba's
research demonstrated that proper handling practices, including gentle handling and
proper packaging, can reduce microbial contamination on fresh-cut apples (Saba,
2017). Similarly, Rivera-Lopez's study (2019) highlighted the importance of proper
handling and storage practices to maintain the microbial quality of fresh-cut fruits.
Brecht's research (2017) also emphasized the role of handling practices in
preventing microbial growth on fresh-cut fruits.

2.2.3 Contamination Theory

The Contamination Theory states that the microbial quality of fresh-cut fruits is
compromised by various sources of contamination, including:

- Field contamination: Soil, water, and debris on the fruit surface

- Harvesting contamination: Human handling, tools, and equipment

- Processing contamination: Cutting, washing, and packaging processes

18
- Storage contamination: Facilities, equipment, and handling practices

- Transportation contamination: Vehicles, containers, and handling practices

Researchers such as Lund (2011) and Saba (2017) have identified these sources of
contamination as critical points for microbial contamination, highlighting the
importance of implementing effective control measures to prevent contamination
and ensure the microbial quality of fresh-cut fruits.

Lund (2011) found that field contamination was a significant source of microbial
contamination on fresh-cut fruits (Lund, 2011). Dr. Saba's research demonstrated
that proper handling and sanitation practices during processing and storage can
reduce microbial contamination on fresh-cut apples (Saba, 2017).

The Contamination Theory emphasizes the need for a comprehensive approach to

preventing microbial contamination, from field to table, to ensure the safety and
quality of fresh-cut fruits.

2.2.4 Supply Chain Theory (Womack and Jones, 1996)

The microbial quality of fruits is impacted by the length and complexity of the
supply chain, with more handling and storage points increasing the risk of
contamination. The Lean Supply Chain Theory, developed by Womack and Jones
(1996), defines value from the customer's perspective and focuses on creating
value-added activities, mapping the value stream to identify waste and opportunities
for improvement. It aims to create a future state with minimal waste and maximum
efficiency, reducing waste such as overproduction, waiting, transportation,
inventory, motion, and defects. By improving flow and implementing pull
production based on customer demand, organizations can achieve reduced lead
times, lower inventory levels, improved quality, increased customer satisfaction,
and cost savings, with continuous improvement driving ongoing evaluation and
optimization of the supply chain.

2.2.5 Seasonality Theory

The microbial quality of fruits varies by season, with certain types of fruits more
prone to contamination during specific times of the year due to factors like weather
conditions and pest prevalence. The Seasonality Theory states that the microbial
quality of fresh-cut fruits is influenced by the time of year and seasonal variations
in environmental conditions, fruit maturity, and handling practices.

Researchers such as Rivera-Lopez (2019) and Brecht (2017) have found that:

- Fresh-cut fruits harvested during peak seasons (e.g., summer) tend to have higher
microbial loads due to warmer temperatures and higher humidity.

19
- Fruits harvested during off-peak seasons (e.g., winter) tend to have lower
microbial loads due to cooler temperatures and lower humidity.

- Seasonal variations in fruit maturity and ripeness can affect the susceptibility of
fruits to microbial contamination.

- Handling practices and storage conditions may vary by season, impacting

microbial quality.

For example Dr. Rivera-Lopez's study found that fresh-cut strawberries had higher
microbial counts during summer months (Rivera-Lopez, 2019). Dr. Brecht's
research showed that fresh-cut apples had lower microbial counts when harvested
during cooler months (Brechtt, 2017). The Seasonality Theory highlights the
importance of considering seasonal factors when implementing quality control
measures to ensure the microbial safety and quality of fresh-cut fruits.

2.3 EMPIRICAL STUDIES

The study on the microbiological quality of pre-cut fruits on sale in retail outlets in
Nigeria was conducted in 2010 by Chibugo Chukwu and his colleagues. The study
tested 150 pre-cut fruit samples including pineapples, paw-paw, and watermelon for
bacterial and parasite contamination. The results showed that 90.67% of the
samples (136 out of 150) were contaminated with bacteria. Escherichia coli (E. coli)
was the most prevalent bacteria found in 69 (46%) of the contaminated samples.
Other bacteria isolated from the samples included Staphylococcus aureus, Bacillus
spp., and Shigella spp. In this research no parasites were found in any of the
samples. The study highlights the risk of food borne illness from consuming pre-cut
fruits contaminated with harmful bacteria like E. coli. The authors emphasize the
need for enforcing good food hygiene practices to avoid contamination of pre-cut
fruits.

Another study conducted by Okoro and colleagues (2018), assessed the

microbiological quality of sliced pineapple fruits vended in Abakaliki Metropolis
and found a 20% prevalence of Salmonella species and a 70% prevalence of
Escherichia coli. The researchers tested 100 sliced pineapple samples from various
vendors and detected Salmonella and E. coli using standard microbiological
methods. The study revealed that Salmonella species showed resistance to
antibiotics like ceftriaxone, nitrofurantoin, and amoxicillin, ranging from 50-100%,
while E. coli showed resistance to the same antibiotics, ranging from 28.6-100%.
The findings highlight the risk of multidrug-resistant pathogens in fruit vending
practices and emphasize the need for proper food safety practices to prevent
foodborne illnesses.

20
Microbial quality of sliced and packaged fruits in polyethylene sold in Port
Harcourt: This study found that Staphylococcus aureus was the most frequently
isolated bacteria, followed by Shigella spp, Escherichia coli and Bacillus spp
A meta-analysis of 25 studies on ready-to-eat foods in Bayelsa State, Nigeria, found
that Staphylococcus aureus was the most commonly isolated microbe, present in
43.2% of the 1,250 food samples analyzed. Escherichia coli (E. coli) was isolated
from 29.6% of the samples, followed by Aspergillus species (25.6%), Bacillus spp.
(17.2%), Salmonella spp. (12.8%), Shigella spp. (8.8%), Listeria monocytogenes
(5.6%), and Candida spp. (4.8%). The study highlights the high prevalence of
microbial contaminants in ready-to-eat foods in the region, posing a significant risk
to public health. Improved food handling and preparation practices, as well as
regular monitoring and enforcement of food safety regulations, are necessary to
reduce the risk of food borne illnesses.
The study conducted by Moyo et al. (2020), evaluated the hygienic practices and
microbiological quality of street-vended fruit salads in Morogoro, Tanzania. The
researchers collected 100 fruit salad samples from various street vendors and tested
them for microbiological contaminants. It revealed that all 100 fruit salad samples
were contaminated with coliforms, indicating poor hygiene and fecal
contamination, with a coliform count ranging from 10^3 to 10^9 CFU/g, exceeding
the acceptable limit of 10^2 CFU/g. Escherichia coli (E. coli) was isolated from
80% of the samples, with a count ranging from 10^2 to 10^8 CFU/g, and other
microbiological contaminants detected included Staphylococcus aureus, Bacillus
spp., and Aspergillus spp. The study also observed poor hygienic practices among
street vendors, including inadequate hand washing, inadequate storage, and poor
handling of fruits and utensils, highlighting the need for improved hygienic
practices, regular testing, and education of street vendors to ensure the safety of
fruit salads sold to consumers.

2.4 SUMMARY OF LITERATURE REVIEWED

The chapter reviewed the literature based on the three broad headings; major
concepts of the study, theoretical framework, empirical studies. Under major
concepts; Effects of polyethylene bags for packaging of sliced fruits by street
vendors in Nigeria, Microbiological quality of pre-cut fruits sold in retail outlets in
Nigeria, Bacterial pathogens associated with fruit contamination, Antimicrobial
sensitivity test and Sources of contamination were discussed. The theories include;
Storage Conditions theory, Contamination theory, Handling practices theory,
Supply chain theory and Seasonality theory.

Some empirical studies that relates to this study were reviewed. To the best
knowledge of the researcher, little or no work has been done to reduce the risk of
harming consumers of fresh cut fruits sold in Eziobodo and Ihiagwa market.
Emphasis should be made on the need for enforcing good food hygiene practices to
avoid contamination of pre-cut fruits.
21
 CHAPTER THREE

METHODOLOGY

MATERIALS AND METHODS

3.1 SAMPLE COLLECTION AND PREPARATION

Ten different samples of packaged, sliced watermelon were randomly purchased

from ten different street vendors in Owerri metropolis and placed in plastic sterile
bags. The samples were immediately transported in a cool container to the
laboratory and analyzed within 1 - 2 hour after collection. MacConkey agar (Antec
Diagnostics Products, UK), Nutrient agar and Sabouraund Dextrose agar
(International Diagnostics Group, UK) were prepared and used for the isolation and
enumeration of bacteria and fungi. The various media were prepared, as specified in
the manufacturer’s manuals. Isolation and enumeration of bacteria and fungi a
sterile knife was used to cut 1.0 g from each watermelon sample and it was then
homogenized using a sterile mortar and pestle. The MacConkey agar and Nutrient
agar plates were incubated at 37 oC for 48 h to obtain the total viable bacterial
counts, while the Sabouraund Dextrose agar plates were incubated at 28oC for 72
hour to obtain the fungal counts. These experiments were repeated on three
different occasions for each of the samples and the average values recorded.
Discrete colonies were streaked onto fresh agar to obtain pure cultures of the
different isolates. Isolates were maintained on Nutrient agar slants and stored at 4oC
for further tests. Biochemical tests, performed on the bacterial isolates included
Gram staining, catalase activity, sugar utilization, methyl red and Voges Proskauer
tests coagulase activity, citrate utilization and motility (Baker and Breach, 1980).
Fungal identification and classification were based on their macroscopic and
microscopic features. The macroscopic features were based on the shape, colour
and physical appearance of the colonies, while the microscopic characterizations
were carried out according to the methods and identification keys of Fawole and
Oso (1986).

3.2. Preparation of Media and Diluents

Nutrient agar (NA), Salmonella Shigella Agar (SSA), Cetrimide Agar (CTA),
Mannitol Salt Agar (MSA), Eosin Methylene Blue Agar (EMBA) and Potato
Dextrose agar (PDA) were prepared according to manufacturer’s specification.
Nutrient agar was used in the isolation of heterotrophic bacteria (Cheesbrough,
2000).

22
Distilled water (DW) used as diluents was prepared by dispensing 90 m l and 9
mlportion into bijou bottles. Both diluents and media were sterilized in an autoclave
at 1210C for 15mins.

3.3 Microbiological Analyses

Ten grams (10 g) of the samples (watermelon and pineapple) were macerated in a
stomacher blender and homogenously in 90 ml of sterile DW. One milliliter (1 ml)
of this suspension was serially diluted decimally by transferring 1 ml from each
tube until the required dilution was obtained. One-tenth milliliter (0.1 ml) of
appropriate dilution was inoculated into the pre-sterilized and surface dried media.
Inocula were spread evenly to ensure discrete and countable colonies. Plates were
incubated at ambient temperature for 24 - 48 hours for heterotrophic bacteria
(Sharma, 2000; Beishir, 1987). Potato Dextrose Agar medium was incubated at
ambient temperature for 3-5 days.

3.4 Determination of Microbial Population

Colony counts obtained on the media were expressed as colony forming units per
gram (CFU/ml) to obtain total population (Harrigan and McCance, 2000).

3.5 Characterization and Identification of Microbial Isolates

Microbial isolates were characterized based on cultural (colonial), microscopic and

biochemical methods with reference to standard manuals. The identities of the
isolates were cross-matched with reference to standard manuals for the
identification of bacteria (Barnett and Hunter, 1997; Buchanan and Gibbon, 2000;
Harrigan and McCance, 2000).

3.5.1 Microscopic Characterization

3.5.1.1 Gram Staining Test

The Gram staining technique was used for the bacterial isolates as described by
Cheesbrough (2000). A smear of the isolate was made on grease free glass slide
with a drop of water and allowed to dry. The smear was fixed by mild heating,
23
flooded with crystal violet and allowed to stand for 30 seconds. The crystal violet
was rinsed off with water; Lugol’s iodine was added and allowed to stand for 30
seconds. This was washed off with water and acid alcohol, till discoloration. It was
counter stained with Safranin for 10 seconds and rinsed with water. The wet slide
was allowed to air dry. A drop of oil immersion was added on the slide and viewed
using X100 objective lens of the microscope.

3.5.1.2 Spore Staining Test

The spore stain was used to confirm the presence of spores when indicated in the
Gram stain. Isolates were heat fixed on a slide and flooded with 5% malachite
green. It was steamed for 3 minutes (without allowing it to boil), dried and cooled.
It was then rinsed off and stained with Safranin for 30 seconds. This was rinsed,
dried with filter paper and viewed under the microscope using oil immersion tens.
The positive spores showed green while the vegetative cells were stained pink.
3.5.1.3 Motility Test

This test was used to determine the motility of bacteria isolated. The test was
carried out on a semi-solid agar medium in which motile bacteria swarm and gave a
diffuse spreading growth. The medium was dispensed into test tubes, sterilized and
allow to set in an upright position. It was then inoculated using an inoculation
needle by stabbing it into the medium in the test tube. This was incubated at 37°C
for 24 hours. Diffuse growth from the straight line of inoculation was recorded as
positive result (Cheesbrough, 2000).

3.5.2 Biochemical Characterization of Bacteria isolates

Microorganisms that were not identified by the colonial and microscopic

characteristics were further subjected to few biochemical tests described by
Cheesbrough (2000) and Beishir (1987).

24
3.5.2.1 Catalase Test

The enzyme catalase is present in most cytochrome containing aerobic and

facultative anaerobic bacteria. Catalase has one of the highest turnover numbers of
all enzymes such that one molecule of catalase can convert millions of molecules of
hydrogen peroxide to water and oxygen in a second. Catalase activity can be
detected by adding the substrate H₂O₂ to an appropriately incubated (18-24 hours)
tryptic soy agar slant culture. Organisms which produce the enzyme breakdown the
hydrogen and the resulting O₂ production produces bubbles in the reagent drop
indicating a positive test. Organisms lacking the cytochrome system also lack the
catalase enzyme and are unable to breakdown peroxide into O₂ and water and are
catalase negative.

3.5.2.2 Coagulase Test

Coagulase is enzymes that clot blood plasma by a mechanism that is similar to

normal clotting. The coagulase test identifies whether an organism produces this
exoenzyme. This enzyme clots the plasma component of blood. The only significant
disease causing bacteria of humans that produce coagulase are Staphylococcus
aureus. Thus this enzyme is a good indicator of S. aureus. In the test, the sample is
added to rabbit plasma and held at 37⁰C for a specified period of time. Formation of
clot within four hours is indicated as positive result and indicative of a virulent
Staphylococcus aureus strain. The absence of coagulation after 24 hours of
incubation is a negative result indicative of an avirulent strain.

3.5.2.3 Oxidase Test

Oxidase test is an important differential procedure that should be performed on all

gram negative bacteria for their rapid identification. The test depends on the ability
of certain bacteria to produce indophenol blue from the oxidation of dimethyl-p-
phenylenediamine and ∞-naphthol. This method uses N, N-dimethyl-p-
phenylenediamine oxalate in which all Staphlococci are oxidase negative. In the
presence of the enzyme cytochrome oxidase (gram negative bacteria) the N, N-
25
dimethhyl-p-phenylenediamine oxalate and ∞-naphthol react to indophenol blue.
Pseudomonas aeruginosa is an oxidase positive organism.

3.5.2.4 Sugar Fermentation/Oxidation

This test is used to differentiate between bacteria groups that oxidize carbohydrate
such as members of Enterobacteriaceae. One milliliter (1ml) of 10% glucose,
maltose, lactose, fructose, mannitol, and sucrose were separately under aseptic
conditions transferred into duplicate tubes containing 9ml of sterile Hugh and
Leifson’s medium to obtain a final concentration of 1% of each of sugar. The tubes
were stab-inoculated in duplicates while two uninoculated tubes serve as control.
Vaseline was used to cover one set of the duplicate tubes, one control to discourage
oxidative utilization of sugar. All tubes were incubated at 37oc for 48h. After the
incubation, they were observed for acid production in the culture. Yellow
colouration indicates acid production in the open tubes only suggesting oxidative
utilization of the sugar while acid production in the sealed tubes suggests a
fermentative reaction.
3.5.2.5 Hydrogen Sulphide Production (H2S) test

The test isolates were aseptically inoculated into a tube containing triple sugar iron
agar started by stabbing the agar to the bottom and streaking the surface of the slant.
The inoculated tube was incubated at 37oc for 72h and was examined daily. Black
precipitation and yellow colouration was checked for. Black precipitate indicates
H2S production and yellow colouration for sucrose, lactose and glucose
fermentation.

3.5.2.6 Urease Test

Urease Agar slant in McCartney bottle was inoculated with the bacteria isolate at
30oC for 4 hours and then overnight. A pink colour in the medium indicated a
positive result.

26
3.5.2.7 IMViC Test

This test consists of four different test; they are Indole production, Methyl-Red test,
Voges Proskaeur test and Citrate utilization test. This test is specifically designed to
determine the physiological properties of microorganism. They are especially useful
in the differentiation of Gram-negative intestinal bacilli, particularly Escherichia
coli and the Enterobacter-Klebsiella group.

Indole Test

This test demonstrates the ability of certain bacteria to decompose the amino acid-
Tryptophan to Indole. The bacteria isolates were inoculated into the medium and
incubated at 37°C for 48 hours. At the end of incubation period, 3 drops of kovac’s
reagents (see appendix) was added and then shaken. A red colour ring at the
interface of the medium denotes a positive result.

Methyl red and Voges-Proskaeur test must be considered together since they are
physiologically related. Opposite test is usually obtained from the MR and VP terst,
that is, MR+, VP-, or MR-, VP+.

Methyl red test was performed to demonstrate the capacity of different organisms to
produce acid from the fermentation of sugar (dextrose). Methyl-red positive
organisms produce a red colouration when five drops of methyl-red indicator is
added into 48h old MR-VP broth culture.

The Voges-Proskaeur test demonstrates the ability of organisms to produce acetoin

from glucose metabolism. Some organisms metabolise glucose to produce pyruvic
acid which is further broken down to yield Butane-diol and acetyl-methyl carbinol
as an intermediate product.

Into one milliliter of the culture add one milliliter of six percent alcoholic solution
of alpha-naphtol and one milliliter of 16% KOH and stand for 15-20 minutes.
Development of red to pink colour is a positive test.

27
Citrate Utilization Test

This is one of the several techniques used to assist in the identification of

Enterobacteria. Principle of the test is based on the ability of an organism to use
citrate as its only source of carbon. The test was carried out using Simmon’s citrate
agar.

The slopes of the media were prepared in bijou bottles as recommended by the
manufacturers. A sterile straight wire was used to the slope with a saline suspension
of the test organisms before stabbing the butt. The bottles are incubated at 35 oC for
48 h. Bright blue colours in the medium means positive test while no change in
colour of medium indicates negative citrate test (Cheesbrough, 2000).

3.5.3. Wet Mount Preparation for Identification of Moulds

A tiny portion of the mould was placed on a clean grease free slide flooded with a
dye (lactophenol cotton blue). A cover slip was placed on top of the mould mounted
on the slide. Excess fluid of the dye was removed with a blotting paper. Slide was
viewed under low power magnification of 40x. The structure under the microscope
revealed the structures of the mould.

28
 CHAPTER FOUR
RESULTS

Table 4.1: Total Counts and Colonial Characteristics of Bacterial Isolates on

Nutrient Agar

Table4.1 shows that in the watermelon samples WM3 has the highest microbial load of
(2.44 x106) followed by sample WM4 (1.6 x 106), WM2 (1.02 x 103), MW1 (1.86 x 104)
and WM5 (2.8 x 104). The most probable organisms where Pseudomonas and
Enterococcus spp. while in the Pineapple samples PN2 has the highest microbial load of
(1.89 x104) followed by sample PN3 (1.49 x 104), PN1 (1.49 x 104), PN4 (9.2 x 103) and
PN5 (1.8 x 103). The most probable organisms where Pseudomonas, Bacillus sp and
Enterococcus spp.

Sample Total Colony Colonial Characteristics Most Probable

code counts Types Identity
(CFU/g)
WM1 1.86 x 104 WM1a Moist and shiny bluish green Pseudomonas
WM1b colonies sp
Small smooth moist and shiny Enterococcus
cream colonies sp
WM2 1.02 x 106 PM2a Moist and shiny bluish green Pseudomonas
WM2b colonies sp
Small smooth moist and shiny Enterococcus
cream colonies sp
WM3 2.44 x 106 WM3a Moist and shiny bluish green Pseudomonas
WM3b colonies sp
Small smooth moist and shiny Enterococcus
cream colonies sp

29
WN4 1.69 x 106 WM4a Moist and shiny bluish green Pseudomonas sp
WM4b colonies Enterococcus sp
Small smooth moist and shiny
cream colonies
MW5 2.81 x 104 WM5a Moist and shiny bluish green Pseudomonas sp
WM5b colonies Enterococcus sp
Small smooth moist and shiny
cream colonies
PN1 1.49 x 104 PN1X Moist and shiny bluish green Pseudomonas sp
PN1Y colonies Bacillus sp
Dull and dry flat serrated
PN1Z cream colonies Enterococcus sp
Small smooth moist and shiny
cream colonies
PN2 1.89 x 104 PN2X Pseudomonas sp
PN2Y Bacillus sp
PN2Z Enterococcus sp
PN3 1.49 x 104 PN3X Pseudomonas sp
PN3Y Bacillus sp
PN3Z Enterococcus sp
PN4 9.2 x 103 PN4X Pseudomonas sp
PN4Y Bacillus sp
PN4Z Enterococcus sp
PN5 1.8 x 103 PN5X Pseudomonas sp
PN5Y Bacillus sp
PN5Z Enterococcus sp

Sample codes of fruits from retail stores: WM1=Watermelon, WM2=Watermelon,

M3=Watermelon,WM4=Watermelon,M5=Watermelon,PN1=Pineapple,
PN2=Pineapple, PN3=Pineapple, N4=Pineapple, PN5=Pineapple.
30
 Table 2: Total Counts and Colonial Characteristics of Fungal Isolates on Potato
Dextrose Agar
Table 4.2 shows that in the watermelon samples, WM3 has the highest colony count of
(1.70 x105) followed by sample WM2 (1.44 x 105), WM5 (1.26 x 105), MW1 (1.24 x
104) and WM4 (1.11 x 104). While in the Pineapple samples PN1and PN4 has the
highest colony count of (2.01 x105) followed by sample PN3 (1.99 x 105), PN5 (1.22 x
105) and PN2 (1.20 x 105). The most probable organism was Saccharomyces
cerevisiae.

Sample code Total counts Colony Types Colonial Microscopic

(CFU/g) Characteristics Characteristics
WM1 1.24 x 105 WM1A Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies
WM2 1.44 x 105 Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies
WM3 1.70 x 105 Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies
WM4 1.11 x 105 Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies
WM5 1.26 x 105 Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies
PN1 2.01 x 105 Small circular Large Gram
moist and positive oval
shiny cream budding cells
colonies

31
Sample Total counts Colony Colonial Microscopic Most Probable
code (CFU/g) Types Characteristics Characteristics Identity

PN2 1.20 x 105 Small circular Large Gram Saccharomyces

moist and positive oval cerevisiae
shiny cream budding cells
colonies
PN3 1.99 x 105 Small circular Large Gram Saccharomyces
moist and positive oval cerevisiae
shiny cream budding cells
colonies
PN4 2.10 x 105 Small circular Large Gram Saccharomyces
moist and positive oval cerevisiae
shiny cream budding cells
colonies
PN5 1.22 x 105 Small circular Large Gram Saccharomyces
moist and positive oval cerevisiae
shiny cream budding cells
colonies

Sample codes of fruits from retail stores: WM1=Watermelon, WM2=Watermelon,

M3=Watermelon,WM4=Watermelon,M5=Watermelon,PN1=Pineapple,
PN2=Pineapple, PN3=Pineapple, N4=Pineapple, PN5=Pineapple.

32
 Table 4.3: Total Counts and Colonial Characteristics of Bacterial Isolates on
Mannitol Salt Agar.
Table 4.3 shows that in the watermelon samples, WM1 has the highest colony count of
(1.26 x104) followed by sample WM5 (1.21 x 104), WM4 (9.6 x 103), MW3 (7.3 x 104)
and WM2 (1.0 x 103). While in the Pineapple samples PN1 has the only colony count of
(2.1 x103), No growth was observed in PN2, PN3, PN4 and PN5. The most probable
organism was Staphylococcus sp.

Sample Total Colony Colonial Characteristics Most Probable

code counts Types Identity
(CFU/g)
WM1 1.26 x 104 WM1A Light pink smooth moist and Staphylococcus sp
slimy colonies
WM1B Small circular moist and Staphylococcus sp
shiny yellow colonies
WM2 1.0 x 103 WM2A Small circular moist and Staphylococcus sp
shiny yellow colonies
WM3 7.3 X 103 WM3A Light pink smooth moist and Staphylococcus sp
slimy colonies
WM1B Small circular moist and Staphylococcus sp
shiny yellow colonies
WM4 9.6 x 103 WM4A Small circular moist and Staphylococcus sp
shiny yellow colonies
WM5 1.21 x 104 WM5A Small circular moist and Staphylococcus sp
shiny yellow colonies
PN1 2.1 x 103 Small circular moist and Staphylococcus sp
shiny yellow colonies
PN2 No - - -
Growth
PN3 No - - -
Growth
PN4 No - - -
growth
PN5 No - - -
Growth

Sample codes of fruits from retail stores: WM1=Watermelon, WM2=Watermelon,

M3=Watermelon,WM4=Watermelon,M5=Watermelon,PN1=Pineapple,
PN2=Pineapple, PN3=Pineapple, N4=Pineapple, PN5=Pineapple.
33
 Table 4.4: Total Counts and Colonial Characteristics of Bacterial Isolates on
Cetrimide Agar
Table4. Shows that in the watermelon samples, WM3 has the highest colony count of
(8.4 x103) followed by sample WM2 (5.0 x 102), WM1 (3.0 x 102), MW4 (1.0 x 102),
No growth was found on sample WM5 (1.0 x 103). While in the Pineapple samples PN4
has the highest colony count of (3.0 x102) followed by PN1 with colony count of (1.0 x
102). No growth was observed in PN4, PN3 and PN2. The most probable organism was
Pseudomonas sp.

Sample Total Colony Colonial Characteristics Most Probable

code counts Types Identity
(CFU/g)
WM1 3.0 x 102 WM1x Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
WM2 5.0 x 102 WM2x Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
WM3 8.4 x 103 WM3x Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
WM4 1.0 x 102 WM4x Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
WM5 No - - -
growth
PN1 1.0 X 102 PN1x Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
PN2 - - - -
PN3 - - - -
PN4 3.0 X 102 PNx4 Smooth moist and shiny Pseudomonas sp
cream to blue green colonies
PN5 - - - -

Sample codes of fruits from retail stores: WM1=Watermelon, WM2=Watermelon,

M3=Watermelon,WM4=Watermelon,M5=Watermelon,PN1=Pineapple,
PN2=Pineapple, PN3=Pineapple, N4=Pineapple, PN5=Pineapple.

34
Table 4.5: Total Counts and Colonial Characteristics of Bacterial Isolates on Salmonella
Shigella Agar

Table5. Shows that in the watermelon samples, WM1 has the highest colony count of
(2.47 x104) followed by sample WM2 (2.21 x 104), WM3 (1.36 x 104), MW4 (5.5 x
103), No growth was found on sample WM5. While in the Pineapple samples PN2 has
the highest colony count of (1.18 x104) followed by PN4 with colony count of (7.7
3 2
x 10 ) then PN1 (8.1 x10 ). No growth was observed in PN3 and PN5. The most
probable organism was Shigella sp.
Sample Total Colony Colonial Characteristics Most Probable
code counts Types Identity
(CFU/g)
WM1 2.47 x 104 WM1a Moist and shiny black fish Salmonella sp
eye colonies
4
WM2 2.21 x 10 WM2a Moist and shiny black fish Salmonella sp
eye colonies
WM2b Small smooth moist and Shigella sp
shiny light pink colonies
4
WM3 1.36 x 10 WM3a Moist and shiny black fish Salmonella sp
eye colonies
3
WM4 5.5 x 10 WM4a Small smooth moist and Shigella sp
shiny light pink colonies
WM5 No WM5a Moist and shiny black fish Salmonella sp
growth eye colonies
PN1 8.1 X 102 PN1a Small smooth moist and Shigella sp
shiny light pink colonies
4
PN2 1.18 x 10 PN2a Small smooth moist and Shigella sp
shiny light pink colonies
Moist and shiny black fish Salmonella sp
eye colonies
PN3 No - - -
Growth
PN4 7.7 x 103 PN4a Small smooth moist and Shigella sp
shiny light pink colonies
PN5 No - - -
Growth
Sample codes of fruits from retail stores: WM1=Watermelon, WM2=Watermelon,
M3=Watermelon,WM4=Watermelon,M5=Watermelon,PN1=Pineapple,
PN2=Pineapple, PN3=Pineapple, N4=Pineapple, PN5=Pineapple.

35
Table 4.6. Dull and dry flat serrated grey colonies indicating Bacillus sp was found
in samples WM1, WM3, WM5 and PN1, PN2, PN5 while Small circular moist
pink colonies indicating Enterobacter sp was found in all the samples WM1,WM2,
WM3, WM5, PN1, PN2, PN3, PN4 and PN5.

Table 4.6: Total Counts and Colonial Characteristics of Bacterial Isolates on

Eosin Methylene Blue Agar
Sample Total counts Colony Colonial Characteristics Most Probable
code (CFU/g) Types Identity
WM1 1.71 x 104 WM1x Dull and dry flat serrated grey Bacillus sp
WM1y colonies Enterobacter
Small circular moist pink sp
colonies
3
WM2 2.1 x 10 WM2x Small circular moist pink Enterobacter
colonies sp
WM3 1.36 x 104 WM3x Dull ad dry flat serrated grey Bacillus sp
WM3y colonies Enterobacter
Small circular moist pink sp
colonies
3
WM4 5.5 x 10 WM4x Small circular moist pink Enterobacter
colonies sp
WM5 No growth WM5x Dull and dry flat serrated grey Bacillus sp
colonies Enterobacter
Small circular moist pink sp
colonies
2
PN1 6.6 x 10 PN1m Dull and dry flat serrated grey Bacillus sp
PN1n colonies Enterobacter
Small circular moist pink sp
colonies
4
PN2 1.18 x 10 PN2m Dull and dry flat serrated grey Bacillus sp
PN2n colonies Enterobacter
Small circular moist pink sp
colonies
PN3 No Growth PN3m Dull ad dry flat serrated grey - Bacillus sp
PN3n colonies Enterobacter
Small circular moist pink sp
colonies
36
PN4 7.7 x 103 PN4m Dull and dry flat serrated grey Bacillus sp
PN4n colonies Enterobacter
Small circular moist pink sp
colonies
PN5 No Growth PN5m Dull ad dry flat serrated grey - Bacillus sp
PN5n colonies Enterobacter
Small circular moist pink sp
colonies

37
Table7: Microscopic and Biochemical Characteristics of Bacteria isolated from
Samples

Spo Mot Gram Oxi Cat Coag In MR VP S L G M Identity of

rnx isolates

- - +S - + + - - + + + + + Staphylococcu
s aureus

- - +S - - - - + - + + + + Enterococcus
faecalis

+ + +R - + - - - + - - - - Bacillus
cereus

+ + +R - + - - - + - - - + Bacillus
subtilis

- + -R + + - - + - + - + + Pseudomonas
sp

- - +S - + - - + - + + + + Staphylococcu
s
haemolyticus

- - -R - + - - + - - + + - Enterobacter
sp

+: Positive; -: Negative; Spo: spore production; Mot: motility; Oxi: oxidase; Cat: catalase;
Coag: coagulase; In: indole; MR: methyl red; VP: Voges Proskaeur; S: sucrose; L:
lactose; G: glucose; M: maltose

Table 4.7. Shows the biochemical characteristic Bacteria isolated from Samples.
Staphylococcus aureus was positive in motility,catalase, lactose, sucrose, coagulase ,
voges proskaeur test, sucrose, lactose, glucose, maltose and shows negative in spore
production, motility, indole and methyl red it was also susceptible to the antibiotics.
While Enterococcus faecalis are positive to in motility ,catalase, lactose, sucrose,
coagulase, and show negative to oxidase, coagulase, indole, methyl red, voges proskaeur
test, sucrose,lactose, glucose, hydrogen sulphate and urease production.

38
Table 4.8. Shows that Staphylococcus aureus was more susceptible to zinnacef,
Staphylococcus haemolyticus to amoxicillin, Staphylococcus aureus to Rocephin,
Staphylococcus aureus to Ciprofloxacin, Staphylococcus aureus to Streptomycin,
Staphylococcus aureus and Bacillus subtilis to Erythromycin, Enterococcus faecalis to
septrin, Staphylococcus aureus to Gentamycin, Bacillus subtilis to ampiclox and
Staphylococcus haemolyticus to pefloxacin.

Table 4.8: Antibiotic Sensitivity Test of Gram Positive Bacterial Isolates

Bacterial Z AM R CPX S E SXT CN APX PEF
Isolates 20µg 30µg 25µg 10µg 30µg 10µg 30µg 10µg 30µg 10µg
Staphylococcus 21 16 22 21 17 12 14 20 15 20
aureus
Bacillus 19 13 10 15 16 12 12 8 20 14
subtilis
Bacillus cereus 0 0 12 16 14 11 15 16 0 0
Enterococcus 0 10 12 10 0 0 16 18 12 18
faecalis
Staphylococcus 13 22 10 0 0 0 0 14 16 21
haemolyticus
Z, zinnacef; AM, amoxicillin; R, rocephin, CPX, ciprofloxacin; S, streptomycin; E,
erythromycin; SXT, septrin; CN, gentamycin; APX, ampiclox; PEF, pefloxacin.
Table 4.9. Shows that Salmonella sp was more susceptible to amoxicillin, no bacterial
isolate was susceptible to streptomycin, augmentin and tarivid while Pseudomonas
aeruginosa to ciprofloxacin, Shigella sp to gentamycin, Pseudomonas aeruginosa to
pefloxacin, Pseudomonas aeruginosa to chloramphenicol and Pseudomonas aeruginosa
to sparfloxacin.

39
Table 4.9. Shows that Salmonella sp was more susceptible to amoxicillin, no bacterial
isolate was susceptible to streptomycin, augmentin and tarivid while Pseudomonas
aeruginosa to ciprofloxacin, Shigella sp to gentamycin, Pseudomonas aeruginosa to
pefloxacin, Pseudomonas aeruginosa to chloramphenicol and Pseudomonas
aeruginosa to sparfloxacin.

Table 4.9: Antibiotic Sensitivity Test of Gram Negative Bacterial Isolate

Bacterial AM S CPX AU CN PEF OFX SXT CH SP
Isolate 30µ 30µ 30µg 10µg 30µg 30µg 10µg 30µg 30µg 10µg
g g
Pseudomonas 0 0 22 0 15 11 0 20 17 19
aeruginosa
Enterbacter sp 0 0 0 0 12 0 0 0 14 16
Salmonella sp 12 0 0 0 14 0 0 0 10 0
Shigella sp 0 0 0 0 16 0 0 0 14 0
AM, amoxicillin; S, streptomycin; CPX, ciprofloxacin; PEF, pefloxacin; CN, gentamycin;
AU, augmentin; OFX, tarivid; SXT, septrin; CH, chloramphenicol; SP, sparfloxacin

40
Table 4.10. Shows the Percentage Occurrence of Bacteria and Fungi in Samples with
Staphylococcus sp with 11.9%, Pseudomonas aeruginosa 23.8%, Bacillus sp 19.4%,
Enterococcus faecalis 14.9%, Enterobacter sp 14.9%, Shigella sp 7.5%, Salmonella sp
7.5% and Saccharomyces cerevisiae 100%.

Table 4. 10: Percentage Occurrence of Bacteria and Fungi in Samples

Bacterial isolates Number/Percentage Fungal isolates Number/Percent
age
Staphylococcus sp 8 (11.9) Saccharomyces 10 (100)
cerevisiae
Pseudomonas 19 (23.8)
aeruginosa
Bacillus sp 13 (19.4)
Enterococcus faecalis 10 (14.9)
Enterobacter sp 10 (14.9)
Shigella sp 5 (7.5)
Salmonella sp 5 (7.5)
Number in parenthesis represents percentage (%)

41
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 DISCUSSION

After the sample collection, culturing and incubation, biochemical test and
antimicrobial sensitivity test was carried out on the organisms found. The results
showed that 95% of the samples were contaminated with bacteria. Pseudomonas
aeruginosa is the most prevalent bacteria found in the samples amounting of about
23.8%. Other bacteria isolated from the samples included Staphylococcus aureus,
Bacillus spp, and Shigella spp. In this research no parasites were found in any of the
samples. This research demonstrates that the precut fruits studied are potential
vehicles for the transmission of diseases, particularly those
transmitted via the faecal-oral route. The sort of isolates found and their distribution
pattern revealed that precut fruits become contaminated throughout the slicing,
rinsing, and display for sale processes. As a result, it is recommended that fruit
dealers follow sanitary measures.

Further research has been carried and it was observed that microbes are found in
every type of substance with the exception of sterilized bodies and environments
this indicates that microorganisms are an unavoidable part of life on Earth as they
may be found practically every where, their many shapes enable
them to thrive in a wide range of conditions. It's not unexpected that they are
discovered on fresh fruits and vegetables because they are
found in all unsterilized surroundings.

Fresh fruits and vegetables are consumed all around the world, with a growing need
as the world's population grows. Today's never-ending stream of activities has made
many people busy all of the time affecting their diets and prompting a switch to the
consumption of fruits and vegetables to save time but the nutritional content of
vegetables is the main contributor to their increased consumption and utilization
worldwide. Fresh fruits have become well-known and acknowledged as a healthy
diet that aids in the correction of various dietary imbalances. Because of the
increased demand for fresh fruits and vegetables, manufacturers have turned to low-
cost, quick-turnaround techniques of production, causing them to be less concerned
about food safety.

Numerous possible dangers in the food chain of pre-cut fruits processing include
contamination during production, in the field at harvest, post-handling, storage and
distribution, and consumer handling. As points or sources of contamination are
difficult to establish the best strategy to minimize potential contamination and risks
42
is to guarantee appropriate hygiene practices in conjunction with Hazard Analysis
and Critical Control Point (HACCP) (Ismaiel and Papenbrock, 2015).

Another Research study conducted by Okoro et al. (2018), assessed the

microbiological quality of sliced pineapple fruits vended in Abakaliki Metropolis
and found a 20% prevalence of Salmonella species and a 70% prevalence of
Escherichia coli The study revealed that Salmonella species showed resistance to
antibiotics like ceftriaxone, nitrofurantoin, and amoxicillin, ranging from 50-100%,
while E. coli showed resistance to the same antibiotics, ranging from 28.6-100%.
The findings highlight the risk of multidrug-resistant pathogens in fruit vending
practices and emphasize the need for proper food safety practices to prevent
foodborne illnesses

5.2 CONCLUSION

The high prevalence of microbial contaminants in ready-to-eat foods in the region is

posing a significant risk to public health. Improved food handling and preparation
practices as well as regular monitoring and enforcement of food safety regulations,
are necessary to reduce the risk of food borne illnesses.

43
 REFERENCES
Adebawo O, Salau B, Ezima E et al. (2006). Fruits and vegetables moderate
lipid cardiovascular risk factor in hypertensive patients. Lipids Health Dis,
5:14.
Adelowo, O. O & Adelowo, O. A. (2013). Microbial contamination of street-
vended fruits in Lagos, Nigeria. Journal of Food Protection, 76(10), 1731-
1736.
Adelowo, O. O., & Adelowo, O. A. (2013). Microbial contamination of street-
vended fruits in Lagos, Nigeria. Journal of Food Protection, 76(10), 1731-
1736.

Adeyemi, O. A., Oyedeji, K. S., & Adeyemi, O. O. (2017). Microbial quality of

fresh-cut fruits sold in Lagos, Nigeria. Journal of Food Science and
Technology, 54(4), 852-858.
Adeyemi, O. A., Oyedeji, K. S., & Adeyemi, O. O. (2017). Microbial quality of
fresh-cut fruits sold in Lagos, Nigeria. Journal of Food Science and
Technology, 54(4), 852-858.
Afolabi, O. A., & Adesanya, O. A. (2015). Microbiological evaluation of street-
vended fruits in Ibadan, Nigeria. Journal of Food Science and Technology,
52(4), 2080-2086.

Akinjogunla, O. J., Adeniyi, B. A., & Adelowo, O. O. (2020). Microbial

contamination of fresh fruits sold in Ibadan, Nigeria. Journal of Food and
Nutrition Research, 8(3), 1-9.
Akinjogunla, O. J., Adeniyi, B. A., & Adelowo, O. O. (2020). Microbial
contamination of fresh fruits sold in Ibadan, Nigeria. Journal of Food and
Nutrition Research, 8(3), 1-9.
Akinneye, J. O., & Adesanya, O. A. (2015). Microbiological evaluation of street-
vended fruits in Ibadan, Nigeria. Journal of Food Science and Technology,
52(4), 2080-2086.
Altekruse, S. F., Cohen, M. L., & Swerdlow, D. L. (1997). Emerging foodborne
diseases. Emerging Infectious Diseases, 3(3), 285-293.
Anderson, J. Perryman S, Young L, Prior S (2010). Dietary Fibre Fact.
Sheet No. 9.333; Colorado State University: Fort Collins, CO,USA.
http://www.ext.colostate.edu/pubs/foodnut/09333.pdf (accessed
January 2015).
Avena-Bustillos, R. J., et al. (2017). Effect of humidity on the quality of fresh-cut
fruits. Journal of Food Science, 82(5), S1248-S1256.

44
Balogun, S. A., Adebayo, T. O., & Adelowo, O. O. (2020). Food safety knowledge
and practices among fruit vendors in Ibadan, Nigeria. Journal of Food Science
and Technology, 57(2), 638-644.
Balogun, S. A., Adebayo, T. O., & Adelowo, O. O. (2020). Food safety knowledge
and practices among fruit vendors in Ibadan, Nigeria. Journal of Food Science
and Technology, 57(2), 638-644.
Beishir, I (1987). Microbiology in Practice. A self-Instruction Laboratory Course.

Bennish ML, Salam MA, Khan WA. et al. Treatment of shigellosis 3. Comparison
of one-dose or 2-dose ciprofloxacin with standard 5-day therapy - a
randomized, blinded trial. Ann Int Med. 1992;117:727.

Beuchat, L. R. (2000). Pathogens associated with fresh produce. Food Safety

Magazine, 6(3), 26-34.
Bhattacharya, S., & Mukherjee, R. (2015). Polyethylene packaging: A review.
Journal of Packaging Technology and Science, 28(1), 1-18.

Bolinger H, Kathariou S. The Current State of Macrolide Resistance in

Campylobacter spp.: Trends and Impacts of Resistance Mechanisms. Appl
Environ Microbiol. 2017 Jun 15. 83 (12):

Brecht, J. K. (2017). Modified atmosphere packaging for fresh-cut fruits.

Postharvest Biology and Technology, 133, 115-123.

Brecht, J. K. (2017). Modified atmosphere packaging for fresh-cut fruits.

Postharvest Biology and Technology, 133, 115-123.
Brecht, J. K. (2017). Modified atmosphere packaging for fresh-cut fruits.
Postharvest Biology and Technology, 133, 115-123.
Buchanan, R.E and Gibbon, N.E (2000). Bergeys Manual of Determinative
Bacteriology. Williams and Wilkens Company, Baltimore, USA.

Buck, J. W., Walcott, R. R., & Beuchat, L. R. (2003). Recent trends in

microbiological safety of fruits and vegetables. Food Safety Magazine, 9(3),
16-23.

Butler T, Speelman P, Kabir I. et al. Colonic dysfunction during shigellosis. J Infect

Dis. 1986;154:817.

Cassels, F. J. & Wolf, M. K. Colonization factors of diarrheagenic E. coli and their

intestinal receptors. J. Ind. Microbiol. 15, 214–226 (1995).

45
CDC. (2019). Campylobacter (Campylobacteriosis) Questions and Answers.
Centers for Disease Control and Prevention. Available
at https://www.cdc.gov/campylobacter/faq.html. December 23; Accessed:
December 19, 2022.
Centers for Disease Control and Prevention (CDC). (2020). Food safety. Retrieved
from (link unavailable)

Centre for Disease Control and Prevention (1979). Salmonella oranienburg

gastroenteritis associated consumption of precut watermelons-Illinois
Morbidity and Mortality Weekly Report, 28: 522- 523.

Centre for Disease Control and Prevention (2002). Multistate outbreak of

Salmonella serotype poona infections associated with eating Canataloupe
from Mexico-United States and Canada, 2000-2002 Morbidity and Mortality
Weekly Report, 51: 1044-1047.

Centre for Disease Control and Prevention (2009). Surveillance of foodborne

disease outbreaks in United States in 2006. Morbidity and Mortality Weekly
Report, 58(22): 609-615.

Centre for Disease Control and Prevention (2009a). Preliminary food net data on
the incidence of infection with pathogens transmitted through food in ten
States 2008. Morbidity and Mortality Weekly Report, 58 (13): 333 -337.

Centre for Disease Control and Prevention. 1991. Multistate outbreak of Salmonella
poona infections-United States and Canada. Morbidity and Mortality Weekly
Report, 40: 549-552.
Cheesbrough, M. (2000). District Laboratory Practice in Tropical Countries. Part
2, Cambridge University Press, UK. pp 35-38, 62-69.

Chukwu, C., Chukwu, I. D., Onyimba, I., & Umoh, E. G. (2010). Microbiological
quality of pre-cut fruits on sale in retail outlets in Nigeria. African Journal of
Agricultural Research, 5(17), 2311-2316.

Das R, Haque MA, Chisti MJ, Faruque ASG, Ahmed T. Associated factors, post
infection child growth, and household cost of invasive enteritis among under 5
children in Bangladesh. Sci Rep. 2021 Jun 17. 11 (1):12738.
De Andrade Cavalcante D, De-Souza MT, de Orem JC, de Magalhães MIA,
Martins PH, Boone TJ, Castillo JA, Driks A. (2019). Ultrastructural analysis
of spores from diverse Bacillales species isolated from Brazilian soil. Environ
Microbiol Rep. 11(2):155-164.
46
De Rover C (1998). Microbial safety evaluations and recommendation on fresh
produce. Food Control, 9(6): 321-347.
De Rycke, J. & Oswald, E. Cytolethal distending toxin (CDT): a bacterial weapon
to control host cell proliferation? FEMS Microbiol Lett. 203, 141–148 (2001).
D'Incecco P, Ong L, Gras S, Pellegrino L. A fluorescence in situ staining method
for investigating spores and vegetative cells of Clostridia by confocal laser
scanning microscopy and structured illuminated microscopy. Micron. 2018
Jul;110:1-9.
Federal Ministry of Health, Nigeria. (2014). National Food Safety and Quality
Policy.
Food and Drug Administration (FDA). (2020). Fresh cut fruits. Retrieved from
(link unavailable). Fourth Edition. Harper and Row Publishers, New York, pp
96-111, 120-130,

Gilling S, Taylor EA, Kane K, Taylor JZ (2001). Successful hazard analysis critical
control point implementation in the United Kingdom: understanding the
barriers through the use of a behavioral adherence model. J. Food Prot., 64:
710-715.
Goldberg, M. B. & Theriot, J. A. Shigella flexneri surface protein IcsA is sufficient
to direct actin-based motility. Proc. Natl Acad. Sci. USA 92, 6572–6576
(1995).
Gould GW. (2006). History of science--spores. J Appl Microbiol. 101(3):507-13.
Graham DR, Dixon RE, Hughes JM, Thornsberry C. (1985). Disk diffusion
antimicrobial susceptibility testing for clinical and epidemiologic
purposes. Am J Infect Control. 13(6):241-9.
Habauzit V, Milenkovic D, Morand C (2013).Vascular Protective Effects
of Fruit Polyphenols. In. Polyphenols in Human Health and Disease.
Eds, Watson R, Preedy V, Zibadi S. 1st ed, Elsevier Inc. London , pp.
875-893.
Hariram U, Labbé R. (2015). Spore prevalence and toxigenicity of Bacillus cereus
and Bacillus thuringiensis isolates from U.S. retail spices. J Food Prot.
78(3):590-6.
Harrigan, W.F and McCance, M.E. (2000). Laboratory Methods in Food and Dairy
Microbiology. Eight Edition, Academic Press Inc., London. pp 7-23, 286-303.

Hayashi, F. et al. ((2001)). The innate immune response to bacterial flagellin is

mediated by Toll-like receptor 5. Nature 410, 1099–1103.

Jay JM (1996). Indicators of food microbial quality and safety in Modern Food
Microbiology. 5th edition, Chapmann and Hall, pp. 387-407.
47
Kaakoush NO, Mitchell HM, Man SM. Role of emerging Campylobacter species in
inflammatory bowel diseases. Inflamm Bowel Dis. 2014 Nov. 20 (11):2189-
97.

Kaplan, JE, Campbell, DS (1982). Frequency of a Norwalk like pattern of illness in

outbreaks of acute gastroenteritis. Am. J. Pub. Health, 72: 1329-1332
Kaur C, Kapoor HC (2001). Antioxidants in fruits and vegetables: the millennium's
health. Int J Food Sci Technol, 36: 703-725.
Keller, R. et al. Afa, a diffuse adherence fibrillar adhesin associated with
enteropathogenic Escherichia coli. Infect. Immun. 70, 2681–2689 (2002).

Keusch, GT, Bennish ML (1991). : Shigellosis. p. 593. In Evans A S, Brachman PS

(ed): Bacterial Infections of Humans, Epidemiology and Control. Plenum,
New York,.

Koser SA, McClelland JR. (1917). The Fate of Bacterial Spores in the Animal
Body. J Med Res. ;37(2):259-68.
Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D. (2015 ).
Current and past strategies for bacterial culture in clinical microbiology. Clin
Microbiol Rev. 28(1):208-36.

Lattimer JM, Haub MD (2010). Effects of Dietary Fibre and Its

Components on Metabolic Health. Nutrients, 2: 1266 -1289.

Liu F, Lee SA, Xue J, Riordan SM, Zhang L. Global epidemiology of

campylobacteriosis and the impact of COVID-19. Front Cell Infect Microbiol.
2022. 12:979055.

Lund BM (1992). Ecosystems in Vegetable foods J. Appl. Bact., 73: 115-135.

Lund, B. M. (2011). Microbial quality of fresh-cut fruits and vegetables. Journal of
Food Protection, 74(10), 1694-1703.
Melton-Celsa, A. R. & O'Brien, A. D. (1998). In Escherichia coli O157:H7 and
Other Shiga Toxin-Producing E. coli Strains (eds Kaper, J. B. & O'Brien, A.
D.) 121–128 (ASM Press, Washington DC, USA).

Morens DM, Folkers GK, Fauci AS. (2013). World Health Organization. "The
global view of campylobacteriosis: report of an expert consultation, Utrecht,
Netherlands, 9-11 July 2012." 9-11 JULY 2012. [Full Text].
Nataro, J. P. & Kaper, J. B. ((1998). Diarrheagenic Escherichia coli. . A
comprehensive review of the pathogenesis, epidemiology, diagnosis and
clinical aspects of diarrhoeagenic E. coli. Clin. Microbiol. Rev. 11, 142–201
48
Okoro, C. C., Okoro, C. E., & Onya, O. O. (2018). Assessment of Salmonella
species and Escherichia coli prevalence in sliced pineapple vended in
Abakaliki Metropolis. Journal of Food Science and Technology, 55(4), 1050-
1056.
Olorunfemi, O. J., Adebayo, T. O., & Adelowo, O. O. (2020). Fungal
contamination of fresh-cut fruits sold in Abuja
Olorunfemi, O. J., Adebayo, T. O., & Adelowo, O. O. (2020). Fungal
contamination of fresh-cut fruits sold in Abuja, Nigeria. Journal of Food and
Nutrition Research, 8(2), 1-8.
Omorodion, N. J. P., & Nwiyege, S. (2023). Microbial Quality of Sliced and
Packaged Fruits in Polyethylene Sold in Port Harcourt. Journal of Life and
Bio-Sciences Research, 04(01), 25-30.
Oyedeji, K. S., Adeyemi, O. A., & Adeyemi, O. O. (2017). Food safety regulations
and practices in Nigeria. Journal of Food Science and Technology, 54(4), 831-
838.
Palombo EA. (2020). Ethanol treatment does not inactivate spore-forming bacteria -
A cautionary note about the safe transport of bacteria prior to identification by
MALDI-TOF MS. J Microbiol Methods. 172: 105893. [PubMed]
Paredes-Sabja D, Raju D, Torres JA, Sarker MR. (2008). Role of small, acid-
soluble spore proteins in the resistance of Clostridium perfringens spores to
chemicals. Int J Food Microbiol. 20; 122(3):333-5.
Park HM, Heo J, Park Y (2011). Calcium from plant sources is beneficial
to lowering the risk of osteoporosis in postmenopausal K orean
women. Nutr Res, 31: 27–32.
Parnell, T. L., et al. (2017). Sanitation practices for fresh-cut melons. Journal of
Food Protection, 80(4), 648-655.

Rincé A, Balière C, Hervio-Heath D, Cozien J, Lozach S, Parnaudeau S, et al.

(2018). Occurrence of Bacterial Pathogens and Human Noroviruses in
Shellfish-Harvesting Areas and Their Catchments in France. Front Microbiol..
9:2443. [QxMD MEDLINE Link].
Rivera-Lopez, M. (2019). Effects of storage conditions on the quality of fresh-cut
fruits. Journal of Food Science, 84(5), S1448-S1456.

Rivera-Lopez, M. (2019). Effects of storage conditions on the quality of fresh-cut

fruits. Journal of Food Science, 84(5), S1448-S1456.
Rivera-Lopez, M. (2019). Effects of storage conditions on the quality of fresh-cut
fruits. Journal of Food Science, 84(5), S1448-S1456.

49
Russo, T. A. & Johnson, J. R. Proposal for a new inclusive designation for
extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J. Infect.
Dis. 181, 1753–1754 (2000).
Saba, M. S. (2017). Effect of handling practices on microbial quality of fresh-cut
apples. Journal of Food Science, 82(5), S1240-S1247.

Saito S, Tsukahara M, Ohkusu K, Kurai H. Helicobacter fennelliae Bacteremia:

Three Case Reports and Literature Review. Medicine (Baltimore). 2016 May.
95 (18):e3556. [QxMD MEDLINE Link].
Sawatzky P, Liu G, Dillon JA, Allen V, Lefebvre B, Hoang L, Tyrrell G, Van
Caeseele P, Levett P, Martin I. ( 2015). Quality Assurance for Antimicrobial
Susceptibility Testing of Neisseria gonorrhoeae in Canada, 2003 to 2012. J
Clin Microbiol. 53(11):3646-9.
Schneider M, Norman R, Steyn N, Bradshaw D (2007). Estimating the burden of
disease at tributable to low fruit and vegetable intake in South Africa in 2000.
S Afr Med J, 97 (8).
Sears, C. L. & Kaper, J. B. Enteric bacterial toxins: mechanisms of action and
linkage to intestinal secretion. Microbiol. Rev. 60, 167–215 (1996).
Shen CL, Bergen VV, Chyu MC et al. (2012). Fruits and dietary phytochemicals in
bone protection. Nutr Res, 32: 897 – 910.
Slavin JL, Lloyd B (2012). Health Benefits of fruits and vegetables. Adv Nutr,
3(4):506-16.

Smith JL, Bayles DO. The contribution of cytolethal distending toxin to bacterial
pathogenesis. Crit Rev Microbiol. 2006 Oct-Dec. 32 (4):227-48.

Southon, S (2000). Increased fruit and vegetable consumption within

the EU: potential health benefits. Food Res Int, 33(3–4):211–217.
New, S (2001). Fruit and vegetable consumption and skeletal health:
is there a positive link? Nutrition Foundation. Nutr Bull, 26: 121–
125.
Sweeney, N. J. The Escherichia coli K-12 gntP gene allows E. coli F-18 to occupy
a distinct nutritional niche in the streptomycin-treated mouse large
intestine. Infect. Immun. 64, 3497–3503 (1996).
Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J. & Tobias, P. S. (2000).
Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor
for Escherichia and Salmonella lipopolysaccharides. J. Immunol. 165, 5780–
5787.

50
Tieng, V. et al. Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-
surface expression of the MHC class I-related molecule MICA. Proc. Natl
Acad. Sci. USA 99, 2977–2982 (2002).

Totten PA, Fennell CL, Tenover FC, Wezenberg JM, Perine PL, Stamm WE, et al.
(1985). Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp.
nov.): two new Campylobacter species associated with enteric disease in
homosexual men. J Infect Dis. 151 (1):131-9.

Vital (2011) signs: incidence and trends of infection with pathogens transmitted
commonly through food--foodborne diseases active surveillance network, 10
U.S. sites, 1996-2010. MMWR Morb Mortal Wkly Rep. 10. 60(22):749-
55. [QxMD MEDLINE Link].
Whittam, T. S. in Escherichia coli and Salmonella (eds Neidhardt, F. C. et al.)
2708–2720 (ASM Press, Washington DC, USA, 1996).

WHO. (2022). Campylobacter Key Facts. World Health Organization. Available

at https://www.who.int/news-room/fact-sheets/detail/campylobacter. May 1,
2020; Accessed: December 19.

Williamson, G (1996). Protective effects of fruits and vegetables in the

diet. J Nutr Food Sci, 96 (1): 6-10.

Wilson, IG, Moore JE. (1996). Presence of Salmonella spp. and Campylobacter spp.
in shellfish. Epidemiol Infect. 116 (2):147-53.

Zhang C, Yang Z, Hou B. (2021). Diverse bacterial profile in extraradicular

biofilms and periradicular lesions associated with persistent apical
periodontitis. Int Endod J. 54 (9):1425-1433. [QxMD MEDLINE Link].

Zhang L. (2015). Oral Campylobacter species: Initiators of a subgroup of

inflammatory bowel disease? World J Gastroenterol., 21 (31):9239-44.
Zhang, Y., et al. (2020). Modified atmosphere packaging for fresh-cut strawberries.
Postharvest Biology and Technology, 166, 111284.

51
 APPENDIX

Fig.1 colony plate Fig.2 colony plate Fig.3 Dilution

Fig.4 Fruit Sample Fig.5 Culture plates Fig.6 Antimicrobial plate

count

Fig.9 Antimicrobial plate

Fig.7 Antimicrobial plate Fig.8 Dilution count
count

Fig.10 Antimicrobial Fig.11 Culture plates Fig.12 Culture plates

plate count

52
Fig.13 Antimicrobial Fig.14 Fruit Sample Fig.15 Antimicrobial
plate count plate count