CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

Food borne diseases are an important cause of morbidity and mortality worldwide. There are

over 200 different types of illness that may be transmitted by food. The causes of foodborne

illness are bacteria, viruses, parasites, and chemicals: Bacterial contamination is the most

common cause of illness (Lynch et al., 2006). Most food borne bacterial infections cause self-

limiting diarrhoea, however, systemic infection and death can occur, particularly in vulnerable

groups such as the elderly, people with diminished immunity or infants and young children

(Kennedy et al., 2004). Bacteria have accounted for more than 70% of deaths associated with

foodborne transmission (Lynch et al., 2006; Hughes et al., 2007).

The family Enterobacteriaceae comprises a large group of Gram-negative non-spore forming

bacteria typically 1-5 μm in length. They are facultative anaerobes and with the exception of

Saccharobacter fermenters and some strains of Yersinia and Erwinia, they share the ability to

reduce nitrate to nitrite. These bacteria are generally motile by peritrichous flagella except for

Shigella and Tatumella and some other non-motile members of this family. For example,

Salmonella are typically motile, notable exceptions being the Salmonella serotypes Pullorum and

Gallinarum.

1
 A common feature of the Enterobacteriaceae, which helps to differentiate them from

other closely related bacteria, is the lack of cytochrome Coxidase, although there are exceptions

such as Plesiomonas spp. Enterobacteriaceae are catalase positive with the exception of Shigella

dysenteriae and Xenorhabdus species. Enterobacteriaceae ferment a variety of carbohydrates, but

their ability to produce acid and gas from the fermentation of D-glucose is one characteristic that

remains an important diagnostic property and is commonly used as a basis for their detection and

enumeration. Some members of the Enterobacteriaceae (e.g., Enterobacter spp, Escherichia coli,

Citrobacter spp and Klebsiella spp.) can be recognized using methods that exploit their ability to

ferment lactose rapidly (usually within 24-48 h) producing acid and gas (Tortora and Funke,

2009).

Enteric bacteria are microbes that reside in the guts of animals and humans. However

there are some among them that reside in intestinal tract of animals that can cause diseases and

harsh reactions when human become infected with them (Singh et al., 2013). They can cause a

mild infection, such as food poisoning or severe community infections like diarrhea. Such

examples of enteric bacteria include Salmonella, Escherichiacoli, Shigella, Klebsiella,

Campylobacter, Enterobacter Yersinia, Vibrio and Citrobacter (Kim et al., 2015).

2
The human gut is therefore the natural habitat for various bacteria species and majority of them

participate in metabolic activities that salvage energy and absorbable nutrients protecting the

colonized host against invasion by alien microbes and important atrophic effects on intestinal

epithelia and immune structure and function. An estimated 9.4 million food borne illness caused

by a known pathogen occur annually in United State (Scallan et al., 2011). It has been reported

that about 2 million diarrhea disease patients die per year throughout the world (Flint et al.,

2005). Considering the public importance of acute diarrhea disease, laboratory surveillance of

acute diarrhea is utilized in many countries for safety and prevention efforts (Kendall et al.,

2012).3

1.2 Statement of the Problem

Enteric bacterial pathogens are one of the major causes of foodborne gastroenteritis in humans

and remain an important health problem worldwide. The family of enterobacteriaceae cause

primary infections of the human gastrointestinal tract. Members of this family are major causes

of opportunistic infection (including septicemia, pneumonia, and meningitis and urinary tract

infections). Hence, there is need to carryout antibiogram with sensitive antibiotics that will help

to combat the infections of the enteric bacterial pathogens.

3
1.3 Justification of the Study

The outcome of this research work will provide additional baseline data on determining the

isolates and the antibiogram of some enteric bacterial pathogens which cause food borne

gastroenteritis in humans and the possible dynamic routes that facilitates the transmission of the

disease in the study area and possible means of prevention of the disease. The research work will

also help to assess and provide information on the antibiogram of some enteric bacterial

pathogens.

1.4 Aim
The aim of this research is to isolates and determines the antibiogram of some enteric bacteria
pathogens from cow intestine sold in Wukari market, North - Eastern Nigeria.

1.5 Objectives

The objectives of this research are to:

isolates and identify some enteric bacterial pathogens from cow intestine of study area.

determine the prevalence of some enteric bacterial pathogens in cow intestine of study area.

determine antibiotic resistance pattern of enteric bacterial pathogens.

4
 CHAPTER TWO

2.0 Literature Review

Enteric pathogens have been implicated in most food and water-borne infections that have been
responsible for rising morbidity and mortality globally especially in Africa (Payment and Riley,
2002; Nma and Oruese, 2013; Ogunleye et al., 2013). Though some of the organisms in this
group are normal microbiota of gut of man and other higher animals, they get their way into the
food through environmental contamination (Karshima et al., 2013; Ogunleye et al., 2013;
Kemal, 2014).

They are mainly Gram-negative bacteria which cause different gastrointestinal diseases in
man while quite a number of other animals serve as either carrier or secondary host.
Gastrointestinal tract (GIT) infection in humans usually originates from pets, other humans and
through the ingestion of contaminated water or animal food products, most often eggs, poultry,
and raw meat (Bhan et al., 2005; Centers for Disease Control and Prevention, 2005; Swanson et
al., 2007; Smith et al., 2012; Karshima et al., 2013). Following ingestion of the organisms, the
likelihood of infection developing, as well as the severity of infection, is related to the dose and
virulence of the organism in question or its strain and the status of host’s defense mechanisms
(Payment and Riley, 2002).

In most cases, diagnosis of GIT infection is often missed or delayed, which is a reflection
of the multi-system nature of the diseases. Consequent upon development and availability of
modern sewage and water treatment facilities, these diseases have become rare in developed
countries but remain a serious health challenge in low resource countries with inadequate
sanitation and safe water supply. Although enteric fever is a major global public health problem;
data on the relative risk of contracting travel-associated enteric fever is not documented in most
developed world, while adequate epidemiological data are grossly inadequate in developing
countries (Crump et al., 2004). Vending of street food, particularly in urban areas, is a growing
and global phenomenon and today street vended foods are important sources of daily meals for
massive urban populations as well as in African. However, food poisoning, food borne diseases
and food safety have been declared a major public health concern by international health
agencies, while in many studies; street vended foods have been associated with microbiological
contamination and low hygienic standards (WHO, 2006).
5
 Hence, street food vendors play a significant role in public health since this group of
individuals alone influences the life and health of thousands of people daily. Food handlers have
been reported to greatly contribute to the dissemination and distribution of pathogens due in most
cases to their low level of education, poor personal and environment hygiene (Nkere et al., 2011;
CDC, 2013). Moreover, some of the food vendors are carriers of most of the enteric bacterial
pathogens and consequently introduce the pathogens into the food they handle (Chukwu et al.,
2010; Oranusi and Olorunfemi, 2011). Food from local vendors, though most of the time are
prepared under unhygienic conditions and by people with very low knowledge of hygiene, they
still enjoy high patronage due essentially to their affordability, easy accessibility and claimed
palatability or organoleptic quality (Karshima et al., 2013).

Animal gut is therefore the natural habitat for various bacteria species and majority of them
participate in metabolic activities that salvage energy and absorbable nutrients protecting the
colonized host against invasion by alien microbes and important atrophic effects on intestinal
epithelia and immune structure and function. An estimated 9.4 million food borne illness caused
by a known pathogen occur annually in United State (Scallan et al., 2011). It has been reported
that about 2 million diarrhea disease patients die per year throughout the world (Flint et al.,
2005). Considering the public importance of acute diarrhea disease, laboratory surveillance of
acute diarrhea is utilized in many countries for safety and prevention efforts (Kendall et al.,
2012).

2.1 Socio-economic Effects of Foodborne Outbreaks

Regarding the complexity of farming and food system, in addition to production, social and
ecological aspects should be taken in to account (Breland et al., 2007). 6

Food safety is an issue which can disrupt markets and cause significant losses to farmers,
consumers and marketers. When an outbreak happens it tends to affect all produce consumption.
Even when the outbreak occurs far from where people live, consumers will refuse to consume
the product for a while. In fact they will reduce their consumption of all “fresh produce” (Fan et
al., 2009). 6

This fact that how quick producers can manage the contamination problem and persuade buyers
that their product doesn’t have a risk anymore is an important factor in the economic effect of

6
foodborne illness outbreak. Human infections related to E. coli O157:H7 mostly occur in
summer-time from May through September. Foodborne diseases (FBD) can also have a
devastating impact on the economy of different countries by affecting their tourism industry
since tourists do not want to return to a country where they have become sick (Bach et al., 2002).
7

Furthermore there has been an expansion in pathogen range. As has been mentioned in Wallace
et al. (2011) about 50 years ago there were four main foodborne pathogens while today there are
around 30 foodborne pathogens. There are some factors responsible for the spread of these
pathogens: 7

Modern transportation systems which allow the transport of large quantities of crops, animals
and people.7

The ability of microorganisms to adapt themselves to the changing environment.

Climate change; as the planet warms, the geographic range of pathogens expand from tropical to
temperate regions.

War, poverty and famine which occurs around the world put more stress on human populations,
increasing their susceptibility to infectious disease.

The lack of will at governmental and intergovernmental levels to take action in order to better
protect public health. 7

2.2 Transmission of Enteric Pathogens in the Environment

Although the exact mechanism of contamination in most produce-related outbreaks is not clear,
lots of field research has attempted to explain the ecology of the growing environment. There are
different sources associated with the transmission of bacteria in the field, for instance farm
workers, machinery and equipment used in the farm. Pre-harvest stage is the main cause of leafy
vegetables’ contamination such as lettuce and spinach in many outbreaks (Islam et al., 2004).

Also, irrigation water, faeces, soil amendments, organic fertilizers and wild and domestic
animals are considered to be a potential source of contamination at the pre-harvest stage. Due to
more intensive agriculture these days, and consequently the proximity of fields of produce to

7
animal production zones, the ecological connections between wild animals, farm animals, and
produce have become closer (Lynch et al., 2008). Vegetables can become contaminated with
pathogens at the pre-harvest stage through the use of inadequately composted manure in fields as
fresh fertilizer. Both conventional and organic vegetable producers usually add animal manure to
fields as fertilizer (Islam et al., 2004). Furthermore water is always a well-recognized factor in
the transmission of bacteria. For bacteria such as Salmonella and E. coli O157 it has been
reported that contamination has occurred mostly through irrigation water and animal manure
(Buck et al., 2003). In fact the contamination of water mainly in lettuce and spinach has been
mentioned as a major cause of E. coli outbreaks (Ottson et al., 2011).

Waste water is another important source of enteric pathogens among soil in developing
countries due to its common use in agricultural irrigation (Santamaria et al. 2003). Moreover
bacterial pathogens can enter into plant tissues through different routes. Bacteria can easily enter
at a scar, bruise or wound in the fruit or leaf‘s surface through irrigation water with “capillary
action” at the pre-harvest stage (Fan et al., 2007). 8

CHAPTER THREE

3.0 Materials and Methods

8
3.1 Study Area

The study was conducted in Wukari local government area of Taraba State, which is located in
north-eastern region of Nigeria at southern guinea Savana which coordinate's latitude 7 o,51'N-
7.85oN and longitude 9o, 47'E-9.783OE, annual precipitation of 1205 mm and it has an average
temperature of 26.8o. The research took place during the dried season 2022. Wukari is dominated
by Jukun tribe, and other tribes are the Tiv, and Hausa. Majority of the people are farmers and
hunters. Most of these people eat food in the premitive way with bare hands.

3.2 Materials and Chemicals used

3.2.1 Glass wares and others instrument

Test tube

Test tube holder/ rack

Conical flask

Spirit lamp

Cotton, foal paper

Pipette

Micropipette

Measuring cylinder

Electric balance machine

Glass spreader

Streaking loop

Incubator

Refrigerator9

9
3.2.2 Chemical Reagents

PBS (Phosphate buffer solution)

70% Alcohol

Distilled Water

3% Hydrogen per Oxide (H2O2)

Normal saline solution

50% Buffered Glycerol Saline

Other common laboratory chemicals10

3.3 Sample Collection

A total number of five (5) samples were purchased from three (3) locations of the Wukari old
market. The samples were collected in sterile containers directly from the sellers and
immediately taken to the Microbiology laboratory Federal University Wukari for standard
microbial analysis as described by Ji deani, (2006).

3.3.1 Sterilization of Materials

All glass ware including conical flasks, beakers, test tubes and bottles were washed thoroughly
with detergents, rinsed with distilled water. They were dried and sterilized in analytical oven at
temperature of 1600C for 1 hour as described byJudeani (2003). The media was also sterilized in
an autoclave at a temperature of 1210C for 15 minutes.

3.4 Media Preparation

All media used for this research work were prepared according to manufacturer's instructions.

3.5 Isolation of the Bacterial species

10
A loopfull of the sample preparations will be inoculated on to Salmonella Shigella agar (SSA),
MacConkey and Eosin Methylene Blue (EMB) agar plates by streaking and then will be
inoculated at 37oC for 24 hours.

3.6 Gram Staining

Gram staining technique will be carried out using a technique described by Cheese brought
(2009). From a 24 hours culture, a colony will be picked and emulsified in sterile distilled water
on a slide and will be heat fixed. The fixed smear will be covered with crystal violet stain for 60
sec. The stain will be washed off rapidly with distilled water, and the slide will be tilted off to
remove the water and smear will be covered with Lugol's iodine for 60 sec. The iodine will be
washed off with distilled water. Rapid decolourization will be done with acetone-alcohol and
washed immediately with distilled water. The smear will be covered with Safranine for 60 Sec.
The stain will be washed off with clean water and air dried. Morphology and Gram reactions of
the isolates will be determined by use of a microscope. Isolates, which appeared as Gram
positive, rod shaped organisms, will further be screened for biochemical tests such as catalase,
indole and motility tests.

3.7 Biochemical test

3.7.1 Catalase Tests.

A drop of 3% hydrogen peroxide (H 2O2) will be placed on a clean dry slide. Then a fresh (24 h)
colony on nutrient agar medium will be placed on the drop of hydrogen peroxide, positive result
will be indicated by air bubbles as a result of oxygen production, absence of air bubbles
indicated negative result (Usman et al., 2016b).

3.7.2 Indole Test

The colony of the 24 h culture of the test organism will be inoculated in a bijou bottle containing
3ml of sterile tryptone water. It was inoculated at 37 oC for up to 48 h and then will be tested for
production of indole by dispensing 0.5 ml of Kovac's reagent. After it will gently be mixed, a

11
negative result will show no red colour in the surface layer within 10 mins (den Bakker et al.,
2013).

3.7.3 Motility Test

Small suspension of the pathogen from 24 h culture will be dropped on a slide, emulsified with
distilled and covered with a cover glass and then sealed with molten petroleum jelly to avoid
drying. The preparation will be examined microscopically for motile organisms using 10× and
40× objectives lenses (Cheesbrough, 2009).

3.7.4 Antibiotic Susceptibility Test

The antibiotic susceptibility pattern will be determined using Kirby-Bauer-National Committee

for Clinical Laboratory Standards (NCCLS) modified disc diffusion technique (Cheesbrough).
The standardised inocula will be inoculated by streaking on prepared Mueller-Hinton agar
(Oxoid, CM0337) using sterile swab stick; the antibiotic disc will be placed on the inoculated
medium aseptically with the help of sterile forceps and inoculated at 37 oC for 24 h. The zones of
inhibition cleared by each of the antibiotics against the test organisms were measured and the
results will be interpreted using the guideline from Clinical and Laboratory Standards Institute
(CSLI) (2016) and all the results will be recorded appropriately.

12
 CHAPTER FOUR

4.0 RESULTS

The Cultural characteristics of the recovered isolates on MacConkey agar is presented in

Table4.1. The result showed how the recovered isolate were characterized on the basis of colony

morphology and staining characteristics. It was observed that all the isolates were Gram negative

rods i.e. pink colored and morphologically are small rod in shape which are arranged in single or

paired under the microscopic examination. The cultural characteristic ranges from mucoid

pink, non-mucoid darker pink, colorless colony with jagged edge and transparent colorless

colony. The Biochemical characterization of the recovered isolates is presented in Table 4.2. The

result showed how recovered isolates were characterized on the basis of biochemical

identification. Biochemical test include Indole, Motility, Citrate utilization test, Oxidase and

Catalase test. number of isolates recovered from each cow intestine sample is presented in

Table4.3. A total of fourteen (14) isolates were recovered from five (5) cow intestine samples

with highest number of isolates in sample 1, 2, 3 and 5 (3 isolates each) while least number of

isolates was recorded in sample 4 (2 isolates).13The Number and percentage occurrence of the

isolates recovered is presented in Table4.4. The results obtained from the data shows that the

bacteria found in the cow Intestine samples were Escherichia coli, Salmonella spp, Shigella spp

and Klebsiella spp and their prevalence was 36%, 29%, 21% and 14% respectively.

13
Table 4.1: Cultural characteristics of the recovered isolates

S/N Isolate code Colony Morphology (EMB)

1. S1 Non-mucoid darker pink

colony

2. S2 Transparent colorless smooth

colony

3. S3 Transparent colorless colony

with jagged edge

4. S4 Mucoid pinkish colony

The Biochemical characterization of the recovered isolates is presented in Table 2. The result
showed how recovered isolates were characterized on the basis of biochemical identification.
Biochemical test include Indole, Motility, Citrate utilization test, Oxidase and Catalase test.

14
Table 4.2: Biochemical characterization of the recovered isolates
S/N Isolate IN M CI OX CA Suspected
Code
Organism

1. S1 + - - - Escherichia
coli

2. S2 - + - - Salmonella
spp

3. S3 - - - - Shigella
spp

4. S4 - + - - Klebsiella
spp

IN=Indole,M=motility,CI=Citrateutilization,OX=O
xidase, CA = Catalase.

15
The number of isolates recovered from each cow intestine sample is presented in Table 3. A total

of fourteen (14) isolates were recovered from five (5) cow intestine samples with highest number

of isolates in sample 1, 2, 3 and 5 (3 isolates each) while least number of isolates was recorded in

sample 4 (2 isolates).

16
4.3: Number and suspected isolates recovered from each cow intestine sample

S/N Sample code No. Of isolates Suspected isolates

1 S1 3 E.coli, Salmonella, and Shigella

2 S2 3 E.coli, shigella and klebsiella

3 S3 3 E.coli, Salmonella and Shigella,

4 S4 2 E.coli, and salmonella

5 S5 3 E.coli, salmonella and

Klebsiella

17
The Number and percentage occurrence of the isolates recovered is presented in Table 4.4. The

results obtained from the data shows that the bacteria found in the cow Intestine samples were

Escherichia coli, Salmonella spp, Shigella spp and Klebsiella spp and there prevalence was 36%,

29%, 21% and 14% respectively.

Table 4 4: Number and percentage occurrence of the microorganisms recovered

S/N Microorganisms No. Of occurrence % Occurrence

1 Escherichia coli 5 36

2 Klebsiella 2 14

3 Salmonella spp 4 29

4 Shigella 3 21

Total 14 100

18
The antibiotic susceptibility of the isolate shows that all the isolates were susceptible to
Streptomycin as presented in Table 4.5 and 80% of the isolates were resistant to ciprofloxacin.

Table4.5:
Antibiotic susceptibility of the isolates from Cow
intestine

19
 No. Of isolates = 5

Antibiotic S I R

S 5(100%) 0(0%) 0(0%)

PN 3(60%) 1(20%) 1(20%)

CEP 4(80%) 1(20%) 0(0%)

NA 4(80%) 1(20%) 0(0%)

PEF 3(60%) 2(40%) 0(0%)

CN 4(80%) 0(0%) 1(20%)

AU 2(40%) 0(0%) 2(40%)

CPX 0(0%) 1(20%) 4(80%)

SXT 2(40%) 0(0%) 2(40%)

20
Key: S = Streptomycin, PN = Penicillin, CEP = Ceporex, NA = Nalidixic Acid, PEF =
Pefloxacin, CN = Gentamycin, AU = Augumentin, CPX = Ciprofloxacin, SXT = Septrin

Table 4.6 showed the resistance pattern and frequency of enteric isolates from Cow intestine.
Ciprofloxacin and Septrin had the high frequency of three while Penicillin, Augumentin,
Ceporex and Gentamycin had the least frequency of one respectively.

21
 Table 4.6: Antibiotic resistance pattern of enteric isolates from cow intestine

S/N Resistance pattern Frequency

1 PN 1

2 CPX 3

3 AU 1

4 CEP 1

5 CN 1

6 CPX, SXT 3

22
 CHAPTER FIVE

5.0 DISCUSSION

23
Enteric bacteria are microbes that reside in the guts of animals and humans. However, there are

some among them that reside in intestinal tract of animals that can cause diseases and harsh

reactions when human become infected with them (Singh et al., 2013). They can cause a mild

infection, such as food poisoning or severe community-infections like diarrhea. Such examples

of enteric bacteria include Salmonella, Escherichia coli, Shigella, Klebsiella, Campylobacter,

Enterobacter Yersinia, Vibrio and Citrobacter (Kim et al., 2015). Adeleye et al.(2017) reported

that the prevalence of enteric bacteria pathogens in the intestines of cows sold in the markets was

significantly higher than the prevalence in the cows from other sources such as diary farms.

Therefore, the selling of cow intestine in the markets of Nigeria is a potential source of enteric

bacteria pathogens. Related study was conducted by Obi et al. (2007) on enteric bacterial

pathogen in stool of residents of urban and rural regions of Nigeria, the results shows the most

frequently encountered pathogens in rural area are E. coli (28%), followed by Salmonella (16%),

Shigella (14%), Aeromonas (9%) and Campylobacter (8%). Similarly, the result of this research

was in conformity with the study conducted by Kim et al., (2015) on enteric bacteria isolated

from diarrhea patients in Korea which reveals that Escherichia coli was the most prevalent

isolated accounted for 22%, this is followed by Clostridium 14% and Salmonella 13.5%. The

resistance of the pathogens against some of the Antibiotics could be as a result of drug abuse or

indiscriminate use of drugs by cattle rearers as reported by Hu et al. (2009). Reports have

demonstrated that strains of enterobacteriaceae found in the cow Intestine are highly resistant to

antibiotics commonly use in veterinary as reported by Xu et al. (2008). In Nigeria Oyeleke et al.,

(2015) also reported similar case

CHAPTER SIX

6.0 CONCLUSION AND RECOMMENDATION

24
6.1 Conclusion

Intestine samples from different cows sold in Wukari market, North - Eastern Nigeria were

tested for identification of enteric bacteria. Isolates were confirmed as Escherichia coli,

Salmonella, Shigella and Klebsiella by Cultural characteristic, Gram staining and Biochemical

tests. This study shows Escherichia coli as the most frequent isolate recovered. The findings

point out that the enteric bacteria are associated with the food borne gastroenteritis infection in

humans.

6.2 Recommendation

It is therefore, recommended that, health education should play a role to create awareness about

food borne infection linked with unhygienic food handling and preparation.

Further research is needed to identify the potential sources of these pathogens and the potential

risks associated with consuming these cow Intestines.

Cow Intestines should thoroughly be wash and cook before consumption to avoid infection cause

by these pathogens.

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