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ISOLATION OF
ESCHERICHIA IN FOOD SOLD IN IMO STATE
POLYTECHNIC



BY

OBINWA JOSEPH .N.

MB/2006/130



A PROJECT RESEARCH ON MICROBIOLOGY AND
BIOTECHNOLOGY

FACULTY OF NATURAL SCIENCE



CARITAS UNIVERSITY, AMORJI
-
NIKE

EMENE, ENUGU STATE

IN PARTIAL FULFILLMENT

FOR THE AWARD OF

BACHELOR OF SCIENCE (B. SC) IN

MICROBIOLOGY AND BIOTECHNOLOGY




AUGUST, 2010









TABLE OF CONTENT

Title page

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i

Certification

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ii

Dedication

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iii

Acknowledgement

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iv

Table of content


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v

List of tables

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ix

Abstract

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CHAPTER ONE:

1.0

Introduction

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1

1.1

Aim and objectives

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3

CHAPTER TWO:

2.0

Literature Review

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4

2.1

Escheric
hia

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4

2.11

Strains

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5

2.12

Biology and Biochemistry

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6

2.13

Role as normal microbiota

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8

2.14

Therapeutic use of non pathogen
Escherichia

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8

2.2

Role in Disease

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8

2.21

Gastroin
testinal infection

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8

2.22

Epidemiology of gastrointestinal infection

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13

2.23

Urinary tract infection

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14

2.24

Neonatal meningitis

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15

2.3

Antibiotic therapy and resistance

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16

2.31

Beta


lactamase

strains

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17

2.32

Phage therapy

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17

2.33

Vaccination

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18

2.4

Role in biotechnology

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19

2.41

Environmental quality

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20

2.42

Model organism


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20

2.5

Escherichia

and
food

cont
amination

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21

2.51

How does
Escherichia

enter our
food

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22

2.52

What can individual do to help

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22

2.6

Bacteriological analysis of
food

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23

2.61

Approach

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24

2.7

Methodologies

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25

2.71

Mul
tiple tube method

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25

2.72

ATP testing.

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26

2.73

Plate count

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26

2.74

Membrane filtration

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28

2.75

Pour plates

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28

2.8

Pathogen analysis.

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29

2.9

Types of nutrient medi
a used in analysis.

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29

CHAPTER THREE:

3.0

MATERIALS AND METHOD

3.1

Sample collection

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31

3.2

Sample analysis

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31

3.3

Bacteriological analysis of the sample

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31

3.3.1

Membrane filtration technique

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31

3.4

Bacteria identification

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32

3.41

Grams staining

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32

3.42

Motility test

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33

3.5

Biochemical tests


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33

3.51

Urease test


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33

3.52

Catalase test

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34

3.53

Methyl red
test

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34

3.54

Indole test

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35

3.55

Citrate utilization test

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36

3.56

Voges


proskeur test (V.P. test)

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37

3.57

Sugar fermentation

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38

CHAPTER FOUR:

4.0

Results of Analysis

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40

CHAPTER FIVE:

5.0

Discussion

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42

5.1

Conclusion

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43

References


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Appendix 1


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Appendix 2


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50





LIST OF TABLES

TABLE 1:

Virulence properties of
Escherichia
.

TABLE 2:

Isolation of Bacteria from
food

samples

TABLE 3:

Physiochemical properties of bacteria isolated































ABSTRACT

Presence of
Escherichia

from
food sold in Imo State Polytechnic Umuagwo

was
determined. Out of eight different
food
examined which were designated
alphabetically as A, B, C, D, E, F, and H.
Escherichia

was isolated from four
food
sellers
. The membrane filtration technique was used and the presumptive test
showed other bacteria such as Klebsiella spp and Bacillus spp we
re seen growing
in the MacConkery agar. The pink colonies on MacConkey plates and metallic
blue sheen colour on EMB agar were isolated and subcultured in fresh agar plates
incubated at 370c for 24
-
hrs. The pure cultures were then identified using
biochemic
al tests.


























CHAPTER ONE

1.0

INTRODUCTION

Food is supposed to constitute a complex ecosystem for various microorganisms
including

Bacteria. food products like
fruit,

grain
s and
vegetable

are widely
consumed and market

in Imo poly for
them has existed in many parts of the world
for many generations. There is an increase demand by the

consumer for high
quality natural food, free from artificial preservatives, and contaminating
microorganisms. Contamination of food and food products, wi
th pathogenic
bacteria is largely due to processing, handling, and unhygienic conditions. This
paper describes the presence of
Escherichia
, Staphylococcus Aureus
, and
Listeria
monocytogenes
in cottage cheese and curd available at shops comprising the
unorg
anized sectors in Agra.

Escherichia

frequently contaminates food organism and it is a good indicator of
fecal pollution

(Diliello, 1982; Soomro
et al.,
2002; Benkerroum
et al.,
2004).
Presence of
Escherichia

in food products indicates the presence of
enter
opathogenic microorganisms, which constitute a public health hazard.
Enteropathogenic
Escherichia

can cause severe diarrhoea and vomiting in infants,
and
young children (Anon.,
1975).

Of late
L. monocytogenes
has been recognized
as a food born pathogen (Ka
clikova,
et al.,
2001) that can contaminate dairy
products (Menendez
et al.,
2001). Its virulent strain can cause a

serious disease
called listeriosis, particularly the risk populations including pregnant women,

newborns, the very old, and people who are i
mmune compromised (Fleming
et al.,
1985; Bille,

1989). Illness through
S. aureus
range from minor skin infection such
as pimples, boils, cellulites, toxic shock syndrome, impetigo, and abscesses to life
threatening disease such as pneumonia, meningitis, en
docarditis, and septicemia.
(Soomro
et al.,
2003; Masud
et al.,
1988).

Escherichia

have been identified as an indicator microorganism for food
safety (Adams and Moss, 2000). Pathogenic
Escherichia

have been
recognized as an increasingly important human
diarrheagenic pathogen in
all parts of the world, especially in young children in developing countries
(Porat
et al
, 1998). Five major categories of diarrheagenic
Escherichia

have been defined on the basis of their pathogenic mechanisms:

enteropathogenic
E
scherichia

(EPEC), enteroinvasive
Escherichia

(EIEC),
enterotoxigenic
Escherichia

(ETEC), enterohemorrhagic
Escherichia

(EHEC) or shiga toxin
-
producing
Escherichia

(STEC) and
enteroaggregative
Escherichia

(EAEC) (Porat
et al
, 1998). Traditionally,
diarrhea
genic
Escherichia

belong to a number of distinct serogroups and
were once defined solely on the basis of their sero types (Gomes
et al
,
1989). Recently, molecular biology and the knowledge of specific genes
encoding for characteristic virulence factors suc
h as
invE
for EIEC,
LTh
and
STh
for ETEC,
eaeA

for EPEC, and
stx
for STEC, have been used to
categorize these
Escherichia

(Porat
et al
, 1998; Rosa
et al
, 1998).
Increasing antimicrobial resistance of
Escherichia

in both humans and
animals over the world ha
s been reported (Schroeder
et al
, 2002).

Extensive studies on food
-
borne pathogens, such as
Salmonella
, have
been conducted in Thailand, but not much has been reported on the
characterization of diarrheagenic
Escherichia

(especially O157) and the
antimicro
bial susceptibilities of
Escherichia

strains isolated from food. Our
study therefore investigated the serogroups and the virulence genes of
diarrheagenic
Escherichia

isolated from foods, and their antimicrobial
susceptibilities.




1.1

AIM AND OBJECTIVE

To isolate
Escherichia

from domestic
food sold

in
Imo Poly
.
















CHAPTER TWO

2.0


LITERATURE REVIEW


While
food

of all types contains microorganisms that are harmless both from
sanitary and from technical points of view, the quality of
food

may depend
decisively on the microbial content.
Food

can be rendered unsatisfactory by the
microorganisms it contains. One of the most common faecal indicator organism
found in
food

is the
Escherichia
.

2.1

Escherichia
.

Domain

-

Bacteria

Plylum

-

Proteoba
cteria

Class

-

Gamma Proteobacteria

Order


-

Coccidioides

Family

-

Enterobacteriaceae

Genus

-

Eschericia

Species

-

Escherichia


Escherochia coli commonly abbreviated
Escherichia
, named after Theodor
Escherich a German pediatrician and bacteriologist i
s a gram
-
negative rod
-
shaped
bacterium that is commonly found in the lower intestine of warm
-
blooded
organisms (endotherms). Most
Escherichia

strains are harmless, but some, such as
serotype 0157:H7, can cause serious food poisoning in humans and are
occas
ionally responsible for product recalls (Feng et al, 2002). The harmless
strains are part of the normal flora of the gut, and can benefit their hosts by
producing vitamin K2 (Bently and Meganathan, 2007) and by preventing the
establishment of pathogenic ba
cteria within the intestine.
Escherichia

are not
always confined to the intestine, and their ability to survive for brief periods
outside the body makes them an ideal indicator organism to test environmental
samples for feacal contamination (Thompson and A
ndrea, 2007).



The bacteria can also be grown easily and their genetics are comparatively
simple and easily manipulated or duplicated through a process of metagenics,
making it one of the best


studied procaryotic model organisms and an important
species

in biotechnology and microbiology

2.11

STRAINS

A strain of
Escherichia

is a sub
-
group within the species that has unique
characteristics that distinguish it from other Enterobacteria. These differences are
often detectable only at the molecular level; how
ever, they may result in changes
to the physiology or lifestyle of the bacteria. For example, a strain may gain
pathogenic capacity the ability to use a unique carbon source, the ability to take
upon a particular ecological niche or the ability to resist a
ntimicrobial agents.
Different strains of
Escherichia

are often host


specific, making it possible to
determine the source of faecal contamination in environmental samples (Thompson
and Andrea, 2007). For example, knowing which
Escherichia

strains are present in
a
food

sample allows to make assumptions about whether the contamination
originated from a human, another mammal or a bird (Bach et al, 2002).


New strains of
Escherichia

evolve through the natural biological process of
mutation and

through horizontal gene transfer (Lawrence and Ochman, 1998).
Some strains develop traits that can cause a bout of diarrhea that is unpleasant in
healthy adults and is often lethal to children in the developing world. More virulent
strains, such as 0157:H
7 cause serious illness or death in elderly, the very young or
the immuno
-
compromised (Institute of Medicine of the National Academy, 2002).

2.12

BIOLOGY AND BIOCHEMISTRY


Escherichia

is gram
-
negative, facultative anaerobic and non speculating cells
are typically rod
-
shaped and are about 2 micrometers (µm) long and 0.5 µm in
diameter, with a cell volume of 0.6


0.7 um3, it can live on a wide variety of
substrates.
Escherichia

uses mix
ed acid fermentation in anaerobic conditions,
producing lactate, succinate, ethanol, acetate and carbon dioxide. Some many
pathways in mixed
-
acid fermentation produce hydrogen gas, these pathways
require the levels of hydrogen to be low, as is the case whe
n
Escherichia

lives
together with hydrogen


consuming organisms such as methanogens or surface
reducing bacteria (Madigan and Markinko, 2006).


Optimal growth of
Escherichia

occurs at 370c (98.60f) but some laboratory
strains can multiply at temperatures
of up to 490c (120.20f) (Fotadar et al, 2005).
Growth can be driven by aerobic or anaerobic respiration using a large variety of
redox pairs, invading the oxidation of pyruvic acid, formic acid, hydrogen and
amino acids, and the reduction of substrates suc
h as oxygen, nitrate, dimethyl
sulfoxi and trimethylamine noxide (Ingledew and Poole, 1984).


Strains that posses flagella can swim and are motile. The flagella have a
peritrichous arrangement.
Escherichia

and related bacteria posses the ability to
transfe
r DNA via bacterial conjugation, transudation or transformation, which
allows genetic material to spread horizontally through an existing population. This
process led to the spread of the gene encoding shiga toxin from stigella to
Escherichia

0157:H7, carr
ied by a bacteriaphage (Brussow etal, 2004).

2.13

ROLES AS NORMAL MICROBIOTIOTA


Escherichia

normally colonizes an infant’s gastrointestinal tract within 40
hours of birth, arriving with food or
food

or with the individuals handling the child.
In the bowl,

it adheres to the mucus of the large intestine. It is the primary
facultative anaerobic of the human gastro intestinal tract. As long as these bacteria
do not acquire genetic elements encoding for virulence factors, they remain bening
commensals.



2.14

T
HERAPEUTIC USE OF NON PATHOGENIC
Escherichia

Nonpathogenic
Escherichia

strain Nissle 1917 also know as Mutaflor is used as a
probiotic agents in medicine, mainly for the treatment of various
gastroenterological diseases, including inflammatory bowel diseas
es.



2.21

GASTROINTESTINAL INFECTION

Certain strains of
Escherichia

such as 0157:H7, 0121 and 0104:H21 produce
potentially lethal toxins. Food poisoning caused by
Escherichia

is usually caused
by eating unwashed vegetables or undercooked meat. 0157:H7 i
s also notorious for
causing serious and even life


threatening complications such as hemolytic


uremic syndrome (HUS). This particular strains is linked to the 2006 united states
Escherichia

outbreak due to fresh spinach. Severity of the illness varies
considerably; it can be fatal, particularly to young children, the elderly or the
immune
-
compromised, but is more often mild. Earlier, poor hygienic method of
preparing meat in Scotland killed seven people in 1996 due to
Escherichia

poisoning, and left hun
dreds more infected.
Escherichia

can habour both heat
-
stable and heat
-
labile enterotoxins. The latter, termed LT, contains one A subunit
and five B subunits arranged into one holotoxin and is highly similar in structure
and function to cholera toxins. The
B subunits assist in adherence and entry of the
toxin into host intestinal cells, while the A subunit is cleared and prevents cells
from absorbing
food
, causing diarrhea. LT is secreted by the type 2
-
secretion
pathway (Chalmers etal, 2000).


If
Escherichia

bacteria escape the intestinal tract through a perforation for
example from an ulcer, a ruptured appendix or due to a surgical error and enter the
abdomen, they usually cause peritonitis that can be fatal without prompt treatment.
However,
Escherichia

are

extremely sensitive to such antibiotic as streptomycin or
gentromicin. This could change since, as noted below,
Escherichia

quickly
acquires drug resistance. Recent research suggests that treatment with antibiotics
does not improve the outcome of the dise
ase, and may infact significantly increase
the chance of developing heomolytic
-
uraemic syndrome (Wong etal, 2000)


Intestinal mucosa


associated
Escherichia

are observed in increased
numbers in the inflammatory bowel diseases, crohn’s disease and
ulcerati
v
Escherichia
tis. Invasive strains of
Escherichia

exist in high numbers in the
inflamed tissue and the number of bacteria in the inflamed region correlates to the
severity of the bowel inflammation (WHO, 2009).

Table 1:

VIRULENCE PROPERTIES Of
Escherichia


Enteric
Escherichia
are classified on the basis of serological characteristics and
virulence properties. Virotypes include:

NAME

HOST

DESCRIPTION


Enterotoxigenic
Escherichia


(ETEC)

Causative agent of diarrhea (without fever) in humans, pigs, sheep, goa
ts, cattle,
dogs and horses

ETEC uses fimbrial adhesions (projections from the bacteria cell surface) to bind
enterocyte cells in the small intestine.

The larger of the two proteins, LT enterotoxin in structure and function.

The smaller protein, ST entroto
xin causes CGMP accumulation in the target cells
and a subsequent secretive of fluid and electrolytes into the intestinal lumen.

ETEC strains are non


invasive, and they do not leave the intestinal lumen. ETEC
is the leading bacterial causes of diarrhea in children in the developing world, as
well as the most common cause of traveler’s diarrhea. Each year, ETEC cases of
diarrhea an
d 380, 000 deaths, mostly in children in developing countries.


Enteropathogenic
Escherichia


(EPEC)

Causative agent of diarrhea in humans, rabbits, dogs, acts and horses.

Like ETEC, EPEC also causes diarrhea, but the molecular mechanisms of
colonizati
on and etiology are different. EPEC lack fimbriae, ST and LT toxins, but
they utilize an adhesion known as intimin to bind host intestinal cells. This biotype
has an array of virulence factors that are similar to those found in shigella, and may
posses a s
higa toxin Adherence to the intestinal mucosa causes a rearrangement of
action in the host cell, causing significant deformation EPEC cells are moderately
invasive (i.e. they enter host cells) and elicit an inflammatory in response


changes in intestinal

cell ultras
-

structure due to “attachment and effacement” is
likely the prime cause of diarrhea in those afflicted with EPEC.


Enteroinvasive
Escherichia


(ELEC)

Found only in humans

ELEC infection causes a syndrome that is identical to shigellosis, w
ith profuse
diarrhea and high fever.


Enterohemorrhagic
Escherichia


(EHEC)

Found in human cattle, and goats,

The most famous member of this virotype is strain 0157:H7, which causes bloody
diarrhea and no fever, EHEC can cause hemolytic


uremic syndrome a
nd sudden
kidney failure. It uses bacterial fimbriae for attachment (
Escherichia

common piles

ECP) is moderately invasive and possesses a phage
-
encoded shiga toxin that can
elicit an intense inflammatory responds.


Entroaggregative
Escherichia


(EAEC)

F
ound only in humans

So named because they have fimbriae which aggregate tissue culture cells, EAEC
bind to the intestinal mucosa to cause
food
y diarrhea without fever


EAEC are
non invasive. They produce a hemolysis and an ST enterotoxin similar to that
of
ETEC




2.22

EPIDEMIOLOGY OF GASTROINTESTINAL INFECTION

Transmission of pathogenic
Escherichia

often occurs via fecal
-

oral transmission.
Common routes of transmission include.

Unhygienic food preparation

Farm contamination

Irrigation of crops with contaminated gray
food

or raw sewage, feral pigs on
cropland, (Thomas etal, 2007).

Direct consumption of sewage contaminated
food
.

Diary and beef cattle are primary reservoirs of
Escherichia

0157:H7 and they can
carry it symptomat
ically and shed it in feces. Food products associated with
Escherichia

outbreaks include raw ground beef, raw seed sprouts or spinach, raw
food
, unpasteurized juice, unpasteurized cheese and food contaminated by infected
food workers via fecal
-

oral route.

The fecal
-
oral cycle of transmission can be
disrupted by cooking food properly, preventing cross contamination, instituting
brazier such as gloves for food workers, instituting health care policies so food
industry employees seek treatment when trap are i
ll, pasteurization of juice or
dairky products and proper hand washing requirements. (Bach et al, 2002) shiga
toxin
-
producing
Escherichia

(STEC) specifically serotype 0157:H7, have also been
transmitted by flies, as well as directs contact with farm animal
s, and airborne
particles found in animal rearing environments. (Pearson, 2007).

2.23

URINARY TRACT INFECTION

Uropathogenic
Escherichia

(UPEC) is responsible for approximately 90% of unary
tract infections (UTI) seen in individuals with ordinary anatomy (T
odar, 2007) in
ascending infections, fecal bacteria colonize the urethra and spread up the urinary
tract to the bladder as well as to the wings causing pyelonephritis or the prostate in
males. Because women have a shorter urethra than men, they are 14
-
time
s more
likely to suffer from an ascending UTI. (Todar, 2007) uropathogenic
Escherichia

utilize fimbriae (pyelonepnritis associated pill) to bind urinary tract endothelial
cells and colonize the bladder. These adhesions specifically bind D
-
gelatos
-

D
-
gelato
s moieties on the p blood group antigen of erythrocytes and uroepithelial
cells (Todar, 2007) Approximately 1% of the human population lacks this receptor
and it’s presence or absence dictates an individual’ susceptibility to
Escherichia

unary tract infect
ions. Uropathogenic
Escherichia

products alpha and beta
Hemolysins which cause lyses of unary tract cells.

UPEC can invade the body’s innate immune defenses (e.g the complement system)
by invading superficial umbrella cells to form intracellular bacterial

communities
(IBLS). (Justice et al, 2006) They also have the ability to form k antigen, capsular
polysaccharides that contribute to biofilm formation. Biofilm producing
Escherichia

are recalcitrant to immune factors and antibiotic therapy and are often
re
sponsible for chronic unary tract infections antigen producing
Escherichia

infections are commonly found in the upper unary tract (Todar, 2007).

Descending infections, though relatively rare, occur when
Escherichia

cells enter
the upper unary tract organs
(kidneys, bladder or urethras) from the blood stream.


2.24

NEONATAL MENINGITIS



It is produced by a stereotype of
Escherichia

that contains a capsular antigen
called k1. The colonization of the newborn’s intestines with these stems, that are
present in the mother’s vagina, lead to bacteriemia, which leads to meningitis and
because of the absence of the I gm antibodies from the m
other, plus the fact that
the body recognizes as self the k1 antigen, as it resembles the cerebral
glycopeptides, this leads to severe meningitis in the neonates.

2.3

ANTIBIOTIC THERAPY AND RESISTANCE


Bacterial infections are usually treated with antibiot
ics. However, the
antibiotic sensitivities of different strains of
Escherichia

vary widely as gram
-
negative organisms,
Escherichia

are resistant to many antibiotics that are effective
against gram
-
positive organisms. Antibiotics which may be used to treat
Escherichia

infection include amoxillin as well as other semi
-
synthetic penicillins,
many cephalosporin, carbapenems, aztreonam, trimethoprim
-
sulfamethoxa
-
zole,
ciprofloxacin nitrofurantoun and the aminoglycosides.


Antibiotic resistance is a growing probl
em. Some of this is due to overuse of
antibiotics in humans but some of it is probably due to the use of antibiotics as
growth promoters in food of animals (Johnson et al, 2006). Antibiotic
-
resistant
Escherichia

may also pass on the genes responsible for a
ntibiotic resistance to
other species of bacteria, such as staphylococcus aureus.
Escherichia

often carry
multi drug resistant plasmids and under stress readily transfer those plasmids to
other species. Indeed,
Escherichia

is a frequent member of biofilms
where many
species of bacteria exist in close proximity to each other. This mixing of species
allows
Escherichia

strains that are paliated to accept and transfer plasmids from
and to other bacteria. Thus
Escherichia

and the other enterobacteria are importa
nt
reservoirs of transferable antibiotic resistance.

2.31

BETA


LACTAMASE STRAINS

Resistance to beta
-
lactam antibiotics has become a particular problem in recent
decades, as strains of bacteria that produce extended
-
spectrum beta
-
lactames have
become more
common. These beta
-
lactamase enzymes make many, if not all, of
the penicillin’s and cephalosporin’s ineffective as therapy. Extended
-
spectrum
beta
-
lactamase

producing
Escherichia

are highly resistant to an array of antibiotics
and infections by these strai
ns are difficult to treat. In many instances, only two
oral antibiotics and a very limited group of intravenous antibiotics remain
effective.

2.32

PHAGE THERAPY

Phage therapy
-
viruses that specially target pathogenic bacteria has been developed
over the las
t 80
-
years primary in the former soviet union where it was used to
prevent diarrhea caused by
Escherichia

presently, phage therapy for humans is
available only at the phage therapy center in the republic of Georgia and in Poland
(Girard et al, 2006). Howev
er, on January 2, 2007 the united states FDA gave
omnilytics approval to apply it’s
Escherichia

0157:H7 killing phage in a mist,
spray or wash on live animals that will be slaughtered for human consumption. The
bacteriophage T4 is highly studied phage that

targets
Escherichia

for infection.

2.33

VACCINATION

Researchers have actively been working to develop safe, effective vaccines to
lower the worldwide incidence of
Escherichia

infection. In March 2006, a vaccine
eliciting an immune response against the
Esc
herichia

0157:H7 specific
polysaccharide conjugated to recombinant exotoxin A of Pseudomonas aerugiosa
(0157
-
rEPA) was reported to be safe in children two to five years old. Previous
work had already indicated that it was safe for adults (Ahmed et al, 2006
). A phage
111 clinical trial to verify the large
-
scale efficacy of the treatment is planned.


In January 2007, the Canadian bio
-
pharmaceutical company, Bioniche
announced it has developed a cattle vaccine which reduces the number of 0157:H7
shed in manure

by a factor of 1000, to about 1000 pathogenic bacteria per gram of
manure.


In April 2009 a Michigan state university researcher announced that he has
developed a working vaccine for a strain of
Escherichia
.

2.4

ROLE IN BIOTECHNOLOGY

Because of it’s long

history of laboratory culture and ease of manipulation,
Escherichia

also plays an important role in modern biological engineering and
industrial microbiology (Lee, 1996). The work of Stanley Norman Cohen and
Herbert Boyer in
Escherichia
, using plasmids an
d restriction enzymes to create
recombinant DNA became a foundation of biotechnology (Russo, 2003).


Considered a very versatile host for the production of heterogonous preteens,
researchers can introduce genes into the microbe using plasmids, allowing fo
r the
mass production of proteins in industrial fermentation processes. Genetic systems
have also been developed which allow the production of recombinant proteins
using
Escherichia
. One of the first useful applications of recombinant DNA
technology was th
e manipulation of recombinant of
Escherichia

to produce human
insulin. Modified
Escherichia

have been used in vaccine development,
bioremediation and production of immobilized enzymes.
Escherichia

cannot
however be used to produce some of the more large, c
omplex proteins which
contain multiple disulfide bonds and on particular, unpaired thiols, or proteins that
also require post
-
transnational modification for activity.

2.4 1

ENVIRONMENTAL QUALITY

Escherichia

bacteria have been commonly found in recreational
food

and their
presence of used to indicate the presence of recent fecal contamination, but
Escherichia

presence may not be indicative of human waste.
Escherichia

are
harbored in all warm
-
blooded animals: birds and mammals alike.
Escherichia

bacteria have also been found in fish and turtles. Sand and soil also harbor
Escherichia

bacteria and some strains of
Escherichia

bacteria have become
naturalized. Some geograp
hic areas may support unique populations of
Escherichia

and conversely some
Escherichia

strains are cosmopolitan. (Feng et al, 2002)

2.4 2 MODEL ORGANISM

Escherichia

is frequently used as a model organism in microbiology studies.
Cultivation strains
e.g.
Escherichia

k12 are well adapted to the laboratory
environment and unlike wild type strains, have lost their ability to thrive in the
intestine. Many lab strains lose their ability to form biofilms (Fux etal, 2005).
These f
eatures protect wild type strains from antibodies and other chemical attacks,
but require a large expenditure of energy and material resource.


In 1946, Joshua lederberg and Edward tatum first described the phenomenon
known as bacterial conjugation using
Escherichia

as a model bacterium, and it
remains the primary model to study conjugation.
Escherichia

was an integral part
of the first experiments to understand phage genetics, and early researchers such as
Seymour Benzer used
Escherichia

and phage T4 to
understand the topography of
gene structure. Prior to Benzer’s research, it was not known whether the gene was
a linear structure, or if it had a branching pattern.

Escherichia

was one of the first organisms to have it’s genome sequenced, the
complete geno
me of
Escherichia

k12 was published by science in 1997 (Frederich
et al, 1997)

2.5

ESCHERICHIA

AND
FOOD

CONTAMINATION

Escherichia

is a specie of bacteria naturally present in human and animal
excrement and is part of th
Escherichia
form group of bacteria. Th
is group of
bacteria and
Escherichia

in particular has been used as an indicator of the
bacteriological safety of
food

since it was first isolated from faeces in the late 19th
century.

Escherichia

is used as a
food

quality indicator because large numbers
of the
bacteria are always present in the feces of humans and other warm blooded
animals, but are not naturally found in
food
. Since these bacteria don’t live long in
food

once outside the intestine, their presence in
food

means there has been recent
conta
mination through sewage discharge or other sources.

2.51

HOW DOES
Escherichia

ENTER OUR
FOOD
?

Food

can be contaminated in a variety of ways. Main sources of
Escherichia

are
municipal

sewage discharges or run off from failing septic systems, animal feed
operations. Farms and faeces deposited in woodlands from warm blooded animals
(such as pets in a park or on street) may be washed into creeks, river, streams,
lakes, or ground
food

durin
g rainfalls or snow melts. The contamination in
food

is
often highest immediately following a storm, because of the runoff. In addition,
infected bathers can unknowingly contaminate
food
, or contamination can occur
from boaters discharging wastes directly
unto the
food
. When these
food

are used
as sources of drinking
food

and the
food

is not treated or inadequately treated,
Escherichia

may end up in drinking
food
.


2.52

WHAT CAN INDIVIDUAL DO TO HELP?

To help avoid
Escherichia

from reaching our
food

suppl
y

Avoid going in the
food

if you have an often wound or an infection

Pick up pet dropping and dispose of item hygienically.

Avoid using fertilizers near recreational
food
.

Ensure septic systems are operating properly

Practice pollution
-
free boating by

disposing of human waste hygienically.

Make sure livestock are maintained a safe distance away from drinking
food

sources.

Ensure that drinking wells are properly maintained and capped to avoid
contamination

Encourage proper waste
food

treatment by your
municipality and local industries.

2.6

BACTERIOLOGICAL ANALYSIS OF
FOOD

There are methods to detect bacterial contamination of
food
. The chief objective is
to identify coliform organisms as
Escherichia
.

Their presence indicates that
food

contains fecal pol
lution and is unsafe for
consumption. Bacteriological
food

analysis is a method of analyzing
food

to
estimate the numbers of bacteria present and, if needed, to find out what sort of
bacteria they are. It is a microbiological analytical procedure which use
s samples
of
food

and from these samples, determines the concentration of bacteria. It is then
possible to draw inferences about the suitability of the
food

for use from these
concentrations. This process is used, for example, to routinely confirm that
food

is
safe for human consumption or that bathing and recreational
food
s are safe to use.



The interpretation and the action trigger levels for different
food
s vary
defending on the use made of
food
. Very stringent levels apply to marine bathing
food

whe
re much lower volumes of
food
s are expected to be ingested by users.


2.61

APPROACH

The common feature of all these routine screening procedures is that the primary
analysis is for indicator organisms rather than the pathogens that might cause
concern. Indicator organisms are used because even when a person is infected with
a more pathoge
nic bacteria, they will still be excreting many millions times more
indicator organisms than pathogens. It is therefore reasonable to surmise that if
indicator organism’s levels are low, then pathogen levels will be very much lower
or absent.

Judgments as
to suitability of
food

for use are based on very extensive precedents
and relate to the probability of any sample population of bacteria being able to be
infective at a reasonable statistical level of confidence.


Analysis is usually performed using cul
ture, biochemical and some times
optical methods (Wikipidia, 2010). When indicator organisms levels exceed pre
-
set
triggers, specific analysis for pathogens may then be undertaken and these can be
quickly detected (where suspected) using specific culture m
ethods or molecular
biology.

2.7

METHODOLOGIES


Because the analysis is always based on a very small sample taken from a
very large volume of
food
, all method reply on statistical principals.

2.71

MULTIPLE TUBE METHOD



One of the oldest methods is called

multiple tube method. In this method a
measured sub sample (perhaps 10ml) is diluted with 100ml of stente growth
medium and an aliguot of 10ml is then decanted into each of ten tubes. The
remaining 10ml. is then diluted again and the process repeated. At
the end of 5
dilutions this produces 50 tubes covering the dilution range of 1:10 through to
1:10000. The tubes are then incubated at a pre
-
set temperature for a specified time
and at the end of the process the number of tubes with growth in is counted fo
r
each dilution. Statistical tables are then used to derive the concentration of
organisms in the original sample. This method can be enhanced by using indicator
medium which changes colour when acid forming species are present and by
including a tiny tube

in each sample tube. This inverted tube catches any gas
produced. The production of gas at 37oc is a strong is a indication of the presence
of
Escherichia
.


2.72

ATP TESTING

An ATP test is the process of rapidly measuring active microorganism in
food

thro
ugh detection of a molecule called Adenosine Troposphere, or ATP.

ATP is a molecule found only in and around living cells and as such it gives a
direct measure of biological concentration and health. ATP is quantified by
measuring the light produced throug
h its reaction with be naturally occurring
firefly enzyme luciferase using a illuminometer. The amount of light produced is
directly proportional to the amount of biological energy present in the sample
(Luminultra. com, 2010)


2nd generation ATP test are
specifically designed for
food
, waste
food

and
industrial applications where, for the most part, sample contain a variety of
components that can interfere with the ATP assay.

2.73

PLATE COUNT


The plate count method relies on bacteria growing a colony on
a nutrient
medium so that the colony becomes visible to the naked eye and the number of
colonies on a plate can be counted. To be effective, the dilution of the original
sample must be arranged so that on average between 10 and 100 colonies of the
target b
acterium are grown. Fewer than 10 colonies makes the interpretation
statistically unsound whlist greater than 100 colonies often results in over lapping
colonies and imprecision in the count. To ensure that an appropriate number of
colonies will be generat
ed several dilutions are normally cultured.


The laboratory procedure involves making serial dilutions of the sample
(1:10,1:100, 1:1000 etc) in sterile
food

and cultivating these of nutrient agar in a
dish that is sealed and incubated. Typical media incl
ude plate count agar for a
general count or macCounkey agar to count gram
-
negative bacteria such as
Escherichia
. Typically one set of plates is incubated at 22oc and for 24 hours and a
second set at 37oc for 24 hours. The composition of the nutrient usuall
y includes
reagents that resist the growth of non
-
target organism and make the target
organism easily identified, often by a colour change in the medium. Some recent
methods include a fluorescent agent so that counting of the colonies can be
automated. At
the end of the incubation period the colonies are counted by eye, a
procedure that takes a few moments and does not require a microscope, as the
colonies are typically a few millimeters across.

2.74

MEMBRANE FILTRATION


Most modern laboratories use a refin
ement of total plate count in which
serial dilutions of the sample are vacuum filtered through purpose made membrane
filters and these filters are themselves laid on nutrient medium within sealed plates.
The methodology is otherwise similar to conventional

total plate counts.
Membranes have a printed millimeter grid printed on and can be reliably count a
much greater number of colonies under a binocular microscope.

2.75

POUR PLATES


When the analysis is looking for bacterial species that grow poorly on air
,
the initial analysis is done by mixing serial dilutions of the sample in liquid
nutrient agar, which is then poured into bottles, which are sealed and laid on their
sides to produce a sloping agar surface. Colonies that develop in the body of the
medium
can be counted by eye after incubation.


The total number of colonies is referred to as the total viable count (TVC)
the unit of measurement is cfu/ml (or colony forming units per millimeter) and
relates to the original sample. Calculation of this is a mul
tiple of the counted
number of colonies multiplied by the dilution used.

2.8

PATHOGEN ANALYSIS


When samples show elevated levels of indicator bacteria further analysis is
often undertaken to look for specific pathogenic bacteria. Species commonly
investig
ated in the temperate zone include Salmonella typhi and Salmonella
typhimurrium Depending on the likely source of contamination investigation may
also extend to organism’s such as Cryptosporidium spp. In tropical areas analysis
of vibro cholerae is also

routinely undertaken.

2.9

TYPES OF NUTRIENT USED IN ANALYSIS


MacConkey agar is culture medium designed to grow Gram
-
negative
bacteria and stain them for lactose fermentation. (wikipedia 2010). It contains bile
salt (to inhibit most gram
-
positive bacteria
), Crystal violet dye (which also inhibit
certain gram
-
positive bacteria), neutral red dye (which stains microbes fermenting
lactose), lactose and peptone.

Alfred
-

Theodore MacConkey developed it while working as a biologist for he
Royal Commission on se
wage Disposal in the united Kingdom. (wikipedia, 2010).


ENDO Medium contains peptone, lactose, dipotassium phosphate, agar,
sodium sulfite, and basic fuchsine and was originally developed for the Isolation of
salmonella typhi, but is now commonly used in
food

analysis. As in macConkey
agar, coliform organisms ferment the lactose and the colonies become red. Non
-
lactose
-

fermenting organisms produce clear, colourless colonies against the faint
pink background of the medium.


MFC medium is a medium used in m
embrane filtration, which contains
selective and differential agents. These include Rosolic acid to inhibit bacterial
growth in general, except for faced coliforms, Bile salts inhibit non
-
enteric bacteria
and Aniline blue indicates the ability of faecal co
liforms to ferment lactose to acid
that causes a pit change in the medium.


TYEA Medium contains tryptone, yeast extract, common salt and L
-
arabinose per liter of glass distilled
food

and is a nonselective medium usually
cultivated at two temperatures (22
oc and 36oc) to determine a general level of
contamination also know as colony count.







CHAPTER THREE

3.0

MATERIAL AND METHODS

The materials, media as well as their method of preparation and the list of reagents
used in this work are shown in the
appendix 1.

3.1

SAMPLE COLLECTION

Food

samples were collected from eight different tanker
food

vendors using sterile
universal sample bottles. The 20ml samples were collected in duplicates and eight
sample source were designated as A,B,C,D,E,F,G and H. The

sample were
analyzed within 6 hours from the time of collection

3.2

SAMPLE ANALYSIS

3.3

BACTERIOLOGICAL ANALYSIS OF THE SAMPLES


The membrane filtration" (MF) method was used to analyze the
food

samples

3.31

MEMBRANE FILTERATION TECHNIQUE

The undiluted
f
ood

sample was filtered through the Millipore membrane filter
(0.45µm). To do the filtration, sterile forceps was used to remove the sterile filter
paper from it’s packet and carefully placed on the base of the Millipore filtration
apparatus which had been

previously swabbed with ethanol and then placed when
the membrane had been put in place, the funnel is then sieved in position over the
membrane of the base of the section apparatus.
Food

was then poured into the
funnel up to 100ml. Margin. Section pressu
re was then applied and
food

filtered
from the funnel through the membrane filter into a collecting chamber.

After filtration the sterile forceps was used to remove the filter membrane and
placed on the surface of an already gelled MacConkey and Eosine Met
hylene Blue
(EMB) Agar. The plates were then incubated at 44oc for 24
-
hour. The pink
colonies on MacConkey plates and metallic blue sheen colonies on EMB agar were
isolated and sub
-
cultured in fresh agar plates incubated at 37oc for 24
-
hours to
obtain pure

culture. The pure cultures were then identified using biochemical tests.

3.4

BACTERIAL IDENTIFICATION

3.41

GRAMS STAINING:



The method used was that described by carpenter (1977) and Thomas
(1973). Smears of the isolates were prepared and heat fixed on

clean grease free
slides. The smears were stained for one minute with crystal violet. This was
washed out with a gentle running tap
food
. The slides were flooded with dilute
gram’s iodine solution. This was washed off with
food

and the smears were
decolor
ized with 95% alcohol till the blue colour no more dripped out (about 30
seconds).

The smears were then counter stained with Saffranin solution for about 10 seconds.
Finally, the slides were washed with tap
food

air
-
dried and observed under
immersion objec
tive.


3
.42

MOTILITY TEST


This test was used to determine which of the isolates were motile. Motility
test is usually used to differentiate motile organisms from non
-
motile ones. For this
test, the hanging drop technique was employed and the technique was

carried out
as described by Kirk. (Kirk et al, 1975).


A little Vaseline jelly was rubbed around the cavity of a hanging drop slide.
a drop of peptone
food

containing the pure culture was placed on a cover slide. The
hanging drop slide was then placed ove
r the drop of peptone
food

in such a way
that the center of the depression lies over the drop. The slide was quickly inverted
and viewed under the microscope, using oil immersion objective.






















CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.0

DISCUSSION

Escherichia

were isolated from four of the eight
food sold

examined. The isolation
of
Escherichia

from the
food

sample indicates that the source from where the
tanker vendors fetch the
food

they supply is feacally contaminated. The continued
su
pply of
food

by the tanker from the source is a great danger to public health. The
tanker itself is a potential danger to the public because even if he collects
food

from an uncontaminated
food

source, the tanker will contaminate the
food

because
the tanke
r vendors do not wash their tankers in between two deliveries. The
tendency is that once the tanker is contaminated it will continue to contaminate any
food

if fetches and supplies.


According to the work done in India by Siya Ram, Poormma and Rishi in
20
07, coliform bacteria were enumerated in portable
food

samples collected from
six locations in Lucknow, a major city in Northern India, using the most probable
number method.
Escherichia

(n=81) randomly isolated by membrane filtration
technique from four
sites, were identified by biochemical characterization.
Escherichia

were not detected in samples from two other sites. (Siya et al, 2007).


A similar situation was observed in the Netherlands in private drinking
food

supplies contaminated by
Escherichia

d
ue to defective sewage lines (schets et al,
2005).

5.1

CONCLUSION




The presence of
Escherichia

in potable
food

collected from domestic
food

suppliers may pose health risks to people using the domestic
food

supply for
drinking and other domestic purposes. Therefore, the presence of
Escherichia

in
drinking
food

distribution systems in
Imo Poly

requires increased surveillance for
risk assessment and prevention strategies to protect public health.









RE
FERENCES


Ahmed, A; Robbins,S and Szu, S. (2006). Safety And Immunogenicity

Of
Escherichia

01570 Specific Polysaccharide Conjugate Vaccine in 2
-
5
-
year old
Children. Journal of Infections And Disease. 193(4): 515
-
521.


Bach, S.J; Allister, M.C; Veira, T.A

and Cannon, V. (2002). Transmission

And Control Of
Escherichia

0157:H7. Cannadian Journal Of Animal Science.
82:475
-
490.


Barker, F.J and Breach, M .R.. (1976). Microbiology. 4th Edition. W.B.

Sounders Company, Philadelphia. Pp 57, 401


402.


Bentley,

R and Meganathan, R. (1982). Biosynthesis Of Vitamin

In Bacteria. Microbiology Journal. Rev. 46.241
-
280.


Brussow, H; Canchanya, C and Hardt, W. (2004). Phages And The

Evolution Of Bacterial Pathogens From Genomic Rearrangements To Lysogenic
Conversion.

Molecular Biology Journal. 68 (3): 560
-
602.


Chalmers, R; Aird, H and Botton, F.J. (2000).
Food

Borne Escherichia

Coli. Society for Applied Microbiology Symposium Series. (29):124


132.


Chukwura, M.O; Edema, M.O; Omemu, A.M and Fapetu, O.M. (2001).

Microbiology and Physiochemical Analysis of Different Sources of Drinking
food

in Abeokuta, Nigeria. Nigeria Journal of Microbiology. 15(1) 57


61.


Evans, J; Doyle, J; Dolores, G. (2007).
Escherichia
. Medical

Microbiology. 4th Edition. The University of

Texas.


Feng, P; Weagant, S; And Grant, M.(2002). Enumeration of Escherichia

coli and th
Escherichia
form Bacteria. Journal for food Safety and Applied Nutrient.
Retrieved 2007
-
01
-
25.


Fotadar, U; Zaveloff, P; And Terraco, L, (2005). Growth of Escherichia


coli at Elevated Temperatures. Journal for Basic Microbiology. 45(5): 403


404.


Frederick, R; Blathner, G; Craig, B. (1997). The Complete Genome

Sequence of
Escherichia
. Science Journal 277. (5331): 14553
-
1462.


Fux, C.A; Shirtliff, M; Costerton, J.W.

(2005). “Can Laboratory

Reference Strains Mirrors “real


world” Pathongenesis?” Trends Microbiology .
13(2): 58


63.


Girrard, M; Steel, D; Chaignat, C; Kieny, M. (2006). A Review Of

Vaccine Research And Development. Human Enteric Infections. Vaccine
Journal.
24. (15): 2732


2750.


Ingledew, W.J And Poole, R.K. (1984). The Respiratory Chains of

Escherichia
. Journal for Microbiology. 48 (3): 222


271.


Institute Of Medicine Of The National Academies. (2002). Escherichia

coli

0157:H7 In Ground Beef: Review of a Draft risk Assessment. Washington,
DC. The National Academics press.


Johnson, J; Kushowski, M; Menard, M; Gajewshi, A; xercavins, M;

Garau, J. (2006). Similarity between Human and Chicken
Escherichia

Isolated in
Relat
ion to Ciproploxacin Resistance Status. Journal of Infections and Diseases.
194. (1): 71


78.


Justice, S; Hunstad, D; Seed, P; Hultgrens, S; (2006). Famentation By

Escherichia

Subverts Innate Defenses During Urinary Tract Infection. Aced
Scrence Journa
l. U.S.A. 103. (52) 19884


19889.


Kirk, C.J; Feel, R. N; Dershew, R.J. (1975). Basic Medical Laboratory.

Pitman Medical Publishing Ltd, London. Pp. 121


122.


Lawrence, J.G. and Ochman, H, (1998). Molecular Archaelogy of the

Escherichia

genome. Natl A
ced Sci. U.S.A. 95: 9413


9417.


Lee, S.Y; (1996). High Cell


Density Culture of
Escherichia
. Trends

Biotechnology Journal. U.S.A. 14(3): 98


105.


Luminuttra.com. (2010). Methods of
Food

Analysis.


Maligan, M.T; and Markinko, J.M. (2006). Brock Biology

of

Microorganisms. 11th Edition. Person. U.S.A. 133


139.


Nataro, J.P and Kaper, J.P. (1998). Diarrheagenic
Escherichia
.

Clinical Microbiology Journal. Rev. 11(1): 142


201.


Okonko, I,O; Adeboye, O.D; Ogunnusi, T.A; Fajobi, E.A and Shiflit ,

O.B. (
2008). Microbiology and Physiochemical Analysis of Different
Food

Samples used for Domestic Purposes in Abeokuta and Ojota, Lagos, State, Nigeria.
Agricultural Journal of Biotechnology. 7(3) 6/7
-
621.

Pearson, H. (2007). The Dark Side of
Escherichia
.

Nature Journal

445. (7123): 8

9.


Russo, E. (2003). The Birth of Biotechnology. Nature Journal 421.

(6921): 456
-
457.


Salyers, A,A; Gupta, A and wang, V. (2004). Human Intestinal Bacteria

As Resources For Antibiotics Resistance Genes. Trends Microbiolo
gy. 12 (1): 412


416.


Schets, F.M; During, M; Italiander, R; (2005).
Escherichia

In

Drinking
Food

From Private
Food

Suppliers In The Netherlands. Wat Res Journal
.39:4485


4493.


Siya Ram; Poornima, V and Rishi, S. (2007). Contamination of Potable

Fo
od

Distribution Systems by Mutlcant Microbial Resistance Enterohemorrheagic
Escherichia
. Environmental Health Perspect. 116 (4): 448


452.


Speck, M.L. (1976). Compendium of Methods For The Microbiological

Examination Of Foods. American Public Health Ex
amination Association Inc.
Washington D.C. Pp 563


567.


Thomas, C.G. (1973). Medical Microbiology. 3rd Edition. Balicore

Tindali, London. Pp 8
-
9.


Thomas, R And Degregun. (2007). CHFI, Maddening Media

Misinformation on Biotech and Industrial Agricultu
re. Retrieved 2007
-
12
-
08.


Thompson and Andrea. (2007).
Escherichia

Thrives In Beach Sands. Live

Science Journal. Retrieved 2007.


Todar, U. (2007). Pathogenic
Escherichia
. Online Textbook Of Bacteriology.

University of Wisconsin. Madison Department
of Bacteriology. Retrieved 2007
-
11
-
30.


Wikipedia com. (2010). Bacteriological Analysis of
Food
.


Wong, C.S; Jelacic, S; Itabeeb, R.L. (2000). The Risk Of The Hemolytic



Uremic Syndrome After Antibiotic Treatment Of
Escherichia

0157:H7
Infections.England

Medical Journal. 342. (26): 1930


1936.


World Health Organization. (2009). Enterotoxigenic
Escherichia


(ETEC).


World Health Organization.(2005). The WHO Report. Make Every

Mother And Child Count. Genevia.










APPENDIX I

Instruments:

Autoclave

Microscope

Bijou Bottles

Conical flasks

Test tubes

Glass slides

Measuring cylinder

Pasture pipette

Durham tubes

Wire loop

Petri dishes



APPENDIX II

MEDIA AND REAGENTS. NUTRIENT AGAR

This is used in maintain pure culture of isolates in slants. It is a gen
eral media
capable of supporting growth of many microbial species.


Composition


Lab
-
Iemco
powder


Yeast
extracts



Peptone


Nacl

Agar

pH


Distilled
food





g/Litre



1g


2g


5g



5g


15g



7.4


100ml





Preparation

2.8g of the commercial available agar was added to 100ml of distilled
food
. This
was then heated to dissolve completely, then autoclaved at 121°C for 15 minutes at
the end of which the flask containing the medium was allowed to cool and then
poured in 20ml

amount into Petri dishes. The plates rotated to ensure even
distribution of the medium and then allowed to solidify.

MAcCONKEY BROTH



Composition



Sodium
taurocholate


Peptone

Lactose

Nacl

Bromocresol
indicator

Distilled
food

(single
strength)


pH


Distilled
food

(double
strength)

g/Litre

5g

20g

10g

5g


5g


100ml


7.4


500ml












Preparation

45g of the commercial available broth was added to 100ml of distilled
food
. The
ingredients contained in the flask were dissolved by shaking properly. No heat was
applied. The medium was then disposed into test tubes containing Durham tubes
and autoclaved at 121°C for 15 minutes. The doubles strength was the same
amount of ingre
dients dissolved in 500ml of distilled
food
.


.


PEPTONE
FOOD

MEDIUM

Used in inoculums enrichment and development for motility test


Composition



Peptone



Nacl



Distilled
food



pH



g/Litre



10g



5g



100ml


7.6


Preparation

For 100ml of medium, 1.5g of media is dissolved in 100ml of sterile
food

and
autoclaved at 121°C for 15
-
minutes. It is allowed to cool and then used.


EOSINE METHYLENE BLUE [EMB] AGAR

This is confirmatory test media



Composition



Peptone


Lactose


KH2P04



Eosine
Y


Methylene
blue

Agar
powder


g/Litre


10g

10g


2g



0.4g



0.0659



15g



Preparation

For 100ml of the media is dissolved in 10ml of
food

and the suspension is
dissolved by heating and sterilized in autoclave at 121°C for 15 minutes. The
molten media is allowed to cool to 45°C and then dispensed into sterile Petri
dishes. This solidified and it then used.


KOVAC's Reagent

A reagent used for

the Indole test, which is used to determine which organism has
the ability to split Indole from tryptophan present in buffered peptone
food
.


Composition


Amyl or 150 amyl
alcohol


P
-
dimethvl
-
amino
benzalidehvlole



Concentrated
hydrochloric acid
(H
CL)



g/Litre



150ml


10g


50ml




Preparation

Dissolve the aldehyde in the alcohol slowly add the acid prepare in small quantities
and store in refrigeration shake gently before use. 0.5ml kovac's reagent is added
on the 48
-
hrs peptone '
food

containing the isolates for organism.



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