ABSTRACT
The susceptibility patterns of E. coli and salmonella species isolated from pork intestine in Umuahia, Abia state, were used for this research work. A total of 49 samples were collected, 47 isolates were positive for E. coli while 29 isolates were positive for Salmonella species. The Kirby Bauer disc diffusion method was used for the antibiotic susceptibility test. A multi disc containing eight different antibiotic, Ceftazidime (CAZ), Cefuroxime (CRX), Gentamicin (GEN), Cefixime (CXM), Ofloxacin (OFL), Augmentin (AUG),Nitrofuratoin (NIT) and Ciprofloxacin (CPR) were used for this study. E.coli had the highest susceptibility (93.6%)) to Ofloxacin followed by Gentamycin (87.2%). All the E. coli isolates were resistant to Cefixime and Augmentin. For Salmonella species, the highest susceptibility was recorded against Gentamycin (68.9%) while the least susceptibility was recoreded against Nitrofuratoin (0%). The highest resistance was recorded against Ceftazidime and Cefixime with 44.8% each. All the Salmonella isolates were resistant to more than one antibiotic confirming their multi drug resistance. This result has public health significance with Salmonella and E. coli infections being zoonotic in nature
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of contents v
List of tables viii
Abstract ix
CHAPTER ONE
1.1 Introduction 1
1.2 Aims and Objectives 2
1.3 literature Review 2
1.4 History of E. coli 3
1.5 Diversity 5
1.6 Serotype 5
1.7 Genome plasticity 5
1.8 Neotype strain 6
1.9 Phylogeny 6
1.10 Roles as Normal Microbiota 6
1.11 Role in Disease 7
1.12 Uses of Non Pathogenic E. coli 7
1.13 Salmonella 7
1.13.1 History of Salmonella. 8
1.13.2 Salmonella
Nomenclature 8
1.13.3 Salmonella
as Disease Causing Agent 9
1.13.4 Durability 11
1.13.5 Source of Infection 11
CHAPTER TWO
2.1 Collection of Sample 13
2.2 Isolation and Identification of Isolates 13
2.3 Isolation and Confirmation of Salmonella 13
2.4 Procedure for Media Preparation 14
2.5 Biochemical Test and Procedures 14
2.6 Antibiotic Susceptibility Test 16
CHAPTER THREE
Results 18
CHAPTER FOUR:
4.0 Discussion 22
4.1 Conclusion 23
4.2 Suggestion for Further Studies 23
LIST OF TABLES
Table Title Page
1.
Identification
and Characterization of the Isolates 19
2.
The
Percentage Occurrence of the Sensitivity and Resistance of E. coli 20
Isolates
3.
The
Percentage Occurrence of the Sensitivity and Resistance Pattern of 21
Salmonella spp.
CHAPTER ONE
INTRODUCTION AND
LITERATURE REVIEW
1.1 Introduction
Pigs
are a major reservoir of bacterial zoonotic pathogens of which E. coli and Salmonella are the major occurring bacteria (Mc Dowell et al., 2007). Except for self consumption
and scientific research, pigs are slaughtered at slaughter houses which are
sometimes contaminated with the fecal material. E. coli and Salmonella
belong to a group of prioritized, extended list of food and water-borne
zoonoses (Cardoen et al., 2009)
making it necessary for food safety authorities to focus on them as most
relevant hazards in the food chain.
Food-borne
diseases are an important cause of morbidity and mortality worldwide. The contamination of meat with pathogens
constitutes a major public health concern (Cohen et al., 2007). In Nigeria, processing procedures and monitoring of
critical points in the meat production are not fully developed. Abattoir has
become a source of infection and pollution attracting domestic and wild carnivores
and rodents due to adequate slaughtering and disposal facilities (Adeyemo,
2002). Apart from incorrect processing procedures, marketing practice of meat
consumed by most populace is a major area of concern (Okoli et al., 2006a).
However,
previous studies have documented contamination of raw meat by bacterial
pathogen (Uche and Agbo, 1985). Epidemiological reports suggest that of diarrheal
illness which account for 36% of mortality cases in Nigeria (FAO/WHO, 2002).
Food
contamination with antibiotic resistant bacteria can also be a major threat to
public health. This is because the antibiotic resistance determinant can be
transferred to other pathogenic bacteria, potentially compromising the
treatment of severe bacterial infections. The susceptibility pattern and the
prevalence of antimicrobial resistance among food borne pathogens have
increased during recent decades (Van et
al., 2007). This increase is attributed to the selection pressure created
by using antimicrobial in food producing animals in addition to unregulated use
of antibiotics by humans in developing countries (Vanden Bogard et al., 2000).
1.2 Aims and Objectives
The
aim and objective of this study is:
·
To investigate the
prevalence of E. coli and Salmonella isolated
from pork intestines in Umuahia, Abia State.
·
To determine the
antibiotic susceptibility pattern of the E.
coli and Salmonella isolates.
1.3 Literature Review
E.
coli is a gram-negative, facultative
anaerobic, rod-shaped bacterium that is commonly found in the lower intestine
of warm-blooded organisms (endotherms).The cells are about 2.0 microns (µm)
long and 0.5 µm in diameter with a cell volume of 0.6-0.7µm (Kubitschek, 1990).
It can live on wide variety of substrates. E.
coli uses mixed acid fermentation in anaerobic conditions. Since many
pathways in mixed-acid fermentation produce hydrogen gas, these pathways
require the levels of hydrogen to be low, as in the case when E. coli lives together with hydrogen consuming
organisms such as methanogens or sulphate reducing bacteria (Madigan and Martinko,
2006). The optimal growth of E. coli
occurs at 370C (98.60F) but some laboratory strains can
multiply at temperatures up to 490C (120.20F) (Fotador et al., 2005). Strains that possess
flagella are motile. Their flagella have a peritrichous arrangement (Darnton et al., 2007).
Most E.
coli strains are harmless but some serotypes can cause serious food
poisoning in humans and are occasionally responsible for product recall due to
food contamination and hazard. The harmless strains are part of the normal flora
of the gut, and can benefit their host by producing Vitamin K2 (Bentley et al.,, 1982), and by preventing the
establishment of pathogen bacteria within the intestine (Hudault et al., 2001).
E.coli
and related bacteria constitute about 0.1% of gut flora (Eckburg et al., 2003), and fecal oral
transmission is the major route through which pathogenic strains, which are
strains that have acquired certain genetic materials the body for a limited
amount of time, which makes them ideal indicator organisms to test
environmental samples for fecal contaminations (Feng et al., 2002). There is however a growing body of research that has
examined environmentally persistent E.
coli which can survive for extended period outside the host (Ishii and Sadowsky,
2008).
The
bacterium can also be grown easily and inexpensively in a laboratory setting,
and has been intensively investigated for over 60 years. E. coli is the most widely studied prokaryotic model organisms and
an important species in the fields of biotechnology and microbiology where it
has served as the host organism for the majority of work with recombinant DNA.
1.4 History of E. coli
The
genera Escherichia and Salmonella diverged around 102 million years ago, which
coincides with the divergence of their host: the former being found in mammals
and latter in birds and reptiles (Battlistuzi et al., 2004). This was followed by a split of the Escherichian ancestor into five species
(E. alberti, E. coli, E. Fergusonii, E.
hermannii and E. vulneria).
In
1885, a German Pediatrician called Theodor Escherich, discovered this organism
in the faeces of healthy individuals and called it “Bacterium coli commune” due
to the fact it is found in the colon and early classification of prokaryotes
placed these genera based on their shape and motility (Haeckel et al., 1867), during this period, Erbst
Haeckel’s classification of bacteria in the kingdom monera was in place.
Bacterium coli was the type species of the now invalid genus bacterium when it
was revealed that the former type species “Bacterium triloculare” was missing
following the reclassification as Bacillus
coli by Migula in 1895 and later
reclassified in the newly created genus Escherichia
named after its original discoverer (Castellani and Chalmers, 1919). The genus
belongs in a group of bacteria informally known as “coliforms” and is a member
of the Enterobacteriaceae family (enterics) of the gamma proteobacteria (George
and Garrity, 2005).
Certain
strains of E. coli are a major cause
of food borne illness (diarrheal illness) such as enterotoxigenic E. coli
(ETEC), enteropothogenic E. coli
(EPEC), entero hemorrhagic E. coli (EHEC) also called shiga toxin
producing E. coli or (STEC), enteroaggregative
E. coli (EAEC or EAggEc), however E. coli (EHEC) bacteria can lead to
hemolyticurenic syndrome (HUS), a medical emergency that requires urgent
treatment (Rohde et al., 2011).
E. coli
and related bacteria posses the ability to transfer DNA via bacteria
conjugation, transduction or transformation, which allows genetic material to
spread horizontally through an existing population. This process of the gene
encoding shiga toxin from shigella to E.
coli 0157.H7 carried by a bacteriophage.
1.5 Diversity
E. coli
encompasses and enormous population of bacteria that exhibit a very high degree
of both genetic phenotypic diversity, and remains one of the most diverse
bacterial species which only 20% of the genome is common to all strains (Lukjencenko
et al., 2010). A strain is a
sub-group within the species that has unique characteristics that distinguish
it from other strains. These differences are often detectable only at the
molecular level; however, they may result in changes to the physiology or
lifecycle of the bacterium. 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 antimicrobial agents.
Different strains of E. coli are
often host specific, making it possible to determine the source of fecal
contamination in environmental samples (Feng et al., 2002). Such as knowing which E. coli strains are present in a water sample allows researchers to
make assumptions about whether the contamination originates from a human,
another mammal or a bird.
1.6 Serotype
The
most common subdivision system of E.coli that,
is not based on the evolutionary relatedness, is by serotype, which is based on
major surface antigens such as E.coli
0157:H7. (Orskor et al., 1977). It is
however common to cite only the serogroup i.e the O-antigen. At present, about
190 serogroups are known (Stenutz et al.,
2006) the common laboratory strain has a mutation that prevents the formation
of an o-antigen and is thus non-typeable.
1.7 Genome Plasticity
New
strains of E.coli evolve through the
natural biological process of mutation, gene duplication and horizontal gene
transfer, in particular 18% of the genome of the laboratory stain MG 1655 was
horizontally acquired since the divergence from salmonella (Lawrence and Ochman,
1998). However, in microbiology all strains of E.coli derive from E.coli
k-12 (B strains), some strains develop traits that can be harmful to host
animal. These virulent strains typically cause a significant diarrhea that is
unpleasant in healthy adults and is often lethal to children in the developing
world (Nataro and Kaper, 1998). More virulent strains, such as 0157:H7 cause
serious illness or death in the elderly, the very young or the immune
compromised (Hudault et al., 2001).
1.8 Neotype Strain
E. coli
is the type species of the genus (Escherichia) and in turn Escherichia is the
type genus of the family Enterobacteriaceae and should be noted that the family
name does not stem from the genus Enterobacter, but from enterobacterium
(though enterbacterium being not a genus, but an alternative trivial name to
enteric bacterium) (George et al.,
2005).
1.9 Phylogeny
E. coli
is species. A large number of strains belonging to this species have been
isolated and characterized. In addition to serotype, they can be classified
according to their phylogeny i.e the inferred evolutionary history where the
species is divided into six group (Brzuszkiewicz et al., 2011), namely; Group B2, Group D, Group E, Group B1, Group
A and Group B strain derivatives.
1.10 Roles as Normal Microbiota
E. coli
normally colonizes an infant’s gastrointestinal tract within 40hrs of birth,
arriving with food or water or with the individuals handling the child. In the
bowel, it adheres to the mucus of the large intestine. It is the primary
facultative anaerobe of the human gastro intestinal tract (Todar, 2007). Facultative
anaerobes are organisms that can grow in either the presence or absence of
oxygen. As long as these bacteria do not acquire genetic elements encoding for
virulence factors, they remain commensals (Evans et al., 2007).
1.11 Role in Disease
Virulent
strains of E. coli can cause
gastroenteritis, urinary tract infections, and neonatal meningitis. In rare
cases, virulent strains are also responsible for hemolytic-uremic syndrome,
peritonitis, mastitis, and septicemia and gram negative pneumonia (Todar, 2007).
UPEC (uropathogenic E.coli) is one of
the main causes of urinary tract infections. It is part of the normal flora in
the gut and can be introduced in many ways. In particular for females, the
direction of wiping after defecation (wiping back to front) can lead to fecal
contamination of the urogenital orifices. Anal sex can also introduce this
bacterium into the male urethra and in switching from anal to vaginal
intercourse the male can also introduce UPEC to the female urogenital system.
1.12 Uses of Nonpathogenic E. coli
Nonpathogenic
E. coli strain also known as Mutaflor
is used as a probiotic agent in medicine, mainly for the treatment of various
gastroenterological diseases including inflammatory bowel disease (Grozdanov et al., 2004).
1.13 Salmonella
Salmonella
is a genus of rod-shaped gram-negative, non-spore-forming, predominantly motile
enterobacteria with diameters of about 0.7 to 1.5μm, lengths from 2 to 5μm, and
flagella that grade in all directions (i.e peritrichous). They are
chemoorganotrophs obtaining their energy from oxidation and reductions using
organic source, and are facultative anaerobes. Most species produce hydrogen
sulphide, (Clark and Barret, 1987) which can readily be detected by growing
them on media containing ferrous sulphate, such as Triple Sugar Iron (TSI) or Salmonella
Shigella Agar (SSA). Most isolates exist
in two phases a motile phase 1 and a non -motile phase II. Cultures that are
non motile upon primary culture maybe switched to the motile phase using a
cragie tube.
Salmonella
is closely related to the Escherichia genus and are found worldwide in cold and
warm blooded animal (including humans) in the environment. They cause illness
such as typhoid fever, paratyphoid fever, and foodborne illness (Salmonellosis) (Ryan and Ray, 2004)
1.13.1 History of Salmonella
The
genus Salmonella was named after
Daniel Elmer Salmon, an American veterinary pathologist. While Theobald Smith
was the actual discoverer of the type bacterium (Salmonella enteric var. choleraesuis) in 1885, Dr. Salmon was the
administrator of the United States disease control Agency research program, and
thus the organism was named after him by Smith (Food safety A to Z reference
Guide, 2009). Smith and Salmon had been searching for the cause of common hog
cholera and proposed this organism as the causal agent. Later research, however
now show this organism (now known as Salmonella
enterica) rarely causes enteric
symptoms in pigs, and was thus not the agent they were seeking which was
eventually shown to be a virus. However, related bacteria in the genus Salmonella were shown to cause other
important infectious diseases. The genus Salmonella
was finally and formally adopted in 1900 by J. Lignieres for the many species of
Salmonella, after Smith first type
strain Salmonella cholera.
1.13.2 Salmonella Nomenclature
Initially,
each Salmonella species was named
according to clinical considerations (Kanffmann, 1941). Example; Salmonella typhimurium (Mouse typhoid
fever), S. choleraesuis (hog
cholera). After it was recognized that host specificity did not exist for many
species, new strains (Serovar or Serological Variants) received species names
according to the location at which the new strain was isolated later, molecular
findings led to the hypothesis that Salmonella
consisted of only one species (Le Minor and Popoff, 1987), S. enterica, and the serovar were classified into six groups,
(Evins et al., 1989), two of which
are medically relevant. But as this now formalized nomenclature (Tindal et al., 2005) is not in harmony with the
traditional usage familiar to specialist in microbiology and infectologists,
the traditional nomenclature is common. Currently, there are three recognized
species. S. enteric, S bongori and S. subterranean, with six main
subspecies: enteric (1), Salamae (II), Arizonae (IIIa), Diarrizonae (IIIb), Houtenae
(IV), and Indica (VI). Historically serotype V was bongori, which is now
considered its own species (Janda and Abbott, 2006). The serovar (i.e Serotype)
is a classification of Salmonella into
subspecies based on antigens that the organism presents. It is based on the
Kauffman white classification scheme that differentiates serological varieties
from each other (Porwollik, 2011). Serotypes are usually put into subspecies
groups after the genus and species, with the serovars capitalized but not
italicized: an example is Salmonella enteric
Serovar Typhimurium Newer methods for
Salmonella typing and subtyping
include genome-based methods such as pulsed field gel electrophoresis (PFGE),
multiple Loci VNTR Analysis (MLVA), Multilocus sequence typing (MLST) and
(multiplex) PCR-based methods (Achtman et
al., 2012).
1.13.3 Salmonella as Disease Causing Agent
Salmonella
infections are zoonotic and can be transferred between humans and non human
animals. Many infections are due to ingestion of contaminated food (Jantsch et al., 2011). In speaking of other Salmonella serotypes, Salmonella enteritis, typhoid and paratyphoid. S. enteritis can cause serious illness because of its special virulence
factors while Salmonella typhi is
adapted to human and does not occur in other animals. Salmonella species are facultative intracellular pathogens that
enter cells via macropinosomes (Kerr et
al., 2010). Enteritis Salmonellosis
or food poisoning Salmonella consist
of potentially every other serotype (over a thousand) of the Salmonella bacteria, most of which have
never been found in humans. These are encountered in various Salmonella species; most having never
been linked to a specific host, but can also infect humans. It is therefore a
zoonotic disease. The organism enters through the digestive track and must be
ingested in large numbers of before it can cause diseases in healthy adults.
Gastric acidity is responsible for the destruction of the majority of ingested
bacteria. Salmonellosis is a disease
caused by raw or undercooked food. Infection usually occurs when a person
ingests foods that contains a high concentration of the bacteria, similar to a
culture medium, the symptoms are usually mild, normally, no sepsis occurs, but
it can occur exceptionally as a complication in elderly or weakened patients
(Example, those with Hodgkin’s disease).
However,
infant and young children are much more susceptible to infection easily
achieved by ingesting a small number of bacteria. In infants, contamination
through inhalation of bacteria, laden dust is possible. After a short
incubation period of a few hours to one day, the bacteria multiply in the
intestinal lumen, causing an intestinal inflammation with diarrhea that is
often mucopurulent and bloody. In infants, dehydration can cause a state of
severe toxicosis. Extra intestinal localizations are possible, especially Salmonella meningitis in children,
osteitis etc.
Salmonella enteritis
(example enterica subspecies enteritidis) can cause diarrhea, which usually
does not require antibiotic treatment. However, in people at risk such as
infants, small children, the elderly, Salmonella
infections can become very serious, leading to complications. If these are not
treated, HIV patients and those with suppressed immunity can become seriously
ill. Children with sickle cell anemia who are infected with Salmonella may develop osteomyelitis.
Most
people with Salmonellosis develop
diarrhea, fever, vomiting, and abdominal cramps 12 to 72 hrs after infection
(Brown and Brown 1978). In most cases, the illness lasts four to seven days,
and most people recover without treatment. In some cases through, the diarrhea
maybe so severe, the patient becomes dangerously dehydrated and most be taken
to a hospital. At the hospital, the patient may receive intravenous fluids to
treat the dehydration and may be given medication to provide symptomatic relief
such as fever reduction. In severe cases, the Salmonella infection may spread
from the intensives to the blood stream, and then to other body sites and can
cause death, unless the person is treated promptly with antibiotics. The
elderly, infants and those with impaired immune systems are more likely to
develop severe illness.
1.13.4 Durability
Salmonella
bacteria can survive for weeks outside a living body, and they are not
destroyed by freezing (Sorrells et al., 1970). Ultraviolet radiation and heat
accelerate their demise; they perish after being heated 550C for
90mins, or 600C for 12 mins (Beuchat and Heaton, 1975). To protect
against Salmonella infection heating
food for at least then minutes at 750C is recommended, so the centre
of the food reaches this temperature.
1.13.5 Source of Infection
Injected
food, often gaining an unusual look or small, then is introduced into the
stream of commerce; (Mermin et al.,
1997)
-
Poor kitchen hygiene,
especially problematic in institutional kitchens and restaurants because this
can lead to a significant outbreak.
-
Excretion from either
sick or infected but apparently clinically healthy people and animals
(especially endangered are caregivers and animals).
-
Polluted surface water
and standing water (such as in shower hoses or unused water dispensers).
-
Unhygienically thawed
fowl (the meltwater contains many bacteria)
-
An association with
reptiles (pet tortoises, snakes, iguanas and frogs) but primarily aquatic
turtles is well described.
Salmonella
can survive for some time without a host, thus, they are frequently found in
polluted water, contamination from the excrement of carrier animal being
particularly important.
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