This study was conducted to determine the
antibiotic resistance profile of Escherichia coli isolated from apparently
healthy domestic livestock viz, cow, goats, and chicken from Akure, Ondo State
Nigeria, E. coli was isolated using Eosin methylene Blue Agar (EMB) and
identified by conventional microbiology technique. The isolate were tested
against 14 antibiotics using the disc diffusion method. A total of 42 different
antibiotics resistance profile were observed with each isolate showing
resistance to at least four or more drugs tested. Generally the E. coli
isolates showed resistance rates of 93.8% to ampicillin, 16.% to
chloramphenicol, 57.5% to cloxacillin, 75.5% to Erythromycin, 20% to
Gentamicin, 60.5% to penicillin, 19.5% to streptomycin, 25.8% to Ceftazidine,
45.8% to Cefuroxine,22.2% to cefixine 30.6% to Loxacin, 65.9% to augmentin, 26%
nitrofurantoin, 29.3% to ciprofloxacin, 70.3% to tetracycline. This study
showed that averages number of resistance phenotypes per isolate was
significantly higher for goat and cow compared with poultry. A significant
public health concern observed in this
study is that multi drug resistance commena E. coli strains may constitute a
potential reservoir of resistance genes that could be transferred to pathogenic
TABLE OF CONTENTS
Title page ii
Literature review 1
History of Escherichia coli 3
Escherichia coli in the gastrointestinal tract 3
Pathogenesis of Escherichia coli 3
Genome plasticity 5
Phylogeny of Escherichia coli strains 6
Therapeutic use of nonpathogenic E. coli 6
Role of normal micobiota 6
Roles in disease 6
Role in biotechnology 7
Model organism 7
Characteristics of Escherichia coli 8
History of antibiotics 8
Alexander fleming and the discovery of penicillin 9
Antibiotic resistance profile of Escherichia coli 10
Materials and Methods 12
Sample collection 12
Media preparation 12
Cultivation of Escherichia coli 13
Characterization and identification of E. coli 13
Indole test 13
Methyl red test 14
Citrate test 14
Fermentation of sugars 14
Antibiotics susceptibility test 14
Table 3.1 15
Table 3.2 16
Results analysis 17
are naturals substances secreted by bacterial and funji to kill other bacteria that
are competing for limited nutrients (Bud, 2007). The term antibiotic was first used
in 1942 by Selman Waksman and his collaborators in journal articles
to describe substance produced by a microorganism that is cribe substances
produced by a microorganism in high dilution. Many antibacterial components are
relatively small molecules with a molecular weight of less than 2000 atomic mass
units (Dorlands, 2010).
advances in medicinal chemistry, most of today antibacterial chemically are semi
synthetic modifications of various nautral compound (Nussbaum, 2006). These
include, for example, the beta-lactam antibacterial, which include the penicillins
(produced by fungi in the genus penicillum), the cephalosporins and the carbapenems.
In accordance with thus many antibacterial compound are classified on the basis
of chemical biosynthetic origin into natural, semisynthetic, and synthetic.
classification system is based on biological activity, in this classification, antibacterial
are divided into two broad group according to their biological effect micro-organism
bactericidal agent that kill bacteria and bacteriostatic agents that slow down
or stall bacteria growth (Nussbaum, 2006).
the first natural antibiotics discovered by Alexander fleming in 1928.
coli is the head of the bacterial family, entero bacteriaceae, the enteric
bacteria, which are facultatively anaerobic aram-negative rods that live in the
intestinal tracts of animal in health and disease. The entero bacteriaceae are among
the most important bacteria medically. A number of genera (e.g. salmonella, shigella,
yersinia). Several others are normal colonists of the human gastro intestinal tract
(e.g. Escherichia, Enterobacter, Klebsiella) but the bacteria
as well, may occasionally be associated with diseases of humans. (Kubitschek,
Escherichia coli is versatile and well-adapted to its characteristic
habitats. It can grow in media with glucose as the sole organic constituent
wild-type Escherichia coli has no growth factor requirements, and
metabiologically it can transform glucose into all of the macromolecular components
that make up the cell. The bacterium can grow in the presence or absence of oxygen
(O2) under anaerobic conditions it will grow by means of fermentation,
producing characteristic “mixed acids and gas” as end product. However, it can grow
by means of anaerobic respiration, since it is able to utilize No3,
No2 or fumarate as final election acceptors for respiratory election
transport processes. In part, E. coli to its intestinal (anaerobic)
and its extraintestinal (aerobic or anaerobic habitats) (Kubitschek, 1990).
coli can respond to environmental signals such as chimerical, pH,
temperature, osmolarity etc. in a number of very remarkable ways considering its
is a unicellular organism. For example, it can sense the presence or absence of
chemicals and gases in its environment and swim towards or away from them. Or it
can stop swimming and grow fimbriae that will specifically attach it to a cell
or surface receptor. In response to change in temperature and osmolarity, it can
vary the pore diameter of its outer membrane porins to accommodate larger molecules
(nutrients) or to exclude inhibitory substances. With its complex mechanisms
for regulation of metabolism the bacterium can survey the chemical contents in
its environment in advance of synthesizing any enzymes that metabolized these compounds.
It does not wastefully produce enzymes for degradation of carbon sources unless
they are available, and it does not produce enzymes for synthesis of metabolites
if they are available as nutrients in the environment (Ishii et al., 2009).
coli is a consistent inhabitant of the human intestinal tract, and it is
the predominate facultative organism in the human gastro intestinal tract, however,
it makes up a very small proportion of the total bacterial content. The
anaerobic bacteriodles species in the bowel out number E. coli by
at least 20:1. However the regular presence of E. coli. In the human
intestine and faces has led to tracking the bacterium in nature as an indicator
offaecal pollution and water contamination. As such, it is taken to mean that,
wherever E. coli is found there may be faecal contamination by intestinal
parasites of human (Fotadar et al.
SCIENTIFIC CLASSIFICATION OF ESCHERICHIA
Binomial name Escherichia
HISTORY OF ESCHERICHIA COLI
coli was first described by Theodor Escherich in 1885, as bacterium coli
commune, which he isolated from the faces of new borns. It was later renamed Escherichia
coli, and for many years the bacterium was simply considered to be a
commensal organisms of the large intenstine. It was not until 1935 that a strain
of Escherichia coli was shown to be the cause of an outbreak of diarrhea
among infants. (Bread et al., 2000).
gastrointestinal tract of most warm-blooded animals is colonized by Escherichia
coli within hours or a few days after birth. The bacterium is ingested
in foods or water or obtained directly from other individuals handling the infant
(Hudaults et al., 2001).
human bowel is usually colonized within 40 hours of birth. Escherichia can
adhere to the mucus overlying the large intestine once established, an Escherichia
coli strain may persist for months or years. Resident strains shift over
a long period (weeks to month), and more rapidly after enteric infection or antimicrobial
chemotherapy that perturbs the normal flora. The basis of these shifts and the
ecology of Escherichia coli in the intestine of humans are poorly
understood despite the vast amount of information on almost every other aspect
of the organism existence. The entire DNA base sequence Escherichia coli
genome has been known since 1997. (Hudaults et
Escherichia Coli in the Gastrointestinal
commensal E. coli strains that inhibit the large intestine of all
humans and warm-blooded animals comprise no more than 1% of the total bacterial
biomass. The E. coli flora is apparently in constant flux one
study on the distribution of different E. coli strains colonizing
the large intestine of women during a one year period (in a hospital setting)
showed that 52.1% yielded one serotype, 34.9% yielded two, 4.4% yielded three,
and 0.6% yielded four. The most likely source of new serotype of E-coli is acquisition
by the oral route. (Hudauits et al.,
Pathogenesis of Escherichia coli
700 antigenic types (serotype) of E. coli are recognized based on
O, H and K antigens. At one time stereotyping was important in distinguishing
the small number of strains that actually cause disease. Thus, the serotype
0157:H7 (0 refers o somatic antigen; H refers to flagelar antigen) is usually responsible
for causing Hus (hemolytic uremic syndrome). Nowadays, particularly for diarrheagenic
strains (those that cause diarrhea) pathogenic E. coli are classified
based on their unique virulence factors and can only be identified by these traits.
Hence analysis for pathogenic E. coli usually requires that the isolates
first be identified as E. coli before testing for virulence markers
(Brussow et al., 2004). Pathogenic
strains of E. coli are responsible for three types of infections in
humans. Urinary tract infection (UTI), neonatal meningitis, and intestinal diseases
(gastroenteristis). The diseases caused (or not caused) by a particular strain of
E-coli depend on distribution and expression of an array of virulence determinants,
including adhensins, invasins, toxins and abilities to withstand host defenses.
(Brussow et al., 2004).
coli encompasses an enormous population of bacteria that exhibit a very high
degree of both genetic and phenotypic diversity. Genome sequencing of a large number
of isolates of E. coli and related bacteria shows that a taxonomic
reclassification would be desirable. However, this has not been done, largely
due to its medical importance (Krieg et
al., 1984) and E. coli remains one of the most diverse bacterial
species: only 20% of the genome is common to all strains. (Lukjancenko et al., 2010).
strain is a sub-group within the species that has unique characteristic 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, 2002). For example,
knowing which E. coli strains are present in a water sample allow
researchers to make assumptions about whether the contamination originated from
a human, another manual or a bird.
common subdivision system of E. coli, but not based on evolutionary
relatedness, is by serotype, which is based on major surface antigens (O antigen:
part of lipopolysacchar ride layer: H: Flagellin; K antigen: capsule), e.g. 0157:H7
first complete DNA sequence of an E. coli genome (Laboratory strain
K-12 derivative mG 1655) was published in 1997. It was found to be a circular
DNA molecule 4.6 million base pair in length, containing 4288 annotated protein-coding
genes (organized into 2584 operons) seven ribosomal RNA (rRNA operons and 86 transfer RNA (tRNA) genes. The
coding density was found to be very high, with a mean distance between genes of
only 118 base pairs. The genome was observed to contain a significant number of
transposable genetic elements, repeat elements, (ryptic prophages and bacteriophage
remnants) (Blattner et al 1997).
over 60 complete genomic sequences of Escherichia and shigella
species are available. Comparison of these sequences shows a remarkable amount of
diversity; only about 20% of each genone represents sequences that are present
in every one of the isolates while approximately 80% of each genome can vary
among isolates (Lukjancenko et al., 2010).
Each individual genome contains between 4,000 and 5,500 genes, but the total number
of different gene among all of the sequenced E. coli strains (the
pan-genome exceed 16,000. This very large variety of component, genes has been interpreted
to mean that two-third of the E. coli pan-genome originated in
other species and arrived through the process of horizontal gene transfer. (Zhanjbayeva
et al., 2011).
all life forms new strain of E. coli evolve through the natural biological
processes of mutation, gene duplication and horizontal gene transfer in particular
18% of the genome of the laboratory strain MG1655 was horizontal acquired since
the divergence from salmonella. (Lawrence et
al., 1998). In microbiology all strains of E. coli derive
from E. coli K-12 or E. coli B strain some strain develop
traits that can be harmful to a host animal (Nataro et al., 1998)
coli is the type species of genus (Escherichia and in turn Escherichia
is the type genus of the family Enterobacter + “1” (Sic) + “aceae”,
but from “enterobacterium” + “aceae” (enterobacteriaium
being not a genus, but an alternative trrural name to enteric bacterium). (George
al, 2005). The original strain described by Escherichia is believed
to be lost, consequently a new type strain (neotyped was chosen as a representative;
the neotype) strain is ATCC 11775, also known as NCTC 9001 which is pathogenic to
chickens has an 01: K1: H7 serotype. However, in most studies either 0157:H7 or
K-12MG1656 or K-12 W3110 are used as a representative E. coli (Migula,
Phylogeny of Escherichia coli
coli is a 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 that is the inferred evolutionary history as shown
below where the species is divided into six groups (Sims et al., 2011).
link between phylogenic assistance and pathology is small e.g. the 0157:H7
serotype strains, which form an exclusive group. Group E are all enterohaerogic
strains (EHEC), but not all EHEC strains are closely related. In fact four different
species of shigella are nested among E-coli strains while Escherichia
alberti and Escherichia fergusoni are outside of this group.
All commonly used research strains of E-coli belong to group A and are derived mainly
from (lifton’s K – 12 strain and to a lesser) degree from d’iterelle’s Bacillus
coli strain (B strain) (07).
Therapeutic use of Nonpathogenic E. coli
E. coli strain Nissle 1917 also know as mutaflor is used as
probiotic agent in medicine, mainly for the treatment of various gastro enterological
diseases, including inflammatory bowel disease (Kamada, 2005).
Role as Normal
coli normally colonizes an infant’s gastro intestinal tract with 40
hours 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
facilitative anaerobe of the human gastrointestinal tract (Todar K, 2007). (Facultative
anaerobes are organisms that can grow in either the presence or absence of oxygen).
As long as these bacteria do not acquire elements encoding for virulence
factors, they remain benign commensals. (Kamada, 2005).
Roles in Disease
strains of E. coli can cause gastroenteritis, urinary tract
infections and neonatal meningitis. In rarer cases, virulent strains are also responsible
for hemolytic-uremic syndrome, peritonitis mastitis, septicemia and Gram-negative
pneumonia (Todar, 2007).
(uropathogenic E. coli) is one of the main causes of urinary tract
infection. It is part of the normal flora in the gut and can be introduced many
ways. In particular for females, the direction of wiping after defecation can lead
to fecal contamination of the urogenital oritices. Anal sex can also introduce these
bacteria into the male urethra, and in switching from ancel to vaginal intercourse
the male can also introduce UPEC to the female urogenital system.
Role in Biotechnology
coli play an important role in modern biological engineering and industrial
microbiology (Research 2004). The work of stanely Norman cohen and Herbert Boyer
in E. Coli using plasmids and restriction enzymes to create recombinant
DNA become a foundation of biotechnology (Lee 1996).
coli is a very versatile host for the production of heterologous proteins
(Russo 2003) and various protein expression systems have been developed which allow
the production of recombinant proteins in E. coli one of the
first useful applications of recombinant DNA technology was the manipulation of
E. coli to produce human insulin (Cornelis, 2000)
proteins previously thought difficult or impossible to be expressed in E.
coli in olded form have also been successfully expressed in E. coli
E. coli cells have been used in vaccine development,
bioremediation and production of immobilized enzymes. (Ruso, 2003).
coli is frequently used as a model organism in microbiology studies cultivated
strains (e.g. E. coli K 12) are well adapted to the laboratory
environment, and unlike wild type strains, have lost their ability to thrive in
the intestine (Fredrick, 1997). Many lab strains lose their ability to form biofilms.
These features protect wild type strains from antibodies and other chemical attracts,
but require a large expenditure of energy and material resources.
1946, Joshua Lederberg and Edward Tatum first described the phenomenon known as
bacterial conjugation using E. coli as a model bacterium (Prigent 1998 and it remains
the primary model to study conjugation. E. coli was an integral part
of the experiments to understand phage genetics (Prigent 1998).
evaluating the possible combination of nanotechnologies with landscape ecology complex
habitat landscapes can be generated with details at the nanoscale. On such synthetic
eco-systems, evolutionary experiments with E. coli have been performed to study
the spatial biophysics of adaptation in an Inland biogeography on chip (Fredrick
Characteristics of Escherichia coli
coli commonly abbreviated as E. coli is a Gram-negative,
rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded
organisms (endotherms). Most E. coli strains are harmless,
but some serotype can cause serious food poisoning in human. (Escherichia
coli et al 2012). The harmless
strains are part of the normal flora of the gut, and can benefit their hosts by
producing vitamin K2 (Bentley and Meganathan, 2001).
is about 2 micro meter long and 0.5μm in diameter with a cell volume of 0.6 – 0.7(μm)3
(Kubitschek HE 1990). It can live on a wide variety of substrates. E. coli uses
mixed acid fermentation in anaerobic conditions, producing lactate, succinate,
ethanol, acetate and carbon dioxide. (Madigan et al 2006).
growth of E. coli occurs at 370c (98.6F) but some laboratory
strains can multiply at temperatures of up to 490C (120.20F)
Fotadar et al 2005).
can be driven by aerobic or anaerobic respiration, using a large variety of redox
pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino
acids, and the reduction of substances such as oxygen, nitrate, fumarate, dimethyl
sulfoxide and trimethylamine N-oxide (Ingedew 1984).
coli and elated bacteria posses the ability to transfer DNA via bacterial
conjugation, transduction or transformation which allow genetic material to spread
horizontally through an existing population. This process led to the spread of
the gene encoding shigatoxin from shigella to E. coli 0157:H7,
carried by a bacteriophage (Brussow H. 2004).
HISTORY OF ANTIBIOTICS
term antibiosis meaning against life was introduced by the French bacteriologist
Vuillemin as a descriptive name of the phenomenon exhibited by these early antibacterial
drugs. Antibiosis was first described in 1877 in bacteria when Louis Pasteur
and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus
anthraxis. These drugs were later renamed antibiotics by Selman Waksman,
an American microbiologist in 1942. (Dorland, 2010).
search for antibiotics began in the late 1800s, with the growing acceptance of
the germ theory of disease, a theory which linked bacteria and other microbes
to the causation of a variety of ailments. As a result, scientists began to devote
time to searching for drugs that would kill these disease causing bacteria. The
goal of such research was to find so called magic bullet’s that would destroy microbes
without toxicity to the person taking the drug (Grigorgan et al 2006).
1877, Louis Pasteur showed that the bacterial disease anthrax, which can cause
respiratory failure, could be rendered harmless in animals with the injection
of soil bacteria. In 1887, Rudolf Emmerich showed that the intestinal infection
cholera was prevented in animals that had been previously infected with the
streptococcus bacterium and then injected with the cholera bacillus. While
these scientist showed that bacteria could treat disease, it was until a year
later, in 1888, that the German Scientist E-de Fredenreich isolated an actual
product from a bacterium that had antibacterial properties. Freudenreich found
that the blue pigment released in culture by the bacterium bacillus pyocyanase
arrested the growth of other bacteria in the cell culture.
Alexander Fleming and the Discovery of Penicillin
the early 1920s, the British scientist Alexander reported that a product in human
tears could lyse bacterial cells. Fleming findings, which he called lysozyme,
was the first example of an antibacterial agent found in humans. Like pyocyanse.
Lysozyme would also prove to be a dead end in the search of an efficacious antibiotic
flaming generally receives credit for discovering penicillin, in fact technically,
fleming rediscovered the substance. Through follow-up work, fleming showed experimentally
that the mold produced a small substance that diffused through the agar of the plate
to lyse the bacteria. He named this substances penicillin after the penicillium
mold that had produced it. (Mcnulty et al.,
was not until about ten years after penicillin’s rediscovery in 1939, that Howard
florey. Ernst chain and Norman hearley picked up the project. Fleming were able
to overcome the technical difficulties that had plagued him, in the process spectacularly showing penicillin’s efficacy in the clinical
setting cross-continent cooperation in the early 1940s a resulted in the increased
scale of penicillin production.
is not surprising that initially penicillin was used almost exclusively to teat
soldiers injured during the war. That would change, though, with one fateful
penicillin’s most important clinical trial occurred after a fore at a Boston club, which resulted
in numerous burn victims being sent to Boston-area hospitals. At that time, it
was common for severe burn victims to die of bacterial infections, such as
those from staphylococcus. The success that physicians had in treating severely
burned victims that night was largely attributed to the effects of penicillin. (Goossens
et al, 2006).
1932, the German Gerhard Domage turned his attention away from natural antibiotics
and towards synthetic ones. Domagk who investigated the effects of different
channel dynes for their effects on bacterial infections, found that the protosil
cured diseases caused by the streptococcus bacteria when injected into infected
the time that florey and coworkers picked up the work on penicillin, the antibiotic
gramicidin was isolated from a soli-inhabiting microbe. Gramicidin, then first natural
antibiotic extracted from soil bacteria was able to arrest the growth of staphylococcus
but proved highly toxic.
1943, Selman Waksman and his group isolated another antibacterial agent from a
soil bacterium, streptomyces griseus. Waksman’s antibiotic, streptomycin,
proved effective against several common infections the microbe causing
tuberculosis, which had to that point resisted numerous methods of treatment. Streptomycin,
through, carried with it highly toxic side effects and a fast rate of mutation,
making it not a viable clinical option. (H. Gooszens and D. Guillemot, 2006).
Antibiotic Resistance Profile of Escherichia
are natural substances secreted by bacteria and fungi to kill other bacteria
that are competing for limited nutrient. The introduction of antibiotics after World
War I resulted in aromatic decrease of numbers of death due to bacteria infections
(R. Bud, 2007). As early as 1945,
fleming warned that the misuse of penicillin could lead to selection of resistant
forms of bacteria. Infact, fleming had already experimentally derived such strains
by varying the dosage and conditions upon which the added antibiotic to
bacteria. Fleming posted that resistance to penicillin could be conferred in
two ways either through the strengthening of the bacterial cell wall which the drug
destroyed or through the selection of bacteria expressing mutant proteins
capable of degrading penicillin.
usage is possibly the most important factor that promotes the emergence, selection
and dissemination of antibiotic – resistant microorganism, in both veterinary
and human medicine (Daniels et al,
2009). This acquired resistance occurs not only in pathogenic bacteria but also
in the endogenous flora of exposed individual. It is known that genes responsible
for antibiotic resistance are present in microorganism, providing them with self
protection to the antibiotic compounds they produce ass defence mechanism
against other microorganisms. Similarities among the genes and resistance mechanisms
found in the antibiotic producers and in human pathogenic bacteria are the pools
of origin of antibiotic resistance genes (Courvalin, 2008).
antibiotic selection pressure for bacteria drug resistance in the animal is
high and invariably their faecal flora contains a relatively high proportion of
resistant bacteria (Whitworth et al.,
2008). Colonization of the intestinal tract with resistant Escherichia coli
from chicken has been shown in human volunteers and there is historical evidence
that animals are a reservoir for E. coli found in humans in various countries has
been reported (Frang et al., 2008).
to Kurenbach et al, 2003 transfer of antibiotic
resistance genes from Gram positive Gram negative bacteria invitro is very rare
event. In vitro transfer of a naturally occurring Gram positive plasmid PIPSOI
in E. faecals to E. coli has been described. Widespread reliance on
antimicrobial in food animal production has resulted in a considerable rise of antimicrobial
resistant strains of bacteria, complicating the treatment of infectious diseases
in livestock, companion animals and humans. The selective pressure from the use
of antimicrobial agents at sub therapeutic level in dairy cattle could result
in the selection of those strains that contain genres for antimicrobial
resistance (Call et al 2008).
tools have been used to correlate animal associated pathogens with similar pathogens
affecting humans and to clearly demonstrate transferable resistance genes carried
by plasmids common to both animals and humans (Pitout et al., 2009).
the developed world, the extensive use of antibiotics in agriculture, especially
for prophylactic and growth promoting purposes has generated much debate as to whether
thus practice contributes significantly to increased frequencies and dissemination
of resistance genes into other ecosystems. In developing countries like Nigeria,
antibiotics are used only when necessary, especially if the animals fall sick,
and only the sick ones are treated in such cases. According to John and Fishman
(1997) will provide information on resistance trends including emerging antibiotic
resistance which are essential for clinical practice. This work was therefore, undertaken
to investigate the antibiotic resistance profile of E. coli
isolates from apparently healthy domestic livestock viz: cow, goat and chicken.
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