ABSTRACT
Colibacillosis is considered important in the poultry industry because it generates economic losses due to the disturbance of growth, the decline in production, an increase number of culled chicken, and reduced quality of carcasses and eggs. It is caused by the Avian Pathogenic E. coli (APEC). To overcome this, antibiotics have been widely used to eliminate E. coli infection in poultry farms in recent years. Treatment with antibiotics has been considered as a vital regimen to control E. coli infection at the farm level for many years. However, high frequency of antibiotic resistance of E. coli isolates from chicken has become the center of attention due to public health importance. The aim of the present study is to determine antimicrobial susceptibility among a collection of avian pathogenic E. coli strains isolated from chicken. Multidrug resistant profiles against 10 different antibiotics of 24 E. coli isolates were determined by using disk diffusion method according to Clinical Laboratory Standard Institute (CLSI). Antibiogram revealed that 81.6% of the E. coli isolates showed multidrug resistant profiles to different antibiotics. Most of the E.coli isolates were highly resistant to cefuroxime (91.7%), followed with amoxiciline (87.5%), imipenem and cefotaxime (79.1%), cefexime (58.3%), ceftriaxone and nalidixic acid (25%). Out of 24 isolates tested, 5.9% were resistant to six antibiotics. These findings also demonstrated that most of the isolates were susceptible to antibiotics commonly used for E.coli infections treatment in poultry with lowest resistant score against ofloxacin (12.5%) and gentamycin (12.5%). Moderate resistant profiles were observed towards nalidixic acid and ceftriaxone (25%). High percentage of multidrug resistance was found among the E. coli isolated from chicken as an indicator to more serious problems in animal health. Therefore, continuous surveillance of antibiotic resistance profiles in chicken and other food animals is crucial to ensure food chain safety.
TABLE OF CONTENTS
Title
Page i
Certification ii
Dedication iii
Acknowledgements iv
Table
of Contents v
List
of Tables vii
Abstract viii
CHAPTER ONE: INTRODUCTION
1.1.
Aims and Objectives 7
CHAPTER TWO: REVIEW
OF RELATED LITERATURE 9
2.1. Antimicrobials and Antimicrobial resistance
9
2.2. Scope of antimicrobial resistance in Bacteria
10
2.3. Modes of action of Antimicrobial
11
2.4. Mechanisms of antimicrobial resistance in bacteria
12
2.5. Predisposing factors for development of antimicrobial
resistance 12
2.6.1 Phenotypic method 13
2.7. Diffusion Technique 13
2.8 Growth and Inactivation 14
2.9 Usage of chicken as the study animal 14
CHAPTER THREE: MATERIALS AND METHODS
3.1. Sample
Collection 16
3.2.
Isolation and identification 16
3.3. Antimicrobial
susceptibility testing 16
3.4.
Data management and statistical analysis 17
CHAPTER FOUR: RESULTS 18
CHAPTERFIVE: DISCUSSION AND CONCLUSION
21
Conclusion
24
Recommendation
25
References 26
LIST OF TABLES
Table
Title Page
4.1 Antibiotic susceptibility profile of
the E. coli isolates. 19
4.2 Antibiotic discs used to test E. coli and their respective
concentration. 20
CHAPTER ONE
1.1
INTRODUCTION
Escherichia coli is a Gram
negative bacteria belonging to the family Enterobacteriacae. Escherichia
coli found in the environment, intestinal tracts of humans and animals.
Most E. coli strains are harmless and are important part of healthy
human and animal intestinal tracts. However, some strains of E. coli have
acquired virulence attributes and are called pathogenic E. coli (Hessain
et al., 2013; Javadi et al., 2016). Pathogenic E.
coli are often grouped into one of six pathotype
categories: enteropathogenic, enterotoxigenic, enterohemorrhagic,
enteroinvasive, enteroaggregative and diffuse-adhering. While extra-intestinal
pathogenic E. coli (ExPEC) can be categorized as uropathogenic and
meningitis associated (Manges and Johnson, 2012). Extra-intestinal pathogenic E.
coli (ExPEC) are responsible for most community and hospital onset human
infections such as urinary tract and bloodstream infections, sepsis (Manges and
Johnson, 2012).
Escherichia coli is a Gram-negative, facultative
anaerobic, rod shaped bacterium of the genus Escherichia
that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Escherichia coli is one
of the most versatile bacterial species. It alternates between its primary
habitat, the gut of vertebrates, where it lives as a commensal (Tenaillon
et al., 2010),
and its secondary habitat, water and sediment. High density cultures of
several strains of E. coli are currently used to produce recombinant
proteins due to its high volumetric productivity, (Huang et al. 2012).
This, in practice, has transformed E. coli into a “factory” of
recombinant proteins and many pharmaceutical products are produced in this way.
Emergence
of multidrug resistant E. coli in food animals including chicken arise
due to the improper use of antibiotics, thereby reducing the clinical efficacy
to antibiotics commonly used in human and veterinary medicine (Wang et al., 2013).
The
extensive use of antibiotics in poultry as growth promoters and most
importantly for the control
and treatment of diseases have been attributed as the cause of the emergence of
bacteria with multidrug resistance associated with poultry. The emergence of
resistance has the potential to impact on the treatment and management of
infectious diseases in both animals and humans (Mamza et al., 2010).
The
discovery of antibiotics was a revolutionary achievement for both human and
veterinary medicine. Since the discovery, antimicrobials have cured humans and
animals from bacterial infections (Byarugaba, 2010). Antimicrobials can be
defined as any natural, synthetic or semisynthetic origin substance which kill
or inhibit the growth of microorganisms (Guardabassi and Maartens et al., 2011 Giguère, 2013). In
contrast, antibiotic refers to a low molecular weight substance produced by
microorganisms which act against another microorganism at low concentrations.
The term antibiotic has been used interchangeably with the term antimicrobial
in many instances (Giguère, 2013).
The
first antibiotic was penicillin discovered by Alexander Fleming in 1928 and was
first used therapeutically in the 1940s. However, treatment failures and
bacteria resistant to penicillin were first noticed immediately after the
discovery of penicillin, (Aminov, 2010; Byarugaba, 2010; Ventola, 2015). Even
Alexander Fleming warned that bacteria could become resistant to these
remarkable drugs in his Nobel Prize speech in 1945 (WHO, 2014). Indeed, each
new antibacterial drug development has been followed by the detection of
resistance to it. Antibiotic resistance is defined as the ability of microbes
to resist the effects of drugs, as a result the drugs become ineffective to
neither kill nor inhibit the microbes (CDC, 2015; WHO, 2014). Antimicrobial
resistance in bacteria results from selection pressures applied by
antimicrobial use (WHO, 2014). Use of antibiotic can trigger the antimicrobial
resistance by exerting selection pressure on bacterial strains.
The
emergence and rapid spread of antimicrobial resistance is now a global concern
(Laxminarayan et al., 2013; Ventola,
2015; WHO, 2014). In addition to the prospect of untreatable infections,
antimicrobial resistance results in higher economic costs due to longer
hospital stays in infected patients, the requirement for additional diagnostics
and more expensive drugs.
Prompt
diagnosis and treatment of colibacillosis are crucial to ensure optimal
productivity in poultry farms.
E.
coli is
a Gram-negative, facultative anaerobic (that makes ATP by aerobic respiration
if oxygen is present, but is capable of switching to fermentation or anaerobic
respiration if oxygen is absent) and nonsporulating bacterium. Cells are
typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25–1.0 μm
in diameter, with a cell volume of 0.6–0.7 μm3.
E. coli stains Gram-negative because its cell wall is composed
of a thin peptidoglycan layer and an outer membrane.
During the staining process, E. coli picks up the color of the
counterstain safranin and stains pink. The outer membrane surrounding the cell
wall provides a barrier to certain antibiotics such that E. coli is not damaged by penicillin. Strains that possess flagella
are motile. The flagella have a peritrichous arrangement.
Antimicrobial
resistance has emerged in the past few years as a major problem and many
programs have been set up for its surveillance in human and veterinary
medicine. These programs are aimed mainly at human pathogens, agents of zoonosis,
and indicator bacteria of the normal intestinal flora from animals. However,
little attention has been paid to the resistance in specific animal pathogens.
Limited studies on the ecology of E. coli have been reported,
particularly from developing countries (Rahimi and Nayebpour, 2012).
The
magnitude of the public health burden due to resistant food borne pathogens is
complex and is influenced by a number of variables such as antimicrobial use
practices in farming, process control at slaughter, storage and distribution
systems, the availability of clean water, and proper cooking and home hygiene,
among others. The major concern on the public health threat of food borne
illness is infection by antimicrobial resistant strains that lead to more intractable
and severe disease.
This situation is further complicated by the potential of
resistant bacteria to transfer their resistance determinants to resident
constituents of the human microflora and other pathogenic bacteria (Olatoye et
al., 2012). Several studies have suggested that foods might be a source of
human acquired antimicrobial-resistant E. coli. The food supply is an
established vehicle for certain other antimicrobial resistant and/or pathogenic
bacteria including E. coli (Oliver et al., 2011; Rahimi and
Nayebpour, 2012).
In developing countries of the world, where there is still an
alarming rate of insanitary conditions, malnutrition and poor health
facilities, there is an urgent need to study this organism and its
characteristics with an aim to reduce the human hazard caused by this emerging
pathogen (Isibor et al., 2013). Investigations of antibiotic resistance of E. coli are
useful to identify types of resistance present in the region and better
understand the challenge to establish appropriate and effective interventions
(Omulo et al., 2015). Unfortunately,
in Nigeria, little is known on the extent of antibiotic resistant strains of E.
coli in poultry farms, hence the risk of antibiotic resistance spreading
from poultry to humans or other animals cannot be estimated.
Poultry farms are considered to be among the most
profitable agricultural projects in the world. They are often referred to as
"the farms of the future" because of the large variety of possible
products and their expected high profit potential. Currently commercial farming
is growing up in about 100 countries in all continents and regions. Poultry
farming has been rapidly expanding in Nigeria to produce usable products such
as meat, hides, feathers, and eggs.
The sector of poultry production continues to
grow and to be more industrialized in many parts of the world. The amount of
chicken meat produced per year has been increasing worldwide for the last
decade (FAOSTAT, 2017).
An increasing human population, greater
purchasing power and urbanization have been strong drivers of this growth (FAO,
2016). Moreover, commercial poultry farms play an important role in meeting the
protein supply through the supply of eggs and meat (Jabir and Hague, 2010).
To alleviate poverty
and ensure sustainable food security, the efficient use of the available land
through poultry and small livestock production could be one of the options
(Mbuza et al., 2016).
The poultry industry has been developing quickly in the country,
with a rise in commercial poultry farms in Nigeria. This may imply a widespread
use of antibiotics in poultry productions to control and prevent bacterial
diseases but also to promote growth in poultry (Manishimwe et al., 2015).
Unfortunately, since the use of antibiotics in animals is poorly
regulated in Nigeria and poultry farmers lack trainings on animal health
management (Mbuza et al., 2016; Mbuza
et al., 2017), antibiotic resistance
could emerge from poultry farms. Normal intestinal flora of animals constitutes
an enormous reservoir of resistance genes that can be transferred to
pathogenic bacteria (Carlet, 2012). The emergence of resistant bacteria in the
gut may be an indicator for selection pressure exerted by antibiotic use in
each animal population (Carlet, 2012).
In Nigeria, studies on antibiotic resistance in poultry (Omulo et al., 2015) are scarce. But in other
African countries, several studies have established the prevalence of resistant
E. coli strains in poultry farms (Majalija et al., 2010; Naliaka, 2011; Hamisi et al., 2012; Alonso et al.,
2017). Other studies showed differences in the antibiotic resistance of E.
coli between various farming systems (Naliaka, 2011; Rugumisa et al., 2016).
Broiler and local chickens serve as the major sources of poultry
meat in Nigeria with consumption occurring independently of religious and
ethnic backgrounds (Omodele & Okere, 2014). Broiler chickens (Gallus
gallus domesticus) are gallinaceous domesticated fowl, bred and raised
mainly for meat production while local or indigenous chickens are free range
gallinaceous fowl which are generally hardy and have the ability to adapt to
adverse weather conditions and fluctuations in feed availability (Ajayi, 2010).
Broilers reach maturity in terms of size and weight faster than local chickens.
Poultry production, especially broiler and local chickens, accounts for a high
percentage of quality protein and in addition serves as a sources of revenue
for farmers and traders in Nigeria, (Emaikwu et al., 2011).
Escherichia
coli (E. coli) is one of the most
economically important bacteria which is responsible for early chick mortality
in poultry farms. Escherichia coli is normal microflora in the digestive
tract of animals and human, but certain strains that are pathogenic in birds
are called avian pathogenic E. coli (APEC). They are able to spread to
various organs and cause systemic and fatal colibasillosis (Barnes et al. 2013).
In
general, pathogenic E. coli in poultry has specific serotypes, mostly
consisted of serotype as follows: O78, O1, and O2, and under certain condition
includes O15 and O55 (Barnes et al.,
2013).
The
disease remains a major problem in the poultry industry because it generates
economic losses due to the disturbance of growth, the decline in production, an
increase in the number of culled chicken, and reduce quality of carcasses and
eggs (Barnes et al. 2013). Recently,
it has have been reported that E. coli serotype O1, O2 and O78
were identified from colibacillosis cases in poultry farms (Wahyuwardani et al. 2014). Serotyping of the antigens
is a very useful method for detecting pathogenic E. coli strains in
clinical specimens, foods, and environmental samples and for understanding the
epidemiology of the pathogen (Wang et al., 2010).
Avian
pathogenic E. coli is responsible for a disease syndrome referred to as
Colibacillosis in chickens. Colibacillosis occur as an acute fatal septicemia,
fibrinous lesions (airsacculitis, peritonitis and pericarditis) salphingitis
and swollen head syndrome (Raji et al., 2007; Messai et al.,
2013). Colibacillosis
account for high economic losses from decreased egg production, mortality,
morbidity, decreased feed conversion, decreased carcass weight, carcass
condemnation and costs incurred from prevention, control and treatment (Messai et
al., 2013).
In
this study, E. coli strains isolated from chickens droppings from
different poultry Farms in Umudike were analyzed to determine their
susceptibilities to antimicrobial agents used in veterinary and human.
1.2.
Aims and Objectives
1. To isolate E. coli, from
environmental specimens collected from poultry farms in Umudike.
2. To determine antimicrobial susceptibility among a collection of
avian pathogenic E. coli, isolated from environmental specimens
collected from poultry farms in umudike.
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