ABSTRACT
Antimicrobial resistance in bacterial pathogens of food animals has become a public health problem around the world. This is of particular concern Salmonella species isolated from poultry. The aim of this project was to investigate the antibiotic susceptibility profile and resistance genes from Salmonella sp. isolates from chickens in poultry farms in Umuahia, Abia State. A total of 200 specimens consisting of 100 cloacal swabs and 100 samples of fecal droppings were collected from broiler chicken using from 10 poultry farms in Umuahia, Abia State, Nigeria. The Specimens were inoculated in peptone water and subsequently enriched in Selenite F broth. Isolation was done by inoculating the enriched samples onto Salmonella Shigella agar. Confirmation of the presumptive Salmonella sp. isolates was carried out using different biochemical tests. Antibiotic susceptibility testing was performed using Kirby-Bauer disc agar diffusion method. Salmonella sp. isolates that showed resistance against third generation cephalosporin by disc diffusion method were selected for further detection of ESBL production using double disc synergy test. Detection of resistance genes was done with the Polymerase Chain Reaction for the following resistance genes: aada1, aac3-iv, bla-TEM, bla-CMY, qnra. A total number of 26 (13%) of Salmonella isolates were obtained. Of this, of 18(69.2%) were susceptible to Ofloxacin and 12(46.2%) to Ciprofloxacin. All the isolates were resistant to Augmentin, Cefuroxime while 18(69.2%) were resistant to Chloramphenicol and 20(76.9%) to Ceftriaxone. Nine (34.6%) isolates were extended spectrum beta lactamase (ESBL) producers. Multiple Antimicrobial Resistance Index values ranging from 0.2 to 0.6 with 21(80.8%) exhibiting resistance to two or more of the antimicrobial agents tested). Out of the 9 isolates screened for antibiotic resistance genes, bla-TEM was detected in 2(22.2%) isolates and aac-iv was detected in 1(11.1%). None of the isolates was positive for qnra, bla-CMY and aada1. This study showed a prevalence of the 13% of antibiotic resistance among Salmonella sp. isolates from poultry farms in Umuahia. Resistance to multiple antibiotics was high among the resistant isolates. However, the prevalence of the resistance genes tested was low among the isolates. This study suggests the need for surveillance of the emerging antimicrobial resistance in Salmonella species in poultry farms in Umuahia and to control the use of antibiotics in poultry production.
TABLE
OF CONTENTS
Title
Page i
Declaration
ii
Certification iii
Dedication iv
Acknowledgements
v
Table
of Contents vi
List
of Tables x
List
of Figure xi
Abstract
xii
CHAPTER 1:
INTRODUCTION
1.1 Background
of the Study 1
1.2 Statement
of the Problem 5
1.3 Objectives
of the Study 5
1.4
Justification of the Study
6
CHAPTER 2:
LITERATURE REVIEW
2.1 Salmonella 7
2.2 Taxonomy
of Salmonella 8
2.3 Epidemiology
and Pathogenesis of Salmonella
Species 9
2.4 Infections
Caused by Salmonella 10
2.4.1 Salmonellosis
11
2.4.2 Enteric
fever 12
2.4.3 Enterocolitis
12
2.4.4 Bacteraemia
13
2.5 Salmonella
Detection 13
2.5.1 Pre-enrichment
media 13
2.5.2 Enrichment
media 14
2.6 Molecular
Detection of Antimicrobial Resistance Genes 15
2.7 Salmonella
Incidence in Animals 16
2.8 Transmission
of Salmonella 18
2.9 Public
health significance of Salmonellosis 19
2.10 Treatment
of Salmonellosis 20
2.11
Antibiotics and Antimicrobial Resistance 21
2.11.1 Antibiotic
resistant Salmonella 22
2.11.2 Global
trends in resistance pattern 23
2.11.3 Antibiotic
resistance pattern of Salmonella in
Nigeria 23
2.12 Classification
of Resistance 24
2.12.1 Innate
(intrinsic) resistance 25
2.12.2 Acquired
(extrinsic) resistance 26
2.13 Mechanisms
of Antibiotics Resistance in Salmonella 26
2.13.1 Plasmid
mediated resistance 27
2.13.2 Reduced
membrane permeability 28
2.13.3 Modification of the target site 29
2.13.4 Rapid
extrusion or efflux pump 29
2.13.5 Chromosome
mediated-resistance 30
CHAPTER 3: MATERIALS AND
METHODS
3.1 Study Area 31
3.2 Collection of Samples 31
3.3 Isolation
and Identification of Salmonella Species 31
3.3.1 Culture
of the specimens 32
3.3.2 Gram
staining and microscopy 32
3.4 Biochemical Screening Test of Salmonella
Species 32
3.4.1 Triple
sugar fermentation test (TSI) 33
3.4.2 Urease
test 33
3.4.3 Citrate
utilization test 33
3.4.4 Indole
production 33
3.4.5 Methyl
red test 34
3.4.6 Voges
proskauer test 34
3.5 Susceptibility
Test of Salmonella Isolates 34
3.5.1 Antibiotic
susceptibility test 34
3.5.2 Standardization
of the inoculum 35
3.5.3 Inoculation
of test plates 35
3.5.4 Application
of discs to inoculated agar plates 35
3.5.5 Examination
of plates and interpretation of results 36
3.6 Screening for ESBLS Producing Salmonella
Strains 36
3.6.1 Phenotypic disc diffusion method for ESBLs
confirmation 36
3.7 Determination of Multiple Antibiotic
Resistance (MAR) index
37
3.8 Detection of Antibiotics Resistance Genes in Salmonella 37
Isolates by PCR
3.8.1 Bacteria
cell preparation 37
3.8.2 DNA
extraction using zymo research kits 38
3.8.3 PCR
Amplification 38
3.8.4 Agarose
gel electrophoresis 41
CHAPTER
4: RESULTS
4.1 Occurrence of Salmonella Isolates from Cloacal and
Fecal 42
Samples of Poultry Farms in Umuahia
4.2 Cultural and Biochemical Characteristics
of Isolates 44
4.3 Antimicrobial Susceptibility of Salmonella Isolates Obtained 46
from
Poultry Samples
4.4 Multiple antimicrobial resistance Profiles
of Salmonella 48
isolates
(n=26)
4.5 Extended Spectrum Beta Lactamases (ESBL)
Screening 50
and
Confirmation
4.6 Molecular Detection of Resistance Genes 52
CHAPTER 5: DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1
Discussion 56
5.2
Conclusions 63
5.3
Recommendations 64
References 65
LIST OF TABLES
3.1 Primers and the corresponding sequences
for 40
Salmonella isolates
4.1 Occurrence of Salmonella isolates from
cloacal and fecal 43
samples of poultry
4.2 Biochemical test of the isolates 45
4.3 Antimicrobial
susceptibility of isolates obtained from 47
poultry samples
4.4 Multiple antimicrobial resistance profiles
of Salmonella 49
Isolates
(n=26)
4.5
Frequency of multidrug resistance genotype patterns
exhibited 51
by the Salmonella isolates.
4.6 ESBL
profile of the Salmonella isolates 53
LIST OF FIGURES
1 Mechanisms
of antibiotics resistance 27
2 Efflux
pump 29
3 Agarose Gel
electrophoresis of genomic DNA 54
extracted from the isolates
4 Gel Electrophoresis of PCR amplicon of bla-TEM gene 54
in
Salmonella isolates
5 Gel
electrophoresis of PCR amplicon of aac(3)-iv
gene 54
in Salmonella isolates
6: Gel electrophoresis of PCR
amplicon of aac(3)-iv gene in Salmonella
Isolates 55
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Salmonella
species is an anaerobic bacteria with a gram-negative rod-shaped appearance.
They are constituent of varied group of bacteria that have been separated into
two groups, namely Salmonella enterica
and Salmonella bongori, consisting in
numerous serovars of many subspecies (Malorny et al., 2011). On the basis of biochemical and genomic
modifications, Salmonella enterica
can be further divided into enterica,
salamae, arizonae, diarizonae, houtenae and indica subspecies. Most Salmonella
are fermenters of lactose, consumers of hydrogen sulfite, negative to oxidase,
and positive to catalase.
Salmonella serovars
gallinarium and pullorum were a common cause of production losses to the
poultry industry in the first half of the 20th century (Cogan and
Humphrey 2003). However, disease associated with these two serovars in poultry
was successfully controlled through vaccination and led to their virtual
eradication by the mid 1970s. It has been postulated that the decrease in these
serovars in poultry provided a vacant niche which allowed Salmonella serovar
enteriditis to establish a foothold. In contrast to serovars gallinarium and
pullorum, enteriditis is not associated with disease in poultry but is very
commonly associated with human disease. By 1997, foodborne illness associated
with Salmonella serovar
enteriditis had risen from 10,000 to over 30,000 cases per year in the United
Kingdom and accounted for about 70% of human Salmonella infections.
It was shown that salmonellosis was associated with the consumption of poultry
and that phage type 4 of Salmonella serovar
enteriditis-related disease was specifically associated with the consumption of
shelled eggs. Similar enteriditis-associated epidemics were also observed in
other European countries and the USA at this time (Braden 2006, Patrick et al. 2004, Poirier et al. 2008, Wegener et al. 2003).
The primary habitat of Salmonella serovars is the intestinal tract of human and the farm
animals. It may also be found in intestinal tracts of wild birds,
reptiles, and occasionally insects. Feedstuffs, soil, bedding, litter and fecal
matter are commonly identified in farms as sources of contamination with Salmonella (Akoachere et al.
2009). At the other side, Salmonella
spp., in particular Salmonella serovar pullorum and Salmonella serovar gallinarum,
are collectively responsible for systemic acute and chronic diseases of chicks
and mature birds. Salmonellosis and colibacillosis are responsible for high
economic losses resulting from decreased egg production, mortality, morbidity,
reduced feed conversion, decreased carcass weight, carcass condemnation, and
prevention, control and treatment costs (Messai et al., 2013). Usually, infection is caused by predisposing factors
such as stress, mycoplasma or viral infections and adverse environmental
conditions (Kaniz et al., 2014). An estimated 1.3 billion
cases each year resulting in around 3 million deaths due to salmonellosis alone
occur worldwide in poultry farms (Nchawa and Bassey, 2015). Despite the
importance of poultry as the key ingredient in the human food chain, it has
also been identified as one of the most significant causes of food poisoning
due to Salmonella serovars causing
the majority of food born outbreaks around the world (Akond 2012; Akoachere et al.
2009; Kabir 2010). Products from poultry
are recognized as the major modes of Salmonella
species transmission that cause Salmonellosis. Efficient use of antimicrobials
has contributed to the growth of resistant Salmonella
species in poultry production.
In addition to promoting sanitation and hygiene, as
well as immunization and good nutrition, the use of antibiotics has given
substantial advantages in the human life expectancy. Nevertheless, the enhanced
use of antibiotics in both public and veterinary settings has contributed to
the rise of antibiotic resistance, posing a significant threat to the
protection of public health (Abdullahi et
al., 2014). The continued use of
drugs typically initiates selective pressure that facilitates the production of
antibiotic-resistant pathogens. One of the really significant reasons possible
for the creation of antibiotic-resistant microorganism strains is the excessive
use of these antibiotics in animal production settings, which has resulted in
the development of bacterial strains that were once contagious only to animals
and are now contagious to humans due to the provision of antibiotic-resistant
traits (Akond et al., 2012).
The dramatic and persistent rise in drug-resistant Salmonella strain production in recent
years has been recorded frequently and is of great importance in both
industrialized and emerging nations (Yemisi et
al., 2014). Animals contaminated with
Salmonella antibiotic-resistant
strains are major components of resistant determinants that find space for
human infection for the Salmonella serovars
(Akoachere et al., 2009). Salmonella
antibiotic resistant strains have been regularly recovered from foods of animal
origin in which poultry is of serious importance (Nchawa and Bassey, 2015).
While different techniques have been recommended for Salmonella antibiogram testing, the Kirby-Bauer disc diffusion
method is the traditional method used widely according to the Clinical and
Laboratory Standards Institute (CLSI, 2014) (Tsegaye et al., 2016). In order
to identify the effective drug of choice to destroy pathogens (bacteria),
antibiotic sensitivity testing against a pathogen such as bacteria is always
required. This term is essential since trends of vulnerability to
antimicrobials cannot be estimated and the development of drug resistance is
widely publicized in the world (Yemisi et
al., 2014; Akond et al., 2012).
However, non typhoid Salmonella infections are
self-limiting, but if illness persists and may become life-threatening the infected individual will be treated using
drug of choice obtained after performing antimicrobial susceptibility test
using (Nchawa and Bassey, 2015
).The routine practice of antibiotic use in domestic animals as a
mechanism of preventing and treating diseases, as well as promoting
development, is a big issue in the proliferation of antibiotic-resistant
bacteria, which are subsequently transmitted to humans across the food chain
(Akond et al., 2012). A large rise in the incidence of antimicrobial drug
resistance in Salmonella strains
seems to be of serious concern in developed and developing countries in modern
times (Akond et al., 2012; Abdullahi et al.,
2014).
Traditional identification methods
including phenotyping and serotyping are time consuming and labor intensive.
For these reasons, the use of PCR for identification of Salmonella serovars is an
attractive alternative to the most traditional techniques. Serotyping is a
basic biomarker to investigate the epidemiological situation of Salmonella infections and it is
commonly used to trace back the contamination sources during outbreaks. White et
al in 2001 developed the serotyping scheme that was based on the
flagella H, somatic O antigens and the observed phase-shift in flagella
antigen. This method is worldwide and it is considered as the standard method
for Salmonella
serotypes identification. The advantages of identifying Salmonella serotypes include
providing information about the disease severity, contamination source and the
resistance -pattern (Molbak et al.,
2006). Moreover, molecular techniques have been used to differentiate the
strains of Salmonella
isolates including repetitive intergenic consensus (ERIC) PCR, Random
Amplification of Polymorphic DNA (RAPD), Single Strand Conformation
Polymorphism (SSCP), hybridization and ribotyping-PCR (Anjay et al., 2015). Hence, this work was
designed to determine the molecular characterization and antibiotic resistance
of Salmonella species from poultry
farms in Umuahia.
1.2 STATEMENT OF PROBLEM
Salmonella
enterica has been established worldwide as the
primary cause of human and animal salmonellosis, with Salmonella serovar typhimurium
causing primarily human salmonellosis (Ifeanyi et al., 2014). Poultry has been recognized as a primary host of
animal salmonellosis caused by Salmonella
enterica serovar gallinarum (Ruban et
al., 2010). Sub-therapeutic use of antibiotics as feed additives has been
referenced as one of the selective forces for the development of antibiotic
resistance (Chashni et al.,
2009). The rise in antibiotics
resistance has been reported in the past years in Umuahia study area and still
remain a global problem today (Emmanuel et al., 2020.). The level of
drug susceptibility to Salmonella species in Umuahia is poorly
understood because antibiotic susceptibility testing is not often done.
1.3 OBJECTIVES OF THE STUDY
The main
objective of this study is to determine the antibiotic susceptibility profile
and characterize the resistance genes of Salmonella species isolated
from poultry farms in Umuahia.
Specific objectives
i.
To
isolate and identify Salmonella species from poultry farms in Umuahia
ii.
To
determine antimicrobial susceptibility profile of Salmonella isolates
obtained from poultry farms in Umuahia
iii.
To
determine the multiple antibiotic index of the isolates
iv.
To
evaluate the production of Extended Spectrum Beta-Lactamases by the resistant
isolates
v.
To
identify the resistance genes of Salmonella isolates
1.4 JUSTIFICATION OF THE STUDY
The motivation of this study lies in the urgent
need to document the attributable risk of antibiotic resistance of Salmonella
species and to
develop evidence based protocols for monitoring, preventing and managing
antibiotic resistance among poultry farms as it affects public health. There is
currently a paucity of published studies on antimicrobial susceptibility profile
of Salmonella
species from
poultry farms in Umuahia, Abia State, Nigeria. Findings from this study,
therefore, will provide a valuable reference to the scientific community and
body of knowledge at large as far as the right antimicrobial to be use in order to manage antibiotic resistance. Investigation from this study
will provide a critical appraisal of the current protocols for monitoring
antibiotic resistance of Salmonella species in poultry farms and will generate
recommendations to improve these protocols.
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