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
3.8 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
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
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
CHIGOZIE, E (2023). Antibiotic Susceptibility Profile And Resistant Genes Of Salmonella Species Isolated From Poultry Farms In Umuahia, Abia State. Repository.mouau.edu.ng: Retrieved Sep 08, 2024, from https://repository.mouau.edu.ng/work/view/antibiotic-susceptibility-profile-and-resistant-genes-of-salmonella-species-isolated-from-poultry-farms-in-umuahia-abia-state-7-2
EBUKA, CHIGOZIE. "Antibiotic Susceptibility Profile And Resistant Genes Of Salmonella Species Isolated From Poultry Farms In Umuahia, Abia State" Repository.mouau.edu.ng. Repository.mouau.edu.ng, 10 Aug. 2023, https://repository.mouau.edu.ng/work/view/antibiotic-susceptibility-profile-and-resistant-genes-of-salmonella-species-isolated-from-poultry-farms-in-umuahia-abia-state-7-2. Accessed 08 Sep. 2024.
EBUKA, CHIGOZIE. "Antibiotic Susceptibility Profile And Resistant Genes Of Salmonella Species Isolated From Poultry Farms In Umuahia, Abia State". Repository.mouau.edu.ng, Repository.mouau.edu.ng, 10 Aug. 2023. Web. 08 Sep. 2024. < https://repository.mouau.edu.ng/work/view/antibiotic-susceptibility-profile-and-resistant-genes-of-salmonella-species-isolated-from-poultry-farms-in-umuahia-abia-state-7-2 >.
EBUKA, CHIGOZIE. "Antibiotic Susceptibility Profile And Resistant Genes Of Salmonella Species Isolated From Poultry Farms In Umuahia, Abia State" Repository.mouau.edu.ng (2023). Accessed 08 Sep. 2024. https://repository.mouau.edu.ng/work/view/antibiotic-susceptibility-profile-and-resistant-genes-of-salmonella-species-isolated-from-poultry-farms-in-umuahia-abia-state-7-2