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

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