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Product Code: 00007202

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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-CMYqnra. 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.


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



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



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


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



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


5.1              Discussion                                                                                                       56

5.2              Conclusions                                                                                                     63

5.3              Recommendations                                                                                          64

References                                                                                                      65



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     









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.


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.


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


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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