BETA LACTAMASE PRODUCTION BY ANTIBIOTIC RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM POULTRY BIRDS

ABSTRACT

The inappropriate use of antibiotic on poultry birds has made Staphylococcus aureus resistant to antibiotic which has become a problem in the medical sector. This study was aimed at evaluating the beta-lactamase production potentials of S. aureus isolates associated with various poultry birds. Swabs from the different birds studied (quail, pullet, Local birds, turkey and guinea fowl) were inoculated into Mannitol salt agar plate. A total of Eighty-two (82) S. aureus was recovered from a total of 100 birds sampled. A greater percentage of the S. aureus was obtained from turkey (100%) and guinea fowl (85%) respectively. The results of the antibiotic susceptibility profile revealed that the most effective antibiotic against the isolates was Oflaxacin with 100% susceptibility rate follow closely by gentamycin, recording 80% susceptibility. High level of resistance to Augmentin, Ceftazidime, Cloxacillin, Cefuroxime and Cefotaxime was recorded amongst the S. aureus isolates of the turkey S aureus isolates recovered from the birds, 57 (72.15%) were found positive to beta-lactamase production while 25 (31.64%) were negative to beta-lactamase production. The findings of this study therefore calls for proper caution to prevent transmission of antibiotic resistant S. aureus from poultry to humans which could further add to the already elevated antibiotic resistance problem.

TABLE OF CONTENTS

Table page                                                                                                                              i

Certification                                                                                                                            ii

Acknowledgements                                                                                                                iii

Abstract                                                                                                                                   iv

Table of Contents                                                                                                                   vii

List of Tables                                                                                                                          viii

CHAPTER ONE

INTRODUCTION

1.1       Background of study                                                                                                   1

1.2       Aim and objectives                                                                                                     4

CHAPTER TWO

LITERATURE REVIEW     

2.1       Staphylococcus aureus: characteristics and pathogenicity                                         5

2.2       Antibiotics resistance in poultry birds                                                                        7

2.3       Antibiotic resistance                                                                                                   8

2.4       Causes of antibacterial resistance                                                                               9

2.5       Top line view of β-lactamases                                                                                    9

2.6       β-lactamase origins                                                                                                     10

2.7       β-lactamases in gram-positive bacteria                                                                      14

2.8       β-lactamases classification                                                                                         16

2.9       Beta-lactamase-producing bacteria and their role in infection                                   17

2.10     Beta-lactamase inhibitors                                                                                           17

2.11     Beta-lactamase and beta-lactamase inhibitors                                                            18

CHAPTER THREE

3.1       Materials and methods                                                                                                19

3.2       Sterilization of materials                                                                                            19

3.3       Preparation of culture media                                                                                      19

3.4       Isolation of antibiotics-resistant Staphylococcus aureus                                            19

3.5       Purification of isolates                                                                                                19

3.6       Identification of the antibiotics-resistant Staphylococcus aureus                              20

3.7       Gram staining                                                                                                             20

3.8       Biochemical test                                                                                                         20

3.8.1    Catalase Test                                                                                                               20

3.8.2    Coagulase Test                                                                                                            21

3.8.3    DNase Test                                                                                                                 21

3.19     Antibiotics susceptibility testing                                                                                21

3.10     Test for beta-lactamase production                                                                            22

3.10.1  Beta-lactamase determination using acidimetric method                                           22

CHAPTER FOUR

RESUITS                                                                                                                               23

CHAPTER FIVE            

DISCUSION, CONCLUSION AND RECOMMENDATION

5.1       Discussion                                                                                                                   34

5.2       Conclusion                                                                                                                  36

5.3       Recommendation                                                                                                        36

LIST OF TABLES

Table                                Title                                            Page

4.1                  Frequency of isolation of S. aureus from poultry birds                                    27

4.2                Cultural Morphology and Biochemical characteristics of the isolates               28

4.3                 Beta-lactamase profile of S. aureus isolated from different birds                      35

LIST OF FIGURES

Figure                              Title                                             Page   

4.1                      Antibiotic susceptibility profile of the Quail bird isolates                    29

4.2                     Antibiotic susceptibility profile of the Pullet bird isolates                    30 

4.3                     Antibiotic susceptibility profile of the Local bird isolates                    31

4.4                     Antibiotic susceptibility profile of the Broiler isolates                         32

4.5                     Antibiotic susceptibility profile of the Guinea fowl isolates                 33

4.6                     Antibiotic susceptibility profile of the Turkey isolates                         34

INTRODUCTION

1.1       Background of study

Staphylococcus aureus is a normal nasal flora of humans and animals, although they are generally considered commensal bacteria; they have the potential to cause a number of infections which constitute health concerns for woman, new born, elderly, and immune compromised individuals (Gardete and Tomasz, 2014). In human, Staphylococcus aureus has been implicated in disease such as dermatitis, pneumonia, septicaemia, osteomyelitis and meningitis in both humans and swine, as well as bovine mastitis in cattle and bumble-foot disease in poultry (Quinn et al., 2000) In poultry, the disease conditions associated with Staphylococcus  vary with the site and route of inoculation in hatchery and poultry farms, and can infect the bones, joints, tendon sheaths, skin, sterna bursa navel, and yolk sac through breakage of the skin and mucosal membrane of the birds, The immune compromised ones are often more prone to Staphylococcal infections. Once in the host, Staphylococcus aureus invade the blood stream, resulting in systemic infection in multiple organs, there by influencing economic losses, which accrued as a result of weight loss, decreased egg production, lameness, mortality, and condemnation at slaughter (Altahat et al., 2012). Reports have shown that prevalence of enter toxigenic Staphylococcus aureus in  food  handlers that serves as vehicle for zoonotic dissemination of pathogenic Staphylococcus aureus among poultry from workers, communities and hospitals varies in industries and countries. In Japan, a retail survey performed between 2002 and 2003 found 17.6% of raw chicken meat infested with enter toxigenic Staphylococcus aureus; an indication of future and possible Staphylococcosis outbreak, which could influence increased mortality and morbidity. To control and manage these disease/pathogenic Staphylococcus aureus, inappropriate use of antibiotics is employed in both poultry farms and clinical setting. In poultry management, antibiotics are often used in animal food production for growth promotion and routine disease prevention without prescription or control measures. This has necessitated the development of drug resistant superbug such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin intermediate Staphylococcus aureus (VISA), and vancomycin resistant Staphylococcus aureus (VRSA), which are now known as major emerging public health problem (Bala et al., 2016). With increase in population density within a particular geographical location, the incidence of both community associated and hospital associated multidrug resistant Staphylococcus aureus have been observed to increase with time, regardless of hospitals size and control measures due to drug abuse and zoonotic transfer of resistance gene mainly located on mobile element, such as plasmids or prophages and transferable through horizontal gene transfer (Igwe et al., 2013).

In Nigeria, the antibiotics use by poultry farmers from various regions in Nigeria varied among studies. Among the most commonly used agents reported in literature include neomycin and gentamycin (Van et al., 2007) enrofloxacin and chlortetracycline (Adelowo et al., 2009), tetracycline and sulphonamide (Nahar et al., 2014) fluoroquinoloes (Oluwasile et al., 2014), as well as gentamycin and tetracycline (Adebowale et al., 2016). Based on responses received from respondents, it is evident that most of the poultry famers do not obtain information on antibiotics they use from qualified personnel.

             Staphylococcus aureus is a major pathogen that can cause various forms of diseases varying from simple to life-threatening infection in human population (lowy 1998, Diekema et al., 2001). The invasion of the host tissue by Staphylococcus aureus apparently involves the production of a formidable array of extracellular enzymes (invasion) which facilitates the invasive process. Some may occur also as cell associated proteins by breaking down primary and secondary defences of the host which can facilitate the growth and spread the pathogen. The damage of the host as a result of this invasive activity may become part of the pathology of an infection (Noble, 2010).                      

            Beta-lactam compounds such as penicillin continues to be one of the most frequently used drugs in veterinary medicines (Pitkala et al., 2007). Two primary resistance mechanisms to beta-lactams are noteworthy of Staphylococcus spp. The expression of beta-lactamase enzymes encoded by the blaz gene, and production of the penicillin-binding protein 2a (Pbp2a), resulting in a higher-level of resistance encoded by the mecA gene (Fuda et al., 2005). Prevalence of penicillin resistance in Staphylococci causing animal disease is most commonly due to the blaz gene (Pitkala et al., 2007).

Beta-lactam antibiotics are among the most frequently prescribed antibiotics worldwide in the control of Staphylococcus-aureus infection. They act on peptidoglycan synthesis by molecularly acting on transpeptidase and carboxypeptidase thereby disrupting cell wall formation of the pathogen. However, the efficacy of antibiotics for therapy have suffered a setback due to growing trend of multidrug resistant strains observed in the organism to Beta-lactam and other antibiotics (Lowy et al.,2003, Deurenberg and Stobberingh, 2008).

Different test can be performed to evaluate beta-lactamase production in Staphylococci. A qualitative procedure for detecting production of beta-lactamase is the usage of Nitrocefin disks. The reaction is based on the production of a coloured compound when the substrate (nitrocefin) is exposed to a beta-lactamase-producing- bacteria. The clover leaf test (CLT) is an alternative with high sensitivity and specificity for investigating beta-lactamase production in staphylococci (Bergan et al., 2007).

 In addition extended spectrum beta-lactamase (ESBL.) capable of hydrolyzig penicillins, broad spectrum cephalosporins and monobactams in enterobacteriaceae (David and Bonomo, 2005) are often located on plasmids that are transferable from strain to strain and between bacteria species (Rupp and Fey, 2003). Extended spectrum beta-lactamase (ESBL) producers have continued to draw attention globally with their attendant clinical failure to new generation antibiotics and nosocomial spread (Olowe et al., 2012). In addition rapid change over time in extended spectrum beta-lactamase has been observed with variations within geographic areas. Clinical outcomes data indicates that extended spectrum beta-lactamase are clinically significant and when detected, suggest the need for the use of appropriate antibacterial agents. Hence, this study was designed to isolate antibiotics resistant Staphylococcus aureus from poultry birds in Michael Okpara University of Agriculture, Umudike, and also evaluate B-lactamase production by the antibiotics resistant Staphylococcus aureus isolate.

1.2                  AIM AND OBJECTIVES

The aim of this study was to isolate antibiotic resistant Staphylococcus aureus from poultry birds in Michael Okpara University Of agriculture, Umudike, and also evaluate Beta-lactamase production by the Staphylococcus aureus isolated while the specific objectives were;

1.              To isolate and identify Staphylococcus aureus from the swabbed samples of different   

             Poultry birds.                               

3.         To determine the antimicrobial susceptibility profile of the isolate from the swabbed sample.

4.         To screen the Staphylococcus aureus isolate for beta lactamase production.

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