STUDIES ON Β-LACTAMASE PRODUCTION BY ANTIBIOTIC RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM HEALTH WORKERS.

ABSTRACTS

The aim of this study was to determine the prevalence of β-lactamase producing antibiotic resistance Staphylococcus aureus from health workers in Umudike. A total of hundred (100) nasal samples were collected from from male and female health workers in Umudike using sterilized swab sticks. The swabs were streaked directly on media plates, the plates were duplicated then incubated at 37°C for 24 to 48 hours. Staphylococcus aureus was identified using colonial morphology and biochemical features. From the results of 100 samples, Staphylococcus aureus isolates from nasal swabs showed highest frequency occurrence in female within the age of 21-30yrs with a frequency of 10(16.4 %) while the lowest occurred in male within the age of 51-60yrs with a frequency of 1(2.6 %). The antibiotic susceptibility profile of Staphylococcus aureus isolates was carried out using different antibiotic. The results showed that all the isolates were resistance to Ceftazidime and Augement 100%. While susceptibility to Ofloxacin and Gentamicin was 85.7% and 83.39%. Beta lactamase production among the isolates was highest in female works with 22 (52.4). The tube method and Filter paper method was effective and superior to the agar method. The iodometric tube method is simple, rapid and can be performed in any bacteriological laboratory.

TABLE OF CONTENTS

Title page                                                                                                                                ii

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of content                                                                                                                      vi

List of tables                                                                                                                           ix   

Abstract                                                                                                                                  x

CHAPTER ONE

1.0 Introduction                                                                                                                      1

1.1 Aim and Objectives                                                                                                          4

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1. Staphylococcus aureus                                                                                                    5

2.1.1 Epidemiology of Staphylococcus aureus                                                                      5

2. 1.2 Staphylococcus aureus Carriage and Disease                                                              6

2.1.3. Virulence Factors of S. aureus                                                                                     7

2.1.4 Pathogenesis of S. aureus Infections                                                                             7

2.1.5 Treatment and Prevention of S. aureus Infections                                                        8

2.2 Antibiotics                                                                                                                        9

2.2.1 Mechanism of Action of Antibiotics and Its Resistance                                               9

2.2.1.1 Enzymatic Inactivation                                                                                              10

2.2.1.2 Permeability Barrier                                                                           11

2.2.1.3 Altered Targets                                                                                   11

2.2.1.4 Metabolic Bypass                                                                               11

2.2.2 Resistance Based On Altered Receptors for a Drug                              12

2.2.3 Emergence of Antibiotic Resistance in Staphylococcus Aureus            13

2.3. Extended – Spectrum Beta-Lactamase                                                    15

2.4. Characteristics of ESBL and Their Groups                                             15

2.7. Detection of ESBL                                                                                   16

2.8 The Prevalence of S aureus Nasal Carriage                                             17

CHAPTER THREE

3.0     Materials and Methods                                                                         20

3.1 Collection of Samples                                                                              20

3.2 Media Used                                                                                               20

3.4 Bacteriological Analysis                                                                           20

3.4.1 Isolation of Staphylococcus aureus                                                       20

3.4.2 Identification Of Staphylococcus aureus                                                                                                       20

3.3.1 Gram Staining                                                                                       21

3.4 Biochemical Tests                                                                                    21

3.4.1 Catalase Test                                                                                          21

3.4.2 Coagulase Test                                                                                      21

3.5 Antibiotic Sensitivity Test                                                                        22

3.6 βeta-Lactamase Determination Using Acidometric Method                    22

CHAPTER FOUR

4. 0 Results                                                                                                     23                                                                                                                              

5.0. CHAPTER FIVE

5.1       Discussion, Conclusion and Recommendation                                  28                   

5.2       Discussion                                                                                           28                   

5.3       Conclusion                                                                                          29                   

5.4       Recommendations                                                                              30                                                                                                       

Reference

LIST OF TABLES

CHAPTER ONE

1.0 INTRODUCTION

Staphylococci are Gram positive bacteria that range from 0.5-1.5 μm in diameter and exist as irregular grape like clusters (Harris et al., 2002). These are one of the most prevalent bacteria in both human and animals (Persoons et al., 2009; Suleiman et al., 2013). Staphylococcus aureus is one of the most important pathogens colonizing the skin and mucus membranes of the nares in human and animals incriminated in different disease conditions ranging from minor skin infections, such as furunclosis and carbunclosis to severe and highly debilitating conditions such as pneumonia and endocarditis (Jensen and Lyon, 2009). The pathogen is endowed with a great variety of virulence markers, which include both structural and secreted products participating in pathogenesis of infection (Plata et al., 2009).

Staphylococcus aureus are part of human flora, and are primarily found in the nose and skin. Many strains of Staphylococcus aureus cause disease through the production of enterotoxins or through direct invasion and destruction of tissue. Infections caused by S. aureus remain a significant cause of mortality and morbidity in tropical countries (Parsek and Sigh 2003).  The principal site of staphylococcal colonization is the anterior nares. It has been observed that if repeated cultures are performed, up to 80% of adults are found to harbor S. aureus in the nose at one time or the other. However, in most persons, the carrier state is transient, but 20 to 40% of adults remain colonized for months or even years (Pantosti et al.,2007).

 Increased nasal colonization rates have been noted in insulin dependent diabetes, individuals on haemodialysis, those on ambulatory peritoneal dialysis, intravenous drug understand patients receiving routine allergy injections (Jensen and Lyon, 2009).

Staphylococci have a record of developing resistance quickly and successfully to antibiotics. This defensive response is a consequence of the acquisition and transfer of antibiotic resistance plasmids and the possession of intrinsic resistance mechanisms (Esan et al., 2009). Three species of clinical importance are Staphylococcus aureus, Staphylococcus epidermis and Staphylococcus saprophyticus. Of all these, Staphylococcus aureus is the major pathogen for humans. It’s pathogenic effect is characterized by it’s ability to hemolyse blood, coagulate plasma(a biochemical test that differentiates it from other non-pathogenic Staphylococcus, and produces a variety of extra cellular enzymes and toxins. Staphylococcus aureus is present in the nasal passages and throat, on the hair and skin of healthy individuals. Almost every person will have some type of Staphylococcus infection during a life time, ranging from severity of food poisoning or minor skin infections to severe life threatening infections.

Staphylococcus aureus has been recognized as a very important virulent and frequently encountered pathogen in clinical practice. It is an endogenous microorganism colonizing the nasal cavity, skin, gastrointestinal, anus and vaginal vulvae of healthy women (Onanuga et al.,2005). The capacity to produce human diseases had not diminished even with the introduction of antibiotics (Waldvogel, 1990).

S. aureus has been associated with different clinical conditions. For instance, it is still one of the most frequently encountered single bacterial species in hospitals and continues to be frequent cause of burns and wounds sepsis. It produces pustules, carbuncles, boils and impetigo. It frequently causes septicaemia, osteomyelitis, bacteraemia and otitis media (Emmerson, 1994; Shaposhnikbova et al., 1995). S. aureus exhibits remarkable versatility in their behavior towards antibiotics (Grassi,  2007).

Therefore, the insight into the antibiotic susceptibility of clinical isolates profile in any community is very imperative and desirable for effective management of the clinical conditions considering the relative differences in the pattern of susceptibility and resistance of S aureus to antibiotics from one locality to another. Also the susceptibility and resistance of S.aureus to antibiotics is known to be altered at relatively higher temperatures. For instance, May et al. (2007) observed that clinical isolates of S. aureus which was resistant to streptomycin at 37^oC became sensitive when cultured at 44^oC.

 Extended spectrum β-lactamase (ESBLs) are a group of enzymes encoded by genes described predominantly on plasmid that are common among Enterobacteriaceae (Garcia-Graells et al., 2010). Although most ESBLs are mutants of temoneira (TEM) and sulfhydryl variable (SHV) enzymes, the cefotaximase (CTX-M) type-lactamases which have become important, originated from β-lactamases found in environmental species of the genus Kluyvera, and this enzyme hydrolyzes cefotaxime and cefriaxone but is weakly active against ceftazidime (Geser et al., 2011). At present, there are more than 300 different ESBL variants, and these have been clustered into nine different structural and evolutionary families based on amino acid sequence. TEM and sulphydryl variable SHV were the major types. However, CTX-M type is more common in some countries (Geser et al., 2011).

Extended spectrum β-lactamase (ESBL) isolates were first detected in Western Europe in the mid-1980s. Since then, their incidence has been increasing steadily. ESBLs are able to hydrolyze 3 and 4 generation cephalosporins and monobactams. ESBL producing strains are inhibited by β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) (Patterson and Bonomo, 2005). A large number of outbreaks of infections due to ESBL producing organisms have been described on every continent of the globe. In some hospitals, initial outbreaks of infections have been supplanted by endemicity of the ESBL producing organisms. This may lead to increased patient mortality when antibiotics inactive against ESBL producers are used. Therefore, control of the initial outbreak of ESBL producing organisms in a hospital or specialized unit of a hospital is of critical importance (Patterson and Bonomo, 2005).

1.1 AIM AND OBJECTIVES

The aim of this study was to determine the prevalence of β-lactamase producing antibiotic resistant Staphylococcus aureus from health workers.

The objectives are:

1.     To isolate and identify Staphylococcus aureus from health workers.

2.     Determine the prevalence of Staphylococcus aureus from health worker.

3.     To determine the antibiogram of Staphylococcus aureus isolated from

4.     To determine the prevalence of β-lactamase producing Staphylococcus aureus.

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