Β-LACTAMASE PRODUCTION BY ANTIBIOTIC RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM WOUND PATIENTS

ABSTRACT

The present study was undertaken to investigate the β-lactamase production by antibiotic resistant Staphylococcus aureus isolated from wound samples from patients at the Federal Medical Center Umuahia, Abia State. A total of 50 wound swabs from different sites including burns, skin infection and accident wounds were analysed in this study. The swabs were streaked directly onto mannitol salt agar plates. A total of 29 (58.0%) of the samples analysed harbored Staphylococcus aureus. Distribution of the S. aureus among the sample types showed that 83.3% of the skin swabs had S. aureus, 60% from accident wound and 38.9% was from burns. The results of the antibiotic susceptibility profile of the S. aureus isolates showed that 79.3% and 72.4% of the isolates were susceptible to Ciprofloxacin and Ofloxacin respectively while Gentamicin had 62.1% susceptibility. The isolates showed 100% resistance to Cefuroxime, Cefepime and had 34.4% susceptibility to Azithromycin. It was also found that 16(55.2%) of the total isolates tested positive to β-lactamase production whereas 44.8% were negative. The study concludes that S. aureus is the constantly isolated pathogen in wound patients and regular intervention is required for the control of infection caused by this organism. With high susceptibility of S. aureus isolates to Levofloxacin and Ciprofloxacin there is therefore, an indication that these antibiotics can be used for empirical treatment of infections on patients wounds.

TABLE OF CONTENTS

Cover Page                                                                                                                             i

Title Page                                                                                                                                ii

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Table                                                                                                                           viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.1.INTRODUCTION                                                                                                    1

1.2.Aim and Objective of the Study                                                                                 3

CHAPTER TWO

Literature Review

2.1 Staphylococcus aureus                                                                                                     4

2.2 Bacteriology                                                                                                                     4

2.2.1. Microscopic morphology of S. aureus                                                                         4

2.2.2. General cultural and biochemical characteristics of S. aureus                                     5

2.3. Medical laboratory diagnosis                                                                                          5

2.4. General pathogenesis and clinical diseases                                                                     5

2.4.1 Pathogenesis                                                                                                                  5

2.4.2. Hospital and community infections                                                                             6

2.4.3. Virulence factors                                                                                                          6

2.5 Epidemiology of infections                                                                                              7

 2.5.1 Nasal carriage                                                                                                               7

2.5.2 Emergence and evolution of MRSA                                                                              7

2.5.3 Health care-associated and community MRSA                                                             9

2.5.3.1 Health care-associated MRSA (HA-MRSA)                                                              9

2.5.3.2 Community-associated MRSA (CA-MRSA)                                                             10

2.6 Antibiotic resistance                                                                                                         11

2.6.1      Beta-lactam resistance                                                                                                11

2.6.2 Penicillin resistance                                                                                                       11

2.6.3 Methicillin resistance                                                                                                    12

2.6.4 Quinolones resistance                                                                                                    12

2.6.5 Vancomycin resistance                                                                                                  13

2.6.6 Vancomycin intermediate S. aureus                                                                              14

2.6.7 Vancomycin-resistant S. aureus                                                                                                15

2.6.8 Resistance to other antibiotics                                                                                       15

2.7     Therapeutic approach                                                                                             16

2.8     Topical anti-MRSA drugs                                                                                      16

2.8.1       Mupirocin                                                                                                                  16

2.8.2       Fusidic acid                                                                                                               16

2.9      Systemic anti-MRSA drugs                                                                                   17

2.9.1      Vancomycin                                                                                                               17

2.9.2      Newer anti-MRSA drugs                                                                                            19

2.10  Alternative therapeutic approach                                                                           19

2.11  Anti-virulence agents                                                                                             19

2.11.2    Plants                                                                                                                         20

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study area and Sample collection                                                                                    25

3.2 Sample Collection                                                                                                            25

3.3 Preparation of Media for Identification of Staphylococcus Species                                    25

3.4 Inoculation and Isolation                                                                                                  25

3.5 Identification of The Isolates                                                                                            26

3.5.1 Gram Staining                                                                                                               26

3.5.2 Biochemical Test                                                                                                           26

3.6 Antibiotic Susceptibility Testing                                                                                      27

3.7 Tests for Beta Lactamase Production                                                                               27

3.7.1 Beta Lactamase Determination Using Acidimetric Method                                         27

CHAPTER FOUR

4.1 RESULTS                                                                                                                        28

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

4.2 Discussion                                                                                                                         33

5.1 Conclusion                                                                                                                        34

5.2 Recommendation                                                                                                              35

REFERENCES                                                                                                                       37

List of Tables

Table                                    Title                                             Page

2.1:                  Newer anti-MRSA drug                                               18

4.1:                  Frequency of occurrence of Staphylococcus aureus from Various Wound Types                                                                24

4.2:                  Colonial Description and Biochemical Characteristics of Isolates                        25

4.3:                  Antibiotics Susceptibility profile of the Staphylococcus aureus Isolates                                                                          26

4.4:                  Beta-lactamase Production Profile of the Isolates         27

CHAPTER ONE

1.1  INTRODUCTION

Wound is a breach in the skin, which can lead to infections with the presence of replicating microorganisms with the discharge of pus (Dulon et al. 2011). Wound infections result after the active interactions that takes place between a host, a potential pathogen and the surrounding extrinsic factors. The intensity of wound infections may range from a simple self-healing to a severe and life threatening (Moet et al., 2010). Tissue invasion by bacterial pathogens is determined by the location of wound (Oluwatosin, 2010). The common bacterial pathogens isolated from wound infections are Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pyogenes, coagulase negative staphylococci (CoNS), Acinetobacter species, Pseudomonas species, Escherichia coli, Klebsiella species, Proteus species, Enterobacter species, Citrobacter species, and anaerobes such as Clostridium species and Peptostreptococcus species (Dulon et al. 2011).

Staphylococcus aureus is a Gram-positive bacterium and the causative agent of a wide range of infectious diseases such as skin infections, bacteremia, endocarditis, pneumonia and food poisoning. The organism was originally a leading nosocomial pathogen and afterwards epidemiologically distinct clones emerged in community settings. S. aureus expresses a number of virulence factors which help to establish infection by facilitating tissue attachment, tissue invasion and evading from host immune response. Although S. aureus is typically a commensal organism, it has been known to be opportunistic. Invasive infections due to wound invasion can lead to numerous diseases, including scalded skin syndrome, abscesses, septicaemia, pneumonia, food poisoning, and toxic shock syndrome (Boyd, and Brüssow, 2008).

S. 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 tissues by S. aureus apparently involves the production of a formidable array of extracellular enzymes (invasins) which facilitate the actual invasive process. Some may occur also as cell associated proteins by breaking down primary or secondary defenses of the host which can facilitate the growth and spread of the pathogen. The damage of the host as a result of this invasive activity may cause woods and become part of the pathology of an infection (Noble, 1998).  

Staphylococcus aureus has been recognized as an important cause of disease around the world ranging from relatively mild infections of the skin and soft tissue to life-threatening sepsis. The emergence of strains resistant to methicillin and other antimicrobial agents has become a major concern, especially in the hospital environment (Spagnolo et al. 2014). The emergence of resistance to antibiotics in Gram positive pathogens has become a major international problem (Patel et al., 2011). Antibiotic therapy is typically started before susceptibility information is available, but inappropriate initial therapy is associated with adverse clinical outcomes (Rodriguez-Bano et al., 2009). The pathogen S. aureus may use a variety of strategies to resist antibiotic therapy; these antibiotic-resistant strains are called Methicillin-Resistant Staphylococcus aureus (MRSA) (Khorvash et al., 2008). Infections caused by resistant pathogens are associated with higher morbidity and mortality than those caused by susceptible pathogens (Vaghasiya and Chanda, 2007).

β-lactam antibiotics are among the most frequently prescribed antibiotics worldwide in the control of S. aureus infection. They act on peptidoglycan synthesis by molecularly acting on trans-peptidases and carboxypeptidases thereby disrupting cell wall formation of the pathogen. However, the efficacy of antibiotics for therapy have suffered a setback due to the growing trend of multiply resistant strains observed in the organism to β-lactam and other antibiotics (Lowy et al., 2003; Deurenberg and Stobberingh, 2008; Jensen and Lyon, 2009). 

Resistance to β-lactam group of antibiotics in S. aureus is mediated through a variety of β-lactamases or the expression of low-affinity penicillin binding protein PBP2a. The chromosomally mediated penicillin binding protein 2a initiates resistance to methicillin which confers a low affinity for all β -lactams and other unrelated group of antibiotics, thereby limiting choice for treatment (Woodford and Livermore, 2009).  β-lactamase is the predominant extracellular enzyme synthesized after exposure of S. aureus to β-lactam antibiotics (Chopra, 2013). The enzyme is encoded in the plasmid or chromosome and its expression can either be constitutive or inductive. It deactivates the drug by cleaving the β-lactam ring. The hydrolytic ability of β-lactamase in conferring resistance in S. aureus largely depends on its location, kinetics, quantity Physiochemical conditions and interplay of determinants (Anderson and Gums, 2008). In addition, selective pressure from excess antibiotic use accelerates the emergence of resistance. β-lactamase has been observed to be responsible for resistance in β-lactam, βlactamase inhibitors and extended spectrum cephalosporins (Anderson and Gums, 2008).  Antibiotic resistance in S. aureus have an adverse effect on healthcare management of infections. In response to the increasing rate of antibiotic resistance in S. aureus, this study aims to analyze the β-lactamase production by antibiotics resistant Staphylococcus aureus.

1.2 Aim and Objective of the Study

The aim of this research work was to investigate β-lactamase production by antibiotic resistant Staphylococcus aureus isolated from wound patients in Federal Medical Centre Umuahia Abia State with the following specific objectives:

i.               To isolate and Identify S aureus from different wound types

ii.              To determine the antibiotics susceptibility profile of the isolates.

iii.            To screen the isolate for β-lactamase production

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