STAPHYLOCOCCAL CASSETTE CHROROMOSOME MEC (SCCmec) TYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) ISOLATES FROM PATIENTS

ATTENDING TENGKU AMPUAN AFZAN HOSPITAL (HTAA) IN KUANTAN, PAHANG,

MALAYSIA

BY

NASREEN MOHAMMAD ALAROSI

A dissertation submitted in fulfilment of the requirement for the degree of Master of Medical Sciences

Kulliyyah of Medicine International Islamic University

Malaysia

AUGUST 2011

ii

ABSTRACT Staphylococcus aureus is a major human pathogen. It causes a wide range of infections in the hospital setting as well as in the community environment. A broad variety of infections, ranging from minor pyodermas to life-threatening infections can be caused by S. aureus.The adaptive power of S. aureus to antibiotics lead to the emergence of methicillin-resistant S. aureus (MRSA) in the early 1960s. The cause of resistance to methicillin is the acquisition of the mecA gene, which is situated on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). Five major variants of SCCmec, types I to V, are distinguished. One of the most important techniques used to investigate the molecular epidemiology of S. aureus is SCCmec typing. This technique has been used to study the evolution of the MRSA and to study their subsequent worldwide dissemination.This study was conducted to determine the molecular typing of methicillin-resistant S.aureus (MRSA) organisms isolated by the Bacteriology Laboratory in Hospital TengkuAmpuanAfzan (HTAA), Kuantan, Pahang, Malaysia from inpatients admitted during the period from 1stApril to 30th September, 2010. A total of 28 MRSA isolates were re-identified by known bacteriological methods and the minimal inhibitory concentration (MIC) of oxacillin was determined by E-test. The antibiotic susceptibility was tested, by disc diffusion method to 7 different antibiotics as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).Resistance to oxacillin was 100%,erythromycin 82.1%, gentamicin75%, tetracycline 78.6%, and trimethoprim-sulfamethoxazole78.6%. None of the isolates was resistant to vancomycin or chloramphenicol. All isolates showed an MIC >4μg/mL. All 28 MRSA isolates were subjected to SCCmec typing by duplex real time PCR, 78.5%(22/28) were shown to possess SCCmec-III and 21.5% (6/28) were of type IV. This confirms observations in several other neighboring Far Eastern countries and corroborates the epidemicity of this type in Kuantan, Malaysia.This work represents a valuable foundation study for future work on the antibiotic susceptibility/resistance profile and the epidemiology of MRSA SCCmec types in Kuantan, Malaysia.

iii

البحث خلاصة

مهمة الممرضهال بكتريامن ال هبة المقاومة للمتتيسلين تعتبرذالمات العنقوديه لمكوراافي المستشفيات وآذلك المجتمع بشكل الأخماجتسبب مجموعة واسعة من حيثجدا, الى الخّراجات وأخماج جروح بين اصابات جلدية طفيفة هذه الأخماج تتدرجالعام.

. قدرة هده البكتيريا علي التكيفيه للمضادات وتجرثم الدم وغيرها. العمليات الجراحيهيسلين في بدايات ثهبة المقاوم للمذالم المكورةالحيويه ادت الي ظهور نوع من

وجود هو ثيسيلين. السبب الرئيسي الذي جعل هذه البكتريا مقاومة للمي1960الستافيلوآوآال آاسيت , متحرك وراثي عنصر على الدي يقع ميك ا، جين

أهم آروموسوم ميك. خمسة انواع مختلفة من هده الكاسيت تم اآتشافها. منهبة ذالم المكورةالجزيئية للبكتريا علم الأوبئة للتحقيق ولدراسة المستخدمة التقنيات

ومسومورآ ايلوآوآس ستف ة تنميطهي استخدام طريقفي انتشارها ودراسة هذه الجرثومة تطور لدراسة التقنية هذه استخدمت وقد .آاسيت

لمكورةل الجزيئي التصنيف هذه الدراسة أجريت لتحديد لاحقا. جميع أنحاء العالم 1 خلال الفترة من علم الجراثيم المقاومة للمثيسيلين، التي عزلت في معمل هبةذالم

امبوان كومستشفى تن من المرضى النزلاء في 2010 سبتمبر 30 -أبريل المقاومه هبةذالم المكورة من عزله 28العدد الاجمالي آان باهانج. آوانتان، افزانالمعروفة وبواسطه بواسطة الطرق البكتريولوجية تحديدهاعزلها و تميسلين ثللم

ترآيزالمثبط للاوآسيسلين (ام اي سي). ال الأدنى من الحد تم تحديد (E test) اختباروآذلك تم .مل / ميكروغرام 4اآتر من جميع البكتريا المعزولة اظهرت ام اي سي

المضادات الحيوية باستخدام طريقة انتشار قرص المضاد الحيوي حساسية اختبارووجد ان هده .الوطنية للمختبرات الطبية اللجنة وفقا للمعايير الموصى بها من قبل

، %82.1الاريثروميسين ،%100بنسبة البكتريا مقاومه لمضاد الاوآسيسلين سلفاميثوآسازول – ميثوبريموتراي ، % 78.6% والتتراسيكلين 75والجنتاميسين

البكتريا المعزولة مقاومة من لم تكن أيوان النتائج اظهرت ايضا انه . % 78.6الكلورامفينيكول. جميع العينات خضعت لتصنيف اس سي أو لمضاد الفنكومايسين

) 22/28( % 78.5 وجد ان سي ميك باستخدام التفاعل المتسلسل البوليماراز الثنائ) تحمل 6/28% (21تحمل النوع الثالث من اس سي سي ميك بينما من العزلات دان أخرى بل عدة في لبحوت سابقةنتائج مشابهة هذه الدراسة اآدت النوع الرابع.

آوانتان هذا النوع من البكتريا في وبائيةتوضيح عززتو الأقصى الشرق في يه حول مستقبلتشكّل نواة لدراسات قيمة أساسيه دراسة العمل هذا. يعتبرماليزيا ،

قاومه ا المالبكتري انتشار وآيفية حدوث العدوى بواسطة هذا النوع من .ماليزيا ،آوانتان الحيوية في للمضادّات

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.

............................................... Mohammed Imad Al-Deen Mustafa Supervisor .............................................. Nasser Muhammad Amjad Co Supervisor

I certify that I have read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.

................................................. Nasuruddin Hj Abdullah Examiner

This dissertation was submitted to the Department of Pharmacology and is accepted as a fulfilment of the requirement for the degree of Master of Medical Sciences.

.................................................. Pakeer Oothuman Sayed Ahmed Head, Department of Basic Medical Sciences.

This dissertation was submitted to the Kulliyah of Medicine and is accepted as a fulfilment of the requirement for the degree of Master of Medical Sciences.

............................................... Mohammed Fauzi Abdul Rani Dean, Kulliyyah of Medicine

v

DECLARATION

I hereby declare that this dissertation is the result of my own investigations, except

where otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Nasreen Mohammad Alarosi Signature …………………………………… Date ……………………..

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright © 2011 by NasreenAlarosi. All rights reserved.

STAPHYLOCOCCAL CASSETTE CHROMOSOME MEC (SCCMEC) TYPING OF METHICILLIN-RESISTANT STAPH.AUREUS (MRSA)

ISOLATES FROM INPATIENTS ATTENDING HOSPITAL TENGKU AMPUAN AFZAN (HTAA) IN KUANTAN, PAHANG, MALAYSIA

No part of this unpublished research may be reproduced, stored in retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior permission of the copyright holder except s provided below.

1. Any material contained in or derived from this unpublishedresearch may only be used by others in their writing with dueacknowledgement.

2. IIUM or its library will have the right to make and transmit copies(print

or electronic) for institutional and academic purposes. 3. The IIUM library will have the right to make, store in a retrievalsystem

and supply copies of this unpublished research if requestedby other universities and research libraries.

Affirmed by Nasreen Mohammad Alarosi.

…………………….. …………………….. Signature Date

vii

To;

My husband,

a light that guides my foot steps

My mother and father,

no reward is good enough for you

viii

ACKNOWLEDGMENTS

In The Name Of Allah, The Beneficent, The Most Merciful.

All perfect praise is for Allah, the Most Gracious who has made this work possible. I extend my sincere appreciation to my country for sponsoring my study at the

IIUM. Special thanks to my supervisor Assoc. Prof Dr. Mohammed Imad AlDeen Mustafa and co-supervisor, Prof. Dr. Nasser Muhammad Amjad for the knowledge and guidance they have provided throughout the course of this project.

Many thanks to my colleagues Sr.Rabia Merihil, Dr. Farah Ibrahim Khalil, Dr. Ahmad A. Elbadri, Dr Wisam Nabil, for the timely help and knowledge shared.

A heartfelt gratitude to my family and to all my dear friends for being there for me every time I need them.

ix

TABLE OF CONTENTS Abstract .................................................................................................................... ii Abstract in Arabic .................................................................................................... iii Approval Page .......................................................................................................... iv Declaration ............................................................................................................... v Copyright page ......................................................................................................... vi Dedication ................................................................................................................ vii Acknowledgement ................................................................................................... viii List of Figures ......................................................................................................... xii List of Tables ........................................................................................................... xiv List of Abbreviation ................................................................................................. xv CHAPTER ONE: INTRODUCTION .................................................................. 1

1.1General Objective .................................................................................... 2 1.2 Specific Objective .................................................................................. 3

CHAPTER TWO: LITERATURE REVIEW ..................................................... 4

2.1 Staphylococcus aurous ........................................................................... 4 2.1.1 Discovery and identification ........................................................ 3 2.1.2 Structure and virulence factors .................................................... 5

2.2 Antibiotic treatment of staphylococcal infections .................................. 7 2.2.1 β-lactam antibiotics ..................................................................... 8 2.2.2 Mechanisms of resistance ............................................................ 9

2.2.2.1 Beta-lactamase production................................................... 9 2.2.2.2 Methicillin resistance ........................................................... 11

2.3 Classification of SCCmec elements ...................................................... 12 2.4 Staphylococcal cassette chromosome mec ............................................. 14 2.5 Community acquired-MRSA ................................................................. 16 2.6 Epidemiology ......................................................................................... 18

2.6.1 Prevalence in Malaysia ................................................................ 19 CHAPTER THREE: MATERIALS AND METHODS ..................................... 21

3.1 Collection and storage of bacterial samples ........................................... 21 3.2 Bacterial reference strains ...................................................................... 21 3.3 Re-identification ..................................................................................... 22

3.3.1 Microorganisms’ Reactivation from Cryobeads .......................... 22 3.3.2 Bacterial suspension ..................................................................... 22 3.3.3 Gram Staining .............................................................................. 23 3.3.4 Catalase test .................................................................................. 25 3.3.5 Coagulase test .............................................................................. 26 3.3.6 Mannitol salt agar ......................................................................... 29 3.3.7 BrillianceMRSA agar ................................................................... 32

x

3.4 Antimicrobial susceptibility ................................................................... 33 3.4.1 Preparation of Müeller-Hinton Agar ............................................ 34 3.4.2 Procedure for Performing the Disc Diffusion Test ...................... 34 3.4.3 Quality control microorganisms ................................................... 35

3.5 The E test ................................................................................................ 36 3.5.1 Principle of the test ...................................................................... 37 3.5.2 Procedure for Performing the E Test ........................................... 37 3.5.3 Quality control strains of Staphylococcus aureus ........................ 38

3.6 DNA extraction from cultures of gram- positive bacteria ..................... 38 3.7 Gel electrophoresis ................................................................................. 40

3.7.1 Gel Electrophoresis Procedure ..................................................... 40 3.8 Real-time PCR ....................................................................................... 41

3.8.1 Primers and Probes Reconstitution .............................................. 41 3.8.2 Testing primers performance using SYBR Green melt curve Analysis ................................................................................................. 41 3.8.3Testing Primers–Probe Sets Performance in Monoplex Real-Time PCR ....................................................................................................... 44 3.8.4 Testing Primers–Probe Sets Performance in Multiplex Real-Time PCR ....................................................................................................... 48 3.8.5 Duplex Real-Time PCR Optimization ......................................... 49 3.8.6 DuplexReal-time PCR Testing of MRSA isolates ....................... 51

CHAPTER FOUR: RESULTS ............................................................................. 52

4.1 Bacterial isolates .................................................................................... 52 4.2 Demographic data .................................................................................. 52 4.3 Distribution of MRSA isolates within HTAA ....................................... 52 4.4 Sites of infection .................................................................................... 53 4.5 Antimicrobial susceptibility pattern of S.aureus by disk diffusion test . 54 4.6 Determination of minimal inhibitory concentration (MIC) to oxacillin by E test ............................................................................................................. 57 4.7 Hospital acquired MRSA (HA-MRSA) and community acquired MRSA (CA-MRSA) ................................................................................................. 57 4.8 Determination of quality of extracted DNA ........................................... 58 4.9 Optimization of the SCCmec Real-Time PCR assay ............................. 58 4.10 Application of duplex Real-TimePCR on the clinical samples ........... 59

CHAPTER FIVE: DISCUSSION ......................................................................... 60

5.1 Antibiotic susceptibility pattrens ............................................................ 60 5.2 MRSA as a vital health problem ............................................................ 62 5.3 Staphylococcal cassette chromosome mec (SCCmec) typing of methicillin-Resistant Staphylococcus aureus strains ................................... 63

 

CHAPTER SIX: CONCLUSION ......................................................................... 68  

BIBLIOGRAPHY .................................................................................................. 70

xi

LIST OF TABLES

Table No. Page No.

3.1 Disk diffusion test, inhibition zone diameter interpretation according

to NCCLS guidelines 35

3.2 SYBR Green Real-time PCR Reaction Mixture 43

3.3 SYBR Green Real-time PCR Reaction conditions 43

3.4 MonoplexReal-time PCR Reaction Mixture 45

3.5 Monoplex Real-time PCR Reaction Conditions 47

3.6 Duplex Real-time PCR Reaction Mixture 49

3.7 Duplex Real-time PCR Reaction conditions 51

4.1 Patient’s data 54

4.2 Antimicrobial susceptibility pattern of S. aureus by disk diffusion test 56 4.3 SCCmec types 59  

xii

LIST OF FIGURES

Figure No. Page No.

2.1 The induction of β -lactamase synthesis in the presence of staphylococcal penicillins.

10

2.2 A schematic drawing of SCCmec types I to VI in MRSA 13

3.1 Staph. aureus grown on blood agar after 24 hours incubation

under aerobic conditions

22

3.2 Gram-stained staphylococci 25

3.3 Positive catalase test 26

3.4 Negative catalase test 26

3.5 Tube coagulase 29

3.6 Unineculated Mannitol Salt Agar 31

3.7 S. epidermidisare grown onMannitol Salt Agar 31

3.8 S. aureusare grown onMannitol Salt Agar 31

3.9 MRSA are grown on Brilliance MRSA agar 33

3.10 No growth of S. aureus ATCC®25923 on Brilliance MRSA Agar

33

3.11 Staphylococcus aureus ATCC®25923, the zone of inhibition for oxacillin more than 13mm

36

3.12 Staphylococcus aureus(NCTC10442)Kirby-Bauer disk diffusion antibiotic test on Mueller-Hinton Agar

36

3.13

3.14

E-Test Antibiotic Susceptibility Test

Amplification curves of eight primers for SCCmec typing from bacterial reference strains.

38

44

xiii

3.15

4.1

4.2

4.3

DNA electrophoresis of eight genes for SCCmec typing from bacterial reference strains Distribution of sits of infections

Agarose gel electrophoresis (1%) of extracted DNA from MRSA sample number 1. Duplex real-time PCR showing amplification profile of some samples

44

53

58

59

xiv

LIST OF ABBREVIATIONS AND SYMBOLS

µM Micromolar µL Microliter µg/ml Microgram per milliliter MRSA Methicillin-resistant S.aureus PBP Penicillin binding protein CDC Center For Disease Control CA-MRSA Community acquired MRSA HA-MRSA Hospital acquired MRSA MIC Minimal Inhibitory Concentration   

1

CHAPTER ONE

INTRODUCTION Staphylococcus aureus is a major pathogen that causes a wide spectrum of clinical

manifestations ranging from common skin infections such as impetigo and cellulitis to

the more serious manifestations of necrotizing fasciitis, abscess formation,

osteomyelitis, pneumonia, sepsis, and food poisoning.

Over the last 20 years, the incidence of both community-acquired-(CA) and

hospital-acquired (HA) S. aureus infections have increased, with a worldwide increase

in the spread of S. aureus strains that are resistant to multiple antibiotics, including

methicillin (Ghebremedhin et al., 2009). This increase could be an apparent one due

to the greater awareness and vigilance in testing and reporting these organisms and/or

a real increase due to injudicial control of antibiotic usage acting as a selective

pressure in favor of these resistant forms. Other factors, include urbanization with the

increased number of population accompanied by the rapid growth of cities in many

developing countries tending to concentrate large numbers of people into crowded

areas with poor sanitation. These conditions foster transmission of contagious

diseases. Modern transport that can spread diseases to far away destinations, and the

increase number of people with risk factors such as patients with chronic diseases also

play a significant role (Daszak et al., 2002; Krauss et al., 2003).

Methicillin-resistant S. aureus (MRSA) was first reported in 1961 (Enright,

2002) and subsequently spread throughout hospitals worldwide (François et al., 2004).

2

More recently, highly virulent community-associated MRSA (CA-MRSA) strains and

vancomycin-resistant or intermediate-resistant S. aureus strains have emerged and

pose further challenges (Mukesh Patel et al., 2007).

The genetic basis of the methicillin resistance of MRSA isolates is the

presence of mecA, a gene coding for the low-affinity penicillin-binding protein PBP2′,

which is located on a mobile genetic element designated SCCmec (staphylococcal

cassette chromosome mec) inserted into the chromosome.. Currently, five SCCmec

types (I–V) have been distinguished, and several variants of these SCCmec types have

been described. In addition, SCCmec contains site-specific cassette chromosome

recombinases (ccr) responsible for the integration of SCCmec into the bacterial

genome. The ccr genes exist in 2 forms, ccrAB and ccrC, and 4 main allotypes of

ccrAB have been described. (Ito et al., 2001; Katayama et al., 2003; Oliveira et al.,

2006; Takano et al., 2008; Zhang et al., 2009).

The aim of this study is to detect and characterize the staphylococcal cassette

chromosome mec (SCCmec) in methicillin-resistant Staphylococcus aureus (MRSA)

isolates from patients attending Hospital Tengku Ampuan Afzan (HTAA) in Kuantan.

The study will help in better understanding of the molecular types and antibiogram of

local MRSA isolates. This information will constitute the nucleus for further studies

aiming at improving the control of MRSA infections in the hospital.

General objective

• To understand the regional evolution of virulent and antibiotic-resistant

strains of MRSA and thereby improving the institution of control

measures for limiting the spread of these infections.

3

• To establish a foundation for future investigation trends in molecular

epidemiology of MRSA in HTAA in the face of a constantly increasing

number of reported cases.

Specific objectives

1. To determine the molecular characteristics of local MRSA strains by

typing the SCCmec chromosome using multiplex real-time PCR and then

compare the obtained data with those published in Malaysia and Asia.

2. To analyze the clinical data of MRSA infections, in terms of hospital

wards involved, type of infection, site of infection, age of the patients, and

other biodata.

4

CHAPTER TWO

LITERATURE REVIEW 2.1 STAPHYLOCOCCUS AUREUS

Taxonomically, the genus Staphylococcus is in the family Staphylococcaceae, which

includes three lesser known genera, Gamella, Macrococcus and Salinicoccus. The

genus consists of more than 40 species and subspecies (Todar, 2011).

Staphylococcus aureus (S. aureus) is the most important and most studied

human pathogen of thegenus Staphylococcus and it is capable of causing a broad

variety of infections ranging from minor skin infections to serious conditions such as

osteomyelitis, bacteremia and infective endocarditis. However, S. aureus is usually a

harmless colonizer of about one third of healthy humans without causing infections

and it is most likely found in the nares (Mainous et al., 2002). Moreover, S. aureus is

capable of producing a wide range of enzymes and toxins that are associated with

specific clinical conditions, such as food poisoning or toxic shock syndrome (Arch et

al., 1998).

2.1.1 Discovery and identification

Sir Alexander Ogston, a Scottish surgeon, was the first one to describe S. aureus in

1880 following the examination of pus from surgical abscesses. He discovered that a

cluster-forming bacteria was involved in causing abscess formation in humans.

Therefore, the name “Staphylococcus” was given based on the Greek term staphyle,

which meant, “bunch of grapes”. The term “aureus” comes from the Latin meaning

5

“golden” describing the golden-yellow pigmentation of the colonies when grown on a

nutrient agar medium. (Leonard and Markey, 2008).

In addition to its unique golden pigmentation, S. aureus can be

distinguishedfrom other staphylococcal species by the production of coagulase, an

enzyme that induces fibrin clot formation. All other members of the staphylococcal

species unable to produce free coagulase are named as coagulase-negative

staphylococci. S.aureus may also be identified by its growth on mannitol salt agar

medium, which is supplemented with 7.5% sodium chloride, to inhibit the growth of

most other organisms. The sugar is fermented by S. aureus (but not other

staphylococci), producing characteristic yellow colonies.

Furthermore, using deoxyribonuclease test, in which a clear zone around

colonies appears after the addition of 1N HCL, differentiates S.aureus from other

staphylococcal species (Leonard&Markey, 2008).

2.1.2 Structure and virulence factors

Staphylococcus aureus is a facultative aerobic Gram positive, catalase positive coccus

living alongside humans and animals as an opportunistic pathogen.

S. aureus consists of cytosol, a single cytoplasmic membrane, and the

surrounding cell wall. The cell wall of S. aureus is a homogeneous structure of 20 to

40 nm thickness. The major component of the cell wall is peptidoglycan, composed of

repeating disaccharide N-acetylglucosamine-N-acetylmuramic acid (GlcNAc

MurNAc) units with attached teichoic acids. Glycan chains are cross linked by

tetrapeptides consisting of L-alanine, D-glutamate, L-lycine, and L-alanine, to

pentaglycine interbridge, linked to wall peptide. This cross linking is catalyzed by the

enzyme transpeptidases which belong to a group of cell wall proteins known as

6

penicillin binding proteins (PBP) (Hiramatsu et al., 2001). The main function of

peptidoglycan is to provide a rigid envelope for the cell contents and osmotic stability.

Teichoic acids are another cell wall component, which is important for establishing

nasal colonization and contributes, together with peptidoglycan, to the severity of

staphylococcal sepsis.

Most S. aureus strains produce a slimy, extracellular capsular polysaccharide.

The capsule protects bacteria by inhibiting phagocytosis of the organisms by

polymorphonuclear leukocytes. In addition it facilitates adherence to foreign bodies

such as catheters. A total of 11 capsular serotypes have been identified in S. aureus.

Serotypes 5 and 7 account for the majority of isolates found in humans (Brooks,

Carroll, Butel, and Morse, 2007a).

The adherence of S. aureus to host tissue is an important step in its

pathogenesis as well as in colonization. Surface proteins such as protein A, clumping

factors, fibronectin-binding proteins, and collagen-binding proteins can adhere to

extracellular matrix components of the host (Foster & Hook, 1998). The main function

of protein A, however, is to bind the IgG Fc-domain and thus inhibits antibody-

mediated clearance.

Almost all strains produce and secrete enzymes and exotoxins including

hemolysins (alpha, beta, gamma, and delta), proteases, lipases, nucleases,

hyalonuridase, and collagenase, which have important roles in invasion and tissues

damage. Some toxins, such as toxic shock syndrome toxins (TSST-1) or enterotoxins

(A-E, G-I) produced by certain strains, are sufficient to cause specific diseases (toxic

shock syndrome and food poisoning respectively). Exfoliative toxins (ETA and ETB)

and leukocidins such as Panton-Valentine leukocidin (PVL) are also produced by

some strains. These toxins are an important cause of tissue damage. PVL genes

7

encode a leukotoxin, which comprises pore-forming staphylococcal toxins together

with alpha and gamma-haemolysins. These toxins may be required to break the tissue

down into nutrient components for bacterial growth but their primary function is to

inhibit the host immune response by killing the leukocytes, although they also have

potent effects on cells such as the induction of calcium influx in the PMNs that

triggers a cascade of signal transduction that will initiate the production of cytokines

and other inflammatory mediators. (Genestier, et al., 2005).

No single virulence factor has been shown to cause an inflammatory process.

For example, to cause systemic diseases such as bacteremia, S. aureus must produce

components capable of attaching to cells or tissues, factors that decrease phagocytosis

in order to escape the host immune system, and to produce proteases, exotoxins, and

enzymes, causing tissue damage, and thus, allowing the dissemination of the organism

(Archer, 1998).

2.2 ANTIBIOTIC TREATMENT OF STAPHYLOCOCCAL INFECTIONS

Since staphylococci cause a wide range of infections, the choice of antibiotic

treatment should be assessed according to location and severity of the infection and

the mechanism of action of the antimicrobial agent which could be either

bacteriostatic or bactericidal. The type of infection is also important in determining

which kind of drug to use. Furthermore, the treatment could be complicated, for

example in cases of infections associated with foreign bodies when the staphylococci

are often adhering to the foreign material and embedded in a biofilm which protects

the organism from the blood stream immune defense and consequently also to the

antibiotics. In addition, it is difficult to treat localized or focal infections such as

8

abscesses or osteomyelitis in which penetration of antibiotics is poor. The duration of

treatment also varies depending on type and site of the infection (Jones, 2008).

There is wide range of antibiotics available for the treatment of staphylococcal

infections. The semisynthetic penicillins such as oxacillin are usually the most

effective and appropriate choice for treatment of infections caused by susceptible

staphylococci. Other antibiotics, such as vancomycin, clindamycin and fusidic acid

are used predominantly for treatment of resistant staphylococci or in cases of allergy

to beta-lactam antibiotics (Jones, 2008). However resistance to these antibiotics may

develop as with vancomycin where the first case of vancomycin intermediate

resistance was reported in France in 1995. In 2002 the first vancomycin resistance in

S, aurues was reported in USA (Srinivasan et al., 2002).

2.2.1 β-lactamantibiotics

Beta-lactam antibiotics or penicillins interfere with cell wall synthesis by inactivating

the membrane-bound enzymes responsible for catalyzing the final stages in the

biosynthesis of the cell wall. This results in inhibition of the covalent binding of the

antibiotic to one or more penicillin sensitive enzymes, termed penicillin-binding

proteins (PBPs). The compromised cell wall structure leads to the decline of cell wall-

based metabolism, cell division, and autolysis will eventually occur.

In the pre-antibiotic era in the 1940s, invasive staphylococcal infections often

resulted in mortality of approximately 80% of patients. When penicillin was

introduced in 1941, the mortality rate was markedly reduced. However, the first

strains resistant to this antibiotic were isolated in 1942, first in hospitals and later in

the community. (Stewart and Holt, 1963; Lowy, 1998; Sampathkumar, 2007).

9

The prevalence of penicillin-resistant S. aureus within hospitals began to

increase after World War II because of the increased availability and perhaps

inappropriate use of penicillin, which positively selected the resistant bacteria and

allowed these to spread. Initially, penicillin-resistant strains were solely a nosocomial

problem in the 1950s, whereas the S. aureus circulating in the community remained

susceptible (Chambers, 2001). However, penicillin was considered as an effective

agent for the treatment of staphylococcal infections until the early 1970s when a

Danish study reported that the prevalence of penicillin resistance in hospital was 85%

to 90%, and 65 % to 70 % in the community. The high prevalence of penicillin

resistance among community isolates was not only limited to Denmark, in United

States the prevalence of penicillinase-producing strains was found to be 70% to 85%

regardless of location in inner cities, suburbs or rural areas in 2001 (Chambers, H. F,

2001).

2.2.2 Mechanisms of resistance

2.2.2.1 Beta-lactamase production

The first reported mechanism of resistance to penicillin is mediated by the enzyme β-

lactamase (penicillinase) that inactivates the penicillin molecule by hydrolyzing the β-

lactam ring before it reaches its target on the bacterial cell. β-lactamase is encoded by

the blaZ gene, which is carried on a large plasmid. The expression of blaZ is under the

tight regulation of two adjacent regulatory genes, the blaR1 (transmembrane signal

transducer) and blaI (repressor). When the penicillin binds the blaR1 on the surface of

the cell membrane, an intracellular signaling pathway will be initiated and this lead to

the cleavage of the blaI repressor on the operator region, which will allow

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transcription of the blaZ gene and hence production of β-lactamase (Clarkeand Dyke,

2001).

An illustration of the induction of -lactamase synthesis and regulation is

shown in Fig 2.1.

Figure 2.1: The induction of β-lactamase synthesis in the presence of staphylococcal penicillins. (A) In the absence of penicillin, the BlaI binds to the operator region and thus repress the RNA transcription from blaZ as well as blaR1 and blaI and the β -lactamase is expressed only at low levels. (B) Once the penicillin binds to the transmembrane sensor-transducer BlaR1 it will lead to BlaR1 autocatalytic activation. (C-D) The active BlaR1 will cleave and thus inactivate the BlaI, either directly or via a second BlaR2 protein, and allow transcription of blaZ, blaR1 and blaI. (E) The β -lactamase, which is encoded by blaZ, hydrolyzes the β -lactam ring and thereby inactivates the penicillin (F-G). (Adapted from Clarke, S. R., and K. G. Dyke. 2001)

Since penicillin was no longer effective in treatment of most S. aureus

infections, semisynthetic -lactamase-resistant penicillins such as methicillin and

oxacillin were developed to treat methicillin-sensitive S. aureus. However, within a