Title page for ETD etd-02182010-172959

Document Type Master's Dissertation
Author Makgotlho, Phuti Edward
Email Makgotlho.edward@gmail.com
URN etd-02182010-172959
Document Title Molecular characterisation of methicillin-resistant Staphylococcus aureus strains
Degree MSc
Department Medical Microbiology
Advisor Name Title
Dr M M Kock Co-Supervisor
Prof M M Ehlers Supervisor
  • soft tissue infections
  • MRSA
  • skin
Date 2009-11-27
Availability unrestricted

Methicillin-resistant Staphylococcus aureus (MRSA) is a pandemic human pathogen accounting for most of health-care associated infections throughout the world. However, in recent years, a more virulent strain of MRSA has emerged in the community defined as community-associated MRSA (CA-MRSA). These emerging strains of CA-MRSA are described to have different antibiotic susceptibility profiles, possess the SCCmec type IV element and usually produce the Panton-Valentine leukocidin (PVL) toxin. The majority of these CA-MRSA strains are associated with skin and soft tissue infections and necrotising pneumonia, with a 34% mortality rate.

Identification and characterisation of MRSA isolates is mainly performed using phenotypic methods, which are time consuming. Little information exists on the prevalence and characteristics of MRSA isolates including antibiotic susceptibility patterns, PVL-producing CAMRSA strains, the SCCmec types and genotypes that might be circulating in the Steve Biko Academic Hospital. Identification and characterisation of MRSA isolates based on these criteria are important in controlling possible outbreaks in the clinical setting.

In this study, 97 clinical MRSA isolates from the Steve Biko Academic Hospital, South Africa were collected between April 2006 to February 2007. These isolates were analysed and characterised using multiplex PCR (M-PCR), real-time PCR as well as staphylococcal protein A (spa) and hyper-variable region (HVR) typing. The aim of this study was to determine the antibiotic profiles, prevalence of MRSA isolates, the SCCmec types and the genotypes.

Antibiotic susceptibility determination was performed using the disk diffusion susceptibility method as guidelined by the CLSI. Six distinct antibiotypes were identified with a total of 73%, 71%, 70% and 7% of MRSA isolates resistant to clindamycin, erythromycin, gentamicin and fusidic acid, respectively.

The presence of Staphylococcus aureus specific 16S rRNA, the mecA and PVL genes was determined using a modified M-PCR assay. A total of 4% of the MRSA isolates possessed the PVL gene. Real-time PCR analysis also showed a 100% prevalence of the PVL gene in the same 4% MRSA isolates confirming the results of the first M-PCR assay. The second M-PCR was used to determine the SCCmec type prevalence and to distinguish between health-care associated MRSA (HA-MRSA) and CA-MRSA. SCCmec typing showed 67% of the isolates belonged to SCCmec type II and 14.4% SCCmec type III, both types belonging to HA-MRSA. A total of 4% of the MRSA isolates were CA-MRSA belonging to SCCmec type IVd. Genotyping results showed three distinct spa clusters whilst HVR showed six distinct clusters.

Molecular-based assays proved to be useful tools to determine the prevalence and monitoring of MRSA outbreaks as well as to identify the SCCmec types, subtypes and genotypes of MRSA strains that might be circulating in the hospital. The determination of the different antibiotypes of MRSA can assist in the monitoring of the antibiotic resistant profile trends in the Steve Biko Academic Hospital, thus assisting with the correct implementation of antibiotic regimens for suspected MRSA infections. In an endeavour to assess the dissemination of MRSA strains especially PVL expressing CA-MRSA strains, it is of paramount importance to continuously monitor the emergence of these strains in clinical settings.

Copyright 2009, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Makgotlho, PE 2009, Molecular characterisation of methicillin-resistant Staphylococcus aureus strains, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02182010-172959/ >


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