Molecular detection and characterization of antibiotic resistance genes in clinical Malaysian S. aureus isolates

Shafiei Sabet, Negar (2008) Molecular detection and characterization of antibiotic resistance genes in clinical Malaysian S. aureus isolates. PhD thesis, University of Malaya.

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Official URL: http://dspace.fsktm.um.edu.my/handle/1812/481

Abstract

This study aimed to utilize molecular techniques to develop rapid molecular methods for detection of Staphylococcus aureus (S. aureus) and subsequently to study important antibiotic determinants and characterize S. aureus isolates from Malaysia. 152 clinical isolates from University Malaya Medical Center (UMMC) were used in this study. Conventional and real-time multiplex PCR assays for simultaneous identification and detection of major antibiotic resistant genes were developed to enable rapid confirmation of resistance patterns within these isolates. The first assay was a conventional gel-based quadruplex PCR assay that targeted four genes mecA (methicillin resistance) Coa (coagulase) Sa442 fragment (species specific) and 16S rRNA (internal control). Inclusion of CoA gene along with the Sa442 marker, which is specific for S. aureus, increased the specificity of the assay by being able to simultaneously detect two specific markers for the identification of S. aureus. Subsequently, a triplex real-time PCR assay was developed using three of the primer pairs utilized in conventional assay which enabled a more curate comparison on the specificity and sensitivity of these assays. Both assays enabled the detection of Sa442 fragment in all S. aureus isolates. The next part of the study focused on designing the more complete assays for simultaneous identification of the organism and detection of antibiotic resistance genes and this resulted in the development of two new triplex real-time PCR assays. The first of these assays enabled identification of S. aureus using femA gene and detection of two important antibiotic resistant genes mecA, (methicillin resistance) and aacA-aphD (aminoglycoside resistance) thereby ombining the detection of MRSA (methicillin-resistant S. aureus) and aminoglycoside resistance in single real-time PCR assay. Evaluation of this assay correlated well with the results of the phenotypic antibiotic susceptibility testing. The second real-time PCR assay combined the detection of MRSA (mecA and femA), and erythromycin resistance (ermA) in a single assay. All the three genes were detected in all MRSA isolates tested. The triplex real-time PCR assays that were developed are rapid (performed within three hours) sensitive and specific assays which enable the detection of S. aureus as well as antibiotic resistance pattern. The high incidence of MRSA isolates in our center led to the next part of our study which was to determine the clonality, if any, of these isolates. The coagulase gene, which was used in the identification of S. aureus and other staphylococci, was used in a simple typing assay using restriction fragment length polymorphism (RFLP) to analyze 40 S. aureus isolates. This coagulase gene RFLP analysis showed that all the MRSA that were digested with two restriction enzymes, AluI and CfoI, showed the same pattern for each enzyme used which is suggestive of the presence of a single clone of MRSA in our hospital setting. The MSSA (methicillin-sensitive S. aureus) strains, however, showed seven distinct patterns, none of which resembled the pattern generated by the MRSA strains. The next part of our study focused on whether the transfer of mecA occurred from MRSA to MSSA strains in vitro. Liquid cultures consisting of donor strains harboring the mecA gene (mecA+) and recipient strains (mecA-) were combined prior to being plated out on selective antibiotic plates. Two types of ransconjugants (transconjugants A and B) were obtained which were analyzed using PCR to detect the presence of mecA and ccrAB (transfer factor) genes. The mecA gene was present in donor and both transconjugants A and B. The ccrAB gene was present in the donor, and both the transconjugants A and B. To study molecular basis of the mechanisms involved in development of resistance to the cell wall active antibiotic oxacillin, microarray study were performed. The retrospective analysis helped to identify a subset of genes that were differentially expressed in methicillin-resistant strains upon exposure to antibiotic oxacillin. Genetic determinants involved in various functional groups such as stress response, regulatory function, cell envelope/cell wall biogenesis, DNA replication, recombination and repair mechanism were found to be strongly expressed in MRSA strain during oxacillin treatment. These findings indicate a possible change in regulation of transcription as contributory factor in the development of antibiotic resistance in S. aureus.

Item Type: Thesis (PhD)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine, Health and Life Sciences > School of Medicine
Depositing User: MS SITI NUR ATIKAH MOHAMAD RUSDI
Date Deposited: 23 Jul 2013 06:26
Last Modified: 23 Jul 2013 06:26
URI: http://repository.um.edu.my/id/eprint/1004

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