Molecular identification of methicillin resistance and virulence marker in staphylococcus aureus
Loading...
Date
2009
Authors
Gunawardena, N. D.
Journal Title
Journal ISSN
Volume Title
Publisher
University of Peradeniya
Abstract
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial and community-acquired pathogen of worldwide concern. In Sri Lanka, MRSA was first described in 1989 and have emerged subsequently as a major nosocomial pathogen in hospital environments. Therefore, accurate methods to detect methicillin resistance among S. aureus strains are essential to ensure optimal antimicrobial therapy and management of infection control caused by these strains. Objectives To detect the S. aureus species marker-the femB gene, mecA gene encoding the penicillin binding protein2a which is associated with staphylococcal methicillin resistance and a key virulence factor, Panton-Valentine Leukocidin(PVL) gene which causes leukocyte destruction and tissue necrosis and is responsible for severe infection with S. aureus. Design: Prospective laboratory analysis Setting: GENETECH, Colombo 8; Department of Microbiology, University of Peradeniya. Methods and results A clinical MRSA isolate, confirmed by disc diffusion method was obtained from Apollo Lanka Hospital in order to establish the PCR assay for the detection of femB , mecA and PVL genes. Forty-three clinical isolates of S. aureus were identified by the conventional laboratory methods of colony morphology, Gram stain, catalase and slide and tube coagulase. Using the PCR, femB gene was detected in 36 of the 43 isolates. The MICs of oxacillin for all 43 isolates were determined by Mueller-Hinton Agar dilution method. 31 isolates had MIC’s of ≤2 mg/L (Methicillin sensitive S. aureus-MSSA) and 12 isolates were MRSA with values of 4 and ≥ 128 mg/L. Using PCR, all 12(28%) MRSA strains gave positive results for mecA gene. Furthermore, 10 of the 31 isolates identified as MSSA were mec positive. All 7 femB gene negative isolates were mecA positive. Eleven out of the 36 (30%) S. aureus isolates were positive for the PVL gene. None of the JSemB negative isolates carried the PVL gene. 10 of them were MSSA and 1 was MRSA. A further 43 clinical specimens from which S. aureus was isolated and ABST performed in the routine clinical microbiology laboratory-THP were directly tested in order to compare the time required to detect MRSA using conventional methods vs. PCR assay. Among 43 S. aureus isolates from patients, 18 (42%) were MRSA by conventional disc diffusion testing. Test results were obtained in 48-72hrs. All 43 clinical specimens were directly examined for femB, mecA and PVL genes using PCR. 15 specimens were failed to detect in PCR assay. Of the 28 specimens, femB gene was detected in 24(86%). Again indicating a discrepancy between conventional and molecular identification of S aureus. The mecA gene was detected in all 14 of the 18 (78%) specimens from which MRSA were isolated. In addition, 2 specimens from which MSSA had been reported were positive for mecA, Four specimens which expressed the mecA gene were negative for femB. Fourteen out of 43 specimens tested directly yielded positive results for PVL gene. 7 of them were MSSA and 7 were MRSA. The PVL gene was not detected in any of the femB negative specimens. Direct testing by PCR required 4 hours for DNA preparation, PCR performance and detection of PCR products into the gel, contrasting with the 48-72 hours required for conventional testing. A minimum of 25-26 bacterial cells in 11 of specimens were detectable using this assay. This study shows that conventional methods for detection of methicillin resistance like disc diffusion and MIC are time consuming. PCR offers a rapid and accurate identification of species, methicillin resistance and virulence detection for clinical use. However, its use may be limited to molecular diagnostic laboratories as technical expertise is required and cost is high (currently ≈ Rs.3600).
Description
Keywords
Plant Sciences , Staphylococcus Aureus