Molecular Characterization of Nosocomial Methicillin Resistant Staphylococci aureus by Rep-PCR

摘要

Nosocomial methicillin-resistance Staphylococcus aureus (MRSA) poses problem to clinicians and hospital administrators for its management. In the present study isolation, identification molecular characterizations of methicillin-resistance Staphylococcus aureus were performed at Hospital Kuala Lumpur, Malaysia during December 2005 to April 2007. Twenty seven MRSA positive samples were identified based on cultural, biochemical and antibiotic sensitivity assay. These were, 51% from blood samples, 33.3% from tracheal aspirate and 11,1% from nasal swab and pus. Molecular method rep-PCR was used to characterize the MRSA positive strains. Results of rep-PCR showed 5 different pattern of bands based on their genomic nature. Thus, rep-PCR was proved to be potential tool to determine genomic differences of nosocomial MRSA in resource limited setting. Introduction Staphylococcus aureus is considered as a major nosocomial pathogen which causes a range of diseases, including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food poisoning, carbuncles, and boils.Infection with nosocomial methicillin-resistance Staphylococcus aureus (MRSA) in hospitals poses significant problems to clinicians managing patients, as well as to hospital administration. Since the introduction of methicillin in the 1960s, prevalence of nosocomial MRSA infections increased globally. Many countries have reported periodic outbreaks of nosocomial infections with MRSA especially in the intensive care units. In the United States, the National Nosocomial Infection Surveillance System reported the increase of MRSA in the intensive care units from 3% to 53% over 20 years. In Malaysian hospitals the rate of MRSA in 1988 reported to be between 10-25%.3 Further data obtained from the National Surveillance on Antibiotic Resistance program showed increase in the percentages of MRSA from 28% in 1992 to 36% in 1996. However, in the past four years, the rates of MRSA infections in 17 major hospitals showed decreased in trend. Detection and characterization MRSA strains during outbreaks are important to monitor trace and control the intrahospital and interhospital spread or evolution of bacterial strains. Molecular methods have been proven to be cost-effective for the study of nosocomial outbreaks and give genomic nature of infected organisms. However, PFGE is time consuming, tedious and the reagents are expensive, hence may not be practical in resource limited settings. Rep-PCR seems to be a promising technique in view of its adequate discriminatory power, good reproducibility and at a low cost in detecting MRSA.5,6 The present communication deals with use of rep-PCR to characterize genetic relatedness among the MRSA strains isolated and identified from clinical samples. Materials And Methods Study placeThe present research was conducted on the patients admitted to Hospital Kuala Lumpur (HKL), Malaysia from December 2005 to April, 2007. SpecimensBlood, tracheal aspirate, nasal swab and pus specimens obtained from the suspected patients were processed in the Microbiology laboratory for the isolation and identification of Methicillin Resistant Staphylococcus aureus(MRSA) organisms.Isolation and IdentificationMethicillin Resiatant Staphylococcus aureus(MRSA) organisms were isolated and identified based on colonial morphology, Gram’s stain, Coagulase test, Antibiotic resistance to methicillin by oxacillin 1 μg, disk susceptibility test following the guidelines of the Clinical and Laboratory Standards Institute.7 The MRSA identified bacteria were kept in microbank bead and stored at -70°C. These were sub-cultured on blood agar before use.Antibiotic sensitivity testAntibiotic susceptibility tests were performed by disc diffusion method using the Mueller-Hinton agar which was inoculated with a suspension adjusted to 0.5 McFarland turbidity standards. Plates were incubated overnight at 35°C. CLSI guidelines were used as reference for determination of susceptibility. In addit