Background:Phenotypic methods for the detection of methicillin resistance are inadequate, due to presence of hetero-resistant population and dependence of environmental factors that may affect the phenotypic expression of resistance.Aims:Present study was conducted, to evaluate the efficacy of phenotypic methods for the identification of species and mec-A mediated resistance in S. aureus with polymerase chain reaction (PCR), and to assess the prevalence of the Panton-Valentine leukocidin (pvl) toxin in methicillin resistant S. aureus (MRSA) and overall S.aureus population.Materials and Methods:A total of 200 clinical isolates of Staphylococci were subjected to phenotypic and genotypic methods for the species identification and detection of MRSA.Results:The specificity and sensitivity of conventional methods in the detection of S.aureus, was found to be 100 and 97.59% respectively. However, the performance of phenotypic methods in the detection of MRSA were: Oxacillin disc diffusion (DD)-sensitivity 70.58%, specificity 75.75%; cefoxitin DD-sensitivity 86.27%, specificity 83.33%; and oxacillin agar dilution-sensitivity 92.15%, specificity 90.90%. PVL gene was detected in all mec-A positive isolates irrespective of their types.Conclusion:Phenotypic methods still preferred for the species identification, but for the reliable detection of MRSA an algorithm should include a combination of tests and apply a genotypic method for confirmation of resistance isolates showing discrepant results. Considering the high prevalence of PVL-MRSA, we recommend PCR as assay, as it has an advantage of simultaneous detection of mec-A and pvl genes by multiplex PCR.

Background & objectives:The two major genotypic markers that distinguish community acquired (CA) from hospital acquired (HA) methicillin resistant Staphylococcus aureus (MRSA) isolates are the architecture of mobile genetic element (SCCmec type) and presence of panton valentine leukocidin (PVL) toxin. This study was conducted to determine the molecular characteristics of CA- and HA- MRSA and methicillin sensitive S. aureus (MSSA) isolates in Sikkim.Methods:A total of 150 clinical isolates of S. aureus isolated from various clinical specimens were subjected to duplex (mec-A and pvl gene) and multiplex (SCCmec typing) PCR.Results:Of the 150 isolates, 53 (35.33%) and 66 (44%) were positive for mec-A (MRSA) and pvl genes, respectively. Thirty eight (25.33%) met the definition of CA-MRSA and 15 (10%) of HA-MRSA and the remaining 63 (42%) and 34 (22.66%) as CA- and HA-MSSA, respectively. No significant difference was seen in the distribution of PVL toxin in MRSA and MSSA isolates, but it was significantly (P<0.001) high in overall MRSA isolates than in MSSA. The majority of the MRSA isolates showed a double amplification band of SCCmec type III plus V (54.71%), and only a fewer isolates were amplified by single DNA fragments of type I (1.88%), III (3.77%), IVa (1.88%) and V (11.32%). SCCmec types I, III, IVa, were found only in HA-MRSA isolates, whereas type V in both the CA- and HA-MRSA. AST pattern showed that 18.42 per cent (7/38) and 46.66 per cent (7/15) were multidrug resistant (MDR)-CA-MRSA and MDR-HA-MRSA, respectively.Interpretation & conclusions:The present results show that SCCmec type V MRSA has been on the rise, and genotypic markers such as pvl gene detection used for the differentiation of these clinically distinct isolates of MRSA may not be reliable.