Tohoku J. Exp. Med., 2010, 220(2)

Genetic Diagnosis of Community-Acquired MRSA: A Multiplex Real-Time PCR Method for Staphylococcal Cassette Chromosome mec Typing and Detecting Toxin Genes

MAIKO MOTOSHIMA,¹ KATSUNORI YANAGIHARA,¹ YOSHITOMO MORINAGA,¹ JUNICHI MATSUDA,¹ KAZUYUKI SUGAHARA,¹ YASUAKI YAMADA,¹ SHIGERU KOHNO² and SHIMERU KAMIHIRA¹

¹Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, Nagasaki, Japan
²Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, Nagasaki, Japan

Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of infections in health care settings and community environments. In particular, community-acquired MRSA (CA-MRSA) is important for clinicians because many fatal cases in healthy populations have been reported. Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element and carries the central determinant for broad-spectrum beta-lactam resistance encoded by the mecA gene. The emergence of MRSA is due to the acquisition and insertion of the SCCmec element into the chromosome. CA-MRSA is characterized as SCCmec type IV. Thus, we aimed to establish a novel multiplex real-time PCR method to distinguish SCCmec type, which enables us to evaluate the pathogenicity of MRSA. A total of 778 MRSA were isolated at Nagasaki University Hospital from 2000 to 2007. All isolates were subjected to minimal inhibitory concentration testing and PCR for SCCmec typing and detecting genes of toxins: tst (toxic shock syndrome toxin 1), sec (encoded enterotoxin type c), etb (exfoliative toxin type b), and lukS/F-PV (Panton-Valentine leukocidin). PCR was performed to amplify a total of 10 genes in the same run. The 667 MRSA clones detected from pus in 778 clones were classified as SCCmec type II (77.7%), type IV (19.2%), and type I (3.0%). 87.5% of SCCmec type II clone had tst and sec genes. No isolate was lukS/F-PV positive. The present study indicates the high rate of lukS/F-PV-negative SCCmec type IV in Nagasaki. Our PCR method is convenient for typing MRSA and detecting toxins in Japan.

keywords —— CA-MRSA; SCCmec type; pathogenicity; real-time PCR; rapid typing