Multilocus Sequence Typing of Methicillin-Resistant Staphylococcus aureus from an Area of Low Endemicity by Real-Time PCR

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Abstract
A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates ( n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCC mec ) type identified 27 different genotypes, and type IV SCC mec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCC mec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCC mec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCC mec -typing results when investigating the origins of MRSA.

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