Rapid detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive BacT/Alert blood culture bottles using real-time polymerase chain reaction: evaluation and comparison of 4 DNA extraction methods.

Although the introduction of automated blood culture systems has dramatically reduced laboratory personnel bench time, 48 to 72 h is still required for the identification of pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) and the accurate determination of antimicrobial sensitivities for prompt optimal patient therapy and infection control initiatives. The following 4 DNA blood culture extraction methods were compared: (a) organic, (b) differential centrifugation and lysis, (c) alkali wash/lysis, and (d) Qiagen lysis/filtration (QIAGEN, West Sussex, UK). The benzyl alcohol extraction method (a) was found to be the most optimal method having a reasonable extraction time of 1.8 h and 100% correlation with the "gold standard" laboratory culture. A "dual locus" real-time polymerase chain reaction (PCR) targeting the S. aureus-specific thermonuclease nuc gene and the staphylococcal methicillin resistance determinant mecA gene were used as a reliable indicator of the presence of MRSA. In conjunction with the DNA extraction method (a), detection time for MRSA/MSSA isolates from positive blood cultures was dramatically reduced from at least 24 to 48 h to approximately 3 h.