• Maite Ruiz-Pérez de Pipaón, María José Torres-Sánchez, Luis Antonio Arroyo-Pedrero, Trinidad Prados-Blanco, José Carlos Palomares-Folía, and Javier Aznar-Martín.
• Servicio de Microbiología, Hospital Universitario Virgen del Rocío, Sevilla, Spain. maite.ruiz.sspa@juntadeandalucia.es
• Enferm. Infecc. Microbiol. Clin. 2005 Apr 1; 23 (4): 208-12.

IntroductionThe aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system.MethodsOne hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene.ResultsAll the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively.ConclusionThis is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.

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