Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System

• Published in: Enfermedades infecciosas y microbiología clínica Vol. 23; no. 4; pp. 208 - 212
• Main Authors: Ruiz-Pérez de Pipaón, Maite, Torres-Sánchez, María José, Arroyo-Pedrero, Luis Antonio, Prados-Blanco, Trinidad, Palomares-Folía, José Carlos, Aznar-Martín, Javier
• Format: Journal Article
• Language: Spanish
• Published: Spain 01-04-2005
• Subjects: Automation; Bacteremia - drug therapy; Bacteremia - microbiology; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Bacteriological Techniques - instrumentation; Blood Specimen Collection - instrumentation; Computer Systems; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Humans; Methicillin Resistance - genetics; Micrococcal Nuclease - genetics; Micrococcal Nuclease - physiology; Penicillin-Binding Proteins; Polymerase Chain Reaction - instrumentation; Sensitivity and Specificity; Staphylococcal Infections - drug therapy; Staphylococcal Infections - microbiology; Staphylococcus - drug effects; Staphylococcus - genetics; Staphylococcus - isolation & purification; Staphylococcus aureus - drug effects; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification
• ISSN: 0213-005X
• DOI: 10.1157/13073146

The 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.
One 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.
All 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.
This 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.