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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Ruiz-Pérez de Pipaón
Maite
M
Servicio de Microbiología, Hospital Universitario Virgen del Rocío, Sevilla, Spain. maite.ruiz.sspa@juntadeandalucia.es
Torres-Sánchez
María José
MJ
Arroyo-Pedrero
Luis Antonio
LA
Prados-Blanco
Trinidad
T
Palomares-Folía
José Carlos
JC
Aznar-Martín
Javier
J
D001331:Automation; D016470:Bacteremia / Q000188:drug therapy / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001431:Bacteriological Techniques / Q000295:instrumentation; D001800:Blood Specimen Collection / Q000295:instrumentation; D003199:Computer Systems; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000502:physiology; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000382:microbiology*; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; D008836:Micrococcal Nuclease