title: Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates.
abstract: Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature "AT repeat" sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3' end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of > or =6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had > or =6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for > or =6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA.
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Bonnstetter
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wolter
authors.forename: Fred C
authors.initials: FC
authors.lastname: Tenover
authors.forename: Linda K
authors.initials: LK
authors.lastname: McDougal
authors.forename: Richard V
authors.initials: RV
authors.lastname: Goering
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D015373:Bacterial Typing Techniques; D002876:Chromosomes, Bacterial; D017714:Community-Acquired Infections / Q000382:microbiology*; D017931:DNA Primers; D005098:Exotoxins / Q000235:genetics; D006801:Humans; D007956:Leukocidins / Q000235:genetics; D016106:Methicillin Resistance / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D013045:Species Specificity; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin