Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates

Kristin K. Bonnstetter, Daniel J. Wolter, Fred C. Tenover, Linda K. McDougal, Richard V. Goering

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥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.

Original languageEnglish
Pages (from-to)141-146
Number of pages6
JournalJournal of Clinical Microbiology
Volume45
Issue number1
DOIs
StatePublished - Jan 2007

Fingerprint

Multiplex Polymerase Chain Reaction
Methicillin-Resistant Staphylococcus aureus
Chromosomes
Conserved Sequence
Pulsed Field Gel Electrophoresis
Cytotoxins
DNA Sequence Analysis
Genes
Panton-Valentine leukocidin
Infection

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)
  • Microbiology

Cite this

Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates. / Bonnstetter, Kristin K.; Wolter, Daniel J.; Tenover, Fred C.; McDougal, Linda K.; Goering, Richard V.

In: Journal of Clinical Microbiology, Vol. 45, No. 1, 01.2007, p. 141-146.

Research output: Contribution to journalArticle

Bonnstetter, Kristin K. ; Wolter, Daniel J. ; Tenover, Fred C. ; McDougal, Linda K. ; Goering, Richard V. / Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates. In: Journal of Clinical Microbiology. 2007 ; Vol. 45, No. 1. pp. 141-146.
@article{00e7491893584462936355d315f11f33,
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 ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥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.",
author = "Bonnstetter, {Kristin K.} and Wolter, {Daniel J.} and Tenover, {Fred C.} and McDougal, {Linda K.} and Goering, {Richard V.}",
year = "2007",
month = "1",
doi = "10.1128/JCM.01228-06",
language = "English",
volume = "45",
pages = "141--146",
journal = "Journal of Clinical Microbiology",
issn = "0095-1137",
publisher = "American Society for Microbiology",
number = "1",

}

TY - JOUR

T1 - Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates

AU - Bonnstetter, Kristin K.

AU - Wolter, Daniel J.

AU - Tenover, Fred C.

AU - McDougal, Linda K.

AU - Goering, Richard V.

PY - 2007/1

Y1 - 2007/1

N2 - 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 ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥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.

AB - 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 ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥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.

UR - http://www.scopus.com/inward/record.url?scp=33846240796&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846240796&partnerID=8YFLogxK

U2 - 10.1128/JCM.01228-06

DO - 10.1128/JCM.01228-06

M3 - Article

C2 - 17093011

AN - SCOPUS:33846240796

VL - 45

SP - 141

EP - 146

JO - Journal of Clinical Microbiology

JF - Journal of Clinical Microbiology

SN - 0095-1137

IS - 1

ER -