Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton-Valentine leucocidin (PVL), mecA and homologue mecALGA251

J Antimicrob Chemother. 2012 Oct;67(10):2338-41. doi: 10.1093/jac/dks221. Epub 2012 Jun 11.

Abstract

Background: The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing.

Methods: In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin].

Results and discussion: The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Toxins / genetics*
  • Bacteriological Techniques / methods*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Denmark
  • Exotoxins / genetics*
  • France
  • Humans
  • Leukocidins / genetics*
  • Methicillin-Resistant Staphylococcus aureus / genetics
  • Methicillin-Resistant Staphylococcus aureus / isolation & purification*
  • Micrococcal Nuclease / genetics*
  • Molecular Sequence Data
  • Multiplex Polymerase Chain Reaction / methods*
  • Penicillin-Binding Proteins
  • Real-Time Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA
  • Staphylococcal Infections / microbiology
  • United Kingdom
  • Virulence Factors / genetics

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • DNA, Bacterial
  • Exotoxins
  • Leukocidins
  • Panton-Valentine leukocidin
  • Penicillin-Binding Proteins
  • Virulence Factors
  • mecA protein, Staphylococcus aureus
  • nuc protein, staphylococcus
  • Micrococcal Nuclease

Associated data

  • GENBANK/JN794592
  • GENBANK/JN794593
  • GENBANK/JN794594
  • GENBANK/JN794595
  • GENBANK/JN794596
  • GENBANK/JN794597
  • GENBANK/JN794598
  • GENBANK/JN794599
  • GENBANK/JN794600