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


CONCLUSIONS
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.
Laboratory of Healthcare Associated Infection, Microbiology Services Colindale, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK. bruno.pichon@hpa.org.uk
Bruno
B
Pichon
Robert
R
Hill
Frederic
F
Laurent
Anders Rhod
AR
Larsen
Robert L
RL
Skov
Mark
M
Holmes
Giles F
GF
Edwards
Christopher
C
Teale
Angela M
AM
Kearns
D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003718:Denmark; D005098:Exotoxins / Q000235:genetics*; D005602:France; D006801:Humans; D007956:Leukocidins / Q000235:genetics*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D008836:Micrococcal Nuclease / Q000235:genetics*; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D046915:Penicillin-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013203:Staphylococcal Infections / Q000382:microbiology; D006113:United Kingdom; D037521:Virulence Factors / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D046915:Penicillin-Binding Proteins; D037521:Virulence Factors; C487789:mecA protein, Staphylococcus aureus; C074002:nuc protein, staphylococcus; D008836:Micrococcal Nuclease