Abstract
BackgroundStaphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution.ResultsA new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour.ConclusionThe speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0490-9) contains supplementary material, which is available to authorized users.
Highlights
Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health
Since S. aureus and coagulasenegative staphylococci (CoNS) harbor the mecA gene in common, Huletsky and colleagues highlighted the need for a discriminatory assay capable of identifying the cohabitation of S. aureus and coagulase-negative staphylococci (CoNS) especially in non-sterile sites such as the nares and the skin [6]
This allows the simultaneous detection of markers specific for S. aureus and CoNS within the same PCR tube without recourse to the conventional elimination approach allowing mixtures of S. aureus and CoNS to be identified without cumber and ambiguity
Summary
Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Since S. aureus and coagulasenegative staphylococci (CoNS) harbor the mecA gene in common, Huletsky and colleagues highlighted the need for a discriminatory assay capable of identifying the cohabitation of S. aureus and coagulase-negative staphylococci (CoNS) especially in non-sterile sites such as the nares and the skin [6]. They discriminated S. aureus from CoNS by detecting DNA from the staphylococcal cassette chromosome (SCC) using oligonucleotides which hybridised to the S. aureus open reading frame encoding unknown function (orfX) [6]. Commercial assays inspired by the orfX oligos attracted numerous reports of assay failure because some SCC types which lacked the mecA gene were misidentified as MRSA [7, 8]
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