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Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Okolie, Charles Emeka; Wooldridge, Karl G.; Turner, David P.J.; Cockayne, Alan; James, Richard

Authors

Charles Emeka Okolie

Karl G. Wooldridge

David P.J. Turner

Alan Cockayne



Abstract

Background
Staphylococcal 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.

Results
A 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.

Conclusion
The 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.

Journal Article Type Article
Publication Date Aug 5, 2015
Journal BMC Microbiology
Electronic ISSN 1471-2180
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 15
Issue 1
Article Number 157
APA6 Citation Okolie, C. E., Wooldridge, K. G., Turner, D. P., Cockayne, A., & James, R. (2015). Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes. BMC Microbiology, 15(1), https://doi.org/10.1186/s12866-015-0490-9
DOI https://doi.org/10.1186/s12866-015-0490-9
Keywords Differential diagnosis; tuf gene polymorphism; Mixed infection; Therapy-refractory
Publisher URL http://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-015-0490-9
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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