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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 CE, Wooldridge KG, Turner DP, Cockayne A, James R - BMC Microbiol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Healthcare Associated Infections, Centre for Biomolecular Sciences Building, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK. okolie.charles@lmu.edu.ng.

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 1.0x10(3) CFU/mL for the 16S rRNA marker and 1.0x10(4) 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.

No MeSH data available.


Related in: MedlinePlus

Validation of the new heptaplex PCR assay. Lanes 1 and 8: 100 bp DNA ladder (NEB, UK); Lane 2: MRCoNS strain S. epidermidis NRS8 showing the markers cns, 16S, mecA, and tuf; Lane 3: Nottingham local MSCoNS showing the markers cns, 16S, and tuf; Lane 4: Group A Streptococcus showing only the 16S marker for bacterial 16SrRNA gene; Lanes 5 and 11: PCR negative control (Candida albicans); Lane 6: VRSA strain VRS1 showing the markers 16S, mecA, tuf, vanA, and spa; Lanes 7 and 14: mixed template comprising vancomycin-resistant S. aureus strain VRS1, methicillin susceptible CoNS strain S. lugdunensis NCTC12217 and PVL-positive MSSA strain NRS157 and showing all the seven markers (cns, 16S, mecA, tuf, pvl, vanA, and spa); Lane 9: PVL-negative MRSA strain Sanger252 showing the markers 16S, mecA, tuf, and spa; Lane 10: PVL-negative MSSA strain Sanger476 showing the markers 16S, tuf, and spa; Lane 12: PVL-positive MSSA strain NRS157 showing the markers 16S, tuf, pvl, and spa; Lane13: PVL-positive S. aureus strain USA400 (MW2) showing the markers 16S, mecA, tuf, pvl, and spa
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Fig2: Validation of the new heptaplex PCR assay. Lanes 1 and 8: 100 bp DNA ladder (NEB, UK); Lane 2: MRCoNS strain S. epidermidis NRS8 showing the markers cns, 16S, mecA, and tuf; Lane 3: Nottingham local MSCoNS showing the markers cns, 16S, and tuf; Lane 4: Group A Streptococcus showing only the 16S marker for bacterial 16SrRNA gene; Lanes 5 and 11: PCR negative control (Candida albicans); Lane 6: VRSA strain VRS1 showing the markers 16S, mecA, tuf, vanA, and spa; Lanes 7 and 14: mixed template comprising vancomycin-resistant S. aureus strain VRS1, methicillin susceptible CoNS strain S. lugdunensis NCTC12217 and PVL-positive MSSA strain NRS157 and showing all the seven markers (cns, 16S, mecA, tuf, pvl, vanA, and spa); Lane 9: PVL-negative MRSA strain Sanger252 showing the markers 16S, mecA, tuf, and spa; Lane 10: PVL-negative MSSA strain Sanger476 showing the markers 16S, tuf, and spa; Lane 12: PVL-positive MSSA strain NRS157 showing the markers 16S, tuf, pvl, and spa; Lane13: PVL-positive S. aureus strain USA400 (MW2) showing the markers 16S, mecA, tuf, pvl, and spa

Mentions: The 111bp vanA marker was amplified from all (100%) VRSA reference strains listed in Additional file 1. Upon these findings, it was inferred that useful as in silico bioinformatics work-up is in PCR primer prediction, wet experimentation is still needed to confirm the reliability of PCRs developed from such predictions. This is particularly important as very powerful bioinformatics tools are entering the oligo-design research arena, including those capable of aligning nearly a million sequences of the bacterial 16S rRNA gene [31]. Also, the lack of amplification in the negative control PCRs containing C. albicans template (Fig. 1 lane 2 and Fig. 2 lanes 5 and 11) further attests to the specificity of the new assay which is illustrated using numerous reference strains and their combinations (Fig. 2).Fig. 2


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

Okolie CE, Wooldridge KG, Turner DP, Cockayne A, James R - BMC Microbiol. (2015)

Validation of the new heptaplex PCR assay. Lanes 1 and 8: 100 bp DNA ladder (NEB, UK); Lane 2: MRCoNS strain S. epidermidis NRS8 showing the markers cns, 16S, mecA, and tuf; Lane 3: Nottingham local MSCoNS showing the markers cns, 16S, and tuf; Lane 4: Group A Streptococcus showing only the 16S marker for bacterial 16SrRNA gene; Lanes 5 and 11: PCR negative control (Candida albicans); Lane 6: VRSA strain VRS1 showing the markers 16S, mecA, tuf, vanA, and spa; Lanes 7 and 14: mixed template comprising vancomycin-resistant S. aureus strain VRS1, methicillin susceptible CoNS strain S. lugdunensis NCTC12217 and PVL-positive MSSA strain NRS157 and showing all the seven markers (cns, 16S, mecA, tuf, pvl, vanA, and spa); Lane 9: PVL-negative MRSA strain Sanger252 showing the markers 16S, mecA, tuf, and spa; Lane 10: PVL-negative MSSA strain Sanger476 showing the markers 16S, tuf, and spa; Lane 12: PVL-positive MSSA strain NRS157 showing the markers 16S, tuf, pvl, and spa; Lane13: PVL-positive S. aureus strain USA400 (MW2) showing the markers 16S, mecA, tuf, pvl, and spa
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Related In: Results  -  Collection

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Fig2: Validation of the new heptaplex PCR assay. Lanes 1 and 8: 100 bp DNA ladder (NEB, UK); Lane 2: MRCoNS strain S. epidermidis NRS8 showing the markers cns, 16S, mecA, and tuf; Lane 3: Nottingham local MSCoNS showing the markers cns, 16S, and tuf; Lane 4: Group A Streptococcus showing only the 16S marker for bacterial 16SrRNA gene; Lanes 5 and 11: PCR negative control (Candida albicans); Lane 6: VRSA strain VRS1 showing the markers 16S, mecA, tuf, vanA, and spa; Lanes 7 and 14: mixed template comprising vancomycin-resistant S. aureus strain VRS1, methicillin susceptible CoNS strain S. lugdunensis NCTC12217 and PVL-positive MSSA strain NRS157 and showing all the seven markers (cns, 16S, mecA, tuf, pvl, vanA, and spa); Lane 9: PVL-negative MRSA strain Sanger252 showing the markers 16S, mecA, tuf, and spa; Lane 10: PVL-negative MSSA strain Sanger476 showing the markers 16S, tuf, and spa; Lane 12: PVL-positive MSSA strain NRS157 showing the markers 16S, tuf, pvl, and spa; Lane13: PVL-positive S. aureus strain USA400 (MW2) showing the markers 16S, mecA, tuf, pvl, and spa
Mentions: The 111bp vanA marker was amplified from all (100%) VRSA reference strains listed in Additional file 1. Upon these findings, it was inferred that useful as in silico bioinformatics work-up is in PCR primer prediction, wet experimentation is still needed to confirm the reliability of PCRs developed from such predictions. This is particularly important as very powerful bioinformatics tools are entering the oligo-design research arena, including those capable of aligning nearly a million sequences of the bacterial 16S rRNA gene [31]. Also, the lack of amplification in the negative control PCRs containing C. albicans template (Fig. 1 lane 2 and Fig. 2 lanes 5 and 11) further attests to the specificity of the new assay which is illustrated using numerous reference strains and their combinations (Fig. 2).Fig. 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Healthcare Associated Infections, Centre for Biomolecular Sciences Building, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK. okolie.charles@lmu.edu.ng.

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 1.0x10(3) CFU/mL for the 16S rRNA marker and 1.0x10(4) 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.

No MeSH data available.


Related in: MedlinePlus