Development of New Pentaplex PCR Assay for Differentiating Staphylococci from Other Bacteria with Simultaneous Detection of Staphylococcus aureus Genes Encoding Panton-Valentine Leukocidin and Methicillin Resistance

In some human infections including those of blood, skin and respiratory tract, the causative bacterial agents tend to overlap, especially Staphylococcus spp. and Streptococcus spp. Such overlaps constitute difficulties in the choice of diagnostic tools, antibacterial chemotherapy and infection control strategies. To resolve this challenge, we developed a pentaplex PCR assay which simultaneously detects sequences for the recognition of bacteria (bacterial 16S rRNA), the genus staphylococcus translation elongation factor Tu (tuf), Staphylococcus aureus (spa), mecA-encoded staphylococcal methicillin resistance (mecA) and the S. aureus Panton-Valentine leukocidin (PVL) virulence factor (pvl). The new pentaplex PCR assay was validated using standard bacterial strains (N=377) including strains from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), the National Collection of Type Cultures (NCTC), and the National Collection of Industrial and Marine Bacteria (NCIMB). The new pentaplex PCR assay enables inference of bacterial presence/absence, differentiates between the genus Staphylococcus spp. and other bacteria, separates S. aureus from other Staphylococcus spp., differentiates between methicillin-susceptible and methicillin-resistant staphylococci, and detects the S. aureus PVL gene locus. The negative predictive value was 100% while the positive predictive value was 100%. Using a 96-well plate, the time to result was 2.5 hours against ≥24 hours by bacteriological culture. The new pentaplex PCR assay can easily be integrated into routine diagnostic microbiology workflow especially for laboratories with slim budgets which are unable to incorporate next generation sequencing at the moment.