The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82–99.87%) and 96.15% (95% CI, 87.02–99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.

Background: Fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus (MRSA) involves multiple mechanisms, including quinolone resistance-determining region (QRDR) mutations and efflux pump activity. However, the relationship between these resistance mechanisms and virulence factors (VFs) remains unclear. This study investigated the relative contributions of QRDR mutations and carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-responsive mechanisms to fluoroquinolone resistance in MRSA and their associations with VF profiles. Methods: Sixty ciprofloxacin-resistant MRSA bloodstream isolates were analyzed. The minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) and levofloxacin (LEV) were determined before and after treatment with the efflux pump inhibitor CCCP. The isolates were classified as CCCP responsive or non-responsive based on a ≥4-fold reduction in CIP MICs. The associations of QRDR mutations in gyrA, gyrB, parC, and parE and virulence-associated genes with CCCP responsiveness were evaluated. Results: CCCP significantly reduced both CIP and LEV MICs, although changes in LEV MICs were limited to a subset of isolates. Of the 60 isolates, 15 were CCCP responsive, whereas 45 were non-responsive. The non-responsive isolates had significantly more QRDR mutations, particularly gyrA nucleotide mutations and GyrA amino acid substitutions. fnbA-positive isolates showed greater CCCP-mediated CIP MIC reductions and fewer QRDR nucleotide mutations, whereas seg-, sei-, and tst-positive isolates showed lower CCCP-mediated CIP MIC reductions and greater QRDR nucleotide mutation numbers. Conclusions: QRDR mutations and CCCP-responsive mechanisms were differentially associated with fluoroquinolone resistance phenotypes in MRSA. Their associations with distinct VF profiles suggest a potential relationship between CCCP responsiveness, QRDR mutation profiles, and the virulence genetic background of MRSA.