Development of a Single-nucleotide Polymorphism Genotyping Assay for the Rapid Detection of Vancomycin-intermediate Resistance in Staphylococcus aureus Epidemic Lineage ST5
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Background: Vancomycin is a treatment option for patients with severe methicillin-resistant Staphylococcus aureus (MRSA) infection. Unfortunately, reduced susceptibility to vancomycin in S. aureus is becoming increasingly common. We developed a method for the rapid and accurate diagnosis of vancomycin-intermediate resistant S. aureus (VISA). Methods: We performed a microbial genome-wide association study to discriminate between VISA and vancomycin-susceptible S. aureus (VSSA) using 42 whole-genome sequences. A TaqMan single-nucleotide polymorphism (SNP) genotyping assay was developed to detect target SNPs in VISA strains. Results: Four SNPs in the VISA strains resulting in nonsynonymous amino-acid substitutions were associated with reduced susceptibility to vancomycin: SA_RS01235 (G203S), SA_RS09725 (V171A), SA_RS12250 (I48F), and SA_RS12550 (G478A). These four SNPs were mainly detected in the typical hospital-associated sequence type (ST)5 clonal lineage. The TaqMan assay successfully detected all four SNPs using as little as 0.2 ng DNA per reaction. Using 10 VSSA and VISA clinical strains each, we validated that the assay accurately discriminates between VISA and VSSA. Conclusions: The TaqMan SNP genotyping assay targeting four SNPs may be an alternative to current standard methods for the rapid detection of vancomycin-intermediate resistance in S. aureus epidemic lineage ST5.
Abstract
Background: Vancomycin is a treatment option for patients with severe methicillin-resistant Staphylococcus aureus (MRSA) infection. Unfortunately, reduced susceptibility to vancomycin in S. aureus is becoming increasingly common. We developed a method for the rapid and accurate diagnosis of vancomycin-intermediate resistant S. aureus (VISA). Methods: We performed a microbial genome-wide association study to discriminate between VISA and vancomycin-susceptible S. aureus (VSSA) using 42 whole-genome sequences. A TaqMan single-nucleotide polymorphism (SNP) genotyping assay was developed to detect target SNPs in VISA strains. Results: Four SNPs in the VISA strains resulting in nonsynonymous amino-acid substitutions were associated with reduced susceptibility to vancomycin: SA_RS01235 (G203S), SA_RS09725 (V171A), SA_RS12250 (I48F), and SA_RS12550 (G478A). These four SNPs were mainly detected in the typical hospital-associated sequence type (ST)5 clonal lineage. The TaqMan assay successfully detected all four SNPs using as little as 0.2 ng DNA per reaction. Using 10 VSSA and VISA clinical strains each, we validated that the assay accurately discriminates between VISA and VSSA. Conclusions: The TaqMan SNP genotyping assay targeting four SNPs may be an alternative to current standard methods for the rapid detection of vancomycin-intermediate resistance in S. aureus epidemic lineage ST5.
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