Ann Lab Med.  2023 Jul;43(4):355-363. 10.3343/alm.2023.43.4.355.

Development of a Single-nucleotide Polymorphism Genotyping Assay for the Rapid Detection of Vancomycin-intermediate Resistance in Staphylococcus aureus Epidemic Lineage ST5

• Kim JW
• Lee KJ

• KMID: 2551953
• DOI: http://doi.org/10.3343/alm.2023.43.4.355

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.

Keyword

Vancomycin-intermediate resistant Staphylococcus aureus; Single-nucleotide polymorphism; TaqMan SNP genotyping assay; Genome-wide association study

Figure

• 

  Fig. 1 Bioinformatics workflow. Each box represents a series of tasks related to a certain functionality (indicated in bold). The bioinformatics software packages used in each module are also mentioned (indicated in italics). Abbreviation: SNP, single-nucleotide polymorphism.

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  Fig. 2 Allelic discrimination plots for the four SNPs using the TaqMan genotyping assay on 20 samples (10 VSSA and 10 VISA). Red (VISA) and blue (VSSA) dots represent the homozygous genotypes. The square at the bottom left of the plot is the no-template control. Abbreviations: SNP, single-nucleotide polymorphism; VISA, vancomycin-intermediate resistant Staphylococcus aureus; VSSA, vancomycin-susceptible S. aureus.

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