Dissecting Vancomycin-Intermediate Resistance in Staphylococcus aureus Using Genome-Wide Association

Abstract

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes.

Novel Mutation Sites in the Development of Vancomycin- Intermediate Resistance in Staphylococcus aureus

“…Alam et al reported that rpoB H481 is the predominant locus associated with an increased vancomycin MIC (Alam et al, 2014). The mutations H481Y/N in rpoB play a dual role in rifampin and vancomycin resistance (Watanabe et al, 2011; Gao et al, 2013).…”

Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains

“…It is now appreciated that large numbers of microbial genome sequences can be used for robust genome-wide association studies (GWAS), enabling the detection of genetic factors underlying phenotypic variation (Falush and Bowden 2006;Farhat et al 2013;Sheppard et al 2013;Alam et al 2014;Laabei et al 2014). Here, in light of the open nature of the E. coli pan-genome, we observed a significant number of novel sequences present neither in reference genomes nor previous isolates with each additional strain that was sequenced.…”

A genome-wide association study identifies a horizontally transferred bacterial surface adhesin gene associated with antimicrobial resistant strains

“…In contrast, the machine-learning approach does not consider the inter-dependence of individuals, which is not necessarily required if the purpose is simply to build a machine-learning classifier. In order to conduct a GWAS rather than a machine-learning approach, controlling for the inter-dependence of strains, or for population structure, is necessary to avoid inflation of the type I error, as described in previous reports of bacterial GWAS approaches12323334, one of which also took a machine-learning approach and succeeded in building a classifier of virulence32. We utilized the method implemented in the bugwas package, which, unlike a pioneering bacterial GWAS method based on clonal phylogeny12, does not involve splitting the data into each clonal complex for separate analysis, thus reducing the sample size for GWAS discovery.…”