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 = 8.78 E-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.

Keywords: bacteria; genomics; phylogeny; whole-genome sequencing.

Fig. 1.—

(A) Manhattan plot showing the significance of association between 55,977 SNPs and the Etest-based VISA phenotype using QROADTRIPS. The x axis shows SNP positions (Mb) in order of N315 reference genome coordinate and the y axis shows −log₁₀ of the P values (−log₁₀ [P]) resulting from the association test. Each dot in the plot represents an SNP, and SNPs within the most frequently mutated genes walK (yellow), walR (maroon), rpoB (red), graR (lime), graS (navy), vraF (black), vraG (pink), stp1 (aqua), SA1063 (blue), vraR (green), vraS (blueish-green), and yvqF (navy) are highlighted. (B) Quantile–quantile (QQ) plot comparing distribution of observed P values against expected P values distribution under the null hypothesis of no association. If the two distributions are similar, the points should fall roughly on the 45-degree reference line. Genomic control inflation factor = 0.60. The rpoB H481 position is indicated on the plot. The horizontal red dashed line in both plots indicates Bonferroni-corrected significance threshold of 0.05 for 55,977 independent tests (−log₁₀ [P] = 6.05).

Fig. 2.—

Neighbor-joining cladogram based on 1,512 core protein clusters describing genetic relationships among 124 Staphylococcus aureus strains. The 75 strains highlighted as blue text are from this study, whereas the remaining 49 strains are from the NCBI RefSeq database (details provided in supplementary tables S1 and S2, Supplementary Material online). The genomes from the RefSeq database are labeled either by their popular strain name (e.g., COL, Newman, N315, Mu50, and MW2) or accession numbers (those with NC prefix, such as NC_002953). Gray dots represent VSSA strains, red dots represent VISA, and those without dots are strains with unknown vancomycin susceptibility profiles. The blue star indicates strains harboring rpoB H481Y/L/N mutation. The MLST type (outer ring) represented by more than two strains is indicated, whereas others are singletons. The MLST types and accession number of all 49 completed genomes are provided in supplementary table S2, Supplementary Material online.

Fig. 3.—

Most frequently described SNPs in the rpoB (1,183 amino acids). The SNPs with asterisk were found in our study and in other studies. The RRDR (amino acids 463–550) is highlighted. The H481Y mutation significantly associated with VISA phenotype is shown in red.

Fig. 4.—

Relationship between the number of ECM+RMCG model loci and vancomycin resistance for each of the 75 strains in the study measured by (A) Etest and (B) PAP-AUC. The regression lines are shown in red. The dashed blue horizontal line is the threshold for a VISA phenotype, 3 µg/ml in the case of Etest and 0.9 PAP-AUC ratio. In (A), the equation of the regression line is y = 2.08+0.83x. The adjusted R² is 0.30 and the F-statistic P value is 2.52e⁻07. In (B), the equation is y = 0.92+0.25x. The adjusted R² is 0.39 and the F statistic P value is 1.33e⁻09.