Comparative Genomics Identifies Novel Genetic Changes Associated with Oxacillin, Vancomycin and Daptomycin Susceptibility in ST100 Methicillin-Resistant Staphylococcus aureus

Abstract

Infections due to vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) represent a serious concern due to their association with vancomycin treatment failure. However, the underlying molecular mechanism responsible for the hVISA/VISA phenotype is complex and not yet fully understood. We have previously characterized two ST100-MRSA-hVISA clinical isolates recovered before and after 40 days of vancomycin treatment (D1 and D2, respectively) and two in vitro VISA derivatives (D23C9 and D2P11), selected independently from D2 in the presence of vancomycin. This follow-up study was aimed at further characterizing these isogenic strains and obtaining their whole genome sequences to unravel changes associated with antibiotic resistance. It is interesting to note that none of these isogenic strains carry SNPs in the regulatory operons vraUTSR, walKR and/or graXRS. Nonetheless, genetic changes including SNPs, INDELs and IS256 genomic insertions/rearrangements were found both in in vivo and in vitro vancomycin-selected strains. Some were found in the downstream target genes of the aforementioned regulatory operons, which are involved in cell wall and phosphate metabolism, staphylococcal growth and biofilm formation. Some of the genetic changes reported herein have not been previously associated with vancomycin, daptomycin and/or oxacillin resistance in S. aureus.

Keywords: IS256; MRSA; S. aureus; ST100; WGS; hVISA/VISA.

Figure 1

Oxacillin population analysis profile and area under the curve (PAP-AUC) of bacterial strains included in this study. Serial 10-fold dilutions of cultures were plated onto Mueller–Hinton agar with oxacillin (OXA). Each point represents the viable count (log10 CFU/mL) after 48 h against increasing concentrations of OXA.

Figure 2

(A) Transmission electron microscopy (TEM) of representative cells; images obtained at 50,000×. A: D1; B: D2; C: D23C9; D: D2P11. (B) Cell diameter measured by TEM (30 cells per strain). The results are expressed in nanometers (nm) as violin plots showing median (dashed line) and interquartile range (dots) and were compared with Kruskal–Wallis’s test (p < 0.0001). The horizontal bars show significant differences between individual groups detected by Dunn’s multiple comparison test. ** p < 0.001; *** p < 0.0001. (C) Growth curves. Each point represents the mean and standard error from three independent assays.

Figure 3

Clinker gene cluster comparison of the genomic region between graX and SA0625. Homologous CDSs are in the same color and linked through grey bars with the percentage amino acid identity, as indicated in the legend. IS256 insertions are marked as aqua circles (in case of contig ends), or annotated CDS (confirmed by PCR).

Figure 4

Biofilm production. Results are represented as final OD for each strain with median value (line) and interquartile range (whiskers) and are compared with Kruskal–Wallis’s test (p < 0.0001). The horizontal bars show significant differences between individual groups detected by Dunn’s multiple comparison test. * p < 0.05; *** p < 0.001. NRS101 (S. epidermidis NRS101), strong biofilm producer; Δica (S. aureus Newman Δica), ica independent-biofilm producer strain. The bottom picture shows a representative microtiter plate of the biofilm assay for each strain.

Figure 5

Genes harboring genetic changes in hVISA/VISA strains analyzed in this study and their relationships with cellular processes (lines). Blue boxes: Genes with mutations related to hVISA/VISA shared by D1, D2, D23C9 and D2P11. Yellow boxes: Genes with mutations differing between D2 and D1. Red boxes: Genes differing between the in vitro-selected mutants (D23C9 and D2P11) and their parental strain (D2). Δ ≈8 kb IS256-mediated deletion. * SNP/INDEL. Purple boxes: Key regulatory operons related to vancomycin resistance in hVISA/VISA (not mutated in these strains). R: Resistance. VAN: vancomycin. OXA: oxacillin. DAP: daptomycin.