"Slow VISA," a novel phenotype of vancomycin resistance, found in vitro in heterogeneous vancomycin-intermediate Staphylococcus aureus strain Mu3

Abstract

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1×10(-6)). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase β-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

FIG 1

Heterogeneity in the sizes of Mu3-6R-P colonies formed on a drug-free agar plate. About 10³ CFU of 6R-P after 2 days of passages in BHI broth were spread on a BHI agar plate. The photos were taken after incubation at 37°C for 30, 48, 72, and 96 h. Inset, magnification of the area in the square. Three sizes of colonies were judged after 30 h of incubation: large (L), small (S) and pinpoint (P). The L and S variants maintained their colony sizes in subsequent passages on the BHI agar plate.

FIG 2

Analysis of vancomycin-resistant subpopulation profiles (population analysis [PA]). (A) Profiles of Mu3, 6R-P, and Mu50 observed after 48, 72, 96, 120, and 144 h of incubation at 37°C. (B) 6R-P and its phenotypic revertant strains L1, L2, and L3 as observed after 96 h of incubation at 37°C. (C) ΔIP and H14 strains and their derivatives introduced with the rpoB(R512P) mutation. About 10⁷ CFU of the overnight culture of each strain were inoculated on BHI agar plates containing various concentrations of vancomycin. The numbers of colonies formed after incubation of 96 h at 37°C were counted and plotted in a semilogarithmic graph. Note that the shape of the PA curve of H14 is reminiscent of hVISA; however, its resistant subpopulation, as judged by the growth on the plate containing 4 mg/liter vancomycin, is much smaller than that of Mu3, which is >1 × 10⁻⁶. We call such an S. aureus strain with reduced susceptibility to vancomycin pre-hVISA.

FIG 3

The effect of rpoB(R512P) mutations on the growth of S. aureus strains. (A) Mu3 and Mu3-derived strains in comparison with Mu50; (B) ΔIP-derived strains, and Mu3 as a control. H14 is ΔIPvraS(S329L), having a pre-hVISA phenotype (see Fig. 2C legend). Ten milliliters of BHI broth was inoculated with 10⁷ cells of each strain, followed by incubation with shaking at 37°C for up to 1,000 min. The OD was monitored every 2 min during the cultivation.

FIG 4

Reduction of autolytic activity in rpoB(R512P) mutant strains. Triton X-100 (0.05%)-stimulated autolysis of two wild-type rpoB strains, Mu3 and ΔIP, and four rpoB mutant strains, 6R-P, L1, L2, and ΔIPrpoB(R512P), and VISA strain Mu50 were tested. Mu50 possesses the rpoB(H481Y) mutation. Autolysis was measured as the decline in optical density versus time and is expressed as the percentage of the initial optical density.

FIG 5

Volcano plot analysis of transcription profiles. The −log₁₀ (P value for a t test) values are plotted against the log ratios (log₂ fold change). (A) Comparison of the level of gene expression of 6R-P, its two LC-sized variants L1 and L2, and Mu50 with that of clinical strain Mu3. (B) Comparison of the transcription profiles of 6R-P and Mu50 with those of Mu3 and ΔIP. The genes which are upregulated (red) or downregulated (blue) more than two times in Mu3 relative to VSSA strain ΔIP were downregulated and upregulated, respectively, in Mu50 relative to Mu3. However, this reversal of gene expression was not seen in 6R-P. As a result, 6R-P showed a much more scattered distribution of gene expression than Mu50 relative to VSSA strain ΔIP. The gray plots show the genes having a P value of ≥0.05 with t test.