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A Single Amino-Acid Substitution of RNA Polymerase ß subunit Confers A Novel Class of VISA phenotype on hVISA Strain Mu3

ABSTRACT: hVISA clinical strain Mu3 spontaneously generates VISA at an extremely high frequency (1x10-6 or greater) within its cell population. The VISA-converted mutant strains generally grow slower than their parent hVISA strain, but they usually form colonies on vancomycin-containing agar plates after 48 hours of incubation on population analysis. However, we noticed a curious group of VISA mutants whose colony formation is much more delayed and observed as discrete colonies only after 96 hours incubation. They have extremely prolonged lag-phase and doubling-times, but recorded vancomycin MICs of 8 to 24 mg/L when determined after 48 to 96 hours of incubation. We established strain Mu3-6RS having vancomycin MIC of 12 mg/L (at 72h) as a representative of the ‘slow VISA’ (sVISA) strains. Its cell wall was thickened, and autolytic activity was decreased as compared with those of the parent strain Mu3. Whole genome sequencing of Mu3-6RS revealed only one mutation, which substituted the 512th amino-acid sequence of RNA polymerase ß-subunit from Arg of Mu3 to Proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant loss of small colony morphology, cell-wall thickness and reduced autolytic activity. Sequencing of rpoB genes of the reverted strains revealed mutations affecting the 512th codon replacing the Proline of Mu3-6RS to Leucine, Serine, or Histidine. This study proposes sVISA as a discrete class of VISA phenotype, which can serve as a temporary shelter for S. aureus to survive relatively high concentrations of vancomycin. The sVISA spontaneously returns to hVISA when the threat of vancomycin passes by. The rpoB(R512P) mutation may be regarded as a ‘regulatory mutation’ switching on and off the reversible sVISA phenotype. 5 sample analysis using 60mer-oligo microarray, 3 rpoB mutant(R512P,R512L,R512S), 2 wild-type strain(Mu3, ΔIP)

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Keiichi Hiramatsu   Yuki Katayama  tomomi hishinuma  Michie Saito  Yoshifumi Aiba  Longzhu Cui  Tomomi Hishinuma  Nanae Aritaka 

PROVIDER: E-GEOD-50826 | ArrayExpress | 2014-03-31



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