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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.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE50826 | GEO | 2014/03/31

SECONDARY ACCESSION(S): PRJNA219096

REPOSITORIES: GEO

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Project description:The phenotype “intermediate vancomycin resistance” in Staphylococcus aureus (CLSI: MIC = 4-8 mg/L) has been assigned to changes that lead to alterations in cell wall synthesis and morphology. Most vancomycin intermediately resistant S. aureus (VISA) strains are characterised by an increased cell wall thickness as a consequence of an activated cell wall biosynthesis and decreased autolysis. The purpose of this study was to analyse the genetic basis of the vancomycin resistance mechanism of the clinical VISA isolate SA137/93A and its spontaneous mutant strain SA137/93G. The methicillin-resistant S. aureus (MRSA) SA137/93A was isolated from a tracheal secretion and displays heterogeneous intermediate vancomycin resistance (hVISA strain, MIC: 8 mg/L in BHI). Subculturing in presence of 6 mg/L vancomycin generated a mutant with homogeneous intermediate vancomycin resistance, that showed an MIC value of 16 mg/L in BHI and was designated SA137/93G. PFGE profiles and phage typing of the strains showed that they were members of the Iberian clone (ST247), which was prevalent in Germany in the early nineties under the designation “Northern German epidemic strain”. Both strains possess a thickened cell wall. However, the vancomycin resistance of strain SA137/93A is most probably enhanced by an increased amount of free D-Ala-D-Ala termini in the cell wall, which is due to decreased crosslinking, whereas the mutant strain SA137/93G shows normal crosslinking. Moreover, strain SA137/93A displays an increased expression of the essential two-component system yycFGHI as a consequence of an IS256 insertion in the promoter region, while strain SA137/93G is characterised by an insertion of IS256 into the gene tcaA. Although both insertions were shown to correlate with a decrease in susceptibility to vancomycin, the difference in the vancomycin resistance level of the strain pair could be mainly attributed to the disruption of tcaA in the mutant.This study was conducted to identify resistance mechanisms that both strains might have in common. To this end we compared the transcriptomes of both strains with that of the closely related vancomycin susceptible MRSA/VSSA strain SA1450/94 (MIC: 2 mg/L). We found that the genes of the capsule biosynthesis were the only genes with higher expression in both VISA strains. Keywords: strain comparison

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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

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