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Comparison of Clinical hVISA Parent and Laboratory-Derived VISA Mutants

ABSTRACT: The mechanisms leading to the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype are incompletely understood. In an effort to identify VISA determinants, clinical hVISA strain MM66 and its 1st-step VISA mutants were compared. S. aureus microarrays, array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center), as well as comparative genomic sequencing (CGS) were utilized to determine VISA transcriptome alterations and mutations that define the MM66 VISA mechanism. In addition, whole cell autolysis, antimicrobial susceptibility tests, growth in the presence of salt, coagulase and hemolysis assays were performed to confirm VISA transcriptional alterations. Overall, 2 MM66 VISA demonstrated 85 up-regulated and 8 down-regulated genes in common. SAMMD (Staphylococcus aureus Microarray Metadatabase) analysis identified a significant differential gene expression overlap between the global stress response regulator sigB-controlled genes in MM66-3 and MM66-4. CGS analysis on a MM66 VISA revealed two mutations in: a sensory box histidine kinase (yycG) and a putative adenine phosphoribosyltransferase (apt). The yycFG two-component system has been reported to regulate whole cell autolysis and MM66 VISA demonstrated reduced whole cell autolysis. Five ssaA homologues (putative amidases) and a gene that expresses a bifunctional autolysin (atlA) controlled by YycFG were downregulated in MM66 VISA. One MM66 VISA mutant demonstrated drastic downregulation of a gene cluster (betA, gbsA, betB, and cudT) required for the osmoprotective response of S. aureus and MM66 VISA mutants demonstrated reduced growth in high salt media compared to MM66. Virulence genes were also downregulated (coa, hla, hlb, hlgA, hlgB, and hlgC) in 2 MM66 VISA mutants which also demonstrated reduced coagulase and hemolytic activities compared to MM66. pur operon genes were also upregulated in MM66 VISA and it is interesting that we have also found an apt mutation, since Apt activity can be inhibited by high AMP levels and reduces the conversion of AMP into adenine. Conclusion: Mutations in yycG and apt allow hVISA to acquire the VISA phenotype and yycFG is involved with altered autolytic activity in MM66 VISA.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE16479 | GEO | 2010/03/25

SECONDARY ACCESSION(S): PRJNA116317

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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Comparison of Clinical hVISA Parent and Laboratory-Derived VISA Mutants

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