Staphylococcus aureus
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PROVIDER: PRJNA273731 | ENA |
REPOSITORIES: ENA
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Project description:Various mutations in the rpoB gene, which encodes the RNA polymerase beta subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate S. aureus (VISA) and hetero-VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found slow VISA (sVISA) phenotype (Saito et al 2014 AAC). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (72.2 min) and high vncomycin MIC (16 mg/L). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of slow growth rate, cell wall thickness, and reduced autolysis. Whole genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation F562L in rpoC. Introduction of the wild-type rpoC gene using multi-copy plasmid resolved the sVISA phenotype of V6-5, indicating that rpoC(P440L) mutation expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated additional 10 revertants to hVISA and VISA. In subsequent whole genome analysis, we identified compensatory mutations in the genes of three distinct functional categories; rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ which is involved in stringent response. It appears that rpoC(P440L) mutation causes sVISA phenotype by augmenting cell-wall peptidoglycan synthesis, and through the control of stringent response. 9 analysis using 60 mer-oligo microarray, 4 rpoC mutant, 2 relQ mutant, 2 mutations associated with the peptidoglycan, 1 wild-type strain
2015-05-08 | E-GEOD-65338 | biostudies-arrayexpress
Project description:Various mutations in the rpoB gene, which encodes the RNA polymerase beta subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate S. aureus (VISA) and hetero-VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found slow VISA (sVISA) phenotype (Saito et al 2014 AAC). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (72.2 min) and high vncomycin MIC (16 mg/L). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of slow growth rate, cell wall thickness, and reduced autolysis. Whole genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation F562L in rpoC. Introduction of the wild-type rpoC gene using multi-copy plasmid resolved the sVISA phenotype of V6-5, indicating that rpoC(P440L) mutation expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated additional 10 revertants to hVISA and VISA. In subsequent whole genome analysis, we identified compensatory mutations in the genes of three distinct functional categories; rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ which is involved in stringent response. It appears that rpoC(P440L) mutation causes sVISA phenotype by augmenting cell-wall peptidoglycan synthesis, and through the control of stringent response.
2015-05-08 | GSE65338 | GEO
Project description:Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of Mu50 was achieved by sequentially introducing mutations into five genes of a vancomycin-susceptible S. aureus (VSSA) strain ∆IP. Introduction of mutation Ser329Leu into vraS encoding the sensor histidine kinase of vraSR two-component regulatory (TCR) system and another mutation Glu146Lys into msrR, encoding putative methionine sulfoxide reductase regulator, raised vancomycin resistance to the level of heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, graR (Asn197Ser) of graSR TCR system and rpoB(His481Tyr) encoding ß subunit of RNA polymerase, converted the hVISA strain into a VISA strain having the level of vancomycin resistance of Mu50. Surprisingly, however, the constructed quadruple mutant strain did not have thickened cell wall, a cardinal feature of VISA phenotype. Subsequent study showed that cell-wall thickening was an inducible phenotype with the mutant strain as opposed to that of Mu50, which is a constitutive one. Finally, introduction of mutation Ala297Val into the orf SAV2309 of the mutant strain converted the inducible cell-wall thickening into a constitutive one. SAV2309 encodes a putative formate dehydrogenase (designated Fdh2). Though not a transcription regulator, the mutation of the fdh2 caused a significant change in transcriptome. Thus, all of the five mutated genes required for VISA phenotype acquisition were directly or indirectly involved in the regulation of cell physiology. VISA seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.
2016-04-19 | GSE43643 | GEO
Project description: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.
2014-03-31 | GSE50826 | GEO
Project description: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 M-bM-^@M-^Xslow VISAM-bM-^@M-^Y (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 M-CM-^_-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 M-bM-^@M-^Xregulatory mutationM-bM-^@M-^Y 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, M-NM-^TIP)
2014-03-31 | E-GEOD-50826 | biostudies-arrayexpress
Project description:Daptomycin (DAP) is the last-resort treatment for heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-S.aureus (VISA), and DAP-resistance onset which is also linked to reduced vancomycin susceptibility, is an increasing public health problem. To have more insight into the mechanisms of daptomycin resistance, the comparative transcriptomes of two DAP-R (1C-3B) clinical isogenic isolates vs their DAP-S (1A-3A) counterparts were investigated by Illumina RNA-seq, the Rockhopper tool, computational filtering analyses and bioinformatic tools.
2020-08-07 | GSE121797 | GEO
Project description:90 S. aureus isolates were analyzed for protein biomarker discovery, including MSSA, vancomycin-susceptible S. aureus (VSSA), hVISA, and VISA strains. Label-free data-independent acquisition proteomics was used to identify protein biomarkers that allow for discrimination among MSSA, hVISA, and VISA strains.
2020-07-18 | MSV000085776 | MassIVE
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
2013-03-12 | GSE10529 | GEO
Project description: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.
2010-03-25 | GSE16479 | GEO
Project description:To determine if significant genomic changes are associated with the development of vancomycin intermediate Staphylococcus aureus, genomic DNA microarrays were performed to compare the initial vancomycin susceptible Staphylococcus aureus (VSSA) and a related vancomycin intermediate Staphylococcus aureus (VISA) isolate from five unique patients (five isolate pairs). Keywords: comparative genomic hybridization
2006-11-03 | GSE5047 | GEO