Dataset Information

Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.

ABSTRACT: Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in the walKR 5' untranslated region (5' UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

SUBMITTER: McEvoy CR

PROVIDER: S-EPMC3697332 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.

McEvoy Christopher R E CR Tsuji Brian B Gao Wei W Seemann Torsten T Porter Jessica L JL Doig Kenneth K Ngo Dung D Howden Benjamin P BP Stinear Timothy P TP

Antimicrobial agents and chemotherapy 20130429 7

Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin ...[more]

PMID: 23629723

Similar Datasets

Project description:Vancomycin (VAN)-intermediate-resistant Staphylococcus aureus (VISA) is continually isolated globally, with a systematic review suggesting a prevalence of 2% in all blood culture samples. Most VISA strains exhibit common characteristics, such as a thickened cell wall, reduced autolysis, and attenuated virulence. Here, based on multi-omics approaches, we have characterized clinical VISA isolates obtained through prolonged antimicrobial treatment in a single patient. All VISA isolates were isogenic, based on multi-locus sequence typing (MLST) ST5, SCCmec type II (2A), and spa type t17639. Core-genome single nucleotide variations (SNVs) found among thirteen isolates during the patient's hospitalization, indicated clonality, but not notable genetic features of the VISA phenotype. We determined the complete genome sequence of VAN-susceptible strain KG-03 (minimum inhibitory concentration [MIC] 0.5 ?g/mL) and two VISA strains, KG-18 and KG-22 (MIC 8.0 and 4.0 ?g/mL, respectively). Comparative genome analysis showed remarkable strain-specific IS256 insertions. RNA-Seq transcriptome analysis revealed IS256-mediated overexpression of the walKR two-component system in VISA KG-18, possibly leading to modulation of cell wall integrity (lytM and sceD) and surface charge (mprF and dltABCD). In addition, secretome analysis indicated that cell wall-anchored proteins (Protein A, SasG, and SdrD) were significantly decreased. KG-18 and KG-22 exhibit thickened cell wall, and are relatively resistant to lysostaphin, which cleaves a staphylococcus-unique pentaglycine chain in the peptidoglycan. We conclude that KG-18 achieved reduced susceptibility to VAN by IS256-mediated WalKR overexpression, leading to a markedly thickened cell wall for trapping free VAN molecules with redundant D-Ala-D-Ala targets. In addition, a positively charged surface with lysyl-phosphatidylglycerol and depolarization of wall teichoic acid could contribute to inhibiting cationic daptomycin and VAN antimicrobial activity. Comparative omics approaches in this study strongly suggest that fully complete and annotated genome sequences will be indispensable for characterizing overall VISA phenotype.

Project description:The increased prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) is an emerging health care threat. Genome-based comparative methods hold great promise to uncover the genetic basis of the VISA phenotype, which remains obscure. S. aureus isolates were collected from a single individual that presented with recurrent staphylococcal bacteremia at three time points, and the isolates showed successively reduced levels of vancomycin susceptibility. A population genomic approach was taken to compare patient S. aureus isolates with decreasing vancomycin susceptibility across the three time points. To do this, patient isolates were sequenced to high coverage (~500×), and sequence reads were used to model site-specific allelic variation within and between isolate populations. Population genetic methods were then applied to evaluate the overall levels of variation across the three time points and to identify individual variants that show anomalous levels of allelic change between populations. A successive reduction in the overall levels of population genomic variation was observed across the three time points, consistent with a population bottleneck resulting from antibiotic treatment. Despite this overall reduction in variation, a number of individual mutations were swept to high frequency in the VISA population. These mutations were implicated as potentially involved in the VISA phenotype and interrogated with respect to their functional roles. This approach allowed us to identify a number of mutations previously implicated in VISA along with allelic changes within a novel class of genes, encoding LPXTG motif-containing cell-wall-anchoring proteins, which shed light on a novel mechanistic aspect of vancomycin resistance. IMPORTANCE The emergence and spread of antibiotic resistance among bacterial pathogens are two of the gravest threats to public health facing the world today. We report the development and application of a novel population genomic technique aimed at uncovering the evolutionary dynamics and genetic determinants of antibiotic resistance in Staphylococcus aureus. This method was applied to S. aureus cultures isolated from a single patient who showed decreased susceptibility to the vancomycin antibiotic over time. Our approach relies on the increased resolution afforded by next-generation genome-sequencing technology, and it allowed us to discover a number of S. aureus mutations, in both known and novel gene targets, which appear to have evolved under adaptive pressure to evade vancomycin mechanisms of action. The approach we lay out in this work can be applied to resistance to any number of antibiotics across numerous species of bacterial pathogens.

Dataset Information

Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.

Publications

Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets