title: Different bacterial gene expression patterns and attenuated host immune responses are associated with the evolution of low-level vancomycin resistance during persistent methicillin-resistant Staphylococcus aureus bacteraemia.
abstract: BACKGROUND
  Low-level vancomycin resistance in Staphylococcus aureus (vancomycin-intermediate S. aureus (VISA) and hetero-VISA [hVISA]) emerges during persistent infection and failed vancomycin therapy. Up-regulation of genes associated with the "cell wall stimulon" and mutations in the vraSR operon have both been implicated in the development of resistance, however the molecular mechanisms of resistance are not completely understood. To further elucidate the mechanisms leading to resistance transcriptome comparisons were performed using multiple clinical pairs of vancomycin-susceptible S. aureus (VSSA) and hVISA/VISA (n = 5), and three VSSA control pairs from hospitalized patients with persistent bacteraemia that did not develop hVISA/VISA. Based on the transcriptome results multiple genes were sequenced and innate immune system stimulation was assessed in the VSSA and hVISA/VISA pairs.
  
  
  RESULTS
  Here we show that up-regulation of vraS and the "cell wall stimulon" is not essential for acquisition of low-level vancomycin resistance and that different transcriptional responses occur, even between closely related hVISA/VISA strains. DNA sequencing of vraSR, saeSR, mgrA, rot, and merR regulatory genes and upstream regions did not reveal any differences between VSSA and hVISA/VISA despite transcriptional changes suggesting mutations in these loci may be linked to resistance in these strains. Enhanced capsule production and reduced protein A expression in hVISA/VISA were confirmed by independent bioassays and fully supported the transcriptome data. None of these changes were observed in the three control pairs that remained vancomycin-susceptible during persistent bacteremia. In a macrophage model of infection the changes in cell surface structures in hVISA/VISA strains were associated with significantly reduced NF-kappaB activation resulting in reduced TNF-alpha and IL-1beta expression.
  
  
  CONCLUSIONS
  We conclude that there are multiple pathways to low-level vancomycin resistance in S. aureus, even among closely related clinical strains, and these can result in an attenuated host immune response. The persistent infections associated with hVISA/VISA strains may be a consequence of changes in host pathogen interactions in addition to the reduced antibiotic susceptibility.
authors.affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, Victoria, Australia. Ben.Howden@med.monash.edu.au
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Howden
authors.forename: Danielle J
authors.initials: DJ
authors.lastname: Smith
authors.forename: Ashley
authors.initials: A
authors.lastname: Mansell
authors.forename: Paul D R
authors.initials: PD
authors.lastname: Johnson
authors.forename: Peter B
authors.initials: PB
authors.lastname: Ward
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Stinear
authors.forename: John K
authors.initials: JK
authors.lastname: Davies
mesh_terms: D016470:Bacteremia / Q000235:genetics / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016106:Methicillin Resistance; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012097:Repressor Proteins / Q000235:genetics; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D020713:Vancomycin Resistance / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D016328:NF-kappa B; D012097:Repressor Proteins; C478169:VraS protein, Staphylococcus aureus; C408643:rot protein, Staphylococcus aureus