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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.

Howden BP, Smith DJ, Mansell A, Johnson PD, Ward PB, Stinear TP, Davies JK - BMC Microbiol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, Victoria, Australia. Ben.Howden@med.monash.edu.au

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.

Conclusion: 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.

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Heat map analysis of selected genes. Heat map analysis of selected genes which were differentially expressed in at least 2 isolate pairs. The results are expressed as fold ratio of gene expression for hVISA/VISA compared to VSSA. Results are presented for all 5 isolate pairs without antibiotic exposure and all 5 isolate pairs after exposure to vancomycin. This figure shows the upper quartile of these selected genes, for the full image please see additional file 1.
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Figure 1: Heat map analysis of selected genes. Heat map analysis of selected genes which were differentially expressed in at least 2 isolate pairs. The results are expressed as fold ratio of gene expression for hVISA/VISA compared to VSSA. Results are presented for all 5 isolate pairs without antibiotic exposure and all 5 isolate pairs after exposure to vancomycin. This figure shows the upper quartile of these selected genes, for the full image please see additional file 1.

Mentions: For the VSSA and hVISA/VISA pairs a list of genes of interest was generated as described in the methods section. These results are presented as the fold ratio of hVISA/VISA compared to the related VSSA isolate. There were 54 genes that were down-regulated, and 89 genes that were up-regulated in at least two isolate pairs. Selected genes from this list which were of particular interest are highlighted in figure 1 and additional file 1. The complete gene list is also provided in additional file 2. Down-regulation of the quorum sensing global regulator agr (agrA, agrB) was prominent across three pairs. This would be expected to have secondary effects on exotoxin production and expression of cell adhesion molecules, and a number of genes involved in pathogenesis and toxin production, and cell surface adhesion molecules were indeed significantly down-regulated (spa, fnbA, fnbB, efb, putative exotoxin SACOL0478, fibrinogen-binding protein related proteins and precursors SACOL1164 and SACOL1169). Of particular note, marked down-regulation of the spa gene SACOL0095 was demonstrated in four of the five hVISA/VISA isolates, and another IgG-binding protein, SACOL2418 was down-regulated in all five isolates.


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.

Howden BP, Smith DJ, Mansell A, Johnson PD, Ward PB, Stinear TP, Davies JK - BMC Microbiol. (2008)

Heat map analysis of selected genes. Heat map analysis of selected genes which were differentially expressed in at least 2 isolate pairs. The results are expressed as fold ratio of gene expression for hVISA/VISA compared to VSSA. Results are presented for all 5 isolate pairs without antibiotic exposure and all 5 isolate pairs after exposure to vancomycin. This figure shows the upper quartile of these selected genes, for the full image please see additional file 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289824&req=5

Figure 1: Heat map analysis of selected genes. Heat map analysis of selected genes which were differentially expressed in at least 2 isolate pairs. The results are expressed as fold ratio of gene expression for hVISA/VISA compared to VSSA. Results are presented for all 5 isolate pairs without antibiotic exposure and all 5 isolate pairs after exposure to vancomycin. This figure shows the upper quartile of these selected genes, for the full image please see additional file 1.
Mentions: For the VSSA and hVISA/VISA pairs a list of genes of interest was generated as described in the methods section. These results are presented as the fold ratio of hVISA/VISA compared to the related VSSA isolate. There were 54 genes that were down-regulated, and 89 genes that were up-regulated in at least two isolate pairs. Selected genes from this list which were of particular interest are highlighted in figure 1 and additional file 1. The complete gene list is also provided in additional file 2. Down-regulation of the quorum sensing global regulator agr (agrA, agrB) was prominent across three pairs. This would be expected to have secondary effects on exotoxin production and expression of cell adhesion molecules, and a number of genes involved in pathogenesis and toxin production, and cell surface adhesion molecules were indeed significantly down-regulated (spa, fnbA, fnbB, efb, putative exotoxin SACOL0478, fibrinogen-binding protein related proteins and precursors SACOL1164 and SACOL1169). Of particular note, marked down-regulation of the spa gene SACOL0095 was demonstrated in four of the five hVISA/VISA isolates, and another IgG-binding protein, SACOL2418 was down-regulated in all five isolates.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, Victoria, Australia. Ben.Howden@med.monash.edu.au

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus