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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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NF-κB activation results for VSSA and hVISA/VISA isolate pairs. Stably transfected RAW cells with an ELAM-NF-kB reporter construct were exposed to formaldehyde killed VSSA and hVISA/VISA pairs for 6 hours. Results are presented as fold NF-κB activation for hVISA/VISA compared to VSSA and are the result of multiple replicates. (* p < 0.05)
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Figure 5: NF-κB activation results for VSSA and hVISA/VISA isolate pairs. Stably transfected RAW cells with an ELAM-NF-kB reporter construct were exposed to formaldehyde killed VSSA and hVISA/VISA pairs for 6 hours. Results are presented as fold NF-κB activation for hVISA/VISA compared to VSSA and are the result of multiple replicates. (* p < 0.05)

Mentions: Because we detected a number of significant changes in staphylococcal surface structures that interact with the innate immune system during the evolution of hVISA/VISA, we next wished to establish if hVISA/VISA displayed a decreased activation of the prototypic inflammatory transcription factor NF-κB. As shown in Figure 5, there was a statistically significant decrease in NF-κB activation demonstrated with 4 of the 5 hVISA/VISA isolates compared to their parent VSSA strain, particularly at lower concentrations. This appeared to correlate with the vancomycin MIC result, with reduced NF-κB activation detected with all the strains with a vancomycin MIC of 4 mg/L, but only with one of those with an MIC of 2 mg/L. To determine the biological impact of this effect, a single pair (JKD6021 and JKD6023) were selected to determine if the reduced NF-κB activation resulted in a diminished expression of the NF-κB-dependent inflammatory cytokines TNF-α and IL-1β. As demonstrated in Figure 6, after 6 hours exposure to both strains, there was a significant decrease in TNF-α expression when comparing JKD6021 to JKD6023 (p < 0.001), while IL-1β expression was substantially decreased.


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)

NF-κB activation results for VSSA and hVISA/VISA isolate pairs. Stably transfected RAW cells with an ELAM-NF-kB reporter construct were exposed to formaldehyde killed VSSA and hVISA/VISA pairs for 6 hours. Results are presented as fold NF-κB activation for hVISA/VISA compared to VSSA and are the result of multiple replicates. (* p < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: NF-κB activation results for VSSA and hVISA/VISA isolate pairs. Stably transfected RAW cells with an ELAM-NF-kB reporter construct were exposed to formaldehyde killed VSSA and hVISA/VISA pairs for 6 hours. Results are presented as fold NF-κB activation for hVISA/VISA compared to VSSA and are the result of multiple replicates. (* p < 0.05)
Mentions: Because we detected a number of significant changes in staphylococcal surface structures that interact with the innate immune system during the evolution of hVISA/VISA, we next wished to establish if hVISA/VISA displayed a decreased activation of the prototypic inflammatory transcription factor NF-κB. As shown in Figure 5, there was a statistically significant decrease in NF-κB activation demonstrated with 4 of the 5 hVISA/VISA isolates compared to their parent VSSA strain, particularly at lower concentrations. This appeared to correlate with the vancomycin MIC result, with reduced NF-κB activation detected with all the strains with a vancomycin MIC of 4 mg/L, but only with one of those with an MIC of 2 mg/L. To determine the biological impact of this effect, a single pair (JKD6021 and JKD6023) were selected to determine if the reduced NF-κB activation resulted in a diminished expression of the NF-κB-dependent inflammatory cytokines TNF-α and IL-1β. As demonstrated in Figure 6, after 6 hours exposure to both strains, there was a significant decrease in TNF-α expression when comparing JKD6021 to JKD6023 (p < 0.001), while IL-1β expression was substantially decreased.

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