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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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Related in: MedlinePlus

Capsule immunoblot comparison of VSSA to hVISA/VISA. Capsule immuoblot using anti-capsule type 8 antibody. Serial 3-fold dilutions of crude capsule extracts were loaded onto nitrocellulose membrane. Positive control, capsule type 8 positive strain P1; negative control, capsule type 5 positive strain Newman.
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Figure 4: Capsule immunoblot comparison of VSSA to hVISA/VISA. Capsule immuoblot using anti-capsule type 8 antibody. Serial 3-fold dilutions of crude capsule extracts were loaded onto nitrocellulose membrane. Positive control, capsule type 8 positive strain P1; negative control, capsule type 5 positive strain Newman.

Mentions: To determine if the transcriptional changes that were detected in hVISA/VISA isolates resulted in changes in protein A and capsule production, as well as urease activity, expression was determined for all isolates. A significant decrease in protein A production was demonstrated for 4 of the 5 pairs (Figure 3). For the 5th pair, protein A production was low in both isolates. Urease activity was higher for the hVISA/VISA isolates compared to the VSSA isolates for 4 of the 5 pairs (Table 2). These results correlated well with spa and ureA gene expression (Table 2, Figure 3, additional file 2). A PCR was designed to determine the capsule genotype of isolates (capsule type 5 or capsule type 8). All isolates from pairs 1 to 5 were capsule type 8 strains by PCR. Capsule immunoblot confirmed the PCR capsule typing results, and demonstrated a significant increase in capsule expression in 4 of the 5 pairs (Figure 4). The last pair (no 5) had minimal capsule expression in the VSSA or hVISA strain, and these results also correlated well with microarray data.


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)

Capsule immunoblot comparison of VSSA to hVISA/VISA. Capsule immuoblot using anti-capsule type 8 antibody. Serial 3-fold dilutions of crude capsule extracts were loaded onto nitrocellulose membrane. Positive control, capsule type 8 positive strain P1; negative control, capsule type 5 positive strain Newman.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Capsule immunoblot comparison of VSSA to hVISA/VISA. Capsule immuoblot using anti-capsule type 8 antibody. Serial 3-fold dilutions of crude capsule extracts were loaded onto nitrocellulose membrane. Positive control, capsule type 8 positive strain P1; negative control, capsule type 5 positive strain Newman.
Mentions: To determine if the transcriptional changes that were detected in hVISA/VISA isolates resulted in changes in protein A and capsule production, as well as urease activity, expression was determined for all isolates. A significant decrease in protein A production was demonstrated for 4 of the 5 pairs (Figure 3). For the 5th pair, protein A production was low in both isolates. Urease activity was higher for the hVISA/VISA isolates compared to the VSSA isolates for 4 of the 5 pairs (Table 2). These results correlated well with spa and ureA gene expression (Table 2, Figure 3, additional file 2). A PCR was designed to determine the capsule genotype of isolates (capsule type 5 or capsule type 8). All isolates from pairs 1 to 5 were capsule type 8 strains by PCR. Capsule immunoblot confirmed the PCR capsule typing results, and demonstrated a significant increase in capsule expression in 4 of the 5 pairs (Figure 4). The last pair (no 5) had minimal capsule expression in the VSSA or hVISA strain, and these results also correlated well with microarray data.

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