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.

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.

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.

Figure 2

Heat map analysis of cell wall genes. Heat map analysis of "core cell wall genes" and additional cell wall genes which have been commonly shown to be important in cell wall biosynthesis [56]. 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.

Figure 3

Western blot analysis of protein A production. Analysis of protein A production by Western Blot in hVISA/VISA and VSSA pairs. A. Pair 1 (lane 1, JKD 6000; lane 2, JKD 6001), pair 2 (lane 3, JKD 6009; lane 4, JKD 6008), pair 3 (lane 5, JKD 6021; lane 6, JKD 6023), pair 4 (lane 7, JKD 6052, lane 8, JKD 6051), pair 5 (lane 9, JKD 6004; lane 10, JKD 6005). B. Fold ratios for protein A production and protein A (spa) gene expression for 5 hVISA/VISA and VSSA pairs.

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.

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)

Figure 6

TNF-α and IL-1β expression. TNF-α and IL-1β expression from RAW264.7 cells after 6 hours exposure to formaldehyde killed JKD6021 (VSSA) and JKD6023 (VISA). (** p < 0.001).