Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach

Abstract

Objectives: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined.

Methods: We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques.

Results: Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid.

Conclusions: Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.

Figure 1.

Biofilms of strains 616 and 701 under oleic acid stress and bacterial quantification. (a) Serial dilutions of planktonic cells spotted on agar plates. (b) Quantification of planktonic cells by OD₅₄₀ for two independent experiments. (c) Biofilms stained with crystal violet in a 6-well multititre plate. The box shows a magnification of strain 701 grown in the presence of oleic acid and the arrows show spots stained with crystal violet. (d) Adherence in the presence of oleic acid during two independent experiments. Error bars show the range of duplicate experiments.

Figure 2.

Molecular genotyping of the set of strains. Genotyping tree obtained using microarray covering whole S. aureus genomes of eight sequenced strains revealed that our isolates are genetically comparable while N315 appears as the more related reference strain. Each probe is represented by a single row of grey-scaled boxes and each sample corresponds to a single column. The grey areas correspond to genes present, whereas white bars indicate missing genes (N315 used as the reference). The dendrogram (top and left part of the figure) represents the similarity matrix of probe sets.

Figure 3.

Potential pathways leading to DAP^R. Cell wall metabolism and regulators appear as major targets of mechanisms leading to DAP^R acquired in vivo during therapy. The biosynthesis of bacterial envelopes is involved in the process leading to major phenotypic changes between DAP^S and DAP^R S. aureus. Black bars represent inhibition.