Emergence of daptomycin resistance in daptomycin-naïve rabbits with methicillin-resistant Staphylococcus aureus prosthetic joint infection is associated with resistance to host defense cationic peptides and mprF polymorphisms

Abstract

Background: Previous studies of both clinically-derived and in vitro passage-derived daptomycin-resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R).

Methods: In the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles.

Results: In comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P<0.05); (ii) thicker cell walls (P<0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype.

Conclusions: THESE RESULTS SUGGEST: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDP-DAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined.

Figure 1. DAP population analysis of the parental vs two animal passage strains.

Figure 2. qRT-PCR analyses of mprF (A) and dltA (B) expression in the study strains.

RNA samples were isolated from exponential-phase cultures of the strains and were subjected to qRT-PCR to detect relative transcription levels of mprF and dltA. Expression levels of mprF and dlt genes in the parental strain 271 were normalized to 1.

Figure 3. Flow cytometric analyses of the relative amounts of MprF protein content within the parental vs two of the animal passage strains.