Heterogeneity of mprF sequences in methicillin-resistant Staphylococcus aureus clinical isolates: role in cross-resistance between daptomycin and host defense antimicrobial peptides

Abstract

Over the past several years, single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been proposed to be associated with a gain-of-function phenotype in terms of daptomycin (DAP) nonsusceptibility (referred to as daptomycin resistance [DAP-R] herein for ease of presentation) in Staphylococcus aureus. We investigated the frequencies of SNPs within the mprF ORF and the relationships of such SNPs to cross-resistance between DAP and cationic host defense peptides (HDPs). Thirty-five well-characterized, unique DAP-susceptible (DAP-S) and DAP-R methicillin-resistant S. aureus (MRSA) isolates of the clonal complex 5 genotype were used. In addition to mprF SNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i) mprF expression, (ii) membrane phospholipid content, (iii) positive surface charge, (iv) DAP binding, and (v) cell wall thickness profiles. A number of DAP-S strains (MICs of ≤ 1 μg/ml) exhibited mprF SNPs, occasionally with high-level mprF sequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicled mprF hot spot locations associated with DAP-R in S. aureus. In contrast, all 8 DAP-R isolates demonstrated SNPs within such known mprF hot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhanced mprF expression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array of mprF SNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrent mprF dysregulation, were associated with the DAP-R phenotype.

FIG 1

Transcription of mprF genes among the study strains during exponential growth phase (A) and stationary phase (B). Total cellular RNA samples from the strains grown in Trypticase soy broth were isolated at 2 h (exponential growth phase) or 12 h (stationary phase) postinoculation and subjected to qRT-PCR analyses. Means ± SDs are shown. *, P < 0.05 versus results for group with DAP MICs of ≥2 μg/ml; **, P < 0.01 versus results for group with DAP MICs of ≥2 μg/ml.

FIG 2

Representative binding of fluorescent DAP (Bodipy-DAP) to S. aureus bacteria of different DAP MIC groups. S. aureus strains from groups with DAP MICs of ≤0.5 μg/ml (A), 1 μg/ml (B), and ≥0.5 μg/ml (C) were stained with Bodipy-labeled DAP. (D) Intensity of fluorescence from cells stained with Bodipy-DAP. Mean results ± SD are shown. *, P < 0.05 versus results for DAP-S groups.