Heterogeneity ofmprFSequences in Methicillin-Resistant Staphylococcus aureus Clinical Isolates: Role in Cross-Resistance between Daptomycin and Host Defense Antimicrobial Peptides

Abstract

ABSTRACTOver the past several years, single-nucleotide polymorphisms (SNPs) within themprFopen 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) inStaphylococcus aureus. We investigated the frequencies of SNPs within themprFORF 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-resistantS. aureus(MRSA) isolates of the clonal complex 5 genotype were used. In addition tomprFSNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i)mprFexpression, (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) exhibitedmprFSNPs, occasionally with high-levelmprFsequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicledmprFhot spot locations associated with DAP-R inS. aureus. In contrast, all 8 DAP-R isolates demonstrated SNPs within such knownmprFhot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhancedmprFexpression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array ofmprFSNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrentmprFdysregulation, were associated with the DAP-R phenotype.