Mechanisms of in-vitro-selected daptomycin-non-susceptibility in Staphylococcus aureus

Abstract

Daptomycin is highly active against Staphylococcus aureus, including multidrug-resistant strains and those with reduced susceptibility to vancomycin. However, daptomycin-non-susceptible (Dap^NS) strains [minimum inhibitory concentration (MIC) >1 mg/L] have been derived clinically and in vitro. The mechanism(s) by which this occurs is incompletely understood, but existing data indicate that it is multifactorial. Dap^NS derivatives of one laboratory and three clinical strains of S. aureus produced using gradient plates were evaluated. The Dap^NS phenotype included increases in glycopeptide and nisin MICs and in some instances defective autolysis and reduced susceptibility to lysostaphin lysis. Amino acid substitutions in MprF, YycG (WalK), or both, were identified in all Dap^NS strains. Reduced cytochrome c binding and ability of daptomycin to depolarise whole cells correlated with the Dap^NS phenotype, consistent with an increase in cell surface positivity. Gene expression data revealed increased expression of vraS, one member of a two-component system involved in the regulation of cell wall biosynthesis, in three of five Dap^NS strains. The pathway to the Dap^NS phenotype is not linear, as variable genetic and phenotypic changes may result in identical increases in MICs.

Introduction

Daptomycin is a cyclic lipopeptide antimicrobial agent that is rapidly bactericidal against Staphylococcus aureus, including meticillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains [1]. Daptomycin exerts its antimicrobial effect by a calcium-dependent interaction with the cytoplasmic membrane resulting in depolarisation, ion loss and rapid cell death [2]. Unfortunately, loss of daptomycin susceptibility in S. aureus in the clinical setting associated with treatment failures has been described.

A full understanding of the mechanism(s) of daptomycin-non-susceptibility [minimum inhibitory concentration (MIC) >1 mg/L] in S. aureus remains elusive. Mutations in MprF (lysylphosphatidylglycerol synthetase), YycG (a histidine kinase also known as WalK), and RpoB and RpoC (subunits of RNA polymerase) correlate with daptomycin-non-susceptibility [3]. MprF mutations appear to be gain of function in nature, resulting in increased surface positive charge favouring repulsion of the functional daptomycin–Ca²⁺ complex [4]. Changes in expression and/or function of other gene products associated with cell surface charge such as dltA (part of an operon involved in d-alanylation of wall teichoic acids) may also be involved in reduced drug binding and the emergence of daptomycin-non-susceptibility [5].

Modifications in cytoplasmic membrane fluidity also occur in daptomycin-non-susceptible (Dap^NS) strains, but the mechanism(s) by which this contributes to the phenotype is not known [4], [6]. Dap^NS strains frequently have thickened cell walls, commonly accompanied by defective autolysis [6]. Increases in daptomycin MICs commonly correlate with reduced susceptibility to glycopeptides, implying an overlap in resistance mechanisms. Genetic changes in common between VISA and Dap^NS strains include increased expression of vraFG and vraSR, encoding, respectively, an ATP-dependent transporter involved in cell surface charge and possibly cationic antimicrobial peptide transport and a two-component system implicated in the regulation of cell wall biosynthetic processes. In addition, mutations in graRS, another two-component system modulating the expression of vraFG, may affect both vancomycin and daptomycin susceptibility [7], [8]. A great deal remains to be learned about these processes.