Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus

Abstract

Daptomycin is a lipopeptide antibiotic with potent activity against gram-positive bacteria. Complete-genome comparisons of laboratory-derived Staphylococcus aureus with decreased susceptibility to daptomycin and their susceptible parent were used to identify genes that contribute to reduced susceptibility to daptomycin. Selective pressure of growth in sublethal concentrations of daptomycin resulted in the accumulation of mutations over time correlating with incremental decreases in susceptibility. Single point mutations resulting in amino acid substitutions occurred in three distinct proteins: MprF, a lysylphosphatidylglycerol synthetase; YycG, a histidine kinase; and RpoB and RpoC, the beta and beta' subunits of RNA polymerase. Sequence analysis of mprF, yycF, yycG, rpoB, and rpoC in clinical isolates that showed treatment-emergent increases in daptomycin MICs revealed point mutations in mprF and a nucleotide insertion in yycG, suggesting a role for these genes in decreased susceptibility to daptomycin in the hospital setting.

FIG. 1.

Serial-passage experiments with S. aureus MW2 selected for increasing resistance to daptomycin. Each line indicates a serial-passage experiment. The results for a control passage experiment with drug-free medium are included (the dark blue line labeled MW2).

FIG. 2.

MIC analysis of daily archived colonies from three specific serial-passage experiments, CB1616, CB1617, and CB1618. Each spot on the graph represents the average MIC from more than six daptomycin MIC assays for an isolated representative of the daily population.

FIG. 3.

Time lines representing three serial-passage experiments. Panels: A, CB1616; B, CB1617; C, CB1618. The daptomycin MICs are divided into five ranges, as indicted by colored bars, with daptomycin MIC in micrograms per milliliter. Black arrows above the bars indicated the first day that a mutation resulting in an amino acid change was identified. The protein name and the amino acid change found in each protein are indicated directly above the respective arrow. Gray arrows below the bars represent the first day that a mutation was identified in the intergenic regions (a, 2694054; b, 1512174; c, 1654504; d, 2041052; e, 2705330; see Table 4 for additional details). Once present, the mutation was found on all subsequent days of the experiment.