Abstract
Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.
Highlights
As therapeutic strategies have been developed for treating bacterial infections over the past century, there has been a subsequent rise in antibiotic resistant bacteria
To determine whether E. faecalis OG1RF stationary phase cells have increased tolerance, i.e., increased survival, to daptomycin when compared to exponential phase cells, we examined sensitivity to daptomycin using cell survival assays
We demonstrate that daptomycin tolerance, i.e., increased survival following treatment above the minimal inhibitory concentration (MIC), is dependent on several factors: growth phase, the ability of cells to synthesize proteins and fatty acids, and the presence/absence of peptidoglycan
Summary
As therapeutic strategies have been developed for treating bacterial infections over the past century, there has been a subsequent rise in antibiotic resistant bacteria. Persister cells can arise through varying mechanisms–including induction of metabolic dormancy through varying expression of the toxin component of toxin/antitoxin systems [32,33,34,35,36,37] and Peptidoglycan removal causes daptomycin tolerance induction of stress responses [38,39,40,41,42,43,44] In each of these cases, the persister cells in question are not genetically resistant, as they do not grow during treatment with the drug [45]: rather, they are able to survive antibiotic pressure through differential expression of genes which can be stochastic [32,46] or triggered by environmental changes [38,39,40,41,42,43,44]. We demonstrate that inhibition of active growth of E. faecalis serves to increase its ability to survive high doses of daptomycin, supporting a model for daptomycin targeting peptidoglycan biosynthesis
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