A Novel Point Mutation Promotes Growth Phase-Dependent Daptomycin Tolerance in Staphylococcus aureus

Abstract

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus. Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (P i ) transporter gene pitA. Drug tolerance caused by this allele, termed pitA6, was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of P i and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.

Should we develop screens for multi-drug antibiotic tolerance?

“…Tolerance levels in E. coli and S. aureus increase tremendously upon repeated exposure to ampicillin and daptomycin [22,23]. Second, we confirmed the longstanding prediction of a direct correlation between the evolved persister level and the frequency of antibiotic treatments in E. coli [24].…”

“…Their prevalence in the genome is correlated with the level of persisters [31] and highpersistence mutations in HipA are abundant among clinical isolates of uropathogenic E. coli [32]. Mutations resulting in increased tolerance in experimental evolution studies represent other promising genetic markers [22][23][24].…”

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon

“…In contrast to resistant cells, tolerant strains are unable to grow in the presence of DAP; however, they survive the bactericidal action of DAP at least for a restricted period of time (7).…”

“…In a recent study, we selected a mutant of S. aureus strain HG003 with a 10,000-fold-increased DAP tolerance (7). The tolerance of the corresponding mutant HG003pitA6 differs from that of the wild-type strain as it fulfills all criteria of genetic tolerance with a rather slow but linear decrease of viability during DAP treatment (8,12).…”

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus

“…Larvae of Galleria mellonella in the final stage (R. J. Mous Live Bait, EX Balk, Netherlands) weighing between 300 and 700 mg were infected with 1 ϫ 10 6 cells of S. aureus JE2 either untreated or with exogenously applied GAPDH and FbaA at 20 g/ml each (37,38). For each experiment, 10 larvae weighing between 300 and 700 mg were infected.…”