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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 (Pi) 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 Pi 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.

SUBMITTER: Mechler L

PROVIDER: S-EPMC4538524 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

Mechler Lukas L Herbig Alexander A Paprotka Kerstin K Fraunholz Martin M Nieselt Kay K Bertram Ralph R

Antimicrobial agents and chemotherapy 20150622 9

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. T ...[more]

PMID: 26100694

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Project description:It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

Project description:It has been shown recently in a number of in vitro laboratory evolution experiments that under repetitive antibiotic exposure, bacterial populations can adapt quickly to the treatment condition by becoming tolerant and/or resistant to the drug. The repeated killing and regrowth cycles hasten the selection for tolerant/resistant mutants with survival advantages. Due to the random nature of mutagenesis and the large target size of tolerance mutations, this dynamic evolutionary process appears to be highly unpredictable, generating distinct mutants even under identical, well-controlled laboratory conditions. Here, we utilized an adaptive laboratory evolution (ALE) experiment to hunt for novel tolerance and resistance mutations by subjecting multiple lineages of methicillin-resistant Staphylococcus aureus (MRSA) to repetitive daptomycin treatment. By sequencing multiple isolates along the course of evolution, we obtained three tolerant mutants that have different tolerance levels and identified novel daptomycin resistance mutations in the mprF gene. In addition, we found that tolerance/resistance development is more rapid if the population is treated in the exponential phase than if it is treated in the stationary phase, which is likely attributable to the more effective killing of growing cells by the antibiotic. Through competition assays, we found that whether or not the resistant mutants can take over the population heavily depends on the relative survival advantages conferred by the tolerance and resistance mutations. This study reports novel daptomycin resistance and tolerance mutations and offers new insights into the dynamics of the development of tolerance and resistance in bacterial populations under antibiotic exposure. IMPORTANCE Although the phenotype of increased tolerance and/or resistance was commonly observed in evolved populations from typical adaptive laboratory evolution (ALE) experiments, a wide variety of mutations that underlie those phenotypes have been discovered. Therefore, performing ALE experiments in multiple populations in parallel would serve the purpose of mining for different tolerant/resistant mutants and would be useful to explore the diverse population dynamics of evolution. In this study, we performed in vitro evolution in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using a lethal concentration of a drug that is frequently used in the clinic, daptomycin. Using this strategy, we obtained three distinct daptomycin-tolerant mutants and identified six daptomycin resistance mutations in different locations on the mprF gene, collectively adding to our current knowledge of this important pathogen. In addition, we found out that in most cases, the daptomycin-resistant mutant outcompetes other susceptible and tolerant mutants and becomes established in the final population. Follow-up competition experiments offered an explanation; the resistant mutant cannot invade populations of tolerant mutants that confer higher survival advantages than itself. In summary, we demonstrated an experimental strategy to explore the landscape and dynamics of the evolution of tolerance and resistance in MRSA toward daptomycin and made observations that will guide future ALE experiments.

Project description:Adaptive laboratory evolution (ALE) is a useful tool to study the evolution of antibiotic tolerance in bacterial populations under diverse environmental conditions. The role of population bottlenecks in the evolution of tolerance has been investigated in Escherichia coli, but not in a more clinically relevant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used ALE to evolve MRSA under repetitive daptomycin treatment and incorporated population bottlenecks following antibiotic exposure. We observed that the populations finally attained a tolerance mutation in the yycH gene after 2 weeks of evolution with population bottlenecks, and additional mutations in yycI and several other genes further increased the tolerance level. The tolerant populations also became resistant to another glycopeptide antibiotic, vancomycin. Through proteomics, we showed that yycH and yycI mutations led to the loss of function of the proteins and downregulated the WalKR two-component system and the downstream players, including the autolysin Atl and amidase Sle1, which are important for cell wall metabolism. Overall, our study offers new insights into the evolution of daptomycin tolerance under population bottlenecking conditions, which are commonly faced by pathogens during infection; the study also identified new mutations conferring daptomycin tolerance and revealed the proteome alterations in the evolved tolerant populations. IMPORTANCE Although population bottlenecks are known to influence the evolutionary dynamics of microbial populations, how such bottlenecks affect the evolution of tolerance to antibiotics in a clinically relevant methicillin-resistant S. aureus (MRSA) pathogen are still unclear. Here, we performed in vitro evolution of MRSA under cyclic daptomycin treatment and applied population bottlenecks following the treatment. We showed that under these experimental conditions, MRSA populations finally attained mutations in yycH, yycI, and several other genes that led to daptomycin tolerance. The discovered yycH and yycI mutations caused early termination of the genes and loss of function of the proteins, and they subsequently downregulated the expression of proteins controlled by the WalKR two-component system, such as Atl and Sle1. In addition, we compared our proteomics data with multiple studies on distinct daptomycin-tolerant MRSA mutants to identify proteins with a consistent expression pattern that could serve as biological markers for daptomycin tolerance in MRSA.

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A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

Publications

A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

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