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Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance.

ABSTRACT: With increasing numbers of hospital-acquired antibiotic resistant infections each year and staggering health care costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about the relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. Here we used quantitative experimental evolution of a single polymorphic population in continuous culture with whole-genome sequencing and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin-resistant clinical pathogen Enterococcus faecalis S613. Importantly, we sustained both planktonic and nonplanktonic (i.e., biofilm) populations in coculture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. Quantitative experimental evolution revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Despite the relative simplicity of this ex vivo approach compared to the ecological complexity of the human body, we showed that experimental evolution allows for rapid identification of clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.

SUBMITTER: Miller C

PROVIDER: S-EPMC3811304 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance.

Miller Corwin C Kong Jiayi J Tran Truc T TT Arias Cesar A CA Saxer Gerda G Shamoo Yousif Y

Antimicrobial agents and chemotherapy 20130819 11

With increasing numbers of hospital-acquired antibiotic resistant infections each year and staggering health care costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about the relative importance of genomic changes, their order of appearance, or potential epistatic r ...[more]

PMID: 23959318

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Project description:Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid.IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

Project description:UnlabelledEnterococcus faecalis is a commensal bacterium of the gastrointestinal tract that can cause nosocomial infections in immunocompromised humans. The hallmarks of this organism are its ability to survive in a variety of stressful habitats and, in particular, its ability to withstand membrane damage. One strategy used by E. faecalis to protect itself from membrane-damaging agents, including the antibiotic daptomycin, involves incorporation of exogenous fatty acids from bile or serum into the cell membrane. Additionally, the response regulator LiaR (a member of the LiaFSR [lipid II-interacting antibiotic response regulator and sensor] system associated with cell envelope stress responses) is required for the basal level of resistance E. faecalis has to daptomycin-induced membrane damage. This study aimed to determine if membrane fatty acid changes could provide protection against membrane stressors in a LiaR-deficient strain of E. faecalis We noted that despite the loss of LiaR, the organism readily incorporated exogenous fatty acids into its membrane, and indeed growth in the presence of exogenous fatty acids increased the survival of LiaR-deficient cells when challenged with a variety of membrane stressors, including daptomycin. Combined, our results suggest that E. faecalis can utilize both LiaR-dependent and -independent mechanisms to protect itself from membrane damage.ImportanceEnterococcus faecalis is responsible for a significant number of nosocomial infections. Worse, many of the antibiotics used to treat E. faecalis infection are no longer effective, as this organism has developed resistance to them. The drug daptomycin has been successfully used to treat some of these resistant strains; however, daptomycin-resistant isolates have been identified in hospitals. Many daptomycin-resistant isolates are found to harbor mutations in the genetic locus liaFSR, which is involved in membrane stress responses. Another mechanism shown to increase tolerance to daptomycin involves the incorporation of exogenous fatty acids from host fluids like serum or bile. This improved tolerance was found to be independent of liaFSR and suggests that there are additional ways to impact sensitivity to daptomycin. Thus, further studies are needed to understand how host fatty acid sources can influence antibiotic susceptibility.

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Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance.

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Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance.

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