Whole-Genome Analysis of a Daptomycin-Susceptible Enterococcus faecium Strain and Its Daptomycin-Resistant Variant Arising during Therapy

Abstract

i Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the "susceptible" cls allele from S447 for the "resistant" one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains. The rise of Enterococcus faecium as an important nosocomial pathogen in the last few decades has led to treatment challenges in clinical settings, particularly after the emergence of multidrug-resistant isolates. The current FDA-approved therapies for vancomycin-resistant E. faecium (VRE), namely, quinupristindalfopristin and linezolid, have important limitations for the treatment of serious E. faecium infections due to their intrinsic bacteriostatic effects, toxicity profiles, problems with administration, and emergence of resistance (1). Daptomycin (DAP) is a lipopeptide antibiotic with potent in vitro bactericidal activity against VRE. Many clinicians use DAP as first-line off-label therapy in VRE endovascular infections despite the lack of FDA approval, and that efficacy is supported only by retrospective data (2-4). However, the main challenge when using DAP against VRE appears to be development of resistance during therapy, which has been extensively documented (5-10) (although the accepted term is "DAP nonsusceptibility," we use the term "DAP resistance" throughout for ease of presentation).The main mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner (11), and our initial genomic studies in Enterococcus faecalis (12) provided ...

The action mechanism of daptomycin

“…In Enterococcus faecium, mutations in the yycFG operon have also been found in daptomycin-resistant strains. 101,114 Another line of evidence that connects yycG to daptomycin's MOA has been pointed out by Baltz. 11 YycG is localized at the cell division septum, and inhibition of YycG causes aberrant cell division septa.…”

“…99 Some of these mutants have been shown to have increased activity, 100 but their substitution into the genome of a daptomycin-susceptible strain did not detectably increase resistance. 101 A study that comprehensively characterized the membrane lipid compositions of daptomycin-resistant Staphylococcus aureus strains found the expected decrease in PG, as well as an increase in lysyl-PG, but no significant change in CL. 94 Since CL is formed from PG, it seems possible that CL synthase gain-of-function mutants merely compensate for the reduction in substrate levels.…”

Fosfomycin Enhances the Activity of Daptomycin against Vancomycin-Resistant Enterococci in an In Vitro Pharmacokinetic-Pharmacodynamic Model

“…VRE resistance to daptomycin has previously been linked to genetic changes in genes that regulate cell envelope homeostasis and membrane lipid metabolism (liaFSR, yycFG, cls, and cfa); however, there appears to be a variety of genetic pathways to daptomycin nonsusceptibility (49)(50)(51)(52)(53)(54)(55). In addition to altered cell surface charge, an alternative mechanism that has been proposed is related to redistribution of membrane lipids that drive daptomy-cin binding away from division septa, where it is believed to be more devastating to the organism (53,55,56). Mutations of the phosphotransferase system (PTS), previously associated with bacteriocin resistance in E. faecium and E. faecalis, may also contribute to daptomycin nonsusceptibility in E. faecium (48,57).…”

A Current Perspective on Daptomycin for the Clinical Microbiologist

“…Fewer data exist to describe the mechanism of daptomycin-NS phenotypes in the enterococci compared to those described above for S. aureus. However, whole-genome sequencing of daptomycin-susceptible and -NS E. faecium (157,160,162) and E. faecalis (163,164) strain pairs suggests that limited mutations in the bacterial genome are sufficient to elicit daptomycin MICs above the CLSI breakpoint of 4 g/ml. Like in S. aureus, mutations to both cell envelope stress response system and genes specifically involved in phospholipid biosynthesis have been identified in daptomycin-NS Enterococcus.…”