Whole-genome analysis of a daptomycin-susceptible enterococcus faecium strain and its daptomycin-resistant variant arising during therapy

Abstract

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.

Fig 1

The Yyc system in E. faecium. (A) Structural organization of the orthologous yyc gene cluster in E. faecium; transmembrane coding regions are marked as black bars. Annotations are based on a published report by Türck et al. (49). (B) Predicted domain architecture of the putative YycG histidine kinase and changes found in E. faecium R446, TX0133a, and TX0133a.4, with black arrows denoting the localization of the predicted amino acid changes. Numbers indicate the corresponding amino acids spanning each domain. The HAMP domain is predicted to promote communication between the input and output domains; the PAS sensor domain was named for homology to the PER-ARNT-SIM proteins of D. melanogaster; HisKA, phosphoacceptor domain of histidine kinases; HATPase_c, histidine kinase-like ATPase. The scheme was based in part on published data from Dubrac et al. (56).