Genetic basis for daptomycin resistance in enterococci

Abstract

The emergence of multidrug-resistant enterococci as a leading cause of hospital-acquired infection is an important public health concern. Little is known about the genetic mechanisms by which enterococci adapt to strong selective pressures, including the use of antibiotics. The lipopeptide antibiotic daptomycin is approved to treat Gram-positive bacterial infections, including those caused by enterococci. Since its introduction, resistance to daptomycin by strains of Enterococcus faecalis and Enterococcus faecium has been reported but is still rare. We evolved daptomycin-resistant strains of the multidrug-resistant E. faecalis strain V583. Based on the availability of a fully closed genome sequence for V583, we used whole-genome resequencing to identify the mutations that became fixed over short time scales (~2 weeks) upon serial passage in the presence of daptomycin. By comparison of the genome sequences of the three adapted strains to that of parental V583, we identified seven candidate daptomycin resistance genes and three different mutational paths to daptomycin resistance in E. faecalis. Mutations in one of the seven candidate genes (EF0631), encoding a putative cardiolipin synthase, were found in each of the adapted E. faecalis V583 strains as well as in daptomycin-resistant E. faecalis and E. faecium clinical isolates. Alleles of EF0631 from daptomycin-resistant strains are dominant in trans and confer daptomycin resistance upon a susceptible host. These results demonstrate a mechanism of enterococcal daptomycin resistance that is genetically distinct from that occurring in staphylococci and indicate that enterococci possessing alternate EF0631 alleles are selected for during daptomycin therapy. However, our analysis of E. faecalis clinical isolates indicates that resistance pathways independent from mutant forms of EF0631 also exist.

Fig. 1.

Daptomycin serial passage experiments. The highest daptomycin concentration demonstrating growth (in μg/ml) is shown on the y axis for each day of passage. Diamonds, experiment A; squares, experiment B; triangles, experiment C.

Fig. 2.

EF0631 predicted cardiolipin synthase. The EF0631 protein sequence with predicted transmembrane (TM) helices and phospholipase D (PLD) domains is shown. The location of DAP-A N77_Q79del is boxed in green. The location of the DAP-B and DAP-C R218Q substitution is indicated by an asterisk. Transmembrane helices were predicted by TMHMM, and PLD domains were predicted by Pfam-A.

Fig. 3.

EF1797 hypothetical protein. The EF1797 protein sequence with predicted transmembrane (TM) helices is shown. Transmembrane helices were predicted by TMHMM. The location of the DAP-A frameshift is denoted by a blue asterisk. The location of the DAP-B IS256 insertion is denoted by a red asterisk. The location of the DAP-C G130_F154 deletion is shown.