Genetic basis for in vivo daptomycin resistance in enterococci

Abstract

Background: Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. However, the emergence of resistance to daptomycin during therapy threatens its usefulness.

Methods: We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 μg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712.

Results: Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 μg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential.

Conclusions: Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).

Figure 1. Transmission Electron Microscopy of the Cell Envelope of Enterococcus faecalis Isolates, One Susceptible to Daptomycin (S613) and the Other Resistant (R712)

At high magnification, cells in the S613 isolate have symmetric septa, with only a single septum observed between two cells, and cell separation is easily detected, with single cells visible (Panels A, B, and G). In contrast, multiple septal structures can be seen before complete cell separation in the R712 isolate (Panels A and B, red arrowheads). There are also prominent distortions in the cell envelopes of R712 cells. First, the envelopes appear to be altered at the point of cell contact (which is likely to be the point at which the cells separate) (Panels B, D, and E; blue arrowheads), and the surfaces of the R712 cells lack the smooth appearance of the S613 cells, even when they are in close proximity (a feature observed in all R712 cells analyzed) (Panel E). Second, several structures appear to be connecting the R712 cells (Panels B, C, and F; purple arrowheads); these connecting projections, which were not observed in S613 cells, appear to originate in the cell envelope and maintain the contact between adjacent cells even after the cells have separated. Third, localized protrusions of the cell envelope are a common feature of R712 cells (Panels G and H, black arrowheads). These protrusions were usually observed in proximity to a septal stricture that appeared to originate from the cell envelope. On the other hand, the surface of S613 cells is smooth and symmetric, without any obvious projections or protrusions (more than 100 cells analyzed) (Panels G and H).