The off-label use of daptomycin occurs often in the treatment of severe enterococcal infections, including infections with vancomycin-resistant enterococci or those species exhibiting high-level resistance to aminoglycosides. However, a major drawback for the successful use of this antibiotic is the emergence of resistance during therapy. In addition, in vitro, enterococci are less susceptible to daptomycin than S. aureus,
with a clinical threshold for sensitivity that is four times as high (≤4 μg per milliliter, vs. ≤1 μg per milliliter for S. aureus
An essential event for the activity of daptomycin is calcium-mediated interaction with the cell membrane, a property that this antibiotic shares with related cationic antimicrobial peptides that are part of the human host defense against microbes. The change in the bacterial surface also appears to play an important role in the interaction of daptomycin with the cell membrane, and it has been postulated that a more positively charged cell envelope “repels” the cationic daptomycin from the cell membrane, contributing to the development of resistance.15
A major factor in the cell-envelope charge is the phospholipid composition of the inner and outer cell-membrane leaflets, such as the negatively charged phospholipid cardiolipin and the positively charged amino derivatives of phosphatidylglycerol. In some S. aureus
isolates, reduced susceptibility to daptomycin has been attributed to a decrease in the negative surface charge of the cell membrane as a result of modifications in phospholipid content, mainly through increased synthesis and translocation (“flipping”) of the positively charged lysyl-phosphatidylglycerol from the inner to the outer leaflet of the cell membrane.15,26
It has also been shown that lysyl-phosphatidylglycerol attenuates membrane perturbations caused by cationic antimicrobial peptides.27
Our findings in this study indicate that the development of resistance to daptomycin in the vancomycin-resistant E. faecalis
isolate R712, like the development of resistance in S. aureus
, is associated with alterations in the cell envelope and biophysical properties of the cell membrane. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus
. Indeed, in the R712 isolate, none of the genes associated with the emergence of resistance to daptomycin in S. aureus20–24
differed from those in the daptomycin-susceptible parental isolate, S613. Instead, our data provide direct evidence that changes in two genes — namely, liaF
— are sufficient for the development of resistance to daptomycin in the E. faecalis
clinical strain pair.
The alteration of the LiaFSR system is probably a pivotal initial event in the development of resistance, since replacement of only the liaF allele in the S613 isolate with that from the R712 isolate decreased the susceptibility of the S613 isolate to daptomycin.
LiaF is part of the three-component LiaFSR regulatory system, which is known to orchestrate the response of the cell envelope to antibiotics and antimicrobial peptides in some gram-positive bacteria. The LiaFSR system has been well characterized in B. subtilis,8,16 Streptococcus mutans
In B. subtilis
and S. mutans
, the LiaFSR system is usually activated by the presence of antibiotics that disrupt cell-membrane and peptidoglycan synthesis through alterations of lipid-II metabolism (i.e., bacitracin, daptomycin, ramoplanin, nisin, and vancomycin).16,19
In B. subtilis
, LiaF is a membrane-anchored, negative regulator of LiaS (which is the sensor protein of the system and also functions as a histidine kinase that phosphorylates the cognate-response regulator, LiaR).8
Therefore, it is predicted that mutations in liaF
may release the inhibitory effect of LiaS, resulting in activation of this system.
Nonetheless, our genetic experiments indicated that mutations in liaF
are not sufficient for full expression of the resistant phenotype. Indeed, the subsequent introduction of a mutation in gdpD
was sufficient to increase the MIC to a level similar to that in the daptomycin-resistant R712 isolate, indicating that both genes are needed for the full expression of the resistant phenotype. Thus, it appears that resistance to daptomycin in enterococci requires two major steps. First, an initial activation of the LiaFSR system occurs through mutations in liaF
or other components of the LiaFSR system (which might be selected by means of exposure to antibiotics that alter lipid-II metabolism); activation of the system may influence cell-envelope homeostasis by affecting the transcription of several genes that can help mitigate the damage caused by the antibiotic. Second, a subsequent alteration in the cell membrane occurs through changes in enzymes involved in phospholipid metabolism (e.g., GdpD or Cls), leading to critical and compensatory changes in the composition or distribution of phospholipids in the cell membrane. Indeed, the bacterial GdpD has been shown to be important in glycerol metabolism, hydrolyzing several cell-membrane glycerophosphodiesters28
that affect phospholipid metabolism. Similarly, cardiolipin has been found to play several key roles in cell-membrane physiology, such as in bacterial cell division,29,30
transporter localization (in Escherichia coli
and the triggering of compensatory changes in the phospholipid composition of the cell membrane, which affect bacterial adaptive responses.32
In support of our hypothesis that the changes discussed above are also important in other daptomycin-resistant isolates of enterococci, we found changes in genes encoding the LiaFSR system and Cls in three additional clinical isolates of daptomycin-resistant enterococci. In four other daptomycin-resistant clinical isolates, only one of these genes appeared to be altered, suggesting that additional loci involved in cell-wall homeostasis or phospholipid metabolism may be important in these enterococcal isolates. Indeed, unlike the S. aureus
cell membrane, the enterococcal cell membrane has several amino acid–containing phospholipids, apart from lysyl-phosphatidylglycerol (including arginyl-phosphatidylglycerol and alanyl-phosphatidylglycerol33
). In addition, there are other two-component systems present in enterococci that can potentially modulate the response to the antimicrobial challenge.
It is also of interest that the amino acid changes in the LiaF and GdpD proteins of the R712 isolate occurred in a region that harbors repeats of Ile; this suggests that these in-frame changes may have originated from recombination between adjacent repetitive nucleotide sequences. Mutations that occur by means of this mechanism were observed to alter the function of LiaF in B. subtilis8
and the histidine kinase VanSB
involved in E. faecium
resistance to vancomycin,34
and these mutations suggest the presence of an underlying genetic mechanism for the development of resistance to daptomycin in E. faecalis
In summary, our data indicate that the emergence of resistance to daptomycin is the result of concomitant alterations in genes (liaF and gdpD) encoding proteins that are probably involved in regulating the stress response to antimicrobial agents acting on the cell envelope and enzymes that are responsible for phospholipid metabolism in the cell membrane.