Enterococci are leading causes of nosocomial infections in the US [1], causing a variety of life-threatening syndromes such as bacteremic infections (including endocarditis), urosepsis and meningitis, among others. Enterococcal disease occurs frequently in patients that are seriously ill and/or with important degrees of immunosuppression. Two species are responsible for the vast majority of enterococcal infections, E. faecalis and E. faecium. The treatment of such infections is often impacted by the increased prevalence of multidrug resistance in these isolates. Indeed, ampicillin and vancomycin resistance is now present in more than 80% of E. faecium isolates [1], making these compounds almost obsolete for the treatment of this pathogen. Moreover, E. faecium is one of the “no ESKAPE” pathogens (E. faecium, Staphylococus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter spp.), highlighted by the Infectious Diseases Society of America as nosocomial organisms requiring new therapeutic approaches, both because of their commonality and challenging clinical presentations, but importantly also due to multiple antibiotic resistances [2]. Although most E. faecalis isolates remain susceptible to ampicillin, the increased prevalence of resistance to aminoglycosides and vancomycin within this species also limits the therapeutic alternatives, as well as the ability to achieve bactericidal killing of this organism [1], [3].

Daptomycin (DAP) is a lipopeptide antibiotic approved by the Food and Drug Administration (FDA) in 2003 for the treatment of skin and soft tissue infections caused by susceptible Gram-positive organisms and, subsequently in 2006, for S. aureus bacteremia and right-sided endocarditis [4]. DAP has potent in vitro bactericidal activity against vancomycin-resistant enterococci (VRE), although it does not have an FDA approval for VRE infections. Nonetheless, many clinicians often use this compound for the treatment of severe enterococcal infections, particularly those caused by E. faecium. Two retrospective studies have shown that outcomes of patients with serious enterococcal infections treated with DAP were similar to those treated with linezolid (an FDA-approved antibiotic for VRE infections) [5], [6]. However, there are several recent reports of patients failing DAP mono-therapy in association with emergence of DAP resistance during therapy [7], [8], [9].

The mechanism of action of DAP involves a calcium-dependent interaction with the bacterial cell membrane (CM) [10], [11]. The insertion of the drug into the CM causes disruption in its homeostasis that is associated with a leakage of potassium ions from the cytoplasm of the bacterial cell [10]. These alterations of the CM lead to bacterial cell death by mechanisms that are not fully elucidated but are likely to involve alterations in cell division homeostasis [11]. Emergence of DAP non-susceptibility has been described in both S. aureus and enterococci. Several genes have been associated with this phenomenon and include: i) genes encoding two or three-component regulatory systems involved in cell envelope homeostasis: yvqF-vraSR in S. aureus, and liaFSR in enterococci and Bacillus subtilis [12], [13]; ii) genes coding for enzymes involved in CM phospholipid metabolism: mprF (multiple peptide resistance factor) [12], [14], [15], cls (cardiolipin synthase) [14], pgsA (phosphatidylglycerol synthase) [14] in S. aureus; gdpD (glycerophosphodiesterphosphodiesterase) [13], cls [13], [16] in enterococci, and pgsA [17] in B. subtillis; and iii) genes encoding for β subunits of RNA polymerase in S. aureus (rpoB/rpoC) [14], [18].

In the present study, we delineate the following CM characteristics of both two enterococcal clinical strain-pairs (DAP-susceptible and DAP-resistant), each isolated from a patient during failed DAP therapy: i) phospholipid repertoire, ii) fatty acid composition, iii) fluidity, and iv) DAP-induced permeabilization-depolarization profiles.

DAP is one of the few antibiotics that exhibit in vitro bactericidal activity against enterococci, including those that are vancomycin- and ampicillin-resistant [20]. This antibiotic is now commonly used “off-label” for the treatment of serious enterococcal infections due to the lack of other therapeutic alternatives. A challenging issue for the use of DAP against enterococci is the emergence of resistance during therapy. Indeed, several cases of patients infected with E. faecalis and E. faecium who developed resistance during the course of DAP treatment have now been documented [7], [8], [9]. Furthermore, enterococci are less susceptible to DAP in vitro than staphylococci, and this relatively reduced ability of DAP to kill enterococci may contribute to the development of DAP-resistance in vitro, and subsequent failure during DAP therapy of serious infections.

The mechanism of action of DAP involves interactions with the bacterial CM in a calcium-dependent manner [11]. We have previously shown that the development of DAP-resistance in a clinical-strain pair of E. faecalis was associated with important ultrastructural changes in the cell envelope, including an increase in cell-wall thickness, as well as notable perturbations in cell surface charge and a reduced ability of DAP to depolarize and permeabilize the CM [13]. In the current work, we extended the above observations to a clinical strain-pair of E. faecium and, most importantly, focused our studies on a comparative analysis of the CM PL profile of both clinical-strain pairs. A number of interesting themes emerged from our investigations. Firstly, as we observed previously in E. faecalis, development of DAP resistance in E. faecium was associated with marked structural and functional changes in the cell envelope, and in the ability of DAP to alter the CM integrity. Thus, development of DAP resistance in E. faecium was accompanied by structural perturbations within the cell envelope (e.g., peri-septal CM protrusions) and in the cell wall (thickening). Also, biophysically, the CMs of both DAP-resistant enterococci became less fluid. Our findings confirm that structural alterations of the cell envelope and changes in the biophysical properties of the CM are strongly associated with the mechanism of DAP resistance, as previously described in S. aureus [13], [19] and B. subtillis [11].

Secondly, detailed analysis of CM PL content in both clinical strain-pairs revealed important differences between E. faecalis and E. faecium. Indeed, whereas in E. faecium, the only amino-containing PL detected was L-PG (similar to S. aureus) [19], E. faecalis CMs contained at least three amino-PLs; one was identified as L-PG (∼15% of total PLs), with two other amino-PLs of unknown identity. To our knowledge, a study that comprehensively characterized the amino-containing PLs of E. faecalis was published over 40 years ago [21], and provided compelling evidence that lysyl-, alanyl- and arginyl-containing PLs were present in their CM extracts. A recent study also noted the presence of alanyl- and arginyl-containing PLs in their membrane lipid extract [22]. Although our experimental growth conditions for PL extraction were different from those used in the former study (BHI vs medium containing 1% tryptone, 0.5% yeast extract, 0.5% dipotassium phosphate and 1% glucose, respectively), it is tempting to speculate that the other amino-containing PLs identified in our E. faecalis strain-pair are likely to represent alanyl-PG and arginyl-PG. Of interest, we did not find any significant difference in the amino-containing PL content or L-PG “flipping” between the DAP-resistant E. faecalis R712 compared to its parental DAP-susceptible S613. In contrast, in our E. faecium strain-pair, a trend towards increased content of L-PG in the outer CM of the DAP-resistant isolate was observed, although this did not quite reach statistical significance. Our findings suggest that differences in content and distribution (“flipping”) of amino-containing PLs are not a major determinant of DAP resistance in enterococci. This is in contrast to data from S. aureus where increased L-PG translocation to the outer CM leaflet (likely mediated by gain-in-function mutations in the CM lipid-modifier protein, MprF) appears to play a pivotal role in DAP-R in selected strains [23], [24].

Thirdly, a striking finding in our studies was that the in vivo development of DAP resistance in enterococci was associated with a significant decrease in PG content. Such striking reductions in CM PG have been previously associated with resistance to DAP and other cationic antimicrobial peptides in Gram-positive bacteria [14], [25]. For example, using an in vitro passage strategy for developing DAP-resistance, Hachmann et al. [17] demonstrated that development of DAP resistance was associated with a marked decrease in CM PG content in a laboratory strain of B. subtilis, although changes in other CM PLs were not described. The reduction in PG was correlated with the presence of a single nucleotide polymorphism (A64V) in pgsA, a gene encoding a key PG synthase. Of interest, mutations in pgsA have also been recently associated with DAP-resistance in S. aureus [14]. Similarly, characterization of E. faecium mutants resistant to the class II bacteriocin, mundticin KS (a 43 amino acid peptide produced by E. mundtii and active against E. faecium), was associated with significant reductions in PG (and also CL) [25]. PG is an abundant anionic PL of bacterial CMs in both Gram-positive and Gram-negative bacteria [26]. In E. coli, the balance of zwitterionic and acidic (negatively-charged) PLs is the result of the formation of PG, which is tightly coupled with the regulation of the synthesis of other PLs [26]. Maintaining the PL balance is crucial for the homeostasis of several bacterial processes including CM protein topology [27] and cell division [28], among others. Interestingly, we previously showed that a codon deletion in a gene encoding a putative glycerophosphodiesterphosphodiesterase (GdpD, an enzyme that hydrolyzes PLs) was necessary (but not sufficient) to confer DAP resistance in E. faecalis R712 [13]. Therefore, one could hypothesize that the decrease in PG associated with development of DAP resistance in enterococci could be due to the rapid recycling of PG; of note, the glycerol phosphate moiety of PG undergoes rapid turnover in bacteria [26], [29]. It is, thus, tempting to speculate that changes in the “PG pools” are important in the development of DAP resistance by providing the necessary substrates for synthesis of other PLs or glycerol-containing molecules (e.g., lipoteichoic acids) [26], [30] in order to avoid killing by cationic antimicrobial molecules. Of interest, we did not observe any major changes in CL content associated with development of DAP-resistance in E. faecalis or E. faecium despite the fact that mutations in the CL synthase gene (cls) have been previously linked to DAP resistance in E. faecalis [16], E. faecium [13] and S. aureus [14]. Nonetheless, as PG is a substrate for Cls, it is plausible that changes in Cls activity or specificity may also contribute to the shift in PG pools.

Fourthly, a significant increase in a negatively charged glycerophosphoglycolipid ultimately identified as GP-DGDAG by LC/ESI-MS/MS was found in both DAP-resistant enterococci studied. It has been previously shown that E. faecalis contains several glycolipids including glycerophosphoryl-diglucosyl-diglyceride [31], phosphatidyldiglucosyldiglyceride [32], [33] and glycerophosphoryl-phosphatidylkojibiosyl-diacylglycerol [34]. Interestingly, some of the above glycolipids appear to be the precursors of enterococcal lipoteichoic acids (LTA), which are important constituents of the cell surface of Gram-positive organisms [35]. Indeed, Ganfield & Pieringer showed that PG is the donor of sn-glycerol 1-phosphate moieties to phosphatidyl-kojibiosyldiacylglycerol in the in vitro synthesis of enterococcal membrane LTA [36]. Furthermore, alanylation of wall teichoic acids (TA) has been shown to play a role in the defense against antimicrobial peptides in S. aureus [37] and overexpression of the dlt operon, which encodes 4 genes involved in the alanylation of wall TAs in S. aureus, has been linked to DAP resistance [38]. Thus, our findings suggest that the emergence of DAP-resistance in enterococci is associated with increase production of membrane glycerophosphoglycolipids which may serve as precursors of LTA using PG as the donor of glycerol-phosphate moiety, which, upon alanylation, might increase relative positive surface charge and contribute to DAP resistance.

Fifthly, changes in the fatty acid composition of bacterial CMs are essential in the optimization of CM function for growth and response to modifications in environmental conditions (including the presence of antimicrobial molecules) [26]. Fatty acid composition is also a major determinant of CM order (fluidity-rigidity) that affects the homeostasis of the bacterial cell. In the current study, the development of DAP resistance in both enterococcal isolates was associated with less fluid (more rigid) membranes. Of interest, this is in contrast to previous reports in clinically-derived DAP-resistant S. aureus strains, whose CMs were significantly more fluid than their respective DAP-susceptible parental strains [19]. Conversely, a modest decrease in CM fluidity was observed in a DAP-resistant S. aureus strain selected by serial in vitro passages in DAP [39]. These apparent paradoxes in CM order responses indicate that such CM changes may well be either strain-specific and/or dependent on diverse DAP exposure conditions [40]. Additionally, factors that influence the development of DAP resistance in vivo are highly likely to be different from those in vitro, including a cadre of host factors such as serum components, host immune cells and innate host defense peptides [39]. Interestingly, the decrease in CM fluidity observed in our DAP-resistant E. faecalis strain could not be linked to changes in the proportion of saturated vs unsaturated (or cyclic) fatty acids, and, thus, other factors may be important. In contrast, a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate (mostly due to a decrease in the C18:1ω7c species), representing a potential explanation for the observed reduction in CM fluidity in this strain. It should be noted that there was a significant increase in cyclopropane fatty acids observed in this same DAP-resistant E. faecium isolate as compared to its DAP-susceptible parent. Cyclopropane fatty acids have been shown to stabilize CMs to a variety of environmental challenges [41].

In summary, we present evidence that development of DAP resistance in clinical enterococcal strains is associated with a significant shift in PL profiles (i.e., decreases in PG and increases in GP-DGDAG). A common feature of DAP resistance in both E. faecalis and E. faecium include marked ultrastructural changes in the cell envelope, as well as increases in the net surface positive charge and increased rigidity of the CMs. Our findings suggest that important biochemical and biophysical modifications in CM lipid metabolism occur in response to DAP exposures in enterococci.