Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

Daptomycin is a lipopeptide antibiotic that is used as a last resort in the treatment of infections caused by methicillinresistant Staphylococcus aureus (MRSA), vancomycinintermediate S. aureus (VISA) and vancomycin-resistant enterococci (VRE) [1][2][3]. The use of daptomycin is becoming more common, with prescriptions increasing by 72 % between 2012 and 2015 in the UK [4]. Daptomycin is the only lipopeptide antibiotic that is used clinically and it functions in a similar manner to antimicrobial peptides [5]. The antibiotic inserts into the membrane of Gram-positive bacteria by targeting phosphatidylglycerol, where it forms oligomeric complexes [6][7][8]. The precise mechanism by which the antibiotic kills bacteria is unclear, but it involves depolarization of the bacterial membrane and inhibition of cell wall biosynthesis without causing lysis [8][9][10][11][12][13]. Although daptomycin resistance is rare, treatment failure occurs in up to 30 % of staphylococcal infections and 23 % of enterococcal infections [14,15]. The failure rates are highest in invasive infections such as bacteraemia or osteomyelitis, with rates of 24 and 33 % respectively, resulting in poor patient prognoses [14]. Understanding the reasons for this treatment failure is crucial to improving the effectiveness of daptomycin treatment.
We recently discovered that S. aureus has a transient defence mechanism against daptomycin, which contributed to treatment failure in a murine model of invasive infection [16]. In response to the antibiotic, phospholipids were released from the cell membrane, which sequestered daptomycin and abrogated its bactericidal activity [16]. Phospholipid release occurred via an active process, which was blocked by the lipid biosynthesis inhibitor platensimycin [16,17]. In addition to daptomycin, phospholipid release also provided protection against the antimicrobial peptides nisin and melittin, suggesting a general defence against membrane-targeting antimicrobials [16].
It is currently unknown whether other Gram-positive bacteria release phospholipids in response to daptomycin, although membrane vesicles have been observed on the surface of Enterococcus faecalis cells exposed to daptomycin [18]. In addition, there is growing evidence that other Gram-positive pathogens, including group A streptococci (GAS) and group B streptococci (GBS), release phospholipids from their surfaces in the form of extracellular vesicles [19,20]. Production of these membrane vesicles is increased in the presence of antimicrobials and, at least for GAS, they are rich in phosphatidylglycerol, which was shown to be essential for daptomycin inactivation by S. aureus [16,19,21]. Therefore, we hypothesized that phospholipid release is a common strategy amongst Gram-positive pathogens to resist membrane-acting antimicrobials.
Given the increasing use of daptomycin to treat enterococcal infections, the primary aim of this work was to determine whether enterococci release membrane phospholipids that inactivate the antibiotic. We also examined pathogenic streptococci and S. epidermidis, as the rising tide of antibiotic resistance may necessitate the use of daptomycin to tackle these bacteria in the future.
For all strains, there was a dose-dependent decrease in survival after 8 h exposure to daptomycin, as assessed by c.f.u. counts ( Fig. 1a-h). Broadly, the survival of strains exposed to supra-MIC concentrations of daptomycin correlated with the MIC values, with survival of the two enterococcal strains, the staphylococci and S. gordonii, being greater than the survival of the GAS or GBS strains at each of the concentrations of daptomycin examined (Fig. 1a-h).
Next, we explored whether streptococci and enterococci released phospholipids in response to daptomycin challenge, and how this related to the susceptibility of the strains to the antibiotic. Using the phospholipid-reactive fluorescent dye FM-4-64 (Life Technologies), we confirmed our previous observation that wild-type staphylococci released phospholipids in the absence of daptomycin, but this was significantly increased for bacteria exposed to daptomycin (Fig. 1i, j) [16]. By contrast, neither enterococci nor streptococci released phospholipids in the absence of daptomycin ( Fig. 1k-p). Upon exposure to daptomycin, however, all of the streptococci and enterococci released phospholipids, albeit to differing levels. The quantity of phospholipid released was much greater for staphylococci than for the other species examined (Fig. 1i-p). However, for both staphylococci and streptococci, the quantity of phospholipid released was lowest when the daptomycin concentration was highest, suggesting that the antibiotic may have killed the bacteria before they could release the lipid (Fig. 1i-p). By contrast, the enterococci released high levels of phospholipid in the presence of the highest concentrations of daptomycin (Fig. 1o, p). Therefore, unlike survival, phospholipid release did not correlate with daptomycin MIC, and this may indicate different daptomycin concentration thresholds for the triggering of phospholipid release.
To determine whether phospholipid release resulted in the inactivation of daptomycin, the activity of the antibiotic in the culture supernatants was measured using a previously described zone of inhibition assay [16] (Fig. 1q-x). Daptomycin was inactivated to varying degrees by the bacteria, depending on the concentration of the antibiotic used. However, both staphylococcal strains, both enterococcal strains, S. gordonii and the GAS strain completely inactivated daptomycin at 5 µg ml À1 , but GBS strains only partially inactivated the antibiotic at this concentration. At 10 µg ml À1 daptomycin, only the staphylococci, S. gordonii and the enterococci showed significant inactivation of the antibiotic, while at a concentration of 20 µg ml À1 daptomycin, only staphylococci and enterococci inactivated the antibiotic to any significant degree, with a loss of 30-60 % of antibiotic activity. However, despite triggering phospholipid release, at 40 µg ml À1 daptomycin there was relatively little (<20 %) inactivation of the antibiotic by any of the bacteria tested. Therefore, phospholipid release is finite and can be overcome with a sufficiently high dose of daptomycin.
The predominant phospholipid in the membrane of Grampositive bacteria is phosphatidylglycerol, with much smaller quantities of cardiolipin and/or lysyl-phosphatidylglycerol also present [31]. Our previous work using purified phospholipids revealed that, at physiologically-relevant concentrations, phosphatidylglycerol is the only component of the membrane that can inactivate daptomycin [16]. Therefore, whilst we did not identify the particular species of phospholipid released from enterococci or streptococci, the ability of released lipids to inactivate daptomycin demonstrates the presence of phophatidylglycerol.
These data extend our previous finding that S. aureus releases phosphatidylglycerol in response to daptomycin, and that this results in inactivation of the antibiotic by revealing a very similar phenotype for S. epidermidis. Further, these findings also support the previous observation that E. faecalis releases phospholipids in response to daptomycin [18], and show that this phospholipid release correlates with daptomycin inactivation and bacterial survival. Streptococci, particularly S. gordonii, also released phospholipids and inactivated daptomycin, albeit less efficiently than E. faecalis. Therefore, daptomycin-induced phospholipid release appears to be a conserved mechanism across Gram-positive pathogens.
Next, we wanted to explore whether the mechanism of phospholipid release and daptomycin inactivation by enterococci and streptococci was similar to that of S. aureus. Therefore, we undertook further experiments with E. faecalis, which was the most efficient of the enterococci and streptococci at releasing phospholipids and inactivating daptomycin, and S. aureus, in which daptomycin-triggered phospholipid release has been well characterized [16].
In S. aureus, daptomycin-triggered phospholipid release is an active process that requires energy, as well as protein and lipid biosynthesis [16]. To determine whether phospholipid release by E. faecalis exposed to daptomycin was occurring via an active process, or simply as a consequence of membrane damage caused by the antibiotic, bacteria were exposed to the antibiotic in the presence or absence of a sub-inhibitory concentration of the phospholipid biosynthesis inhibitor, platensimycin [17]. As described previously, the exposure of S. aureus to daptomycin (10 µg ml À1 ) resulted in increased phospholipid in the supernatant, but this was significantly reduced in the presence of platensimycin at half the MIC (0.25 µg ml À1 ) (Fig. 2a). Similarly, phospholipid was released upon the exposure of E. faecalis to daptomycin (10 µg ml À1 ), but this was blocked when platensimycin was present at half the MIC (0.5 µg ml À1 ) (Fig. 2b). The presence of platensimycin prevented S. aureus from inactivating daptomycin (Fig. 2c) and significantly reduced the ability of E. faecalis to inactivate daptomycin (Fig. 2d). This confirmed that daptomycin-induced phospholipid release by E. faecalis is an active process that requires de novo lipid biosynthesis and is not simply a consequence of membrane damage caused by the antibiotic. The ability of platensimycin to block phospholipid release and prevent daptomycin inactivation by E. faecalis also provided strong evidence that, as for S. aureus, daptomycin activity is blocked by the phospholipid in the supernatant. However, it was necessary to rule out an alternative hypothesis; that the loss of daptomycin activity was simply due to binding of the antibiotic to the bacterial surface.
To measure the binding of daptomycin to bacteria, daptomycin was labelled with the BODIPY fluorophore (Life   Technologies) as described previously [11,16]. As reported previously, a killing assay with E. facealis indicated that the labelled antibiotic had slightly reduced bactericidal activity relative to unlabelled daptomycin [11] (Fig. 3a). However, as described above for unlabelled antibiotic (Fig. 1q, x), the activity of the antibiotic decreased after incubation with E. faecalis or S. aureus (Fig. 3b), confirming that the BOD-IPY label does not significantly affect the interaction of the antibiotic with the bacteria studied.
After 8 h incubation with BODIPY-daptomycin, the bacterial cells were pelleted and the fluorescence of both the cells and the supernatants was measured separately using a Tecan microplate reader with excitation at 502 nm and emission at 510 nm. Antibiotic attachment to the E. faecalis cellular fraction was similar for both BODIPY-daptomycin concentrations examined, suggesting saturated binding to cells (Fig. 3c). However, most of the antibiotic remained in the supernatant (Fig. 3d). By comparison, BODIPY-daptomycin bound S. aureus more strongly than E. faecalis, with higher levels of fluorescence associated with bacterial cells and a corresponding drop in the fluorescence of the supernatant (Fig. 3c, d). This difference in antibiotic binding may explain why the daptomycin MIC of the E. faecalis strains used here (2-4 µg ml À1 ) is higher than that of the S. aureus strain examined (1 µg ml À1 ), and why daptomycin triggers greater phospholipid release from staphylococci than enterococci.
Together, these data confirmed that the loss of daptomycin activity in E. faecalis cultures was not due to binding of the antibiotic to the bacterial surface or the plastic vessels used in the assays. However, as a final confirmation that phospholipids released from E. faecalis inactivated daptomycin, we exposed the bacterium to daptomycin (5 µg ml À1 ) to trigger phospholipid release, collected the cell-free culture supernatant and added a second dose of the antibiotic (5 µg ml À1 ). The culture supernatant containing the released phospholipids significantly reduced the activity of the second dose of daptomycin (by~25 %; Fig. 3e). Therefore, as described for S. aureus, the release of phospholipids by E. faecalis in response to daptomycin inactivates the antibiotic. The data described above also indicate that several species of streptococci release phospholipids in response to daptomycin, and these inactivate the antibiotic, albeit to a lesser extent than for E. faecalis or S. aureus.
Streptococci and enterococci cause a range of serious diseases, including septicaemia and endocarditis, which can be treated by daptomycin, especially when the pathogen is multi-drug resistant (e.g. VRE or VISA) or the patient has a b-lactam allergy [1,32]. Our finding that this defence mechanism is present in a variety of clinically relevant Grampositive bacteria indicates that it is conserved and could be a viable target to improve the effectiveness of daptomycin therapy against these pathogens. However, it should be noted that this study employed drug-sensitive strains and so further work is required to explore how the phospholipid release system works in bacteria that are resistant to vancomycin or daptomycin, which typically results in alterations to the cell membrane or wall [1,2]. Changes to the membrane may also occur in vivo due to the utilization of hostderived fatty acids or in response to environmental stress, such as the presence of antimicrobial peptides, and so these factors will also need to be considered [31,33,34].
In this work, we focussed on daptomycin because it is a lastresort antibiotic and is associated with high rates of treatment failure. However, whilst daptomycin use is increasing, it is very unlikely to have provided the selection pressure for the evolution of the phospholipid release defence mechanism described here and previously [16]. Since cationic antimicrobial peptides (CAMPs) act via a similar mechanism to daptomycin in targeting the Gram-positive cell membrane [5] we hypothesize that these host defence molecules have likely driven the evolution of phospholipid release as a defence mechanism.
The discovery of phospholipid release in several Gram-positive pathogens has expanded our growing appreciation of the broad-spectrum extracellular defence mechanisms that protect bacteria against antibiotics or host defences. For example, previous work has shown that the production of outer-membrane vesicles by Escherichia coli can protect against membrane-acting antimicrobials such as polymixin E and colistin [35], whilst another report revealed that lipochalins released by Burkholderia can sequester several different antibiotics [36]. These findings underline the complex nature of innate antibiotic resistance, but also provide opportunities for mechanistic insights and improved therapeutic approaches. For example, in this report and previously, we have shown that inhibition of phospholipid biosynthesis using platensimycin prevents the inactivation of daptomycin by both S. aureus and E. faecalis [16]. Although platensimycin has not entered clinical trials due to poor pharmacokinetic properties [17,37], other inhibitors of lipid biosynthesis are in clinical development [38]. Therefore, the use of daptomycin in combination with lipid biosynthesis inhibitors may provide an effective way of enhancing treatment outcomes compared to the lipopeptide antibiotic alone.
In summary, we have demonstrated that E. faecalis releases phospholipids in response to daptomycin via an active mechanism requiring de novo lipid biosynthesis and that these phospholipids inactivate daptomycin. Pathogenic streptococci also appear to be capable of inactivating daptomycin by releasing phospholipids, indicating that this mechanism is conserved amongst Gram-positive pathogens.