Bactericidal synergism between phage endolysin Ply2660 and cathelicidin LL-37 against vancomycin-resistant Enterococcus faecalis biofilms

Abstract

Antibiotic resistance and the ability to form biofilms of Enterococcus faecalis have compromised the choice of therapeutic options, which triggered the search for new therapeutic strategies, such as the use of phage endolysins and antimicrobial peptides. However, few studies have addressed the synergistic relationship between these two promising options. Here, we investigated the combination of the phage endolysin Ply2660 and the antimicrobial peptide LL-37 to target drug-resistant biofilm-producing E. faecalis. In vitro bactericidal assays were used to demonstrate the efficacy of the Ply2660-LL-37 combination against E. faecalis. Larger reductions in viable cell counts were observed when Ply2660 and LL-37 were applied together than after individual treatment with either substance. Transmission electron microscopy revealed that the Ply2660-LL-37 combination could lead to severe cell lysis of E. faecalis. The mode of action of the Ply2660-LL-37 combination against E. faecalis was that Ply2660 degrades cell wall peptidoglycan, and subsequently, LL-37 destroys the cytoplasmic membrane. Furthermore, Ply2660 and LL-37 act synergistically to inhibit the biofilm formation of E. faecalis. The Ply2660-LL-37 combination also showed a synergistic effect for the treatment of established biofilm, as biofilm killing with this combination was superior to each substance alone. In a murine peritoneal septicemia model, the Ply2660-LL-37 combination distinctly suppressed the dissemination of E. faecalis isolates and attenuated organ injury, being more effective than each treatment alone. Altogether, our findings indicate that the combination of a phage endolysin and an antimicrobial peptide may be a potential antimicrobial strategy for combating E. faecalis.

Fig. 1. Bactericidal activity of LL-37 and/or Ply2660 against E. faecalis strains.

a Enterococcus faecalis strains were treated with LL-37, Ply2660, or a mixture of both and incubated for 1 h at 37 °C. Tenfold serial dilutions of each sample after different treatments were spotted onto BHI agar and incubated at 37 °C for 18 h. Controls were treated with PBS alone. These assays were repeated three times on independent occasions with similar results, and representative experiments are shown. b Time-dependent killing efficacy of LL-37 or/and Ply2660 against E. faecalis V583. Bacterial cells were washed with PBS and treated with 4 μM of LL-37, 1.6 μM of Ply2660, or a mixture of both for different times (30, 60, 90, and 120 min); the viable cell number after each treatment is determined by plating on brain heart infusion (BHI) agar. c Dose-dependent killing efficacy of LL-37 or/and Ply2660 against E. faecalis V583. Bacterial cells were washed with PBS and treated with 2–8 μM of LL-37, 0.8–3.2 μM of Ply2660, or a mixture of both for 60 min, the viable cell number after each treatment is determined by plating on BHI agar. The data are presented as the means ± SD from three independent assays; error bars represent the standard deviation. Statistical significance was calculated using two-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 2. Transmission electron micrographs of E. faecalis after LL-37 and/or Ply2660 treatment.

a TEM analysis of E. faecalis V583 in the mid-logarithmic phase or after exposure to LL-37, Ply2660, or a mixture of both was conducted. Controls were treated with PBS alone. Varying degrees of lysed morphology and leakage of contents are shown in E. faecalis after the different treatments. Scale bar, 500 nm. b Averages and standard deviations are shown for three independent counts, and the number of cells for each count was 100 (n = 100); error bars represent standard deviation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. ***P < 0.001; ns, not significant.

Fig. 3. Effects of LL-37 and/or Ply2660 on cell walls and cytoplasmic membranes of E. faecalis.

a Degradation of cell walls of E. faecalis V583 after different treatments. Cell walls were prepared from E. faecalis in the logarithmic growth phase and diluted with fresh PBS. LL-37, Ply2660, a mixture of both, or mutanolysin, was added into the cell wall suspension, and samples were incubated at 37 °C. Controls were treated with PBS alone. OD₆₀₀ was measured at various time points to determine turbidity. The data are presented as the means ± SD from three independent assays; error bars represent the standard deviation. b Membrane damage of E. faecalis V583 after different treatments. Enterococcus faecalis was treated with LL-37, Ply2660, a mixture of both, or mutanolysin, and incubated at 37 °C for 1 h. Controls were treated with PBS alone. Membrane permeability was measured by detecting the fluorescence intensity of PI. The data are presented as the means ± SD from two independent assays with three biological replicates in each assay; error bars represent standard deviation. Statistical significance was calculated using Brown–Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparison test. ***P < 0.001; ns, not significant.

Fig. 4. Inhibition of E. faecalis biofilm formation by LL-37 and/or Ply2660.

Biofilm inhibition was assessed by incubating LL-37 and/or Ply2660 with E. faecalis isolates in the wells of 96-well polystyrene microtiter plates for 24 h. Bacteria without any treatment were used as a control. The adhered biofilms of a V583 and b Y15 isolates were measured using crystal violet staining. OD₆₀₀ values are the average of two independent assays with three biological replicates in each assay. The number of viable bacteria in the biofilms of c V583 and d Y15 isolates was examined. Results expressed as [log₁₀ CFU/well]. The data are presented as the means ± SD from two independent assays with three biological replicates in each assay; error bars represent standard deviation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 5. Treatment of preformed biofilms and bacterial viability of LL-37 and/or Ply2660.

Bactericidal activity of LL-37 and/or Ply2660 against preformed biofilms of E. faecalis isolates a V583 and b Y15. Biofilms were allowed to develop for 24 h and then treated with LL-37 and/or Ply2660 for 6 h at 37 °C. Bacteria without any treatment served as a control. Results are expressed as the number of viable bacteria [in log₁₀ CFU/well]. The data are presented as the means ± SD from two independent assays with three biological replicates in each assay; error bars represent standard deviation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. c Representative confocal microscope images of LIVE/DEAD-stained E. faecalis Y15 in 24-h-established biofilms after treatment with the indicated concentrations of LL-37, Ply2660, or a mixture of both at 37 °C for 6 h.

Fig. 6. Bacterial loads in the organs of E. faecalis-infected mice treated with LL-37 and/or Ply2660.

a Survival curves of mice infected intraperitoneally with E. faecalis V583 (high dose, 1.8 × 10⁹ CFU) and treated with a single dose of LL-37, Ply2660, a combination of LL-37 and Ply2660, or PBS via intraperitoneal injection (n = 12 for each group). For examination of bacterial loads in the b lung, c liver, and d kidney, mice were infected intraperitoneally with a lower dose (8 × 10⁷ CFU) of E. faecalis V583 and treated as above (n = 5 for each group). The bacterial load was calculated by plating the samples on BHI agar. The data are presented as the means ± SD from five mice; error bars represent the standard deviation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. **P < 0.01; ***P < 0.001; ns, not significant. e Bacterial detection (arrows) in tissue sections with H&E staining. Scale bars, 20 μm.

Fig. 7. Histological evaluation of organ injuries in E. faecalis-infected mice treated with LL-37 and/or Ply2660.

a Samples of the lung, liver, and kidney from mice with different treatments were fixed in 4% formalin, and tissue sections were prepared for H&E staining. Scale bars, 50 μm. Pathological scores of the b lung, c liver, and d kidney were evaluated from three random fields by two independent scientists. The data are presented as the means ± SD; error bars represent the standard deviation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 8. Schematic representation of the lysis of E. faecalis by the phage endolysin Ply2660 and the antimicrobial peptide LL-37.

Enterococcus faecalis is protected against LL-37 by a thick mesh of peptidoglycan. However, Ply2660 degrades the cell wall peptidoglycan of E. faecalis, making the cytoplasmic membrane more accessible to LL-37. Subsequently, LL-37 interacts with the cytoplasmic membrane and inserts itself into lipid bilayers, leading to pore formation in the lipid membranes and lysis of E. faecalis. Thus, the combination of Ply2660 and LL-37 exerts synergistic antimicrobial effects against E. faecalis.