Fig 1.
Periapical lesions were induced in a rat model.
Dental pulp of the maxillary incisor teeth of male Wistar rats were exposed and infected with E. faecalis (VRE ATCC 700802). Standard root canal treatment was conducted using instrumentation and one of the following treatments: group A: saline irrigation; group B: EFDG1/EFLK1 phage cocktail irrigation (109 PFU/mL); group C: EFDG1/EFLK1 poloxamer-phage formulation (109 PFU/mL).
Fig 2.
Poloxamer-phage cocktail releases active anti-E. faecalis phages for a month.
PFU/mL of the EFDG1/EFLK1 phage cocktail released from 30% poloxamer-phage formulation measured after 1, 2, 8, 14, 21 and 28 days. Phage release was measured for a month. A high concentration of phage release was observed during the test period, and the phage concentration of the solution gradually decreased from 109 to 103 PFU/mL. The results are based on 5 independent biological replicates.
Fig 3.
Poloxamer-phage formulation kills logarithmic and stationary E. faecalis.
(A) Treatment of bacteria in the logarithmic growth phase with phages and poloxamer-phage formulation reduced bacterial growth. Treatment with phages and a poloxamer-phage formulation (dashed curves: EFDG1/EFLK1 phage cocktail) reduced bacterial growth of logarithmic E. faecalis compared with the control (untreated bacteria and poloxamer alone), as depicted by OD changes. The results are based on 6 independent biological replicates.(B) Treatment of E. faecalis in the stationary growth phase with phages and poloxamer-phage formulation reduced bacterial growth. Treatment with phages and a poloxamer-phage formulation (dashed curves: EFDG1/EFLK1 phage cocktail) reduced bacterial growth of stationary E. faecalis compared with that of cells in stationary phase treated with poloxamer alone (PBS based) and untreated bacterial control as depicted by OD changes. The results are based on 6 independent biological replicates.
Fig 4.
Sustained-release of phages targets E. faecalis biofilm.
(A) E. faecalis biofilm biomass decreased following treatment with sustained-release EFDG1/EFLK1 phage cocktail. Treatment with phages and a poloxamer-phage formulation decreased bacterial biofilm biomass, as evaluated by crystal violet staining after 0 h (dots), 24 h (white), 72 h (light gray), 1 week (dark gray), and 4 weeks (black). After 24 h, the phage suspension showed the best antibacterial activity, but after 4 weeks of treatment, the poloxamer-phage formulation reduced biofilm mass most efficiently. The results are presented as percentages, normalized to the biofilm biomass controls. Statistical significance was calculated by Student’s t-test (significance level: p < 0.05) compared to the untreated control. The results are based on 8 independent biological replicates. (B) E. faecalis biofilm viable bacterial counts decreased following treatment. The viable counts of E. faecalis biofilm bacteria (CFU/ mL) after 0 h (dots), 24 h (white), 72 h (light gray), 1 week (dark gray) and 4 weeks (black) after treatment with phage suspension and poloxamer-phage formulation are described. Treatment with phage suspension reduced the viable bacterial counts by more than 90% during all treatment periods. Treatment with poloxamer-phage formulation reduced the bacterial counts by more than 90% after 72 h of treatment. The results are presented as percentages, normalized to the viable counts controls. The CFU/mL values of the viable counts and the standard deviation are detailed in the table below. Statistical significance was calculated by Student’s t-test (significance level: p < 0.05) compared to the untreated control. The results are based on 8 independent biological replicates. (C) Treatment with EFDG1/EFLK1 poloxamer-phage formulation and EFDG1/EFLK1 phage suspension targeted E. faecalis after 4 weeks. Confocal microscopy of live/dead cells in stained biofilm: [A] E. faecalis untreated biofilm shows bacterial clusters. [B] EFDG1/EFLK1 phage suspension-treated E. faecalis biofilm shows lysis of the bacteria. [C] EFDG1/EFLK1 poloxamer-phage formulation-treated E. faecalis biofilm shows lysis of the bacteria. (D) Decrease in green-stained cells of E. faecalis biofilm following treatment with EFDG1/EFLK1 poloxamer-phage formulation and EFDG1/EFLK1 phage suspension. Treatment with phage suspension and poloxamer-phage formulation decreased green stained bacteria, as depicted by live/dead cell staining after 24 h and 4 weeks visualized by confocal microscopy (Zeiss). The results are presented as percentages, normalized to the untreated controls. Statistical significance was calculated by Student’s t-test (significance level: p < 0.05) compared to the untreated control. The results are based on 8 independent biological replicates.
Fig 5.
Poloxamer-phage formulation is not toxic to cells.
XTT viability indicator shows that RAW macrophage cell viability was unaffected by poloxamer-phage formulation treatment compared to an immune system stimulator (heat killed Pg). The decrease in the OD450 of the tested group was insignificant (p > 0.05). White = immune system stimulator, gray = untreated control, black = phages-poloxamer. Statistical significance was calculated by Student’s t-test (significance level: p < 0.05) compared to the untreated control. The results are based on 8 independent biological replicates.
Fig 6.
Phage treatment against VRE periapical infection in rats.
(A) Intracanal viable enterococci counts after treatment with EFDG1/EFLK1 phage suspension and EFDG1/EFLK1 poloxamer-phage formulation. Viable counts of enterococci show a significant decrease following phage treatment. Control = saline irrigation, EFLK1+EFDG1 = EFDG1/EFLK1 phage suspension irrigation (109 PFU/mL), PLX-EFLK1+EFDG1 = EFDG1/EFLK1 poloxamer-phage formulation (109 PFU/mL). Statistical significance was calculated by Student’s t-test (significance level: p < 0.05) compared to the untreated control (n = 14–16 teeth in every tested group). (B) Scanning electron microscopy: no survival of bacteria in rat dentinal tubules. (magnification: X5000) [A] control: E. faecalis-infected tooth shows biofilm inside the dentinal tubules; [B] E. faecalis-infected tooth treated by poloxamer-phage formulation does not exhibit bacteria inside the dentinal tubules. (C) Histology of E. faecalis-infected rat incisors followed by root canal treatment with poloxamer-phage formulation showing reduced inflammation. Histology of sagittal cross-sections of the periapical area of Wistar rat incisors that were decalcified, and H&E stained. [A] E. faecalis-infected teeth show enlarged peri-radicular connective tissue and osteoclasts suggesting inflammation, bone resorption and apical periodontitis. [B] E. faecalis-infected teeth that were irrigated with saline show enlarged highly cellular peri-radicular connective tissue and necrosis. Osteoclasts are present in the alveolar bone. [C] E. faecalis-infected tooth that was treated with poloxamer-phage formulation shows highly vascularized dental pulp, reduced peri-radicular connective tissue, and a woven bone with osteoclast presence. White arrows indicate infiltration of osteoclasts. DP = dental pulp. RD = root dentin. PR-CT = peri-radicular connective tissue. AB = alveolar bone (magnification: × 10). (D) Changes of the relative abundance of intracanal phyla due to root canal treatments. The changes in the intracanal microbiota were evaluated using 16S genome sequencing. Intracanal samples were collected 4 weeks after E. faecalis infection (before treatment) and 3 weeks after root canal treatment. Root canal treatment included instrumentation and treatment with one of the following: group A: Saline irrigation; group B: EFDG1/EFLK1 phage cocktail irrigation (109 PFU/mL); or group C: EFDG1/EFLK1 poloxamer-phage formulation (109 PFU/mL). The most abundant phyla were Firmicutes, Bacteroidetes and Proteobacteria. Phage cocktail suspension and poloxamer-phage formulation treatments caused a decrease in the relative abundance of Firmicutes and an increase in the relative abundance of Proteobacteria. (n = 14–16). (E) Alpha diversity dot plot representing taxa richness changes before and after root canal treatment (Shannon index) and differences between the treatment groups. Standard root canal treatment with instrumentation and group A: saline irrigation; group B: EFDG1/EFLK1 phage cocktail irrigation (109 PFU/mL); or group C: EFDG1/EFLK1 poloxamer-phage formulation (109 PFU/mL). Higher values of alpha diversity (via Shannon index) were obtained following poloxamer-phage formulation and phage cocktail suspension treatment compared to the other groups. (F) Relative enterococcal abundance changes before and after poloxamer-phage formulation treatment. Representative data from 16S genome sequencing at the genus level reveals an enterococcal decrease following poloxamer-phage formulation treatment. 16 incisor teeth are presented in each group before and after treatment. Gray = 4 weeks after E. faecalis infection. Black = 3 weeks after poloxamer-phage formulation treatment (7 weeks after infection).
Table 1.
Histological analysis of the antibacterial activity on host inflammation in the periapical area—Periapical Index (PAI*) (n = 7 each group).