To evaluate the in vitro antibacterial effect of freshly-prepared slightly acidic electrolyzed water (SAEW) against Enterococcus faecalis (E. faecalis), a common and persistent pathogen in root-canal therapy, and to preliminarily explore its underlying mechanism.

Freshly-prepared SAEW was produced by on-site electrolysis. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SAEW against planktonic E. faecalis were determined. An ex-vivo infected single-rooted tooth model was established. Infected teeth were assigned into four groups by simple randomization, and root canals were irrigated with 0.9% sodium chloride solution, low-concentration SAEW (MIC), high-concentration SAEW (2×MIC), and 5.25% sodium hypochlorite solution, respectively. Immediate antibacterial efficacy was assessed by bacterial sampling and colony counting before and after irrigation. Root-canal irrigants were collected. Scanning electron microscopy (SEM) was used to observe bacterial ultrastructural alterations. Confocal laser scanning microscopy combined with SYTO 9/PI fluorescent staining was performed to quantitatively analyze the effect of SAEW on bacterial membrane integrity. The cytotoxicity of SAEW toward human gingival fibroblast cells (HGFCs) was detected using the CCK-8 assay. Statistical analyses were conducted with SPSS 26.0. Normality and homogeneity-of-variance tests were performed for quantitative data. Normally distributed data with homogeneous variances were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by the least-significant-difference (LSD) post-hoc test was applied for multi-group comparisons. A two-sided P<0.05 was considered statistically significant.

Freshly-prepared SAEW exhibited distinct concentration-dependent antibacterial activity against both planktonic and intracanal E. faecalis. The MIC and MBC values of SAEW for planktonic E. faecalis were 6.25 and 12.50 mg/L, respectively. The overall difference in the colony reduction rates of Enterococcus faecalis among the four groups was statistically significant (F = 306.46, P<0.001). The colony reduction rates of groups A, B, C, and D were (25.69 ± 7.41) %, (73.38 ± 1.69) %, (85.89 ± 2.09) %, and (96.60 ± 1.33) %, respectively. Compared with group A, groups B, C, and D exhibited significantly higher colony reduction rates. Moreover, group C showed a significantly higher antibacterial effect than group B but a lower bactericidal efficacy than group D (all P<0.001). These results indicated that SAEW exerts a concentration-dependent antibacterial effect, while its immediate bactericidal potency is inferior to 5.25% sodium hypochlorite. Scanning electron microscopy revealed varying degrees of bacterial ultrastructural damage in each group. Quantitative confocal laser scanning microscopy with SYTO 9/PI staining showed bacterial membrane-damage rates of 0.84% for the 0.9% sodium-chloride group, 74.39% for the low-concentration-SAEW group, 87.38% for the high-concentration-SAEW group, and 92.62% for the 5.25% sodium-hypochlorite group, with significant inter-group differences (F = 2 056.72, P<0.001). These findings indicated that SAEW exerted antibacterial effects by disrupting bacterial membrane integrity in a concentration-dependent manner. At effective antibacterial concentrations, the viability of HGFCs remained above 90% in SAEW-treated groups, whereas cell viability was below 10% in the sodium-hypochlorite-treated group.

This study demonstrated that SAEW could effectively inhibit and kill E. faecalis. Although its immediate in vitro bactericidal potency may be lower than that of high-concentration sodium hypochlorite, SAEW showed promising potential as a supplementary or alternative root-canal irrigant owing to its favorable antibacterial performance and biocompatibility. Further validation using complex biofilm and in vivo models is required.