[In vitro and in vivo antibacterial effects of the mitochondrial antioxidant Visomitin against Enterococcus]

With the increase in antibiotic resistance among Enterococcus species, the discovery of novel antimicrobial agents has become a major research focus. This study aims to investigate the in vitro and in vivo antibacterial activity of the mitochondria-targeted antioxidant Visomitin against Enterococcus through a drug repurposing strategy.

Clinical Enterococcus isolates were obtained from hospitalized patients at The Third Xiangya Hospital of Central South University. Antibacterial susceptibility and bactericidal efficacy of Visomitin against Enterococcus were first evaluated using broth microdilution assays, disk diffusion assays, and time-kill curve analyses. Resistance induction by sub-inhibitory concentrations of Visomitin was assessed through serial passage experiments. Protease quantification assays were performed to evaluate the inhibitory effect of Visomitin on protease production in Enterococcus. Subsequently, crystal violet staining and SYTO9 fluorescent probe assays were used to determine the inhibitory and eradication effects of Visomitin on Enterococcus biofilms. Finally, a mouse subcutaneous abscess model was established to evaluate the in vivo antibacterial activity of Visomitin.

Visomitin exhibited potent antibacterial activity against Enterococcus with the MIC and minimal bactericidal concentration of 4 to 8 μg/mL and 4 to 16 μg/mL, respectively. Disk diffusion assay revealed dose-dependent growth inhibition activity against Enterococcus by Visomitin, while the concentration- and time-dependent bactericidal effects were observed by time-kill curves. For instance, 2×MIC of Visomitin could reduce the viable bacterial counts of E. faecium ATCC 19434 and E. faecalis ATCC 29212 to the detection limit after 2 h and 12 h treatment, respectively. Unlike vancomycin, sub-MIC of Visomitin did not induce obvious resistance occurrence even after 14 serial passages. At 1/8×MIC, visomitin reduced the protease production of ATCC 19434 from 100%±2.01% to 96.25%±1.16% (q=3.661, P<0.05). At 1/2×MIC, visomitin decreased the protease production of ATCC 29212 from 100%±2.95% to 41.49%±0.45% (q=35.790, P<0.001). Furthermore, as determined by crystal violet staining, 1×MIC to 4×MIC of Visomitin could inhibit the Enterococcus biofilm formation, while it could also partially eradicate the preformed biofilms at the concentrations of 2×MIC to 4×MIC, as confirmed by SYTO9 staining. Furthermore, Visomitin significantly reduced both abscess size and bacterial load in mice. Compared to the vehicle control group, treatment with Visomitin decreased the bacterial load from [2.90 (2.52, 3.18)]×10⁷ CFU/abscess to [1.56 (0.35, 4.78)]×10⁶ CFU/abscess (U=0.000, P<0.01).

Visomitin demonstrates potent antibacterial activity against Enterococci both in vitro and in vivo, with a low risk of inducing resistance and potential for repurposing, providing key preclinical evidence for its development as a novel therapeutic candidate against refractory enterococcal infections.