Carbon monoxide-releasing molecule-3 eradicates mature Enterococcus faecalis biofilms and inhibits recolonization

Objective: To evaluate the effects of carbon monoxide-releasing molecule-3 (CORM-3) on Enterococcus faecalis biofilm inhibition and eradication, and assess its biosafety. Methods: CO release kinetics were measured by UV spectrophotometry. Mature biofilm eradication was assessed using 72-h biofilm models with crystal violet staining and XTT assay. Colonization inhibition was observed by scanning electron microscopy (SEM). Expression of biofilm-associated genes ( esp , gelE , fsrB , cylL ) was quantified by RT-qPCR. Cytocompatibility was evaluated in human oral keratinocytes (HOK) and human gingival epithelial cells (HGE). Systemic toxicity was assessed in SD rats through hematological and histopathological analyses. Results: CORM-3 released CO time-dependently with a half-release time of approximately 1.5 min. Treatment with 200 μM and 400 μM CORM-3 achieved biofilm clearance rates of 42.8 ± 5.8% and 65.3 ± 4.7%, with metabolic activity reductions of 38.5 ± 6.2% and 61.7 ± 5.3%, respectively. SEM revealed significantly reduced bacterial adhesion in treated groups. RT-qPCR showed 400 μM CORM-3 downregulated esp , gelE , fsrB , and cylL expression by 64.2%, 68.7%, 55.3%, and 42.1%, respectively. Cell viability remained above 87% after 24 h exposure to 400 μM CORM-3. No hematological abnormalities or organ damage were observed following 7-day intraperitoneal administration. Conclusion: CORM-3 exhibits dual anti-biofilm activity against E. faecalis through controlled CO release, inhibiting bacterial colonization and preventing recolonization while eradicating mature biofilms, with favorable biocompatibility at effective concentrations, supporting its potential as an adjunctive agent in endodontic treatment.