In vitro effect of mouthwash containing chlorhexidine and chlorine dioxide against halitosis pathogens

Objective: This study evaluated the antibacterial efficacy of chlorhexidine-chlorine dioxide (CHX-CDO) mouthwash formulations against major halitosis-related pathogens: Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), and Eikenella corrodens (Ec). Materials and methods: Five bacterial strains were cultured anaerobically. Experimental mouthwashes with varying concentrations of CHX (0.01–0.2%) and CDO (0.05–0.1%), labeled solution A–J, were tested. Agar diffusion assays measured inhibition zones. MICs were determined by broth microdilution with 24 h anaerobic incubation and OD600 measurement. The time-kill test quantified CFUs from serial dilutions at time points (0, 1, 3, 6, 12, and 24 h). Results: All CHX-CDO mouthwash combinations inhibited bacterial growth, with A (0.05% CHX + 0.05% CDO), C (0.2% CHX + 0.05% CDO), F (0.2% CHX + 0.1% CDO), and J (CHX 0.02/CDO 0.1) showing the largest inhibition zones and G (CHX 0.01/CDO 0.05) and H (CHX 0.02/CDO 0.05) exhibiting the most favorable MIC values. Time-kill assays confirmed sustained bactericidal effects for low concentration-CHX/0.05% CDO formulations. Significant differences in antibacterial activity were observed among the formulations (p < 0.05). Conclusion: Low-dose CHX combined with 0.05% CDO mouthwash maintains antibacterial efficacy against halitosis-associated pathogens, suggesting a promising CHX dose reduction while preserving effectiveness.