An oxygen-releasing mouthwash shows species-dependent antibacterial and antibiofilm activity against periodontal pathogens

This study evaluated whether an oxygen-releasing mouthwash formulation (O 2 MW) produces species-dependent antibacterial, antibiofilm, and gene expression responses in Aggregatibacter actinomycetemcomitans and Prevotella intermedia compared with 0.12% chlorhexidine under planktonic and biofilm conditions in vitro. Minimum inhibitory concentrations (MICs) were determined using a resazurin-based broth microdilution, and minimum bactericidal concentration (MBC) by agar subculture. Agar diffusion assessed growth inhibition. Biofilm biomass was quantified using crystal violet staining, while pre-formed biofilm structure and membrane integrity were examined by confocal laser scanning microscopy (CLSM). Quantitative PCR assessed adhesion-, toxin-, oxidative-stress-, and iron-associated genes. Molecular docking used sodium perborate as a representative oxygen-releasing constituent. O 2 MW MICs were 3.13% for A. actinomycetemcomitans and 50% for P. intermedia . MBC was detected only for A. actinomycetemcomitans . Agar diffusion showed inhibition zones from 0.78% O 2 MW for A. actinomycetemcomitans , whereas P. intermedia showed zones only at 50% and 100% O 2 MW. O 2 MW reduced biofilm biomass, with minimum biofilm inhibitory concentration (MBIC) values of 1.56% and 25%, respectively. CLSM showed reduced biomass and thickness at higher concentrations. Sub-MIC O 2 MW increased expression of adhesion- and virulence-associated genes, particularly ltxA in A. actinomycetemcomitans and oxidative-stress and iron-associated genes, including ahpF , ftnA , and feoB in P. intermedia . Docking suggested pathway-level involvement only. O 2 MW showed species-dependent antibacterial and antibiofilm activity, with greater effects at lower concentrations against A. actinomycetemcomitans . The findings support further preclinical and host-cell evaluation before clinical translation. O 2 MW may have potential as an adjunct for periodontal biofilm control, but pathogen-specific responses warrant further evaluation.