## 4 Discussion
The CAP group exhibited the lowest bacterial viability (Ratio\({}_{\text{G/G+R}}\)), indicating its potential superiority in inhibiting bacterial proliferation within oral microcosm biofilms, as well as weakened bacterial aggregation compared with that in the DW and CHX groups (Figure 7). RONS produced during CAP treatment are known to disrupt bacterial morphology, thereby weakening bacterial aggregation [39]. A previous study also reported shorter streptococcal chains of \(S\). _mutans_ after CAP treatment compared with the pre-treatment length [11]. During plasma production, oxidants like hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) accumulate at the vacuum-water boundary and within the water itself [40], implying that oxidants can infiltrate liquids. Thus, the RONS generated by CAP may reach the biofilm, potentially undermining the structural integrity of EPSs and leading to cell damage [41]. Moreover, a previous study reported that CAP destabilized amyloid-beta peptide aggregation by inducing oxidation [39], which may affect EPS aggregation in biofilms in a similar manner. Although this previous study did not focus directly on biofilms, the similarities in the aggregation processes of peptides and EPSs suggest that the oxidative properties of CAP could potentially hinder the structural integrity of EPSs in biofilms. The present study evaluated total and aciduric bacterial counts to assess bacterial adhesion and growth, and revealed a significant decrease between the CAP and DW groups (\(p<0.05\), Table 1), consistent with the findings of a previous study that reported reduced adherent bacterial counts following nitrogen-based CAP jet pre-treatment in \(S\). _mutans_ biofilms [42]. However, the CAP and CHX groups did not show significant variances in both total and aciduric bacterial counts (Table 1), aligning with a previous study that reported no significant difference in bactericidal activity between the CAP and 2% CHX on in vitro endodontic biofilms [43]. Despite the lack of statistically significant differences between the CAP and CHX groups in terms of bacterial counts, the confocal laser scanning micrographs revealed a significant difference in bacterial viability (Ratio\({}_{\text{G/G+R}}\)) between these two groups (Figure 7). Previous studies have also suggested that plasma-derived RONS can oxidize bacterial walls and EPSs, thereby disrupting bacterial aggregation [41; 44]. These resultssuggest that CAP may inhibit biofilm formation [11], thus explaining the diminished biofilm pathogenicity where adhesion and growth was limited, and the weakened aggregation compared with the CHX group. Overall, these findings suggest that CAP effectively reduced the pathogenicity of oral microcosm biofilms by decreasing the oral bacterial count and weakening their ability to aggregate. The plasma pipette used in the present study is a small jet device capable of compacting plasma through a nozzle with a diameter of approximately 1 mm, enhancing accessibility within the oral cavity and enabling localized application at clinical sites. Using this device, CAP can be effectively applied to control oral biofilms that accumulate in areas where removal is challenging with physical brushing alone, such as deep periodontal pockets, pits, and fissures. Unlike CHX, which must be used as a mouthwash, localized CAP treatment could prevent normal oral microbiota disruption, thereby reducing opportunistic infections. This is crucial in a clinical setting, where long-term management of oral biofilms is often necessary. Moreover, the ability of CAP to penetrate mature biofilms, as demonstrated in the present study, holds significant practical benefits in oral healthcare, since CHX has limited efficacy in reaching the inner layers of advanced biofilms. Moreover, this benefit is particularly crucial for addressing oral conditions involving biofilms that have developed resistance to conventional antibacterial treatments. This study has some limitations.