## 3 Results
aeruginosa_ biofilms (cultured on HA) produced in the observed field of visions a higher amount of extracellular matrix compared to their counterparts grown on the agar surface. In the case of the last type mentioned, the biofilm took the form of a multi-cellular structure in which cells were interconnected by adhesin-like structures rather than embedded within the extracellular matrix. The opposite trend was observed in the case of _Streptococcus pyogenes_ (higher amount of matrix on soft agar compared to the hard surface of HA). The _C. albicans_ and _A. actinomycetemontans_ biofilms took similar forms regardless of the applied growth surface; however, the share of extracellular matrix in the biofilm formed by the bacterium was distinctively higher than what was observed in the fungal biofilm (applied in an in vitro setting). When the capability of the tested microorganisms to form a biofilm on soft agar and hard HA surfaces (imitating to a certain extent the surfaces of soft tissue and bones, respectively) was proven, the main part of the analysis was performed to assess the antibioticfilm activity of cotenidine dihydrochloride-containing lozenges (Figures 3 and 4) and an cotenidine dihydrochloride-containing liquid antiseptic (comparator) (Figures 5 and 6). Figure 3: The reduction [%] in _S. aureus_, _S. pyogenes_, _P. aeruginosa_, _C. albicans_, _A. actinomycetemontans_ biofilms pre-formed on Hydroxyapatite disks as a result of OCT activity. a, b—statistically significant (\(p>0.05\)) differences between values of reduction. Figure 4: The reduction [%] in _S. aureus_, _P. aeruginosa_, _C. albicans_, _A. actinomycetemcomitans_ biofilms pre-formed on agar disks as a result of OCT activity. a, b, statistically significant (_p_ > 0.05) differences between values of reduction. Figure 5: The reduction [%] in _S. aureus_, _P. aeruginosa_, _C. albicans_, _A. actinomycetemcomitans_ biofilms pre-formed on hydroxyapatite disks as a result of OL (comparator) activity. a, b—statistically significant (_p_ > 0.05) differences between values of reduction. In this setting, Artificial Saliva (AS) was applied in the fluid immersing the formed biofilm to bring this in vitro model closer to the real conditions of the oral cavity. The patterns of biofilm reduction [%] did not differ with regard to the type of surface (HA vs. agar) or with regard to the antiseptic formulation (OCT vs. OL) applied, while the biofilm-forming species was a differentiating factor regarding the level of biofilm reduction. The aforementioned values, presented in descending order, with regard to the species forming biofilms are as follows: _A. actinomycetemcomitants_\(>\)_S. aureus_\(>\)_P. aeruginosa_\(>\)_C. albicans_. The measured reduction [%] in the C. _albicans_ biofilm was always significantly lower (\(p>\) 0.05) than the measured reduction [%] in other biofilm-forming species used in this research, while the measured reduction [%] in _A. actinomycetemcomitants_ biofilm was always significantly higher (\(p>\) 0.05) than the measured reduction in other biofilm-forming species. The comparison of average values of biofilm reduction [%] measured between specific biofilm-forming strains with regard to the antiseptic formulations (comparison of data presented in Figures 3 and 4 vs. Figures 5 and 6) shows no impactful (\(p<\) 0.05) differences (the antib Biofilm effects achieved were comparable). The observed differences between the biofilms' reduction [%] did not exceed, on average, a value of 7%. Finally, by means of Confocal Laser Microscopy, the visualization of biofilms treated with OCT was performed and compared to the biofilms not treated with an antimicrobial (Figure 7). As presented in Figure 7, the allotment of compromised (dead) cells in the structures of biofilms treated with OCT increased markedly compared to the non-treated biofilms. Moreover, as shown in representative images in Figure 7, the highest intensity of red fluorescence (\"dead cells\") was observed in _A. actinomycetemcomitans_, while the lowest one was in the _C. albicans_ biofilms.