Abstract
Oral bacterial pathogens, including Bacillus species, form biofilms that enhance antibiotic resistance, promote bacterial adherence, and maintain structural integrity. The ability of bacteria to form biofilms is directly linked to several oral diseases, including gingivitis, dental caries, periodontitis, periapical periodontitis, and peri-implantitis. These biofilms act as a predisposing factor for such infections. Nanoparticles, known for their strong antibacterial properties, can target specific biofilm-forming microorganisms without disturbing the normal microflora of the oral cavity. This study focuses on the biofilm-forming ability and clindamycin (CM) resistance of Bacillus species found in the oral cavity. It aims to evaluate the antibacterial and antibiofilm properties of zinc oxide nanoparticles (ZnO-NPs) against oral Bacillus species and assess the effectiveness of combining CM with ZnO-NPs in reducing antibiotic resistance. The antibacterial susceptibility of Bacillus isolates was tested using ZnO-NPs and CM, demonstrating synergistic effects that reduced the minimum inhibitory concentrations by up to 8-fold. The fractional inhibitory concentration (FIC) index indicated a significant synergistic effect in most strains, with FIC values ranging from 0.375 to 0.5. It was found that the majority of Bacillus strains exhibited significant biofilm-forming capabilities, which were reduced when treated with the ZnO-NPs and CM combination. The study also evaluated the cytotoxicity of ZnO-NPs on cancer cells (CAL27) and normal fibroblasts (HFB4). CAL27 cells showed stronger cytotoxicity, with an IC(50) of 52.15 µg/mL, compared to HFB4 cells, which had an IC(50) of 36.3 µg/mL. Genetic analysis revealed the presence of biofilm-associated genes such as sipW and tasA, along with antibiotic resistance genes (ermC), which correlated with the observed biofilm phenotypes. Overall, this study demonstrates the potential of combining ZnO-NPs with CM to overcome antibiotic resistance and biofilm formation in the oral bacterial pathogens, Bacillus species. These findings suggest new approaches for developing more effective dental treatments targeting oral biofilm-associated infections and antibiotic resistance.