Bacterial Community Shift on Implant Threads Treated with Different Modalities of Ozone Therapy on an in vitro Multispecies Biofilm Related to Peri-Implantitis

This study aimed to evaluate the modulation of the bacterial community on implant threads through various ozone (O₃) therapy modalities using an in vitro multispecies biofilm model associated with peri-implantitis. A total of 72 conical titanium implants were randomly allocated into six groups: G1) O₃ oil (ozonated sunflower oil, peroxide value: 510–625 meq/kg); G2) O₃ water (60 µg/mL); G3) O₃ gas (50 µg/mL); G4) combined O₃ therapy (O₃ water, oil, and gas); G5) phosphate-buffered saline (PBS); and G6) chlorhexidine digluconate (CHX, 0.12%). Following treatment, biofilm metabolic activity and bacterial composition were analyzed using DNA–DNA checkerboard hybridization. Significant alterations in the bacterial community structure were observed in the groups treated with combined O₃ therapy, O₃ water, and O₃ gas, which exhibited markedly lower proportions of pathogenic species from Socransky’s orange and red complexes (combined O₃: 2.32%; O₃ water: 3.57%; O₃ gas: 5.87%) compared to O₃ oil (26.91%), CHX (29.01%), and PBS (27.09%) (p < 0.00001). Furthermore, these groups showed a significant reduction in the total bacterial load derived from these complexes (p < 0.00001). In conclusion, both the combined and individual applications of O₃ water and gas demonstrated a substantial ability to alter the microbial profile on implant surfaces, significantly reducing the presence of periodontopathogenic bacteria.