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%) (<i>p</i> &lt; 0.00001). Furthermore, these groups showed a significant reduction in the total bacterial load derived from these complexes (<i>p</i> &lt; 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. This in vitro multispecies biofilm model aimed to evaluate the bacterial shifts on implant threads following the application of different ozone therapy modalities, either alone or in combination. Microbiological analysis revealed that ozonated water, ozone gas, and their combined application effectively reduced the presence of periodontopathogenic bacterial species on implant surfaces. Ozone demonstrated notable antimicrobial activity and may serve as a valuable adjunct to mechanical debridement in reducing biofilm activity associated with peri-implantitis. However, further studies are necessary to assess whether these effects support cellular attachment to the implant surface and result in clinically meaningful benefits in vivo.