Resilience of multispecies biofilm communities: Comparative analysis of a Veillonella dispar-dominant oral biofilm under diverse oxygen conditions in vitro

Oral multispecies biofilms developing on dental implants drive peri-implant mucositis and peri-implantitis. They are shaped by the physicochemical conditions of their surrounding environment, among which oxygen availability is an important influential factor. However, while oxygen tension fluctuates significantly within the peri-implant pocket, whether these environmental shifts are sufficient to influence the structure and composition of an established microbial community remains unclear. For this purpose, we evaluated the structure and composition of a four-species oral biofilm model comprising the species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, which was cultivated under normoxic (21% O2), hypoxic (1% O2) and anoxic (0% O2;) conditions for a period of 21 days. Biofilm 3D architecture, volume and membrane integrity was analyzed by LIVE/DEAD staining combined with confocal laser scanning microscopy. Community composition was assessed by quantitative real-time PCR and complemented by Fluorescence In Situ Hybridization (FISH) to provide structural context. While normoxia transiently promoted higher early biofilm volume and membrane integrity, the community exhibited remarkable long-term resilience, consistently maintaining a V. dispar-dominated structure across all oxygen gradients. Crucially, the obligate anaerobic pathogen P. gingivalis persisted as a viable minority (~1%) even under 21-day normoxic cultivation, sheltered within the deeper layers of the biofilm. These results suggest that clinical oxygen levels alone are not able to drastically modulate established oral biofilm communities and that oxygen-sensitive pathogens persist in a subclinical sanctuary protected by the surrounding non-pathogenic species.