The interactive relationship between Fusobacterium necrophorum, Bacteroides pyogenes, and Porphyromonas levii in driving inflammatory uterine disease

BACKGROUND: Bovine metritis is associated with uterine microbiota dysbiosis, characterized by the proliferation of Gram-negative anaerobes, including Fusobacterium necrophorum, Bacteroides pyogenes, and Porphyromonas levii. However, the mechanisms by which these opportunistic pathogens proliferate together and contribute to disease remain unclear. This study aimed to identify the interactions among these bacteria and to elucidate their role in the development of metritis. METHODS: F. necrophorum, B. pyogenes, and P. levii were isolated from the uteri of cows with metritis and cultured anaerobically in chopped meat carbohydrate broth. Bacterial growth, coaggregation, biofilm formation, endotoxin production, protease activity, adhesion to bovine endometrial epithelial (BEND) cells, and cytotoxicity were evaluated under single-, dual-, and triple-species culture conditions. RESULTS: Among the three species, F. necrophorum grew faster and acted as a key species that coaggregated with B. pyogenes and P. levii, promoting multi-species biofilm formation and increasing bacterial adhesion to BEND cells. The growth of B. pyogenes was enhanced by metabolites from P. levii, whereas the growth of P. levii was delayed by metabolites from F. necrophorum. The multi-species biofilm formed by these three bacterial species generated the highest biomass, with P. levii predominantly occupying the basal layer and F. necrophorum and B. pyogenes co-localizing in the upper layer. This spatial arrangement likely reflects the strong biofilm-forming ability of P. levii, which rapidly produces matrix and provides a scaffold for cohabiting species. Endotoxin levels were highest in both F. necrophorum and P. levii, and protease activity was highest in P. levii under mono-culture conditions. However, no synergistic effects were observed for either parameter under co-culture conditions. Interestingly, co-culture with B. pyogenes lowered the endotoxin levels of the other bacteria and attenuated the cytotoxicity of P. levii. CONCLUSIONS: Our findings suggest that these opportunistic uterine pathogens interact synergistically to promote bacterial persistence in the uterus rather than exacerbating disease severity, which likely contributes to uterine microbiota dysbiosis and chronic inflammation. Video Abstract.