Actinomyces naeslundii GroEL-dependent initial attachment and biofilm formation in a flow cell system

• Both butyric acid and low pH were required to form significant biofilms of A. naeslundii in the flow cell system.
• Butyric acid induced GroEL-dependent initial attachment and colonization of A. naeslundii resulting in significant biofilm.
• This is the first report that shows the role of stress proteins in initial attachment, and biofilm of A. naeslundii.
• A useful new model was established to investigate various issues in biofilm of A. naeslundii in the flow cell system.

Actinomyces naeslundii is an early colonizer with important roles in the development of the oral biofilm. The effects of butyric acid, one of short chain fatty acids in A. naeslundii biofilm formation was observed using a flow cell system with Tryptic soy broth without dextrose and with 0.25% sucrose (TSB sucrose). Significant biofilms were established involving live and dead cells in TSB sucrose with 60 mM butyric acid but not in concentrations of 6, 30, 40, and 50 mM. Biofilm formation failed in 60 mM sodium butyrate but biofilm level in 60 mM sodium butyrate (pH 4.7) adjusted with hydrochloric acid as 60 mM butyric media (pH4.7) was similar to biofilm levels in 60 mM butyric acid. Therefore, butyric acid and low pH are required for significant biofilm formation in the flow cell. To determine the mechanism of biofilm formation, we investigated initial A. naeslundii colonization in various conditions and effects of anti-GroEL antibody. The initial colonization was observed in the 60 mM butyric acid condition and anti-GroEL antibody inhibited the initial colonization. In conclusion, we established a new biofilm formation model in which butyric acid induces GroEL-dependent initial colonization of A. naeslundii resulting in significant biofilm formation in a flow system.