Central role of the early colonizer Veillonella sp. in establishing multispecies biofilm communities with initial, middle, and late colonizers of enamel

Abstract

Human dental biofilm communities comprise several species, which can interact cooperatively or competitively. Bacterial interactions influence biofilm formation, metabolic changes, and physiological function of the community. Lactic acid, a common metabolite of oral bacteria, was measured in the flow cell effluent of one-, two- and three-species communities growing on saliva as the sole nutritional source. We investigated single-species and multispecies colonization by using known initial, early, middle, and late colonizers of enamel. Fluorescent-antibody staining and image analysis were used to quantify the biomass in saliva-fed flow cells. Of six species tested, only the initial colonizer Actinomyces oris exhibited significant growth. The initial colonizer Streptococcus oralis produced lactic acid but showed no significant growth. The early colonizer Veillonella sp. utilized lactic acid in two- and three-species biofilm communities. The biovolumes of all two-species biofilms increased when Veillonella sp. was present as one of the partners, indicating that this early colonizer promotes mutualistic community development. All three-species combinations exhibited enhanced growth except one, i.e., A. oris, Veillonella sp., and the middle colonizer Porphyromonas gingivalis, indicating specificity among three-species communities. Further specificity was seen when Fusobacterium nucleatum (a middle colonizer), Aggregatibacter actinomycetemcomitans (a late colonizer), and P. gingivalis did not grow with S. oralis in two-species biofilms, but inclusion of Veillonella sp. resulted in growth of all three-species combinations. We propose that commensal veillonellae use lactic acid for growth in saliva and that they communicate metabolically with initial, early, middle, and late colonizers to establish multispecies communities on enamel.

FIG. 1.

Quantification of lactic acid in reservoir saliva (A) and effluent saliva (B to D) collected from flow cells after 4 h and 18 h of biofilm growth. (A) Sterile reservoir saliva; (B) single-species biofilms of S. oralis (So), A. oris (Ao), and Veillonella sp. (Va); (C) Two-species biofilms of S. oralis plus Veillonella sp. and A. oris plus Veillonella sp.; (D) three-species biofilms of S. oralis plus Veillonella sp. plus F. nucleatum (Fn), S. oralis plus Veillonella sp. plus P. gingivalis (Pg), S. oralis plus Veillonella sp. plus A. actinomycetemcomitans (Aa), and A. oris plus Veillonella sp. plus P. gingivalis. Error bars indicate standard deviations.

FIG. 2.

Confocal micrographs of biofilms formed in flow cells inoculated with one species after 4 h (left column) and 18 h (right column) of growth on 25% saliva. Top panels, S. oralis; middle panels, A. oris; bottom panels, Veillonella sp. Colonization (4 h) and growth (18 h) of each species were monitored. Bacterial cells are stained with species-specific fluorophore-conjugated immunoglobulin G.

FIG. 3.

Time-resolved changes in biovolumes (μm³ per field of view) of S. oralis (So), A. oris (Ao), and Veillonella sp. (Va) following 4 h and 18 h of incubation in 25% saliva-fed flow cells. The biovolume values are for the single-species-inoculated flow cells (see Fig. 2). An asterisk indicates statistically significant increases (P < 0.05) in bacterial growth. Error bars indicate standard deviations.

FIG. 4.

Representative confocal micrographs of 4-h (left column) and 18-h (right column) biofilms showing growth in two-species-inoculated flow cells. Top panels, Veillonella sp. plus A. oris; bottom panels, Veillonella sp. plus S. oralis. Bacterial cells were stained with species-specific fluorophore-conjugated immunoglobulin G (blue, Veillonella sp.; green, A. oris and S. oralis) and show cell-cell contact.

FIG. 5.

Time-resolved changes in biovolumes (μm³ per field of view) of Veillonella sp. (Va), A. oris (Ao), S. oralis (So), F. nucleatum (Fn), and A. actinomycetemcomitans (Aa) following 4 h and 18 h of incubation in 25% saliva-fed flow cells. (A and B) Two-species-inoculated flow cells (see Fig. 4); (C and D) three-species-inoculated flow cells (see Fig. 6). An asterisk indicates statistically significant increases (P < 0.05) in bacterial growth. Error bars indicate standard deviations.

FIG. 6.

Representative confocal micrographs of biofilms from three-species-inoculated flow cells. Mixed-species communities at 4 h (left column) and at 18 h (right column) show intimate interaction of Veillonella sp. (blue) with S. oralis (green) and F. nucleatum (red) (top panels) and of S. oralis (blue) with A. actinomycetemcomitans (green) and Veillonella sp. (red) (bottom panels).

FIG. 7.

Representative confocal micrographs of biofilms from three-species-inoculated flow cells. Mixed-species communities at 4 h (left column) and at 18 h (right column) show intimate interaction of Veillonella sp. (blue) with S. oralis (green) and P. gingivalis (red) (top panels) and of Veillonella sp. (blue) with A. oris (green) and P. gingivalis (red) (bottom panels).

FIG. 8.

Time-resolved changes in biovolumes (μm³ per field of view) of Veillonella sp. (Va), A. oris (Ao), S. oralis (So), and P. gingivalis (Pg) following 4 h and 18 h of incubation in 25% saliva-fed flow cells (see Fig. 7). An asterisk indicates statistically significant increases (P < 0.05) in bacterial growth. Error bars indicate standard deviations.