Strain-specific colonization patterns and serum modulation of multi-species oral biofilm development

Abstract

Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.

Figure 1

Mono-species and multi-species biofilm formation by three strains of F. nucleatum. Biofilms were allowed to develop for 4 and 16h in flow cells using saliva-supplemented medium as nutritional source. Panel A depicts representative CLSM 3-D image projections and biovolume measurements (in μ³) for mono-species biofilms formed by three strains of F. nucleatum (green). Panel B depicts multi-species biofilm formation by F. nucleatum strains (green) with initial colonizers A. oris (red) and V. parvula (blue). White arrows in 16h CLSM 3-D image projections and 2-D x-z planes slices (bottom) indicate central columnar micro-colonies of A. oris. Yellow arrows indicate three-species inter-digitated structures. F. nucleatum = Fn, A. oris = Ao and V. parvula = Vp. * indicates a P value of less than 0.05 when biovolumes for each species were compared between 4h and 16h. A red star indicates a P value of less than 0.05 when biovolumes of F. nucleatum were compared in mono-species biofilms vs. multi-species biofilms. Bar in all images = 50 μm.

Figure 2

Planktonic growth of F. nucleatum ATCC 10953 in salivary growth medium as a mono-culture or in co-culture with A. oris and V. parvula. Panel A shows planktonic growth curves. Data points represent measurements from three independent replicate experiments. Panel B shows a representative phase contrast micrograph of a mixed-species culture after 10 hours of growth in saliva-supplemented medium. Short rods are A. oris (Ao); coccoid cells are V. parvula (Vp) and thin long rods are F. nucleatum (Fn). Scale bar = 10 μm. * indicates a P value of less than 0.05 when colony forming units (c.f.u) mL⁻¹ of Fn were compared between mono-culture and co-culture conditions at each time point.

Figure 3

Mono-species biofilm formation by four strains of P. gingivalis. Biofilms were allowed to develop for 4 and 16h in flow cells using saliva-supplemented medium as nutritional source. P. gingivalis (Pg) appears in red in CLSM 3-D image projections. * indicates a P value of less than 0.05 when biovolumes were compared between 4h and 16h. Left scale bar in all images = 50 μm. Scale bar in inset digital zoom images = 3 μm.

Figure 4

Ability of virulent P. gingivalis strains W50 and W83 to integrate into 4h-old biofilm communities of health-associated species. In panel A, biofilms of A. oris and V. parvula or A. oris, V. parvula and F. nucleatum were allowed to develop for 4h in flow cells using saliva-supplemented medium as nutritional source. After 4h, P. gingivalis was inoculated, and biofilms were allowed to develop for an additional 4h and 16h. In CLSM 3-D image projections A. oris (Ao) and V. parvula (Vp) appear in blue, F. nucleatum (Fn) appears in green and P. gingivalis (Pg) in red. A. oris and V. parvula were quantified as a combined biovolume (Ao + Vp). * indicates a P value of less than 0.05 when biovolumes were compared between 4h and 16h. Bar in all images = 50 μm.

Figure 5

Ability of avirulent P. gingivalis strains ATCC 33277 and 381 to integrate into 4h-old biofilm communities of health-associated species. Biofilms of A. oris and V. parvula or A. oris, V. parvula and F. nucleatum were allowed to develop for 4h in flow cells using saliva-supplemented medium as nutritional source. After 4h, P. gingivalis was inoculated, and biofilms were allowed to develop for an additional 4h and 16h. In CLSM 3-D image projections A. oris (Ao) and V. parvula (Vp) appear in blue, F. nucleatum (Fn) appears in green and P. gingivalis (Pg) in red. A. oris and V. parvula were quantified as a combined biovolume (Ao + Vp). * indicates a P value of less than 0.05 when biovolumes were compared between 4h and 16h. Bar in all images = 50 μm.

Figure 6

Effect of serum on mono-species and multi-species biofilm communities. Left image and biovolume data represent the control condition in which no serum was added to the salivary growth medium. In the three central images and graphs serum was present in the salivary medium during inoculum resuspension, substratum conditioning, attachment and in the medium flow. In right image and biovolume data graph, serum was only added to the salivary medium during flow. * indicates a P value of less than 0.05 when biovolumes were compared between 4h and 16h. Bar in all images = 65 μm. A. oris (Ao) and V. parvula (Vp) appear in blue, F. nucleatum ATCC 10953 (Fn) appears in green and P. gingivalis 381 (Pg) in red.

Figure 7

Effect of serum on viability of A. oris (Ao), V. parvula (Vp), F. nucleatum ATCC 10953 (Fn) and P. gingivalis 381 (Pg). Planktonic growth of microorganisms was evaluated in their appropriate nutrient-rich medium supplemented with 10% native human serum or with PBS as control. Data represent measurements from three independent replicate experiments.