Development of a multispecies oral bacterial community in a saliva-conditioned flow cell

Abstract

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.

FIG. 1.

Confocal micrographs of planktonic cultures containing coaggregates of S. gordonii, A. naeslundii, V. atypica, and F. nucleatum processed for FISH with fluorescein isothiocyanate-labeled probes (green) and counterstained with general nucleic acid stain Syto 59 (red). Colocalization of both fluorescent markers appears yellow to yellow-green. (A) Streptococcus-specific probe targeting S. gordonii. (B) Species-specific probe labeling short rod-shaped A. naeslundii cells. (C) Clustered V. atypica cells hybridized with Veillonella-specific probe. (D) Long slender rod-shaped F. nucleatum cells labeled with F. nucleatum-specific probe. Bar, 5 μm.

FIG. 2.

Growth of mono-species biofilms. (A to D) Confocal micrographs of mono-species biofilms at 1 h stained with nucleic acid stain Syto 59. (A) S. gordonii (bar, 40 μm); (B) A. naeslundii; (C) V. atypica; (D) F. nucleatum. (E to H) Confocal micrographs of 14-h mono-species biofilms stained with Syto 59. (E) S. gordonii; (F) A. naeslundii; (G) V. atypica; (H) F. nucleatum. (I) Graph depicting biovolumes of mono-species biofilms. Abbreviations: Sg, S. gordonii; An, A. naeslundii; Va, V. atypica; Fn, F. nucleatum.

FIG. 3.

Comparison of sequentially and coaggregate-inoculated biofilms. (A to D) Confocal micrographs of four-species biofilms containing S. gordonii, A. naeslundii, V. atypica, and F. nucleatum. Biofilms were stained with general nucleic acid stain Syto 59. (A) One-hour biofilms inoculated in a sequential order demonstrating the widespread coverage of flow cell surface. Bar, 40 μm. (B) Fourteen-hour sequentially inoculated biofilm. (C) One-hour coaggregate-inoculated biofilm. (D) Fourteen-hour biofilm inoculated with a coaggregate of the four species. (E) Biovolumes of sequentially and coaggregate-inoculated biofilms.

FIG. 4.

Abundance of S. gordonii in four-species biofilm using FISH with Cy3-labled probes. (A to D) Confocal micrographs of mixed-species communities hybridized with Streptococcus-specific probe (red) and counterstained with nucleic acid stain Syto 9 (green). Colocalization of both fluorescent probes appears yellow. (A) One-hour biofilms inoculated in a sequential order. Bar, 40 μm. (B) Sequentially inoculated biofilm at 14 h. (C) One-hour biofilm inoculated with a coaggregate of mixed species. (D) Coaggregate-inoculated biofilm at 14 h. (E). Graph indicating the total biovolumes of the four-species biofilms (hatched bars) and biovolumes of S. gordonii (Sg) (stippled bars) in sequentially and coaggregate-inoculated biofilms at 1 and 14 h.

FIG. 5.

The presence of A. naeslundii in four-species biofilm using FISH with Cy3-labled probes. (A to D) Confocal micrographs of mixed-species biofilms hybridized with an A. naeslundii-specific probe (red) and counterstained with general nucleic acid stain Syto 9 (green). Colocalization of both probes appears yellow. (A) One-hour sequentially inoculated biofilm. Bar, 40 μm. (B) Fourteen-hour sequentially inoculated biofilm. (C) One-hour coaggregate-inoculated biofilm. (D) Fourteen-hour coaggregate-inoculated biofilm. (E) Graph indicating the total biovolumes of the four-species biofilms (hatched bars) and biovolumes of A. naeslundii (An) (stippled bars) in sequentially and coaggregate-inoculated biofilms.

FIG. 6.

Prevalence of V. atypica in four-species biofilm using FISH with Cy3-labled probes. (A to D) Confocal micrographs of four-species biofilms labeled with a Veillonella-specific probe (red) and counterstained with general nucleic acid stain Syto 9 (green). Areas of yellow indicate colocalization of both fluorescent markers. (A) One-hour sequentially inoculated biofilm. Bar, 40 μm. (B) Fourteen-hour sequentially inoculated biofilm. (C) One-hour coaggregate inoculated biofilm. (D) Fourteen-hour coaggregate-inoculated biofilm. (E) Graph indicating the total biovolumes of the four-species biofilm (hatched bars) and biovolumes of V. atypica (Va) (stippled bars) in sequentially and coaggregate-inoculated biofilms.

FIG. 7.

Examination of F. nucleatum in a four-species biofilm using FISH with Cy3-labled probes. (A to D) Confocal micrographs of four-species biofilms labeled with a F. nucleatum-specific probe (red) and counterstained with general nucleic acid stain Syto 9 (green). Areas of yellow indicate colocalization of both fluorescent markers. (A) One-hour sequentially inoculated biofilm. Bar, 40 μm. (B) Fourteen-hour sequentially inoculated biofilm. (C) One-hour coaggregate-inoculated biofilm. (D) Fourteen-hour coaggregate-inoculated biofilm. (E) Graph indicating the total biovolumes of the four-species biofilm (hatched bars) and biovolumes of F. nucleatum (Fn) (stippled bars) in sequentially and coaggregate-inoculated biofilms.