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. 2020 Oct 15;86(21):e01262-20.
doi: 10.1128/AEM.01262-20. Print 2020 Oct 15.

Competition and Caries on Enamel of a Dual-Species Biofilm Model with Streptococcus mutans and Streptococcus sanguinis

Natalia Díaz-Garrido  1 , Carla P Lozano  2 , Jens Kreth  3 , Rodrigo A Giacaman  4
Affiliations

Competition and Caries on Enamel of a Dual-Species Biofilm Model with Streptococcus mutans and Streptococcus sanguinis

Natalia Díaz-Garrido et al. Appl Environ Microbiol. .

Abstract

Imbalances within the dental biofilm trigger dental caries, currently considered a dysbiosis and the most prevalent noncommunicable disease. There is still a gap in knowledge about the dynamics of enamel colonization by bacteria from the dental biofilm in caries. The aim, therefore, was to test whether the sequence of enamel colonization by a typically commensal and a cariogenic species modifies biofilm's cariogenicity. Dual-species biofilms of Streptococcus mutans and Streptococcus sanguinis on saliva-coated enamel slabs were inoculated in different sequences: S. mutans followed by S. sanguinis (Sm-Ss), S. sanguinis followed by S. mutans (Ss-Sm), S. mutans and S. sanguinis inoculated at the same time (Sm=Ss), and the single-species controls S. mutans followed by S. mutans (Sm-Sm) and S. sanguinis followed by S. sanguinis (Ss-Ss). Biofilms were exposed to 10% sucrose 3 times per day for 5 days, and the slabs/biofilms were retrieved to assess demineralization, viable cells, biomass, proteins, polysaccharides, and H2O2 production. Compared with Sm-Sm, primary inoculation with S. sanguinis reduced demineralization (P < 0.05). Both Ss-Sm and Sm=Ss sequences showed reduction in biomass, protein, and polysaccharide content (P < 0.05). The highest S. sanguinis viable count and H2O2 production level and the lowest acidogenicity were observed when S. sanguinis colonized enamel before S. mutans (P < 0.05). Initial enamel adherence with commensal biofilms seems to induce more intense competition against more typically cariogenic species, reducing cariogenicity.IMPORTANCE The concept of caries as an ecological disease implies the understanding of the intricate relationships among the populating microorganisms. Under frequent sugar exposure, some bacteria from the dental biofilm develop pathogenic traits that lead to imbalances (dysbiosis). Depending on which microorganism colonizes the dental surface first, different competition strategies may be developed. Studying the interactions in the entire dental biofilm is not an easy task. In this study, therefore, we modeled the interplay among these microorganisms using a caries-inducing species (S. mutans) and a health-associated species (S. sanguinis). Initial enamel adherence with S. sanguinis seems to induce more intense competition against typically caries-inducing species. Besides continuous exposure with sugars, early colonization of the enamel by highly cariogenic species like S. mutans appears to be needed to develop caries lesions as well. Promoting early colonization by health-associated bacteria such as S. sanguinis could help to maintain oral health, delaying dysbiosis.

Keywords: Streptococcus mutans; Streptococcus sanguinis; cariogenicity; competition; dental caries; oral biofilm; primary colonization.

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Figures

FIG 1
FIG 1
(A) Biofilm acidogenicity. Biofilms were exposed to 10% sucrose for 5 min, 3 times per day under different colonization sequences (as indicated) on enamel slabs: Ss=Sm, Ss-Sm, Sm-Ss, Ss-Ss, and Sm-Sm. The medium pH was measured twice per day during the 5 days of the experiment. Each point in the plot depicts the means for two independent experiments, each in triplicate (n = 6). Different letters represent significant differences across all colonization sequences (P < 0.05). These data are adapted from reference with permission of the publisher (Karger Publishers, Basel, Switzerland [copyright 2018]). (B) Enamel demineralization. Enamel slabs from each biofilm exposed to cariogenic challenges with 10% sucrose were retrieved from the orthodontic wire and cleaned of the adhered biomass. Initial and final surface microhardness (SH) were measured before and after the experiment, respectively, to assess percentage of SH loss (%SHL). Bars denote mean values of two independent experiments in triplicate (n = 6). Error bars show the standard deviation. Different letters (a to d) represent significant differences across all colonization sequences (P < 0.05).
FIG 2
FIG 2
(A) Viable microorganisms. Mean counts of S. mutans (black bar) and S. sanguinis (gray bar) expressed as CFU per milliliter were determined in each colonization sequence. Bars represent mean values of two independent experiments in triplicate (n = 6). Error bars show the standard deviation. Different letters represent significant differences across all colonization sequences (P < 0.05). These data are adapted from reference with permission of the publisher (Karger Publishers, Basel, Switzerland [copyright 2018]). (B) H2O2 concentration. Production of H2O2 (μM) in each biofilm condition as described in Materials and Methods. Bars show mean values of two independent experiments in triplicate (n = 6). Error bars show the standard deviation. Different letters (a to d) represent significant differences across all colonization sequences (P < 0.05).
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