A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production

Abstract

Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans within a seven species panel. This phenotype was common amongst all assayed isolates of S. mutans, but was specific to a single strain of S. mitis, ATCC 49456. The growth inhibitory factor was not effectively carried in spent supernatants of S. mitis. However, we documented ATCC 49456 to accumulate 4-5 times more hydrogen peroxide (H₂O₂) than other species tested, and 5-18 times more than other S. mitis strains assayed. The S. mutans biofilm formation inhibitory phenotype was reduced when grown in media containing catalase or with a S. mitis mutant of pyruvate oxidase (spxB; pox), confirming that SpxB-dependent H₂O₂ production was the main antagonistic factor. Addition of S. mitis within hours after S. mutans inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms. Transcriptome analysis revealed responses for both S. mitis and S. mutans, with several S. mutans differentially expressed genes following a gene expression pattern previously described, while others being unique to the interaction with S. mitis. Finally, we show that S. mitis also affected coculture biofilm formation of several other commensal streptococci. Our study shows that strains with abundant H₂O₂ production are effective at inhibiting initial growth of caries pathogens like S. mutans, but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.

Figure 1.. S. mitis Inhibits S. mutans Biofilm Formation.

(A) Representative image of a crystal violet (CV) biofilm biomass assay where S. mutans is cocultured with different oral streptococci species (listed down right y-axis). S. mutans monoculture (mono) is shown at the top for reference. (B) CV quantification (Abs 575 nm) of the experiment shown in A. Data is expressed as the percentage of biomass formed in comparison to S. mutans monoculture (i.e., monoculture values set to 100%). B’ is same data on a smaller y-axis with S. cristatus and S. sobrinus coculture data removed. n = 6. (C) Merged representative maximum intensity 40X Z-projection of 24 h S. mutans monoculture biofilm. S. mutans constitutively expresses GFP (green), eDNA was probed with labeled antibodies (yellow), and glucans visualized with labeled dextran (red). Scale bar (100 μm) is shown in the bottom right corner. (D) Merged representative maximum intensity 40X Z-projection of 24 h S. mutans cocultured biofilm with S. mitis. (E) Quantification of individual S. mutans microcolony volumes, (F) number of S. mutans microcolonies per field of view, (G) glucan biomass, and (H) eDNA biomass from the microscopy data shown in C and D. n = 4. Quantification was completed using Gen5 Image+ software. (I) S. mutans colony forming units (CFUs) returned from 24 h biofilms, with enumeration of cells in either biofilm or planktonic growth phase, grown with or without S. mitis. n = 4. (J) S. mitis CFUs returned. Data graphing and two-way analysis of variance (ANOVA) with multiple comparisons or student’s T-Test was completed in GraphPad Prism software.

Figure 2.. Strain ATCC 49456 Specifically Inhibits S. mutans Biofilm Formation.

(A) CV quantification (Abs 575 nm) of biomass formed by various S. mutans isolates grown in monoculture (− S. mitis) or in coculture with S. mitis (+ S. mitis). (B) Representative image of a CV biofilm biomass assay where S. mutans is cocultured with different strains of S. mitis (listed down right y-axis). Coculture with S. oralis is included as a reference. (C) CV quantification (Abs 575 nm) of the experiment shown in B. Data is expressed as the percentage of biomass formed in comparison to S. mutans monoculture. (D) Merged representative maximum intensity 40X Z-projection of 24 h S. mutans cocultured biofilms with different strains of S. mitis. S. mutans constitutively expresses GFP (green), eDNA was probed with labeled antibodies (yellow), glucans visualized with labeled dextran (red), and a total cell strain applied to visualize S. mitis within the biofilms (Hoechst 33342; blue). Scale bar (100 μm) is shown in the bottom right corner. (E) Quantification of individual S. mutans microcolony volumes, (F) biofilm thickness, and (G) glucan biomass from the microscopy data shown in D. n = 4. Quantification was completed using Gen5 Image+ software. Data graphing and one-way analysis of variance (ANOVA) with multiple comparisons was completed in GraphPad Prism software.

Figure 3.. S. mitis ATCC 49456 Produces High Levels of Hydrogen Peroxide.

(A) Representative image of a crystal violet (CV) biofilm biomass assay where S. mutans is grown in fresh medium, cocultured with S. mitis (cells), or grown in extracted 24 h supernatants of various cultures (listed down right y-axis). (B) CV quantification (Abs 575 nm) of the experiment shown in A. Data is expressed as the percentage of biomass formed in comparison to S. mutans grown in fresh medium (i.e., monoculture values set to 100%). n = 6. (C) Quantification of hydrogen peroxide present in culture supernatants of different oral species. Values were normalized to culture density (OD_{600 nm}) prior to centrifugation and extraction of culture supernatants. S. mitis (ATCC 49456) is shown on the left. C’ is same data on a smaller y-axis with S. mitis data removed. n = 4. (D) Quantification of hydrogen peroxide present in culture supernatants of different S. mitis strains. Data graphing and one-way analysis of variance (ANOVA) with multiple comparisons was completed in GraphPad Prism software.

Figure 4.. S. mitis ATCC 49456 Hydrogen Peroxide Production Inhibits S. mutans Biofilm Formation.

(A) Quantification of hydrogen peroxide present in culture supernatants of S. mitis ATCC 49456 wild-type (WT), with addition of 100 U mL⁻¹ catalase to the growth medium (+ Catalase), and with an spxB mutant (ΔspxB). n = 3. (B) Representative image of a CV biofilm biomass assay where S. mutans is cultured in different conditions (listed down right y-axis). (C) CV quantification (Abs 575 nm) of the experiment shown in B. Data is expressed as the percentage of biomass formed in comparison to S. mutans monoculture alone (i.e., monoculture values set to 100%). n = 6. (D) Merged representative maximum intensity 40X Z-projection of 24 h S. mutans biofilms grown in monoculture (− S. mitis), in coculture with S. mitis (+ S. mitis), with S. mitis and addition of 100 U mL⁻¹ catalase (+ S. mitis + Catalase), and with the S. mitis spxB mutant (+ S. mitis ΔspxB). S. mutans constitutively expresses GFP (green), eDNA was probed with labeled antibodies (yellow), and glucans visualized with labeled dextran (red). Scale bar (100 μm) is shown in the bottom right corner. (E) Quantification of individual S. mutans microcolony volumes, (F) biofilm thickness, and (G) glucan biomass from the microscopy data shown in D. n = 4. Quantification was completed using Gen5 Image+ software. Data graphing and one-way analysis of variance (ANOVA) with multiple comparisons was completed in GraphPad Prism software.

Figure 5.. S. mitis Disruption of Forming and Pre-formed Biofilms of Caries Pathogens.

(A) Representative image of a CV biofilm biomass assay where either fresh medium (FM) or medium containing S. mitis ATCC 49456 replaced the original growth medium of either S. mutans or S. sobrinus biofilms at the time point indicated on the left y-axis. 0 indicates addition during the inoculation of S. mutans or S. sobrinus. Biofilms were grown for a total of 24 h. (B) CV quantification (Abs 575 nm) of the experiment shown in A. Data is expressed as the percentage of biomass remaining in the S. mitis coculture condition compared to FM addition at each specific time point. (C) Representative image of a CV biofilm biomass assay where medium from 24 h pre-formed S. mutans biofilms is replaced with either 1X PBS, FM, or S. mitis ATCC 49456 wild-type (WT) or spxB mutant (ΔspxB) at different optical densities (OD_{600 nm} = 1.0, 0.4, or 0.1). The biofilms were then grown for another 24 h prior to CV staining. (D) CV quantification (Abs 575 nm) of the experiment shown in C. Data is expressed as the percentage of biomass remaining in comparison to the 1X PBS control (i.e., biofilm formed at 24 h without additional growth). FM refers to the addition of fresh medium (i.e., no S. mitis added). n = 8. (E) Representative maximum intensity 40X Z-projection of 48 h S. mutans biofilms grown in the absence of (− S. mitis), or with the addition of, S. mitis WT or ΔspxB at different optical densities at 24 h. Biofilms were then grown for another 24 h prior to imaging. S. mutans constitutively expresses GFP (green). Scale bar (100 μm) is shown in the bottom right corner. (F) Quantification of S. mutans biomass from the microscopy data shown in E. FM refers to the – S. mitis control (i.e., no S. mitis added). n = 4. Quantification was completed using Gen5 Image+ software. Data graphing and two-way analysis of variance (ANOVA) with multiple comparisons was completed in GraphPad Prism software.

Figure 6.. Transcriptomes of S. mutans and S. mitis ATCC 49456 during Coculture Growth.

(A) Principal component analysis (PCA) from RNA-Seq expression data (n = 3) of S. mitis grown in monoculture (black circles) or coculture with S. mutans (red hexagons). The proportion of variance for either PC1 (x-axis) or PC2 (y-axis) are listed. (B) Volcano plot of changes within individual S. mitis genes (circles) between monoculture and coculture with S. mutans. Differentially expressed genes (DEGs; = genes with ≥4 Log10 p value and Log2 fold change ≥ (−)1) are shown in either red (upregulated, right) or blue (downregulated, left). Individual gene identifier, name and/or characterized function are displayed, if able. (C) Stacked bar chart of upregulated S. mitis DEGs from the dataset grouped by pathway/operon/function. (D) Stacked bar chart of downregulated S. mitis DEGs. (E) PCA from RNA-Seq expression data of S. mutans grown in monoculture (black circles) or coculture with S. mitis (red hexagons) (n = 3). (F) Volcano plot of changes within individual S. mutans genes between monoculture and coculture with S. mitis. (G) Stacked bar chart of upregulated S. mutans DEGs from the dataset grouped by pathway/operon/function. (H) Stacked bar chart of downregulated S. mutans DEGs. (I) Venn diagram of the number of upregulated S. mutans DEGs from growth in quadculture (included S. gordonii, S. oralis and S. sanguinis; grey) or coculture with S. mitis (red). (J) Venn diagram of the number of downregulated S. mutans DEGs. Data graphing and PCA calculations were completed in GraphPad Prism software.

Figure 7.. S. mitis ATCC 49456 Impacts Biofilm Formation of other Oral Streptococci.

(A) Representative image of a CV biofilm biomass assay of different oral Streptococcus species listed on the right y-axis, grown in monoculture (−) or in coculture with S. mitis (+) in medium lacking (−Sucrose) or containing (+Sucrose) sucrose. (B) CV quantification (Abs 575 nm) of the experiment shown in A. Data is expressed as the percentage of biomass remaining in the S. mitis coculture condition compared to the monoculture condition, which lacks S. mitis. Dark grey bars with green circles represents TYG medium lacking sucrose (−Sucrose), while light grey bars with blue triangles represents TYG medium containing sucrose (+Sucrose). n = 12. Data graphing were completed in GraphPad Prism software.