Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Abstract

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.

FIG. 1.

Inhibition ability of S. sanguinis, S. gordonii, and S. mutans when grown with or without oxygen. (A) S. gordonii or S. sanguinis was inoculated first and grown for 16 h at 37°C with (+O₂) or without oxygen (−O₂), S. mutans was then inoculated next to the pioneer colonizer, and the plates incubated overnight. (B) S. mutans was inoculated first and grown for 16 h at 37°C with (+O₂) or without oxygen (−O₂), and then S. gordonii or S. sanguinis was inoculated next to the pioneer colonizer and the plates were incubated overnight.

FIG. 2.

Inhibition ability and competitiveness of wild-type and Pox⁻ mutant of S. sanguinis and S. gordonii. (A) S. sanguinis or S. gordonii or their derivatives were inoculated first and incubated aerobically at 37°C. S. mutans was inoculated 16 h later, and the plates were incubated overnight. a, SK36; b, JKH2 Pox⁻; c, JKH2/pDL276-pox; d, DL1; e, JKH1 Pox⁻; f, JKH1/pDL276-pox. (B) Growth of S. mutans in sterile conditioned medium from S. sanguinis or S. gordonii cultures. The conditioned medium was prepared from exponentially growing S. sanguinis or S. gordonii cells or their derivatives, filter sterilized, supplemented with 0.25% glucose, and immediately inoculated with S. mutans at a titer of 10⁷ bacteria ml⁻¹. Cells were incubated aerobically at 37°C, and 1 ml was removed to determine the A₆₀₀ at the indicated time points. The data presented are representative of two independent experiments with similar results. (C) Competitiveness of S. sanguinis or S. gordonii wild-type and Pox⁻ mutants in submerged dual-species biofilms with S. mutans. Dual-species biofilms were grown in BHI. After overnight growth, the cells were dispersed by vigorous pipetting, serially diluted, and plated. The CFU values for each strain and the standard deviations were calculated from three independent experiments performed on different days. *, P ≤ 0.05. Gray bars, S. sanguinis or S. gordonii; black line, S. mutans.

FIG. 3.

Influence of glucose on the competitiveness of S. sanguinis or S. gordonii. (A) S. mutans growth inhibition assay in the presence or absence of 1% glucose. (B) Growth of S. mutans in sterile conditioned medium from S. sanguinis or S. gordonii cultures grown with or without 1% added glucose. The conditioned medium was prepared from cells growing to the mid log growth phase and immediately inoculated with S. mutans. Cells were incubated aerobically at 37°C, and 1 ml removed to determine the A₆₀₀ at the indicated time points. The data presented are representative of two independent experiments with similar results. (C) Competitiveness of S. sanguinis or S. gordonii in submerged dual-species biofilms with S. mutans. Dual-species biofilms were grown in BHI plus 1% glucose when indicated. After overnight growth, the cells were dispersed by vigorous pipetting, serially diluted, and plated. CFU values and standard deviations were calculated from three independent experiments performed on different days. *, P ≤ 0.05. Gray bars, S. sanguinis or S. gordonii; black line, S. mutans.

FIG. 4.

Production of H₂O₂ during growth of S. sanguinis or S. gordonii. (A) SK36 or DL1 wild type with or without 1% glucose or catalase (30 μg), Pox⁻ mutants JKH1 and JKH2, and complemented strains grown for 3 h until mid-exponential phase in TSB medium. (B) Time course of DL1 H₂O₂ production with or without glucose. (C) Time course of SK36 H₂O₂ production with or without glucose. Hydrogen peroxide concentration was adjusted to the absorbance at 600 nm. Gray bars, TSB; white bars, TSB plus 1% glucose. *, P ≤ 0.05 (n = 3).

FIG. 5.

Differential expression of nlmA, comC, and comE from S. mutans during aerobic and anaerobic growth. Gene expression was compared from cells growing on BHI agar plates for 7 h with or without oxygen. The values are given as relative cDNA abundance, with the expression under aerobic conditions set to 100%. Transcript levels were measured by real-time PCR using the 16S RNA as a housekeeping control. cDNA abundance was normalized against the 16S cDNA. The data presented represent the means and standard deviations of two (comC and comE) or three (nlmA) independent experiments performed on different days.

FIG. 6.

Quantification of DNA release and cell densities from mixed cultures of S. sanguinis and S. gordonii during growth under aerobic and anaerobic conditions. (A) Real-time PCR quantification of DNA release from mixed cultures of wild-type SK36/DL1 and Pox⁻ mutants JKH1/JKH2 after overnight incubation with or without oxygen using universal primers for 16S RNA genes of S. gordonii and S. sanguinis (16S strep F and 16S strep R). The DNA release in each individual sample is reported relative to a serial dilution of chromosomal DNA from S. sanguinis (see Materials and Methods for details) by comparison of the C_T values for the standard curve and the samples. (B) CFU counts and A₆₀₀ values of the overnight cultures prior to DNA quantification. The data presented represent the means and standard deviations of three independent experiments performed on different days. Gray bars, CFU; black line, absorbance (A₆₀₀).