Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions

Abstract

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pH-cycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.

Fig 1. The schema of the biofilm experiment.

Fig 2. Viability of single-species and dual-species 48 h biofilms.

Biofilms were grown under constantly neutral pH or pH-cycling (8 h pH 7.0 and 16 h pH 5.5) conditions. The viability of the biofilms was evaluated by CFU counts. A. Viable cell counts of S. mutans biofilms. Single Sm: S. mutans single-species biofilms; Dual Sm: S. mutans counts in dual-species biofilms. B. Viable cell counts of S. oligofermentans biofilms. Single So: S. oligofermentans single-species biofilms; Dual So: S. oligofermentans counts in dual-species biofilms. The dash line indicates the detection limit. Four replicates were used for viable cell counts in each experiment. The experiment was repeated 3 times. * indicates the significant difference between Single Sm and Dual Sm, p < 0.05.

Fig 3. Lactic acid concentration of single-species and dual-species 48 h biofilms.

Biofilms were grown under constantly neutral pH or pH-cycling conditions. Before quantification of lactic acid concentration, the biofilms were incubated in the assay medium (pH 7.0), supplemented with 1% glucose for 4 h. Single Sm: S. mutans single-species biofilms; Single So: S. oligofermentans single-species biofilms; Dual: dual-species biofilms. Four replicates were used for lactic acid quantification in each experiment. The experiment was repeated 3 times. * indicates the significant difference between Single Sm and the other two groups (Single So and Dual), p < 0.05.

Fig 4. Hydrogen peroxide (HP) production of single-species and dual-species 48 h biofilms.

Biofilms were grown under constantly neutral pH or pH-cycling conditions. The HP production was quantified after the biofilms were incubated in the assay medium (pH 7.0), supplemented with 1% glucose for 4 h. Single So: S. oligofermentans single-species biofilms; Dual: dual-species biofilms. Four replicates were used for HP production in each experiment. The experiment was repeated 3 times. * indicates the significant difference between constantly neutral pH and pH-cycling conditions, p < 0.05.