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In the biofilms representing caries, total counts of bacteria within each biofilm (log₁₀ CFU) differed significantly between the initial pH values at 6 and 24 h (each p < 0.001; Fig. 2a). At 6 h, the lowest values were counted at pH 5 with approximately 5.8 log₁₀ CFU, while the highest counts were recorded at pH 6 with a mean of 7.7 log₁₀ CFU. Post hoc analysis showed differences between each pH 5 and 5.5 on the one hand, and pH 7 on the other (each p < 0.01). At 24 h, the lowest CFU counts were 6.0 log₁₀ when the initial pH was 5; the highest value was 9.7 log₁₀ (pH 7 and 8). At 24 h, the CFU count differences were each statistically significant between pH 5, 5.5, 6, and 7 (each p < 0.01).

At 6 h, there were no statistically significant differences in the percentage of S. gordonii, A. naeslundii, and S. mutans/S. sobrinus between the pH levels. The percentage of L. acidophilus was higher at pH 5 and 5.5 versus pH 7 (p < 0.001, p = 0.017). At 24 h, the percentage of L. acidophilus remained high at pH 5 (p < 0.001 vs. pH 7). Furthermore, there was a significantly higher percentage of S. mutans/S. sobrinus at pH 5 (p < 0.001) and pH 6 (p = 0.016), and a lower percentage of S. gordonii at pH 5, 5.5, and 6 (each p < 0.001) versus pH 7 (Fig. 2b).

The metabolic activity differed between the different pH values at 6 and 24 h (each p < 0.001; Fig. 2c). At both time points, the lowest activities were at pH 5 and 5.5, with each being statistically significantly different to pH 7 (each p < 0.01). However, at 24 h the biofilm metabolic activity was higher for the initial pH level of 6 versus pH 7 (p < 0.001).

Fig. 3. Total bacterial counts (mean and SD; a), bacterial composition (b), metabolic activity (mean and SD; c), and biofilm mass (mean and SD; d) of the eight-species biofilm representing periodontitis in relation to the initial pH. Differences versus pH 7 are presented: * p < 0.05, ** p < 0.01 (the asterisk colors correspond with the composition of biofilms in b).

At 6 h, the biofilm mass was higher at the initial pH level of 6 versus pH 7 (p < 0.001). At 24 h, there was a decreased biofilm quantity at pH 5 and 5.5 in comparison with pH 7 (p = 0.012, p = 0.007; Fig. 2d).

The pH measurements were performed at 6 and 24 h. The gradients were from pH 5.15 and 5.10 (initially pH 5) to pH 7.04 and 7.05 (initially pH 8) at 6 and 24 h. The initial pH value of 7.5 decreased to 6.9 (Table 3).

Table 3. Initial pH values and values (mean ± SD) in the surrounding media after 6 and 24 h of formation of the five-species biofilm representing “caries”