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”
Biofilm Representing Periodontitis
In the biofilms representing periodontitis, the total counts of bacteria (log10 CFU) differed significantly between the initial pH values at 24 and 48 h (each p < 0.001; Fig. 3a). At 24 h, the lowest values were counted at pH 5 and 5.5 with approximately 5.8–5.9 log10 CFU, while the highest counts were detected at pH 7–8 with a mean of 9.3–9.5 log10 CFU. At 48 h, the lowest CFU counts were 6.3 log10 when the initial pH was 5, and the highest values were 9.3–9.4 log10 (pH 7–8). At both time points, post hoc analysis showed differences between each pH 5, 5.5, 6, and 6.5 on the one hand, and pH 7 on the other (each p < 0.01).
At 24 h, the percentages of T. forsythia and T. denticola were higher at pH 5 and 5.5 in comparison with the initial pH 7 (each p < 0.001). At 48 h, the percentage of both species remained high at pH 5 (both p < 0.001). In contrast, the percentage of P. gingivalis increased with increasing pH. The differences for pH 5–6.5 were all statistically significant versus pH 7 (p < 0.001). Apart from P. gingivalis, T. forsythia, and T. denticola, the other bacteria were present at higher percentages at pH 5 (p = 0.004), pH 5.5 (p = 0.017), pH 6 (p = 0.002), and pH 6.5 (p = 0.012) in comparison with the initial pH 7 (Fig. 3b).
The metabolic activity also showed a dependency to the pH level, increasing with elevated pH values at 24 and at 48 h (Fig. 3c). At both time points, the activities were lowest at pH 5 and 5.5, with each being statistically significantly different to pH 7 (each p < 0.001). At 24 h, the biofilm metabolic activity was higher for the initial pH level of 8 versus pH 7 (p < 0.001).
At 24 h, the biofilm mass was higher for the initial pH level of 6.5 versus pH 7 (p < 0.001). At 48 h, there was a decreased mass for pH 5 and 5.5 in comparison with pH 7 (both p < 0.001; Fig. 3d).
The pH measurements were performed at 24 and 48 h. The gradients were from pH 5.21 and 5.24 (initially pH 5) to pH 7.45 and 7.48 (initially pH 8) at 6 and 24 h. It is of interest to note that the initial pH 7 changed to 7.08 (Table 4).
Table 4. Initial pH values and values (mean ± SD) in the surrounding media after 24 and 48 h of formation of the eight-species biofilm representing “periodontitis”
Discussion
In this in vitro study the influence of an initially defined pH on biofilm formation was investigated. The pH was adjusted on a scale from 5 to 8 by using two buffers. In each case the predefined conditions allowed biofilm formation and bacterial growth but to a differing extent. The follow-up time of the biofilm formation was limited and adjusted according to the clinical situation. In oral health only a thin, young biofilm exists, whereas subgingival biofilm contains several bacterial species that are present only at later stages. Static biofilms were cultivated, which might be a limitation of the study as a certain continuous flow and supply of nutrients and a removal of metabolic bacterial products occurs in vivo. Furthermore, the initial buffer system remained, and in vivo a wider range of buffering of the pH can be assumed. Saliva contains three major buffer systems (bicarbonate, phosphate, and protein); the bicarbonate buffer is most active at pH 6.25–7.25, whereas at a lower pH the protein buffer system contributes most to the buffer capacity [9]. In our study clearly defined laboratory strains were used, allowing a standardized comparison. However, in vivo oral biofilms consist of hundreds of species [10]. Both periodontopathogenic and cariogenic bacteria might be present in distinct oral environments in the same oral cavity [11], where an easy transfer can occur in the case of a changing micromilieu.
It is of interest to note that the initially adjusted pH gradient was only slightly weakened at the end of the experiments. Although a certain gradient remained, there were differences between the three biofilm models which underlines the influence of the included microorganisms on the pH levels. In the “healthy” and “caries” biofilm models, an initially low pH remained (increase from pH 5 to pH 5.15 and 5.10). In these models, an initially alkaline pH dropped, thus only the media initially adjusted to pH 8 were slightly above pH 7 (pH 7.05). In the “periodontitis” biofilm, an initial pH of 5 increased to 5.24, while the pH of the media initially adjusted to pH 7–8 were all above pH 7 at 48 h (with the highest being pH 7.48).
All biofilms at an initial pH of 5 and 5.5 resulted in the lowest quantities of bacteria, CFU counts, and metabolic activities. A low pH may prevent biofilms from growing but could cause other problems. This includes the dissolution of dental hard tissue without the involvement of microorganisms: dental erosion and the combination with mechanical wear. Erosive tooth wear is defined as a chemical-mechanical process which leads to a cumulative loss of hard dental tissue that it is not caused by bacteria [12]. A large study analyzing more than 3,000 young adults aged between 18 and 35 years found a prevalence of erosive tooth wear in 29.4%, which was clearly associated with a high consumption of fresh fruits and juice [13]. Beverages, in particular soft drinks, may have very low pH values (as low as pH 2.4), and the critical pH with respect to hydroxyapatite was calculated at between pH 4.9 and 6.5 for soft drinks [14]. However, it should be noted that although the pH level is the most important factor accounting for the erosive potential of a beverage, other factors, such as the addition of calcium, may counteract this and be protective [15].
Our “healthy” biofilm consisted only of the species S. gordonii and A. naeslundii. At an initial pH