Читать книгу Oral Biofilms - Группа авторов - Страница 49

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 of 5, the percent of A. naeslundii increased in that biofilm. A. naeslundii seemed to be less sensitive to pH 5, but this was probably a short pH window that allowed multiplication of that species. At a lower pH (below pH 4) it was shown that A. naeslundii was killed, but demonstrated greater acid resistance when existing within a biofilm [16]. Interestingly, an initial pH of 5 or 5.5 also decreased the formation of the “cariogenic” biofilm. It can be assumed that biofilm formation begins at slightly higher pH values and the low pH leading to dissolution of enamel is a result of the bacterial metabolism in the deep layer of an advanced biofilm. This supports the different stages of the ecological caries theory [1]. In our study pH was only determined in the surrounding media, while differences within a biofilm are likely. pH values were measured both inside and outside of a single-species biofilm. With pH control (buffering of the media), the pH within the biofilm was about 6.1 and the pH in the surrounding media was 6.7 [17]. Thus, a “cariogenic” biofilm does not have a consistent pH. A recent study measuring pH values in different areas of an in vitro biofilm after sucrose-mediated biofilm formation found acidic regions (below pH 5.5) only in the interior of microcolonies, and in vivo analysis confirmed a spatial heterogeneity of pH, with acidic pH values only close to the enamel surface [18]. The metabolic activity of bacteria depends on the pH value. The expression of glucosyltransferases, which are major genes of S. mutans involved in biofilm formation, was demonstrated to be higher without pH control than in controlled conditions [17]. Also, the competence of S. mutans to internalize DNA from the environment depends on the environmental pH – it is only possible at a pH of around 7 [19]. The biofilm model focused only on enamel caries, and did not consider either root or dentine caries, which involve proteolytic bacteria that contribute to the proteolytic stage of the diseases [20].

A “periodontitis” biofilm is more metabolically active and has a higher quantity at slightly alkaline pH values. The pH of the gingival crevicular fluid increases in vivo from 6.9 to higher values with the severity of inflammation [21]. It was measured to be around pH 8.4 at inflamed sites and it was shown that the bicarbonate buffer system contributes most at that pH value [22]. Measuring the pH in periodontal pockets found levels below 7.0, with lower values in the case of acute inflammation, and alkaline values when the inflammation became chronic [23]. P. gingivalis growth is reduced at an acidic pH [24], which is in accordance with our results showing lower total counts and percentages in the in vitro biofilm assays. P. gingivalis is associated with periodontitis, whereas T. forsythia and T. denticola are present both in gingivitis and periodontitis [25]. In the present in vitro study, the “periodontitis” biofilm had a higher percentage of T. forsythia and T. denticola at lower pH values.

Fig. 4. Effect of different initial pH values on biofilm formation (quantity and metabolic activity), microbial composition, and the pH tendency in the environment of oral biofilms in general (grey), “caries” biofilms (blue), and “periodontitis” biofilms (purple), as well as possible associations with oral disease.

The dependency of biofilm formation on pH offers therapeutic options, and an increase of pH is of interest for preventing caries [26]. In recent years a major focus has been on arginine deiminase, an enzyme synthesized by several oral streptococci which utilizes arginine for alkali production [27]. Conversely, in therapy of periodontal disease a decrease of pH may be beneficial. In general, a relatively low pH seems to be favorable for wound healing by promoting an immune response [28]. However, this should be carefully balanced in periodontal therapy. In monkeys it was shown that short-term etching of root surfaces with a 37% orthophosphoric acid led to more connective tissue and a shorter epithelial junction, whereas a long-term etching for 3 min clearly impaired periodontal healing [29]. Also, the activity of antimicrobials can be affected by the environmental pH. For example, chlorhexidine is more active at an alkaline pH and hypochlorite at a more acidic pH [28]. Thus, investigating the influence of the pH value in periodontal therapy in more detail might be an interesting topic for further research.

Future in vitro research into the influence of pH values on oral biofilm formation should consider more complex models with more microorganisms and a different nutrient supply. More knowledge is needed about the influence of different pH values on the expression of important genes involved in biofilm formation and virulence.

In conclusion (Fig. 4), an initially low pH (pH 5 and 5.5) may suppress biofilm formation and could be associated with the development of erosive tooth wear. A slightly higher pH (around pH 6) might favor the development of caries if respective nutrients (sugar) are available. A pH slightly less than 7 appears to be associated with a higher percentage of Tannerella sp. and Treponema sp., an acute gingivitis, and an increase in the percentage of aciduric bacteria. An initial slightly basic pH contributes to a biofilm associated with periodontitis, and in particular P. gingivalis. Therapeutics leading to a high pH may be helpful in the prevention of caries. In periodontitis, modulation of the pH could be an alternative option in therapy but should also include caries-preventive measures.