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Gunnar Dahlen
Department Oral Microbiology and Immunology, Institute of Odontology
Sahlgrenska Academy, University of Gothenburg, Box 450
SE–405 30 Gothenburg (Sweden)
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 19–29 (DOI: 10.1159/000510196)
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The Impact of the pH Value on Biofilm Formation
Lara B. Schultzea Alejandra Maldonadoa Adrian Lussib, c Anton Sculeana Sigrun Eicka
a Department of Periodontology, Laboratory of Oral Microbiology, School of Dental Medicine, University of Bern, Bern, Switzerland; b School of Dental Medicine, University of Bern, Bern, Switzerland; c Department of Operative Dentistry and Periodontology, University Medical Center, Freiburg, Germany
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Abstract
The pH value of a biofilm influences the pathogenesis and therapy of oral diseases such as caries and periodontitis. This study aimed to investigate the influence of different initial pH values on the microbial composition, bacterial counts, metabolic activity, and quantity of three defined biofilms representing oral health, caries, and periodontal disease. Respective bacterial suspensions in the nutrient broth were initially adjusted to pH values between 5 and 8. Then biofilms were cultured on polystyrene surfaces coated with a proteinaceous solution for 2 h (“healthy” biofilm), 6 h (“healthy,” and “cariogenic” biofilms), 24 h (“cariogenic,” and “periodontitis” biofilms), and 48 h (“periodontitis” biofilm). In all biofilms, total bacterial counts were lower at an initial pH of 5 or 5.5 than at higher pH values. In the biofilm representing caries, the percentage of cariogenic bacteria (Streptococcus mutans, S. sobrinus, Lactobacillus acidophilus) was higher at a low pH, the metabolic activity was highest at pH 6–6.5, and biofilm mass was greatest at pH 7–7.5. In the biofilm representing periodontitis, the percentage of Porphyromonas gingivalis increased with the pH. Also, the metabolic activity was highest at pH 8, whereas mass had the highest value at pH 7. In conclusion, the initial pH value influences biofilm formation. In particular, metabolic activity and the amount of bacteria associated with disease correlated with the respective pH known to be of importance in the development of caries (relatively low pH) and periodontitis (higher pH). Modifying the pH level in oral biofilms might be an alternative concept in (primary) prevention and treatment, not only of caries but also of periodontitis.
© 2021 S. Karger AG, Basel
Two major oral diseases, caries and periodontal disease, are associated with biofilm formation on teeth. Caries is a disease affecting dental hard tissues. A collection of different microorganisms in a biofilm produces acids, decreases the pH, and moves the balance between demineralization and remineralization towards demineralization. The acidogenic stage is followed by an aciduric stage where microorganisms (including mutans streptococci) are still able to survive at low pH values [1]. Treatment options that increase the pH within supragingival biofilms are currently under discussion. For example, arginine as a supplement of toothpaste aims to modify the microbial biofilm composition to be less acidogenic [2].
Table 1. Bacterial strains used in the experimental biofilms
Periodontitis is a disease characterized by the destruction of the tissue surrounding teeth and, if untreated, ultimately leads to tooth loss. Among the many different bacteria found in subgingival biofilm, Porphyromonas gingivalis was postulated as being a keystone pathogen in the development of periodontitis by modifying the host response and finally the composition of the biofilm [3]. A recent analysis in adolescents showed that the severity of periodontitis was inversely associated with enamel caries, whereas the extent of periodontitis (numbers of affected sites) showed a positive association with dentin carious lesions [4]. After initial periodontal therapy, periodontopathogenic bacteria decrease while Streptococcus mutans increases [5]. This suggests a dependency of the composition of the oral biofilm on the surrounding micromilieu. One important factor might be the pH value.
The aim of this study was to investigate the influence of initially different pH values on biofilm formation. For this, nutrient media containing buffers were adjusted to pH values in a range from 5 to 8 in increments of 0.5. Defined microbial strains representing oral health, caries, and periodontal disease were studied for their ability to form biofilms regarding bacterial counts, microbial composition, biofilm mass, and metabolic activity.
Materials and Methods
Defined laboratory strains were used for the different biofilms. Early colonizers represent the biofilm associated with oral health, mutans streptococci belong to the five-species biofilm associated with caries, and P. gingivalis, Tannerella forsythia, and Treponema denticola are additional members of the eight-species biofilm representing periodontal disease (Table 1).
The strains were passaged 48–24 h before the experiments on tryptic-soy agar plates with 5% sheep blood. For T. forsythia, N-acetylmuramic acid (10 mg/L) was added. T. denticola was cultivated in modified mycoplasma broth (BD, Franklin Lakes, NJ, USA) added with 5 mg/mL of cocarboxylase in anaerobic conditions.
Thereafter, the bacteria were suspended in 0.9% w/v NaCl to McFarland 4. The mixed suspension for the “healthy” biofilm consisted of two parts S. gordonii and three parts Actinomyces naeslundii. The cariogenic biofilm was mixed with one part S. gordonii and S. mutans, two parts A. naeslundii and S. sobrinus, and three parts Lactobacillus acidophilus. For the periodontal biofilm, the respective mixture was prepared with one part S. gordonii and three parts each of the other seven bacteria. These mixed suspensions were added 1:20 to the nutrient broth (Wilkins-Chalgren Broth; Oxoid, Basingstoke, UK) with 5 mg/L β-NAD (Sigma-Aldrich, Buchs, Switzerland) which had been adjusted to an approximate pH with two different buffers (citrate buffer and phosphate buffer) and precisely