Читать книгу Oral Biofilms - Группа авторов - Страница 44

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 with NaOH or HCl until pH values of 5, 5.5, 6, 6.5, 7, 7.5, and 8 were achieved.

The biofilms were formed on 96-well plates. First the surface was coated with a proteinaceous solution (25% human serum, 0.27% mucin; both Sigma-Aldrich) for 1 h, before 250 µL/well of the bacteria/nutrient broth suspension was added. Thereafter, the plates were incubated at 37 °C with 10% CO₂ or anaerobically (periodontitis biofilm) for different lengths of time. The “healthy” biofilm was analyzed after 2 and 6 h, the “cariogenic” biofilm after 6 and 24 h, and the “periodontal” biofilm after 24 and 48 h. For the “periodontal” biofilm, 10 µL of microbial suspension consisting of one part each of T. denticola, T. forsythia, and P. gingivalis were added again after 24 h. Each of the three different plates were used in one experimental setting and per time point.

At the respective times, the nutrient broth was removed and the biofilms were careful washed once with 0.9% w/v NaCl. From the first plate, the biofilms were scraped from the surface and suspended in 0.9% w/v NaCl. After intensive mixing by pipetting and vortexing, a serial 10-fold dilution series was made. Each 25 µL were spread on tryptic-soy agar plates with 5% sheep blood (and 10 mg/L NAM). After incubation at 37 °C with 10% CO₂ or anaerobically (the “periodontitis” biofilm) for about 7 days, the total bacterial counts (log₁₀ colony-forming units; CFU) as well as the percent of the different bacteria used were determined. The identification was based on the colony morphology. As this was in part difficult or impossible (T. denticola does not grow on the agar plates), P. gingivalis, T. forsythia, and T. denticola, were counted using real-time PCR as described previously [6].

The second 96-well plate was used for the determination of metabolic activity with the use of Alamar blue reagent as a redox indicator [7]. A total of 5 µL of Alamar blue (alamarBlue®, Thermo Fisher Scientific Inc., Waltham, MA, USA) was mixed with 100 µL of the nutrient media and added to the biofilm. After extensive mixing with the biofilm and an incubation for 1 h at 37 °C, absorbances were measured at 570 nm against 600 nm.

The biofilm mass was quantified from the third 96-well plate according to recently published protocols [8]. First, the biofilms were fixed at 60 °C for 60 min. Thereafter, each 50 µL of 0.06% crystal violet (Sigma-Aldrich) was added. The plate was incubated at 37 °C for 10 min, then washed 3 times with 200 µL of dH₂O. Finally, the plate was read at 600 nm.

Each experiment was performed in quadruplicate in two independent series, resulting in at least eight single values each. ANOVA with post hoc Bonferroni was used for the statistical analysis. The level of significance was set to p = 0.05 and SPSS v.24.0 software (IBM SPSS Statistics, Chicago, IL, USA) was used.