Development of In Vitro Denture Biofilm Models for Halitosis Related Bacteria and their Application in Testing the Efficacy of Antimicrobial Agents

2.1. Subjects and Sampling

Two groups of subjects were included in this study and informed consent was obtained prior to the collection of samples. Group 1 contained ten healthy denture wearers (five women and five men; mean age 69.8±4.7 years); group 2 included ten denture wearers diagnosed with denture stomatitis (five women and five men; mean age 61.6±12.0 years) based on the guideline for denture stomatitis diagnosis [12]. These individuals had not used antibiotics for at least 3 months before sample collection. Subjects had not been treated for any systemic disease nor were they taking any prescription or nonprescription medications.

Subjects were asked to wear dentures for at least 3 hrs before samples were taken. For sample collection, sterile toothpicks (Fisher, USA) were used to scrape the plaque from the surface of the denture that was in contact with the oral mucosa. Collected plaque samples were placed into separate microcentrifuge tubes containing 0.5 ml of 80% ethanol and stored at -20°C until DNA extraction. The QIAamp DNA Micro Kit (Qiagen, USA) was used to extract DNA from collected plaque samples according to manufacturer’s instructions. DNA quality and quantity were checked with a NanoDrop 2000 spectrophotometer (Thermo, USA).

2.2. Detection of Halitosis-related Species by Polymerase Chain Reaction (PCR)

PCR was employed to detect the presence of the four selected halitosis-related bacterial species K. pneumoniae, V. atypica, T. forsythia and F. nucleatum within the plaque samples. Bacterial species-specific primers were designed based on previous studies[13-15].

For the PCR reactions, 10 ng DNA isolated from plaque samples was used as template in a 25 μl reaction mixture containing 1x PCR buffer, 1.5mM MgCl₂, 0.1mM each of the four dNTPs, 1 unit Taq DNA polymerase (Invitrogen, USA) and 1 μM of each primer. The PCR reaction conditions with optimized cycle and annealing temperature for each specific primer sets were as follows: for K. pneumoniae, specific primers Pf (5′-ATT TGA AGA GGT TGC AAA CGA T-3′) and Pr2 (5′-CCG AAG ATG TTT CAC TTC TGA TT-3′) were used; the reaction conditions were 94°C for 10 min; 35 cycles of 94°C for 30 s, 57°C for 20 s, and 72°C for 20 sec, and a final extension at 72°C for 10 min. For V. atypica, specific primer set ATYF (5′- TCT CTT GTT GAA GAA TTA GAA CGC-3′) and VR (5′- GTGTAACAAGGGAGTACGGACC-3′) was used; the PCR conditions were 94°C for 15 min, 20 cycles of 92°C for 1 min, 57°C for 1 min, and 72°C for 1 min, followed by 5 min at 72°C. For T. forsythia, specific primers 5′- GCGTATGTAACCTGCCCGCA-3′ and 5′- TGCTTCAGTGTCAGTTATACCT-3′ were used, and the reaction conditions were 94°C for 3 min, 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min, followed by 72°C for 5 min. For F. nucleatum, specific primer set 5′- AGAGTTTGATCCTGGCTCAG-3′ and 5′-GTCATCGTG CACACAGAATTGCTG -3’ was used; the PCR conditions were 94 °C for 3 min; 23 cycles of 94°C for 1 min, 61.5°C for 1 min, and 72°C for 30 sec, and a final extension at 72°C for 5 min.

PCR products were analyzed by agarose gel electrophoresis (1% w/v) in 1×TAE buffer (40 mM Tris–HCl, 1.18 ml acetic acid, 2 mM EDTA, pH 8.0) and a constant voltage of 100V was applied. Images were digitally recorded using the Molecular Imager Gel Documentation system (Bio-Rad, USA) to evaluate the presence of the amplified DNA product.

2.3. Strains and Growth Conditions

K. pneumoniae IA 565 is a clinical isolate obtained from the University of Iowa Hospitals and Clinics Special Microbiology Laboratory. The strain was grown in LB medium (Tryptone 10g/L Yeast Extract 5g/L NaCl 10g/L) and incubated at 37°C in an aerobic incubator on a rotary shaker operating at 200 rpm. T. forsythia ATCC 43037, V. atypica PK 1910 and F. nucleatum ATCC 23726 were grown anaerobically overnight at 37°C in TF medium, TH medium containing 0.06% (w/v) lactic acid and Columbia broth (Difco, USA), respectively [16].

2.4. DBR Discs

The acrylic DBR discs used in this study were manufactured as previously described[17]. All resin discs were disinfected with 2% (v/v) sodium hypochlorite solution (Sigma-Aldrich, USA) for 10 min and immersed in sterilized ddH₂O overnight prior to microbial inoculation.

2.5. Growth of Biofilms on DBR Disc Surfaces

Exponentially growing cultures of the four halitosis-associated bacterial species were diluted in their respective culture medium to a final OD₆₀₀ of 0.1 Two ml of the respective diluted cultures of the different species were added to each well of 12-well plates (Thermo, USA) containing a DBR disc with the rough surfaces exposed. Wells with growth medium alone served as controls. Plates containing cultures of T. forsythia, F. nucleatum, or V. atypica were incubated at 37°C anaerobically for 24 hrs, while cultures of K. pneumoniae were grown aerobically at 37°C for 24 hrs.

2.6. Crystal Violet Staining of Biofilms on DBR Disc Surfaces

Biofilms formed on the DBR discs were stained with crystal violet as described previously [18]. Briefly, DBR discs were washed twice with phosphate-buffered saline (PBS), air dried for 30 min, and stained with 0.4% (w/v) crystal violet in distilled water for 20 min. The stained discs were gently washed three times with sterilized distilled water and air dried for 30 min before being photographed. Images were taken with a D50 digital camera (Nikon, Japan).

2.7. Observation of Biofilms with Confocal Laser Scanning Microscopy (CLSM)

Different methods have been used to study biofilm formation including CLSM, AFM [19]. In our study, CLSM has been employed to reveal the biofilm structure as well as the cell viability within biofilms. Biofilms grown on the rough surface of the DBR discs were treated with Polident^® experimental formula M138-12 (GlaxoSmithKline, UK) or Efferdent^® antibacterial denture cleanser (Prestige brands, USA) for 5 min according to manufacturer’s recommendations, and untreated controls were stained with 10 μM of the cell-permeable fluorescent stain SYTO 59 (labeling of all cells) and 10 μM of the cell-impermeable fluorescent stain SYTOX Green (labeling of cells with compromised cell walls only) (Invitrogen, USA) in PBS buffer for 20 min at room temperature in the dark before visualization [17]. All biofilm images were collected with a Zeiss LSM 5 PASCAL CLSM using LSM 5 PASCAL software (Zeiss, Germany). Excitation at 633 nm with an argon laser in combination with a 650 nm band-pass emission filter was used for SYTO 59 fluorescence imaging. SYTOX Green signals were visualized using 488 nm excitation with a helium-neon laser and a 503-530 nm band-pass emission filter.

2.8. Evaluation of the Treatment Efficacy of Denture Cleansers in Planktonic and Biofilm Conditions

Biofilms grown on DBR discs were washed three times with PBS buffer before treatment. Two different Polident^® denture cleansing tablets (commercialized formulation 50767 and the experimental formulation M138-12) (GlaxoSmithKline, UK) and Efferdent^® antibacterial denture cleanser (Prestige Brands, USA) were separately dissolved in 150 ml 40°C distilled water according to manufacturer’s instructions. Each DBR disc with attached biofilms was immersed in 50 ml of the treatment solution for 5 min for all three denture cleansers and an additional experiment with a 15 min immersion period for Efferdent^® antibacterial denture cleanser was performed to follow manufacturer’s recommendations. DBR discs with attached biofilms treated with PBS buffer served as negative control. After treatment, the DBR discs were washed three times with PBS buffer and subjected to mechanical disruption of the attached biofilms, serial dilution and plating as previously described to obtain colony forming unit counts[17].

One ml of exponentially growing planktonic cultures (OD₆₀₀ of 1) was washed three times with PBS buffer and pelleted by centrifugation at 16,000 xg for 2 min. One tablet each of the three different denture cleansers tested was predissolved in 150 ml 40°C water for 2 min and 1 ml of the treatment solution was used to resuspend the pelleted cells. After 5 min or 15 min (for the additional treatment with Efferdent^®) of treatment (3 or 13 min incubation with additional 2 min centrifugation at 16,000 xg), cells were washed three times with PBS buffer prior to being subjected to serial dilution and plating.

Treatment efficacy was evaluated by comparing the surviving colony-forming units (CFU) on LB agar plates (for K. pneumoniae), Columbia agar plates supplemented with 5% sheep blood (Colorado Serum company, USA) (for F. nucleatum), TH agar plates supplemented with 0.06% lactic acid (for V. atypica), and TF agar plates supplemented with 5% sheep blood (for T. forsythia) with PBS-treated control group.

3.1. The Prevalence of Four Halitosis-related Strains in Denture Plaque

The aforementioned four halitosis-related bacterial species were chosen as targets for PCR screening among 20 clinical denture plaque samples (10 each for healthy and stomatitis subjects). Among the four targeted species, K. pneumoniae was only detected in two of the ten subjects in the stomatitis group, while the remaining three species were present in both groups (Table 1). Their prevalence in healthy and denture stomatitis plaque was 60% and 40% for F. nucleatum, 30% and 40% for T. forsythia, 30% and 10% for V. atypica, 0% and 20% for K. pneumoniae; respectively (Table 1). Furthermore, 80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque (Table 1). Due to small sample sizes, Fisher-exact chi-square test was used to examine the difference between stomatitis and healthy groups across the species-specific primers (p=0.39).

3.2. Biofilm Formation of Halitosis-related Oral Bacterial Strains on DBR Discs with Rough Surfaces

Crystal violet staining revealed that under the conditions described in Materials and Methods, all four species were capable of forming biofilms on these DBR disc surfaces, although to different extents, as manifested by the different intensities of purple staining (Fig. 1). The biofilm architecture of mono-species biofilms and live/dead distribution of the bacteria within the biofilms was further investigated by CLSM.

CLSM analyses disclosed distinct biofilm architectures for each of the halitosis-related strains tested. F. nucleatum ATCC 23726 formed biofilms with a smooth and homogeneous structure almost completely covering the irregular DBR disc surface (Fig. 2A). A similar surface coverage was observed for T. forsythia ATCC 43037; however, the biofilms formed were less dense compared to F. nucleatum with great variations in overall thickness (Fig. 2B). Surface coverage by V. atypica PK 1910 was incomplete and less compact compared with mono-species biofilms of F. nucleatum and T. forsythia (Fig. 2C). Among the bacterial strains tested K. pneumoniae IA 565 displayed the least biofilm forming ability. Colonization was limited to thin, “patch-like” structures leaving most of the surface uncovered (Fig. 2D). The majority of the bacteria cells within all mono-species biofilms were alive as indicated by predominantly green fluorescent (live cell staining with SYTO59) and very limited red fluorescent (dead cell staining with SYTOX Green) labeling.

3.3. Evaluating the Antimicrobial Efficacy of Denture Cleansers Against DBR Disc Surface-associated Biofilms and Planktonic Cells of Halitosis-related Strains

For a quantitative assessment of treatment efficacy, the DBR disc based biofilm model developed above as well as planktonic cells of K. pneumoniae and F. nucleatum were subjected to treatment as above and CFU counts were used as a read-out for treatment efficacy. F. nucleatum mono-species biofilms grown on DBR discs were most sensitive to the experimental formula Polident^® M138-12 treatment with no viable cells being detected after 5 min incubation. Treatment with Polident^® 50767 for the same time period resulted in 10²-10³ surviving CFU, while 10⁵-10⁶ surviving CFU were still recovered after 5 min and 15 min treatment with Efferdent^® (Fig. 4A). In contrast, planktonic F. nucleatum cells exhibited similar sensitivities to all three formulas, with no surviving CFU being detected after 5 min treatment as well as the additional 15 min treatment with Efferdent^® (Fig. 4A). While Polident^® 50767 and the experimental formula Polident^® M138-12 demonstrated similar killing efficacy toward K. pneumoniae denture biofilms, resulting in about 4 orders of magnitude reduction (from ~10⁸ to ~10⁴) in cell viability (t-test, P<0.01) after 5 min treatment, Efferdent^® manifested about 3 orders of magnitude reduction (from ~10⁸ to ~10⁵) in cell viability (t-test, P<0.01) after 5 min and 15 min treatment (Fig. 4B). The planktonic form of K. pneumoniae was more sensitive than its biofilm counterpart, with no viable cells being detected after a 5 min treatment with Polident^® 50767 or a 15 min treatment with Efferdent^®. Three min treatments with the experimental formula Polident^® M138-12 or Efferdent^® resulted in 10²-10³ and 10³-10⁴ surviving CFU, respectively (Fig. 4B). Quantitative values for killing efficacy were not determined for V. atypica and T. forsythia since CFU recovery on the recommended agar medium plates exhibited large variability under our experimental conditions.

Mono-species DBR-attached biofilms formed by halitosis-related bacterial species were used to evaluate the efficacy of denture cleansers. Biofilms were stained with SYTO59 and SYTOX Green and examined by CLSM to reveal the live/dead cell distribution following the different treatments as described in Material and Methods. Compared to PBS-treated mono-species denture biofilms in which the majority of the cells were alive, treatment with the experimental formula Polident^® M138-12 almost completely killed the bacterial cells within mono-species biofilms formed on DBR discs by F. nucleatum, T. forsythia and V. atypica (Figs. 3A, 3B and 3C). In contrast, the same treatment of K. pneumoniae biofilms yielded less apparent killing (Fig. 3D). Treatment with Efferdent^® did not result in complete killing of bacterial cells within any of the mono-species denture biofilms formed by F. nucleatum, T. forsythia, V. atypica, or K. pneumoniae within 5 min of treatment time (Figs. 3E, 3F, 3G and 3H).