Bacterial strains and culture media
The following archived bacterial strains were used throughout this study: Streptococcus gordonii (JD13A) from healthy oral mucosa, Streptococcus mutans (B4B), Veillonella parvula (10BB), Actinomyces odontolyticus (G3H) and Actinomyces naeslundii [110BT (catalase positive) and CW (catalase-negative)], all originally from dental plaque of a healthy subject; Parvimonas micra (EME), originally from the sub-gingival pocket of a patient with peri-implant disease and the type strain of Porphyromonas gingivalis (W50). Identities of the strains had been confirmed by 16S rRNA gene sequencing. All strains were stored in skim milk (Oxoid) at − 80 °C. Streptococci were grown on blood agar overnight in 5% CO2 in air at 37 °C, whereas all other species were routinely cultured on blood or Brucella agar (P. gingivalis) for up to 7 days at 37 °C under anaerobic conditions (10% H2, 5% CO2 in N2). Multi-species biofilms were grown overnight in protein-rich nutrient medium (PRNM) developed by Naginyte et al. [24] without the porcine gastric mucin.
Preparation of antimicrobial substances
Acetic acid (HAc) solutions (0.125%, 0.25%, 0.5%, 1%, 2%, 6%) were prepared through dilution of glacial acetic acid (Sigma-Aldrich) in distilled water. Solutions were buffered to pH 2.3 or 4.6 using NaOH and/or HCl and kept at room temperature. Solutions of hypochlorous acid (HOCl) (0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 100 ppm, corresponding to 9.53 µM-1.9 mM HOCl) were made by dissolving sodium hypochlorite pentahydrate (Tokyo Chemical Industry) in distilled water. Solutions were buffered to pH 4.6 using perchloric acid (Sigma-Aldrich) and stored in the dark at 4 °C for up to 3 weeks. Confirmation of initial concentrations was performed using spectrophotometry at A236 and comparison against a standard curve. Stabilized HOCl solutions (0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm) in HAc buffer were made on the day of use from a HOCl stock solution (100 ppm) (provided by SoftOx Solutions AB) containing set concentrations of HAc (0.14% or 2%), buffered to pH 4.6. Stock solutions stored in the dark at 4 °C were shown to be stable (< 0.04% reduction in concentration of HOCl) for at least 3 months under these conditions. Chlorhexidine (CHX) solutions [0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 100 ppm (corresponding to 0.99 µM–0.198 mM)] were prepared by dilution in autoclaved distilled water from a stock solution of 20% chlorhexidine gluconate in water (Sigma-Aldrich, St Louis, MI, USA).
Effects of antimicrobial substances on single- and multi-species biofilms
Suspensions (OD600 = 0.1) of individual bacterial species were prepared in 25% Todd-Hewitt (TH) broth (BD Biosciences, NJ, USA). For P. micra, the 25% TH broth was supplemented with 500 µg/mL L-cysteine. Bacterial suspensions were then added to channels in Ibidi® µ-slide VI Ibi-treat flow-cells (Ibidi GmbH, Gräfelfing, Germany) and incubated in a humid chamber under anaerobic/CO2 conditions at 37 °C for 16–24 h to allow biofilm formation on the slide surface.
For biofilms representing supra-gingival (S. mutans, A. odontolyticus and V. parvula) and sub-gingival (S. gordonii, P. gingivalis and P. micra) communities, bacterial species were mixed in approximately equal amounts in phosphate-buffered saline (PBS; 0.15 M NaCl, 0.05 M NaH2PO4, pH 7.4). Suspensions (200 µL) were added to channels in the Ibidi® flow-cells and incubated in a humid chamber under anaerobic conditions for 4 h to allow for bacterial attachment. Following this, the PBS was exchanged for PRNM to allow bacterial growth, and the flow-cells were then incubated for 24 h. All broths and PBS were pre-reduced to anaerobic conditions prior to experimentation. Biofilms were incubated with test solutions at room temperature for 5 min and then slides were rinsed twice with PBS.
Vital fluorescence microscopy and image analysis
Following exposure to the test or control solutions, biofilms were stained in situ for 15 min in the dark using LIVE/DEAD® BacLight™ viability stain (Molecular Probes, Eugene, OR, USA) containing Syto 9 (green) and propidium iodide (red). Syto 9 enters all cells but in those with a compromised membrane, the green staining is quenched out by the entry of propidium iodide turning the dead cells red. Stained biofilms were viewed using a Nikon Eclipse TE200 inverted confocal laser scanning microscope. Image analysis to determine the total biofilm surface coverage and percentage of viable (green) cells was performed using the colour segmentation software bioImage_L [25] on 10 randomly selected images from each experimental condition. All experiments were undertaken in triplicate using independent biological replicates. Test conditions were compared with the control (25% TH broth) using the Kruskal Wallis test in GraphPad Prism with Dunns' post-test for mutliple comparisons. p values < 0.05 were considered statistically significant.
Effect of stabilized HOCl on viability of oral keratinocytes
An MTT assay (Abcam Inc.) was used to determine the effect of stabilized HOCl solutions on the viability of oral keratinocytes. Immortalized human oral keratinocytes (OKF6/TERT-2, p33) were seeded into 96-well tissue culture plates and grown to confluency in keratinocyte serum-free medium (k-sfm) from Thermo Fisher Scientific, Paisley, UK, supplemented with 0.2 ng/mL human recombinant EGF, 25 µg/mL bovine pituitary extract and 0.3 mM CaCl2 containing 1 IU/mL penicillin and 1 µg/mL streptomycin. Cells were treated with the test solutions (1, 5, 10 and 20 ppm HOCl stabilized with 0.14% in k-sfm) for 10 min. Following exposure, solutions were replaced with a 1:1 ratio of MTT reagent in PBS, and incubated for 3 h at 37 °C to allow formazan crystal formation. The MTT reagent was then carefully removed, replaced with MTT solvent and placed on a shaker. Concentrations of formazan were then determined through absorbance at 590 nm. Test conditions were compared with the control (k-sfm) using the Kruskal Wallis test in GraphPad Prism with Dunns' post-test for mutliple comparisons. p values < 0.05 were considered statistically significant.
In-situ quartz crystal microbalance with dissipation (in-situ QCM-D)
Measurement of hydroxyapatite (HA) surface erosion was performed using a Q-sense E4 system (Biolin Scientific AB, Gothenburg, Sweden) equipped with a peristaltic pump from Ismatec (Wertheim, Germany) (Fig. 1). The QCM-D method is based on the change in resonant frequency of a vibrating quartz crystal sensor, a piezoelectric material, according to mass changes of the sensor. The QCM-D instrument monitors real-time changes in the frequency of vibrational modes as well as changes in the vibrational energy dissipation. Chamber temperature was maintained at 22.0 °C ± 0.02 °C and a flow rate of 100 µL/min was used for all the experiments. The AT-cut piezoelectric HA-coated quartz crystal disks used as the sensor chip (Q-sense, Biolin Scientific AB, Gothenburg, Sweden) had a fundamental frequency of 4.95 MHz ± 50 kHz and vibrate in the thickness-shear mode with the overtone n of 1, 3, 5, 7, 9, 11 and 13. Before use, sensor chips were UV-treated for 20 min, then immersed in 99% ethanol for 30 min, rinsed extensively with milli-Q water and blow-dried using nitrogen gas. Finally, the QCM-D sensors were UV-treated for 20 min. Clean QCM-D sensor chips were mounted directly on the QCM-D instrument and used immediately. All solutions were degassed by sonication prior to use. First, ∆f was measured in air to evaluate the correct mounting of the sensor chip and ethanol was then introduced in the cell to eliminate possible air bubbles over the surface. PBS was subsequently flushed through the system until a stable baseline of ∆f and ∆D was observed. Test solutions (1, 5, 10 and 20 ppm HOCl stabilized with 0.14% HAc or 2% HAc) in addition to 0.14% and 2% HAc alone and positive controls; 15% HAc solution (pH 2.3) and 5% HCl (pH 0.3) were flowed over the HA surface for 20 min, followed by a PBS rinse for 15 min. All experiments were performed in duplicate. The data presented correspond to the change in vibrational frequency and its associated dissipation (∆fn = ∆Fn/n and ∆D) of the sensor chip vs. time. The measured frequency shift for the fifth overtone (∆f5) was used to evaluate the effect of the solutions on the HA surfaces.