Culture conditions and standardisation
A selection of laboratory strains of commensal and pathogenic bacteria associated with oral biofilms disease were used in this study, including Porphyromonas gingivalis ATCC 33277 and Fusobacterium nucleatum ATCC 10596, which were maintained at 37°C on fastidious anaerobic agar (FAA [Lab M, Lancashire, UK]) under anaerobic conditions (85% N2, 10% CO2 and 5% H2, [Don Whitley Scientific Limited, Shipley, UK]). Streptococcus mutans 10449, Streptococcus mitis NCTC 12261, Aggregatibacter actinomycetemcomitans OSM 1123 and Enterococcus faecalis NCTC 5957 were grown and maintained at 37°C on Colombia blood agar (CBA [Oxoid, Hampshire, UK] in 5% CO2. All isolates were stored indefinitely in Microbank® vials (Pro-Lab Diagnostics, Cheshire, UK) at −80°C.
P. gingivalis and F. nucleatum were propagated in 10 ml Schaedler’s anaerobic broth (Oxoid), S. mitis and A. actinomycetemcomitans were grown in 10 ml Tryptic Soy Broth (TSB [Sigma-Aldrich, Dorset, UK]) supplemented with 0.6% yeast extract and 0.8% glucose. E. faecalis was grown in TSB with 0.25% glucose, and S. mutans was grown in 10 ml brain heart infusion (BHI [Sigma-Aldrich]), all at 37°C and at appropriate atmospheric conditions. Overnight cultures were washed by centrifugation (1000 xg) and resuspended in 10 ml PBS. All bacteria were then standardised and adjusted to a final working concentration of 5 × 104 and 1 × 107 cells/ml for planktonic and sessile susceptibility testing, respectively.
Antibacterial susceptibility testing of planktonic and biofilm cells
During the course of this study two active compounds from the oral hygiene products Dentracine (Fulhold Ltd, Cape Town, South Africa) and Corsodyl (GlaxoSmithKline Consumer Health Care, UK) were tested, namely CHD-FA and CHX, respectively.
Antimicrobial testing to determine minimum inhibitory concentrations (MICs) of planktonic cells (PMIC) was performed using the CLSI M11-A8 broth microdilution method for anaerobic bacteria  and CLSI M7-A9 for bacteria grown in 5% CO2. Minimum bactericidal concentrations (MBC) were also determined by standard plating methods.
For biofilm testing standardised P. gingivalis, F. nucleatum, S. mitis and A. actinomycetemcomitans were grown for 72 h and E. faecalis for 24 h in their respective media and atmospheric conditions, with the exception of S. mutans which was grown in BHI supplemented with 2% sucrose for 48 h. Biofilms were grown statically in commercially available 96-well flat bottomed microtitre plates (Corning Incorporated, NY, USA) and sessile susceptibility testing was performed as described elsewhere . Following antimicrobial treatment, biofilms were washed twice with PBS and 10% alamarBlue® (Invitrogen, Paisley, UK) was added to the biofilms prior to incubation for 4 h in the dark . Sessile minimum inhibitory concentrations (SMICs) were read visually and no change in colour was defined as the SMIC. Testing of all planktonic and sessile isolates was performed in quadruplicate on two separate occasions.
Antibacterial susceptibility testing of a multi-species periodontal biofilm
A multi-species periodontal biofilm model consisting of P. gingivalis, F. nucleatum, S. mitis and A. actinomycetemcomitans was developed for antimicrobial testing. All bacterial species were standardised to 1 × 107 cfu/mL in artificial saliva (AS) as previously described . This was comprised of porcine stomach mucins (0.25% w/v), sodium chloride (0.35 w/v), potassium chloride (0.02 w/v), calcium chloride dihydrate (0.02 w/v), yeast extract (0.2 w/v), lab lemco powder (0.1 w/v), proteose peptone (0.5 w/v) in ddH2O. Urea was diluted in PBS (40% w/v) and added to a final concentration of 0.05% (v/v) in AS. Biofilms were prepared in 24 well plates (Corning, NY, USA) containing customised Thermanox™ coverslips (13 mm diameter, Fisher Scientific). For the addition of each bacterial species to the biofilm a standardised bacterial suspension was prepared in 500 μL of AS. Initially, S. mitis biofilms were grown for 24 h. Media was then removed and standardised F. nucleatum added, which was incubated anaerobically for a further 24 h. The supernatant was again removed and standardised P. gingivalis and A. actinomycetemcomitans in AS added to the biofilm. This was then incubated at 37°C in an anaerobic chamber for a further 4 days; each day supernatants were replaced with fresh AS. As CHD-FA was shown to be active at 0.5% v/v against all bacterial biofilms tested in this study, this concentration was used in addition to 0.2% v/v CHX to treat multispecies biofilms for 30 min, before carefully washed with PBS, and biofilm viability determined using alamarBlue®. The absorbance was read at 570 nm and the reference wavelength at 600 nm. The percentage reduction in biofilm viability was calculated according to the manufacturer’s instructions. This study was performed on three separate occasions in triplicate.
Following the antimicrobial treatment, biofilms were retained and used to quantify the number of each bacterial species found after CHD-FA and CHX treatment compared to the untreated control. Briefly, biofilms were sonicated in 1 mL of PBS for 10 min and DNA extracted using the MasterPure Gram Positive DNA Purificiation Kit (Epicentre®, Cambridge, UK), following manufacturers instructions. 1 μL of extracted DNA was added to a mastermix containing 12.5 μL SYBR® GreenER™, 9.5 μL UV-treated RNase-free water and 1 μL of 10 μM forward/reverse primers for each bacterial species. The primers used were as follows:
A. actinomycetemcomitans F – 5′GAACCTTACCTACTCTTGACATCCGAA3′, A. actinomycetemcomitans R – 5′TGCAGCACCTGTCTCAAAGC3′, F. nucleatum F – 5′GGATTTATTGGGCGTAAAGC3′, F. nucleatum R – 5′GGCATTCCTACAAATATCTACGAA3′, P. gingivalis F – 5′GCGCTCAACGTTCAGCC3′, P. gingivalis R – 5′CACGAATTCGCCTGC3′, S. mitis F – 5′GATACATAGCCGACCTGAG3′, S. mitis R – 5′CCATTGCCGAAGATTCC3′.
Three independent replicates from each parameter were analysed in triplicate using MxProP Quantitative PCR machine and MxProP 3000 software (Stratagene, Amsterdam, Netherlands). Samples were quantified based upon a previously established standard curve made up of known bacterial counts.
Ultrastructural changes of bacterial biofilms
Scanning electron microscopy (SEM) was performed on S. mutans, E. faecalis, and the multispecies biofilms. Cells were standardised in appropriate media, as described above, and grown directly onto Thermanox™ coverslips (Nunc, Roskilde, Denmark) to allow biofilm formation. Following maturation biofilms were carefully washed with PBS before their respective treatments. Biofilms were then carefully washed twice with PBS and then fixed in 2% para-formaldehyde, 2% gluteraldehyde and 0.15 M sodium cacodylate, and 0.15% w/v Alcian Blue, pH 7.4, and prepared for SEM as previously described . The specimens were sputter-coated with gold and viewed under a JEOL JSM-6400 scanning electron microscope. Images were assembled using Photoshop software (Adobe, San Jose, CA, USA).
Toxicity of CHD-FA upon an oral epithelial cell line
OKF6/TERT2 cells (gifted by the Rheinwald laboratory, Brigham and Woman’s Hospital, Boston, USA), an immortalised human oral keratinocyte cell line, were used for determining the cytotoxicity of CHD-FA. Cells were grown to 90% confluence in keratinocyte serum-free medium (KSFM) at 37°C in 5% CO2 and seeded at a density of 1 × 105 cells/ml in a 24 well plate. Once the cells reached 80-90% confluence, the cells were carefully washed with PBS before treatment with 0.5% (v/v) CHD-FA at the native pH 2.0 and a neutral pH of 7.0 and 0.2% (v/v) CHX for 30 min. After 30 min, the compounds were removed and the cells carefully washed with PBS to remove any residual actives. Cells were incubated in KSFM for 4 and 24 h before cellular viability was assessed using the alamarBlue® assay, as described above. Viability studies were carried out in triplicate, on three separate occasions.
Assessing immunomodulatory properties of CHD-FA
OKF6/TERT2 cells were grown to 90% confluence in 24 well plates in defined-KSFM then pre-treated with 0.5% CHD-FA (pH 7.0) for 30 min. CHD-FA at pH 2.0 was toxic against the cell line used in this study and therefore could not allow us to analyse any potential immunomodulatory properties of this compound. Therefore, CHD-FA buffered to pH 7.0 was used to assess any further biological properties of the compound. 0.5% CHD-FA at pH 2.0 and 0.2% CHX were shown to be toxic to epithelial cells, so were not further investigated. Cells were washed with PBS to remove residual CHD-FA. As an inflammatory agonist we used the multispecies periodontal biofilm, as described above, which was attached to the underside of a hanging cell culture insert (Millipore, Massachusetts, USA) using Vaseline®, then laid adjacent to the cell monolayer. The cells were incubated with the periodontal biofilm for 4 and 24 h at 37°C in 5% CO2. Cells not pre-treated with CHD-FA, or not challenged with biofilms, served as appropriate controls. Following stimulation, supernatants and cell lysates were retained to assess the regulation of a panel of pro-inflammatory mediators.
Initial gene expression analysis was carried out using a custom designed RT2 Profiler PCR Array (Qiagen, Crawley, UK). RT2 Profiler arrays are a SYBR® GreenER™ based real-time PCR that allow for the detection of several genes of interest, simultaneously. Briefly, 24 μl of a mastermix containing SYBR® GreenER™, cDNA synthesised using the RT2 First Strand kit (Qiagen) and RNase-free water was added to each well of the RT2 Profiler plate, which already contained the forward and reverse primers for the genes of interest (IL-1α, IL-1β, IL-6, TNF, CSF2, CSF3, IL-8, CXCL1, CXCL3, CXCL5, CCL1 and GAPDH). Two replicates of each condition were used in the RT2 Profiler, which was carried out on two separate occasions.
IL-8 gene expression was analysed using SYBR® Green based qPCR (Invitrogen), using GAPDH as a housekeeping gene. The primers used were as follows: IL-8 F 5′CAGAGACAGCAGAGCACACAA3′, IL-8 R 5′TTAGCACTCCTTGGCAAAAC3′, GAPDH F 5′CAAGGCTGAGAACGGGAAG3′, GAPDH R 5′GGTGGTGAAGACGCCAGT3′. Briefly, RNA was extracted from cell lysates (Qiagen, Crawley, UK) and 55 ng/μl of cDNA synthesised using the RT2 First Strand cDNA synthesis kit (Qiagen, Crawley, UK), as per manufacturers instructions. 1 μl of synthesised cDNA was added to a mastermix containing 12.5 μl SYBR® GreenER™, 10.5 μl UV-treated RNase-free water and 0.5 μl of forward/reverse primers. Three independent replicates from each parameter were analysed in duplicate using MxProP Quantitative PCR machine and MxProP 3000 software (Stratagene, Amsterdam, Netherlands) and gene expression normalised to the housekeeping gene GAPDH according to the 2
Interleukin 8 (IL-8) release into cell culture supernatants was assessed by ELISA (Invitrogen, Paisley, UK), as per manufacturer’s instructions. Results were calculated using a 4-parameter curve fit, quantifying colometric changes at 630 nm (BMG-Labtech, Ortenberg, Germany).
Graph production, data distribution and statistical analysis were performed using GraphPad Prism (version 4; La Jolla, CA, USA). After assessing whether data conformed to a normal distribution by before and after data transforms, One-way Analysis of Variance (ANOVA) and t tests were used to investigate significant differences between independent groups of data that approximated to a Gaussian distribution. A Bonferroni correction was applied to the p value to account for multiple comparisons of the data. Non-parametric data was analysed using the Mann–Whitney U-test to assess differences between two independent sample groups. Student t-tests were used to measure statistical differences between the ΔCt values of the two independent groups assessed in gene expression studies, although data may be represented as percentage or fold change in the figures. Statistical significance was achieved if P < 0.05.