Subjects: Study protocols were approved by the Ethics Committees of the UWI, St. Augustine. Thirty healthy volunteers from The University of the West Indies (UWI), St. Augustine, Trinidad were enrolled in the study. To be eligible, subjects must have had at least 20 teeth, provided written informed consent and been willing to comply with study procedures. Subjects with systemic, infectious or inflammatory diseases or taking medicines, antibiotics or fluoride in the last month, habitual consumers of xylitol / sorbitol-containing products and mouth rinses, with abnormal salivary flow (\<1ml / min), pregnant, on contraceptive pills, or with abnormal dietary habits were excluded. Consent obtained at the initial visit was verified at the second visit, prior to sample collection. The subjects were examined in the dental chair after thorough medical and dental histories were recorded. The clinical examination involved examination of the soft tissues and then dental hard tissue charting for presence of decayed, missing or filled teeth. No radiographs were used. The presence of untreated dental caries or periodontitis were not used as exclusion criteria. The decayed, missing, and filled teeth for each individual at the initial visit was documented for calculation of the index of decayed-missing-filled permanent teeth (DMFT) score.
Chewing gums: Xylitol gum (Epic Spearmint; 1.5g/pellet) designated Gum X contains 70% xylitol in addition to gum base, natural flavors, soy lecithin, gum arabic, titanium dioxide, carnuba wax. Gum S (Eclipse Spearmint), was similar except that xylitol was replaced by 63% sorbitol and 2% maltitol and aspartame was included. Gums were packed in colour-coded containers. Codes were kept confidential from the participants and researchers who interacted with them until study completion.
Study design: This prospective cross-over, double-blind, randomised study lasted 14 weeks (March - June, 2015). Throughout, subjects were instructed not to use mouthwashes or xylitol products, to consume a normal diet, continue their usual tooth brushing and to report use of antimicrobial medications. Subjects reporting the latter were dropped.
Subjects were randomly allocated to two groups, A and B (see Figure 1). Both groups entered a 4-week "washout period" during which no gum was chewed, followed by a 3-week treatment period (treatment period 1) during which Group A used Gum X and Group B used Gum S (2 gum pieces, 3 times daily after meals for 6 minutes). Both groups then underwent another 4-week washout period before entering treatment period 2 during which Group A used Gum S and Group B used Gum X for 3 weeks.
Sample collection: Saliva and plaque were collected from participants immediately before and after each treatment period (Figure 1). Subjects were instructed not to brush their teeth or use any other oral hygiene procedures at least 24 hours before sample collection, and not to eat or drink at least 1 hour before. For saliva collection, subjects chewed sterile paraffin wax and whole saliva produced was collected for 5 minutes in sterile tubes. The subject was asked to drool into the labelled 50ml collection tube (Falcon sterile, conical polypropylene tube with flat-top screw cap). This process was repeated multiple times in order to collect larger volumes of saliva (2-5 ml). The saliva was transferred via sterile pipettes into labelled sterile centrifuge tubes which were stored at -70˚C until use.
Supragingival plaque was collected with a curette from 2 molars (#16 and #36), 2 premolars (#24 and #44), and 2 incisors (#21 and #41); Plaque collection involved using a Gracey curette to remove all of the supragingival plaque from the buccal surface of the selected index tooth with as many strokes as necessary. The curette tip was immersed in the sterile DNase-free Tris-EDTA (TE) buffer in the centrifuge tube for 4-5 seconds. The face of the curette was wiped on the inside edge of the collection tube and then wiped off with sterile gauze so as to not let the buffer on the tip go into patient's mouth. The site was then immediately sampled again using the same procedure. The lid on the tube was replaced and then the tube shaken for 4-5 seconds in an attempt to maximize dispersion of the specimen in the fluid. The tube was immediately placed on ice in a Ziploc bag and stored at -70˚C until use. Samples were labelled by group (A/B), treatment period (1/2), gum used (X/S in the case of those samples collected at the end of a given treatment period) and type (saliva (s) or plaque (p)). Samples (n=232) were then shipped on dry ice to the J. Craig Venter Institute (JCVI) USA, La Jolla campus for DNA extraction and sequencing.
DNA extraction: Samples were thawed at 4˚C and vortexed thoroughly prior to DNA extraction from 500ul of saliva or plaque suspension using bead beating Lysing Matrix B tubes (MPBio Inc.), then lysozyme digest, phenol/chloroform isoamyl alcohol extraction and ethanol precipitation. Precipitated DNA was resuspended in 1x TE buffer.
Library Preparation and Sequencing: DNA from each sample was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc, Waltham, MA). Amplicons were generated using adaptor and barcode ligated polymerase chain reaction (PCR) primers targeting the V4 region of the 16S rDNA gene (16S) and purified using Qiaquick PCR purification kits (Qiagen, Inc) following manufacturer's instructions. Purified amplicons were quantified using SybrGold (Thermo Fisher Scientific, Inc, Waltham, MA), normalized to ensure equimolar quantities of each sample, and pooled in preparation for Illumina MiSEQ sequencing. The 16S library pool was sequenced using the Illumina MiSEQ dual index 2x250bp V2 chemistry kit according to manufacturer's specifications.
16S RNA sequence data processing: Sequences for each sample were binned according to corresponding dual indices and exported as individual .fastq files using CASAVA v1.8.2 (Illumina Inc. La Jolla, CA). Sequences were processed to ensure that only quality sequences were retained, as stringent settings were kept to ensure no barcode mismatches were permitted during demultiplexing. Processed sequences were applied to the Infernal pipeline \[35\] for additional quality control (QC) checks. Bacterial sequences were taxonomically assigned based on the Genomic-based 16S ribosomal RNA Database (GRD; http://metasystems.riken.jp/grd/).
Statistical analyses. Distribution by age, sex and DMFT score for subjects in groups A and B were compared using independent-samples T- test, Pearson's Chi-Squared test and Wilcoxon rank sum test respectively, with a cut-off of p \<0.05. To avoid possible sequence errors, operational taxonomic unit (OTU) count tables were filtered such that OTUs present in fewer than 0.1% in all samples were discarded. OTU tables were then transformed to relative abundances before community analyses were performed using the R statistical computing language (Cite R). Kruskal-Wallis test was used to assess statistical significance in microbial community composition across treatments. Wilcoxon test was used for pairwise comparison.
