HOMIM was a high-throughput identification array which used 16S rRNA probes to
simultaneously identify the most predominant 300+ bacterial species associated with the oral
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hybridisation probes were printed onto a glass slide (Fouad, 2009; Preza et al., 2009) and
using broad-range primers, 16S rRNA genes within the unknown clinical sample were
amplified and labelled with a cyanine (blue-green) dye molecule (Paster & Dewhirst, 2009;
Preza et al., 2009). The labelled amplicons were hybridised overnight to the slide-printed
probes and then scanned (532nm) to detect Cy3-amplicon hybridised probes. Signals were
then translated into a ‘barcode’ format where intensities could be read within a range of 0 (species unidentified), 1+, 2+, 3+, 4+ and 5+, the lower limit (1+) corresponding to
approximately 104 cells (Colombo et al., 2009).
Depending upon the line of enquiry, HOMIM raw data was sub-categorised into phylum
genera or phylotype and compared at inter- and intra-group levels (i.e. Control, Behavioural
and Fluoride) using Microsoft Excel. The relative abundance (%) was calculated by dividing
the individual hybridisation intensity (HI) by the total (phyla, genera etc.) and then multiplied
by 100. Results were then compared to similar studies in the literature (Crielaard et al., 2011)
for confirmation of category distribution. Statistical analyses were carried out as described in
section 2.8.
2.6.1. Proposed Isohelix protocol and Isohelix-HOMIM trial.
The final stage of the optimisation process was to test human oral samples collected using the
Isohelix system for species-specific bacterial load using HOMIM analysis. This required an
alteration to the final step of the Isohelix gDNA purification protocol, specifically the
resuspension of genomic materials in TE buffer (in readiness for downstream reactions) with
the freezing and lyophilisation of sample materials for overseas transport. This step was
complementary to the HOMIM protocol as resuspension of clinical samples prior to
hybridisation was done so in TE solution. Therefore, the developed Isohelix protocol was
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One swab was taken from four persons (not associated with the Bright Smiles study) aged
approximately 13 y, 8 y, 4 y and 2 y and assigned a unique serial numbers 2000-13.6-13,
2000-8.9-13, 2005-4.10-13, and 2005-2.5-13 respectively; the serial numbers were made up
of three number sets indicating firstly the time of sample harvest (in 24 h), age of the
volunteer (years and months) and then the time (hours and minutes) since previous oral clean.
Samples were harvested from the posterior incisor and lateral incisor tooth surface, targeting
supragingival plaque over a period of no less than 30 s. Samples were collected from the
incisors and lateral incisors only, this was stipulated early in the study as these were the only
erupted teeth available for sampling at Baseline but expanding the sampling sites based on
newly available teeth may introduce numerous variables, reducing comparability between
sample groups, in subsequent years.
The swab head was then submerged in the Isohelix lysis and stabilisation solution (Cooper &
Hole, 2012a), mixed via inversion and stored at room temperature (21°C) for transportation to
the lab. Upon arrival (approximately 72 h later), the swab head was removed from each
sample, which were then mixed thoroughly and split in two; one half was archived at -20°C
and the remainder of each sample was processed under manufacturers guidelines, stored at -
80°C (72 h), freeze-dried (5 d) and despatched to the Forsyth Institute (Cambridge, MA.
USA) for HOMIM analysis.
The process of splitting the samples involved one additional step after gDNA isolation.
Genomic DNA was isolated as described by the manufacturer (Cooper & Hole, 2012a;
Cooper & Hole, 2012b) however, the after the elution step each sample was vortexed briefly
and half was transferred to a sterile tube and stored at -20C. Sample splitting was carried out to ensure against lost or damaged samples or the need to re-assess. If samples posted to the
USA were lost in transit there would be second half archived for back-up or repeat analyses.
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some vital data due to biases caused by handling. In order to identify any potential issues,
internal controls were added to the Bright Smiles samples and analysed via HOMIM; this
included both halves of one sample (see section 5.2), donated by the author which would
indicate any biases in bacterial distribution.
2.6.2. The collection of oral bacteria for HOMIM analysis using the Isohelix DNA
isolation system.
The finalised method for the collection of oral bacteria from the Bright Smiles cohort and
subsequent analyses are as described in sections 2.1.1, 2.1.1.1, 2.1.2, 2.3 (2.3.4-5), 2.6, 2.6.1-
2 and 2.8, however, briefly, Isohelix kits (DDK-50 DNA stabilisation and extraction kit; SK-3
individually wrapped swabs and sterile 2 ml screw cap tubes) were implemented as described
by the manufacturer (Cooper & Hole, 2012a; Cooper & Hole, 2012b). Lysis solution was
prepared, no more than eight hours prior to the sampling appointment. For each sample, 20 µl
PK was mixed with 500 µl LS in a sterile 2 ml crew cap, briefly vortexed and stored at room
temperature. Based on the success of the pilot study, described in sections 3.9., 3.12., 3.13.,
and 3.14, the sampling process was modified to include both the buccal and palatal surfaces of
the upper incisors.
Immediately before sampling, the swab was removed from its packaging, taking care not to
touch the swab head with hands, clothing or other equipment. Holding the swab by the
handle and using firm circular motion, the swab head was rubbed over the inner and outer
surfaces of the incisors and lateral incisors, (covering tooth surfaces and gingival margins) for
no less than 30 seconds. The swab was immediately placed into the lysis solution. Care was
taken not to contaminate the sample head, which was severed from the handle at the ‘break
point’ allowing the tube cap to be screwed shut. The tube was briefly shaken to mix and then stored at room temperature. Finally, prior to HOMIM analysis all samples harvested from the
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Bright Smiles cohort were split into two (section 2.6.1), one half was sent to HOMIM for
composition analysis and the other was archived at -20°C. Species identification and
distribution was carried out by HOMIM as described in section 2.6.
Hybridisation data was converted into three taxonomic levels, the phylum, genus and
phylotype and mean profiles were analysed and compared for trends in biofilm development
that could be linked to the implementation of described interventions. Specific analyses were
carried out on the relative abundances of phyla, genera and phylotype at both inter- and intra-
study categories which included intervention groups (e.g. Behavioural vs Fluoride) and
sampling periods (e.g. Baseline vs. Round 1).