DOI: 10.25349/d9p02h
title: 16S rRNA data for an in vitro model of the human dental plaque bacterial community (3 hosts)

description: The creation of oral microcosms with reproducible composition is important
 for developing model systems of the oral microbiome. Here, we report on
 the outcome of a methodologically simple but scientifically informative
 approach, in which we sample the dental plaque microbiome from 3
 individuals and characterize the variability among the microbiomes after
 storage and subsequent propagation. We use 24-well culture plates with
 artificially generated pellicle under a defined anaerobic atmosphere and
 an undefined medium supplemented by nutrients for fastidious organisms to
 generate the cultures, including the initial, preserved, and propagated
 cultures. Harvested cultures are extracted with the Qiagen PowerSoil kit.
 Culture composition is determined by 16S rRNA sequencing on the Illumina
 MiSeq platform and the mothur pipeline. Data analysis is performed in R
 with the phyloseq, and vegan. Our results show that cultures from
 2 out of 3 individuals cluster into an ‘attractor’ compositional type, and
 the samples from the remaining individual can adopt this compositional
 type after in vitro propagation, even though the
 original composition did not display this type. The results suggest that
 simple selective environments could help create reproducible microcosms
 from different individuals, in this case, reproducible microcosms composed
 of early colonizers of the dental plaque bacterial community. The
 attractor composition also has potential implications for
 synergistic interactions between members of the Streptococcus and
 Veillonella genera, and for antagonism between members of the
 Streptococcus and Prevotella genera. Together, these findings
 show that this dental microbiome model may be a
 promising start of a reproducible in vitro microbiome
 model that captures common "baseline" members of the human oral
 bacterial community.
description: <strong>Plaque Collection and
 Cultures: </strong> Sample collection from
 volunteer hosts took place under protocols 3-18-0189 and 3-19-0119,
 approved by the UCSB Human Subjects Committee. Collection process was
 as follows: Supragingival plaque scrapes of molar teeth from three healthy
 adult hosts were collected with sterile metal curettes, after hosts had
 abstained from food, drink, and dental hygiene practices for 12 - 16
 hours. For consistency, we limited the sites of the scrapes to the mucosal
 supragingival surfaces of 3 molar teeth, though with no restrictions on
 only upper or only lower molars. Plaque from each host was used to
 inoculate 6 mL of SHI media. Each 6mL of inoculated SHI media was equally
 divided among 3 wells in a sterile 24-well plate. Prior to receiving the
 inoculum, all plates were preconditioned with a pellicle layer, formed by
 addition of 150 μL of filtered clarified saliva (BioIVT) incubated at 37°C
 for 1 hour, and then sterilized with UV radiation at 254 nm for 1 hour. A
 separate plate was used for samples from each host. Three negative control
 wells were prepared in a fourth plate by receiving the pellicle layer and
 2.0mL of SHI media without inoculum from host plaque. 
 All plates were incubated at 37°C, in an anaerobic vessel with an
 atmosphere of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide. Every 24
 hours, approximately 1.3 mL of spent media was pipetted from the top of
 each well and replaced with 1.5 mL of fresh SHI medium, followed by a
 supplement of 20 μL of 0.5% sucrose. Plates were then returned to the
 aforementioned anaerobic atmosphere, from which they were extracted and
 processed after a total of 72 hours. These 72-hour cultures were termed
 “initial cultures”. Two of the three inoculated wells were used in the
 preservation experiment described below. The third was pelleted at 16,000
 x g for 5 minutes and flash-frozen in liquid nitrogen for later
 analysis. We investigated several methods of preserving
 the microbial communities derived from the initial cultures. After the
 initial 72 hours, two control wells and two wells from each host were
 selected, and the entire well of 1.75mL of culture was divided into
 five volumes of approximately 0.35mL each for the preservation
 experiments. Sterile glycerol was added to one of the five aliquots to a
 final concentration of 20%, and this aliquot was flash-frozen for later
 analysis and comparison. The remaining four aliquots were subjected to the
 following four preservation conditions: 4°C for 1 day, 4°C for 3
 days, cryopreservation with 40% glycerol at -80°C for 1.5 weeks, and the
 same cryopreservation conditions for 5.5 weeks. After preservation, 
 approximately 170 μL of the 350 μL sample was divided into 2 equal
 aliquots, flash-frozen with 40% glycerol, and stored at -80°C for later
 analysis. To assess microbial communities after revival
 from the preservation process, we used the remaining 180 μL of each 350 μL
 volume to inoculate approximately 6.5 mL of sterile SHI medium. The
 inoculated media was subsequently split into 3 wells in
 plates preconditioned as detailed above. These plates were incubated at
 37°C for 48 hours, under the previously mentioned atmospheric conditions
 with replacement of non-sedimented liquid and supplementation of sucrose.
 From the resulting cultures, termed ‘propagated’ cultures, one well was
 pelleted at 16,000 x g for 5 minutes and flash-frozen as a backup. The
 other two wells were mixed with glycerol to a concentration of 20%,
 pelleted as above, flash-frozen in liquid nitrogen, and stored at -80°C
 until further processing. <strong>DNA
 Extraction:</strong> Initial cultures, preserved
 cultures, and propagated cultures, along with positive and negative
 controls, were processed using the DNeasy PowerSoil kit (Qiagen).
 Negative controls included culture controls mentioned above as well as
 extraction controls, which consisted of 200 μL of fresh 1X PBS. Positive
 controls were 200 μL of ZymoBiomics microbial community standard (D6300).
 Prior to extraction, samples were randomly divided into batches of 12 or
 fewer tubes (which could be processed in a single batch). Extraction was
 begun by incubating frozen pellets on ice and washing pellets with 1X PBS
 three times. Pellets were then resuspended in 200 μL of 1X PBS and DNA
 was extracted according to manufacturer’s instructions. 
 The concentration of extracted DNA was measured using a
 fluorometric kit (QuantIT PicoGreen; Invitrogen). Estimation of bacterial
 biomass post-extraction was performed using the SSoAdvanced Universal SYBR
 Green Supermix qPCR assay (Biorad) using gene-specific primers designed to
 amplify the V4 region from the 16S rRNA gene. Quantitation by qPCR showed
 that experimental samples contained greater than 10,000 copies per μL
 (with the exception of two out of eighty-two samples) and all negative
 controls contained 17 to 2885 copies per μL. These data confirmed that the
 experimental samples contained sufficient bacterial biomass for
 sequencing. The very low copies of 16S rRNA in the negative control
 samples was consistent with the expected low bacterial biomass. All
 samples were included for sequencing.
 <strong>Amplicon Library Construction and Illumina MiSeq
 Sequencing:</strong> 16S rRNA gene amplicon
 libraries were constructed following the dual-index sequencing protocol
 developed by Kozich et al using the specific gene primers (515F-805R)
 designed by the earth microbiome project. This primer set leads to 250 bp
 amplicons, enabling full coverage of the region by the read 1 and read 2
 sequencing primer. Amplicons were generated on 96-well plates using 1 μL
 of template DNA, 1 μL of each index primer and 17 μL of Accuprime Pfx
 Supermix. Each plate contained a negative control well (1 μL of molecular
 grade water) and a positive control well (1 μL of  ZymoBiomics community
 DNA standard). The PCR was performed under the following conditions:
 initial denaturation at 95°C for 2 minutes, followed by 30 cycles of
 denaturation at 95°C for 20 seconds, annealing at 55°C for 15 seconds, and
 extension at 72°C for 1 minute, followed by a final extension at 72°C for
 10 minutes. Amplicon purification using AMPure XP beads was performed,
 followed by normalization and pooling to equimolar amounts of each sample.
 Each sample was sequenced in duplicate on different plates. Illumina MiSeq
 sequencing with PE300 V3 chemistry was performed in the genetics core of
 the Biological Nanostructures Laboratory within the California NanoSystems
 Institute at UCSB. <strong>16S rRNA
 Bioinformatics Analysis:</strong> The mothur
 software package (v. 1.40) was used to process the paired-end Illumina
 16S rRNA gene reads. Briefly, read pairs were assembled into contigs, and
 contigs with ambiguous reads were excluded, to obtain 13,249,077 total
 contigs. The contigs were aligned to version 132 of the SILVA SSU
 reference non-redundant database, and those that did not align were
 removed. A denoising algorithm and chimera identification with UCHIME were
 performed for additional quality control, resulting in 11,662,068 high
 quality sequences. The contigs were clustered at the 3% dissimilarity
 level to generate operational taxonomic units (OTUs). Sequences were
 classified against the Silva SSU reference non-redundant database (version
 132) using a naive Bayseian classifier (80% pseudobootstrap confidence
 score cutoff). The ZymoBiomics Community DNA served as our “mock
 community” to calculate sequencing error rates. When these mock
 communities were examined using the above pipeline, an overall error rate
 of 0.019277% was observed and 8 OTUs were detected (as expected from the 8
 expected bacterial species in the mock community), indicating that the
 read processing pipeline has a low error rate and does not drastically
 overestimate diversity. <strong>Statistical
 Analysis:</strong> All statistical analyses were
 performed with R (v. 4.0.3) in RStudio (v. 1.3.959), with the phyloseq
 package (v. 1.34.0). Before analyzing the cultures, we examined both
 negative controls (culture controls at all stages of preservation and
 propagation and DNA extraction controls) for potential contamination by
 comparing the number of reads and OTUs between negative controls and
 cultures. We also examined positive controls (commercially available mock
 bacterial communities) by comparing the observed microbial DNA community
 (Zymo D6305) and the extracted microbial community (Zymo D6300) with the
 expected species distribution. After verifying that the negative and
 positive controls yielded expected results, we examined sequencing depth
 by host and by sample type. To account for differences in sequencing
 depth, we also examined the number of OTUs by host and sample types, and
 then rarefied samples to a uniform level (240 reads per sample, the lowest
 number of reads among cultures) and observed the number of OTUs detected
 after rarefaction. To investigate potential underestimation of OTUs due to
 a low number of reads, we constructed rarefaction curves for all samples,
 excluding one outlier with more than 700,000 reads and two samples with
 fewer than 1,000 reads (out of eighty-two samples). To determine whether
 any correlation existed between sequencing depth and the number of OTUs
 (taken to represent the number of species), we plotted the number of reads
 against three diversity indices while omitting controls, mock communities,
 samples with fewer than 1,000 reads, and an outlier with more than 700,000
 reads. To determine the most common phyla across these same samples, we
 generated prevalence plots of sequencing depth vs. taxa prevalence
 expressed as a percentage. Rarefied samples were used for the following
 analyses, including OTUs with relative<br> abundances greater than
 0.1%. To examine the distribution of OTUs, we plotted
 relative abundances according to preservation conditions, for each host.
 Principal coordinate analysis (PCoA) was performed using relative
 abundances and the Bray-Curtis dissimilarity metric. Dot plots were used
 to examine the five most prominent OTUs, averaged by preservation
 conditions and categorized by host. Principal component analysis (PCA) was
 also performed on the dataset of initial, preserved, and propagated
 cultures. A scree plot was used to assess the contribution to variation
 from components 3 and beyond in the PCA. To assess the validity of
 performing PCA on relative abundance data, we used centered-log-ratio
 (CLR) and isometric log-ratio (ILR) transformations on the data and
 subjected the transformations to PCA. The mathematical procedure for
 CLR transformations is outlined in Chapter 2. The ILR transformation is
 defined as follows:  ilr(x) = [&lt;x;
 e<sub>1</sub>&gt;<sub>a</sub>; ...;&lt;x;
 e<sub>D-1</sub>&gt;<sub>a</sub>]
 where [e1,...,e<sub>D-1</sub>] is an orthonormal
 basis in the simplex. Alternatively, ILR transformations can be done by
 using CLR basis sets in the following way: ilr(x)
 := V<sup>t</sup>clr(x) where clr(x) is the
 centered log-ratio transform and V is a matrix whose columns form
 an orthonormal basis in the CLR plane. For this analysis, we used the
 default parameters from the ilr function of the compositions package in R
 to transform the relative abundances, and then subjected the transform to
 ordinary PCA procedures. We also performed significance tests using the
 ANOSIM function in the vegan package, with Bray-Curtis distance
 measurements and 999 permutations to test whether and how much
 preservation conditions and/or host differences influence the relative
 abundance differences across samples.