ABSTRACT Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial

community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5 and 7 days with saliva on day 0 were collected from 19 young adults using

a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that

the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a

dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the

plaque bacterial community as well as the inter-individual differences associated with dental caries experience. SIMILAR CONTENT BEING VIEWED BY OTHERS THE RECOVERY OF THE MICROBIAL

COMMUNITY AFTER PLAQUE REMOVAL DEPENDS ON PERIODONTAL HEALTH STATUS Article Open access 07 October 2023 STRUCTURAL CHANGES IN THE ORAL MICROBIOME OF THE ADOLESCENT PATIENTS WITH MODERATE OR

SEVERE DENTAL FLUOROSIS Article Open access 03 February 2021 DENTAL BLACK PLAQUE: METAGENOMIC CHARACTERIZATION AND COMPARATIVE ANALYSIS WITH WHITE-PLAQUE Article Open access 29 September

2020 INTRODUCTION Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species1. The development of biofilm begins with adherence of selected bacteria, including

_Streptococcus_ species, to saliva-bathed surfaces. As bacteria cover the substratum, later colonizers attach to the initial colonizers. The biofilm shows increasing diversity over time2.

Multiplication of the attached organisms results in an increase in biomass, as well as a population shift due to altered environmental conditions within the biofilm. Marsh _et al_.3 stated

that changes in key environmental factors, such as atmosphere, pH and nutrients, drive a compositional shift in the microbiota toward a disease-associated community, leading to a greater

risk of dental caries or periodontitis. Microbial succession in dental plaque development was summarized by Ritz4. It is considered to be an ordered sequence of events, with facultative and

aerobic bacteria, such as _Streptococcus_, _Neisseria_ and _Rothia_, predominantly colonizing the tooth surface in the early stages and the proportions of facultative and anaerobic genera,

such as _Actinomyces_, _Corynebacterium_, _Fusobacterium_ and _Veillonella_, increasing gradually with plaque maturation. Furthermore, a spatiotemporal model of oral bacterial colonization

was proposed by Kolenbrander _et al._5 based on the recognition of salivary pellicle receptors by initial colonizing species and coaggregation by specific bacterial combinations. Conversely,

Langfeldt _et al._6, using a 16S rRNA gene sequencing approach, recently reported that no systematic time-related pattern was detected in the compositional shift of bacterial biofilm formed

on a membrane installed in the oral cavity. Although it is unclear which architecture in the oral cavity was mimicked by the artificial membrane, this result is inconsistent with the

conventional concept of oral biofilm establishment. Therefore, it is reasonable that the development of plaque microbiota on tooth surfaces should also be reconsidered using a comprehensive

molecular approach, including difficult-to-cultivate bacteria. In the present study, we observed the assembly process of dental plaque microbiota on a hydroxyapatite disk in 19 young adults

through molecular approaches using the 16S rRNA gene. We collected plaque samples for 1, 2, 3, 4, 5 and 7 days using a removable resin splint (Figure 1) and the bacterial community structure

was evaluated using a barcoded pyrosequencing approach. In addition, the maturation process of the plaque bacterial community was compared between 9 caries-free (CF) subjects and 10

caries-experienced (CE) subjects. RESULTS Dental plaque samples that accumulated on a hydroxyapatite disk (5-mm diameter) for 1, 2, 3, 4, 5 and 7 days were retrieved from 19 healthy young

adults (Table 1). The total amounts of bacteria on the disks were examined by quantitative PCR analysis of the 16S rRNA gene (Figure 2A). They gradually increased and had almost reached a

plateau at about 109 CFU by day 4. The composition of the bacterial community in accumulating plaque, together with saliva collected just before installing a mouth splint, were investigated

using barcoded pyrosequencing analysis of the 16S rRNA gene. We determined 692,400 bacterial 16S rRNA gene sequences (containing the V1–V2 region), of which 470,423 passed quality-control

steps (822–7,904 reads per sample, Table S1; average length, 346 ± 18 bases). The sequences were assigned to 4,936 species-level operational taxonomic units (OTUs) using a cutoff distance of

0.03. Alpha diversity metrics including the number of detected OTUs, Shannon diversity index and phylogenetic diversity increased over time, showing an especially large increase between

days 5 and 7 (Figure 2B). A principal coordinate analysis (PCoA) plot based on unweighted UniFrac showed a gradual temporal shift of the plaque microbiota to the negative direction of

principal coordinate 1 (Figure 3), suggesting that the community assembly of plaque biofilm occurred in a time-related manner. Permutational multivariate analysis of variance (PERMANOVA)

confirmed that the plaque accumulation time had a significant effect on the composition of the microbiota (_P_ < 0.001). The temporal shift in plaque microbiota composition was also

characterized by changes in the relative abundances of bacterial taxa. Plaque samples on the later days exhibited greater microbial richness and diversity (Figure 2B) and were plotted closer

to the saliva samples in the PCoA UniFrac diagram (Figure 3), suggesting that a greater variety of salivary bacterial taxa was able to grow in the later stages of plaque development. Of the

53 genera identified in the plaque microbiota, _Streptococcus_ species made up most of the microbiota in each subject on all days (Figure 4). Facultative anaerobic bacteria such as

_Neisseria, Abiotrophia_, _Gemella_ and _Rothia_ were predominant in the plaque bacterial community during the earlier days, whereas obligate anaerobes, such as _Porphyromonas_,

_Fusobacterium_, _Prevotella_, _Veillonella_ and _Capnocytophaga_ showed increased dominance on later days, with decreases in facultative members, such as _Streptococcus_ (Figure 4). Of

4,936 species-level OTUs, the representative sequences of 343 OTUs corresponded to the sequences deposited in the Human Oral Microbiome Database (HOMD) with ≥98.5% identity. A total of 31 of

these 343 OTUs were commonly detected (15 of 19 subjects) in the plaque bacterial community on day 7 and they made up large proportions of the community (83.6 ± 9.1% and 68.6 ± 8.2% on days

1 and 7, respectively). A temporal shift in their relative abundances is displayed as a heatmap in Figure 5. The OTUs corresponding to _Streptococcus_ species, such as _S. mitis_, _S.

sanguinis_ and _S. oralis_ were more predominant on earlier days. Other than _Streptococcus_, _N._ _flava, H._ _parainfluenzae_, _A._ _defectiva_, _R. mucilaginosa_ and _G._ _haemolysans_

could also be considered early colonizers. The relative abundance of the OTU corresponding to _Porphyromonas_ sp. OT-279 peaked on days 4 and 5. Subsequently, several OTUs corresponding to

bacterial species such as _G. adiacens_, _Streptococcus_ sp. OT-65, _F. periodonticum_ and _N. flavescens_ appeared and bacterial species, including _V. parvula_, _S. gordonii_ and _G.

morbillorum_ were flourishing on day 7. The total amount of bacteria on the disks and microbial richness and diversity, increased over time in both CF and CE subjects (Figure 6). In

contrast, significantly larger amounts of bacteria were present on the disks of CE subjects than CF subjects on days 1 to 3 (_P_ < 0.05, Student's t-test, respectively), suggesting

that the amount of bacteria reached a plateau later in CF subjects than in CE subjects. A PCoA plot based on UniFrac showed that the developing plaque microbiota of CF subjects localized in

the positive direction of principal coordinate 2, suggesting that the community assembly of their plaque biofilm occurred by a characteristic process (Figure 7). PERMANOVA confirmed that the

dental caries experience also had a significant effect on the microbiota composition (_P_ < 0.001). We performed two-way analysis of variance (ANOVA) with repeated measures for the 343

OTUs to explore the important OTUs responsible for the differences in the assembly process of plaque microbiota between CE and CF subjects (Figure 8). Significant effects of dental caries

experience were observed in the relative abundance shifts of 7 of 343 OTUs, corresponding to the bacterial species deposited in the HOMD with ≥98.5% identity (_P_ < 0.05, Figure 8). The

OTU corresponding to _S. oralis_ OT-707 was observed in a lower proportion of CF subjects than in CE subjects regarding plaque development, particularly on days 3–7. The OTUs corresponding

to _N. flava_ OT-609 and _G. haemolysans_ OT-626 were more predominant in CF subjects than in CE subjects, particularly during the earlier days. The OTUs corresponding to _V. dispar, V.

parvula_ and _G. adiacens_ showed significantly lower proportions in CF subjects than in CE subjects on day 4. The OTU corresponding to _G. adiacens_ showed significantly lower proportions

in CF subjects than in CE subjects on day 5 and the OTUs corresponding to _V. dispar_ and _Actinomyces_ sp. OT-180 were significantly lower on day 7. DISCUSSION Our results using the 16S

rRNA gene sequencing approach revealed microbial succession in dental plaque biofilm established on a hydroxyapatite disk. A plaque microbiota shift was observed in a time-related manner

during maturation (Figures 2, 3, 4 and 5), although inter-individual differences, especially associated with dental caries experience were also observed regarding the composition of

developing plaque microbiota at each stage (Figures 6, 7 and 8). Initially, we expected that the results of this study would differ from the plaque assembly process reported by Ritz4 due to

the difference between their cultivation-based method and our molecular approach, which allowed determination of the composition of the microbiota, including difficult-to-cultivate species.

However, the plaque-development process suggested by our results is unexpectedly consistent with that study. The bacterial communities were assembled in a time-related manner, from an early

biofilm dominated by _Streptococcus_ species to a complex community with high levels of Gram-negative anaerobes (Figure 4). This process demonstrates the effectiveness of a classic

cultivation-based approach for providing an overview of oral bacterial communities, as well as the reliability of our results. In addition, it included more information regarding candidate

early, middle and late colonizer genera (Figures 4 and 5), adding to those reported previously7. Of the bacterial genera missed in Ritz's study4, _Porphyromonas_ species are noteworthy

as a dominant middle colonizer because they displayed similar behavior to _Fusobacterium_ in developing plaque microbiota (Figure 4). _Fusobacterium_ species are considered important

“bridge” organisms that coaggregate initial, early and late colonizers and contribute to an increase in microbial diversity in plaque development8. Our results suggest that _Porphyromonas_

species, particularly _Porphyromonas_ sp. OT-279, may be a new target for the examination of plaque biofilm formation mechanisms. Total amounts of bacteria had almost plateaued by day 4

(Figure 2A). The multiplication and shedding of bacteria presumably kept occurring after the plateau was reached, but about 109 CFU would represent a threshold amount of dental plaque that

could form on the hydroxyapatite disk of 5-mm diameter. Conversely, the alpha diversity metrics increased rapidly between days 5 and 7 (Figure 2B). In particular, phylogenetic diversity

which accounts for species evolutionary relationships did not become elevated until day 4, suggesting that bacteria taxonomically distinct from the early colonizers begin to participate in

the plaque microbiota development after day 4. This is confirmed in Figure 5. These findings suggest that accumulation of a threshold amount of bacteria in plaque biofilm triggers major

shifts in plaque microbiota composition. They also imply that saturation of plaque biomass does not indicate the ultimate state of the plaque microbiota succession and the plaque bacterial

communities at day 7 might not yet have reached their final composition because the complexity of the plaque microbiota at this timepoint was increasing (Figure 2B). When the subjects were

classified into two groups according to dental caries status, notable differences in the plaque bacterial community between CF and CE subjects were observed from day 1. The OTUs

corresponding to _N. flava_ and _G. haemolysans_ made up a higher proportion of the microbiota in CF than in CE subjects, particularly during the early stages (Figure 8). When considering

the lower total amount of bacteria in CF subjects (Figure 6A), the marked growth of _Streptococcus_ during the early stages was inhibited in the more complex plaque microbiota of the CF

subjects. These bacteria were implicated as candidate species that might protect against caries onset and progression in a previous clinical study with a longitudinal design9. Determining

their interaction with acid producers in initial biofilm formation might facilitate the development of novel approaches that might enable assembly of healthy plaque that is less prone to a

shift toward a pathogenic community. Although the differences in the amount of bacteria between CF and CE subjects lessened over time (Figure 6A), the OTU corresponding to _S. oralis_ was

more predominant in CE subjects than in CF subjects (Figure 8). In addition, the growth of non-_Streptococcus_ acid producers, such as _Actinomyces_ and _Granulicatella_ species, during the

later stages was slower in CF subjects (Figure 8)_. Veillonella_ species, which metabolize lactate, were also less predominant in later plaque (Figure 8), in accordance with several previous

studies, indicating that elevated levels of _Veillonella_ were associated with higher acid production within plaque9,10,11,12. The developing plaque in CF subjects likely had a lower

acidogenic potential than that in CE subjects during the later stage of the current study period. Because formation, succession and removal of dental plaque on the tooth surface occur

repeatedly, the differences observed in the assembly process may be involved in the susceptibility to dental caries. Further evaluation of the microbiota shift upon carbohydrate ingestion

and pH measurement within dental plaque would be helpful to clarify their contribution to the progression of dental caries. We performed multiple statistical tests for 343 OTUs to identify

those responsible for the differences in the assembly process of plaque microbiota between CE and CF subjects (Figure 8). We did not adjust for multiple testing, because such adjustments are

not strictly required in exploratory analyses13. As the choice and number of tested hypotheses is data dependent in exploratory analyses and a clear structure in the multiple tests is

missing, an appropriate multiple test adjustment is difficult or even impossible13. Additionally, the number of tests was too large to use Bonferroni adjustment, which is the simplest and

best-known multiple test procedure. With as many as 300 tests, this procedure has insufficient power to detect any true effect13. Actually, no OTU was selected after the adjustment, although

PERMANOVA analysis on UniFrac results confirmed differences in the assembly process between CE and CF subjects (Figure 7, _P_ < 0.001). On the other hand, interpretation of the

significant results in the exploratory analyses needs careful consideration, as the significant relationships shown in this study were only exploratory results. Further, confirmatory studies

with larger sample sizes are required to identify important bacterial species in the plaque assembly process of CF and CE subjects. We observed plaque accumulation on hydroxyapatite disks

instead of natural tooth surfaces. Tooth enamel is mostly (96%) composed of hydroxyapatite and the remainder is organic substances and fluid. The pristine artificial surfaces of the disks

were bathed in saliva immediately after installing a mouth splint and were coated with salivary proteins in the same way as natural teeth in the oral cavity. In addition, considering the

consistency of our results with those of a previous study using natural teeth4, our model is considered to mimic the bacterial community assembly on intact enamel surfaces. However, the

variability in microbial colonization patterns occurring at various ecological niches in the oral cavity must also be considered14. Different processes of plaque development might be

observed in other dental sites, such as gingival sulcus and dental fissures in molars. Our results indicate the bacterial communities were assembled in a time-related manner consistent with

a previous study4 and reconfirm that dental plaque biofilm that forms on tooth enamel is a dynamic microbial community. The microbial composition gradually shifts with accumulation time on

tooth surfaces; therefore, the sampling of dental plaque microbiota as a representative of an individual oral microbiome should be performed with caution. METHODS STUDY POPULATION Subjects

were recruited from among undergraduate students at Kyushu University, Faculty of Dental Science and were aged 20 to 28 years. After an oral examination by a dentist, 9 CF subjects and 10 CE

subjects (who had experienced ≥9 teeth with caries) were enrolled. Although three CE subjects had active caries regions as well as previously filled teeth, all of them were not obvious

cavity but were localized only in the enamel or were secondary caries along the inlay margin. The ethics committee of Kyushu University Faculty of Dental Science approved the study design

and the procedure for obtaining informed consent (Reference number, 20-11 and 26-130). The subjects reported no antibiotic intake in the study period and the preceding 1 month. No subjects

with severe periodontitis or any systemic disease were included in the study population. All experiments were performed in accordance with the approved guidelines. ORAL DEVICE Removable

resin splints were fabricated for each subject to cover the mandibular buccal gingiva and were retained with a wire clasp. On the left and right sides of the buccal surface, six

5-mm-diameter hydroxyapatite disks (Cellyard, Hoya, Japan) were attached to the splint using wax (Figure 1). After paraffin-stimulated saliva collection, subjects wore the splint for 7 days,

except while eating, drinking liquids other than water, or tooth-brushing, when the splint was immersed in phosphate-buffered saline (PBS; pH 7.4). At 1, 2, 3, 4, 5 and 7 days later, one of

the disks was retrieved from each side of the splint after rinsing with PBS. We initially focused on the plaque development process especially in an early stage, therefore we omitted day-6

sample collection due to the limited space on the splint. DNA EXTRACTION Disks covered with plaque biofilm were placed in microcentrifuge tubes containing 500-µl lysis buffer (10 mM Tris, 1 

mM EDTA, 1% SDS). After ultrasonic vibration to completely detach the biofilm, bacterial DNA extraction was performed as described previously15. The left side samples were used for the later

analysis, except when the corresponding disk compromised. DNA extraction from saliva samples was performed according to a protocol described previously15. QUANTIFICATION OF TOTAL BACTERIA

BY REAL-TIME PCR Quantitative PCR was performed using a QuantiFast SYBR green PCR kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The universal bacterial

primers 806F (5′-TTA GAT ACC CYG GTA GTG G-3′) and 926R (5′-CCG TCA ATT YCT YYG AGT TT-3′) were used and details of the procedure have been reported previously16. The amounts of total

bacteria were calculated using the comparative _C_T method and DNA extracted from _Streptococcus mutans_ Xc was used as the real-time PCR control. BARCODED PYROSEQUENCING ANALYSIS In total,

133 samples (7 each from 19 subjects) were assessed by barcoded pyrosequencing analysis of the 16S rRNA gene. The samples were separated into three groups: plaque samples taken on days 1 and

2 and those of CE subjects taken on day 3 (totaling 48 samples); plaque samples taken on days 3 and 4 and those of CF subjects taken on day 3 (totaling 47 samples); and plaque samples taken

on day 7 and saliva samples (totaling 38 samples). The 16S rRNA genes of each sample in each group were amplified using the following primers: 338R with the 454 Life Sciences (Roche, Basel,

Switzerland) adaptor B sequence (5′-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG TGC TGC CTC CCG TAG GAG T-3′) and 8F with the 454 Life Sciences adaptor A and 48 different sample-specific

six-base barcodes (5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG NNN NNN AGA GTT TGA TYM TGG CTC AG-3′). PCR amplification was performed as previously described17. The amplicons were

gel-purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. DNA concentration and quality were assessed using a

spectrophotometer (NanoDrop Technologies, Wilmington, DE) and equal amounts of DNA from each group were pooled together. Pyrosequencing was conducted for each mixture using a 454 Life

Sciences Genome sequencer FLX instrument (Roche) at Hokkaido System Science Co., Ltd. (Sapporo, Japan). DATA ANALYSIS AND TAXONOMY ASSIGNMENT Sequences obtained from all samples were

analyzed together. Sequences were excluded from the analysis using a script written in PHP if they were shorter than 240 bases or had an average quality score <25 and subsequently removed

using a script written in R if they did not include the correct primer sequence, had a homopolymer run >6 nt, or contained ambiguous characters. The remaining sequences were assigned to

the appropriate sample by examining the six-base barcode sequence. Similar sequences were clustered into operational taxonomic units (OTUs) using UCLUST18 in QIIME19, with a minimum pairwise

identity of 97%. The most abundant sequence in each OTU was chosen to represent that OTU. The representative sequences were aligned using PyNAST20 and the Greengenes database21 using a

minimum identity of 75%. Chimeras were removed from the representative set on the basis of identification as being chimeric using Chimera Slayer22. After chimera elimination, a relaxed

neighbor-joining tree was built using FastTree23. The UniFrac metric24 was used to determine the dissimilarity between any pair of bacterial communities. The similarity relationship,

assessed using the UniFrac metric, was presented in a PCoA plot, drawn using R 3.0.1. The taxonomy of representative sequences was determined using the RDP classifier with a minimum support

threshold of 80% and the RDP taxonomic nomenclature (to the genus level). For each representative sequence of the OTUs, nearest-neighbor species with ≥98.5% identity were selected as

candidates using BLAST searches against 831 oral bacterial 16S rRNA gene sequences (HOMD 16S rRNA RefSeq version 13.2) in the Human Oral Microbiome Database25. STATISTICAL ANALYSIS All of

the statistical analyses were conducted using R 3.0.1 (available from URL: http://www.r-project.org/). Student's _t_-test was used for comparison of the total bacterial amount per

sampling time. Number of OTU and Shannon index were calculated using vegan library. Phylogenetic diversity was calculated using pd function in picante library. Permutational multivariate

analysis of variance (PERMANOVA) was used to assess the effects of plaque accumulation time and dental caries experience on plaque microbiota composition using adonis function in vegan

library based on 9,999 permutations. Two-way analysis of variance (ANOVA) with repeated measures was used to assess the effects of plaque accumulation time and dental caries experience on

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ACKNOWLEDGEMENTS This study was supported in part by Grants-in Aid for Scientific Research 25463249 (T.T.), 22592337 (Y.S.) and 25293428 (Y.Y.) from the Ministry of Education, Culture,

Sports, Science and Technology of Japan. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Section of Preventive and Public Health Dentistry, Growth and Development, Division of Oral Health,

Kyushu University Faculty of Dental Science, Fukuoka, Japan Toru Takeshita, Masaki Yasui, Yukie Shibata, Michiko Furuta & Yoshihisa Yamashita * Oral Science Section, Central Laboratory,

Lotte Co., Ltd., Saitama, Japan Yoji Saeki * Department of Biostatistics, Oita University Faculty of Medicine, Yufu City, Oita, Japan Nobuoki Eshima Authors * Toru Takeshita View author

publications You can also search for this author inPubMed Google Scholar * Masaki Yasui View author publications You can also search for this author inPubMed Google Scholar * Yukie Shibata

View author publications You can also search for this author inPubMed Google Scholar * Michiko Furuta View author publications You can also search for this author inPubMed Google Scholar *

Yoji Saeki View author publications You can also search for this author inPubMed Google Scholar * Nobuoki Eshima View author publications You can also search for this author inPubMed Google

Scholar * Yoshihisa Yamashita View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.T., M.Y., Y.S. and Y.Y. designed and supervised the

research project. T.T. wrote the paper. M.Y., T.T. and Y.S. conducted oral examination and sample collection. M.Y. conducted molecular analysis. T.T. conducted data processing. T.T., M.F.

and N.E. conducted statistical analysis. All authors read and approved the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

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plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach. _Sci Rep_ 5, 8136 (2015). https://doi.org/10.1038/srep08136 Download

citation * Received: 23 September 2014 * Accepted: 08 January 2015 * Published: 30 January 2015 * DOI: https://doi.org/10.1038/srep08136 SHARE THIS ARTICLE Anyone you share the following

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