Characterization of bacteriophage communities and CRISPR profiles from dental plaque
© Naidu et al.; licensee BioMed Central Ltd. 2014
Received: 24 September 2013
Accepted: 26 June 2014
Published: 30 June 2014
Dental plaque is home to a diverse and complex community of bacteria, but has generally been believed to be inhabited by relatively few viruses. We sampled the saliva and dental plaque from 4 healthy human subjects to determine whether plaque was populated by viral communities, and whether there were differences in viral communities specific to subject or sample type.
We found that the plaque was inhabited by a community of bacteriophage whose membership was mostly subject-specific. There was a significant proportion of viral homologues shared between plaque and salivary viromes within each subject, suggesting that some oral viruses were present in both sites. We also characterized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in oral streptococci, as their profiles provide clues to the viruses that oral bacteria may be able to counteract. While there were some CRISPR spacers specific to each sample type, many more were shared across sites and were highly subject specific. Many CRISPR spacers matched viruses present in plaque, suggesting that the evolution of CRISPR loci may have been specific to plaque-derived viruses.
Our findings of subject specificity to both plaque-derived viruses and CRISPR profiles suggest that human viral ecology may be highly personalized.
KeywordsOral biofilm Virus Virome Microbiome Dental plaque CRISPR
Much of the study of the human microbiome has concentrated on those indigenous bacterial communities inhabiting different body surfaces [1–4], but relatively little effort has been focused on viruses [5–9]. Recent studies have identified communities of viruses inhabiting the human oral cavity [10, 11], the respiratory tract , skin , and the intestinal tract [5, 7, 13]. While the role of viruses in these communities has yet to be thoroughly examined, a common feature shared among these body surfaces has been that most of the viruses identified have been bacteriophage [5–7, 11, 14]. Because bacteria generally outnumber human cells in these environments, bacteriophage might also be expected to outnumber eukaryotic viruses. Many of the viruses present in these communities have been predicted to have primarily lysogenic lifestyles, carrying gene function that might facilitate the pathogenic functions of their host bacteria [6, 7].
Biofilms contain complex aggregates of microorganisms growing on self-produced solid surfaces, whose constituents and cellular activity may differ substantially from planktonic communities . The oral biofilm is known to be inhabited by numerous species of bacteria and archaea [1, 16–18], but has not been shown to be inhabited by communities of viruses. Because of the potential difficulty in traversing solid surface biofilms, dental plaque has been hypothesized to be relatively devoid of viruses , however, some viruses have previously been identified in dental plaque [19–21]. Given the abundance of bacteria residing within plaque, we hypothesize that dental plaque may have an indigenous viral community.
The human oral cavity contains many microenvironments in which the microbiota are known to differ . There are characteristic differences in the relative abundances of bacteria in subgingival plaque, supragingival plaque, saliva, buccal mucosa and on the tongue. There also are shifts in oral bacteria that can be traced to diet  and oral health status [23–26]. Because of the proximity to tooth surfaces, many have sought to characterize subgingival microbiota in conditions such as chronic periodontal disease [27, 28] and dental caries , as those communities harbor microbes that might contribute to oral inflammation and the subsequent development of disease. Whether viral communities are part of the biofilm microbiota or contribute to oral inflammation has not previously reported.
Characterization of human viral communities has generally been limited by a relative dearth of homologous sequences available to identify metagenome contents [10, 30, 31]. Most of the studies characterizing human viral communities have viromes in which greater than half of the constituents are without homologues [5, 6]. Other studies have used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in bacteria, which acquire short sequences from the viruses to which they are exposed [32–34], as a means to augment analysis of human viral communities. Some dental plaque biota are known to possess CRISPR/Cas systems , suggesting that they can adapt to invading viruses. We believe that there are uncharacterized populations of viruses inhabiting the oral biofilm that may have unique features when compared to planktonic viruses in saliva. In this study, we sought to detail the presence of viral communities populating dental plaque, to determine whether oral viruses might be subject specific or specific to oral sampling site, and to characterize the potential capacity of oral streptococci to counteract their viruses by profiling CRISPRs.
Isolation and sequencing of dental plaque viromes
Although some viruses have previously been isolated [19–21], it is not known whether dental plaque is inhabited by a community of viruses as has been shown for saliva [6, 10, 11]. To determine whether there existed a population of viruses in dental plaque, we evaluated plaque from 4 human subjects with good overall periodontal health (Additional file 1: Table S1). We collected plaque in a biogeographic manner from tooth #3, 9, 12, 19, 25, and 28 (see Additional file 1: Table S2 for international numeration). Virus-like particles (VLPs) were visualized from dental plaque using epifluorescence microscopy and were present at an estimated 1010 VLPs per gram of plaque for all subjects (Additional file 2: Figure S1). Comparatively, there were 108 VLPs per ml of saliva in these same subjects, 108 in the lower respiratory tract of other human subjects, 105 in blood, 107 in the vagina, and 108 in the human gut virome .
Viromes were enriched from the dental plaque of each subject similar to our previously described protocols for isolating DNA viruses from saliva . We sequenced 7,768,251 virome reads from all subjects (3,181,703 from saliva and 4,586,548 from dental plaque) using semiconductor sequencing . All viromes were screened for contaminating cellular nucleic acids by BLASTN analysis against a human reference database and a composite database of 16S rRNA. No homologues were identified among the viromes to 16S rRNA, indicating that these viromes were relatively free of contaminating bacterial DNA (Additional file 1: Table S3). A small number of reads homologous to human DNA were identified in the dental plaque virome of subject #3 (721 reads represented 0.06% of the virome reads), and were removed prior to further analysis.
Characterization of plaque viromes
Viral and bacterial community composition by subject and sample type
We also characterized the bacterial community composition in each subject and sample type by analysis of the V3 region of 16S rRNA. We sequenced 190,720 reads (average of 15,893 per subject and site) from each subject and sample type (Additional file 1: Table S4). Rarefaction analysis demonstrated that the preponderance of bacterial diversity had been sampled in each subject and sample type (Additional file 2: Figure S5). Contrary to the subject-specific results found for viruses in the oral cavity (Figure 3, Panel B), sample type was an important determinant of oral bacterial ecology (Figure 3, Panel C).
Viral homologues between subjects and sites
Percent homologous within subject or sample typea
Percent homologous for comparisons of different subjects or sample typesa
74.11 ± 5.55
36.52 ± 19.80
67.14 ± 3.80
37.76 ± 16.87
44.59 ± 6.75
40.26 ± 19.19
54.26 ± 6.20
36.37 ± 19.92
By sample type
57.50 ± 8.01
56.94 ± 13.92
72.94 ± 3.23
61.00 ± 10.00
Streptococcal CRISPR profiles in dental plaque
We previously profiled streptococcal CRISPRs in the saliva of a cohort of human subjects and identified many matching viral sequences in those same subjects . We evaluated the same Streptococcus Group I (SGI) and Streptococcus Group II (SGII) CRISPRs, both of which represent Type II CRISPR/Cas systems in each species . These repeat motifs have been identified in numerous different streptococcal species (Additional file 1: Table S5) [6, 35]. We sequenced 293,139 SGI and 229,103 SGII CRISPR spacers from each subject and sample type (Additional file 1: Tables S6 and S7), and binned spacers according to their trinucleotide content to account for any potential polymorphisms or sequencing errors . When examining spacer content, only 0.002% of SGI and 0.001% of SGII CRISPR spacers were estimated to have any polymorphisms (Additional file 2: Figure S6).
CRISPR spacer groups shared between subjects and sites
Percent shared within subject or sample typea
Percent shared for comparisons of different subjects or sample typesa
96.74 ± 0.63
50.20 ± 22.18
94.95 ± 0.73
50.00 ± 22.22
96.51 ± 0.50
50.07 ± 22.24
71.68 ± 4.22
50.23 ± 22.28
By sample type
14.12 ± 3.64
23.93 ± 5.27
21.35 ± 3.89
15.77 ± 4.58
95.79 ± 0.70
60.90 ± 19.27
99.48 ± 0.06
60.94 ± 19.20
95.80 ± 0.56
60.67 ± 19.26
89.16 ± 1.76
60.85 ± 19.23
By sample type
15.84 ± 3.40
32.88 ± 4.26
25.64 ± 2.88
20.66 ± 3.44
CRISPR spacers from dental plaque match oral viruses
Our analysis of the viral communities in dental plaque provides insights into relatively unexplored aspects of the microbiota inhabiting the complex oral ecosystem. While the relative paucity of biomass at each tooth precluded analysis of individual teeth, the pooling of dental plaque allowed for analysis of the viruses present. The sampling and analysis of the microbiota in dental plaque and saliva has been performed and reported on for many years [39–41], and the overlap in the viral communities observed between each likely reflects some overlap in the resident bacterial biota from both sites. In support of this hypothesis is the substantial proportion of shared CRISPRs spacers that likely reflect sampling of the same bacteria from both sites in each subject (Figure 4).
The vast majority of the viruses found in this study and others describing human viromes [5, 8–11] have been identified as bacteriophage, with only a few eukaryote viruses including herpesviruses and circoviruses identified. Characterization of bacteriophage from viromes generally has been limited due to a lack of available homologous sequences. The proportion of contigs without homologous sequences in this study was greater than 50% in some viromes, similar to proportions found in other studies [5, 8–10]. We identified numerous homologues to known viruses (Additional file 2: Figure S2) and found that many spanned the entire genome sequences of known viruses (Figure 2), which reinforced that there likely were full-length viral genomes present in dental plaque. Further study with a broader group of participants would be required to define what role viruses may play as members of the dental plaque microbiome.
We explored both bacterial and viral ecology to provide a more comprehensive view of the microbial inhabitants of plaque. While viral ecology was reflective of the subject from which they were derived (Figure 3, Panel B), the bacterial ecology was more reflective of sample type (Panel C). The membership of the dental plaque viral communities differed from planktonic saliva in all subjects, although there were homologous sequences between saliva and plaque in each subject (Figure 3, Panel A; Table 1). The significant proportion of homologous sequences for intra-subject comparisons of viromes and for inter-subject comparisons of dental plaque, suggests that oral viral ecology is influenced by both individual host environment and sample type. There were a significant number of VLPs present in both saliva and dental plaque, which were greater than most other body surfaces. The substantial population of phage present in plaque combined with the high numbers colonizing mucosal surfaces , increases the complexity of comparing relative abundances of oral phage with their putative bacterial hosts.
We studied CRISPRs in the dental plaque of our cohort, as their spacer sequences reveal sequence features of viruses that oral bacteria may counteract. The similar CRISPR profiles in both saliva and plaque likely reflect shared bacterial inhabitants in both niches. The overall trend in shared CRISPR spacers reflected a subject-specific rather than a sample type specific pattern in all subjects (Figure 4, Panels C and D). The CRISPR and virome data together demonstrate distinct ecological differences between subjects, and supports that both oral biogeography and the individual host environment are significant determinants of oral viral ecology. We previously have identified short proto-spacer-adjacent motifs (PAMs) that are used to recognize and select spacers from invading DNA for both SGI and SGII spacers .
As we continue to characterize human microbial communities, we must account for the complexities of biogeography and its potential contribution to an individual’s microbial ecology. Our analysis of dental plaque has uncovered the presence of a community of viruses, whose constituents share some overlap with those of planktonic saliva. Despite that many of the viral contigs identified were unique to either saliva or dental plaque, the overlap observed in the saliva and plaque of individual subjects suggest that there may be shared viruses across each biogeographic site. The analysis presented here provides an additional framework for understanding human oral viral ecology, and demonstrate that oral viruses may be relatively personal features of the human microbiome.
Subject enrollment and sample collection
Subjects were recruited and enrolled from the Western University College of Dental Medicine and were approved by the University of California, San Diego and the Western University Administrative Panels on Human Subjects in Medical Research. All subjects signed an informed consent demonstrating their willingness to participate in the study. Each subject underwent a baseline periodontal examination including measurements of probing depths, clinical attachment loss, Gingival Index, Plaque Index, and gingival irritation , and were all found to be periodontally healthy with no carious lesions. We used the 1999 International Workshop for Classification of Periodontal Diseases and Conditions, where periodontitis including juvenile forms of periodontitis is defined by loss of attachment. For diagnosis of healthy, all sites had to have an attachment level of 0 mm and an absence of bleeding on probing. We excluded attachment levels from sites that were located next to 3rd molars, edentulous areas and sites where attachment loss was clearly caused by factors other than periodontal disease such as chronic toothbrush trauma. Exclusion criteria included antibiotic administration during or for 12 months prior to the beginning of the study and preexisting medical conditions that could result in immunosuppression. Plaque samples were collected first, followed by the patient allowing saliva to pool in his or her mouth for about 5 minutes, followed by collection of pooled saliva into a test tube. Plaque collection was modeled after standard plaque collection procedures used to perform clinical microbial sampling. Teeth were isolated with a rolled sheet of gauze on either side of the tooth, and gently dabbed dry with another piece of gauze. Supragingival plaque was collected with a Gracey curette by scraping the cutting edge of the instrument against the mesial surface of the tooth from the gingival margin and coronal to that, collecting a strip of plaque from the mesiobuccal line angle toward the interproximal contact. For subgingival plaque sampling, the other end of the curette was used to collect plaque below the gingival margin from the mesiolingual line angle towards the contact point. We attempted to performed this process in less than ten seconds to limit exposure of the sample to ambient air. Plaque was collected from the subgingival and supragingival biofilms from tooth #3, 9, 12, 19, 25, and 28 and placed into 200 μl of 0.02-micron filtered phosphate-buffered saline (PBS) (Fisher Scientific, Chico, CA) (See Additional file 1: Table S2 for international enumeration of teeth). Approximately 3 ml of saliva was collected without stimulation from each subject. Both saliva and dental plaque specimens were immediately frozen on dry ice and stored at −80°C until use in this study.
Isolation and analysis of oral viruses
Dental plaque was pooled together by subject, washed twice in 0.02-micron filtered PBS, and spun at 6,000 g for 10 minutes to pellet the biofilm. The biofilm then was incubated at 37°C for 30 minutes, and vortexed vigorously for 10 minutes to separate out viruses. The biofilm was then spun at 6,000 g for 10 minutes, and the supernatant kept for further analysis. A small portion (0.05 g) of the VLPs from each subject were resuspended in 200 μl of 0.02-micron filtered PBS and their counts per gram of plaque determined by epifluorescence microscopy . The remaining supernatant samples then were treated in an identical manner to those of the saliva samples, according to previously described methods for enrichment and extraction of nucleic acids from viruses . The resulting DNA was amplified using the GenomiPhi V2 MDA amplification kit (GE Healthcare, Pittsburgh, PA), fragmented to roughly 100 to 200 bp using a Bioruptor (Diagenode, Denville, NJ), libraries created using the Ion Plus Fragment Library Kit (Life Technologies, Grand Island, NY) according to manufacturer’s instructions, and sequenced using 314 chips on an Ion Torrent Personal Genome Machine (PGM; Life Technologies, Grand Island, NY) . Each resulting read was trimmed according to modified Phred quality scores using CLC Genomics Workbench 4.65 (CLC bio USA, Cambridge, MA), and low complexity reads (where >20% of the length were due to homopolymer tracts), reads with substantial length variation (<50 nucleotides or >200 nucleotides), and reads containing ambiguous characters were removed prior to further analysis. Reads were screened for homology to a composite database of 16S rRNA including the Ribosomal Database Project database , Green Genes database  and Silva database  using BLASTN analysis with an E-score cutoff value of 10−5. Reads also were screened for homology to the Human Reference Database at (ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/) by BLASTN analysis using an E-score cutoff value of 10−5. Any reads homologous to sequences in the human database were removed prior to further analysis. Reads then were assembled using CLC Genomics Workbench 4.65 (CLC bio USA, Cambridge, MA) to construct contigs based on 98% identity with a minimum of 50% read overlap, consistent with criteria developed to discriminate between highly related viruses . Because the shortest reads were 50 nucleotides, the minimum tolerable overlap was 25 nucleotides, and the average overlap was no less than 50 nucleotides depending on the characteristics of each virome. Contigs <200 bp were removed from further study. Specific viral homologues were determined by parsing BLASTX results (E-score cutoff value of 10−5) for known viral genes including replication, structural, transposition, restriction/modification, hypothetical, and other genes previously found in viruses for which the E-score was at least 10−5. Heatmaps were created using JAVA Treeview  based on a database of BLASTX best hits for all virome contigs, and were normalized based on the total number of viral contigs for each virome. Analysis of shared homologues present in each virome was performed by creating custom BLAST databases for each virome, comparing each database with all other viromes using BLASTN analysis (E-score <10−5), and normalization to the size of the smaller virome. Principal coordinates analysis was performed on homologous virome reads with binary Sorensen distances using Qiime . Read mapping of viromes to a combined database of viruses (http://www.phantome.org; ftp://ftp.ncbi.nih.gov/genomes/Viruses/) or to bacterial genomes was performed using CLC Genomics Workbench 4.65 (CLC bio USA, Cambridge, MA), and were mapped using 98% identity over a minimum of 50% of the read length. Many of the virome sequences mapped to CRISPR loci within bacterial genomes, but none matched the CRISPR repeat motifs.
Amplification and sequencing of CRISPRs
From each subject, genomic DNA was prepared from saliva or pooled subgingival or supragingival plaque using the QIAamp DNA MINI Kit (Qiagen, Valencia, CA), with the addition of a bead beating step using Lysing Matrix B (MPBio, Solon, OH) prior to nucleic acid extraction. SGII Primers were designed based on their specificity to the CRISPR repeat motifs present in S. gordonii str. Challis substr. CH1, S. thermophilus LMD-9, S. thermophilus LMG-18311, and S. thermophilus CNRZ-1066, and SGI primers were designed based on their specificity to the CRISPR repeat motifs present in S. mutans UA159, S. thermophilus LMD-9, and S. thermophilus LMG-18311 (Additional file 1: Table S9). Each forward primer contained 10-nucleotide barcode sequences, represented by the ‘X’ in each primer sequence (Additional file 1: Table S10). Reaction conditions included 44 μl Platinum High-Fidelity PCR Mastermix (Invitrogen, Carlsbad, CA), 1 μl of each the forward and reverse primer (10 mmol each), and 4 μl DNA template. The following were used as cycling parameters: 2 minutes initial denaturation at 94°C, followed by 30 cycles of denaturation (15 seconds at 95°C), annealing (15 seconds), and extension (2 minutes at 72°C), followed by a final extension (10 minutes at 72°C). CRISPR amplicons were purified using the MinElute PCR Purification Kit (Qiagen, Valencia, CA) followed by magnetic bead purification using Ampure XP (Agencourt, Beverly, MA). Molar equivalents were determined from each product using a Bioanalyzer HS DNA Kit (Agilent, Santa Clara, CA), and each were pooled into equimolar proportions. Resulting pools were sequenced using an Ion Torrent PGM according to manufacturer’s instructions (Life Technologies, Grand Island, NY) . Barcoded sequences were then binned according to 100% matching barcodes. Each read was trimmed according to modified Phred scores of 0.5 using CLC Genomics Workbench 4.65 (CLC bio USA, Cambridge, MA), and low complexity reads and reads with ambiguous characters were removed from the analysis. Only those reads that had 100% matching sequences to both the 5’ and the 3’ end of the CRISPR repeat motifs were used for further evaluation. Spacers were defined as any nucleotides (length ≥20) in between repeat motifs. Spacers then were grouped according to their trinucleotide content as previously described . For each subject and sample type evaluated, a database of spacer groups was generated, and databases were compared to determine shared spacer groups to create heat maps using Java Treeview . Beta diversity was determined using binary Sorensen distances and was used as input for principal coordinates analysis using Qiime . Spacers from each subject were subjected to BLASTN analysis based on NCBI NR database. Hits were considered significant based on bit scores ≥45, which roughly correlates to 2 nucleotide differences over the 30 nucleotide average length of the spacers, and results displayed using Cytoscape . CRISPR spacers were mapped to each of the bacteriophage, plasmids, and genomes, using CLC Genomics Workbench 4.65 (CLC bio, Boston, MA) using the default parameters for short-read mapping. Circular genome maps were created using CGView  and the mapped reads from each set of CRISPR spacers superimposed to scale on the prophage portions of each genome. CRISPR spacer matches to virome reads were defined as exact matches to any spacer within any spacer group. Matches also could be present on either the sequenced strand for each virome read, or its reverse complement. CRISPR spacers for each subject and biogeographic site were used to search all of the virome reads for matches, and the number of spacer matches per read was used to create heatmaps using Java Treeview .
To assess whether virome reads or spacer groups had significant overlap between different individuals or biogeographic sites, we performed a permutation test. We simulated the distribution of the fraction of overlapping reads between different individuals or biogeographic sites. For each set, we computed the summed fraction of randomly chosen spacer groups or virome reads, and from those computed an empirical null distribution of statistics. The fraction computed resulted from 10,000 iterations for both spacer groups and virome reads. For the CRISPR spacer groups, 1000 spacer groups were sampled in each iteration, and 10,000 reads were sampled in each iteration for the virome reads. The standard deviation was computed from the percentage of homologous virome reads or spacer groups over the 10,000 iterations. For each subject or biogeographic site, an empirical null distribution of statistics was determined. The observed statistic was referred to this distribution, and the p value was computed as the fraction of times the simulated statistic for intra-subject or intra-site comparisons exceeded the simulated statistic for the inter-subject or inter-site comparisons.
Availability of supporting data
Virome and 16S rRNA sequences are available for download in the MG-RAST database (http://metagenomics.anl.gov/) under project #3928, entitled ‘Dental Plaque Study’.
Clustered regularly interspaced short palindromic repeats
Supported by the Robert Wood Johnson Foundation, the Burroughs Wellcome Fund, and the National Institutes of Health 1K08AI085028 to DTP.
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