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Pyrosequencing analysis of the Oral Microflora of healthy adults
1 TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Microbial Genomics Group, Zeist, The Netherlands; Correspondence: * corresponding author, w.crielaard{at}acta.nl
A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably higher than previously reported.
Key Words: pyrosequencing • microflora • plaque • saliva • diversity Abbreviations: GS-20, Genome Sequencer 20 • OTU, Operational Taxonomic Unit • PCR, polymerase chain-reaction • 16S rDNA, small- subunit (16S) ribosomal deoxyribonucleic acid • UV, ultraviolet
Oral health and disease depend on the interplay between the host and the oral microbial community. The impact of the microbial community on shifting the balance from health to disease cannot be understood without a comprehensive view of a healthy community. During the past 40 years, a wealth of knowledge has been gathered: Over 250 oral bacterial species have been isolated and characterized by cultivation, and over 450 species have been identified by culture-independent molecular approaches (Paster et al., 2006). Recent studies where cloning and sequencing approaches of microbial 16S rDNA were applied to the oral microflora have identified still more new species (Aas et al., 2008; Preza et al., 2008; Riggio et al., 2008). This is not surprising, since the number of species detected in a sample is strongly affected by the number of sequences analyzed (Schloss and Handelsman, 2005), and overall diversity is affected by the sample size (Rajili-Stojanovi et al., 2007). This, however, limits the cloning and sequencing method (typically at most a few thousand clones from a low number of individuals) to identification of only the predominant microorganisms in a sample. Detection of low-abundance taxa requires studies on sets of sequences many orders of magnitude larger than those currently reported. Massively parallel pyrosequencing—a next-generation sequencing technique—is a new molecular approach that allows for extensive sequencing of microbial populations in a high-throughput, cost-effective manner (Ronaghi et al., 1998; Von Bubnoff, 2008). This technique has been successfully applied to determine bacterial diversity within various environmental ecosystems, such as hydrothermal vents of a deep marine biosphere (Sogin et al., 2006; Huber et al., 2007) and soil (Roesch et al., 2007). Within the human body, vaginal microflora (Sundquist et al., 2007) and bacteria of chronic wounds (Dowd et al., 2008) have been assessed by this approach, but we are not aware of reports on the oral microbial population. We aimed to estimate the detailed species richness of oral microflora of the healthy adult population, and, more specifically, to determine the number of different phylotypes and their relative abundance in saliva and supragingival plaque.
Samples The study was approved by the Medical Ethical Committee of the Free University Amsterdam. Participants (healthy adults who had not used antibiotics in the preceding 3 mos) were informed verbally and participated on the basis of informed consent. Sampling was performed in the morning before the participants ate breakfast. Saliva was collected from a group of 71 individuals by mouthrinse with 10 mL sterile saline for 30 sec (Boutaga et al., 2007). The saline solution had been UV-irradiated to avoid DNA contamination. Samples were placed on ice immediately and stored at –80°C until further use. We collected supragingival plaque from a group of 98 individuals by sampling buccal dental surfaces using a sterile, DNA-free wooden toothpick. The toothpick tip was cut off, placed in an Eppendorf tube, and stored at –80°C.
Molecular Techniques PCR amplification of the 16S rDNA hypervariable V6-region was performed with 2 forward primers—(909Fw) gcctccctcgcgccatcag-AAACTYAAARRAATTGACGG and (917Fw) gcctccctcgcgccatcag-GAATTGACGGGGRCCCGCA—and 1 reverse primer—(1061Rv) gccttgccagcccgctcag-TCACGRCACGAGCTGACGAC. These primers included the 454 Life Sciences (Branford, CT, USA) Adapter A (for forward primers) and B (for reverse primers) fused to the 5' end of the SSU-rRNA bacterial primer sequence (upper case). Primers were designed to provide the best combination between a high phylogenetic recovery and limited distance to the V6 hypervariable region (Baker et al., 2003; Horz et al., 2005). The PCR amplicon library was created for each individual DNA sample (71 saliva and 98 plaque samples). The amplification mix contained 2 units of Goldstar DNA polymerase (Eurogentec, Liège, Belgium) and Goldstar polymerase (Eurogentec) with 2.5 mM MgCl2. After denaturation (94°C; 2 min), 30 cycles were performed that consisted of denaturation (94°C; 30 sec), annealing (50°C; 40 sec), and extension (72°C; 80 sec). DNA was isolated by means of the MinElute kit (Qiagen, Hilden, Germany), visualized with the Agilent 2100 Bioanalyser, and quantified (Nanodrop ND-1000). Individual amplicon libraries were pooled in equimolar amounts and sequenced unidirectionally in the reverse direction (B-adaptor) by means of the Genome Sequencer 20 (GS-20) system (Roche, Basel, Switzerland) at 454 Life Sciences. Sequences are available in the GenBank Short Read Archive, accession number SRA001159 (ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA001159).
Data analysis
More than 273,000 PCR amplicons were sequenced, of which about 197,600 reads passed quality control (Table 1  ). The individual sequences could be clustered into 28,937 unique V6 tag sequences, representing 22 known phyla or candidate divisions (see also the Appendix Table). The vast majority of sequences (99.6%) belonged to one of the seven major phyla: Actinobacteria, Bacteroides, Firmicutes, Fusobacteria, Proteobacteria, Spirochetes, or candidate division TM7 (Fig. 1). Of the major phyla (Fig. 1), Actinobacteria, Fusobacteria, and Spirochetes were overrepresented in plaque, while Bacteroides, Firmicutes, and Proteobacteria sequences were more abundant in saliva.
At the genus level, sequences from saliva and plaque represented 318 different genera. From these, saliva comprised 185 genera, plaque, 267. About 11% of all sequences could not be identified at the genus level and were classified at the next highest possible resolution level—family, order, class, or phylum. Of all sequences, 97% corresponded to 31 genera and 14 other taxa (Table 2 , marked with *) in plaque and saliva. Three genera (Prevotella, Streptococcus, and Veillonella) constituted about 50% of the salivary microflora. In plaque, 50% of all sequences was occupied by 6 genera: Streptococcus, Veillonella, Corynebacterium, Actinomyces, Fusobacterium, and Rothia.
At the species level, defined as OTUs at 3% difference, about 5600 and 10,000 phylotypes were found in saliva and plaque, respectively, while a more conservative approach—6% difference among OTUs—yielded 3600 and 6800 phylotypes (Table 1  ). The most diverse genus in saliva was the genus Prevotella, while in plaque it was the genus Streptococcus (Table 2). From all sequences, from 0.7 to 2.6% (depending on a similarity distance used) can be considered new, previously unidentified, phylotypes: 5305 sequences differed more than 10% from the reference sequence in the database, while 1328 sequences differed by more than 30% from the nearest reference.
The richness of total bacterial communities of saliva and plaque was estimated by rarefaction analysis. The shapes of the rarefaction curves (Figs. 2A, 2B
The relative abundance of the phylotypes indicates that the majority of OTUs is present at low abundance (Figs. 2C, 2D ). The 1000 most abundant OTUs account for 90–95% of all sequences.
An understanding of the composition of the oral microbial ecosystem in relation to oral health is essential for the prevention and treatment of oral diseases. The introduction of high-throughput pyrosequencing has dramatically increased the resolution at which microbial communities can be analyzed. The main drawback of pyrosequencing is the current length restriction of sequences obtained: 100nt for the GS-20 and 250nt for the current GS-FLX. As a consequence, the method cannot be used to produce full-length 16s rDNA sequences, traditionally used for microbial taxonomic studies. The method does allow for the analysis of small hypervariable regions of the 16s rDNA gene, as has been used in several studies (Sogin et al., 2006; Huber et al., 2007; Sundquist et al., 2007). This approach, however, provides a challenge to the accurate assignment of the bacterial taxonomic groups and to estimation of the microbial richness on the basis of these short sequence tags. First studies have demonstrated the feasibility of this approach (Liu et al., 2007; Sundquist et al., 2007). Tag sequencing has been shown to provide sufficient resolution to explore microbial communities, and conclusions obtained from full-length sequences could be recaptured. However, the taxonomy resolution of the sequence tags obtained may vary between/among the different taxonomic groups. We have used 454 GS-20 pyrosequencing to explore the composition of the oral microbial flora by targeting the 16S rDNA hypervariable V6-region. This region has been used in several similar studies (Huber et al., 2007; Sogin et al., 2006) and has the advantage that the hypervariable part is flanked directly by well-conserved regions that can be used for PCR amplification (Baker et al., 2003). To define the membership of the tags found in species-level phylotypes (OTU), we used a cut-off of 3% difference, facilitating a direct comparison with other studies, as well as a more conservative cut-off of 6%. At a 6% cut-off, our studies revealed 3621 and 6888 species-level phylotypes in saliva and plaque samples, respectively. This number is far greater than the current estimates of oral microbial diversity. Analysis of our data further indicates that the total microbial species richness is in the order of 19,000–26,000, depending on the cut-off chosen (6% or 3%). So far, there are no other metagenomic studies on human microbial flora using pyrosequencing or species-richness estimates available for comparison. Our results on human commensal oral microbial flora showed lower diversity than in the deep marine biosphere, where about 37,000 bacterial and about 3000 archaeal phylotypes have been estimated at 3% difference (Huber et al., 2007), while soil samples showed a somewhat lower diversity (from 5000 to 20,000 OTUs at 3% difference; Roesch et al., 2007) compared with the oral flora. The vast majority of the unexpectedly large number of species-level phylotypes is present at very low levels. This is illustrated by the fact that of the 3600 OTUs identified in saliva, or the 6800 OTUs identified in plaque, the 1000 most common OTUs account for 95% of all sequences. Five percent of sequences thus represented the majority of OTUs identified in saliva or plaque, indicative of a low abundance. In addition, estimations of species richness in the oral microbiota suggest that our data covered approximately 50% of the full microbiota. Additional species are likely to be present at even lower abundance. At this stage, it is hard to predict which role bacteria, present in low numbers, play in oral ecology. However, it is an oversimplification to neglect their presence. The taxonomic distribution of our metagenomic data at the phylum level is in general agreement with previous findings (Munson et al., 2002; Aas et al., 2005; Preza et al., 2008). Firmicutes (genus Streptococcus and Veillonella) and Bacteroidetes (genus Prevotella) were the predominant phyla in saliva, while Firmicutes and Actinobacteria (genus Corynebacterium and Actinomyces) dominated supragingival plaque. The short-read length of 454 pyrosequencing data and the incomplete nature of 16S rDNA databases limit the phylogenetic resolution (Sundquist et al., 2007). In our data, 89% of the sequences could be identified at the genus level. Being aware of the low taxonomic resolution below the genus level, we did not attempt to identify the individual species, and thus report here only the abundance of OTUs at 3% and 6% differences. Based on full-length 16S rDNA sequences, a resolution of 3% OTUs is considered to be the microbial species level (Acinas et al., 2004), but is also believed to underestimate the true number of species (Pedrós-Alió, 2006). One percent cut-off levels have also been used (Munson et al., 2004). It has been demonstrated that short pyrosequencing reads suffice for accurate microbial community analysis (Liu et al., 2007), but how sequence diversity of full-length 16S rDNA sequences relates to that of the hypervariable regions and how this would reflect taxonomic boundaries remains to be determined. Interpretation of the relative abundance of different taxa identified by any molecular method must be done with care. The steps of DNA extraction, PCR amplification (choice of primers), and intrinsic differences in 16S rDNA copy number can/may result in a skewed taxonomic interpretation of the microbial community, and probably result in an underestimation of microbial diversity (Horz et al., 2005). Measures used in this study to avoid technical errors included UV irradiation of solutions and an efficient DNA extraction protocol (Keijser et al., 2007). Implementation of a stringent quality control of the sequences further excluded potential sources for artifacts (Huse et al., 2007). With the increasing read length of the 454 pyrosequencing technology, other hypervariable regions, such as the V3 and V4, become amenable to the technique. These improvements are likely to provide an even more complete view of the composition of microbial communities. By applying this novel approach in oral microbial sample analysis, we created a radically new insight into the richness of the diversity of commensal human oral microflora of both saliva and plaque. We detected diversity at least one order of magnitude higher than previously described and estimated that more than 19,000 species-level phylotypes contribute to the ultimate oral species diversity. This new insight provides a challenge for taxonomists and ecologists to determine the impact of this enormous microbial richness in relation to the physiology of the oral microbiota of individuals, and in relation to the oral health-disease equilibrium.
We thank Mieke Havekes and Louise Nederhoff for helpful discussions and excellent technical assistance. Sue Huse was supported on a subcontract to Mitchell L. Sogin from the Woods Hole Center for Oceans and Human Health, funded by the National Institutes of Health and National Science Foundation (NIH/NIEHS 1 P50 ES012742-01 and NSF/OCE 0430724). We also thank the ACTA Research Institute for financial support.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/11/1016/DC1. Received for publication April 25, 2008. Revision received August 1, 2008. Accepted for publication August 25, 2008.
Journal of Dental Research, Vol. 87, No. 11,
1016-1020 (2008) This article has been cited by other articles:
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