The oral metagenome in health and disease

Abstract

The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.

Figure 1

Bacterial diversity in the oral cavity. The graph on the left shows the relative frequency of different bacterial taxa, based on the assignment of the DNA reads by the PhymmBL software and by 16S rRNA reads extracted from the metagenome, and compared with the PCR results obtained by Bik et al. (2010). The graph on the right indicates the relative contribution of each taxonomic group to the coding potential of the ecosystem, based on the COGs functional classification system. It can be observed that the functional contribution is not equal among taxa.

Figure 2

Correspondence analysis (CoA) of the bacterial diversity in oral samples based on 16S rRNA reads extracted from the metagenomes. The first axis successfully separates healthy from diseased individuals. The graph suggests bacterial genera which are potentially associated with absence of caries.

Figure 3

Functional profiles from oral and adult-gut metagenomic samples. Classification was based on Subsystem hierarchy 2 of MG-RAST. Counts were normalized to the total number of reads per sample and then normalized by function. Blue to red gradient indicates levels of under/over-representation. Large blocks of gene categories are over-represented in each of the two microbiotas, indicating that the gut and the oral cavity are two functionally distinct ecosystems. Within the oral microbiome, some functional roles are over-represented in individuals without caries. A full version of this figure indicating all 101 functional categories is included in Supplementary Figure 6. Sequences from the healthy adult-gut metagenomes were taken from Kurokawa et al. (2007). The age and sex of each individual are indicated below each label.

Figure 4

Searching of bacterial strains with a potential antagonistic effect against cariogenic bacteria. Metagenomic recruitment plots are used to detect the species (a), which are at low frequencies in individuals with caries but are among the most common in caries-free subjects. These species are then selected based on culture conditions and microscopic examination (b). The isolates are grown in solid media to provide an inhibition screening against caries-producing bacteria (c), selecting the strains that display inhibition rings (d), such as the Streptococcus strain 7747. Sequencing the genome of these inhibitory strains and comparing it against the metagenome of caried individuals must confirm that these strains are absent under diseased conditions.