The goal of this study was to characterize the oral microbiome of a general population in the United States using a citizen-scientist model. We used crowdsourced bacterial community data to characterize relationships between microbial composition and individual demographics, oral health attributes, and sweet-liking phenotype. Our data suggests adults and youth share central core oral microbiome genera, but vary in rare taxa abundance and coverage (Fig. 1), where rare taxa were considered genera that constitute less than 3% of the community composition. Youth had higher median alpha diversity than adults, although adults had a higher diversity distance range. Youth also had a lower median UniFrac distance when compared to adults than when adults were compared to each other. This is likely due to youth microbiomes being more similar to each other compared to adults which had higher intra-group (person-to-person) variation and higher dissimilarities (i.e. more personalized microbiomes). Additionally, youth microbiomes contained more genera detected in over 75% of the population (16 in youth compared to 10 in adults) (Fig. 2A) and higher abundances of rare taxa as represented by higher count taxa constituting less than 3% of the community composition such as Granulicatella, Leptotrichia, and Porphyromonas (Fig. 2B). These data indicate that youth oral microbiomes are more diverse than adults as a population, but adult communities have more intra-group variation. These results are largely in agreement with a study by Ling et al. (2013) which compared the salivary microbiomes of healthy Chinese adults and children40.

Although some genera associated with the core oral microbiome, such as Prevotella, varied in abundance across our two populations, they still maintained high coverage (≥75%) (Fig. 2). The presence of these genera in nearly all samples across two distinct populations strengthens the hypothesis of a global core oral microbiome, at least within urban United States, and suggests environmental and health factors may contribute to genera abundances but may not necessarily eliminate core genera from being present at low levels. Currently, it is difficult to assess which specific variables are causing the differences between populations, but previous research suggests culture and sampling locations are likely contributors4,16,25. Twelve genera were differentially abundant between age groups, and are all known inhabitants of the oral cavity (Table 2). Many of them are associated with normal, healthy oral microbes, but some are associated with oral and/or systemic diseases when in dysbiosis4,5,12. Increased abundances of Abiotrophia and Granulicatella, which were found more often in youth communities, have been associated with dental caries, obesity, and endocarditis40,41,42,43,44. Treponema was more often found in adult communities; a genus commonly known for its role in the “red complex” associated with severe forms of periodontal disease and dental caries45. Additionally, Treponema abundance increases have been associated with increasing age29. This may be indicative of differences between the effects of adult and youth habits, diet, and healthcare quality and frequency. Comparing the metadata proportions between age groups further strengthens this idea as adults had statistically higher proportions of oral health issues, less frequent dentist visits, and increased prevalence of obesity (Table 1).

When separating the two age groups and analyzing them individually, we found that each group’s microbiome variation was driven by different factors. The adult oral microbiomes were affected by flossing and time since last dentist appointment (Fig. 3). Previous work comparing twin flossers vs. non-flossers observed periodontal pathogens were overabundant in non-flossers while healthy microbiota were overabundant in flossers over a two-week study7. In the adults of our study, flossers have reduced beta diversity variation potentially caused by the disruption of small niches and physical removal of rare taxa during the flossing process leaving higher relative abundances of core oral microbiome genera behind or by invoking inflammation and activation of the immune system (Fig. 3A). The positive correlation in adults between UniFrac distances and time since last dentist visit suggests dentist visits reduce natural variation and diversity in the oral microbiota, but this variation is gradually reintroduced (Fig. 3B). The oral pathogen Treponema was associated more often with individuals who had not been to the dentist in over 12 months. It is likely, as with flossing, dental cleanings physically remove rare taxa, invoke inflammation, and activate the immune system leading to reduced oral microbiota diversity while helping control pathogen overabundance. Previously, species richness of biofilm redevelopment after dental cleaning has been shown to be increased in periodontitis-affected individuals46. A future in-depth study analyzing oral microbiomes before and after dentist visits with long term longitudinal sampling would be beneficial to determine overall variation and the specific genera that are lost during dental cleanings and if variation reintroduction is caused by the remaining traces of previously present or newly introduced genera.

Youth oral microbiomes were less affected by oral health care habits than adult oral microbiomes (Fig. 4). Youth reported flossing habits similar to adults, but unexpectedly did not show the same microbiome changes (Fig. 4A). This raises the question of whether youth oral microbiota are more resilient to diversity loss due to their increased relative abundances of rarer taxa and higher taxa evenness. Flossing has been determined to affect the oral microbiomes of teenagers, but the effects of flossing on younger children is still unknown7. In this study, ~87% of the youth were 8–9 years of age which suggests flossing may alter diversity differently in prepubescent children. It is also possible youth are more likely to misrepresent how often they floss and may even perform flossing inadequately as compared to teen or adult flossers. Therefore, it is difficult to assess whether the reduced effect of flossing with prepubescent children is correlated to microbial ecology or self-reported data. Youth oral microbiome diversity changed similarly to adults with time since last dentist visit but was not significant (Fig. 4B). This may be due to the fact that more youth had recently been to the dentist, while more adults had not been to see a dentist recently. For example, ~80% of youth had been to the dentist within 6 months and only ~8% had not been to the dentist in over 12 months compared to adults which only ~54% had been to the dentist within 6 months and ~29% had not been to the dentist in over 12 months (Table 1). This made it difficult to appropriately assess the impact of the time since last dentist in youth oral microbiomes.

Gut and oral microbiota dysbiosis has been detected in obese adolescents and children3,44,47. Recently, young children with a high risk of childhood obesity have been associated with the loss of oral microbiota diversity over early growth with microbiota resembling obese adults by age two31. Our study only included youth at least 8 years of age, predominantly children 8–9 years old. Our data show oral microbial communities to be distinct between lean and obese youth with no specific genera being differentially abundant (Fig. 4C), and we observed obese youth to have increased phylogenetic diversity, as opposed to decreasing overall diversity as previously reported31. We also found BMI metrics to have a positive correlation with both unweighted and weighted UniFrac distances. Craig et al. (2018) describe an immediate loss of oral alpha diversity in young children over the first 10 months of growth, but diversity begins to increase again by 25 months as oral microbiota associated with obesity are more prevalent31. This increase in alpha diversity may continue into prepubescent childhood as we see increased diversity in our data, but further longitudinal studies between 2 and 8 years of age are needed. Interestingly, Treponema was determined to be found more often in obese youth, which is the same genera associated with poor oral health found in the adult group. In adults, obesity and poor oral health has been linked with obese individuals having higher prevalence of periodontitis48,49. These data suggest that this link may be observable in youth as well.

Additionally, youth male and female oral microbiomes were determined to be different, but no specific genera were determined to be the cause (Fig. 4D). It is unclear why youth male and female microbiomes differ while adult male and female did not. Possible causes include less attentive hygiene habits, and that standards of cleanliness are generally higher for young females than males50. One limitation to this study is that we cannot consider the impact of puberty or hormones. However, the majority of youth participants were likely prepubescent, and we found no significant differences in the oral microbiomes of adult males and females supporting a limited effect of hormones on the oral microbiome.

Cohabitation and familial relationships have been shown to increase oral microbiome similarity more than host genetics, in particular among rare taxa22,24. Our data suggest this impact on diversity is still observable within a general population (Fig. 5). Participants of the same family have a greater similarity to each other over participants of different families (Fig. 5A). This was also found, albeit to a lesser extent, in mother-child pairs (Fig. 5B). Unsurprisingly, unweighted UniFrac distances were more sensitive to inter-family comparisons than weighted UniFrac distances potentially due to rare taxa variation across families that are not part of core oral microbiome. Same family distances also consistently contained less variation in similarity, suggesting intra-family relationships and cohabitation leads to the sharing of both core and rare taxa24. Weighted differences were still detected between all participants of the same families to different families even though most individuals shared a core set of microbes, which suggests core microbiota differ in abundance across families while still maintaining high coverage within the population.

Sweet-liking phenotypes differed between children and adults. Sweet-liking phenotypes identified for children were “likers” and “dislikers”, while adult phenotypes also included an additional phenotype of “neutral”35,37. These sweet-liking phenotypes are not associated with sex, age, or BMI, but adult sweet “likers” did consume more sweet beverages than “neutral likers”35. The concept of the oral microbiota contributing to food choice and taste perception is currently being investigated since similar associations have been found with the gut microbiota51. One recent study examined relationships between taste perception, food intake, and oral microbiota composition in adults52. The authors reported that bacteria classes, such as Prevotella and Clostridia, were associated with the taste perception of vegetable-rich and protein-rich diets. Additionally, decreased Clostridiales Family XIII relative abundance was the only taxa associated with sweet taste thresholds in adults. Taste sensitivity, as measured by thresholds, are not frequently associated with hedonic evaluations or dietary behaviors, so it is not altogether unexpected that this study did not identify microbiome composition associations with sweet-liking phenotypes or energy intake from beverages (Fig. 6)53. Further work is needed to explore what role the oral microbiome plays on taste sensitivity and perception, food selection, and dietary intake.

In this study, a model of crowdsourcing was used to test oral microbiome associations in a general population. We found that adult and youth oral microbiomes share core taxa, but are distinct from each other in taxa abundances and coverage. Factors affected each age group differently with adults largely impacted by oral health habits, and youth impacted by weight status. The oral pathogen genera Treponema was associated with both poor oral health habits in adults and obesity in youth. Additionally, we found oral microbiomes from participants of the same family were more similar to each other than to oral microbiomes from unrelated individuals. While a number of biological factors were compared, most had no statistical significance, and those that were statistically significant, such as weight status and flossing, appeared to differ minimally. We believe that the detection of these effects in a general population demonstrates that the selective pressures applied to the oral community from each factor are strong enough to overcome interpersonal variations and other background confounding variables, such as genetics, lifestyle, and living situation. Importantly, the small variations detected can be attributed to few select taxa, such as Treponema, which are considered pathogenic and associated with multiple health risks. This leads to seemingly specific oral microbiome differences potentially contributing to major health complications, such as periodontal disease and cardiovascular disease1,2,4,5.

Nonetheless, our study has certain limitations. Firstly, the reduced range of age in the youth population as compared to the adults may limit age comparison conclusions. Secondly, youth are in the transition period of temporal to permanent dentition which may increase inflammation, microbial niches, and create unequal tooth presence/absence across this group. Additionally, the participants sampled in this study were limited to a Western industrialized population living in an urban center, and thus conclusions may not be globally generalizable15,16,17. However, these results further suggest a connection of the oral microbiome to human health, particularly obesity. Lastly, due to the nature of this cross-sectional study, all potential exposures and confounding variables that can affect the microbiome could not be measured (e.g. individual diet, habitat, medications, etc.).

This study was aimed as both a scientific study and a visitor participation experience which engages the participants to “create content” and gather a better understanding of scientific methods34,54,55. These results are relevant to the scientific community because they demonstrate (similar to the American Gut Project) that important human microbiome trends can be recovered using a citizen science model, which can be a powerful and cost-effective means for collecting from a large population, and engages the public in science33. Crowdsourcing in the Genetics of Taste Lab at the Denver Museum of Nature & Science consisted of two forms of community contribution to the scientific process: (1) guests enhanced their science education experience by participating in the study as human research subjects, and (2) enabled volunteer citizen scientists to obtain training, collect data, and support in data analyses.