Citizen science charts two major “stomatotypes” in the oral microbiome of adolescents and reveals links with habits and drinking water composition

In the paper

“Similar to previous oral microbiome surveys [7, 29], we found high alpha (within sample) diversity (mean Shannon diversity 2.5, see Additional file 3) and low beta (between samples) diversity (mean weighted UniFrac distance 0.118).”

What the sources say

• Supported

  The Composition and Diversity of Oral Microbiome section reports high alpha diversity in oral habitats and the lowest beta diversity across subjects compared with other body sites. The citing sentence presents the Shannon and UniFrac means as its authors’ own findings; it cites this paper for the similarity to previous surveys, not for those numerical values. (LLM, conf=0.92)

  cites [7] Moon J-H, Lee J-H. Probing the diversity of healthy oral microbiome with bioinformatics approaches. BMB Rep. 2016;49:662–70. source
• Supported

  The citation supports the comparison to previous oral microbiome surveys, not the citing authors’ own numerical results introduced by “we found.” In “Microbial diversity of healthy humans,” the source reports that saliva had among the highest alpha diversities and one of the lowest beta diversities. (LLM, conf=0.91)

  cites [29] Human T, Project M. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207–214. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3564958&tool=pmcentrez&rendertype=abstract source

In the paper

“An analysis of the oral microbiome in 268 healthy young adults (18–32) classified the samples into five discrete clusters [22], whereas another study of 161 healthy adults found three different clusters [16].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Zaura E, Brandt BW, Prodan A, Teixeira, De Mattos MJ, Imangaliyev S, Kool J, et al. On the ecosystemic network of saliva in healthy young adults. ISME J. 2017;11:1218–31. source
• Supported

  The Results section reports that PAM analysis of saliva samples identified three salivary types, and its Figure 1 caption gives the sample size as 161. The Discussion states that none of the subjects’ salivary microbiota suggested oral disease. The five-cluster finding is attached to a different citation, not this source. (LLM, conf=0.92)

  cites [16] De Filippis F, Vannini L, La Storia A, Laghi L, Piombino P, Stellato G, et al. The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and vegan individuals. PLoS One. 2014;9:e112373. source

In the paper

“The core microbiome comprised typical oral bacteria that are commonly identified as abundant in similar oral microbiome surveys [28].”

What the sources say

• Partly supported

  The Results and Discussion (Sample-specific profiles; Overlap of three individual oral microbiomes, page unavailable) identify taxa present across the three healthy individuals and report that shared higher taxa accounted for 99.8% of reads. The passages do not establish that those particular bacteria were commonly identified as abundant in other oral microbiome surveys. (LLM, conf=0.82)

  cites [28] Zaura E, Keijser BJ, Huse SM, Crielaard W. Defining the healthy “core microbiome” of oral microbial communities. BMC Microbiol 2009;9:259. Available from: http://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-9-259 sourceExperimental — accuracy not yet measured

In the paper

“Previous studies have shown that individual microbiomes from certain niches can be clustered into different types, such as the enterotypes of the gut microbiome [31].”

What the sources say

• Supported

  In “Robust clustering of samples across nations: Identification of enterotypes,” the paper reports that gut microbiome samples formed three distinct clusters, which it designates enterotypes. This supports the cited example of individual microbiomes from a particular niche being clustered into types; the sentence does not attribute findings about other niches to this paper. (LLM, conf=0.94)

  cites [31] Arumugam M, Raes J, Pelletier E, Le P, Yamada T, Mende DR, et al. Enterotypes of the human gut microbiome. Nature. 2011;473:174–80. source

In the paper

“Thus, the abundance covariations observed in both stomatotypes may partly reflect the underlying diversity of biofilm succession stages comprised in our samples and would support the main axis of previously observed core community changes in dental plaque.”

What the sources say

• Partly supported

  The section on dysbiosis describes changes in bacterial proportions as dental plaque accumulates, and the discussion of maturing biofilm links maturation to a shift in the microbiota. That supports the general idea that plaque communities change as biofilms develop. Our copy does not identify a main axis of core-community change or connect it to abundance covariations in two stomatotypes; the latter is the citing authors’ interpretation of their samples. (LLM, conf=0.77)

  cites [1] Kilian M, Chapple ILC, Hannig M, Marsh PD, Meuric V, Pedersen a ML, et al. The oral microbiome – an update for oral healthcare professionals. Bdj. 2016;221:657–66. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [32] Jakubovics NS. Intermicrobial interactions as a driver for community composition and stratification of oral biofilms. J Mol Biol. 2015;427:3662–75. source
• Supported

  The Results report that plaque accumulation time significantly affected community composition and that the microbiota shifted over time along principal coordinate 1. The paper also documents changes in bacterial abundances between early and later stages. The sentence’s interpretation of covariations in its own samples is the citing authors’ inference, not a finding attributed to this source. (LLM, conf=0.89)

  cites [33] Takeshita T, Yasui M, Shibata Y, Furuta M, Saeki Y, Eshima N, et al. Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach. Sci Rep 2015;5:8136. Available from: http://www.nature.com/articles/srep08136 source
• Supported

  Section 2, “Biofilms,” describes sequential colonisation of dental plaque and shifts in its microbial composition as the biofilm matures. That supports the cited background point about previously observed community changes; the interpretation of covariations in the citing authors’ stomatotypes is their own. (LLM, conf=0.88)

  cites [34] Mahajan A, Singh B, Kashyap D, Kumar A, Mahajan P. Interspecies communication and periodontal disease. Sci World J. 2013;2013:765434 source

In the paper

“However, earlier studies using similar collection protocols and including information on plaque status or dental health have found correlations between microbial composition of saliva, the amount of plaque and diseases such as periodontitis, or caries [5, 9, 35, 36].”

What the sources say

• Partly supported

  The Results section ('Salivary abundance of specific oral bacterial species associates with periodontitis and dental caries'; page unavailable) links differences in salivary microbial composition to both diseases. The Methods section ('Study population and sample collection'; page unavailable) describes standardized saliva collection and dental-health classifications, but the paper does not report a correlation with the amount of plaque. (LLM, conf=0.85)

  cites [5] Belstrøm D, Constancias F, Liu Y, Yang L, Drautz-Moses DI, Schuster SC, et al. Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries. NPJ Biofilms Microbiomes 2017;3:23. Available from: http://www.nature.com/articles/s41522-017-0031-4 sourceExperimental — accuracy not yet measured
• Supported

  The Results (Phylogenetic diversity and oral health-related conditions; page not provided) report significant associations between salivary bacterial diversity and plaque index, decayed teeth, and periodontal-pocket depth. The Methods (Saliva collection and DNA extraction; page not provided) describe the saliva collection protocol, while the dental-examination methods document plaque and dental-health assessments. (LLM, conf=0.90)

  cites [9] Takeshita T, Kageyama S, Furuta M, Tsuboi H, Takeuchi K, Shibata Y, et al. Bacterial diversity in saliva and oral health-related conditions: the Hisayama study. Sci Rep. 2016;6:22164. source
• Supported

  The Materials and methods sections describe stimulated-saliva collection and an oral examination recording caries. The Discussion reports an association between salivary bacterial composition and differing levels of caries experience. This supports the caries example for which this paper is cited alongside other studies. (LLM, conf=0.90)

  cites [35] Belstrøm D, Holmstrup P, Fiehn N-E, Kirkby N, Kokaras A, Paster BJ, et al. Salivary microbiota in individuals with different levels of caries experience. J Oral Microbiol Taylor & Francis. 2017;9:1270614. source
• Supported

  The Methods (page not specified) describe collecting unstimulated saliva and recording dental health in caries and caries-free subjects. The Results and Discussion (page not specified) report a significant difference in salivary bacterial composition between those groups. In this multi-citation sentence, the paper supports the caries example; it is not being relied on for the plaque-amount or periodontitis examples. (LLM, conf=0.90)

  cites [36] Zhou J, Jiang N, Wang S, Hu X, Jiao K, He X, et al. Exploration of human salivary microbiomes insights into the novel characteristics of microbial community structure in caries and caries-free subjects. PLoS One. 2016;11:e0147039. source

In the paper

“Most of these genera comprise anaerobic Gram negative species or Gram positives associated to acidic fermentations, which are generally associated to mature biofilm acidification, as well as caries and periodontal disease [3].”

What the sources say

• Partly supported

  The sections on ECC microbiota and the biofilm matrix describe acid-producing bacteria contributing to plaque acidification and caries. The section on other bacteria also discusses Gram-negative Prevotella in severe ECC plaque. Our copy does not establish that most of the citing paper’s genera have the stated Gram-staining or anaerobic characteristics, or associate them with periodontal disease. (LLM, conf=0.84)

  cites [3] Hajishengallis E, Parsaei Y, Klein MI, Koo H. Advances in the microbial etiology and pathogenesis of early childhood caries. Mol Oral Microbiol. 2017;32:24–34. sourceExperimental — accuracy not yet measured

In the paper

“Of note, several of the correlations mentioned in our study coincide with those found in previous studies [16, 22].”

What the sources say

• Supported

  The Results section (‘OTU co-occurrence and/or co-exclusion’; page unavailable) reports several correlations among salivary bacterial genera. This supports citing the paper as a previous study that found correlations; whether they coincide with the citing authors’ results is the citing authors’ comparison, not a finding attributed to this source. (LLM, conf=0.86)

  cites [16] De Filippis F, Vannini L, La Storia A, Laghi L, Piombino P, Stellato G, et al. The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and vegan individuals. PLoS One. 2014;9:e112373. source
• Supported

  The citation points to this paper as a previous study that found correlations; the statement that they coincide with the citing authors’ results is the citing authors’ comparison. The Results sections report correlations among salivary taxa and between microbial taxa and metabolites. The sentence does not specify which correlations overlap, so the overlap itself cannot be checked against this source alone. (LLM, conf=0.88)

  cites [22] Zaura E, Brandt BW, Prodan A, Teixeira, De Mattos MJ, Imangaliyev S, Kool J, et al. On the ecosystemic network of saliva in healthy young adults. ISME J. 2017;11:1218–31. source

In the paper

“This study suggested that environment is the main determinant of the oral microbiome with differences between mono- or di-zygotic twins not being significant.”

What the sources say

• Supported

  The Discussion states that monozygotic twins were somewhat more similar in salivary microbial composition than dizygotic twins, but the difference was not statistically significant. It concludes that environment has a greater impact on overall oral microbiome composition in the studied age range. (LLM, conf=0.94)

  cites [27] Stahringer SS, Clemente JC, Corley RP, Hewitt J, Knights D, Walters WA, et al. Nurture trumps nature in a longitudinal survey of salivary bacterial communities in twins from early adolescence to early adulthood. Genome Res. 2012;22:2146–52. source

In the paper

“In terms of the driving species, our stomatotypes 1 and 2 are similar, respectively, to MIC1.3 and MIC2 of the 268 adults study and to clusters 1 and 2 of the 161 adults study.”

What the sources say

• Supported

  The Results (‘The salivary microbiota is not correlated to dietary habit’) identify this as a study of 161 saliva samples and describe the driving genera of clusters I and II: Neisseria and Fusobacterium for cluster I, and Prevotella for cluster II. The citation identifies the 161-person study and its clusters; the comparison with the citing authors’ stomatotypes is their own interpretation. (LLM, conf=0.92)

  cites [16] De Filippis F, Vannini L, La Storia A, Laghi L, Piombino P, Stellato G, et al. The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and vegan individuals. PLoS One. 2014;9:e112373. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Zaura E, Brandt BW, Prodan A, Teixeira, De Mattos MJ, Imangaliyev S, Kool J, et al. On the ecosystemic network of saliva in healthy young adults. ISME J. 2017;11:1218–31. source

In the paper

“These questions were adapted from questionnaires available at the PhenX toolkit (consensus measures for Phenotypes and eXposures), which provides recommended standard data collection protocols for conducting biomedical research [60] and which has been recommended by the microbiome research community [61].”

What the sources say

• Supported

  The Introduction states that PhenX protocols can be questions for a survey instrument and provide standard procedures for collecting measures in biomedical research. The Key Concepts section also describes the Toolkit’s recommended measures and standard protocols. The separate claim about recommendation by the microbiome research community is attributed to citation [61], not this source. (LLM, conf=0.92)

  cites [60] Hendershot T, Pan H, Haines J, Harlan WR, Marazita ML, CA MC, et al. Using the PhenX toolkit to add standard measures to a study. Curr Protoc Hum Genet. 2015;86:1.21.1–17. source
• Supported

  The citation to this paper [61] supports the statement that the microbiome research community recommends PhenX. In “Assaying and Understanding the Microbiome,” the paper presents PhenX as a model for annotating microbiome metadata and says adherence to its standard would be especially useful. The separate claim about standard data-collection protocols is attributed to [60], not this source. (LLM, conf=0.91)

  cites [61] Huttenhower C, Knight R, Brown CT, Caporaso JG, Clemente JC, Gevers D, et al. Advancing the microbiome research community. Cell. 2014;159:227–30. source

In the paper

“Although 108 students declared smoking occasionally, we did not detect significant differences in their microbiomes.”

In the paper

“The students were more often consuming sweets and chewing gum, while teachers were consuming significantly more coffee and alcohol, reported more dental health problems, and used flossing more frequently.”

In the paper

“Socioeconomic status did correlate significantly with the abundance of some genera, positively with Rhizobium and negatively with Bradyrhizobium, Acinetobacter, and Pseudomonas.”

In the paper

“Measured oral pH in our samples had a median of 7.5 but showed a wide range [see Additional file 9].”

In the paper

“The top ten most abundant genera represented collectively 84.64% of the analyzed sequences and were present in 99.6% of the samples.”

In the paper

“For instance, 20 of our 32 common genera are also common in a recent study of the oral microbiome of 2343 adults in Hisayama (Japan) [9].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [9] Takeshita T, Kageyama S, Furuta M, Tsuboi H, Takeuchi K, Shibata Y, et al. Bacterial diversity in saliva and oral health-related conditions: the Hisayama study. Sci Rep. 2016;6:22164. source

In the paper

“In both stomatotypes, Streptococcus is positively correlated with Gemella and negatively with Prevotella and Fusobacterium (Fig. 3c).”

In the paper

“These patterns show striking similarities with those found in unrelated populations.”

• Also stated in: § Oral microbiome diversity is structured into two major stomatotypes · boosted

  “Importantly, although the studies are performed with different methodologies and have largely different target populations, we noted a strong parallelism between our two stomatotypes and the defined “coinhabiting groups” in the abovementioned Hisayama study [9].”

• Also stated in: § Discussion · plain

  “Importantly, our two defined stomatotypes show notable overlap with the two “coinhabiting” groups of bacteria identified in another large study [9].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [9] Takeshita T, Kageyama S, Furuta M, Tsuboi H, Takeuchi K, Shibata Y, et al. Bacterial diversity in saliva and oral health-related conditions: the Hisayama study. Sci Rep. 2016;6:22164. source

In the paper

“Hygiene habits usually showed high correlations among themselves, so that people who brush their teeth more often tended to use fluoride supplements and floss and were more likely to wash their hands before eating and/or after using the bathroom.”

In the paper

“All genera discussed in the paper with the exception of Rubellimicrobium and Undibacterium have been previously identified in oral samples.”

In the paper

“Notably, however, the two stomatotypes, and some genera, were geographically widespread but showed distinct abundance patterns, which suggest some environmental influence.”

In the paper

“For instance, smokers tend to consume more alcohol, and students who reported having a kissing partner were more likely to smoke, drink alcohol, or chew gum [see Additional file 6].”

In the paper

“Streptococcus was the most abundant genus in most (68%) samples and showed an average relative abundance of 22.3%, followed by Prevotella (11.9%), Haemophilus (11.4%), Neisseria (10.1%), and Veillonella (9%).”

In the paper

“In contrast, we did not find many differences between genders or rural versus urban environment.”

In the paper

“We found no large differences between oral microbiomes of males and females, with only two genera (Actinomyces and Oribacterium) showing significantly different abundances (both higher in males).”

In the paper

“Additionally, people using braces were more often brushing their teeth, and those reporting past nerve extractions drank more alcohol.”

In the paper

“Other differences include higher proportions of Haemophilus in stomatotype 1 and higher proportions of Veillonella and Streptococcus in stomatotype 2.”

In the paper

“Interestingly, students with kissing partners had a higher number of taxa in their microbiomes, which also showed a significantly higher presence of Treponema.”

In the paper

“The stomatotypes are driven mostly by certain abundant genera, but do not explain the variability found in many other genera.”

In the paper

“Although many of the covariations between the two stomatotypes are similar, their strengths can be markedly different. In addition, some covariations appear specific for each stomatotype.”

In the paper

“Overall, we identified 332 operational taxonomic units (OTUs) at the genus level in our dataset.”

In the paper

“Ralstonia abundance was also negatively affected by nearly all other water variables, and it was the genus whose abundance changed the most with tap water quality, followed by Rhizobium, Veillonella, and Pseudomonas.”

In the paper

“Boys and girls had some different habits. While the former tended to drink more milk, coke, or energetic drinks, the latter chewed gum and brushed their teeth more often.”

In the paper

“Overall, the microbiome samples of our study can be clustered into two broad compositional patterns (stomatotypes), driven mostly by Neisseria and Prevotella, respectively.”

• Also stated in: § Oral microbiome diversity is structured into two major stomatotypes · plain

  “The two defined stomatotypes differ in their microbial composition and abundance covariations, and for which Neisseria (stomatotype 1) and Prevotella (stomatotype 2) are the genera driving most differences (Fig. 3).”

• Also stated in: § Discussion · plain

  “We found that the oral microbiome of the studied population can be broadly classified into two different stomatotypes.”

In the paper

“Yet, many parallels can also be found between our stomatotypes, and those in these studies.”

• Also stated in: § Discussion · boosted

  “Although other studies have defined higher number of clusters in the oral microbiome [16, 22], some of these clusters show clear similarities with the two stomatotypes found in this study.”

What the sources say

• Not checked yetcites [16] De Filippis F, Vannini L, La Storia A, Laghi L, Piombino P, Stellato G, et al. The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and vegan individuals. PLoS One. 2014;9:e112373. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [22] Zaura E, Brandt BW, Prodan A, Teixeira, De Mattos MJ, Imangaliyev S, Kool J, et al. On the ecosystemic network of saliva in healthy young adults. ISME J. 2017;11:1218–31. source

In the paper

“The core oral microbiome described in this study is composed of genera commonly identified in other oral microbiome studies.”

In the paper

“However, the two stomatotypes do contain a significant amount of variability and a gradient of variation, sometimes unrelated to the stomatotypes, is observed for the most abundant genera.”

In the paper

“Three hundred thirty-two different genera were identified in these samples.”

In the paper

“Although the presence of the most abundant genera of the oral microbiome such as Streptococcus, Prevotella, or Haemophilus (the top three in our samples) were not significantly affected by tap water, some genera among the ten most abundant were affected, including Veillonella, Porphyromonas, and Gemella.”

In the paper

“The two studies coincide in defining higher proportions of Neisseria, Haemophilus, and Porphyromonas, in one of the types (stomatotype 1, coinhabiting group 2), and those of Prevotella, and Veillonella in the other (stomatotype 2, coinhabiting group 1).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [9] Takeshita T, Kageyama S, Furuta M, Tsuboi H, Takeuchi K, Shibata Y, et al. Bacterial diversity in saliva and oral health-related conditions: the Hisayama study. Sci Rep. 2016;6:22164. source

In the paper

“However, we cannot discard the possibility that some of the low abundance genera identified are not stable components of the oral cavity but result from sporadic colonization from the close environment of the donor (i.e., food, air, or water).”

In the paper

“For instance, in the case of stomatotype 1, we detected positive covariation of Fusobacterium and Capnocytophaga, both anaerobic bacteria implicated in dental plaque progression [33], while in stomatotype 2, we specifically detect antagonism between Streptococcus and Actinomyces, which are known to compete in the initial phases of dental plaque formation [32, 37].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [33] Takeshita T, Yasui M, Shibata Y, Furuta M, Saeki Y, Eshima N, et al. Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach. Sci Rep 2015;5:8136. Available from: http://www.nature.com/articles/srep08136 source

In the paper

“Some of these differences may relate to the fact that some habits, such as smoking or some dietary habits, may have just been recently established, or the habit is more sporadic in adolescents, and the effects in the microbiome will only be apparent after a prolonged period of sustained habit.”

In the paper

“Positive correlations between Porphyromonas and Fusobacterium and negative correlations between Veillonella and Gemella further support this model, while positive correlations between Porphyromonas and Gemella and negative correlations between Porphyromonas and Veillonella would not be explained by the current plaque succession model.”

In the paper

“Microbial compositions in oral rinse samples can only be considered a proxy for plaque communities, as the procedure collects cells from different oral niches.”

In the paper

“In accordance with this, all of our negative controls provided no measurable results and a negligible number of reads when forced into library preparation and sequencing (see the “Materials and samples” section).”

In the paper

“These striking similarities between disparate studies suggest that these two major stomatotypes may be ubiquitous and define global equilibria in the human mouth microbiome.”

• Also stated in: § Discussion · hedged

  “That the two disparate studies agree in the two broadly defined groups strongly suggests that these two stomatotypes define two possible equilibria of oral microbial communities which are globally present.”

In the paper

“It is known that caries is generally associated with an increase of Streptococcus, but also of Granulicatella, and Gemella [21]—although in the latter case, this varies with age [39]—supporting the effect of brushing against primary dental plaque.”

What the sources say

• Not checked yetcites [21] Costalonga M, Herzberg MC. The oral microbiome and the immunobiology of periodontal disease and caries. Immunol Lett. 2014;162:22–38. source

In the paper

“For instance, in the case of stomatotype 1, we detected positive covariation of Fusobacterium and Capnocytophaga, both anaerobic bacteria implicated in dental plaque progression [33], while in stomatotype 2, we specifically detect antagonism between Streptococcus and Actinomyces, which are known to compete in the initial phases of dental plaque formation [32, 37].”

What the sources say

• ?Not checked — no full text of the sourcecites [32] Jakubovics NS. Intermicrobial interactions as a driver for community composition and stratification of oral biofilms. J Mol Biol. 2015;427:3662–75. source
• ?Not checked — no full text of the sourcecites [37] Dige I, Raarup MK, Nyengaard JR, Kilian M, Nyvad B. Actinomyces naeslundii in initial dental biofilm formation. Microbiology. 2009;155:2116–26. source

In the paper

“For instance, strong co-occurrence links Leptotrichia, Actinomyces, and Prevotella, and this latter one with Veillonella, suggesting they may be ecologically related.”

In the paper

“Veillonella species are known to increase their abundance in acidic environments derived from fermentation processes, such as those occurring in mature dental plaque [3].”

What the sources say

• Not checked yetcites [3] Hajishengallis E, Parsaei Y, Klein MI, Koo H. Advances in the microbial etiology and pathogenesis of early childhood caries. Mol Oral Microbiol. 2017;32:24–34. source

In the paper

“Adolescence is a stage with major hormonal and habit changes, which likely impact the oral microbial community.”

In the paper

“It is known that caries is generally associated with an increase of Streptococcus, but also of Granulicatella, and Gemella [21]—although in the latter case, this varies with age [39]—supporting the effect of brushing against primary dental plaque.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [39] Holgerson PL, Öhman C, Rönnlund A, Johansson I. Maturation of oral microbiota in children with or without dental caries. PLoS One. 2015;10:e0128534. source

In the paper

“Selenomonas has been described as one of the most abundant taxa during orthodontic braces treatment and has been linked to common oral diseases such as gingivitis [19].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [19] Koopman JE, van der Kaaij NCW, Buijs MJ, Elyassi Y, van der Veen MH, Crielaard W, et al. The effect of fixed orthodontic appliances and fluoride mouthwash on the oral microbiome of adolescents - a randomized controlled clinical trial. PLoS One 2015;10:e0137318. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26332408%5Cn http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4558009 source

In the paper

“The patterns were sometimes reminiscent of maps of certain public water quality parameters, such as alkalinity or water hardness, which differ significantly across regions in Spain (Fig. 6).”

In the paper

“In addition, the oral microbiome of adolescents may have specificities as a transition phase from childhood to adulthood.”

In the paper

“Admittedly, measurements of saliva pH using pH strips—a limitation imposed in part by our citizen science approach—lack the precision provided by a pH meter (see “Materials and methods” section).”

In the paper

“Higher oral pH was positively correlated with the abundance of Fusobacterium and Porphyromonas, a bacterial genus known to grow optimally in alkaline environments, and able to increase the pH of its medium [38].”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Takahashi N, Schachtele CF. Effect of pH on the growth and proteolytic activity of Porphyromonas gingivalis and Bacteroides intermedius. J Dent Res. 1990;69:1266–9. source

In the paper

“In fact, this period of life is associated with a sharp increase in the incidence and severity of gingivitis [45], which may be related to underlying oral microbiome changes.”

What the sources say

• ?Not checked — no full text of the sourcecites [45] Mombelli A, Gusberti FA, van Oosten MAC, Lang NP. Gingival health and gingivitis development during puberty: a 4-year longitudinal study. J Clin Periodontol. 1989;16:451–6. source

In the paper

“Interestingly, some variables such as body mass index, which is generally associated to alterations in the gut microbiome, and it has been associated to changes in the oral microbiome in adults [44], seemed to have a minor impact on the mouth microbiome of adolescents in our sample.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [44] Shillitoe E, Weinstock R, Kim T, Simon H, Planer J, Noonan S, et al. The oral microflora in obesity and type-2 diabetes. J Oral Microbiol 2012;4:19013. Available from: https://www.tandfonline.com/doi/full/10.3402/jom.v4i0.19013 source

In the paper

“These results suggest that tap water composition may be an important, poorly studied factor shaping the oral microbiome.”

• Also stated in: § Results · plain

  “This points to an important role of drinking water in shaping the oral microbiota, which has been so far poorly explored.”

In the paper

“These large differences may be related to age but also to their understandably different lifestyle.”

In the paper

“As we discuss below, these stomatotypes are not discrete, well separated entities, but rather represent two poles of a gradient of microbial compositions.”

In the paper

“Our results thus raise the question of the role of drinking water in shaping the oral microbiome, suggesting a potentially important role.”

In the paper

“Next, we used the Calinski-Harabasz (CH) index [59] to determine the optimal number of clusters, and we further verified this by calculating the average silhouette width of the samples, which is a measure of the separation of samples within one cluster from those of another cluster, as well as the prediction strength, another measure of the efficiency of clustering.”

What the sources say

• ?Not checked — no full text of the sourcecites [59] Calinski T, Harabasz J. A dendrite method for cluster analysis. Commun Stat - Theory Methods. 1974;3:1–27. source

In the paper

“In fact, this period of life is associated with a sharp increase in the incidence and severity of gingivitis [45], which may be related to underlying oral microbiome changes.”

In the paper

“Furthemore, the adonis function in the vegan package was used to perform a PERMANOVA test on each beta diversity measure to ensure significant separation of stomatotypes.”

In the paper

“As a control for downstream procedures, we also used two DNA samples derived from bacterial mock communities obtained from the BEI Resources of the Human Microbiome Project: Each sample contained genomic DNA of ribosomal operons from 20 bacterial species.”

In the paper

“Saliva samples were collected using a mouth wash and using a protocol that had been previously tested and compared with other procedures during a pilot phase of the project. Of note, this procedure is used in other oral microbiome studies and have been shown to produce consistent results with other sampling procedures [51].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [51] Lim Y, Totsika M, Morrison M, Punyadeera C. The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols. Sci Rep. 2017;7:8523. source

In the paper

“Negative controls of the sample collection buffer, DNA extraction, and PCR amplification steps were routinely performed in parallel, using the same conditions and reagents. Our controls systematically provided no visible band or quantifiable DNA amounts by gel visualization or Bioanalyzer, whereas all of our samples provided clearly visible bands after 20 cycles.”

• Also stated in: § DNA extraction and sequencing · plain

  “Four such controls were subjected to library preparation and sequenced. Expectedly, these sequenced non-template controls systematically yielded very few reads (a range of 30–880 reads per sample), in contrast to an average of 54,000 reads/library in sample-derived libraries.”

In the paper

“Although the use of pH test strips have been validated extensively [50], we validated our chosen strips.”

• Also stated in: § Sample collection · plain

  “The correlation was high (R2 = 0.96), with average absolute differences between the value of the pH meter and that provided by the researcher being 0.33 which is within the range of the limit of detection of the method (0.5).”

What the sources say

• ?Not checked — no full text of the sourcecites [50] Cocco F, Cagetti MG, Lingstrom P, Camoni N, Campus G. The strip method and the microelectrode technique in assessing dental plaque pH. Minerva Stomatol. 2017;66:241–247. source

In the paper

“In this regard, experiments in mice have shown that the composition of tap water can be related with changes in the gut microbiome [47] and have an incidence in the progression of diseases such as diabetes [48].”

What the sources say

• ?Not checked — no full text of the sourcecites [47] Dias MF, Reis MP, Acurcio LB, Carmo AO, Diamantino CF, Motta AM, et al. Changes in mouse gut bacterial community in response to different types of drinking water. Water Res. 2018;132:79–89. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [48] Wolf KJ, Daft JG, Tanner SM, Hartmann R, Khafipour E, Lorenz RG. Consumption of acidic water alters the gut microbiome and decreases the risk of diabetes in NOD mice. J Histochem Cytochem. 2014;62:237–50. source

In the paper

“Clustering was validated using all five distance measures to ensure proper clustering, but analyses here are performed using the clustering based on JSD.”

In the paper

“the described stomatotypes are common and globally distributed does not preclude the possibility that further, clearly distinct, stomatotypes may be found in other populations.”

In the paper

“One hundred eighty samples from 5 of the schools had to be removed due to an apparent batch effect during the sequencing procedure. This batch effect was detected in the initial quality assessment of the comparison of the data.”

• Also stated in: § Microbiome composition profiling · plain

  “Additionally, 33 samples were removed from the analyses because of errors with the sample identifiers, leaving a total of 1319 samples from 35 different schools from around Spain.”

In the paper

“This points to an important role of tap water in shaping the oral microbiome, which has been overlooked in previous studies.”

In the paper

“All participants, and at least one of their parents or legal guardians for those under the age of 18, signed a consent form to use their saliva samples for microbiome research. This consent form and the purpose of this project received approval by the ethics committee of the Barcelona Biomedical Research Park (PRBB).”

In the paper

“Previous studies have analyzed the relationship between the presence of fluoride and the incidence of caries [46], but the overall impact on the human oral microbiota of this and other factors remain unexplored.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [46] Iheozor-Ejiofor Z, Worthington H V, Walsh T, O’Malley L, Clarkson JE, Macey R, et al. Water fluoridation for the prevention of dental caries. Cochrane Database Syst Rev. 2015. https://doi.org/10.1002/14651858.CD010856.pub2 . source

In the paper

“The specific pipeline and parameters were set using sequence reads from both 16S rRNA amplicon and whole genome sequencing of the described mock communities.”