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

It may state two separate claims as one.

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.

“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].”

• 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. sourcePartly 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)

  Experimental — accuracy not yet measured

It may state two separate claims as one.

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

“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.”

• 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. sourceSupported
• 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. sourceCheck pending

It may state two separate claims as one.

Although 108 students declared smoking occasionally, no significant differences were detected in their microbiomes.

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

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

Students were more often consuming sweets and chewing gum, while teachers consumed significantly more coffee and alcohol, reported more dental health problems, and used flossing more frequently.

“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.”

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

Socioeconomic status correlated significantly with abundance of some genera, positively with Rhizobium and negatively with Bradyrhizobium, Acinetobacter, and Pseudomonas.

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

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

Measured oral pH in the samples had a median of 7.5 but showed a wide range.

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

It may state two separate claims as one.

The study found high alpha diversity (mean Shannon diversity 2.5) and low beta diversity (mean weighted UniFrac distance 0.118).

“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).”

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

It may state two separate claims as one.

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

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Twenty of the 32 common genera are also common in a study of 2343 adults in Hisayama, Japan.

“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].”

• 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. sourceCheck pending

It may state two separate claims as one.

In both stomatotypes, Streptococcus is positively correlated with Gemella and negatively with Prevotella and Fusobacterium.

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

It may state two separate claims as one.

The two stomatotypes show striking similarities or notable overlap with the coinhabiting groups found in the Hisayama study of unrelated populations.

“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].”

• 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. sourceCheck pending

It may state two separate claims as one.

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.

“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.”

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The core microbiome comprised typical oral bacteria commonly identified as abundant in similar oral microbiome surveys.

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

• 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 sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

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

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

It may state two separate claims as one.

The two stomatotypes and some genera were geographically widespread but showed distinct abundance patterns.

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

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Smokers tend to consume more alcohol, and students who reported having a kissing partner were more likely to smoke, drink alcohol, or chew gum.

“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].”

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

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

“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%).”

It may state two separate claims as one.

The study did not find many differences between genders or rural versus urban environment.

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

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

There were no large differences between oral microbiomes of males and females, with only Actinomyces and Oribacterium showing significantly different abundances, both higher in males.

“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).”

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

People using braces were more often brushing their teeth, and those reporting past nerve extractions drank more alcohol.

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

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

Other differences between stomatotypes include higher proportions of Haemophilus in stomatotype 1 and higher proportions of Veillonella and Streptococcus in stomatotype 2.

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

It may state two separate claims as one.

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

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

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

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

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

It may state two separate claims as one.

Although many covariations between the two stomatotypes are similar, their strengths can be markedly different, and some covariations appear specific for each stomatotype.

“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.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The dataset contained 332 operational taxonomic units at the genus level.

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Several of the correlations mentioned in the study coincide with those found in previous studies.

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

• 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. sourceNot checked yet
• 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. sourceCheck pending

It may state two separate claims as one.

Ralstonia abundance was 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.

“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.”

• AssumptionRests on an assumption: The chemical composition of tap water in the nearest town represents the tap water consumed by participants.

It may state two separate claims as one.

Boys and girls had some different habits: boys tended to drink more milk, coke, or energetic drinks; girls chewed gum and brushed their teeth more often.

“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.”

• AssumptionRests on an assumption: Questionnaire responses accurately reflect participants' habits and health.

It may state two separate claims as one.

The microbiome samples cluster into two broad compositional patterns (stomatotypes), driven mostly by Neisseria and Prevotella, respectively, and differing in microbial composition and abundance covariations.

“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.”

It may state two separate claims as one.

Many parallels can be found between the study's stomatotypes and those in other studies, including clusters that show clear similarities.

“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.”

• 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. sourceNot checked yet
• 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. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

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

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

It may state two separate claims as one.

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

“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.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

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

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

It may state two separate claims as one.

Although the most abundant genera Streptococcus, Prevotella, or Haemophilus were not significantly affected by tap water, some genera among the ten most abundant were affected, including Veillonella, Porphyromonas, and Gemella.

“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.”

• AssumptionRests on an assumption: The chemical composition of tap water in the nearest town represents the tap water consumed by participants.

It may state two separate claims as one.

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

“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).”

• 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. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The possibility cannot be discarded that some low abundance genera identified are not stable components of the oral cavity but result from sporadic colonization from the close environment of the donor (food, air, or water).

“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).”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Fusobacterium and Capnocytophaga are anaerobic bacteria implicated in dental plaque progression.

“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].”

• 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 sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.

Some of the 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.

“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.”

It may state two separate claims as one.

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

“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.”

Probably not a substantial claim. It may state two separate claims as one.

Other studies of the oral microbiome have found different numbers of clusters: five discrete clusters in 268 healthy young adults and three clusters in 161 healthy adults.

“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].”

• 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. sourceCheck pending
• 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. sourceNot checked yet

Probably not a substantial claim.

Individual microbiomes from certain niches can be clustered into different types, such as the enterotypes of the gut microbiome.

“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].”

• 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. sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it.

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

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

It may state two separate claims as one.

All negative controls provided no measurable results and a negligible number of reads when forced into library preparation and sequencing.

“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).”

It may state two separate claims as one.

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

“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.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Caries is generally associated with an increase of Streptococcus, Granulicatella, and Gemella.

“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.”

• cites [21] Costalonga M, Herzberg MC. The oral microbiome and the immunobiology of periodontal disease and caries. Immunol Lett. 2014;162:22–38. sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Streptococcus and Actinomyces are known to compete in the initial phases of dental plaque formation.

“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].”

• cites [32] Jakubovics NS. Intermicrobial interactions as a driver for community composition and stratification of oral biofilms. J Mol Biol. 2015;427:3662–75. sourceNot checked — no full text of the source
• cites [37] Dige I, Raarup MK, Nyengaard JR, Kilian M, Nyvad B. Actinomyces naeslundii in initial dental biofilm formation. Microbiology. 2009;155:2116–26. sourceNot checked — no full text of the source

It may state two separate claims as one.

The observed abundance covariations in both stomatotypes may partly reflect underlying diversity of biofilm succession stages and would support the main axis of previously observed core community changes in dental plaque.

“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.”

• 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. sourceNot checked yet
• cites [32] Jakubovics NS. Intermicrobial interactions as a driver for community composition and stratification of oral biofilms. J Mol Biol. 2015;427:3662–75. sourceNot checked — no full text of the source
• 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 sourceCheck pending
• cites [34] Mahajan A, Singh B, Kashyap D, Kumar A, Mahajan P. Interspecies communication and periodontal disease. Sci World J. 2013;2013:765434 sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Earlier studies using similar collection protocols and including plaque status or dental health have found correlations between microbial composition of saliva, amount of plaque, and diseases such as periodontitis or caries.

“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].”

• 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 sourceCheck pending
• 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. sourceCheck pending
• 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. sourceCheck pending
• 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. sourceCheck pending

It may state two separate claims as one.

Strong co-occurrence links Leptotrichia, Actinomyces, and Prevotella, and Prevotella with Veillonella, suggesting they may be ecologically related.

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

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

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

• 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. sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.

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

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The association of caries with Gemella varies with age.

“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.”

• 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. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

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.

“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].”

• 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 sourceCheck pending

It may state two separate claims as one.

The geographical 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.

“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).”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The oral microbiome of adolescents may have specificities as a transition phase from childhood to adulthood.

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Measurements of saliva pH using pH strips lack the precision provided by a pH meter.

“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).”

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Porphyromonas is known to grow optimally in alkaline environments and is able to increase the pH of its medium.

“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].”

• cites [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. sourceNot checked — no full text of the source

Probably not a substantial claim. The authors may not put it forward as true. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Environment is the main determinant of the oral microbiome, with differences between monozygotic and dizygotic twins not significant.

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

• 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. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Adolescence is associated with a sharp increase in the incidence and severity of gingivitis.

“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.”

• cites [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. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Body mass index is generally associated with alterations in the gut microbiome and has been associated with changes in the oral microbiome in adults.

“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.”

• 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 sourceCheck pending

It may state two separate claims as one.

Tap water composition may be an important, poorly studied factor shaping the oral microbiome.

“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.”

• AssumptionRests on an assumption: The chemical composition of tap water in the nearest town represents the tap water consumed by participants.

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

The large differences between students and teachers may be related to age but also to their different lifestyle.

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

It may state two separate claims as one.

The stomatotypes are not discrete, well separated entities, but represent two poles of a gradient of microbial compositions.

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

Probably not a substantial claim. It may state two separate claims as one.

The results raise the question of the role of drinking water in shaping the oral microbiome, suggesting a potentially important role.

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

• AssumptionRests on an assumption: The chemical composition of tap water in the nearest town represents the tap water consumed by participants.

Probably not a substantial claim. It may state two separate claims as one.

The optimal number of clusters was determined using the Calinski-Harabasz index and further verified by average silhouette width and prediction strength.

“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.”

• cites [59] Calinski T, Harabasz J. A dendrite method for cluster analysis. Commun Stat - Theory Methods. 1974;3:1–27. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The increase in gingivitis during adolescence may be related to underlying oral microbiome changes.

“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.”

Probably not a substantial claim.

A PERMANOVA test on each beta diversity measure ensured significant separation of stomatotypes.

“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.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Two DNA samples from bacterial mock communities were used as controls for downstream procedures.

“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.”

It may state two separate claims as one.

The saliva collection protocol had been previously tested and compared with other procedures during a pilot phase, and is used in other oral microbiome studies and shown to produce consistent results with other sampling procedures.

“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].”

• 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. sourceCheck pending

It may state two separate claims as one.

Negative controls of sample collection buffer, DNA extraction, and PCR amplification were routinely performed and systematically provided no visible band or quantifiable DNA, and sequenced non-template controls yielded very few reads (30-880) versus average 54,000 reads/library in sample-derived libraries.

“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.”

It may state two separate claims as one.

pH test strips were validated against a pH meter, showing high correlation (R2 = 0.96) and average absolute difference 0.33, within the method's limit of detection (0.5).

“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).”

• cites [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. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Experiments in mice have shown that the composition of tap water can be related with changes in the gut microbiome and have an incidence in the progression of diseases such as diabetes.

“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].”

• cites [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. sourceNot checked — no full text of the source
• 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. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

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

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

It may state two separate claims as one.

The described stomatotypes are common and globally distributed.

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

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

One hundred eighty samples from 5 schools were removed due to an apparent batch effect during sequencing, and 33 samples were removed due to sample identifier errors, leaving 1319 samples from 35 schools.

“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.”

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

The questionnaire questions were adapted from the PhenX toolkit, which provides recommended standard data collection protocols and has been recommended by the microbiome research community.

“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].”

• 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. sourceCheck pending
• 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. sourceCheck pending

It may state two separate claims as one.

Tap water plays an important role in shaping the oral microbiome, which has been overlooked in previous studies.

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

• AssumptionRests on an assumption: The ionic composition of tap water from donors' hometowns, as obtained from national studies, reflects the tap water actually consumed by the donors.

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

All participants and at least one parent or legal guardian for minors signed consent, and the project received ethics committee approval from the Barcelona Biomedical Research Park (PRBB).

“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).”

Probably not a substantial claim. It may state two separate claims as one.

Previous studies have analyzed the relationship between the presence of fluoride and the incidence of caries, but the overall impact on the human oral microbiota of this and other factors remains unexplored.

“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.”

• 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 . sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

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

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