Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy

Luo, Ting; Srinivasan, Usha; Ramadugu, Kirtana; Shedden, Kerby; Neiswanger, Katherine; Trumble, Erika; Li, Jiean J.; McNeil, Daniel W.; Crout, Richard J.; Weyant, Robert J.; Marazita, Mary L.; Foxman, Betsy

doi:10.1128/aem.01132-16

Cited by 34 publications

(26 citation statements)

References 38 publications

(40 reference statements)

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“…Using nitrate supplementation as selective pressure for bacteria capable of nitrate reduction, several studies found proliferation of Veillonella [205], Streptococcus [204], Neisseria [115], Haemophilus [204], and Rothia species[115], all previously identified as high nitrate reducers. Yet, despite these varied attempts, there remains a high degree of variability in results depending on numerous methodological factors[206], including source and sampling of sample (saliva versus oral wash versus tongue scraping), direct sampling versus culture, and choice of 16S variable region for sequencing.…”

Section: The Oral Microbiome and Bacterial Nitrate Reductionmentioning

confidence: 99%

Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health

Koch

Gladwin

Freeman

et al. 2017

Free Radical Biology and Medicine

139

127

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Recent insights into the bioactivation and signaling actions of inorganic, dietary nitrate and nitrite now suggest a critical role for the microbiome in the development of cardiac and pulmonary vascular diseases. Once thought to be the inert, end-products of endothelial-derived nitric oxide (NO) heme-oxidation, nitrate and nitrite are now considered major sources of exogenous NO that exhibit enhanced vasoactive signaling activity under conditions of hypoxia and stress. The bioavailability of nitrate and nitrite depend on the enzymatic reduction of nitrate to nitrite by a unique set of bacterial nitrate reductase enzymes possessed by specific bacterial populations in the mammalian mouth and gut. The pathogenesis of pulmonary hypertension (PH), obesity, hypertension and CVD are linked to defects in NO signaling, suggesting a role for commensal oral bacteria to shape the development of PH through the formation of nitrite, NO and other bioactive nitrogen oxides. Oral supplementation with inorganic nitrate or nitrate-containing foods exert pleiotropic, beneficial vascular effects in the setting of inflammation, endothelial dysfunction, ischemia-reperfusion injury and in pre-clinical models of PH, while traditional high-nitrate dietary patterns are associated with beneficial outcomes in hypertension, obesity and CVD. These observations highlight the potential of the microbiome in the development of novel nitrate- and nitrite-based therapeutics for PH, CVD and their risk factors.

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Section: The Oral Microbiome and Bacterial Nitrate Reductionmentioning

confidence: 99%

Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health

Koch

Gladwin

Freeman

et al. 2017

Free Radical Biology and Medicine

139

127

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“…This approach has exploded in popularity with advances in sequencing throughput such that it is now possible to characterize numerous samples with thousands of sequences per sample. Many factors can impact how a natural community is represented by the sequencing data including the method of acquiring samples (4–8), storage conditions (4–6, 9–12), extraction methods (13), amplification conditions (8, 14, 15), sequencing method (15–17), and analytical pipeline (15, 18–20). The increased sampling depth that is now available relative to previous Sanger sequencing-based methods is expected to compound the impacts of an investigator’s choices and the interpretation of their results.…”

Section: Introductionmentioning

confidence: 99%

The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

Sze

Schloss

2019

Preprint

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1 PCR amplification of 16S rRNA genes is a critical, yet under appreciated step in the generation 2 of sequence data to describe the taxonomic composition of microbial communities. Numerous 3 factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric 4 sequences, and the degree to which the fragments in the product represent their abundance in 5 the original sample (i.e. bias). We compared the performance of high fidelity polymerases and 6 varying number of rounds of amplification when amplifying a mock community and human stool 7 samples. Although it was impossible to derive specific recommendations, we did observe general 8 trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number 9 of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. 10 Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments' 11 fraction of bases that were guanines or cytosines. When analyzing mock community data, the 12 amount that the community deviated from the expected composition increased with rounds of PCR. 13 This bias was inconsistent for human stool samples. Overall the results underscore the difficulty 14 of comparing sequence data that are generated by different PCR protocols. However, the results 15 indicate that the variation in human stool samples is generally larger than that introduced by the 16 choice of polymerase or number of rounds of PCR. 17 Importance 18 A steep decline in sequencing costs drove an explosion in studies characterizing microbial 19 communities from diverse environments. Although a significant amount of effort has gone into 20 understanding the error profiles of DNA sequencers, little has been done to understand the 21 downstream effects of the PCR amplification protocol. We quantified the effects of the choice of 22 polymerase and number of PCR cycles on the quality of downstream data. We found that these 23 choices can have a profound impact on the way that a microbial community is represented in the 24 sequence data. The effects are relatively small compared to the variation in human stool samples, 25 however, care should be taken to use polymerases with the highest possible fidelity and to minimize 26 2 the number of rounds of PCR. These results also underscore that it is not possible to directly 27 compare sequence data generated under different PCR conditions. 28 3

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“…The V6 region of the 16s rRNA gene was amplified in the sample set, using primers and protocols described elsewhere [23,24]. The forward and reverse primer sequences mapped to the 967-985 (CAACGC-GARGAACCTTACC) and 1078-1061 (ACAACACGAGCTGACGAC) on the Escherichia coli 16s rRNA segment, with unique 8-nucleotide error-correcting barcodes added to the reverse primer for each sample.…”

Section: Amplicon Sequencing For Characterization Of Salivary Microbimentioning

confidence: 99%

Co-occurrence of yeast, streptococci, dental decay, and gingivitis in the post-partum period: results of a longitudinal study

Ramadugu

Blostein

Bhaumik

et al. 2020

Journal of Oral Microbiology

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Objective: The interactions between yeast and streptococci species that lead to dental decay and gingivitis are poorly understood. Our study describes these associations among a cohort of 101 post-partum women enrolled in the Center for Oral Health Research in Appalachia, 2012-2013. Methods: All eligible women without dental caries were included (n = 21) and the remainder were randomly sampled to represent the total number of decayed, missing, and filled teeth (DMFT) at enrollment. We used amplicon sequencing and qPCR of saliva from 2, 6, 12 and 24 visits to determine microbiome composition. Results: Active decay and generalized gingivitis were strongly predictive of each other. Using adjusted marginal models, Candida albicans and Streptococcus mutans combined were associated with active decay (OR = 3.13; 95% CI 1.26, 7.75). However, C. albicans alone (OR = 2.33; 95% CI: 0.81, 6.75) was associated with generalized gingivitis, but S. mutans alone was not (OR = 0.55; 95% CI: 0.21, 1.44). Models including microbiome community state types (CSTs) showed CSTs positively associated with active decay were negatively associated with generalized gingivitis. Discussion: C. albicans is associated with active decay and generalized gingivitis, but whether one or both are present depends on the structure of the co-existing microbial community.

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Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy

Cited by 34 publications

References 38 publications

Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health

Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health

The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

Co-occurrence of yeast, streptococci, dental decay, and gingivitis in the post-partum period: results of a longitudinal study

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