scholarly journals Assessing saliva microbiome collection and processing methods

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Abigail J. S. Armstrong ◽  
Veenat Parmar ◽  
Martin J. Blaser
Keyword(s):  
Alpha Diversity ◽  
Shotgun Sequencing ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Shotgun Metagenomics ◽  
Human Dna ◽  
Within Subjects ◽  
Saliva Collection ◽  
Lung Health ◽  
Over Time

AbstractThe oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.

Metagenomic and Metatranscriptomic Responses of Chemical Dispersant Application during a Marine Dilbit Spill

2022 ◽  
Author(s):  
Yiqi Cao ◽  
Baiyu Zhang ◽  
Charles W. Greer ◽  
Kenneth Lee ◽  
Qinhong Cai ◽  
...  
Keyword(s):  
Microbial Community ◽  
Late Stage ◽  
Large Scale ◽  
Community Dynamics ◽  
Early Stage ◽  
Small Scale ◽  
Rrna Gene ◽  
Shotgun Metagenomics ◽  
Microcosm Studies ◽  
Over Time

The global increase in marine transportation of dilbit (diluted bitumen) can increase the risk of spills, and the application of chemical dispersants remains a common response practice in spill events. To reliably evaluate dispersant effects on dilbit biodegradation over time, we set large-scale (1500 mL) microcosms without nutrients addition using low dilbit concentration (30 ppm). Shotgun metagenomics and metatranscriptomics were deployed to investigate microbial community responses to naturally and chemically dispersed dilbit. We found that the large-scale microcosms could produce more reproducible community trajectories than small-scale (250 mL) ones based on the 16S rRNA gene amplicon sequencing. In the early-stage large-scale microcosms, multiple genera were involved into the biodegradation of dilbit, while dispersant addition enriched primarily Alteromonas and competed for the utilization of dilbit, causing depressed degradation of aromatics. The metatranscriptomic based Metagenome Assembled Genomes (MAG) further elucidated early-stage microbial antioxidation mechanism, which showed dispersant addition triggered the increased expression of the antioxidation process genes of Alteromonas species. Differently, in the late stage, the microbial communities showed high diversity and richness and similar compositions and metabolic functions regardless of dispersant addition, indicating the biotransformation of remaining compounds can occur within the post-oil communities. These findings can guide future microcosm studies and the application of chemical dispersants for responding to a marine dilbit spill. Importance In this study, we employed microcosms to study the effects of marine dilbit spill and dispersant application on microbial community dynamics over time. We evaluated the impacts of microcosm scale and found that increasing the scale is beneficial for reducing community stochasticity, especially in the late stage of biodegradation. We observed that dispersant application suppressed aromatics biodegradation in the early stage (6 days) whereas exerting insignificant effects in the late stage (50 days), from both substances removal and metagenomic/metatranscriptomic perspectives. We further found that Alteromonas species are vital for the early-stage chemically dispersed oil biodegradation, and clarified their degradation and antioxidation mechanisms. The findings would help to better understand microcosm studies and microbial roles for biodegrading dilbit and chemically dispersed dilbit, and suggest that dispersant evaluation in large-scale systems and even through field trails would be more realistic after marine oil spill response.

scholarly journals The Oral and Gut Bacterial Microbiomes: Similarities, Differences, and Connections

2020 ◽  
Vol 23 (1) ◽  
pp. 7-20
Author(s):  
Katherine A. Maki ◽  
Narjis Kazmi ◽  
Jennifer J. Barb ◽  
Nancy Ames
Keyword(s):  
Gut Microbiome ◽  
Alpha Diversity ◽  
Oral Microbiome ◽  
Taxonomic Resolution ◽  
Future Research ◽  
Sequencing Analysis ◽  
Systemic Diseases ◽  
Shotgun Metagenomics ◽  
Healthy Humans ◽  
Microbiome Research

Background: The oral cavity is associated with local and systemic diseases, although oral samples are not as commonly studied as fecal samples in microbiome research. There is a gap in understanding between the similarities and differences in oral and gut microbiomes and how they may influence each other. Methods: A scoping literature review was conducted comparing oral and gut microbiome communities in healthy humans. Results: Ten manuscripts met inclusion criteria and were examined. The oral microbiome sites demonstrated great variance in differential bacterial abundance and the oral microbiome had higher alpha diversity as compared to the gut microbiome. Studies using 16S rRNA sequencing analysis resulted in overall community differences between the oral and gut microbiomes when beta diversity was analyzed. Shotgun metagenomics sequencing increased taxonomic resolution to strain level (intraspecies) and demonstrated a greater percentage of shared taxonomy and oral bacterial translocation to the gut microbiome community. Discussion: The oral and gut microbiome bacterial communities may be more similar than earlier research has suggested, when species strain is analyzed through shotgun metagenomics sequencing. The association between oral health and systemic diseases has been widely reported but many mechanisms underlying this relationship are unknown. Although future research is needed, the oral microbiome may be a novel interventional target through its downstream effects on the gut microbiome. As nurse scientists are experts in symptom characterization and phenotyping of patients, they are also well posed to lead research on the connection of the oral microbiome to the gut microbiome in health and disease.

scholarly journals Oral Microbiota Development in Early Childhood

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Beatrice Kennedy ◽  
Sari Peura ◽  
Ulf Hammar ◽  
Silvia Vicenzi ◽  
Anna Hedman ◽  
...  
Keyword(s):  
Early Childhood ◽  
Relative Abundance ◽  
Early Life ◽  
Alpha Diversity ◽  
Antibiotic Use ◽  
Population Based ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Microbiota Composition

AbstractEarly life determinants of the oral microbiota have not been thoroughly elucidated. We studied the association of birth and early childhood characteristics with oral microbiota composition using 16 S ribosomal RNA (rRNA) gene sequencing in a population-based Swedish cohort of 59 children sampled at 6, 12 and 24 months of age. Repeated-measurement regression models adjusted for potential confounders confirmed and expanded previous knowledge about the profound shift of oral microbiota composition in early life. These alterations included increased alpha diversity, decreased beta diversity and alteration of bacterial composition with changes in relative abundance of 14 of the 20 most common operational taxonomic units (OTUs). We also found that birth characteristics, breastfeeding and antibiotic use were associated with overall phyla distribution and/or with the relative abundance of specific OTUs. Further, we detected a novel link between morning salivary cortisol level, a physiological marker of neuroendocrine activity and stress, and overall phyla distribution as well as with decreased abundance of the most common OTU mapped to the Streptococcaceae family. In conclusion, a major part of the maturation of the oral microbiome occurs during the first two years of life, and this development may be influenced by early life circumstances.

scholarly journals Tongue microbiome of smokeless tobacco users

2020 ◽  
Author(s):  
Esam Halboub ◽  
Mohammed Alakhali ◽  
Abdulwahhab H. Al-Amir ◽  
Husham E. Homeida ◽  
Divyashri Baraniya ◽  
...  
Keyword(s):  
Smokeless Tobacco ◽  
Alpha Diversity ◽  
16S Rrna Gene Sequencing ◽  
Cross Sectional Study ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Cross Sectional ◽  
Oral Carcinogenesis ◽  
Rothia Mucilaginosa ◽  
Rrna Gene Sequencing

Abstract Background The possibility that smokeless tobacco may contribute to oral carcinogenesis by influencing the oral microbiome has not been explored. This cross sectional study sought to assess the effect of using shammah, a form of smokeless tobacco prevalent in Arabia, on the tongue microbiome. Tongue scarping samples were obtained from twenty-nine shammah users (SU; 27.34±6.9 years) and 23 shammah non-users (SNU; 27.7±7.19 years) and analyzed with 16S rRNA gene sequencing (V1-V3). Species-level taxonomy assignment of the high-quality, merged reads was obtained using a previously described BLASTn-based algorithm. Downstream analyses were performed with QIIME, LEfSe, and R. Results A total of 178 species, belonging to 62 genera and 8 phyla were identified. Genera Streptococcus , Leptotrichia , Actinomyces , Veillonella , Haemophilus , Prevotella and Neisseria accounted for more than 60% of the average microbiome. There were no differences between the two groups in species richness and alpha-diversity, but PCoA showed significant separation (P=0.015, ANOSIM). LEfSe analysis identified 22 species to be differentially abundant between the SU and SNU. However, only 7 species maintained a false discovery rate of ≤ 0.2 and could cluster the two groups separately: Rothia mucilaginosa , Streptococcus sp. oral taxon 66, Actinomyces meyeri , Streptococcus vestibularis Streptococcus sanguinis and a potentially novel Veillonella species in association with SU, and Oribacterium asaccharolyticum with SNU. Conclusion Shammah use induces tongue microbiome changes that may be relevant to oral carcinogenesis, namely enrichment of species with high acetaldehyde production potential, which warrants further investigation.

scholarly journals Whole-Genome Metagenomic Analysis of the Gut Microbiome in HIV-1-Infected Individuals on Antiretroviral Therapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiangning Bai ◽  
Aswathy Narayanan ◽  
Piotr Nowak ◽  
Shilpa Ray ◽  
Ujjwal Neogi ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Gut Microbiome ◽  
Alpha Diversity ◽  
16S Rrna Gene Sequencing ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Human Gut ◽  
Shotgun Metagenomics ◽  
Human Gut Microbiome ◽  
Hiv 1

Gut microbiome plays a significant role in HIV-1 immunopathogenesis and HIV-1-associated complications. Previous studies have mostly been based on 16S rRNA gene sequencing, which is limited in taxonomic resolution at the genus level and inferred functionality. Herein, we performed a deep shotgun metagenomics study with the aim to obtain a more precise landscape of gut microbiome dysbiosis in HIV-1 infection. A reduced tendency of alpha diversity and significantly higher beta diversity were found in HIV-1-infected individuals on antiretroviral therapy (ART) compared to HIV-1-negative controls. Several species, such as Streptococcus anginosus, Actinomyces odontolyticus, and Rothia mucilaginosa, were significantly enriched in the HIV-1-ART group. Correlations were observed between the degree of immunodeficiency and gut microbiome in terms of microbiota composition and metabolic pathways. Furthermore, microbial shift in HIV-1-infected individuals was found to be associated with changes in microbial virulome and resistome. From the perspective of methodological evaluations, our study showed that different DNA extraction protocols significantly affect the genomic DNA quantity and quality. Moreover, whole metagenome sequencing depth affects critically the recovery of microbial genes, including virulome and resistome, while less than 5 million reads per sample is sufficient for taxonomy profiling in human fecal metagenomic samples. These findings advance our understanding of human gut microbiome and their potential associations with HIV-1 infection. The methodological assessment assists in future study design to accurately assess human gut microbiome.

scholarly journals Tongue microbiome of smokeless tobacco users

2020 ◽  
Author(s):  
Esam Halboub ◽  
Mohammed Alakhali ◽  
Abdulwahhab H. Al-Amir ◽  
Husham E. Homeida ◽  
Divyashri Baraniya ◽  
...  
Keyword(s):  
Smokeless Tobacco ◽  
Alpha Diversity ◽  
16S Rrna Gene Sequencing ◽  
Cross Sectional Study ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Cross Sectional ◽  
Oral Carcinogenesis ◽  
Rothia Mucilaginosa ◽  
Rrna Gene Sequencing

Abstract Background: The possibility that smokeless tobacco may contribute to oral carcinogenesis by influencing the oral microbiome has not been explored. This cross sectional study sought to assess the effect of using hammah, a form of smokeless tobacco prevalent in Arabia, on the tongue microbiome. Tongue scarping samples were obtained from twenty-nine shammah users (SU; 27.34±6.9 years) and 23 shammah non-users (SNU; 27.7±7.19 years) and analyzed with 16S rRNA gene sequencing (V1-V3). Species-level taxonomy assignment of the high-quality, merged reads was obtained using a previously described BLASTn-based algorithm. Downstream analyses were performed with QIIME, LEfSe, and R.Results: A total of 178 species, belonging to 62 genera and 8 phyla were identified. Genera Streptococcus , Leptotrichia , Actinomyces , Veillonella , Haemophilus , Prevotella and Neisseria accounted for more than 60% of the average microbiome. There were no differences between the two groups in species richness and alpha-diversity, but PCoA showed significant separation (P=0.015, ANOSIM). LEfSe analysis identified 22 species to be differentially abundant between the SU and SNU. However, only 7 species maintained a false discovery rate of ≤ 0.2 and could cluster the two groups separately: Rothia mucilaginosa , Streptococcus sp. oral taxon 66, Actinomyces meyeri , Streptococcus vestibularis Streptococcus sanguinis and a potentially novel Veillonella species in association with SU, and Oribacterium asaccharolyticum with SNU.Conclusion: Shammah use induces tongue microbiome changes that may be relevant to oral carcinogenesis, namely enrichment of species with high acetaldehyde production potential, which warrants further investigation.

scholarly journals Microbiome evaluation revealed salivary dysbiosis in addicts of betel nut preparations

2020 ◽  
Author(s):  
Faizan Saleem ◽  
Ghulam Mujtaba ◽  
Junaid Ahmed Kori ◽  
Arshad Hassan ◽  
M. Kamran Azim
Keyword(s):  
Hypervariable Region ◽  
Alpha Diversity ◽  
Oral Microbiome ◽  
P Value ◽  
Rrna Gene ◽  
Betel Nut ◽  
Taxonomic Assignment ◽  
Unifrac Distance ◽  
Genus Level ◽  
Slaked Lime

AbstractBetel nut addiction is recognized as the causative agent of oral microbiome dysbiosis and other systematic disorders. A number of betel nut preparations containing ingredients such as slaked lime, catechu extract and tobacco are being commonly used particularly in South Asia. The underlying variations in the oral microbiome due to usage of betel nut preparations are poorly understood. We evaluated salivary microbiome in response to chewing of betel nut preparation(s). In order to assess the microbiome dynamics, metagenomic analysis of 16S rRNA gene (V3-V4 hypervariable region) from salivary bacteria in chewers of betel nut preparation (n = 16) and non-chewers (n = 55) was carried out by Greengenes and SILVA ribosomal sequence databases. It was observed that Gutka chewers demonstrated lower alpha diversity and number of bacterial genera than the non-chewers. Taxonomic assignment on phylum level revealed Firmicutes (p-value = 0.042 at 95% confidence interval) to be significantly more abundant in Gutka chewers in comparison with non-chewers. Beta diversity analysis at genus level by weighted unifrac distance matrices unveiled both groups to be divergent from each other. On the genus level, Veillonella (p-value = 0.015), Streptococcus (p-value = 0.026), Leptotrichia (p-value = 0.022) and Serratia (p-value = 0.022) species appeared to be significantly more abundant in Gutka chewers in comparison to non-chewers. The present study suggests salivary dysbiosis in response to gutka chewing and concludes that gutka chewers possess higher abundance of acidogenic and aciduric bacteria. This study contributes additional information regarding oral microbiome variations with response to gutka consumption.

scholarly journals Incorporating genome-based phylogeny and trait similarity into diversity assessments helps to resolve a global collection of human gut metagenomes

2020 ◽  
Author(s):  
Nicholas D. Youngblut ◽  
Jacobo de la Cuesta-Zuluaga ◽  
Ruth E. Ley
Keyword(s):  
Microbial Communities ◽  
Large Scale ◽  
Alpha Diversity ◽  
Reference Database ◽  
Rrna Gene ◽  
Human Gut ◽  
Shotgun Metagenomics ◽  
Diversity Measures ◽  
Microbiome Diversity ◽  
A Genome

AbstractTree-based diversity measures incorporate phylogenetic or phenotypic relatedness into comparisons of microbial communities. This improves the identification of explanatory factors compared to tree-agnostic diversity measures. However, applying tree-based diversity measures to metagenome data is more challenging than for single-locus sequencing (e.g., 16S rRNA gene). The Genome Taxonomy Database (GTDB) provides a genome-based reference database that can be used for species-level metagenome profiling, and a multi-locus phylogeny of all genomes that can be employed for diversity calculations. Moreover, traits can be inferred from the genomic content of each representative, allowing for trait-based diversity measures. Still, it is unclear how metagenome-based assessments of microbiome diversity benefit from incorporating phylogeny or phenotype into measures of diversity. We assessed this by measuring phylogeny-based, trait-based, and tree-agnostic diversity measures from a large, global collection of human gut metagenomes composed of 33 studies and 3348 samples. We found phylogeny- and trait-based alpha diversity to better differentiate samples by westernization, age, and gender. PCoA ordinations of phylogeny- or trait-based weighted UniFrac explained more variance than tree-agnostic measures, which was largely a result of these measures emphasizing inter-phylum differences between Bacteroidaceae (Bacteroidota) and Enterobacteriaceae (Proteobacteria) versus just differences within Bacteroidaceae (Bacteroidota). The disease state of samples was better explained by tree-based weighted UniFrac, especially the presence of Shiga toxin-producing E. coli (STEC) and hypertension. Our findings show that metagenome diversity estimation benefits from incorporating a genome-derived phylogeny or traits.ImportanceEstimations of microbiome diversity are fundamental to understanding spatiotemporal changes of microbial communities and identifying which factors mediate such changes. Tree-based measures of diversity are widespread for amplicon-based microbiome studies due to their utility relative to tree-agnostic measures; however, tree-based measures are seldomly applied to shotgun metagenomics data. We evaluated the utility of phylogeny-, trait-, and tree-agnostic diversity measures on a large scale human gut metagenome dataset to help guide researchers with the complex task of evaluating microbiome diversity via metagenomics.

scholarly journals Oral Microbiome Signatures in Hematological Cancers Reveal Predominance of Actinomyces and Rothia Species

2020 ◽  
Vol 9 (12) ◽  
pp. 4068
Author(s):  
Jean-Luc C. Mougeot ◽  
Micaela F. Beckman ◽  
Holden C. Langdon ◽  
Michael T. Brennan ◽  
Farah Bahrani Mougeot
Keyword(s):  
Bacterial Species ◽  
Alpha Diversity ◽  
In Silico Analysis ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Host Immune System ◽  
Linear Discriminant ◽  
Microbiome Composition ◽  
Hematological Cancers ◽  
Healthy Control

The endogenous microbiome of healthy individuals in oral cavities is diverse, representing over 700 bacterial species. Imbalance in oral and gut microbiome composition and associated gene expression has been linked to different forms of hematological (blood) cancers. Our objective is to compare oral microbiome profiles of patients with blood cancers (BC group: N = 39 patients, n = 124 oral samples) to those of healthy control subjects (HC group: N = 27 subjects, n = 100 oral samples). Saliva samples and swabs of buccal mucosa, supragingival plaque, and tongue were collected from blood cancer patients and healthy controls. Next-generation sequencing (16S-rRNA gene V3–V4 region) was used to determine the relative abundance of bacterial taxa present at the genus and species levels. Differences in oral microbiome beta-diversity were determined using multivariate permutational analysis of variance (PERMANOVA). Linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed to identify differentiating bacterial taxa in pairwise comparisons. The PATRICv3.6.7 online tool was used to determine the predominance of potential pathogenicity in the BC group. The oral microbiome beta-diversities of the BC and HC groups differed and corresponded to a reduced alpha-diversity in the BC group. LEfSe analysis showed significant LDA scores for Actinomyces and Rothia spp., differentiating the BC group from the HC group. In silico analysis using PATRICv3.6.7 demonstrated that the groups of bacteria possessing traits of “antibiotic resistance”, “oral pathogen”, and “virulence” was enriched in the BC group. Although 56% of the BC patients received antibiotics within two weeks of the oral bacterial sampling, Actinomyces genus remained the top differentiating feature in the BC group regardless of the administration of antibiotics, while Rothia dentocariosa was detected as the top differentiating feature in the BC patients who did not receive antibiotics, but not in those who received antibiotics. Further investigation is needed to better understand the interactions of certain oral species with the host immune system to better characterize clinically relevant associations with hematological cancers.

scholarly journals Compositional Differences in the Oral Microbiome of E-cigarette Users

2021 ◽  
Vol 12 ◽  
Author(s):  
Jessica Chopyk ◽  
Christine M. Bojanowski ◽  
John Shin ◽  
Alex Moshensky ◽  
Ana Lucia Fuentes ◽  
...  
Keyword(s):  
Buccal Mucosa ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Alpha Diversity ◽  
Aerosol Deposition ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Significant Shift ◽  
Sample Type ◽  
The Impact

Electronic (e)-cigarettes have been advocated as a safer alternative to conventional tobacco cigarettes. However, there is a paucity of data regarding the impact of e-cigarette aerosol deposition on the human oral microbiome, a key component in human health and disease. We aimed to fill this knowledge gap through a comparative analysis of the microbial community profiles from e-cigarette users and healthy controls [non-smokers/non-vapers (NSNV)]. Moreover, we sought to determine whether e-cigarette aerosol exposure from vaping induces persistent changes in the oral microbiome. To accomplish this, salivary and buccal mucosa samples were collected from e-cigarette users and NSNV controls, with additional oral samples collected from e-cigarette users after 2 weeks of decreased use. Total DNA was extracted from all samples and subjected to PCR amplification and sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Our analysis revealed several prominent differences associated with vaping, specific to the sample type (i.e., saliva and buccal). In the saliva, e-cigarette users had a significantly higher alpha diversity, observed operational taxonomic units (OTUs) and Faith’s phylogenetic diversity (PD) compared to NSNV controls, which declined with decreased vaping. The buccal mucosa swab samples were marked by a significant shift in beta diversity between e-cigarette users and NSNV controls. There were also significant differences in the relative abundance of several bacterial taxa, with a significant increase in Veillonella and Haemophilus in e-cigarette users. In addition, nasal swabs demonstrated a trend toward higher colonization rates with Staphylococcus aureus in e-cigarette users relative to controls (19 vs. 7.1%; p = n.s.). Overall, these data reveal several notable differences in the oral bacterial community composition and diversity in e-cigarette users as compared to NSNV controls.

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