scholarly journals Periodontal Pathogens Inhabit Root Caries Lesions Extending beyond the Gingival Margin: A Next-Generation Sequencing Analysis

2021 ◽  
Vol 9 (11) ◽  
pp. 2349
Author(s):  
Shoji Takenaka ◽  
Naoki Edanami ◽  
Yasutaka Komatsu ◽  
Ryoko Nagata ◽  
Traithawit Naksagoon ◽  
...  
Keyword(s):  
Hypervariable Region ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Root Caries ◽  
Periodontal Pathogens ◽  
Gingival Margin ◽  
Abundant Otus ◽  
New Perspective ◽  
Caries Lesions

We performed a comprehensive microbiome analysis of root caries lesions using 22 teeth extracted from patients with severe periodontitis. The carious lesions were mechanically collected and cryo-pulverized following tooth extraction. Differences in the microbiome were compared between independent lesions at the supragingival site (SG) and lesions extending beyond the gingival margin (GCB). DNA was extracted and the microbiome was characterized on the basis of the V3-V4 hypervariable region of the 16S rRNA gene using paired-end sequencing on an Illumina MiSeq device. The microbiota in root caries lesions showed compositionally distinct microbiota depending on the location. The most abundant OTUs in the SG group were Streptococcus (26.0%), Actinomyces (10.6%), and Prevotella (7.6%). GCB presented Prevotella (11.1%) as the most abundant genus, followed by Fusobacterium (9.6%) and Actinomyces (8.7%). The SG group showed a lack of uniformity in microbiota compared with the GCB group. The bacterial profiles of GCB varied considerably among patients, including periodontal pathogens such as Porphyromonas, Selenomonas, Filifactor, Peptococcus, and Tannerella. Periodontal pathogens inhabit root caries lesions that extend beyond the gingival margin. This study provides a new perspective for elucidating the microbial etiology of root caries.

scholarly journals Faecal Microbiota of Dogs Offered a Vegetarian Diet with or without the Supplementation of Feather Meal and either Cornmeal, Rye or Fermented Rye: A Preliminary Study

2020 ◽  
Vol 8 (9) ◽  
pp. 1363
Author(s):  
Julia Hankel ◽  
Amr Abd El-Wahab ◽  
Richard Grone ◽  
Birgit Keller ◽  
Eric Galvez ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Vegetarian Diet ◽  
Rrna Gene ◽  
Feather Meal ◽  
Illumina Miseq Platform ◽  
Faecal Samples ◽  
Miseq Platform ◽  
Preliminary Study

Anthropomorphism of dogs has affected feeding and the choice of components present in diets for dogs. Conflicting trends are present: raw or vegetarian appear more prevalent. Animal-derived proteins seem to have unfavourable impacts on intestinal microflora by decreasing the presence of Bacteroidetes. This preliminary study evaluates whether effects of diets with animal proteins on intestinal microbiota can be compensated by the addition of certain carbohydrates to dog diet. Eight female beagles were included in a cross-over study and fed a vegetarian diet or the same diet supplemented with feather meal (2.7%) and either 20% of cornmeal, fermented or non-fermented rye (moisture content of the diets about 42%). A 16S rRNA gene amplification was performed within the hypervariable region V4 on faecal samples and sequenced with the Illumina MiSeq platform. The Firmicutes/Bacteroidetes ratio tended to shift to the advantage of Firmicutes when feather meal and cornmeal were added (Firmicutes/Bacteroidetes ratio of 5.12 compared to 2.47 when offered the vegetarian diet) and tended to switch back to the advantage of Bacteroidetes if rye: fermented (2.17) or not (1.03) was added. The addition of rye might have the potential to compensate possible unfavourable effects of diets with animal proteins on intestinal microbiota of dogs.

scholarly journals PSXIII-29 Farm of origin has a major influence on the colonic microbiome but alterations due to selection for feed efficiency are also evident

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 475-475
Author(s):  
Stafford Vigors ◽  
Torres Sweeney
Keyword(s):  
Feed Efficiency ◽  
Hypervariable Region ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Nutrient Digestibility ◽  
Major Influence ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Selection For

Abstract While the intestinal microbiota is functionally important in nutrient digestibility and animal performance, the role of the microbiome in influencing feed efficiency is not well characterised. The objective of this experiment was to determine the relative influence of feed efficiency and farm of origin on the pig colonic microbiome. Animals were sourced from two geographically distinct locations in Ireland (farm A + B) and evaluated to identify pigs divergent in feed efficiency. The 8 most efficient (LRFI) & 8 least efficient (HRFI) pigs from farm A and 12 LRFI & 12 HRFI pigs from farm B were slaughtered. Colonic digesta was collected for sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed on the Illumina MiSeq. Alpha diversity differed between the farms in this study with pigs from farm A having greater diversity based on Shannon and InvSimpson measures compared to pigs from farm B (P < 0.05). In agreement with this observation, pigs grouped by farm of origin rather than RFI in the beta diversity analysis. However, despite variation between farms, interesting taxonomic differences were identified between RFI groups. Within the phylum Bacteroidetes, the LRFI pigs had increased abundance of two families BS11 (P < 0.05) and a tendency towards increased Bacteroidaceae (P < 0.10) relative to the HRFI group. At genus level, the LRFI pigs had a tendency towards increased Bacteroides and CF231 (P < 0.10). In conclusion, while farm of origin has a substantial influence on microbial diversity in the pig colon, a microbial signature indicative of feed efficiency status was evident.

scholarly journals Intestinal Microbiota of Fattening Pigs Offered Non-Fermented and Fermented Liquid Feed with and without the Supplementation of Non-Fermented Coarse Cereals

2020 ◽  
Vol 8 (5) ◽  
pp. 638 ◽  
Author(s):  
Sebastian Bunte ◽  
Richard Grone ◽  
Birgit Keller ◽  
Christoph Keller ◽  
Eric Galvez ◽  
...  
Keyword(s):  
Hypervariable Region ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Operational Taxonomic Units ◽  
Liquid Feed ◽  
Faecal Samples ◽  
Genus Lactobacillus ◽  
Miseq Platform ◽  
Small And Large Intestine

Introducing high numbers of lactic acid bacteria into the gastrointestinal tract of pigs via fermented liquid feed (FLF) could have an impact on intestinal bacterial ecosystems. Twenty piglets were allocated into four groups and fed a botanically identical liquid diet that was offered either non-fermented (twice), fully fermented or partially fermented but supplemented with 40% of non-fermented coarse cereals. Microbiota studies were performed on the small and large intestine digesta and faecal samples. A 16S rRNA gene amplification was performed within the hypervariable region V4 and sequenced with the Illumina MiSeq platform. R (version 3.5.2) was used for the statistical analyses. The digesta of the small intestines of pigs fed FLF were dominated by Lactobacillaceae (relative abundance up to 95%). In the colonic contents, the abundance of Lactobacillaceae was significantly higher only in the pigs fed the FLF supplemented with non-fermented coarse cereals. Additionally, the digesta of the small and large intestines as well as in the faeces of the pigs fed the FLF supplemented with non-fermented coarse cereals were significantly enriched for two operational taxonomic units (OTUs) belonging to the genus Lactobacillus and Bifidobacterium. The FLF supplemented with non-fermented coarse cereals had probiotic and prebiotic-like impacts on the intestinal and faecal bacterial composition of pigs.

scholarly journals The Subgingival Microbiome in Patients with Down Syndrome and Periodontitis

2020 ◽  
Vol 9 (8) ◽  
pp. 2482
Author(s):  
Lourdes Nóvoa ◽  
María del Carmen Sánchez ◽  
Juan Blanco ◽  
Jacobo Limeres ◽  
Maigualida Cuenca ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Bacterial Species ◽  
Hypervariable Region ◽  
Alpha Diversity ◽  
Study Groups ◽  
Abundant Species ◽  
Rrna Gene ◽  
Periodontal Pathogens ◽  
Cross Sectional ◽  
The Difference

Objective: To describe the subgingival microbiome of individuals with Down syndrome (DS). Methods: We conducted a cross-sectional observational study that obtained bacterial DNA samples from 50 patients with DS, 25 with periodontitis (PDS) and 25 with a healthy periodontal condition (HDS). The samples were analyzed by sequencing the 16S rRNA gene V3–V4 hypervariable region using the MiSeq System. Taxonomic affiliations were assigned using the naïve Bayesian classifier integrated in QIIME2 plugins. We evaluated the difference in bacteria abundance between the sample groups using Wilcoxon and Kruskal–Wallis tests. We evaluated the alpha diversity of the identified species using the Observed, Chao1metric, ACE and Shannon indices and evaluated beta diversity with principal coordinate analysis (registration code: 2018/510). Results: Twenty-one genera and 39 bacterial species showed a significantly different abundance between the study groups. Among the genera, Porphyromonas, Treponema, Tannerella and Aggregatibacter were more abundant in the PDS group than in the HDS group, as were the less commonly studied Filifactor, Fretibacterium and Desulfobulbus genera. Among the species, Porphyromonas spp. and Tannerella spp. were the most abundant in the PDS group; the most abundant species in the HDS group were Pseudomonas spp., Granulicatella spp. and Gemella spp. Conclusion: Well-recognized periodontal pathogens and newly proposed pathogenic taxa were associated with periodontitis in patients with DS.

scholarly journals Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

scholarly journals PSII-16 Evidence for stratification of rumen wall microbial communities revealed by 16S rRNA based amplicon sequencing

2019 ◽  
Vol 97 (Supplement_2) ◽  
pp. 226-227
Author(s):  
Lucas Koester ◽  
Mark Lyte ◽  
Stephan Schmitz-Esser ◽  
Heather Allen
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Temporal Stability ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rumen Content ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Abundant Otus ◽  
Dorsal Portion

Abstract Rumen content (RC) stratifies based on particle size and density consisting of the less dense forage within the dorsal and the denser particles in the ventral portions of the rumen and is in constant contact with the microbial communities present on the rumen wall (RW) epithelium. Little is known about the nutrient requirements and functional processes of RW microbial communities. Our hypothesis is that the RW microbial communities stratify mirroring the stratification of RC due to different available nutrients. Five fistulated, milking Holstein cows of the same management conditions were sampled at four rumen layers corresponding to the RC stratification. Epithelial biopsies were taken through the fistula; the uppermost aligned with the dorsal portion of the RC (A), and three other sites, each 10 cm ventral to the previous (B, C and D). Each cow and stratification layer was sampled five times over four months to analyze temporal stability of the RW microbial communities. DNA was extracted using the Qiagen Powerlyzer Powersoil kit and used for 16S rRNA gene Illumina MiSeq sequencing. Sequences were clustered into operational taxonomic units (OTU) based on a 99% similarity cutoff using MOTHUR. After quality control, 2.0 million reads remained for 90 samples which were clustered into 5,016 OTUs with 10 or more reads. 99.2% of the reads were bacterial, whereas 0.8% affiliated to Archaea. Statistical analysis revealed that among the 20 most abundant OTUs, phylotypes classified as Desulfobulbus, unclassified_Cardiobacteraceae, Mogibacterium, Lachnospiraceae-UCG008 and Methanobrevibacter were significantly different in abundance between sites A compared to D. On a whole community level, analysis of molecular variance (AMOVA) revealed significant differences between groups A, C and D. Our data reveal first evidence that a stratification of RW microbiota is present in dairy cattle and also reveal high temporal stability of RW microbiota.

scholarly journals The Relationship Between the Cervical Microbiome, HIV Status, and Pre-Cancerous Lesions

2018 ◽  
Author(s):  
Cameron Klein ◽  
Daniela Gonzalez ◽  
Crispin Kahesa ◽  
Julius Mwaiselage ◽  
Nirosh Aluthge ◽  
...  
Keyword(s):  
Hypervariable Region ◽  
Alpha Diversity ◽  
Operational Taxonomic Unit ◽  
Sub Saharan Africa ◽  
Hiv Positive ◽  
Rrna Gene ◽  
High Grade ◽  
Cervical Lesions ◽  
Abundant Otus ◽  
Bacterial Richness

AbstractNearly all cervical cancers are causally associated with Human Papillomavirus (HPV). The burden of HPV-associated dysplasias in Sub-Saharan Africa is influenced by HIV. To investigate the role of the bacterial microbiome in cervical dysplasia, cytobrush samples were collected directly from cervical lesions of 144 Tanzanian women. The V4 hypervariable region of the 16S rRNA gene was amplified and deep-sequenced. Alpha diversity metrics; Chao1, PD whole tree, and operational taxonomic Unit (OTU) estimates, displayed significantly higher bacterial richness in HIV positive patients (P = 0.01) than in HIV negative patients. Within HIV positive patients, there was higher bacterial richness in patients with high grade squamous intraepithelial lesions (HSIL; P = 0.13) than those without lesions. The most abundant OTUs associated with high-grade squamous intraepitheilal lesions (HSIL) were Mycoplasmatales, Pseudomonadales, and Staphylococcus. We suggest that a chronic mycoplasma infection of the cervix can contribute to HPV-dependent dysplasia by sustained inflammatory signals.

scholarly journals Unravelling the Gut Microbiota of Cow’s Milk Allergic Infants, their Mothers and Grandmothers

2020 ◽  
Author(s):  
Leticia Mera-Berriatua ◽  
Elisa Zubeldia-Varela ◽  
Isabel Adoración Martín-Antoniano ◽  
Evangelina López de Maturana ◽  
David Rojo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Illumina Miseq ◽  
Food Allergies ◽  
Cow’S Milk ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Cow's Milk ◽  
Epidemiological Risk

Abstract Background: Cow’s Milk Allergy (CMA) is one of the most prevalent food allergies (FA) among infants. Gut microbiota dysbiosis has been related to the development of FA. The primary colonization of the gut microbiota occurs via maternal route. We hypothesized that a longitudinal influence in the composition of the gut microbiota, transmitted from mothers to offspring, could be directly related to CMA development. Methods: 148 faecal samples of 34 CMA and 16 control 0-8 month-old infants and their respective mothers and grandmothers were studied. Gut microbiota was profiled by 16S rRNA gene sequencing using the Illumina MiSeq platform. 16S rRNA sequencing analysis was performed using the DADA2 pipeline. Descriptive statistics of the epidemiological variables of the three generations were analysed. Statistical analyses were performed with R 3.4.0 software. Results : Mothers allergy status and smoking habits of mothers and grandmothers were associated to infant CMA. We found that adult gut microbiota is richer and more diverse than that of infants. Relative abundance of the Prevotellaceae family was significantly different between infant groups, and between hydrolysate-fed and formula-fed infants. Finally, the Bray-Curtis distance between members of the same family was independent of the allergy status. Conclusions: Microbiota from allergic children do not differ from non-allergic at the onset of allergy. Moreover, microbiota inheritance was similar in healthy and allergic infants. Maternal smoking and allergy status were the most significant epidemiological risk factors associated with CMA. Finally, microbiota composition of infants was influenced by diet and allergy status; however, these were confounded variables.

scholarly journals 206 Characterization of the Rumen Bacterial Communities of Grass-fed and Grain-fed Bison Heifers

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 164-164
Author(s):  
Anlly M Fresno Rueda ◽  
Jason Griffin ◽  
Carter Kruse ◽  
Benoit St-Pierre
Keyword(s):  
North American ◽  
Bacterial Communities ◽  
Sequence Data ◽  
Rumen Fluid ◽  
Illumina Miseq ◽  
Rank Test ◽  
Rrna Gene ◽  
The North ◽  
Abundant Otus ◽  
Blue Creek

Abstract The ruminal bacterial environment of the North American bison has remained largely unexplored. The current study aimed to compare the diversity and composition of ruminal bacteria between bison heifers on two different diets. Stomach tubing was used to collect rumen fluid from lifetime grass-fed heifers between 27 and 30 months of age distributed between 2 ranches located in Standing-Butte (n = 17), SD, and Blue-Creek (n = 17), NE, respectively. A second set of samples was collected after the same individuals had been transitioned to a grain-based diet for 100 days. Bacterial composition was determined by Illumina MiSeq (2×300) sequencing of PCR amplicons generated from the V1-V3 region of the 16S rRNA gene. Next-Generation Sequence data was analyzed using a combination of custom Perl scripts, and publicly available software (Mothur v.1.40, RDP classifier and NCBI Blast). Taxonomic analysis identified Bacteroidetes and Firmicutes as the dominant phyla across all samples analyzed. A total of 61,746 and 68,437 species-level Operational Taxonomic Units (OTUs) were identified in grass-fed heifers, in contrast to 15,003 and 23,458 OTUs that were found in the same animals on a grain-based diet. A comparative analysis using the most abundant OTUs from each group was conducted using the Kruskal-Wallis sum-rank test. In the Standing Butte heifers, 24 abundant OTUs were found to be different between diets (P < 0.05), including BP_31-10446 (µ grass = 0.3% vs µ grain = 3.2%) and BP_25-00404 (µ grass = 0.4% vs µ grain = 4.1%). In the Blue Creek heifers, 20 of the most abundant OTUs were found to be different between diets (P < 0.05), including BP_18-00869 (µ grass = 1.4% vs µ grain = 0.4%) and BCRC_1-00422 (µ grass = 0.07% vs µ grain = 4.4%). Together, these results indicate that the rumen of the North American bison harbors highly diverse bacterial communities that undergo dramatic changes in response to changes in diet.

scholarly journals Comparing Approaches for Capturing Bacterial Assemblages Associated with Symptomatic (Bacterial Blotch) and Asymptomatic Mushroom (Agaricus bisporus) Caps

2020 ◽  
Vol 4 (1) ◽  
pp. 90-99
Author(s):  
Samuel J. Martins ◽  
Ryan V. Trexler ◽  
Fabricio R. Vieira ◽  
John A. Pecchia ◽  
Prem P. Kandel ◽  
...  
Keyword(s):  
Health Status ◽  
Hypervariable Region ◽  
Target Surface ◽  
Operational Taxonomic Unit ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Associated Bacteria ◽  
Pseudomonas Spp ◽  
Shannon Diversity

Bacterial blotch is a complex of diseases, caused by Pseudomonas tolaasii and other Pseudomonas spp., whose growth may be influenced by other microorganisms associated with mushroom caps. The objectives of this work were to (i) compare methods for extracting DNA from mushroom caps to assess bacterial composition, and (ii) describe and quantify the composition of bacteria in and on symptomatic and asymptomatic mushroom caps. Symptomatic and asymptomatic mushrooms were collected at an organic farm in Pennsylvania and processed. Bacteria were removed from mushrooms by sonication to target surface-associated bacteria (washate), from whole mushrooms postsonication to target bacteria within or tightly adhering to the mushrooms (post-washate), or from whole macerated mushrooms to target all mushroom-associated bacteria in one sample (whole). Bacterial composition was determined through sequencing the V4 hypervariable region of the 16S rRNA gene on an Illumina MiSeq. Bacterial composition differed significantly based on mushroom health status and community removal method. Regardless of sampling approach, Proteobacteria and Bacteroidetes appeared as the most dominant phyla. In comparing the three extraction methods, higher Shannon diversity and Pielou’s evenness were observed for the washate and post-washate methods compared with whole mushroom sampling. Additionally, higher Shannon diversity, operational taxonomic unit richness, and Pielou’s evenness were found for the asymptomatic mushrooms when compared with symptomatic mushrooms. Pseudomonas spp. were overrepresented on mushroom surfaces (washate method), regardless of health status. This research made strides in describing the mushroom cap microbiome and established sampling methods that will allow researchers to target studies and isolation of specific bacterial taxa.

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