Is Fluorescence Technology a Promising Tool for Detecting Infected Dentin in Deep Carious Lesions?

2020 ◽  
Vol 54 (3) ◽  
pp. 205-217
Author(s):  
Leslie H. Trippe ◽  
Apoena Aguiar Ribeiro ◽  
M. Andrea Azcarate-Peril ◽  
John S. Preisser ◽  
Rujin Wang ◽  
...  
Keyword(s):  
Rank Correlation ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Human Teeth ◽  
Operational Taxonomic Units ◽  
Promising Tool ◽  
Carious Lesions ◽  
Microbiome Diversity ◽  
Bacterial Microbiome ◽  
Deep Carious Lesions

The purpose of this study was to determine if the degree of fluorescence detected by fluorescence-aided caries excavation (FACE) correlates with dentin bacterial microbiome diversity, as assessed by 16S rRNA gene amplicon sequencing, and with traditional tactile dentin caries assessment. Unidentified human teeth were obtained from a dental facility. The included teeth had a carious lesion two-thirds into the dentin, verified by radiography, and were red-fluorescing (RF) using FACE technology (SIROInspect; Sirona, Bensheim, Germany). Two independent examiners performed visual/tactile assessment of the lesions. RF sites were sampled with a sterile spoon excavator and dentin characteristics were evaluated. Once RF dentin was removed, a second sample of pink-fluorescing (PF) dentin was obtained. After excavation with a sterile round bur to nonfluorescing (NF) dentin, a third sample was collected with a slow-speed round bur. The samples were processed at the UNC (University of North Carolina at Chapel Hill) Microbiome Core Facility. Out of 134 extracted teeth collected, 21 fit the inclusion criteria, yielding 61 dentin samples. RF samples had the highest number of observed operational taxonomic units (n = 154), followed by PF (n = 109) and NF (n = 100). RF carious dentin was primarily “soft,” and NF dentin was assessed as “hard” 100% of the time by both examiners (rank correlation χ2: p < 0.001). However, approximately one-third of the tactile assessments of hard dentin still displayed some fluorescence, either pink or red. We concluded that the sampled fluorescing (RF and PF) and NF carious dentin layers displayed diverse bacterial taxa, and tactile assessments of soft, leathery, and hard corresponded with RF, PF, and NF.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

scholarly journals The Wheat Microbiome Under Four Management Strategies, and Potential for Endophytes in Disease Protection

2017 ◽  
Vol 1 (3) ◽  
pp. 158-168 ◽  
Author(s):  
Kristi Gdanetz ◽  
Frances Trail
Keyword(s):  
Bacterial Communities ◽  
Management Strategy ◽  
Crop Yields ◽  
Management Strategies ◽  
Growing Season ◽  
Amplicon Sequencing ◽  
Antagonistic Activity ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Plant Organ

Manipulating plant-associated microbes to reduce disease or improve crop yields requires a thorough understanding of interactions within the phytobiome. Plants were sampled from a wheat/maize/soybean crop rotation site that implements four different crop management strategies. We analyzed the fungal and bacterial communities of leaves, stems, and roots of wheat throughout the growing season using 16S and fungal internal transcribed spacer 2 rRNA gene amplicon sequencing. The most prevalent operational taxonomic units (OTUs) were shared across all samples, although levels of the low-abundance OTUs varied. Endophytes were isolated from plants, and tested for antagonistic activity toward the wheat pathogen Fusarium graminearum. Antagonistic strains were assessed for plant protective activity in seedling assays. Our results suggest that microbial communities were strongly affected by plant organ and plant age, and may be influenced by management strategy.

scholarly journals A Plant Growth-Promoting Microbial Soil Amendment Dynamically Alters the Strawberry Root Bacterial Microbiome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Siwen Deng ◽  
Heidi M.-L. Wipf ◽  
Grady Pierroz ◽  
Ted K. Raab ◽  
Rajnish Khanna ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Soil Amendment ◽  
Bacterial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Microbiome ◽  
Plant Growth Promoting ◽  
Miseq Platform ◽  
Soil Rhizosphere

AbstractDespite growing interest in utilizing microbial-based methods for improving crop growth, much work still remains in elucidating how beneficial plant-microbe associations are established, and what role soil amendments play in shaping these interactions. Here, we describe a set of experiments that test the effect of a commercially available soil amendment, VESTA, on the soil and strawberry (Fragaria x ananassa Monterey) root bacterial microbiome. The bacterial communities of the soil, rhizosphere, and root from amendment-treated and untreated fields were profiled at four time points across the strawberry growing season using 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. In all sample types, bacterial community composition and relative abundance were significantly altered with amendment application. Importantly, time point effects on composition are more pronounced in the root and rhizosphere, suggesting an interaction between plant development and treatment effect. Surprisingly, there was slight overlap between the taxa within the amendment and those enriched in plant and soil following treatment, suggesting that VESTA may act to rewire existing networks of organisms through an, as of yet, uncharacterized mechanism. These findings demonstrate that a commercial microbial soil amendment can impact the bacterial community structure of both roots and the surrounding environment.

Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

2020 ◽  
Author(s):  
Yingwu Shi ◽  
Hongmei Yang ◽  
Min Chu ◽  
Xinxiang Niu ◽  
Xiangdong Huo ◽  
...  
Keyword(s):  
Community Structure ◽  
Endophytic Bacteria ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Operational Taxonomic Units ◽  
Cotton Sample ◽  
Endophytic Bacterial Community ◽  
V3 Region ◽  
Illumina Amplicon Sequencing

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

scholarly journals Host Species Determines the Composition of the Prokaryotic Microbiota in Phlebotomus Sandflies

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 428
Author(s):  
Christos Papadopoulos ◽  
Panagiotis A. Karas ◽  
Sotirios Vasileiadis ◽  
Panagiota Ligda ◽  
Anastasios Saratsis ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Host Species ◽  
Archaeal Community ◽  
Amplicon Sequencing ◽  
Structural Effect ◽  
Rrna Gene ◽  
Host Genotype ◽  
Operational Taxonomic Units ◽  
Phlebotomine Sandflies ◽  
Leishmania Spp

Phlebotomine sandflies are vectors of the humans’ and mammals’ parasite Leishmania spp. Although the role of gut microbiome in the biological cycle of insects is acknowledged, we still know little about the factors modulating the composition of the gut microbiota of sandflies. We tested whether host species impose a strong structural effect on the gut microbiota of Phlebotomus spp. Sandflies were collected from the island of Leros, Greece, and classified to P. papatasi, P. neglectus, P. tobbi, and P. similis, all being negative to Leishmania spp. The prokaryotic gut microbiota was determined via 16S rRNA gene amplicon sequencing. Phlebotomus species supported distinct microbial communities (p < 0.001). P. papatasi microbiota was the most distinct over-dominated by three Spiroplasma, Wolbachia and Paenibacillus operational taxonomic units (OTUs), while another Wolbachia OTU prevailed in P. neglectus. Conversely, the microbiota of P. tobbi and P. similis was composed of several less dominant OTUs. Archaea showed low presence with the dominant OTUs belonging to methanogenic Euryarcheota, ammonia-oxidizing Thaumarcheota, and Nanoarchaeota. We provide first insights into the composition of the bacterial and archaeal community of Phlebotomus sandflies and showed that, in the absence of Leishmania, host genotype is the major modulator of Phlebotomus sandfly gut microbiota.

scholarly journals Termites Are Associated with External Species-Specific Bacterial Communities

2020 ◽  
Vol 87 (2) ◽  
Author(s):  
Patrik Soukup ◽  
Tomáš Větrovský ◽  
Petr Stiblik ◽  
Kateřina Votýpková ◽  
Amrita Chakraborty ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Negative Effects ◽  
Termite Species ◽  
Positive Effects ◽  
Operational Taxonomic Units ◽  
Wide Range ◽  
Species Specific ◽  
And Control

ABSTRACT All termites have established a wide range of associations with symbiotic microbes in their guts. Some termite species are also associated with microbes that grow in their nests, but the prevalence of these associations remains largely unknown. Here, we studied the bacterial communities associated with the termites and galleries of three wood-feeding termite species by using 16S rRNA gene amplicon sequencing. We found that the compositions of bacterial communities among termite bodies, termite galleries, and control wood fragments devoid of termite activities differ in a species-specific manner. Termite galleries were enriched in bacterial operational taxonomic units (OTUs) belonging to Rhizobiales and Actinobacteria, which were often shared by several termite species. The abundance of several bacterial OTUs, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus, was reduced in termite galleries. Our results demonstrate that both termite guts and termite galleries harbor unique bacterial communities. IMPORTANCE As is the case for all ecosystem engineers, termites impact their habitat by their activities, potentially affecting bacterial communities. Here, we studied three wood-feeding termite species and found that they influence the composition of the bacterial communities in their surrounding environment. Termite activities have positive effects on Rhizobiales and Actinobacteria abundance and negative effects on the abundance of several ubiquitous genera, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus. Our results demonstrate that termite galleries harbor unique bacterial 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 Impact of Helicobacter pylori infection on fluid duodenal microbial community structure and microbial metabolic pathways

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Tadashi Maeda ◽  
Hiroaki Zai ◽  
Yuto Fukui ◽  
Yoshifumi Kato ◽  
Eri Kumade ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Metabolic Pathways ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Commensal Microbiota ◽  
Operational Taxonomic Units ◽  
H Pylori ◽  
The Impact ◽  
16 S Rrna

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16 S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16 S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection changed the relative mean abundance of three phyla (Proteobacteria, Actinobacteria, and TM7) and ten genera (Neisseria, Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

scholarly journals Impact of Helicobacter Pylori Infection on Duodenal Microbial Community Structure and Microbial Metabolic Pathways

2021 ◽  
Author(s):  
Tadashi Maeda ◽  
Hiroaki Zai ◽  
Yuto Fukui ◽  
Yoshifumi Kato ◽  
Eri Kumade ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Metabolic Pathways ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
H Pylori ◽  
The Impact

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection increased the relative mean abundance of Proteobacteria and Neisseria and decreased the abundance of the two other phyla (Actinobacteria and TM7) and nine genera (Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

scholarly journals Response to Edaphoclimatic Conditions and Crop Management of the Bacterial Microbiome of Musa acuminata Rhizosphere Profiled by 16S rRNA Gene Amplicon Sequencing

2021 ◽  
Vol 10 (10) ◽  
Author(s):  
Francisco J. De la Torre-González ◽  
Elisa Fernández-Castillo ◽  
Dailen Azaharez-Llorente ◽  
Jorge Lara ◽  
Enrique Avendaño ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Management Practices ◽  
Crop Management ◽  
Amplicon Sequencing ◽  
Musa Acuminata ◽  
Rrna Gene ◽  
Content Type ◽  
Microbiome Composition ◽  
Bacterial Microbiome

ABSTRACT Bacterial rhizospheric microbiomes of Musa acuminata cultivated in farms close to the west and east Mexican coasts and with different climate, soils, and crop management practices were characterized by 16S rRNA gene amplicon sequencing. Results showed that rhizospheric microbiome composition changed along with seasonal weather but were mostly indifferent to soil type.

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