scholarly journals 16S rRNA Long-Read Sequencing of the Granulation Tissue from Nonsmokers and Smokers-Severe Chronic Periodontitis Patients

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Rebecca Chowdhry ◽  
Neetu Singh ◽  
Dinesh Kumar Sahu ◽  
Ratnesh Kumar Tripathi ◽  
Archana Mishra ◽  
...  
Keyword(s):  
16S Rrna ◽  
Granulation Tissue ◽  
Ketone Bodies ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Differential Abundance ◽  
Streptobacillus Moniliformis ◽  
Increased Risk ◽  
Long Read ◽  
Significant Enrichment

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

scholarly journals Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

2019 ◽  
Author(s):  
Anna Cuscó ◽  
Anna Salas ◽  
Celina Torre ◽  
Olga Francino
Keyword(s):  
16S Rrna ◽  
Bacterial Species ◽  
Draft Genome ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Fecal Samples ◽  
Long Read ◽  
Healthy Dogs ◽  
Pooled Samples

AbstractLong-read metagenomics –using single-molecule sequencers– has the potential to assembly entire genomes, even from complex metagenomics datasets. Using long-read metagenomics with Nanopore sequencing in pooled samples, we aim to improve the individual taxonomic profiles obtained with V4 16S rRNA massive sequencing and to assemble the fecal metagenome of healthy dogs.Fecal samples from healthy dogs were sequenced individually using V4 16S rRNA gene and in pools using a shallow metagenomics approach with Nanopore sequencing. Long-read metagenomics allowed us refining the V4 16S rRNA results up to the species level and determining the main bacterial species inhabiting on fecal microbiota of our cohort of healthy dogs. Among them, the most abundant were Fusobacterium varium; Megamonas hypermegale; Bacteroides vulgatus; Blautia hansenii; Clostridium perfringens; and Clostridoides difficile. Once performed the metagenome assembly, one contig was suggested to be circular and hit to an uncultured crAssphage.To conclude, shallow long-read metagenomics with pooled samples using MinION allowed characterizing the dog fecal microbiota at lower taxonomic level, such as bacterial species. The assembly of the reads retrieved a contig that represents a circular draft genome of an uncultured CrAssphage from dog fecal samples that is one of the most abundant bacteriophages in the human gut.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces

2008 ◽  
Vol 74 (13) ◽  
pp. 3969-3976 ◽  
Author(s):  
Jingrang Lu ◽  
Jorge W. Santo Domingo ◽  
Regina Lamendella ◽  
Thomas Edge ◽  
Stephen Hill
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Diversity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Fecal Dna ◽  
Environmental Waters ◽  
Potential Risks ◽  
Species Specific ◽  
Gut Microbial Community

ABSTRACT In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium, a gram-positive, catalase-negative bacterium. To determine the occurrence of C. marimammalium in waterfowl, species-specific 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird (n = 13) and mammal (n = 26) species. The results showed that both assays were specific to gull fecal DNA and that C. marimammalium was present in gull fecal samples collected from the five locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C. marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters.

scholarly journals Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA

2021 ◽  
Vol 9 (8) ◽  
pp. 1721
Author(s):  
Christian O’Dea ◽  
Roger Huerlimann ◽  
Nicole Masters ◽  
Anna Kuballa ◽  
Cameron Veal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Microbial Diversity ◽  
Surface Waters ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Faecal Contamination ◽  
Hypervariable Regions ◽  
Geographical Regions ◽  
V3 Region

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals’ faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1–V3, V3–V4, and V5–V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3–V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3–V4 (n = 11) and V5–V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

Differentiation of Staphylococcus spp. by high-resolution melting analysis

2010 ◽  
Vol 56 (12) ◽  
pp. 1040-1049 ◽  
Author(s):  
Michal Slany ◽  
Martina Vanerkova ◽  
Eva Nemcova ◽  
Barbora Zaloudikova ◽  
Filip Ruzicka ◽  
...  
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Resolution Melting ◽  
Bacterial Species ◽  
High Resolution Melting Analysis ◽  
Rrna Gene ◽  
Staphylococcus Capitis ◽  
Melting Analysis ◽  
The 16S Rrna Gene

High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains ( Staphylococcus aureus , Staphylococcus capitis , Staphylococcus caprae , Staphylococcus epidermidis , Staphylococcus haemolyticus , Staphylococcus hominis , Staphylococcus intermedius , Staphylococcus saprophyticus , Staphylococcus sciuri , Staphylococcus simulans , Staphylococcus warneri , and Staphylococcus xylosus ) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

Characterization of bacterial community structure dynamics in a rat burn wound model using 16S rRNA gene sequencing

2022 ◽  
Author(s):  
Chen Zheng-li ◽  
Peng Yu ◽  
Wu Guo-sheng ◽  
Hong Xu-Dong ◽  
Fan Hao ◽  
...  
Keyword(s):  
Community Structure ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Community Structure ◽  
Bacterial Species ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sprague Dawley ◽  
Rrna Gene Sequencing

Abstract Burns destroy the skin barrier and alter the resident bacterial community, thereby facilitating bacterial infection. To treat a wound infection, it is necessary to understand the changes in the wound bacterial community structure. However, traditional bacterial cultures allow the identification of only readily growing or purposely cultured bacterial species and lack the capacity to detect changes in the bacterial community. In this study, 16S rRNA gene sequencing was used to detect alterations in the bacterial community structure in deep partial-thickness burn wounds on the back of Sprague-Dawley rats. These results were then compared with those obtained from the bacterial culture. Bacterial samples were collected prior to wounding and 1, 7, 14, and 21 days after wounding. The 16S rRNA gene sequence analysis showed that the number of resident bacterial species decreased after the burn. Both resident bacterial richness and diversity, which were significantly reduced after the burn, recovered following wound healing. The dominant resident strains also changed, but the inhibition of bacterial community structure was in a non-volatile equilibrium state, even in the early stage after healing. Furthermore, the correlation between wound and environmental bacteria increased with the occurrence of burns. Hence, the 16S rRNA gene sequence analysis reflected the bacterial condition of the wounds better than the bacterial culture. 16S rRNA sequencing in the Sprague-Dawley rat burn model can provide more information for the prevention and treatment of burn infections in clinical settings and promote further development in this field.

Streptomyces rhizophilus Causes Potato Common Scab Disease

Plant Disease ◽  
2021 ◽  
Author(s):  
Qi Wei ◽  
Jie Li ◽  
Shuai Yang ◽  
Wenzhong Wang ◽  
Fanxiang Min ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Common Scab ◽  
Bacterial Species ◽  
Economic Losses ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Heilongjiang Province ◽  
Epidemic Disease ◽  
Genus Streptomyces

Common scab (CS) caused by Streptomyces spp. is a significant soilborne potato disease that results in tremendous economic losses globally. Identification of CS-associated species of the genus Streptomyces can enhance understanding of the genetic variation of these bacterial species and is necessary for the control of this epidemic disease. The present study isolated Streptomyces strain 6-2-1(1) from scabby potatoes in Keshan County, Heilongjiang Province, China. PCR analysis confirmed that the strain harbored the characteristic Streptomyces pathogenicity island (PAI) genes (txtA, txtAB, nec1, and tomA). Pathogenicity assays proved that the strain caused typical scab lesions on potato tuber surfaces and necrosis on radish seedlings and potato slices. Subsequently, the strain was systemically characterized at morphological, physiological, biochemical and phylogenetic levels. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 6-2-1(1) shared 99.86% sequence similarity with Streptomyces rhizophilus JR-41T, isolated initially from bamboo in rhizospheric soil in Korea. PCR amplification followed by Sanger sequencing of the 16S rRNA gene of 164 scabby potato samples collected in Heilongjiang Province from 2019 to 2020 demonstrated that approximately 2% of the tested samples were infected with S. rhizophilus. Taken together, these results demonstrate that S. rhizophilus is capable of causing potato CS disease and may pose a potential challenge to potato production in Heilongjiang Province of China.

scholarly journals Impact of preconception vaginal microbiota on women’s risk of spontaneous preterm birth: protocol for a prospective case-cohort study

BMJ Open ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. e035186 ◽  
Author(s):  
Erica M Lokken ◽  
Kishorchandra Mandaliya ◽  
Sujatha Srinivasan ◽  
Barbra A Richardson ◽  
John Kinuthia ◽  
...  
Keyword(s):  
Cohort Study ◽  
Preterm Birth ◽  
Umbilical Cord ◽  
Bacterial Species ◽  
Vaginal Microbiota ◽  
Fetal Membrane ◽  
Fetal Membranes ◽  
Rrna Gene ◽  
Spontaneous Preterm Birth ◽  
Increased Risk

IntroductionBacterial vaginosis (BV) and vaginal microbiota disruption during pregnancy are associated with increased risk of spontaneous preterm birth (SPTB), but clinical trials of BV treatment during pregnancy have shown little or no benefit. An alternative hypothesis is that vaginal bacteria present around conception may lead to SPTB by compromising the protective effects of cervical mucus, colonising the endometrial surface before fetal membrane development, and causing low-level inflammation in the decidua, placenta and fetal membranes. This protocol describes a prospective case-cohort study addressing this hypothesis.Methods and analysisHIV-seronegative Kenyan women with fertility intent are followed from preconception through pregnancy, delivery and early postpartum. Participants provide monthly vaginal specimens during the preconception period for vaginal microbiota assessment. Estimated date of delivery is determined by last menstrual period and first trimester obstetrical ultrasound. After delivery, a swab is collected from between the fetal membranes. Placenta and umbilical cord samples are collected for histopathology. Broad-range 16S rRNA gene PCR and deep sequencing of preconception vaginal specimens will assess species richness and diversity in women with SPTB versus term delivery. Concentrations of key bacterial species will be compared using quantitative PCR (qPCR). Taxon-directed qPCR will also be used to quantify bacteria from fetal membrane samples and evaluate the association between bacterial concentrations and histopathological evidence of inflammation in the fetal membranes, placenta and umbilical cord.Ethics and disseminationThis study was approved by ethics committees at Kenyatta National Hospital and the University of Washington. Results will be disseminated to clinicians at study sites and partner institutions, presented at conferences and published in peer-reviewed journals. The findings of this study could shift the paradigm for thinking about the mechanisms linking vaginal microbiota and prematurity by focusing attention on the preconception vaginal microbiota as a mediator of SPTB.

scholarly journals Biotechnological Potential of Bacteria Isolated from the Sea Cucumber Holothuria leucospilota and Stichopus vastus from Lampung, Indonesia

Marine Drugs ◽  
2019 ◽  
Vol 17 (11) ◽  
pp. 635 ◽  
Author(s):  
Joko T. Wibowo ◽  
Matthias Y. Kellermann ◽  
Dennis Versluis ◽  
Masteria Y. Putra ◽  
Tutik Murniasih ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sea Cucumber ◽  
Bacterial Species ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sea Cucumbers ◽  
Biotechnological Potential ◽  
Rrna Gene Sequencing

In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS1) as well as product/daughter ions (MS2) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential.

scholarly journals Cautionary tale of using 16S rRNA gene sequence similarity values in identification of human-associated bacterial species

2015 ◽  
Vol 65 (Pt_6) ◽  
pp. 1929-1934 ◽  
Author(s):  
Morgane Rossi-Tamisier ◽  
Samia Benamar ◽  
Didier Raoult ◽  
Pierre-Edouard Fournier
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Bacterial Taxonomy ◽  
Associated Species

Modern bacterial taxonomy is based on a polyphasic approach that combines phenotypic and genotypic characteristics, including 16S rRNA sequence similarity. However, the 95 % (for genus) and 98.7 % (for species) sequence similarity thresholds that are currently recommended to classify bacterial isolates were defined by comparison of a limited number of bacterial species, and may not apply to many genera that contain human-associated species. For each of 158 bacterial genera containing human-associated species, we computed pairwise sequence similarities between all species that have names with standing in nomenclature and then analysed the results, considering as abnormal any similarity value lower than 95 % or greater than 98.7 %. Many of the current bacterial species with validly published names do not respect the 95 and 98.7 % thresholds, with 57.1 % of species exhibiting 16S rRNA gene sequence similarity rates ≥98.7 %, and 60.1 % of genera containing species exhibiting a 16S rRNA gene sequence similarity rate <95 %. In only 17 of the 158 genera studied (10.8 %), all species respected the 95 and 98.7 % thresholds. As we need powerful and reliable taxonomical tools, and as potential new tools such as pan-genomics have not yet been fully evaluated for taxonomic purposes, we propose to use as thresholds, genus by genus, the minimum and maximum similarity values observed among species.

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