scholarly journals Consistent metagenome-derived metrics verify and define bacterial species boundaries

2019 ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Bacterial Species ◽  
Sequence Divergence ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power ◽  
Genome Wide ◽  
Gene Level

AbstractLongstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. We compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity, and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of non-synonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, estimates for homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full length 16S rRNA genes were least useful because they were under-recovered from metagenomes, but many ribosomal proteins displayed both high metagenomic recoverability and species-discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination.

scholarly journals Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

scholarly journals Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple rrn Operons

2004 ◽  
Vol 186 (9) ◽  
pp. 2629-2635 ◽  
Author(s):  
Silvia G. Acinas ◽  
Luisa A. Marcelino ◽  
Vanja Klepac-Ceraj ◽  
Martin F. Polz
Keyword(s):  
16S Rrna ◽  
Thermophilic Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Genomes ◽  
16S Rrna Sequence ◽  
Thermoanaerobacter Tengcongensis ◽  
Copy Numbers ◽  
Rrna Sequences ◽  
16S Rrna Sequences

ABSTRACT The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ∼40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to ∼2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.

scholarly journals Treatment nonresponse in children receiving silver diamine fluoride for caries: A pilot study

2020 ◽  
Author(s):  
Ryan Richard Ruff ◽  
Bidisha Paul ◽  
Maria A Sierra ◽  
Fangxi Xu ◽  
Yasmi Crystal ◽  
...  
Keyword(s):  
Pilot Study ◽  
Bacterial Species ◽  
Illumina Miseq ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Lasso Regression ◽  
Silver Diamine Fluoride ◽  
Operational Taxonomic Units ◽  
Acid Tolerant ◽  
Nonsurgical Therapy

AbstractObjectives: Silver diamine fluoride (SDF) is a nonsurgical therapy for the arrest and prevention of dental caries with demonstrated clinical efficacy. Approximately 20% of children receiving SDF fail to respond to treatment. The objective of this study was to develop a predictive model of treatment nonresponse using machine learning. Methods: An observational pilot study (N=20) consisting of children with and without active decay and who did and did not respond to silver diamine fluoride provided salivary samples and plaque from infected and contralateral sites. 16S rRNA genes from samples were amplified and sequenced on an Illumina Miseq and analyzed using QIIME. The association between operational taxonomic units and treatment nonresponse was assessed using lasso regression and artificial neural networks. Results: Bivariate group comparisons of bacterial abundance indicate a number of genera were significantly different between nonresponders and those who responded to SDF therapy. No differences were found between nonresponders and caries-active subjects. Prevotella pallens and Veillonella denticariosi were retained in full lasso models and combined with clinical variables in a six-input multilayer perceptron. Discussion: The acidogenic and acid-tolerant nature of retained bacterial species may overcome the antimicrobial effects of SDF. Further research to validate the model in larger external samples is needed.

scholarly journals Microbial contamination of a University dental clinic in Brazil

2017 ◽  
Vol 15 (4) ◽  
pp. 248
Author(s):  
Gisely Naura Venâncio ◽  
Victor Hugo Marques Coelho ◽  
Thiago Fontanella Cestari ◽  
Maxine Ennata Alves de Almeida ◽  
Carolinie Batista Nobre da Cruz
Keyword(s):  
Culture Media ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dental Clinics ◽  
Dental Office ◽  
Dental Surface ◽  
The University ◽  
The Chairs ◽  
And Staphylococcus Aureus

Pathogens of the oral cavity of a patient can be transferred to the dental office surfaces by direct contact, aerosol instruments and blood or saliva. The objective of this study was to investigate the microbiological contamination presents in the stands, chairs and spittoons in the University Nilton Lins dental clinics, in Manaus, Amazonas. Samples were collected with sterile swabs and seeded in different microbiological culture media for the isolation of microorganisms collected from each room. Then, assays were carried out for identification of strains isolated from each environment, such as: Gram stain, DNA purification, Amplification of 16s rRNA genes and sequencing. All these experiments were performed in the LBS / ILMD / FIOCRUZ. It was found 40 CFU / mL in the stands, 43 on the chairs and 47 in the spittoons and it was also possible to identify microorganisms like Klebsiella pneumoniae, Shigella sonnei and Staphylococcus aureus. The greatest number of CFUs was found in Clinic 3 and it was observed that the spittoon was the dental surface with the highest number of CFUs. Some of the bacterial species isolated are opportunists, suggesting that more severe biosecurity measures must be taken in order to prevent cross-infection.Pathogens of the oral cavity of a patient can be transferred to the dental office surfaces by direct contact, aerosol instruments and blood or saliva. The objective of this study was to investigate the microbiological contamination presents in the stands, chairs and spittoons in the University Nilton Lins dental clinics, in Manaus, Amazonas. Samples were collected with sterile swabs and seeded in different microbiological culture media for the isolation of microorganisms collected from each room. Then, assays were carried out for identification of strains isolated from each environment, such as: Gram stain, DNA purification, Amplification of 16s rRNA genes and sequencing. All these experiments were performed in the LBS / ILMD / FIOCRUZ. It was found 40 CFU / mL in the stands, 43 on the chairs and 47 in the spittoons and it was also possible to identify microorganisms like Klebsiella pneumoniae, Shigella sonnei and Staphylococcus aureus. The greatest number of CFUs was found in Clinic 3 and it was observed that the spittoon was the dental surface with the highest number of CFUs. Some of the bacterial species isolated are opportunists, suggesting that more severe biosecurity measures must be taken in order to prevent cross-infection.

scholarly journals In-depth analysis of Bacillus anthracis 16S rRNA genes and transcripts reveals intra- and intergenomic diversity and facilitates anthrax detection

2021 ◽  
Author(s):  
Peter Braun ◽  
Fee Zimmermann ◽  
Mathias C Walter ◽  
Sonja Mantel ◽  
Karin Aistleitner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Close Relative ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Depth Analysis

Analysis of 16S ribosomal RNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ-, in vitro- and in silico-assays to assess the yet unknown 16S-state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing and bioinformatics we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis genome data-sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy-numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy-numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.

Stool bacteriomic profiling in patients with metastatic renal cell carcinoma suggests an etiology of VEGF TKI-related diarrhea.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 421-421
Author(s):  
Sumanta Kumar Pal ◽  
Sierra Mi Li ◽  
Xiwei Wu ◽  
Manasvi Pinnamaneni ◽  
JoAnn Hsu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
16S Rrna ◽  
Cell Carcinoma ◽  
Metastatic Renal Cell Carcinoma ◽  
Renal Cell ◽  
Clustering Algorithm ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
P Value

421 Background: Vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF TKIs) remain a mainstay of therapy for patients with metastatic renal cell carcinoma (mRCC). Diarrhea represents a pervasive toxicity, with all grade diarrhea affecting roughly 50% of patients receiving VEGF TKIs. The underlying cause of diarrhea in these patients is poorly understood. Methods: Patients with mRCC receiving an FDA-approved VEGF TKI therapy for mRCC were consented. Stool was collected in a standardized fashion and total genomic DNA was isolated using the PowerSoil DNA isolation kit (Mo Bio, USA). A standard PCR protocol was used to amplify bacterial 16S rRNA genes from all samples. PCR primers were used to amplify the V4 and V5 regions of the 16S rRNA. Paired-end of sequencing 2X100bp was performed by Illumina HiSeq 2000, and sequences were clustered using the CD-HIT clustering algorithm. Taxonomy was then assigned using the RDP-II classifier. Non-clustered analyses were also performed, stratifying patients by the presence or absence of diarrhea at the time of stool collection. Results: Of 26 patients consented, 23 patients submitted stool specimens and 20 had sufficient data for the current analysis. Amongst these 20 patients, the median age was 63 and the majority of patients (60%) were intermediate risk by Heng criteria. Eight patients (40%) received VEGF TKI therapy in the first-line setting. Across all lines of therapy, the most commonly used VEGF TKI was sunitinib (44%). A total of 141 bacterial species were identified. With respect to differences in patients who did and did not have diarrhea, the Mann-Whitney U-test identified 7 species with a p value of <0.1. The largest difference was seen in two Bifidobacterium species, B. animalis and B. bifidum, with both bacteria more abundant in patients with no diarrhea. Conclusions: This is the first effort to use stool bacteriomic profiling to ascertain the etiology of VEGF TKI related diarrhea in patients with mRCC. Two Bifidobacterium spp identified in this analysis are commonly found in probiotics. Studies to use probiotics enriched with Bifidobacterium spp to prevent or ameliorate VEGF TKI-related diarrhea are currently in development.

scholarly journals 16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught Anopheles gambiae Sensu Lato and A. funestus Mosquitoes Reveals New Species Related to Known Insect Symbionts

2005 ◽  
Vol 71 (11) ◽  
pp. 7217-7223 ◽  
Author(s):  
Jenny M. Lindh ◽  
Olle Terenius ◽  
Ingrid Faye
Keyword(s):  
16S Rrna ◽  
Anopheles Gambiae ◽  
Bacterial Species ◽  
Anopheles Funestus ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Malaria Vectors ◽  
Rrna Gene ◽  
Species Determination ◽  
Insect Symbionts

ABSTRACT Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

Sputum Microbiota in Coal Workers Diagnosed With Pneumoconiosis as Revealed by 16S Metagenomic Sequencing

2020 ◽  
Author(s):  
Vladimir Druzhinin ◽  
Liudmila Matskova ◽  
Pavel Demenkov ◽  
Elizaveta Baranova ◽  
Valentin Volobaev ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Occupational Activity ◽  
Metagenomic Sequencing ◽  
Coal Miners ◽  
Active Coal ◽  
Coal Workers ◽  
Generation Sequencing

Abstract Objectives: The microbiome of sputum from former and active coal miners diagnosed with coal worker’s pneumoconiosis (CWP) as compared to healthy controls Methods: Next Generation Sequencing of bacterial 16S rRNA genes obtained from the sputum of CWP subjects.Results: Differences were detected between the sputum microbiomes from the healthy and CWP subjects. We noted a significant decrease in Bacteroidetes and an increase in the level of Proteobacteria.Conclusions: The microbiomes found in sputum from CWP subjects are enriched in bacterial species previously reported to induce pro-inflammatory responses. The profile of the microbiomes correlated mainly to the occupational activity and not to the age of the coal miners.

scholarly journals Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

1998 ◽  
Vol 64 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Julian R. Marchesi ◽  
Takuichi Sato ◽  
Andrew J. Weightman ◽  
Tracey A. Martin ◽  
John C. Fry ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Amplification ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr

ABSTRACT We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

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