scholarly journals Genomes from Bacteria Associated with the Canine Oral Cavity: a Test Case for Automated Genome-Based Taxonomic Assignment

2019 ◽  
Author(s):  
David A. Coil ◽  
Guillaume Jospin ◽  
Aaron E. Darling ◽  
Corrin Wallis ◽  
Ian J. Davis ◽  
...  
Keyword(s):  
16S Rrna ◽  
Oral Cavity ◽  
16S Rrna Gene ◽  
Marker Gene ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Test Case ◽  
Bacterial Genomes ◽  
Taxonomic Assignment ◽  
Taxonomic Assignments

AbstractTaxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach was less subjective, and required far less labor. The primary differences in the remaining taxonomic assignments related to proposed taxonomy changes by the GTDB team.

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

scholarly journals Genotyping pattern of the vacuolating cytotoxin A and cytotoxin associated gene A of the Helicobacter pylori strains detected in fecal samples of household dogs

2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Asieh Bolandi ◽  
Saam Torkan ◽  
Iman Alavi
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Fecal Samples ◽  
The Status ◽  
Cytotoxin Associated Gene A ◽  
H Pylori

In despite of the high clinical impact of Helicobacter pylori, its exact sources and routes of transmission are unknown. Dogs may play an imperative role in the transmission of H. pylori to humans. The current investigation was done to study the status of vacA and cagA genotypes in the H. pylori strains of dogs. One-hundred and fifty fecal samples were collected from healthy and complicated household dogs. Genomic DNA was extracted from fecal samples and presence of 16S rRNA gene was studied using the PCR amplification. Distribution of vacA and cagA genotypes were studied by the multiplex PCR. Thirteen out of 150 fecal samples (8.66%) were positive for H. pylori 16S rRNA gene. Prevalence of H. pylori in healthy and complicated dogs were 5.55% and 8.57%, respectively. Male had the higher prevalence of H. pylori (P=0.038). The most commonly detected genotypes among the H. pylori strains were vacAs1A (61.53%), cagA (38.46%), vacAm1a (38.46%), vacAs2 (30.76%) and vacAm2 (30.76%). The most commonly detected combined genotypes were s1aCagA (30.76%), s1am1a (23.07%), s2m1a (23.07%) and s2CagA (23.07%). Iranian household dogs harbor H. pylori in their fecal samples similar in genotypes of the vacA and cagA alleles which suggest that complicated and even healthy dogs may be the latent host of the H. pylori and its genotypes. However, supplementary studies are required to found the exact role of dogs as a definitive host of the H. pylori.

scholarly journals Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

1999 ◽  
Vol 37 (10) ◽  
pp. 3281-3290 ◽  
Author(s):  
Michael M. Tunney ◽  
Sheila Patrick ◽  
Martin D. Curran ◽  
Gordon Ramage ◽  
Donna Hanna ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Immunofluorescence Microscopy ◽  
Joint Infection ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Prosthetic Joint ◽  
Culture Positive ◽  
Bacterial 16S Rrna Gene

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific forPropionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

scholarly journals The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

2020 ◽  
Vol 8 (1) ◽  
pp. 131 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Christian Milani ◽  
Gabriele Andrea Lugli ◽  
Federico Fontana ◽  
Francesca Turroni ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
In Silico Analysis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Test Case ◽  
Bacterial Populations ◽  
Taxonomic Marker ◽  
The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

‘Candidatus Phytoplasma pini’, a novel taxon from Pinus silvestris and Pinus halepensis

2005 ◽  
Vol 55 (1) ◽  
pp. 303-307 ◽  
Author(s):  
Bernd Schneider ◽  
Ester Torres ◽  
María P. Martín ◽  
Manfred Schröder ◽  
Heinz-Dietmar Behnke ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pinus Halepensis ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pinus Silvestris ◽  
Plant Parts ◽  
Lethal Yellowing ◽  
Symptomatic Plant

Pinus silvestris and Pinus halepensis trees grown in Germany and Spain, respectively, showing abnormal shoot branching, dwarfed needles and other symptoms were examined for the presence of plant-pathogenic mollicutes (phytoplasmas). While phytoplasmas could not be detected unambiguously with microscopical methods, PCR amplification using universal phytoplasma primers yielded positive results. Samples collected from symptomatic and non-symptomatic plant parts of both symptomatic Pinus silvestris and Pinus halepensis trees tested positive. Also, surrounding non-symptomatic trees proved to be phytoplasma-infected. Comparisons revealed that the 16S rRNA gene sequences of the phytoplasmas identified in Pinus silvestris and Pinus halepensis were nearly identical. However, the pine phytoplasma is only distantly related to other phytoplasmas. The closest relatives are members of the palm lethal yellowing and rice yellow dwarf groups and ‘Candidatus Phytoplasma castaneae’, which share between 94·5 and 96·6 % 16S rRNA gene sequence similarity. From these data it can be concluded that the phytoplasmas identified in the two Pinus species represent a coherent but discrete taxon; it is proposed that this taxon be distinguished at putative species level under the name ‘Candidatus Phytoplasma pini’.

scholarly journals Prevotella dentasini sp. nov., a black-pigmented species isolated from the oral cavity of donkeys

2010 ◽  
Vol 60 (7) ◽  
pp. 1637-1639 ◽  
Author(s):  
Kazuko Takada ◽  
Kazuhiko Hayashi ◽  
Yutaka Sato ◽  
Masatomo Hirasawa
Keyword(s):  
16S Rrna ◽  
Oral Cavity ◽  
16S Rrna Gene ◽  
Dna Hybridization ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Gene Sequence Analysis ◽  
Rrna Gene Sequencing

Four strains (NUM 1903T, NUM 1904, NUM 1912 and NUM 1925) that were obligately anaerobic, pigmented, Gram-negative-staining rods were isolated from the oral cavity of donkeys. These strains were analysed using the Rapid ID 32A, API 20A and API ZYM systems, by DNA–DNA hybridization with other related species and by 16S rRNA gene sequencing. 16S rRNA gene sequence analysis showed that each of the new isolates was a member of the genus Prevotella and related to Prevotella multiformis PPPA21T, showing about 93 % sequence similarity. Based on phylogenetic and phenotypic evidence, it is proposed that the four strains are representatives of a novel species, for which the name Prevotella dentasini sp. nov. is proposed. The type strain is NUM 1903T (=JCM 15908T=DSM 22229T).

Prevotella aurantiaca sp. nov., isolated from the human oral cavity

2010 ◽  
Vol 60 (3) ◽  
pp. 500-503 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Natsuko Suzuki ◽  
Masaaki Okamoto
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Oral Cavity ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gene Sequence Analysis

Two anaerobic, pigmented, non-spore-forming, Gram-stain-negative, rod-shaped strains isolated from the human oral cavity, OMA31T and OMA130, were characterized by determining their phenotypic and biochemical features, cellular fatty acid profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the new isolates belonged to a single species of the genus Prevotella. The two isolates showed 100 % 16S rRNA gene sequence similarity with each other and were most closely related to Prevotella intermedia ATCC 25611T with 96.4 % 16S rRNA gene sequence similarity; the next most closely related strains to the isolates were Prevotella pallens AHN 10371T (96.1 %) and Prevotella falsenii JCM 15124T (95.3 %). Phenotypic and biochemical characteristics of the isolates were the same as those of P. intermedia JCM 12248T, P. falsenii JCM 15124T and Prevotella nigrescens JCM 12250T. The isolates could be differentiated from P. pallens JCM 11140T by mannose fermentation and α-fucosidase activity. Conventional biochemical tests were unable to differentiate the new isolates from P. intermedia, P. falsenii and P. nigrescens. However, hsp60 gene sequence analysis suggested that strain OMA31T was not a representative of P. intermedia, P. pallens, P. falsenii or P. nigrescens. Based on these data, a novel species of the genus Prevotella, Prevotella aurantiaca sp. nov., is proposed, with OMA31T (=JCM 15754T=CCUG 57723T) as the type strain.

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