scholarly journals Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Petra Videnska ◽  
Kristyna Smerkova ◽  
Barbora Zwinsova ◽  
Vlad Popovici ◽  
Lenka Micenkova ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Isolation ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Illumina Platform ◽  
Gram Positive ◽  
Bacterial Composition ◽  
Rrna Gene Sequencing

Abstract Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.

scholarly journals Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria

2009 ◽  
Vol 58 (8) ◽  
pp. 1030-1036 ◽  
Author(s):  
Patrick C. Y. Woo ◽  
Jade L. L. Teng ◽  
Jeff K. L. Wu ◽  
Fion P. S. Leung ◽  
Herman Tse ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Rrna Gene Sequencing

This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0–2 and 2–13 % of staphylococci, and 0 and 0–10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36–56 and 11–14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S rRNA gene sequencing but are not found in the MicroSeq databases should be included.

Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing

2004 ◽  
Vol 53 (12) ◽  
pp. 1247-1253 ◽  
Author(s):  
Susanna KP Lau ◽  
Patrick CY Woo ◽  
Ami MY Fung ◽  
King-man Chan ◽  
Gibson KS Woo ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Acute Cholangitis ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Gram Positive ◽  
Clinically Significant ◽  
Rrna Gene Sequencing

Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of high pathogenicity. Further studies to look for specific virulence factors are warranted.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Francesco Durazzi ◽  
Claudia Sala ◽  
Gastone Castellani ◽  
Gerardo Manfreda ◽  
Daniel Remondini ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Shotgun Sequencing ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Experimental Conditions ◽  
Rrna Gene Sequencing

AbstractIn this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

scholarly journals Non-Specific Immunity Associated Gut Microbiome in Aristichthys nobilis under Different Rearing Strategies

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 916
Author(s):  
Jianming Yuan ◽  
Zhijian Wang ◽  
Bo Wang ◽  
Huiqing Mei ◽  
Xuliang Zhai ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Bighead Carp ◽  
Live Food ◽  
Rrna Gene ◽  
Food Fish ◽  
Rrna Gene Sequencing

To understand the intestinal microbial diversity and community structure of bighead carp (Aristichthys nobilis) under different feeding strategies, 39 fish from three groups (A: 9 fish, natural live food only; B: 15 fish, natural live food + fish formulated feeds; C: 15 fish, natural live food + fish formulated feed + lactic acid bacteria) were obtained for the high throughput 16S rRNA gene sequencing. We first examined five non-specific immunity indications of the carp—lysozyme (LZM), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD). Interestingly, the composition of gut microbiota and related non-specific immune indices were affected by the feeding treatment of the bighead carp. Notably, all enzyme activity indexes were significantly different (p < 0.01) in the spleen and three enzyme activity indexes (LZM, GSH-PX, and SOD) had significant differences in the hepatopancreas (p < 0.001) of the carp from the three groups. The 16S rRNA gene sequencing showed higher diversity in groups B and C. Compared to group A, the relative abundance of Actinobacteria increased significantly and the relative abundance of Proteobacteria and Firmicutes decreased significantly in groups B and C at the phylum level. Functional analysis revealed the association between non-specific immune indicators and import genera in the hepatopancreas and spleen of bighead carp. This study provides new insights into the gut microbiomes and non-specific immune of bighead carp.

scholarly journals First case of an invasive Bacteroides dorei infection detected in a patient with a mycotic aortic aneurysm—raising a rebellion of major indigenous bacteria in humans: a case report and review

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Takayuki Matsuoka ◽  
Takuya Shimizu ◽  
Tadanori Minagawa ◽  
Wakiko Hiranuma ◽  
Miki Takeda ◽  
...  
Keyword(s):  
Aortic Aneurysm ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Resected Specimen ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
First Case ◽  
Rrna Gene Sequencing

Abstract Background Bacteroides dorei is an anaerobic gram-negative bacterium first described in 2006. Because of the high similarity in mass spectra between B. dorei and Bacteroides vulgatus, discriminating between these species is arduous in clinical practice. In recent decades, 16S rRNA gene sequencing has been a complementary method for distinguishing taxonomically close bacteria, including B. dorei and B. vulgatus, at the genus and species levels. Consequently, B. dorei has been shown to contribute to some diseases, including type 1 autoimmune diabetes mellitus and atherosclerotic diseases. However, there are no reports on invasive infectious diseases caused by B. dorei. This report describes the first case of direct invasion and colonisation of human tissue by B. dorei, thus providing a warning regarding the previously proposed application of B. dorei as a live biotherapeutic for atherosclerotic diseases. Case presentation A 78-year-old Japanese man complained of intermittent chest/back pain and was diagnosed with a mycotic thoracic aortic aneurysm by enhanced computed tomography on admission. Despite strict blood pressure control and empirical antibiotic therapy, the patient’s condition worsened. To prevent aneurysmal rupture and eliminate infectious foci, the patient underwent surgical treatment. The resected specimen was subjected to tissue culture and 16S rRNA gene sequencing analysis to identify pathogenic bacteria. A few days after the surgery, culture and sequencing results revealed that the pathogen was B. dorei/B. vulgatus and B. dorei, respectively. The patient was successfully treated with appropriate antibacterial therapy and after improvement, was transferred to another hospital for rehabilitation on postoperative day 34. There was no recurrence of infection or aneurysm after the patient transfer. Conclusions This report describes the first case of invasive infectious disease caused by B. dorei, casting a shadow over its utilisation as a probiotic for atherosclerotic diseases.

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