scholarly journals Human stool preservation impacts taxonomic profiles in 16S rRNA gene-based metagenomics studies

2020 ◽  
Author(s):  
Anne Plauzolles ◽  
Eya Toumi ◽  
Benoit Goutorbe ◽  
Marion Bonnet ◽  
Guillaume Pénaranda ◽  
...  
Keyword(s):  
Human Health ◽  
Standard Operating Procedure ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Bacterial Composition ◽  
Bacterial Populations ◽  
Composition Variation ◽  
Storage Solutions ◽  
Technological Bias ◽  
Human Stool

Abstract Background: Microbiotas play critical roles in human health, yet in most cases scientists lack standardized and reproducible methods. To date, stool sample preservation remains a source of technological bias in metagenomic sequencing, despite newly developed storage solutions. Studying those biases will help in identifying the appropriate stool preservation approach to promote the implementation of standard operating procedure and improve comparability across multiple microbiome studies. Results: Here, we conducted a comparative study of 10 storage methods for human stool over a 15-day period of storage at fluctuating temperatures. We first compared the performance of each stabilizer with observed bacterial composition variation within the same specimen. Then, we identified the nature of the observed variations to determine which bacterial populations were more impacted by the stabilizer. We found that DNA stabilizers display various stabilizing efficacies and affect the recovered bacterial profiles. Furthermore, our results showed that the bias associated with the stabilizers can be linked to the phenotypical characteristics of the bacterial populations present in the studied samples. Conclusions: Although newly developed storage solutions have improved our capacity to stabilize stool microbial content over time, they are nevertheless not devoid of biases. We therefore recommend considering the nature of these technological biases before claiming whether some microbes are beneficial or even deleterious to human health.

scholarly journals Faecal bacterial composition in dairy cows shedding Mycobacterium avium subsp. paratuberculosis in faeces in comparison with nonshedding cows

2016 ◽  
Vol 62 (6) ◽  
pp. 538-541 ◽  
Author(s):  
Marija Kaevska ◽  
Petra Videnska ◽  
Karel Sedlar ◽  
Iva Bartejsova ◽  
Alena Kralova ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subsp ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Bacterial Composition ◽  
Bacterial Populations ◽  
Faecal Samples ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
The 16S Rrna Gene

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne’s disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

scholarly journals Dead or Alive? Molecular life-dead distinction in human stool samples reveals significantly different composition of the microbial community

2018 ◽  
Author(s):  
Alexandra Perras ◽  
Kaisa Koskinen ◽  
Maximilian Mora ◽  
Michael Beck ◽  
Lisa Wink ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Human Health ◽  
Gene Targeting ◽  
Gut Microbiome ◽  
Rrna Gene ◽  
Stool Samples ◽  
Human Stool

AbstractThe gut microbiome is strongly interwoven with human health. Conventional gut microbiome analysis generally involves 16S rRNA gene targeting next generation sequencing (NGS) of stool microbial communities, and correlation of results with clinical parameters. However, some microorganisms may not be alive at the time of sampling, and thus their impact on the human health is potentially less significant. As conventional NGS methods do not differentiate between viable and dead microbial components, retrieved results provide only limited information.Propidium monoazide (PMA) is frequently used in food safety monitoring and other disciplines to discriminate living from dead cells. PMA binds to free DNA and masks it for subsequent procedures. In this article we show the impact of PMA on the results of 16S rRNA gene-targeting NGS from human stool samples and validate the optimal applicable concentration to achieve a reliable detection of the living microbial communities.Fresh stool samples were treated with a concentration series of zero to 300 μM PMA, and were subsequently subjected to amplicon-based NGS. The results indicate that a substantial proportion of the human microbial community is not intact at the time of sampling. PMA treatment significantly reduced the diversity and richness of the sample depending on the concentration and impacted the relative abundance of certain important microorganisms (e.g. Akkermansia, Bacteroides). Overall, we found that a concentration of 100 μM PMA was sufficient to quench signals from disrupted microbial cells.The optimized protocol proposed here can be easily implemented in classical microbiome analyses, and helps to retrieve an improved and less blurry picture of the microbial community composition by excluding signals from background DNA.

scholarly journals First reported quantitative microbiota in different livestock manures used as organic fertilizers in the Northeast of Thailand

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lampet Wongsaroj ◽  
Ratmanee Chanabun ◽  
Naruemon Tunsakul ◽  
Pinidphon Prombutara ◽  
Somsak Panha ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Organic Fertilizer ◽  
The Other ◽  
Organic Fertilizers ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Physiochemical Properties ◽  
Nutrient Profiles ◽  
Soil Recovery ◽  
Local Farmers

AbstractNortheastern Thailand relies on agriculture as a major economic activity, and has used high levels of agrochemicals due to low facility, and salty sandy soil. To support soil recovery and sustainable agriculture, local farmers have used organic fertilizers from farmed animal feces. However, knowledge about these animal fecal manures remains minimal restricting their optimal use. Specifically, while bacteria are important for soil and plant growth, an abundance and a diversity of bacterial composition in these animal fecal manures have not been reported to allow selection and adjustment for a more effective organic fertilizer. This study thereby utilized metagenomics combined with 16S rRNA gene quantitative PCR (qPCR) and sequencing to analyze quantitative microbiota profiles in association with nutrients (N, P, K), organic matters, and the other physiochemical properties, of the commonly used earthworm manure and other manures from livestock animals (including breed and feeding diet variations) in the region. Unlike the other manures, the earthworm manure demonstrated more favorable nutrient profiles and physiochemical properties for forming fertile soil. Despite low total microbial biomass, the microbiota were enriched with maximal OTUs and Chao richness, and no plant pathogenic bacteria were found based on the VFDB database. The microbial metabolic potentials supported functions to promote crop growth, such as C, N and P cyclings, xenobiotic degradation, and synthesis of bioactive compounds. Pearson’s correlation analyses indicated that the quantitative microbiota of the earthworm manure were clustered in the same direction as N, and conductivity, salinity, and water content were essential to control the microbiota of animal manures.

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 Novel strain-level resolution of Crohn’s disease mucosa-associated microbiota via an ex vivo combination of microbe culture and metagenomic sequencing

2021 ◽  
Author(s):  
J. J. Teh ◽  
E. M. Berendsen ◽  
E. C. Hoedt ◽  
S. Kang ◽  
J. Zhang ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Taxonomic Composition ◽  
Strain Level ◽  
Patient Specific ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Functional Characterisation ◽  
Bacterial Lineages

AbstractThe mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriacea and their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

scholarly journals Structure of Microbial Communities When Complementary Effluents Are Anaerobically Digested

2021 ◽  
Vol 11 (3) ◽  
pp. 1293
Author(s):  
Ana Eusébio ◽  
André Neves ◽  
Isabel Paula Marques
Keyword(s):  
Anaerobic Digestion ◽  
Microbial Communities ◽  
Volatile Fatty Acids ◽  
Olive Mill Wastewater ◽  
Organic Load ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Olive Mill

Olive oil and pig productions are important industries in Portugal that generate large volumes of wastewater with high organic load and toxicity, raising environmental concerns. The principal objective of this study is to energetically valorize these organic effluents—piggery effluent and olive mill wastewater—through the anaerobic digestion to the biogas/methane production, by means of the effluent complementarity concept. Several mixtures of piggery effluent were tested, with an increasing percentage of olive mill wastewater. The best performance was obtained for samples of piggery effluent alone and in admixture with 30% of OMW, which provided the same volume of biogas (0.8 L, 70% CH4), 63/75% COD removal, and 434/489 L CH4/kg SVin, respectively. The validation of the process was assessed by molecular evaluation through Next Generation Sequencing (NGS) of the 16S rRNA gene. The structure of the microbial communities for both samples, throughout the anaerobic process, was characterized by the predominance of bacterial populations belonging to the phylum Firmicutes, mainly Clostridiales, with Bacteroidetes being the subdominant populations. Archaea populations belonging to the genus Methanosarcina became predominant throughout anaerobic digestion, confirming the formation of methane mainly from acetate, in line with the greatest removal of volatile fatty acids (VFAs) in these samples.

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 Faecal bacterial composition in horses with and without free faecal liquid: a case control study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katrin M. Lindroth ◽  
Johan Dicksved ◽  
Erik Pelve ◽  
Viveca Båverud ◽  
Cecilia E. Müller
Keyword(s):  
Relative Abundance ◽  
Case Control Study ◽  
Sampling Period ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Clostridioides Difficile ◽  
Liquid Phases ◽  
And Control ◽  
The 16S Rrna Gene ◽  
Control Study

AbstractFree faecal liquid (FFL) is a condition in horses which manifests as differential defecation of solid and liquid phases of faeces. The etiology of FFL is currently unknown, but deviances in the hindgut microbiota has been suggested to be of importance. The present study aimed to compare the faecal bacterial composition of farm-matched horses with (case, n = 50) and without (control, n = 50) FFL. Samples were collected at three different occasions. The V3 and V4 regions of the 16S rRNA gene were amplified and sequenced using Illumina sequencing. Also, samples were cultivated for detection of Clostridioides difficile and Clostridium perfringens. Analysis revealed similar faecal bacterial composition between case and control horses, but an effect of sampling period (p = 0.0001). Within sampling periods, 14 genera were present in higher or lower proportions in case compared to control horses in at least one sampling period. Compared to controls, case horses had higher relative abundance of Alloprevotella (adjusted p < 0.04) and lower relative abundance of Bacillus spp. (adjusted p < 0.03) in at least two sampling periods. All horses tested negative for C. difficile and C. perfringens by culture of faeces. Further studies are required to establish the clinical relevance of specific bacterial taxa in FFL.

Microbiome population dynamics of cold smoked sockeye salmon during refrigerated storage and after culture enrichment

2021 ◽  
Author(s):  
Karen Jarvis ◽  
Chiun-Kang Hsu ◽  
James B. Pettengill ◽  
John Ihrie ◽  
Hiren Karathia ◽  
...  
Keyword(s):  
Population Dynamics ◽  
Foodborne Pathogens ◽  
Sockeye Salmon ◽  
Pathogen Detection ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Smoked Salmon ◽  
Spoilage Bacteria ◽  
Culture Enrichment ◽  
Processing And Storage

Cold smoked salmon is a ready-to-eat seafood product of high commercial importance. The processing and storage steps facilitate the introduction, growth and persistence of foodborne pathogens and spoilage bacteria. The growth of commensal bacteria during storage and once the product is opened also influence the quality and safety of cold smoked salmon. Here we investigated the microbial community through targeted 16s rRNA gene and shotgun metagenomic sequencing, as means to better understand the interactions among bacteria in cold smoked salmon. Cold smoked salmon samples were tested over 30 days of aerobic storage at 4℃ and cultured at each timepoint in buffered Listeria enrichment broth (BLEB) commonly used to detect Listeria in foods. The microbiomes were comprised of Firmicutes and Proteobacteria namely, Carnobacterium , Brochothrix , Pseudomonas , Serratia , and Psychrobacter . Pseudomonas species were the most diverse species with 181 taxa identified. Additionally, we identified potential homologs to 10 classes of bacteriocins in microbiomes of cold smoked salmon stored at 4°C and corresponding BLEB culture enrichments. The findings presented here contribute to our understanding of microbiome population dynamics in cold smoked salmon, including changes in bacterial taxa during aerobic cold storage and after culture enrichment.  This may facilitate improvements to pathogen detection and quality preservation of this food.

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