scholarly journals Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raiza Hasrat ◽  
Jolanda Kool ◽  
Wouter A. A. de Steenhuijsen Piters ◽  
Mei Ling J. N. Chu ◽  
Sjoerd Kuiling ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Positive Control ◽  
Rrna Gene ◽  
Elution Buffer ◽  
Community Profile ◽  
Microbial Community Profile ◽  
Microbial Dna ◽  
Pcr Conditions

AbstractThe low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

scholarly journals Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

2021 ◽  
Author(s):  
Raiza Hasrat ◽  
Jolanda Kool ◽  
Wouter A.A. de Steenhuijsen Piters ◽  
Mei Ling J.N. Chu ◽  
Sjoerd Kuiling ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Positive Control ◽  
Rrna Gene ◽  
Purification Methods ◽  
Microbial Dna ◽  
Comparability Of Results ◽  
Pcr Conditions ◽  
Respiratory Microbiota

Abstract The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community composition, we amplified the 16S rRNA gene of respiratory samples using various DNA input and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 and V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community composition of low biomass samples. Purification methods and MiSeq reagent kits had only a modest impact on microbiota profiles, while profiles of positive controls were significantly influenced by type of dilution solvent. Microbiota profiles of low biomass samples can be accurately distinguished from DNA blanks. Microbiota profiling of low biomass samples is stable under several PCR conditions, purification methods and MiSeq reagent kits. We recommend to use amplification with 30 PCR cycles. The amplicon pools can best be purified by two consecutive AMPure XP steps and sequenced by V3 MiSeq reagent kit. The benchmarked standardized workflow presented here ensures comparability of results within and between low biomass microbiome studies.

scholarly journals Do initial concentration and activated sludge seasonality affect pharmaceutical biotransformation rate constants?

2021 ◽  
Author(s):  
Tamara J. H. M. van Bergen ◽  
Ana B. Rios-Miguel ◽  
Tom M. Nolte ◽  
Ad M. J. Ragas ◽  
Rosalie van Zelm ◽  
...  
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Community Composition ◽  
Rate Constants ◽  
Wastewater Treatment Plants ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Initial Concentration ◽  
Different Seasons

Abstract Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC–MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. Key points • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change. Graphical abstract

scholarly journals Temporal Dynamics of Cloacal Microbiota in Adult Laying Chickens With and Without Access to an Outdoor Range

2021 ◽  
Vol 11 ◽  
Author(s):  
Janneke Schreuder ◽  
Francisca C. Velkers ◽  
Alex Bossers ◽  
Ruth J. Bouwstra ◽  
Willem F. de Boer ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Intestinal Microbiota ◽  
Environmental Changes ◽  
Temporal Dynamics ◽  
Microbial Community Composition ◽  
Sudden Change ◽  
Rrna Gene ◽  
Poultry House ◽  
Over Time

Associations between animal health and performance, and the host’s microbiota have been recently established. In poultry, changes in the intestinal microbiota have been linked to housing conditions and host development, but how the intestinal microbiota respond to environmental changes under farm conditions is less well understood. To gain insight into the microbial responses following a change in the host’s immediate environment, we monitored four indoor flocks of adult laying chickens three times over 16 weeks, during which two flocks were given access to an outdoor range, and two were kept indoors. To assess changes in the chickens’ microbiota over time, we collected cloacal swabs of 10 hens per flock and performed 16S rRNA gene amplicon sequencing. The poultry house (i.e., the stable in which flocks were housed) and sampling time explained 9.2 and 4.4% of the variation in the microbial community composition of the flocks, respectively. Remarkably, access to an outdoor range had no detectable effect on microbial community composition, the variability of microbiota among chickens of the same flock, or microbiota richness, but the microbiota of outdoor flocks became more even over time. Fluctuations in the composition of the microbiota over time within each poultry house were mainly driven by turnover in rare, rather than dominant, taxa and were unique for each flock. We identified 16 amplicon sequence variants that were differentially abundant over time between indoor and outdoor housed chickens, however none were consistently higher or lower across all chickens of one housing type over time. Our study shows that cloacal microbiota community composition in adult layers is stable following a sudden change in environment, and that temporal fluctuations are unique to each flock. By exploring microbiota of adult poultry flocks within commercial settings, our study sheds light on how the chickens’ immediate environment affects the microbiota composition.

scholarly journals Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Alexander Burkert ◽  
Thomas A. Douglas ◽  
Mark P. Waldrop ◽  
Rachel Mackelprang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Community Composition ◽  
Environmental Conditions ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Subzero Temperatures ◽  
Dormant Cells

ABSTRACTPermafrost hosts a community of microorganisms that survive and reproduce for millennia despite extreme environmental conditions, such as water stress, subzero temperatures, high salinity, and low nutrient availability. Many studies focused on permafrost microbial community composition use DNA-based methods, such as metagenomics and 16S rRNA gene sequencing. However, these methods do not distinguish among active, dead, and dormant cells. This is of particular concern in ancient permafrost, where constant subzero temperatures preserve DNA from dead organisms and dormancy may be a common survival strategy. To circumvent this, we applied (i) LIVE/DEAD differential staining coupled with microscopy, (ii) endospore enrichment, and (iii) selective depletion of DNA from dead cells to permafrost microbial communities across a Pleistocene permafrost chronosequence (19,000, 27,000, and 33,000 years old). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools, how they are influenced by soil physicochemical properties, and whether they change over geologic time. We found evidence that cells capable of forming endospores are not necessarily dormant and that members of the classBacilliwere more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of theClostridia, which were more likely to persist as vegetative cells in our older samples. We also found that removing exogenous “relic” DNA preserved within permafrost did not significantly alter microbial community composition. These results link the live, dead, and dormant microbial communities to physicochemical characteristics and provide insights into the survival of microbial communities in ancient permafrost.IMPORTANCEPermafrost soils store more than half of Earth’s soil carbon despite covering ∼15% of the land area (C. Tarnocai et al., Global Biogeochem Cycles 23:GB2023, 2009, https://doi.org/10.1029/2008GB003327). This permafrost carbon is rapidly degraded following a thaw (E. A. G. Schuur et al., Nature 520:171–179, 2015, https://doi.org/10.1038/nature14338). Understanding microbial communities in permafrost will contribute to the knowledge base necessary to understand the rates and forms of permafrost C and N cycling postthaw. Permafrost is also an analog for frozen extraterrestrial environments, and evidence of viable organisms in ancient permafrost is of interest to those searching for potential life on distant worlds. If we can identify strategies microbial communities utilize to survive in permafrost, it may yield insights into how life (if it exists) survives in frozen environments outside of Earth. Our work is significant because it contributes to an understanding of how microbial life adapts and survives in the extreme environmental conditions in permafrost terrains.

Microbial community shift under exposure of dredged sediments from a tropical eutrophic bay

2020 ◽  
Author(s):  
Juliana Nascimento ◽  
Cole Easson ◽  
Diogo Jurelevicius ◽  
Jose Lopes ◽  
Edison Bidone ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Dredged Sediments ◽  
Community Shift ◽  
Geochemical Parameters ◽  
Enriched Environments ◽  
Microbiome Data

<p>Microbial communities occur in almost every habitat. To evaluate the homeostasis disruption of in situ microbiomes, dredged sediments from Guanabara Bay-Brazil (GB) were mixed with sediments outside the bay (D) in three different proportions (25%, 50% and 75%) which we called GBD25, GBD50 and GBD75. Grain size, TOC and metals -as indicators of complex contamination-; dehydrogenase (DHA) and esterases enzymes (EST) – as indicators of microbial community availability, were determined. Microbial community composition was addressed by amplifying the 16S rRNA gene for DGGE analysis and sequencing using MiSeq platform (Illumina). We applied the Quality Ratio index (QR) to the GB, D and every GBD mixture to integrate geochemical parameters with our microbiome data. QR indicated high environmental risk for GB and every GBD mixture; and low risk for D. The community shifted from aerobic to anaerobic profile, consistent with the characteristics of GB. Sample D was dominated by JTB255 marine benthic group, related to low impacted areas. Milano-WF1B-44 was the most representative of GB, often found in anaerobic and sulfur enriched environments. In GBD, the denitrifying sulfur-oxidizing bacteria, Sulfurovum, was the most representative, typically found in suboxic or anoxic niches. The canonical correspondence analysis was able to explain 60% of the community composition variation and exhibit the decrease of environmental quality as the contamination increases. Physiological and taxonomic shifts of the microbial assemblage in sediments was inferred by QR, which was suitable to determinate sediment risk. The study produced sufficient information to improve dredging plan and management.</p>

scholarly journals Microbial Community Stratification Linked to Utilization of Carbohydrates and Phosphorus Limitation in a Boreal Peatland at Marcell Experimental Forest, Minnesota, USA

2014 ◽  
Vol 80 (11) ◽  
pp. 3518-3530 ◽  
Author(s):  
Xueju Lin ◽  
Malak M. Tfaily ◽  
J. Megan Steinweg ◽  
Patrick Chanton ◽  
Kaitlin Esson ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Community Composition ◽  
Hot Spot ◽  
Microbial Community Composition ◽  
Phosphorus Limitation ◽  
Rrna Gene ◽  
Content Type ◽  
Gene Abundance ◽  
P Limitation

ABSTRACTThis study investigated the abundance, distribution, and composition of microbial communities at the watershed scale in a boreal peatland within the Marcell Experimental Forest (MEF), Minnesota, USA. Through a close coupling of next-generation sequencing, biogeochemistry, and advanced analytical chemistry, a biogeochemical hot spot was revealed in the mesotelm (30- to 50-cm depth) as a pronounced shift in microbial community composition in parallel with elevated peat decomposition. The relative abundance ofAcidobacteriaand theSyntrophobacteraceae, including known hydrocarbon-utilizing genera, was positively correlated with carbohydrate and organic acid content, showing a maximum in the mesotelm. The abundance ofArchaea(primarily crenarchaeal groups 1.1c and 1.3) increased with depth, reaching up to 60% of total small-subunit (SSU) rRNA gene sequences in the deep peat below the 75-cm depth. Stable isotope geochemistry and potential rates of methane production paralleled vertical changes in methanogen community composition to indicate a predominance of acetoclastic methanogenesis mediated by theMethanosarcinalesin the mesotelm, while hydrogen-utilizing methanogens predominated in the deeper catotelm. RNA-derived pyrosequence libraries corroborated DNA sequence data to indicate that the above-mentioned microbial groups are metabolically active in the mid-depth zone. Fungi showed a maximum in rRNA gene abundance above the 30-cm depth, which comprised only an average of 0.1% of total bacterial and archaeal rRNA gene abundance, indicating prokaryotic dominance. Ratios of C to P enzyme activities approached 0.5 at the acrotelm and catotelm, indicating phosphorus limitation. In contrast, P limitation pressure appeared to be relieved in the mesotelm, likely due to P solubilization by microbial production of organic acids and C-P lyases. Based on path analysis and the modeling of community spatial turnover, we hypothesize that P limitation outweighs N limitation at MEF, and microbial communities are structured by the dominant shrub,Chamaedaphne calyculata, which may act as a carbon source for major consumers in the peatland.

scholarly journals Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0116955 ◽  
Author(s):  
Lucas Sinclair ◽  
Omneya Ahmed Osman ◽  
Stefan Bertilsson ◽  
Alexander Eiler
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Illumina Platform

scholarly journals Emergence Shapes the Structure of the Seed Microbiota

2014 ◽  
Vol 81 (4) ◽  
pp. 1257-1266 ◽  
Author(s):  
Matthieu Barret ◽  
Martial Briand ◽  
Sophie Bonneau ◽  
Anne Préveaux ◽  
Sophie Valière ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Practical Applications ◽  
Bacterial Marker ◽  
Species Specific ◽  
Diversity Studies

ABSTRACTSeeds carry complex microbial communities, which may exert beneficial or deleterious effects on plant growth and plant health. To date, the composition of microbial communities associated with seeds has been explored mainly through culture-based diversity studies and therefore remains largely unknown. In this work, we analyzed the structures of the seed microbiotas of different plants from the family Brassicaceae and their dynamics during germination and emergence through sequencing of three molecular markers: the ITS1 region of the fungal internal transcribed spacer, the V4 region of 16S rRNA gene, and a species-specific bacterial marker based on a fragment ofgyrB. Sequence analyses revealed important variations in microbial community composition between seed samples. Moreover, we found that emergence strongly influences the structure of the microbiota, with a marked reduction of bacterial and fungal diversity. This shift in the microbial community composition is mostly due to an increase in the relative abundance of some bacterial and fungal taxa possessing fast-growing abilities. Altogether, our results provide an estimation of the role of the seed as a source of inoculum for the seedling, which is crucial for practical applications in developing new strategies of inoculation for disease prevention.

Temporal and Vertical Variation in Microbial Community Composition in Response to Physicochemical Characteristics in a Water Column of Highly Eutrophied Jinhae Bay, South Korea

2018 ◽  
Vol 28 (2) ◽  
pp. 65-77 ◽  
Author(s):  
Jiyoung Lee ◽  
Jae-Hyun Lim ◽  
Junhyung Park ◽  
Il-Nam Kim
Keyword(s):  
Microbial Community ◽  
South Korea ◽  
Microbial Communities ◽  
Water Column ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Vertical Variation ◽  
Jinhae Bay

Microbial communities play an essential role in marine biogeochemical cycles. Physical and biogeochemical changes in Jinhae Bay, the most anthropogenically eutrophied bay on the coasts of South Korea, are well described, but less is known about the associated changes in microbial communities. Temporal and vertical variation in microbial communities at three depths (surface, middle, and bottom) at seven time points (June to December) at the J1 sampling site were investigated on the MiSeq platform based on the 16S rRNA gene. Overall, the microbial community was dominated by Proteobacteria, Cyanobacteria, and Bacteroidetes from June to November, whereas Firmicutes were dominant in December, especially in the middle and bottom layers. The results indicate that the microbial community composition strongly varied with temporal changes in the physicochemical water properties. Moreover, the community composition differed markedly between the surface and middle layers and the bottom layer in the summer, when the water column was strongly stratified and bottom water hypoxia developed. A redundancy analysis suggested a significant correlation between physicochemical variables (i.e., temperature, salinity, and oxygen concentration) and microbial community composition. This study indicates that temporal changes in water conditions and eutrophication-induced hypoxia effectively shape the structure of the microbial community.

Exploring microbial determinants of apple replant disease (ARD): a microhabitat approach under split-root design

2020 ◽  
Vol 96 (12) ◽  
Author(s):  
Alicia Balbín-Suárez ◽  
Maik Lucas ◽  
Doris Vetterlein ◽  
Søren J Sørensen ◽  
Traud Winkelmann ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Apple Rootstocks ◽  
Apple Replant Disease ◽  
Alpha And Beta Diversity ◽  
Replant Disease ◽  
Split Root ◽  
And Control

ABSTRACT Apple replant disease (ARD) occurs worldwide in apple orchards and nurseries and leads to a severe growth and productivity decline. Despite research on the topic, its causality remains unclear. In a split-root experiment, we grew ARD-susceptible ‘M26’ apple rootstocks in different substrate combinations (+ARD: ARD soil; -ARD: gamma-irradiated ARD soil; and Control: soil with no apple history). We investigated the microbial community composition by 16S rRNA gene amplicon sequencing (bacteria and archaea) along the soil–root continuum (bulk soil, rhizosphere and rhizoplane). Significant differences in microbial community composition and structure were found between +ARD and -ARD or +ARD and Control along the soil–root continuum, even for plants exposed simultaneously to two different substrates (-ARD/+ARD and Control/+ARD). The substrates in the respective split-root compartment defined the assembly of root-associated microbial communities, being hardly influenced by the type of substrate in the respective neighbor compartment. Root-associated representatives from Actinobacteria were the most dynamic taxa in response to the treatments, suggesting a pivotal role in ARD. Altogether, we evidenced an altered state of the microbial community in the +ARD soil, displaying altered alpha- and beta-diversity, which in turn will also impact the normal development of apple rhizosphere and rhizoplane microbiota (dysbiosis), concurring with symptom appearance.

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