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 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.

scholarly journals Distribution of Acetothermia-dominated microbial communities in alkaline hot springs of Baikal Rift Zone.

2017 ◽  
Author(s):  
Svetlana V. Zaitseva ◽  
Elena V. Lavrentieva ◽  
Aryuna A. Radnagurueva ◽  
Olga A. Baturina ◽  
Marsel R. Kabilov ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Hot Springs ◽  
Rift Zone ◽  
Hot Spring ◽  
Baikal Rift Zone ◽  
Rrna Gene ◽  
Acetyl Coa Pathway

Alkaline hot springs are unique extreme habitats resemble the early Earth and present a valuable resource for the discovery of procaryotic community diversity and isolation of the novel thermophilic Bacteria and Archaea. One of the model for the possible origin of biochemistry in alkaline hot springs revealed the acetyl-CoA pathway of CO2 fixation might be the most ancient form of carbon metabolism. Recent phylogenetic studies have suggested that the phylum Acetothermia is one of the deep branches of the Bacteria domain. Firstly Acetothermia (Candidate division OP1) was characterized in a culture independent molecular phylogenetic survey based on the 16S rRNA gene of the sulfide-rich hot spring, Obsidian Pool, a 75 to 95oC hot spring. Two nearly complete genomes of Acetothermia were established based on genome-resolved metagenomic analysis and its capability of implementing acetogenesis through the ancient reductive acetyl-CoA pathway by utilizing CO2 and H2 was revealed. Although genomic, proteomic and metagenomic approaches investigate basic metabolism and potentional energy conservation of uncultivated candidate phyla but ecological roles of these bacteria and general patterns of diversity and community structure stay unclear. General hydrochemical and geological characterization of alkaline thermal springs of the Baikal Rift zone with high silica concentrations and a nitrogen dominated gas phase is provided. Previous microbiogical studies based on culture-dependent methods recovered a large number of bacterial strains from thermal springs located in Baikal Rift zone. We combined microbial communities analysis by using high-throughput 16S rRNA gene sequencing, biogeochemical measurements, sediment mineralogy and physicochemical characteristics to investigate ecosystems of alkaline hot springs located in the Baikal Rift zone. Uncultivated bacteria belonging to the phylum Acetothermia, along with members of the phyla Firmicutes and Proteobacteria, were identified as the dominant group in hydrothermal sediments communities in the alkaline hot springs of Baikal Rift zone. In bottom sediments of the Alla hot spring, about 57% of all classified sequences represent this phylum. Geochemistry of fluids and sample type were strongly correlated with microbial community composition. The Acetothermia exhibited the highest relative abundance in sediment microbial community associated with alkaline thermal fluids enriched in Fe, Zn, Ni, Al and Cr.

scholarly journals Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Zigui Chen ◽  
Pak Chun Hui ◽  
Mamie Hui ◽  
Yun Kit Yeoh ◽  
Po Yee Wong ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Preservation Solution ◽  
Hypervariable Regions ◽  
Preservation Solutions ◽  
Stool Samples ◽  
16S Rrna Gene Primer

ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

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

2018 ◽  
Author(s):  
Alex 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 metagenomic and 16S rRNA gene sequencing. However, these methods do not distinguish between 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 (19K, 27K, and 33K). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools and how they change over geologic time. We found clear evidence that cells capable of forming endospores are not necessarily dormant and that the propensity to form endospores differed among taxa. Specifically, Bacilli are more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of Clostridia, which are more likely to persist as vegetative cells over geologic timescales. We also found that exogenous DNA preserved within permafrost does not bias DNA sequencing results since its removal did not significantly alter the microbial community composition. These results extend the findings of a previous study that showed permafrost age and ice content largely control microbial community diversity and cell abundances.ImportanceThe study of permafrost transcends the study of climate change and exobiology. Permafrost soils store more than half earth’s soil carbon despite covering ∽15% of the land area (Tarnocai et al 2009). This permafrost carbon is rapidly degraded following thaw (Tarnocai C et al 2009, Schuur et al 2015). Understanding microbial communities in permafrost will contribute to the knowledge base necessary to understand the rates and forms of permafrost C and N cycling post thaw. 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 permafrost we can focus efforts searching for evidence of life on cryogenic cosmic bodies. 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 across geologic timescales.

scholarly journals Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Coral Reefs ◽  
2020 ◽  
Vol 39 (6) ◽  
pp. 1817-1827
Author(s):  
Alicia M. Reigel ◽  
Sarah M. Owens ◽  
Michael E. Hellberg
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Dna Amplification ◽  
Community Diversity ◽  
Rrna Gene ◽  
Target Sequence ◽  
Sequence Coverage ◽  
Average Percentage

AbstractEfforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

scholarly journals Distribution of Acetothermia-dominated microbial communities in alkaline hot springs of Baikal Rift Zone.

2017 ◽  
Author(s):  
Svetlana V. Zaitseva ◽  
Elena V. Lavrentieva ◽  
Aryuna A. Radnagurueva ◽  
Olga A. Baturina ◽  
Marsel R. Kabilov ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Hot Springs ◽  
Rift Zone ◽  
Hot Spring ◽  
Baikal Rift Zone ◽  
Rrna Gene ◽  
Acetyl Coa Pathway

Alkaline hot springs are unique extreme habitats resemble the early Earth and present a valuable resource for the discovery of procaryotic community diversity and isolation of the novel thermophilic Bacteria and Archaea. One of the model for the possible origin of biochemistry in alkaline hot springs revealed the acetyl-CoA pathway of CO2 fixation might be the most ancient form of carbon metabolism. Recent phylogenetic studies have suggested that the phylum Acetothermia is one of the deep branches of the Bacteria domain. Firstly Acetothermia (Candidate division OP1) was characterized in a culture independent molecular phylogenetic survey based on the 16S rRNA gene of the sulfide-rich hot spring, Obsidian Pool, a 75 to 95oC hot spring. Two nearly complete genomes of Acetothermia were established based on genome-resolved metagenomic analysis and its capability of implementing acetogenesis through the ancient reductive acetyl-CoA pathway by utilizing CO2 and H2 was revealed. Although genomic, proteomic and metagenomic approaches investigate basic metabolism and potentional energy conservation of uncultivated candidate phyla but ecological roles of these bacteria and general patterns of diversity and community structure stay unclear. General hydrochemical and geological characterization of alkaline thermal springs of the Baikal Rift zone with high silica concentrations and a nitrogen dominated gas phase is provided. Previous microbiogical studies based on culture-dependent methods recovered a large number of bacterial strains from thermal springs located in Baikal Rift zone. We combined microbial communities analysis by using high-throughput 16S rRNA gene sequencing, biogeochemical measurements, sediment mineralogy and physicochemical characteristics to investigate ecosystems of alkaline hot springs located in the Baikal Rift zone. Uncultivated bacteria belonging to the phylum Acetothermia, along with members of the phyla Firmicutes and Proteobacteria, were identified as the dominant group in hydrothermal sediments communities in the alkaline hot springs of Baikal Rift zone. In bottom sediments of the Alla hot spring, about 57% of all classified sequences represent this phylum. Geochemistry of fluids and sample type were strongly correlated with microbial community composition. The Acetothermia exhibited the highest relative abundance in sediment microbial community associated with alkaline thermal fluids enriched in Fe, Zn, Ni, Al and Cr.

scholarly journals Biogeography and Biodiversity in Sulfide Structures of Active and Inactive Vents at Deep-Sea Hydrothermal Fields of the Southern Mariana Trough

2010 ◽  
Vol 76 (9) ◽  
pp. 2968-2979 ◽  
Author(s):  
Shingo Kato ◽  
Yoshinori Takano ◽  
Takeshi Kakegawa ◽  
Hironori Oba ◽  
Kazuhiko Inoue ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Microbial Community Composition ◽  
Organic Content ◽  
Hyperthermophilic Archaea ◽  
Spreading Center ◽  
Rrna Gene ◽  
Hydrothermal Fields

ABSTRACT The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

scholarly journals The diversity of active microbial groups in an activated sludge process treating painting process wastewater

2020 ◽  
Vol 148 ◽  
pp. 01002
Author(s):  
Herto Dwi Ariesyady ◽  
Mentari Rizki Mayanda ◽  
Tsukasa Ito
Keyword(s):  
Wastewater Treatment ◽  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Activated Sludge ◽  
Biological Treatment ◽  
Rrna Gene ◽  
Activated Sludge Process ◽  
Painting Process ◽  
Process Wastewater

Activated sludge process is one of the wastewater treatment method that is applied for many wastewater types including painting process wastewater of automotive industry. This wastewater is well-known to have high heavy metals concentration which could deteriorate water environment if appropriate performance of the wastewater treatment could not be achieved. In this study, we monitored microbial community diversity in a Painting Biological Treatment (PBT) system. We applied a combination of cultivation and genotypic biological methods based on 16S rRNA gene sequence analysis to identify the diversity of active microbial community. The results showed that active microbes that could grow in this activated sludge system were dominated by Gram-negative bacteria. Based on 16S rRNA gene sequencing analysis, it was revealed that their microbial diversity has close association with Bacterium strain E286, Isosphaera pallida, Lycinibacillus fusiformis, Microbacterium sp., Orchobactrum sp., Pseudomonas guariconensis, Pseudomonas sp. strain MR84, Pseudomonas sp. MC 54, Serpens sp., Stenotrophomonas acidaminiphila, and Xylella fastidiosa with similarity of 86 – 99%. This findings reflects that microbial community in a Painting Biological Treatment (PBT) system using activated sludge process could adapt with xenobiotics in the wastewater and has a wide range of diversity indicating a complex metabolism mechanism in the treatment process.

scholarly journals Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis

2014 ◽  
Vol 48 (8) ◽  
pp. 717-728 ◽  
Author(s):  
M. N. Zakaria ◽  
T. Takeshita ◽  
Y. Shibata ◽  
H. Maeda ◽  
N. Wada ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Clone Library ◽  
Apical Periodontitis ◽  
Gene Clone ◽  
Rrna Gene ◽  
Clone Library Analysis
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