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Asset Tag: 16S

September 22, 2019  |  

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3?m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.

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September 22, 2019  |  

Scale-up of sediment microbial fuel cells.

Sediment microbial fuel cells (SMFCs) are used as renewable power sources to operate remote sensors. However, increasing the electrode surface area results in decreased power density, which demonstrates that SMFCs do not scale up with size. As an alternative to the physical scale-up of SMFCs, we proposed that it is possible to scale up power by using smaller-sized individually operated SMFCs connected to a power management system that electrically isolates the anodes and cathodes. To demonstrate our electronic scale-up approach, we operated one 0.36-m2 SMFC (called a single-equivalent SMFC) and four independent SMFCs of 0.09 m2 each (called scaled-up SMFCs) and managed the power using an innovative custom-developed power management system. We found that the single-equivalent SMFC and the scaled-up SMFCs produced similar power for the first 155 days. However, in the long term (>155 days) our scaled-up SMFCs generated significantly more power than the single-equivalent SMFC (2.33 mW vs. 0.64 mW). Microbial community analysis of the single-equivalent SMFC and the scaled-up SMFCs showed very similar results, demonstrating that the difference in operation mode had no significant effect on the microbial community. When we compared scaled-up SMFCs with parallel SMFCs, we found that the scaled-up SMFCs generated more power. Our novel approach demonstrates that SMFCs can be scaled up electronically.

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September 22, 2019  |  

The effects of probiotics administration on the milk production, milk components and fecal bacteria microbiota of dairy cows

Probiotics administration can improve host health. This study aims to determine the effects of probiotics (Lactobacillus casei Zhang and Lactobacillus plantarum P-8) administration on milk production, milk functional components, milk composition, and fecal microbiota of dairy cows. Variations in the fecal bacteria microbiota between treatments were assessed based on 16S rRNA profiles determined by PacBio single molecule real-time sequencing technology. The probiotics supplementation significantly increased the milk production and the contents of milk immunoglobulin G (IgG), lactoferrin (LTF), lysozyme (LYS) and lactoperoxidase (LP), while the somatic cell counts (SCC) significantly decreased (P < 0.01). However, no significant difference was found in the milk fat, protein and lactose contents (P > 0.05). Although the probiotics supplementation did not change the fecal bacteria richness and diversity, significantly more rumen fermentative bacteria (Bacteroides, Roseburia, Ruminococcus, Clostridium, Coprococcus and Dorea) and beneficial bacteria (Faecalibacterium prausnitzii) were found in the probiotics treatment group. Meanwhile, some opportunistic pathogens e.g. Bacillus cereus, Cronobacter sakazakii and Alkaliphilus oremlandii, were suppressed. Additionally, we found some correlations between the milk production, milk components and fecal bacteria. To sum up, our study demonstrated the beneficial effects of probiotics application in improving the quality and quantity of cow milk production.

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September 22, 2019  |  

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling.

In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases.We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

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September 22, 2019  |  

The features of mucosa-associated microbiota in primary sclerosing cholangitis.

Little is known about the role of the microbiome in primary sclerosing cholangitis.To explore the mucosa-associated microbiota in primary sclerosing cholangitis (PSC) patients across different locations in the gut, and to compare it with inflammatory bowel disease (IBD)-only patients and healthy controls.Biopsies from the terminal ileum, right colon, and left colon were collected from patients and healthy controls undergoing colonoscopy. Microbiota profiling using bacterial 16S rRNA sequencing was performed on all biopsies.Forty-four patients were recruited: 20 with PSC (19 with PSC-IBD and one with PSC-only), 15 with IBD-only and nine healthy controls. The overall microbiome profile was similar throughout different locations in the gut. No differences in the global microbiome profile were found. However, we observed significant PSC-associated enrichment in Barnesiellaceae at the family level, and in Blautia and an unidentified Barnesiellaceae at the genus level. At the operational taxa unit level, most shifts in PSC were observed in Clostridiales and Bacteroidales orders, with approximately 86% of shifts occurring within the former order.The overall microbiota profile was similar across multiple locations in the gut from the same individual regardless of disease status. In this study, the mucosa associated-microbiota of patients with primary sclerosing cholangitis was characterised by enrichment of Blautia and Barnesiellaceae and by major shifts in operational taxa units within Clostridiales order.© 2016 John Wiley & Sons Ltd.

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September 22, 2019  |  

Laboratory colonization stabilizes the naturally dynamic microbiome composition of field collected Dermacentor andersoni ticks.

Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species.The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level.In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level.We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

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September 22, 2019  |  

Research benefits of storing genitourinary samples: 16S rRNA sequencing to evaluate vaginal bacterial communities.

Using well-characterised, but old and carefully frozen genital tract research samples may be a cost-effective way of performing metagenomic studies, but risks loss of low abundance (but relevant) bacterial species DNA. Moi et al.1 used 16S rRNA and UreDNA sequencing to detect ureaplasmas in frozen urine samples collected from 362 men with NGU in 2010–2011. They found that urethral inflammatory responses to ureaplasmas were less severe than to Chlamydia trachomatis and Mycoplasma genitalium.

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September 22, 2019  |  

Atmospheric N deposition increases bacterial laccase-like multicopper oxidases: implications for organic matter decay.

Anthropogenic release of biologically available nitrogen (N) has increased dramatically over the last 150 years, which can alter the processes controlling carbon (C) storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan in the United States, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This change occurred concomitantly with compositional changes in Basidiomycete fungi and in Actinobacteria, as well as the downregulation of fungal lignocelluloytic genes. Recently, laccase-like multicopper oxidases (LMCOs) have been discovered among bacteria which can oxidize ß-O-4 linkages in phenolic compounds (e.g., lignin and humic compounds), resulting in the production of dissolved organic carbon (DOC). Here, we examined how nearly 2 decades of experimental N deposition has affected the abundance and composition of saprotrophic bacteria possessing LMCO genes. In our experiment, LMCO genes were more abundant in the forest floor under experimental N deposition whereas the abundances of bacteria and fungi were unchanged. Experimental N deposition also led to less-diverse, significantly altered bacterial and LMCO gene assemblages, with taxa implicated in organic matter decay (i.e., Actinobacteria, Proteobacteria) accounting for the majority of compositional changes. These results suggest that experimental N deposition favors bacteria in the forest floor that harbor the LMCO gene and represents a plausible mechanism by which anthropogenic N deposition has reduced decomposition, increased soil C storage, and accelerated phenolic DOC production in our field experiment. Our observations suggest that future rates of atmospheric N deposition could fundamentally alter the physiological potential of soil microbial communities. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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September 22, 2019  |  

Profiling of metabolome and bacterial community dynamics in ensiled Medicago sativa inoculated without or with Lactobacillus plantarum or Lactobacillus buchneri.

Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiled Medicago sativa (alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation of L. buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed in L. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.

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September 22, 2019  |  

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens.

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9?bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.

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September 22, 2019  |  

Bacterial microbiota and metabolic character of traditional sour cream and butter in Buryatia, Russia.

Traditional sour cream and butter are widely popular fermented dairy products in Russia for their flavor and nutrition, and contain rich microbial biodiversity, particularly in terms of lactic acid bacteria (LAB). However, few studies have described the microbial communities and metabolic character of traditional sour cream and butter. The objective of this study was to determine the bacterial microbiota and metabolic character of eight samples collected from herdsmen in Buryatia, Russia. Using single-molecule real-time (SMRT) sequencing techniques, we identified a total of 294 species and/or subspecies in 169 bacterial genera, belonging to 14 phyla. The dominant phylum was Firmicutes (81.47%) and the dominant genus was Lactococcus (59.28%). There were differences between the bacterial compositions of the sour cream and butter samples. The relative abundances of Lactococcus lactis, Lactococcus raffinolactis, and Acetobacter cibinongensis were significantly higher in sour cream than in butter, and the abundance of Streptococcusthermophilus was significantly lower in sour cream than in butter. Using a pure culture method, 48 strains were isolated and identified to represent seven genera and 15 species and/or subspecies. Among these isolates, Lactococccus lactis subsp. lactis (22.50%) was the dominant LAB species. Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry at elevated energy was used in combination with statistical methods to detect metabolite differences between traditional sour cream and butter. A total of 27,822 metabolites were detected in all samples, and Lys-Lys, isohexanal, palmitic acid, Leu-Val, and 2′-deoxycytidine were the most dominant metabolites found in all samples. In addition, 27 significantly different metabolites were detected between the sour cream and butter samples, including short peptides, organic acids, and amino acids. Based on correlation analyses between the most prevalent bacterial species and the main metabolites in sour cream, we conclude that there may be a connection between the dominant LAB species and these metabolites. This study combined omics techniques to analyze the bacterial diversity and metabolic character of traditional sour cream and butter, and we hope that our findings will enrich species resource libraries and provide valuable resources for further research on dairy product flavor.

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September 22, 2019  |  

Effect of Chinese rice wine sludge on the production of Chinese steamed buns

Chinese rice wine sludge (CRWS), analogous to beer yeast sludge, is the filter cake remaining after squeezing the fermentation mash of Chinese rice wine. CRWS contains high levels of protein (44.74%), nonstructural carbohydrates (37.33%), crude fiber (13.5%), and essential amino acids, which could enhance the trophic value of Chinese steamed buns. In our research, the microbiota of CRWS (mainly Saccharomyces cerevisiae and Lactobacillus sp.) was analyzed at the species level by single-molecule real-time DNA sequencing technology. Interestingly, the microbiota of CRWS was similar to that of the starter dough typically used to prepare Chinese steamed buns. Incorporation of CRWS significantly influenced the pasting properties and farinograph characteristics of the dough, which control the texture of the Chinese steamed buns, and supplementation with 5~30% CRWS caused the properties of the resulting buns to be more similar to those of northern-style steamed buns. CRWS addition also significantly enhanced the content of aroma compounds in the Chinese steamed buns.

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September 22, 2019  |  

Long-term operation of microbial electrosynthesis systems improves acetate production by autotrophic microbiomes.

Microbial electrosynthesis is the biocathode-driven production of chemicals from CO2 and has the promise to be a sustainable, carbon-consuming technology. To date, microbial electrosynthesis of acetate, the first step in order to generate liquid fuels from CO2, has been characterized by low rates and yields. To improve performance, a previously established acetogenic biocathode was operated in semi-batch mode at a poised potential of -590 mV vs SHE for over 150 days beyond its initial development. Rates of acetate production reached a maximum of 17.25 mM day(-1) (1.04 g L(-1) d(-1)) with accumulation to 175 mM (10.5 g L(-1)) over 20 days. Hydrogen was also produced at high rates by the biocathode, reaching 100 mM d(-1) (0.2 g L(-1) d(-1)) and a total accumulation of 1164 mM (2.4 g L(-1)) over 20 days. Phylogenetic analysis of the active electrosynthetic microbiome revealed a similar community structure to what was observed during an earlier stage of development of the electroacetogenic microbiome. Acetobacterium spp. dominated the active microbial population on the cathodes. Also prevalent were Sulfurospirillum spp. and an unclassified Rhodobacteraceae. Taken together, these results demonstrate the stability, resilience, and improved performance of electrosynthetic biocathodes following long-term operation. Furthermore, sustained product formation at faster rates by a carbon-capturing microbiome is a key milestone addressed in this study that advances microbial electrosynthesis systems toward commercialization.

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September 22, 2019  |  

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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September 22, 2019  |  

Long-term changes of bacterial and viral compositions in the intestine of a recovered Clostridium difficile patient after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infections (RCDIs). However, long-term effects on the patients’ gut microbiota and the role of viruses remain to be elucidated. Here, we characterized bacterial and viral microbiota in the feces of a cured RCDI patient at various time points until 4.5 yr post-FMT compared with the stool donor. Feces were subjected to DNA sequencing to characterize bacteria and double-stranded DNA (dsDNA) viruses including phages. The patient’s microbial communities varied over time and showed little overall similarity to the donor until 7 mo post-FMT, indicating ongoing gut microbiota adaption in this time period. After 4.5 yr, the patient’s bacteria attained donor-like compositions at phylum, class, and order levels with similar bacterial diversity. Differences in the bacterial communities between donor and patient after 4.5 yr were seen at lower taxonomic levels. C. difficile remained undetectable throughout the entire timespan. This demonstrated sustainable donor feces engraftment and verified long-term therapeutic success of FMT on the molecular level. Full engraftment apparently required longer than previously acknowledged, suggesting the implementation of year-long patient follow-up periods into clinical practice. The identified dsDNA viruses were mainly Caudovirales phages. Unexpectedly, sequences related to giant algae–infecting Chlorella viruses were also detected. Our findings indicate that intestinal viruses may be implicated in the establishment of gut microbiota. Therefore, virome analyses should be included in gut microbiota studies to determine the roles of phages and other viruses—such as Chlorella viruses—in human health and disease, particularly during RCDI.

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