scholarly journals Towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods

2021 ◽  
Vol 3 (2) ◽  
pp. 231-242
Author(s):  
Haining Hu ◽  
Vengadesh Perumal Natarajan ◽  
Fengping Wang
Keyword(s):  
Marine Sediments ◽  
Bacterial Species ◽  
Molecular Ecology ◽  
Extraction Procedure ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Cell Extraction ◽  
Highly Active ◽  
Microbial Cultivation ◽  
Cultivation Methods

AbstractThe archaea that can be readily cultivated in the laboratory are only a small fraction of the total diversity that exists in nature. Although molecular ecology methods, such as metagenomic sequencing, can provide valuable information independent of cell cultivation, it is only through cultivation-based experiments that they may be fully characterized, both for their physiological and ecological properties. Here, we report our efforts towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods. Initially, cells were retrieved from the sediment samples through a cell extraction procedure and the sediment-free mixed cells were then divided into different size-range fractions by successive filtration through 0.8 µm, 0.6 µm and 0.2 µm membranes. Archaeal 16S rRNA gene analyses indicated noticeable retention of different archaeal groups in different fractions. For each fraction, supplementation with a variety of defined substrates (e.g., methane, sulfate, and lignin) and stepwise dilutions led to highly active enrichment cultures of several archaeal groups with Bathyarchaeota most prominently enriched. Finally, using a roll-bottle technique, three co-cultures consisting of Bathyarchaeota (subgroup-8) and a bacterial species affiliated with either Pseudomonas or Glutamicibacter were obtained. Our results demonstrate that a combination of cell extraction, size fractionation, and roll-bottle isolation methods could be a useful protocol for the successful enrichment and isolation of numerous slow-growing archaeal groups from marine sediments.

scholarly journals Differential carbohydrate utilization and organic acid production by honey bee symbionts

2018 ◽  
Author(s):  
Fredrick J. Lee ◽  
Kayla I. Miller ◽  
James B. McKinlay ◽  
Irene L. G. Newton
Keyword(s):  
Weight Gain ◽  
Organic Acids ◽  
Honey Bee ◽  
Bacterial Species ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Carbohydrate Utilization ◽  
Organic Acid Production ◽  
Highly Active

AbstractThe honey bee worker gut is host to a community of bacteria that primarily comprises 8-10 bacterial species. Collectively, these microbes break down and ferment saccharides present in the host’s diet. The model of metabolism for these gut symbionts is rooted in previous analyses of genomes, metagenomes, and metatranscriptomes of this environment. Importantly, there is a correlation between the composition of the gut microbiome and weight gain in the honey bee, suggesting that bacterial production of organic acids might contribute to the observed phenomenon. Here we identify potential metabolic contributions of symbionts within the honey bee gut. We show significant variation in the metabolic capabilities of these microbes, highlighting the fact that although the microbiota appears simple and consistent based on 16S rRNA gene profiling, strains are highly variable in their ability to use specific carbohydrates and produce organic acids. Finally, we confirm that the honey bee core microbes, especially a clade of γ-proteobacteria (i.e.Gilliamella), are highly activein vivo, expressing key enzymatic genes critical for utilizing plant-derived molecules and producing organic acids. These results suggest thatGilliamella, and other core taxa, may contribute significantly to weight gain in the honey bee, specifically through the production of organic acids.

scholarly journals Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment

2020 ◽  
Vol 58 (12) ◽  
Author(s):  
Sara Rassoulian Barrett ◽  
Noah G. Hoffman ◽  
Christopher Rosenthal ◽  
Andrew Bryan ◽  
Desiree A. Marshall ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nucleic Acids ◽  
Bacterial Species ◽  
False Negative ◽  
Case Series ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Bacterial Dna ◽  
Clinical Specimens

ABSTRACT The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture (“16S Capture”). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.

scholarly journals Deep Investigating the Changes of Gut Microbiome and Its Correlation With the Shifts of Host Serum Metabolome Around Parturition in Sows

2021 ◽  
Vol 12 ◽  
Author(s):  
Hao Fu ◽  
Maozhang He ◽  
Jinyuan Wu ◽  
Yunyan Zhou ◽  
Shanlin Ke ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Gut Microbiome ◽  
Bacterial Species ◽  
Late Pregnancy ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Functional Capacities

Parturition is a crucial event in the sow reproduction cycle, which accompanies by a series of physiological changes, including sex hormones, metabolism, and immunity. More and more studies have indicated the changes of the gut microbiota from pregnancy to parturition. However, what bacterial species and functional capacities of the gut microbiome are changed around parturition has been largely unknown, and the correlations between the changes of gut bacterial species and host metabolome were also uncovered. In this study, by combining 16S rRNA gene and shotgun metagenomic sequencing data, and the profiles of serum metabolome and fecal short-chain fatty acids (SCFAs), we investigated the changes of gut microbiome, serum metabolite features and fecal SCFAs from late pregnancy (LP) to postpartum (PO) stage. We found the significant changes of gut microbiota from LP to PO stage in both 16S rRNA gene sequencing and metagenomic sequencing analyses. The bacterial species from Lactobacillus, Streptococcus, and Clostridium were enriched at the LP stage, while the species from Bacteroides, Escherichia, and Campylobacter had higher abundances at the PO stage. Functional capacities of the gut microbiome were also significantly changed and associated with the shifts of gut bacteria. Untargeted metabolomic analyses revealed that the metabolite features related to taurine and hypotaurine metabolism, and arginine biosynthesis and metabolism were enriched at the LP stage, and positively associated with those bacterial species enriched at the LP stage, while the metabolite features associated with vitamin B6 and glycerophospholipid metabolism had higher abundances at the PO stage and were positively correlated with the bacteria enriched at the PO stage. Six kinds of SCFAs were measured in feces samples and showed higher concentrations at the LP stage. These results suggested that the changes of gut microbiome from LP to PO stage lead to the shifts of host lipid, amino acids and vitamin metabolism and SCFA production. The results from this study provided new insights for the changes of sow gut microbiome and host metabolism around parturition, and gave new knowledge for guiding the feeding and maternal care of sows from late pregnancy to lactation in the pig industry.

scholarly journals The Bovine Foot Skin Microbiota is Associated with Host Genotype and the Development of Infectious Digital Dermatitis Lesions

2021 ◽  
Author(s):  
V. Bay ◽  
A. Gillespie ◽  
E.K. Ganda ◽  
Nicholas Evans ◽  
Stuart Carter ◽  
...  
Keyword(s):  
Bovine Genome ◽  
Bacterial Species ◽  
Significant Proportion ◽  
Subsequent Development ◽  
Rrna Gene ◽  
Skin Microbiome ◽  
Metagenomic Sequencing ◽  
Skin Microbiota ◽  
Host Genotype ◽  
Digital Dermatitis

Abstract Bovine Digital Dermatitis (BDD) is a prevalent infectious disease, causing painful foot skin lesions and lameness in cattle. The polymicrobial nature of this disease has led to the hypothesis that the foot skin microbiota may be associated with occurrence and progression of lesions. We describe herein the bovine foot skin microbiota using 16S rRNA gene amplicon and shotgun metagenomic sequencing on samples from 259 dairy cows from three UK dairy farms. We show differences in the foot skin microbiome profiles of clinically healthy animals that were associated with subsequent development of BDD. We also present the first co-occurrence analysis of the bovine foot skin microbiome showing ecological relationships among bacterial species. Taxonomical and functional differences together with alterations in ecological interactions between bacteria in the normal foot skin microbiome may predispose an animal to develop BDD lesions. Using genome-wide association and regional heritability mapping approaches, we provide first evidence for interactions between host genotype and the foot skin microbiota profiles. We show the existence of genetic variation in the relative abundance of Treponema spp. and Peptoclostridium spp. and identify regions in the bovine genome that explain a significant proportion of this variation.

DIVERSITY OF CULTURABLE BACTERIAL MICROBIOTA OF THE Eisenia foetida DIGESTIVE TRACT

2018 ◽  
Vol 41 (3) ◽  
pp. 255-264 ◽  
Author(s):  
J. Abraham Pérez-Pérez ◽  
David Espinosa-Victoria ◽  
Hilda V. Silva-Rojas ◽  
Lucía López-Reyes
Keyword(s):  
Bacterial Diversity ◽  
Digestive Tract ◽  
Bacterial Species ◽  
Brain Heart Infusion ◽  
Rrna Gene ◽  
Eisenia Foetida ◽  
Bacterial Isolates ◽  
Bacterial Microbiota ◽  
Decomposition Of Organic Matter ◽  
Earthworm Gut

Bacteria are an unavoidable component of the natural earthworm diet; thus, bacterial diversity in the earthworm gut is directly linked to decomposition of organic matter and development of the surrounding plants. The aim of this research was to isolate and to identify biochemically and molecularly the culturable bacterial microbiota of the digestive tract of Eisenia foetida. Earthworms were sourced from Instituto de Reconversión Productiva y Bioenergética (IRBIO) and Colegio de Postgraduados (COLPOS), México. Bacterial isolation was carried out on plates of Brain Heart Infusion (BHI) culture medium. Fifty six and 44 bacterial isolates were obtained from IRBIO and COLPOS, respectively. The population was composed of 44 Gram-negative and 56 Gram-positive isolates. Over 50 % of the bacterial isolates were rod-shaped cells. The 16S rRNA gene was sequenced and nine genera were identified in worms from IRBIO (Bacillus, Paenibacillus, Solibacillus, Staphylococcus, Arthrobacter, Pantoea, Stenotrophomonas, Acinetobacter and Aeromonas) and six in worms from COLPOS (Bacillus, Paenibacillus, Stenotrophomonas, Staphylococcus, Acinetobacter and Aeromonas). Bacillus was the predominant genus, with eight and six species in the oligochaetes from IRBIO and COLPOS, respectively. The most represented bacteria in the worms from both sites were Bacillus sp. and B. subtilis. The predominance of Bacillus was probably due to spore formation, a reproductive strategy that ensures survival and dispersion in the soil and oligochaetes digestive tract. The gut of E. foetida not only harbored bacterial species of agronomic importance but also species potentially pathogenic for humans (Staphylococcus warneri, Pantoea agglomerans and Stentrophomonas sp.). The larger bacterial diversity in worms from IRBIO could be due to their feeding on cattle manure, which is a rich source of bacteria.

scholarly journals Sequential extraction procedure to obtain the composition of terrigenous detritus in marine sediments

MethodsX ◽  
2020 ◽  
Vol 7 ◽  
pp. 100888
Author(s):  
Margit H. Simon ◽  
Daniel P. Babin ◽  
Steven L. Goldstein ◽  
Merry Yue Cai ◽  
Tanzhuo Liu ◽  
...  
Keyword(s):  
Sequential Extraction ◽  
Marine Sediments ◽  
Extraction Procedure ◽  
Sequential Extraction Procedure

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 Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 396
Author(s):  
Ewa Sajnaga ◽  
Marcin Skowronek ◽  
Agnieszka Kalwasińska ◽  
Waldemar Kazimierczak ◽  
Karolina Ferenc ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Entomopathogenic Nematodes ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Bacterial Phyla ◽  
Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

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