Microbial communities of poultry house dust, excreta and litter are partially representative of microbiota of chicken caecum and ileum

Traditional sampling methods for the study of poultry gut microbiota preclude longitudinal studies as they require euthanasia of birds for the collection of caecal and ileal contents. Some recent research has investigated alternative sampling methods to overcome this issue. The main goal of this study was to assess to what extent the microbial composition of non-invasive samples (excreta, litter and poultry dust) are representative of invasive samples (caecal and ileal contents). The microbiota of excreta, dust, litter, caecal and ileal contents (n = 110) was assessed using 16S ribosomal RNA gene amplicon sequencing. Of the operational taxonomic units (OTUs) detected in caecal contents, 99.7% were also detected in dust, 98.6% in litter and 100% in excreta. Of the OTUs detected in ileal contents, 99.8% were detected in dust, 99.3% in litter and 95.3% in excreta. Although the majority of the OTUs found in invasive samples were detected in non-invasive samples, the relative abundance of members of the microbial communities of these groups were different, as shown by beta diversity measures. Under the conditions of this study, correlation analysis showed that dust could be used as a proxy for ileal and caecal contents to detect the abundance of the phylum Firmicutes, and excreta as a proxy of caecal contents for the detection of Tenericutes. Similarly, litter could be used as a proxy for caecal contents to detect the abundance of Firmicutes and Tenericutes. However, none of the non-invasive samples could be used to infer the overall abundance of OTUs observed in invasive samples. In conclusion, non-invasive samples could be used to detect the presence and absence of the majority of the OTUs found in invasive samples, but could not accurately reflect the microbial community structure of invasive samples.

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Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽

10.3390/fuels2020014 ◽

2021 ◽

Vol 2 (2) ◽

pp. 241-252

Author(s):

Dyah Asri Handayani Taroepratjeka ◽

Tsuyoshi Imai ◽

Prapaipid Chairattanamanokorn ◽

Alissara Reungsang

Keyword(s):

Microbial Communities ◽

High Throughput ◽

High Throughput Sequencing ◽

High Salinity ◽

Amplicon Sequencing ◽

Spatial Proximity ◽

Lignocellulosic Waste ◽

Evaporation Pond ◽

Operational Taxonomic Units ◽

Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

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Molecular Approaches to Studying Microbial Communities: Targeting the 16S Ribosomal RNA Gene

Journal of UOEH ◽

10.7888/juoeh.38.223 ◽

2016 ◽

Vol 38 (3) ◽

pp. 223-232 ◽

Cited By ~ 14

Author(s):

Kazumasa FUKUDA ◽

Midori OGAWA ◽

Hatsumi TANIGUCHI ◽

Mitsumasa SAITO

Keyword(s):

Microbial Communities ◽

Ribosomal Rna ◽

16S Ribosomal Rna ◽

Approaches To Studying ◽

Ribosomal Rna Gene ◽

Molecular Approaches ◽

16S Ribosomal Rna Gene

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Molecular profiling of bacterial species in the geese cecum

Czech Journal of Animal Science ◽

10.17221/1433-cjas ◽

2011 ◽

Vol 56 (No. 4) ◽

pp. 192-203 ◽

Cited By ~ 7

Author(s):

B.Y. Liu ◽

Z.Y. Wang ◽

H.R. Wang ◽

P. Hu ◽

D. Xu ◽

...

Keyword(s):

Microbial Diversity ◽

Clone Library ◽

Ribosomal Rna ◽

Related Species ◽

Bacterial Species ◽

Similarity Criterion ◽

Molecular Profiling ◽

Operational Taxonomic Units ◽

Rich Variety ◽

16S Ribosomal Rna Gene

The purpose of this study was to analyse the microbial diversity in the caecum of geese using a 16S ribosomal RNA gene (rRNA) clone library approach. A total of 160 clones and 124 clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a 98% similarity criterion were classified in the contents of goose caecum, as compared to 29 OTUs based on a 97% similarity criterion in the mucosa of goose caecum. The sequences were assigned to 7 and 5 groups in the contents and mucosa of goose caecum, respectively. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum.

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Cloacal swabs and alcohol bird specimens are good proxies for compositional analyses of gut microbial communities of Great tits (Parus major)

10.21203/rs.2.19814/v2 ◽

2020 ◽

Author(s):

Kasun H Bodawatta ◽

Katerina Puzejova ◽

Katerina Sam ◽

Michael Poulsen ◽

Knud A. Jønsson

Keyword(s):

Microbial Communities ◽

Digestive Tract ◽

Wild Bird ◽

Anthropogenic Impacts ◽

Parus Major ◽

Amplicon Sequencing ◽

Cloacal Swab ◽

Non Invasive ◽

16S Rrna Amplicon Sequencing ◽

Microbiome Research

Abstract Background Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for two weeks or two months, respectively. Results We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these community-level differences, swab samples qualitatively captured the majority of the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens. Conclusions Our findings suggest that cloacal swabs, similar to non-invasive fecal sampling, qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. Secondly, the satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.

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In Silico and Experimental Evaluation of Primer Sets for Species-Level Resolution of the Vaginal Microbiota Using 16S Ribosomal RNA Gene Sequencing

The Journal of Infectious Diseases ◽

10.1093/infdis/jiy508 ◽

2018 ◽

Vol 219 (2) ◽

pp. 305-314 ◽

Cited By ~ 10

Author(s):

William J Van Der Pol ◽

Ranjit Kumar ◽

Casey D Morrow ◽

Eugene E Blanchard ◽

Christopher M Taylor ◽

...

Keyword(s):

Ribosomal Rna ◽

Experimental Evaluation ◽

In Silico ◽

Gene Sequencing ◽

Species Level ◽

Ribosomal Rna Gene ◽

Operational Taxonomic Units ◽

16S Ribosomal Rna Gene ◽

Primer Sets ◽

Level Identification

V4 sequence reads clustered at 99% identity and assigned to operational taxonomic units using the 99% clustered, extended Greengenes database provided optimal species-level identification of vaginal bacteria. This method provided results similar to those obtained with DADA2 and/or using the SILVA database.

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Gut Microbial Composition and Predicted Functions Are Not Associated with Feather Pecking and Antagonistic Behavior in Laying Hens

Life ◽

10.3390/life11030235 ◽

2021 ◽

Vol 11 (3) ◽

pp. 235

Author(s):

Daniel Borda-Molina ◽

Hanna Iffland ◽

Markus Schmid ◽

Regina Müller ◽

Svenja Schad ◽

...

Keyword(s):

Microbial Communities ◽

Linear Models ◽

Amplicon Sequencing ◽

Mixed Linear Model ◽

Economic Losses ◽

Microbial Composition ◽

Feather Pecking ◽

Behavioral Traits ◽

Genus Lactobacillus ◽

Lysine Degradation

Background: Feather pecking is a well-known problem in layer flocks that causes animal welfare restrictions and contributes to economic losses. Birds’ gut microbiota has been linked to feather pecking. This study aims to characterize the microbial communities of two laying hen lines divergently selected for high (HFP) and low (LFP) feather pecking and investigates if the microbiota is associated with feather pecking or agonistic behavior. Methods: Besides phenotyping for the behavioral traits, microbial communities from the digesta and mucosa of the ileum and caeca were investigated using target amplicon sequencing and functional predictions. Microbiability was estimated with a microbial mixed linear model. Results: Ileum digesta showed an increase in the abundance of the genus Lactobacillus in LFP, while Escherichia was abundant in HFP hens. In the caeca digesta and mucosa of the LFP line were more abundant Faecalibacterium and Blautia. Tryptophan metabolism and lysine degradation were higher in both digesta and mucosa of the HFP hens. Linear models revealed that the two lines differ significantly in all behavior traits. Microbiabilities were close to zero and not significant in both lines and for all traits. Conclusions: Trait variation was not affected by the gut microbial composition in both selection lines.

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PSII-16 Evidence for stratification of rumen wall microbial communities revealed by 16S rRNA based amplicon sequencing

Journal of Animal Science ◽

10.1093/jas/skz122.398 ◽

2019 ◽

Vol 97 (Supplement_2) ◽

pp. 226-227

Author(s):

Lucas Koester ◽

Mark Lyte ◽

Stephan Schmitz-Esser ◽

Heather Allen

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

Temporal Stability ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Rumen Content ◽

Rrna Gene ◽

Operational Taxonomic Units ◽

Abundant Otus ◽

Dorsal Portion

Abstract Rumen content (RC) stratifies based on particle size and density consisting of the less dense forage within the dorsal and the denser particles in the ventral portions of the rumen and is in constant contact with the microbial communities present on the rumen wall (RW) epithelium. Little is known about the nutrient requirements and functional processes of RW microbial communities. Our hypothesis is that the RW microbial communities stratify mirroring the stratification of RC due to different available nutrients. Five fistulated, milking Holstein cows of the same management conditions were sampled at four rumen layers corresponding to the RC stratification. Epithelial biopsies were taken through the fistula; the uppermost aligned with the dorsal portion of the RC (A), and three other sites, each 10 cm ventral to the previous (B, C and D). Each cow and stratification layer was sampled five times over four months to analyze temporal stability of the RW microbial communities. DNA was extracted using the Qiagen Powerlyzer Powersoil kit and used for 16S rRNA gene Illumina MiSeq sequencing. Sequences were clustered into operational taxonomic units (OTU) based on a 99% similarity cutoff using MOTHUR. After quality control, 2.0 million reads remained for 90 samples which were clustered into 5,016 OTUs with 10 or more reads. 99.2% of the reads were bacterial, whereas 0.8% affiliated to Archaea. Statistical analysis revealed that among the 20 most abundant OTUs, phylotypes classified as Desulfobulbus, unclassified_Cardiobacteraceae, Mogibacterium, Lachnospiraceae-UCG008 and Methanobrevibacter were significantly different in abundance between sites A compared to D. On a whole community level, analysis of molecular variance (AMOVA) revealed significant differences between groups A, C and D. Our data reveal first evidence that a stratification of RW microbiota is present in dairy cattle and also reveal high temporal stability of RW microbiota.

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Nasopharyngeal Microbiota in SARS-CoV-2 Positive and Negative Patients

Biological Procedures Online ◽

10.1186/s12575-021-00148-6 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Phillip A. Engen ◽

Ankur Naqib ◽

Cheryl Jennings ◽

Stefan J. Green ◽

Alan Landay ◽

...

Keyword(s):

Community Structure ◽

Microbial Community ◽

Disease Progression ◽

High Throughput ◽

Ribosomal Rna ◽

Microbial Community Structure ◽

Amplicon Sequencing ◽

Proof Of Concept ◽

Viral Transport ◽

16S Ribosomal Rna Gene

AbstractWe investigated nasopharyngeal microbial community structure in COVID-19-positive and -negative patients. High-throughput 16S ribosomal RNA gene amplicon sequencing revealed significant microbial community structure differences between COVID-19-positive and -negative patients. This proof-of-concept study demonstrates that: (1) nasopharyngeal microbiome communities can be assessed using collection samples already collected for SARS-CoV-2 testing (viral transport media) and (2) SARS-CoV-2 infection is associated with altered dysbiotic microbial profiles which could be a biomarker for disease progression and prognosis in SARS-CoV-2.

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A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis

10.1101/2021.03.01.433496 ◽

2021 ◽

Author(s):

Fangchao Song ◽

Jennifer V. Kuehl ◽

Arjun Chandran ◽

Adam P. Arkin

Keyword(s):

Microbial Communities ◽

Rna Sequencing ◽

Ribosomal Rna ◽

Cost Effective ◽

Community Analysis ◽

Extraction Methods ◽

16S Ribosomal Rna ◽

Direct Pcr ◽

Microbial Composition ◽

Pcr Method

ABSTRACTBacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16s ribosomal RNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial amount of samples becomes necessary to analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which can be biased on the types of species, time-consuming and expensive. Direct PCR methods are a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, ZymoBIOMICS Microbial Community Standards, and groundwater. By comparing direct PCR methods with DNeasy blood and tissue kits and DNeasy Powersoil kits, we found a specific variant of the direct PCR method exhibits a comparable overall accuracy to the conventional DNeasy Powersoil protocol. We also found the method showed higher efficiency for extracting gDNA from the gram negative strains compared to DNeasy blood and tissue protocol. This direct PCR method is 1600 times cheaper ($0.34 for 96 samples), 10 times simpler (15 min hands-on time for 96 samples) than DNeasy Powersoil protocol. The direct PCR method can also be fully automated, and is compatible with small volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.IMPORTANCEUnderstanding bacterial interaction and assembling in complex microbial communities using 16s ribosomal RNA sequencing normally requires a large experimental load. However, the current DNA extraction methods including cell disruption and genome DNA purification are normally biased, costly, time and labor consuming, and not amenable to miniaturization by droplets or 1536 well plates due to the significant DNA loss during purification step for tiny volume and low cell density samples. Direct PCR method could potentially solve these problems. In this study, we demonstrate a direct PCR method which exhibits similar accuracy as the widely used method – DNeasy Powersoil protocol, while 1600 times cheaper and 10 times faster to execute. This simple, cost-effective, and automation friendly direct PCR based 16s ribosomal RNA sequencing method allows us to study the dynamics, microbial interaction and assembly of varying microbial communities in a high throughput fashion.

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The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

PeerJ ◽

10.7717/peerj.7608 ◽

2019 ◽

Vol 7 ◽

pp. e7608

Author(s):

Adam Šťovíček ◽

Smadar Cohen-Chalamish ◽

Osnat Gillor

Keyword(s):

Microbial Communities ◽

High Throughput ◽

Ribosomal Rna ◽

Reverse Transcription ◽

High Throughput Sequencing ◽

Dna Analysis ◽

Amplicon Sequencing ◽

Soil Microbiology ◽

Active Population ◽

Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to six fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing toward a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

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