Buccal Swabbing as a Noninvasive Method To Determine Bacterial,Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants.     

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius)

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.     

Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis)

A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

Rumen Bacterial Diversity of 80 to 110-Day-Old Goats Using 16S rRNA Sequencing

The ability of rumen microorganisms to use fibrous plant matter plays an important role in ruminant animals; however, little information about rumen colonization by microbial populations after weaning has been reported. In this study, high-throughput sequencing was used to investigate the establishment of this microbial population in 80 to 110-day-old goats. Illumina sequencing of goat rumen samples yielded 101,356,610 nucleotides that were assembled into 256,868 reads with an average read length of 394 nucleotides. Taxonomic analysis of metagenomic reads indicated that the predominant phyla were distinct at different growth stages. The phyla Firmicutes and Synergistetes were predominant in samples taken from 80 to 100-day-old goats, but Bacteroidetes and Firmicutes became the most abundant phyla in samples from 110-day-old animals. There was a remarkable variation in the microbial populations with age; Firmicutes and Synergistetes decreased after weaning, but Bacteroidetes and Proteobacteria increased from 80 to 110 day of age. These findings suggested that colonization of the rumen by microorganisms is related to their function in the rumen digestive system. These results give a better understanding of the role of rumen microbes and the establishment of the microbial population, which help to maintain the host’s health and improve animal performance.     

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model

Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.     

Response of rumen microbiota,and metabolic profiles of rumen fluid,liver and serum of goats to high-grain diets

Feeding ruminants a high-grain (HG) diet is a widely used strategy to improve milk yield and cost efficiency. However, it may cause certain metabolic disorders. At present, information about the effects of HG diets on the systemic metabolic profile of goats and the correlation of such diets with rumen bacteria is limited. In the present study, goats were randomly divided into two groups: one was fed the hay diet (hay; n = 5), while the other was fed HG diets (HG; n = 5). On day 50, samples of rumen contents, peripheral blood serum and liver tissues were collected to determine the metabolic profiles in the rumen fluid, liver and serum and the microbial composition in rumen. The results revealed that HG diets reduced (P < 0.05) the community richness and diversity of rumen microbiota, with an increase in the Chao 1 and Shannon index and a decrease in the Simpson index. HG diets also altered the composition of rumen microbiota, with 30 genera affected (P < 0.05). Data on the metabolome showed that the metabolites in the rumen fluid, liver and serum were affected (variable importance projection > 1, P <0.05) by dietary treatment, with 47, 10 and 27 metabolites identified as differentially metabolites. Pathway analysis showed that the common metabolites in the shared key pathway (aminoacyl-transfer RNA biosynthesis) in the rumen fluid, liver and serum were glycine, lysine and valine. These findings suggested that HG diets changed the composition of the rumen microbiota and metabolites in the rumen fluid, liver and serum, mainly involved in amino acid metabolism. Our findings provide new insights into the understanding of diet-related systemic metabolism and the effects of HG diets on the overall health of goats.     

MetGEMs Toolbox: Metagenome-scale models as integrative toolbox for uncovering metabolic functions and routes of human gut microbiome

Investigating metabolic functional capability of a human gut microbiome enables the quantification of microbiome changes, which can cause a phenotypic change of host physiology and disease. One possible way to estimate the functional capability of a microbial community is through inferring metagenomic content from 16S rRNA gene sequences. Genome-scale models (GEMs) can be used as scaffold for functional estimation analysis at a systematic level, however up to date, there is no integrative toolbox based on GEMs for uncovering metabolic functions. Here, we developed the MetGEMs (metagenome-scale models) toolbox, an open-source application for inferring metabolic functions from 16S rRNA gene sequences to facilitate the study of the human gut microbiome by the wider scientific community. The developed toolbox was validated using shotgun metagenomic data and shown to be superior in predicting functional composition in human clinical samples compared to existing state-of-the-art tools. Therefore, the MetGEMs toolbox was subsequently applied for annotating putative enzyme functions and metabolic routes related in human disease using atopic dermatitis as a case study.     

High-grain diet feeding altered the composition and functions of the rumen bacterial community and caused the damage to the laminar tissues of goats

In the current intensive production system, ruminants are often fed high-grain (HG) diets. However, this feeding pattern often causes rumen metabolic disorders and may further trigger laminitis, the exact mechanism is not clear. This study investigated the effect of HG diet feeding on fermentative and microbial changes in the rumen and on the expression of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in the lamellar tissue. In all, 12 male goats were fed a hay diet (0% grain; n=6) or an HG diet (56.5% grain; n=6). On day 50 of treatment, samples of blood, rumen content, and lamellar tissue of hooves of goats were collected. The data showed that compared with the hay group, HG-fed goats had lower (P<0.05) rumen pH but higher (P<0.05) total volatile fatty acids and lactate in the rumen and higher (P<0.05) lipopolysaccharide (LPS) levels in the rumen and blood. HG diet feeding altered the composition of rumen bacterial community, and correspondingly, the results suggested that their functions in the HG group were also altered. HG diet feeding increased (P<0.05) the expression of interleukin-1β, interleukin-6, tumour necrosis factor-α and MMP-2 mRNA in the lamellar tissues compared with the hay group. Correlation analysis indicated that the expression of pro-inflammatory cytokines were positively correlated with MMP-2 expression in lamellar tissues. Overall, these results revealed that HG feeding altered the patterns of rumen fermentation and the composition and functions of rumen bacterial community, and lead to higher levels of LPS in the peripheral blood, and further activated the inflammatory response in lamellar tissues, which may progress to the level of laminar damage.     

Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach

The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using “universal” primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

Isolation of a Gene Encoding a Cellulolytic Enzyme from Swamp Buffalo Rumen Metagenomes and Its Cloning and Expression in Escherichia Coli

Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4–7 and more than 50% maximal activity after incubation at 30–60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.     

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P?相似文献   

Metagenomic analysis of kimchi, a traditional Korean fermented food

Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.     

Invited review: Application of meta-omics to understand the dynamic nature of the rumen microbiome and how it responds to diet in ruminants

Ruminants are unique among livestock due to their ability to efficiently convert plant cell wall carbohydrates into meat and milk. This ability is a result of the evolution of an essential symbiotic association with a complex microbial community in the rumen that includes vast numbers of bacteria, methanogenic archaea, anaerobic fungi and protozoa. These microbes produce a diverse array of enzymes that convert ingested feedstuffs into volatile fatty acids and microbial protein which are used by the animal for growth. Recent advances in high-throughput sequencing and bioinformatic analyses have helped to reveal how the composition of the rumen microbiome varies significantly during the development of the ruminant host, and with changes in diet. These sequencing efforts are also beginning to explain how shifts in the microbiome affect feed efficiency. In this review, we provide an overview of how meta-omics technologies have been applied to understanding the rumen microbiome, and the impact that diet has on the rumen microbial community.     

New insights on diet variability revealed by DNA barcoding and high-throughput pyrosequencing: chamois diet in autumn as a case study

Characterizing the diet of large herbivores and the determinants of its variation remains a difficult task in wild species. DNA-based techniques have the potential to complement traditional time-consuming methods based on the microhistology of plant cuticle fragments in fecal or rumen samples. Recently, it has been shown that a short chloroplast DNA fragment, the P6 loop of the trnL (UAA) intron, can act as a minimalist barcode. Here, we used the trnL approach with high-throughput pyrosequencing to study diet from feces in a wild herbivore, the alpine chamois (Rupicapra rupicapra) and showed that the fine resolution in plant determination obtained with this method allows exploring subtle temporal shifts and inter-individual variability in diet composition. First, we built a DNA barcoding database of 475 plants species. Seventy-two percent of plant species can be unambiguously identified to species level, 79% to genus level and 100% to family level using the P6 loop. Second, we analysed 74 feces collected from October to November. Based on 47,896 P6 loop sequences, we identified a total of 110 taxa, 96 in October and 76 in November, with a clear diet shift between October and November. We recognized four and two clusters of feces composition in October and November, respectively, revealing different diet categories among individuals within each month. DNA-based diet analysis is faster and more taxonomically precise than studies based on microhistology, and opens new possibilities for analysing plant-herbivore interactions in the wild.     

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (−) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.     

Potential of fecal microbiota for early-stage detection of colorectal cancer

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host–microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.     

Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena)

ABSTRACT: BACKGROUND: Animals co-evolve with their gut microbiota; the latter can perform complex metabolic reactions that cannot be done independently by the host. Although the importance of gut microbiota has been well demonstrated, there is a paucity of research regarding its role in foliage-foraging mammals with a specialized digestive system. RESULTS: In this study, a 16S rRNA gene survey and metagenomic sequencing were used to characterize genetic diversity and functional capability of cecal microbiota of the folivorous flying squirrel (Petaurista alborufus lena). Phylogenetic compositions of the cecal microbiota derived from 3 flying squirrels were dominated by Firmicutes. Based on end-sequences of fosmid clones from 1 flying squirrel, we inferred that microbial metabolism greatly contributed to intestinal functions, including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins. Moreover, 33 polysaccharide-degrading enzymes and 2 large genomic fragments containing a series of carbohydrate-associated genes were identified. CONCLUSIONS: Cecal microbiota of the leaf-eating flying squirrel have great metabolic potential for converting diverse plant materials into absorbable nutrients. The present study should serve as the basis for future investigations, using metagenomic approaches to elucidate the intricate mechanisms and interactions between host and gut microbiota of the flying squirrel digestive system, as well as other mammals with similar adaptations.     

The microbiome of Brazilian mangrove sediments as revealed by metagenomics

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2)S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.