Metagenomics Analysis to Investigate the Microbial Communities and Their Functional Profile During Cyanobacterial Blooms in Lake Varese

Toxic cyanobacterial blooms represent a natural phenomenon caused by a mass proliferation of photosynthetic prokaryotic microorganisms in water environments. Bloom events have been increasingly reported worldwide and their occurrence can pose serious threats to aquatic organisms and human health. In this study, we assessed the microbial composition, with a focus on Cyanobacteria, in Lake Varese, a eutrophic lake located in northern Italy. Water samples were collected and used for obtaining a 16S-based taxonomic profile and performing a shotgun sequencing analysis. The phyla found to exhibit the greatest relative abundance in the lake included Proteobacteria, Cyanobacteria, Actinobacteriota and Bacteroidota. In the epilimnion and at 2.5 × Secchi depth, Cyanobacteria were found to be more abundant compared to the low levels detected at greater depths. The blooms appear to be dominated mainly by the species Lyngbya robusta, and a specific functional profile was identified, suggesting that distinct metabolic processes characterized the bacterial population along the water column. Finally, analysis of the shotgun data also indicated the presence of a large and diverse phage population.

RESEARCH OF THE MICROBIOM OF THE GASTROINTESTINAL TRACT OF THE ABERDIN-ANGUS BREED CATTLE

Целью исследования было определение таксономической структуры микробиома кишечника крупного рогатого скота породы Абердин-Ангус с помощью технологии секвенирования нового поколения. 16S метагеномный анализ, позволил определить микробный состав содержимого кишечника, минуя стадию культивирования на питательных средах. Проведена генетическая идентификация и получен таксономический профиль всех присутствующих бактерий, в том числе и некультивируемых форм. The aim of the study was to determine the taxonomic structure of the intestinal microbiome of Aberdeen Angus cattle using a new generation sequencing technology. 16S metagenomic analysis made it possible to determine the microbial composition of the intestinal contents bypassing the stage of cultivation on nutrient media. Genetic identification was carried out and a taxonomic profile of all bacteria present, including non-cultivated forms, was obtained. Key words: microbiome, cattle, Aberdeen Angus, next generation sequencing.

Bifidobacterium animalis subsp. lactis BB-12 Protects against Antibiotic-Induced Functional and Compositional Changes in Human Fecal Microbiome

The administration of broad-spectrum antibiotics is often associated with antibiotic-associated diarrhea (AAD), and impacts gastrointestinal tract homeostasis, as evidenced by the following: (a) an overall reduction in both the numbers and diversity of the gut microbiota, and (b) decreased short-chain fatty acid (SCFA) production. Evidence in humans that probiotics may enhance the recovery of microbiota populations after antibiotic treatment is equivocal, and few studies have addressed if probiotics improve the recovery of microbial metabolic function. Our aim was to determine if Bifidobacterium animalis subsp. lactis BB-12 (BB-12)-containing yogurt could protect against antibiotic-induced fecal SCFA and microbiota composition disruptions. We conducted a randomized, allocation-concealed, controlled trial of amoxicillin/clavulanate administration (days 1–7), in conjunction with either BB-12-containing or control yogurt (days 1–14). We measured the fecal levels of SCFAs and bacterial composition at baseline and days 7, 14, 21, and 30. Forty-two participants were randomly assigned to the BB-12 group, and 20 participants to the control group. Antibiotic treatment suppressed the fecal acetate levels in both the control and probiotic groups. Following the cessation of antibiotics, the fecal acetate levels in the probiotic group increased over the remainder of the study and returned to the baseline levels on day 30 (−1.6% baseline), whereas, in the control group, the acetate levels remained suppressed. Further, antibiotic treatment reduced the Shannon diversity of the gut microbiota, for all the study participants at day 7. The magnitude of this change was larger and more sustained in the control group compared to the probiotic group, which is consistent with the hypothesis that BB-12 enhanced microbiota recovery. There were no significant baseline clinical differences between the two groups. Concurrent administration of amoxicillin/clavulanate and BB-12 yogurt, to healthy subjects, was associated with a significantly smaller decrease in the fecal SCFA levels and a more stable taxonomic profile of the microbiota over time than the control group.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Ecological Risks of Heavy Metals and Microbiome Taxonomic Profile of a Freshwater Stream Receiving Wastewater of Textile Industry

Textile wastewater (TWW) contains toxic metals that are inimical to microbiome, aesthetic quality, and the health of the receiving freshwater. TWW-impacted freshwater (L2) was assessed for metals eco-toxicity and the consequent impact on microbiome taxonomic profile (MTP) compared to a pristine environment (L1). The conductivity (1750 μS/cm), chemical oxygen demand (2,110 mg/L), biochemical oxygen demand (850 mg/L), and salinity (5,250 mg/L) of L2 were far above the permissible limits. Mercury posed very high ecological risks in the water column of L2 as lead, arsenic, and copper exerted high risk in the sediment. The MTP of L2 revealed the dominance of Euryarchaeota (48.6%) and Bathyarchaeota (45.9%) among the Archaea. The relative abundances of Proteobacteria and Bacteroidetes increased from 38.3 to 2.0%, respectively, in the L1 ecosystem to 42.1 and 12.9%, correspondingly, in L2. Unclassified Eukarya_uc_p (50.4%) and Fungi_uc (16.0%) were key players among the fungi kingdom in L2. The impact of the TWW on the microbiome was evident with the extinction of 6,249, 32,272, and 10,029 species of archaea, bacteria, and fungi, respectively. Whereas, 35,157, 32,394, and 7,291 species of archaea, bacteria, and fungi, correspondingly, exclusively found in L2 were assumed to be invading resident communities that combined with dominant autochthonous strains in shaping the ecophysiology dynamics in TWW-impacted freshwater. While the sensitive microorganisms in L2 are suggested bio-indicators of TWW ecotoxicity, the emergent and dominant taxa are pivotal to natural attenuation processes in the contaminated ecosystem that could be adopted for biotechnological strategy in decommissioning the TWW-impacted freshwater.

Metagenomics analysis of marine eukaryotic community in water and sediments at Lang Co - Da Nang sea by throughput 18S rRNA gene sequencing

The present study applied metagenomics to characterize the diversity and relative occurrence of eukaryotic organisms in the sea water (LC05.W and LCDN.W) and sediment (LC05.S and LCDN.S) samples collected at the Lang Co - Da Nang sea in two years 2016 and 2017. The marine DNA metagenomes from water and sediments were isolated and analyzed by using specific primer 18S V4: 528F-706R with the barcode for gene-based metagenomic approach. Total tags were 374,336 (92,864 in LC05.W; 95,742 in LCDN.W; 86,593 in LC05.S and 91,385 in LCDN.S samples) and clustered at a 97% similarity into 5,204 unique operational taxonomic units (936 in LC05.W; 1631 in LCDN.W; 2,259 in LC05.S and 1,631 in LCDN.S). The taxonomic profile obtained by comparison with SILVA SSU database showed predominance of the kingdom: Eukaryote domain (61% in LC05.W; 32% in LCDN.W; 43% in LC05.S and 69% in LCDN.S); Metazoa (26% in LC05.W; 22% in LCDN.W; 37% in LC05.S and 19% in LCDN.S). Fungi in samples collected in 2017 (31% in LCDN.W and 10% in LCDN.S) were dominant as compared to 2016 (6.0% in LC05.W and 0.6% in LC05.S). In addition, 0.4% and 10.0% in water and 19% and 2% in sediment sequences were unclassified. Protalveolata, Annelida, Chlorophyta, Nematoda, Arthropoda, Rotifera, Ascomycota, Diatomea were top ten at the phylum level in Lang Co - Da Nang sea water and sediments. The abundance distribution of 35 dominant genera among all samples was displayed in the species abundance heatmap. The taxonomic assignment based on 18S ribosomal sequences with the SSU base possibly showed the presence of eukaryotic species (191 in LC05.W; 320 in LC05.S; 278 in LCDN.W and 207 in LCDN.S) in the marine water and sediments collected at Lang Co - Da Nang sea.

Probiotics mediated gut microbiota diversity shifts are associated with reduction in histopathology and shedding of Lawsonia intracellularis

Background Clinical intervention during bacterial infections in farm animals such as pigs commonly includes the use of antimicrobials. With the rise of antimicrobial resistance and the attempts to reduce the use of antibiotics in food animals, effective alternatives are urgently needed to reduce or even remove pathogens and disease risks. Improving clinical outcomes and overall pig health by using probiotics appears attractive. However, reliable data sets on the efficacy of probiotics are scarce. The obligate intracellular bacterium Lawsonia intracellularis is widespread in pigs and associated with severe enteropathy, mainly in the ileum, commonly resulting in substantial reduction in weight gain. The impact of three in-feed probiotics and a commercial live L. intracellularis vaccine was compared in a pig challenge model. Probiotic treatment was associated with reduced L. intracellularis fecal shedding and reduced gut lesions. Here, the bacterial microbiota of the ileum of these pigs was characterized with 16S rRNA gene sequencing and was subsequently analyzed with bioinformatics tools. Results The greatest microbial richness was observed in the probiotic treated group T03-LAW, which accounted for 87% of richness observed in the study. Treatment had a significant impact on both the microbiota structure and taxonomic profile in the ileum, explaining between 26 and 36% of the structural variation, with the strongest association in the T03-LAW group. Overall, the largest changes were observed for the pigs treated with in-feed Bacillus pumilus; the microbiota of these pigs had the greatest diversity and highest richness. We also observed depleted and enriched core microbiota amongst the groups; however, there was no correlation with clinical characteristics. The results suggest that an increased diversity of the ileal microbiota is associated with a reduction in shedding, i.e. a unit increase in Shannon diversity index resulted in 2.8 log reduction in shedding. Conclusions Probiotic supplementation of a base feed ration increased ileum microbiota diversity leading to a mitigation of the effects of a pathogenic L. intracellularis challenge. An even and diverse microbiota community benefits pigs infected with L. intracellularis, however, investigations are needed to determine if this is also true for other pathogens. The study unambiguously demonstrates the usefulness of probiotic supplementation in reducing the impact of enteric pathogens and pathogen shedding rates in food animals without the use of antimicrobials.

Trans-ethnic gut microbiota signatures of type 2 diabetes in Denmark and India

Background Type 2 diabetes (T2D), a multifactorial disease influenced by host genetics and environmental factors, is the most common endocrine disease. Several studies have shown that the gut microbiota as a close-up environmental mediator influences host physiology including metabolism. The aim of the present study is to examine the compositional and functional potential of the gut microbiota across individuals from Denmark and South India with a focus on T2D. Many earlier studies have investigated the microbiome aspects of T2D, and it has also been anticipated that such microbial associations would be dependent on diet and ethnic origin. However, there has been no large scale trans-ethnic microbiome study earlier in this direction aimed at evaluating any “universal” microbiome signature of T2D. Methods 16S ribosomal RNA gene amplicon sequencing was performed on stool samples from 279 Danish and 294 Indian study participants. Any differences between the gut microbiota of both populations were explored using diversity measures and negative binomial Wald tests. Study samples were stratified to discover global and country-specific microbial signatures for T2D and treatment with the anti-hyperglycemic drug, metformin. To identify taxonomical and functional signatures of the gut microbiota for T2D and metformin treatment, we used alpha and beta diversity measures and differential abundances analysis, comparing metformin-naive T2D patients, metformin-treated T2D patients, and normoglycemic individuals. Results Overall, the gut microbial communities of Danes and Indians are compositionally very different. By analyzing the combined study materials, we identify microbial taxonomic and functional signatures for T2D and metformin treatment. T2D patients have an increased relative abundance of two operational taxonomic units (OTUs) from the Lachnospiraceae family, and a decreased abundance of Subdoligranulum and Butyricicoccus. Studying each population per se, we identified T2D-related microbial changes at the taxonomic level within the Danish population only. Alpha diversity indices show that there is no significant difference between normoglycemic individuals and metformin-naive T2D patients, whereas microbial richness is significantly decreased in metformin-treated T2D patients compared to metformin-naive T2D patients and normoglycemic individuals. Enrichment of two OTUs from Bacteroides and depletion of Faecalibacterium constitute a trans-ethnic signature of metformin treatment. Conclusions We demonstrate major compositional differences of the gut microbiota between Danish and South Indian individuals, some of which may relate to differences in ethnicity, lifestyle, and demography. By comparing metformin-naive T2D patients and normoglycemic individuals, we identify T2D-related microbiota changes in the Danish and Indian study samples. In the present trans-ethnic study, we confirm that metformin changes the taxonomic profile and functional potential of the gut microbiota.

Probiotics mediated gut microbiota diversity shifts are associated with reduction in histopathology and shedding of Lawsonia intracellularis

Background: Clinical intervention during bacterial infections in farm animals such as pigs commonly consists of antimicrobial use. With the rise of antimicrobial resistance and the attempts to reduce the use of antibiotics in food animals, effective alternatives are urgently needed to reduce or even remove pathogens and disease risk. Improving clinical outcomes and overall pig health by using probiotics appears attractive. However, reliable data sets on the efficacy of probiotics are scarce. The obligate intracellular bacterium Lawsonia intracellularis is widespread in pigs and associated with severe enteropathy, mainly in the ileum, commonly resulting in substantial reduction in weight gain. The impact of three in-feed probiotics and a commercial live L. intracellularis vaccine were compared in a pig challenge model. Probiotic treatment was associated with reduced L. intracellularis fecal shedding and reduced gut lesions. Here, the bacterial microbiota of the ileum of these pigs was characterized with 16S rRNA gene sequencing and was subsequently analyzed with bioinformatics tools. Results: The greatest microbial richness was observed in the probiotic treated group T03-LAW, which accounted for 87% of richness observed in the study. Treatment had a significant impact on both the microbiota structure and taxonomic profile in the ileum, explaining between 26-36% of the structural variation, with the strongest association in the T03-LAW group. Overall, the largest changes were observed for the pigs treated with in-feed Bacillus pumilus; the microbiota of these pigs had the greatest diversity and highest richness. We also observed depleted and enriched core microbiota amongst the groups; however, there was no correlation with clinical characteristics. The results suggest that an increased diversity of the ileal microbiota is associated with a reduction in shedding, i.e. a unit increase in Shannon diversity index resulted in 2.8 log reduction in shedding.Conclusions: Probiotic supplementation of a base feed ration increased ileum microbiota diversity leading to a mitigation of the effects of a pathogenic L. intracellularis challenge. An even and diverse microbiota community benefits pigs infected with L. intracellularis, however, investigations are needed to determine if this is also true for other pathogens. The study unambiguously demonstrates the usefulness of probiotic supplementation in reducing the impact of enteric pathogens and pathogen shedding rates in food animals without the use of antimicrobials.

Fecal Microbiota Changes in NZB/W F1 Mice After Induction of Lupus Disease

Background: Gut microbiota is thought to regulate immune homeostasis. The association between gut microbiota and the development of systemic lupus erythematosus remains unclear. This study aims to investigate the differential alteration of gut microbiota after the induction of lupus in a murine model with viral peptide of human cytomegalovirus (HCMV), which is an established murine model of induced lupus. Results: Specifically, we injected HCMVpp65 peptide in peritoneal space in NZB/W F1 mice to induce lupus. Three arms of animal treatment were prepared: intraperitoneal injection of HCMVpp65 peptide, intraperitoneal injection of adjuvant alone, and none injected. Feces of the study animals were collected before and every two weeks after the lupus induction for 16S rRNA sequencing. At 24 weeks of age, pathological investigation of renal tissue from sacrificed mice was conducted. Statistical analysis for dynamics and alteration of the fecal microbiota and lupus-like-activity-related functional prediction of bacterial communities was performed. HCMVpp65 peptide immunization induced lupus-like activities, with a higher level of anti-dsDNA antibodies, creatinine and proteinuria, and severe glomerular damage, compared to the mice treated with none or adjuvant only. A higher Simpson diversity value was measured in mice with injection of HCMVpp65 peptide than those with injection of adjuvant or none, but there have no difference in the species richness estimates, ACE and Chao1. Statistical Analysis of Metagenomic Profiles (STAMP) showed a higher relative abundance of the family (Saccharimonadaceae, Marinifiaceae, and Desulfovibrionaceae) and genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) that were found in HCMVpp65 peptide treated mice, compared to mice injected with adjuvant or none. The predicted metagenomic taxonomic profile showed statistically significant enrichment of flagellar assembly, bacterial motility, and chemotaxis. Conclusion: A significant correlation between increased related genera abundance (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) and HCMVpp65 peptide immunization induced lupus-like activities was observed. This study confirmed significant changes in gut microbiota after the onset of lupus in a murine animal model.