Fresh Rumen Liquid Inoculant Enhances the Rumen Microbial Community Establishment in Pre-weaned Dairy Calves

The development of the functional rumen in calves involves a complex interplay between the host and host-related microbiome. Attempts to modulate rumen microbial community establishment may therefore have an impact on weaning success, calf health, and animal performance later in life. In this experiment, we aimed to elucidate how rumen liquid inoculum from an adult cow, provided to calves during the pre-weaning period, influences the establishment of rumen bacterial, archaeal, fungal, and ciliate protozoan communities in monozygotic twin calves (n = 6 pairs). The calves were divided into treatment (T-group) and control (C-group) groups, where the T-group received fresh rumen liquid as an oral inoculum during a 2–8-week period. The C-group was not inoculated. The rumen microbial community composition was determined using bacterial and archaeal 16S ribosomal RNA (rRNA) gene, protozoal 18S rRNA gene, and fungal ITS1 region amplicon sequencing. Animal weight gain and feed intake were monitored throughout the experiment. The T-group tended to have a higher concentrate intake (Treatment: p < 0.08) and had a significantly higher weekly weight gain (Treatment: p < 0.05), but no significant difference in volatile fatty acid concentrations between the groups was observed. In the T-group, the inoculum stimulated the earlier establishment of mature rumen-related bacterial taxa, affecting significant differences between the groups until 6 weeks of age. The inoculum also increased the archaeal operational taxonomic unit (OTU) diversity (Treatment: p < 0.05) but did not affect the archaeal quantity. Archaeal communities differed significantly between groups until week 4 (p = 0.02). Due to the inoculum, ciliate protozoa were detected in the T-group in week 2, while the C-group remained defaunated until 6 weeks of age. In week 8, Eremoplastron dilobum was the dominant ciliate protozoa in the C-group and Isotricha sp. in the T-group, respectively. The Shannon diversity of rumen anaerobic fungi reduced with age (Week: p < 0.01), and community establishment was influenced by a change of diet and potential interaction with other rumen microorganisms. Our results indicate that an adult cow rumen liquid inoculum enhanced the maturation of bacterial and archaeal communities in pre-weaning calves’ rumen, whereas its effect on eukaryotic communities was less clear and requires further investigation.

Contrasting patterns of bacterial communities in the rearing water and gut of Penaeus vannamei in response to exogenous glucose addition

Supplementing exogenous carbon sources is a practical approach to improving shrimp health by manipulating the microbial communities of aquaculture systems. However, little is known about the microbiological processes and mechanisms of these systems. Here, the effects of glucose addition on shrimp growth performance and bacterial communities of the rearing water and the shrimp gut were investigated to address this knowledge gap. The results showed that glucose addition significantly improved the growth and survival of shrimp. Although the α-diversity indices of both bacterioplankton communities and gut microbiota were significantly decreased by adding glucose, both bacterial communities exhibited divergent response patterns to glucose addition. Glucose addition induced a dispersive bacterioplankton community but a more stable gut bacterial community. Bacterial taxa belonging to Ruegeria were significantly enriched by glucose in the guts, especially the operational taxonomic unit 2575 (OTU2575), which showed the highest relative importance to the survival rate and individual weight of shrimp, with the values of 43.8 and 40.6%, respectively. In addition, glucose addition increased the complexity of interspecies interactions within gut bacterial communities and the network nodes from Rhodobacteraceae accounted for higher proportions and linked more with the nodes from other taxa in the glucose addition group than that in control. These findings suggest that glucose addition may provide a more stable gut microbiota for shrimp by increasing the abundance of certain bacterial taxa, such as Ruegeria.

Microbial Eukaryotes Associated With Sediments in Deep-Sea Cold Seeps

Microbial eukaryotes are key components of the marine food web, but their distribution in deep-sea chemosynthetic ecosystems has not been well studied. Here, high-throughput sequencing of the 18S rRNA gene and network analysis were applied to investigate the diversity, distribution and potential relationships between microbial eukaryotes in samples collected from two cold seeps and one trough in the northern South China Sea. SAR (i.e., Stramenopiles, Alveolata, and Rhizaria) was the predominant group in all the samples, and it was highly affiliated to genotypes with potential symbiotic and parasitic strategies identified from other deep-sea extreme environments (e.g., oxygen deficient zones, bathypelagic waters, and hydrothermal vents). Our findings indicated that specialized lineages of deep-sea microbial eukaryotes exist in chemosynthetic cold seeps, where microbial eukaryotes affiliated with parasitic/symbiotic taxa were prevalent in the community. The biogeographic pattern of the total community was best represented by the intermediate operational taxonomic unit (OTU) category, whose relative abundance ranged 0.01–1% within a sample, and the communities of the two cold seeps were distinct from the trough, which suggests that geographical proximity has no critical impact on the distribution of deep-sea microbial eukaryotes. Overall, this study has laid the foundations for future investigations regarding the ecological function and in situ trophic relationships of microbial eukaryotes in deep-sea ecosystems.

Microbial associations of shallow-water Mediterranean marine cave Solenogastres (Mollusca)

The first cave-dwelling Solenogastres—marine shell-less worm-like mollusks—were sampled from Mediterranean marine caves floor silt in the Marseille area. The mollusks were 1.5 mm in length, had a transparent body with shiny spicules and appear to represent a new Tegulaherpia species. Electron microscopy revealed a high number of microbial cells, located on the surface of the spicules as well as in the cuticle of Tegulaherpia sp. The observed microbial cells varied in morphology and were unequally distributed through the cuticle, reaching a highest density on the dorsal and lateral sides and being practically absent on the ventral side. Next Generation Sequencing (NGS) of V4 region of 16S rRNA gene amplicons, obtained from the DNA samples of whole bodies of Tegulaherpia sp. revealed three dominating microorganisms, two of which were bacteria of Bacteroidetes and Nitrospirae phyla, while the third one represented archaea of Thaumarchaeota phylum. The Operational Taxonomic Unit (OTU), affiliated with Bacteroidetes was an uncultured bacteria of the family Saprospiraceae (93–95% of Bacteroidetes and 25–44% of the total community, depending on sample), OTU, affiliated with Nitrospirae belonged to the genus Nitrospira (8–30% of the community), while the thaumarchaeal OTU was classified as Candidatus Nitrosopumilus (11–15% of the community). Members of these three microbial taxa are known to form associations with various marine animals such as sponges or snails where they contribute to nitrogen metabolism or the decomposition of biopolymers. A similar role is assumed to be played by the microorganisms associated with Tegulaherpia sp.

Pandora’s Box in the Deep Sea –Intraspecific Diversity Patterns and Distribution of Two Congeneric Scavenging Amphipods

Paralicella tenuipesChevreux, 1908 and Paralicella caperescaShulenberger and Barnard, 1976 are known as widely distributed deep-sea scavenging amphipods. Some recent studies based on genetic data indicated the presence of high intraspecific variation of P. caperesca suggesting it is a species complex. Based on published molecular data from the Pacific and Indian oceans and new material obtained from the North and South Atlantic, we integrated the knowledge on the intraspecific variation and species distribution of the two nominal taxa. The study included analysis of three genes (COI, 16S rRNA, 28S rRNA) and revealed the existence of a single Molecular Operational Taxonomic Unit (MOTU) within P. tenuipes and six different MOTUs forming P. caperesca. The distribution pattern of the recognized lineages varied with three (P. tenuipes, MOTU 1 and MOTU 5 of P. caperesca) being widely distributed. There was evidence of contemporary population connectivity expressed by the share of the same COI haplotypes by individuals from very distant localities. At the same time no signal of recent demographic changes was observed within the studied taxa. The time-calibrated phylogeny suggested the emergence of species to be at the time of Mesozoic/Cenozoic transition that may be associated with global changes of the ocean circulation and deep sea water cooling.

Gut microbiota imbalance in colorectal cancer patients, the risk factor of COVID-19 mortality

Background COVID-19 pandemic is sweeping across the world. Previous studies have shown that gut microbiota is associated with COVID-19, and operational taxonomic unit (OTU) composed of Blautia genus, Lactobacillus genus, and Ruminococcus genus of Firmicutes is correlated with the severity of COVID-19. Gut microbiota imbalance in colorectal cancer patients may lead to the variation of OTU. Results Based on the GMrepo database, the gut microbiota of 1374 patients with colorectal neoplasms and 27,329 healthy people was analyzed to investigate the differences in the abundance of microbes between colorectal neoplasms patients and healthy people. Furthermore, We collected feces samples from 12 patients with colorectal cancer and 8 healthy people in Xiangya hospital for metabolomic analysis to investigate the potential mechanisms. Our study showed that the abundance of Blautia and Ruminococcus was significantly increased in colorectal neoplasms, which may increase the severity of COVID-19. The gender and age of patients may affect the severity of COVID-19 by shaping the gut microbiota, but the BMI of patients does not. Conclusions Our work draws an initial point that gut microbiota imbalance is a risk factor of COVID-19 mortality and gut microbiota may provide a new therapeutic avenue for colorectal cancer patients.

PURC v2.0: a program for improved sequence inference for polyploid phylogenetics and other manifestations of the multiple-copy problem

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatic problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an "operational taxonomic unit" (OTU). Recently, this approach has been improved upon by model-based methods that correct PCR and sequencing errors in order to infer "amplicon sequence variants" (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for identifying allelic or paralogous variants and for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition to preserving original PURC functions, PURC v2.0 allows users to process PacBio CCS/HiFi reads through DADA2 to generate and annotate ASVs for multiplexed data, with outputs including separate alignments for each locus ready for phylogenetic inference. In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that PURC v2.0 (using the ASV approach) is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC, and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output. We expect that PURC v2.0 will provide biologists with a method for generating multi-locus "moderate data" datasets that are large enough to be phylogenetically informative and small enough for manual curation.

Dataset of Oyster Virome and the Remarkable Virus Diversity in Filter-Feeding Oysters

Background:Viruses are the most abundant biological entities, and they play critical roles in entire ecosystems. Nevertheless, current knowledge about them is no more than 1% of the estimated diversity of the Earth’s virosphere. Oysters are filter-feeding molluscan bivalves and are ideal sentinels for marine virus exploration and viral ecology studies. Results: Here we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit (vOTU) contigs and 3,473 high-quality viral genomes, enabling the first comprehensive overview of viral communities in oysters. As in other marine viromes, families Siphoviridae, Podoviridae, and Myoviridae are dominant in the DOV. However, Circoviridae is the most abundant family among the high-quality genomes, indicating that oysters may be their potential hotspots. Despite performing target amplification for RNA genomes, the diversity of RNA viruses was much lower than the diversity of DNA viruses. Notably, most of the vOTUs in the DOV were previously undescribed viruses and could not be clustered with any sequences in three reference datasets. Three approaches (based on references, vOTUs, and auxiliary metabolic genes) consistently showed that host health status, location, and sampling date had potential impacts on virome structures. Conclusions: This study highlights the practicality of oysters for marine virus exploration and provides a new direction to understand the relationship between marine bivalves and the environment.

1064. Treatment Success in Reducing Recurrent Clostridioides difficile Infection with Investigational Live Biotherapeutic RBX2660 Is Associated with Microbiota Restoration: Consistent Evidence from a Phase 3 Clinical Trial

Background Several investigational microbiota-based live biotherapeutics are in clinical development for reducing recurrence of Clostridioides difficile infection (rCDI), including RBX2660 a liquid suspension of a broad consortium of microbiota, which includes Bacteridetes and Firmicutes. RBX2660 has been evaluated in >600 participants in 6 clinical trials. Here we report that RBX2660 induced significant shifts to the intestinal microbiota of treatment-responsive participants in PUNCH CD3—a Phase 3 randomized, double-blinded, placebo-controlled trial. Methods PUNCH CD3 participants received a single dose of RBX2660 or placebo between 24 to 72 hours after completing rCDI antibiotic treatment. Clinical response was the absence of CDI recurrence at eight weeks after treatment. Participants voluntarily submitted stool samples prior to blinded study treatment (baseline), 1, 4 and 8 weeks, 3 and 6 months after receiving study treatment. Samples were extracted and sequenced using shallow shotgun methods. Operational taxonomic unit (OTU) data were used to calculate relative taxonomic abundance, alpha diversity, and the Microbiome Health Index (MHI)—a biomarker of antibiotic-induced dysbiosis and restoration. Results Clinically, RBX2660 demonstrated superior efficacy versus placebo (70.4% versus 58.1%). From before to after treatment, RBX2660-treated clinical responders’ microbiome diversity shifted significantly (Mann-Whitney), and so did microbiome composition (Generalized Wald Test). Post-treatment changes were characterized by increased Bacteroidia and Clostridia and decreased Gammaproteobacteria and Bacilli, changes and were durable to at least 6 months. Repeated measures analysis confirmed that shifts were greater among RBX2660 responders compared to placebo responders (DMRepeat). The majority of responders’ MHI values shifted from a range common to antibiotic dysbiosis to a range common in healthy populations. Figure 1 Left panel. Mean relative abundance taxonomic class level at timepoints for participants in PUNCH CD3 before and after RBX2660 treatment, and for doses of RBX2660 administered in PUNCH CD3. The four taxonomic classes that change most from before to after treatment are shown with the mean and confidence intervals based on fitting OTU data to a Dirichlet multinomial distribution. Right panel, MHI biomarker for the same time points and investigational product groups, shown as median (red) and individual samples. A previously calculated threshold of MHI = 7.2 is shown (dotted line), above which MHI values predict healthy, below which MHI values predict antibiotic-induced dysbiosis. Conclusion Among PUNCH CD3 clinical responders, RBX2660 significantly restored microbiota from less to more healthy compositions, and this restoration was durable to at least 6 months. These clinically-correlated microbiome shifts are highly consistent with results from multiple prior trials of RBX2660. Disclosures Ken Blount, PhD, Rebiotix Inc., a Ferring Company (Employee) Dana M. Walsh, PhD, Rebiotix (Employee)

Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.