Archaea perform critical roles in the microbiome system, including utilizing hydrogen to allow for enhanced microbiome member growth and influencing overall host health. With the majority of microbiome research focusing on bacteria, the functions of archaea are largely still under investigation. Understanding methanogenic functions during the host lifetime will add to the limited knowledge on archaeal influence on gut and host health. In our study, we determined lifelong archaea dynamics, including detection and methanogenic functions, while assessing global, temporal and host distribution of our novel archaeal metagenome-assembled genomes (MAGs). We followed 7 monogastric swine throughout their life, from birth to adult (1–156 days of age), and collected feces at 22 time points. The samples underwent gDNA extraction, Illumina sequencing, bioinformatic quality and assembly processes, MAG taxonomic assignment and functional annotation. MAGs were utilized in downstream phylogenetic analysis for global, temporal and host distribution in addition to methanogenic functional potential determination. We generated 1130 non-redundant MAGs, representing 588 unique taxa at the species level, with 8 classified as methanogenic archaea. The taxonomic classifications were as follows: orders Methanomassiliicoccales (5) and Methanobacteriales (3); genera UBA71 (3), Methanomethylophilus (1), MX-02 (1), and Methanobrevibacter (3). We recovered the first US swine Methanobrevibacter UBA71 sp006954425 and Methanobrevibacter gottschalkii MAGs. The Methanobacteriales MAGs were identified primarily during the young, preweaned host whereas Methanomassiliicoccales primarily in the adult host. Moreover, we identified our methanogens in metagenomic sequences from Chinese swine, US adult humans, Mexican adult humans, Swedish adult humans, and paleontological humans, indicating that methanogens span different hosts, geography and time. We determined complete metabolic pathways for all three methanogenic pathways: hydrogenotrophic, methylotrophic, and acetoclastic. This study provided the first evidence of acetoclastic methanogenesis in archaea of monogastric hosts which indicated a previously unknown capability for acetate utilization in methanogenesis for monogastric methanogens. Overall, we hypothesized that the age-associated detection patterns were due to differential substrate availability via the host diet and microbial metabolism, and that these methanogenic functions are likely crucial to methanogens across hosts. This study provided a comprehensive, genome-centric investigation of monogastric-associated methanogens which will further improve our understanding of microbiome development and functions.

Microbial interactions in natural environments are intricately complex. High numbers and rich diversity of microorganisms, along with compositional heterogeneities complicate the cause. It is essential to simplify these complex communities to understand the microbial interactions. We proposed a concept of "simple state community," which represents a subset of microbes and/or microbial functions of the original population that is necessary to build a stable community. By combining microbial culturing and high-throughput sequencing, we can better understand microbe-microbe and microbe-host interactions. To support our proposed model, we used carbon-based and nitrogen-based media to capture the simple state communities. We used 16S rRNA amplicon sequencing and assigned taxonomic identity to the bacterial populations before and after simple state communities. We showed that simple state communities were a subset of the original microbial communities at both phyla and genera level. We further used shotgun metagenomics to gain insights into the functional potential of the assembled simple state communities. Our proposed model supported the goal of simplifying the complex communities across diverse systems to provide opportunity to facilitate comprehension of both the structure and function of the subset communities. Further applications of the concept include the high-throughput screening of simple state communities using the BIOLOG® system and continuous culturing (Chemostat). This concept has the potential to test diverse experimental hypotheses in simplified microbial communities, and further extend that knowledge to answer the overarching questions at a more holistic level.

The gut microbiome plays important roles in the maintenance of health and pathogenesis of diseases in the growing host. In order to fully comprehend the interplay of the gut microbiome and host, a foundational understanding of longitudinal microbiome, including bacteria and fungi, development is necessary. In this study, we evaluated enteric microbiome and host dynamics throughout the lifetime of commercial swine. We collected a total of 234 fecal samples from ten pigs across 31 time points in three developmental stages (5 preweaning, 15 nursery, and 11 growth adult). We then performed 16S rRNA gene amplicon sequencing for bacterial profiles and qPCR for the fungus Kazachstania slooffiae . We identified distinct bacteriome clustering according to the host developmental stage, with the preweaning stage exhibiting low bacterial diversity and high volatility amongst samples. We further identified clusters of bacteria that were considered core, increasing, decreasing or stage-associated throughout the host lifetime. Kazachstania slooffiae was absent in the preweaning stage but peaked during the nursery stage of the host. We determined that all host growth stages contained negative correlations between K. slooffiae and bacterial genera, with only the growth adult stage containing positive correlates. Our stage-associated bacteriome results suggested the neonate contained a volatile gut microbiome. Upon weaning, the microbiome became relatively established with comparatively fewer perturbations in microbiome composition. Differential analysis indicated bacteria might play distinct stage-associated roles in metabolism and pathogenesis. The lack of positive correlates and shared K. slooffiae -bacteria interactions between stages warranted future research into the interactions amongst these kingdoms for host health. This research is foundational for understanding how bacteria and fungi develop singularly, as well as within a complex ecosystem in the host’s gut environment.

The gut microbiome plays important roles in the maintenance of health and pathogenesis of diseases in the growing host. In order to fully comprehend the interplay of the gut microbiome and host, a foundational understanding of longitudinal bacteriome and mycobiome development is necessary. In this study, we evaluated enteric bacterial, fungal and host dynamics throughout the lifetime of commercial swine. We collected a total of 234 fecal samples from 9 pigs across 31 time points in 3 developmental stages (5 preweaning, 15 nursery, and 11 growth adult). We then performed 16S rRNA amplicon sequencing for bacterial profiles and qPCR for the fungus Kazachstania slooffiae . We identified distinct bacteriome clustering according to the host developmental stage, with the preweaning stage exhibiting low bacterial diversity and high volatility amongst samples. We further recovered clusters of bacterial populations that were considered core, transient and increasing throughout the host lifetime, suggesting distinct yet important roles by which these differing bacterial population clusters played in the different host stages. Kazachstania slooffiae was absent in the preweaning stage but peaked during the nursery stage of the host. We determined that all host growth stages contained negative correlations between K. slooffiae and bacterial genera, with only the growth adult stage containing positive correlates. The lack of positive correlates and shared K. slooffiae-bacteria interactions between stages warrants future research into the interactions amongst these kingdoms for host health. This research is foundational for understanding how the bacteriome and mycobiome develop singularly, as well as within a complex ecosystem in the host’s gut environment.

Background: Climate change will result in more frequent droughts that can impact soil-inhabiting microbiomes (rhizobiomes) in the agriculturally vital North American perennial grasslands. Rhizobiomes have contributed to enhancing drought resilience and stress resistance properties in plant hosts. In the predicted events of more future droughts, how the changing rhizobiome under environmental stress can impact the plant host resilience needs to be deciphered. There is also an urgent need to identify and recover candidate microorganisms along with their functions, involved in enhancing plant resilience, enabling the successful development of synthetic communities. Results: In this study, we used the combination of cultivation and high-resolution genomic sequencing of bacterial communities recovered from the rhizosphere of a tallgrass prairie foundation grass, Andropogon gerardii. We cultivated the plant host-associated microbes under artificial drought-induced conditions and identified the microbe(s) that might play a significant role in the rhizobiome of Andropogon gerardii under drought conditions. Phylogenetic analysis of the non-redundant metagenome-assembled genomes (MAGs) identified a bacterial genome of interest - MAG-Pseudomonas. Further metabolic pathway and pangenome analyses recovered genes and pathways related to stress responses including ACC deaminase; nitrogen transformation including assimilatory nitrate reductase in MAG-Pseudomonas, which might be associated with enhanced drought tolerance and growth for Andropogon gerardii. Conclusions: Our data indicated that the metagenome-assembled MAG-Pseudomonas has the functional potential to contribute to the plant host's growth during stressful conditions. Our study also suggested the nitrogen transformation potential of MAG-Pseudomonas that could impact Andropogon gerardii growth in a positive way. The cultivation of MAG-Pseudomonas sets the foundation to construct a successful synthetic community for Andropogon gerardii. To conclude, stress resilience mediated through genes ACC deaminase, nitrogen transformation potential through assimilatory nitrate reductase in MAG-Pseudomonas could place this microorganism as an important candidate of the rhizobiome aiding the plant host resilience under environmental stress. This study, therefore, provided insights into the MAG-Pseudomonas and its potential to optimize plant productivity under ever-changing climatic patterns, especially in frequent drought conditions.

Background Antimicrobial resistance (AMR) is a significant global public health concern associated with millions of deaths annually. Agriculture has been attributed as a leading factor in AMR and multidrug resistance (MDR) associated with swine production estimated as one of the largest agricultural consumers of antibiotics. Therefore, studying and understanding AMR in swine has global relevance. AMR research has received increased attention in recent years. However, we are still building our understanding of genetic variation within a complex gut microbiome system that impacts AMR and MDR. In order to evaluate the gut resistome, we evaluated genetic variation before, during, and after antibiotic treatments. We studied three treatment groups: non-antibiotic controls (C), chlortetracycline (CTC) treated, and tiamulin (TMU) treated. We collected fecal samples from each group and performed metagenomic sequencing for a longitudinal analysis of genetic variation and functions. Results We generated 772,688,506 reads and 81 metagenome assembled genomes (MAGs). Interestingly, we identified a subset of 11 MAGs with sustained detection and high sustained entropy (SDHSE). Entropy described genetic variation throughout the MAG. Our SDHSE MAGs were considered MDR as they were identified prior to, throughout, and after CTC and TMU treatments as well as in the C piglets. SDHSE MAGs were especially concerning as they harbored relatively high variation. Consistently high variation indicated that these microbial populations may contain hypermutable elements which has been associated with increased chance of AMR and MDR acquisition. Our SDHSE MAGs demonstrated that MDR organisms (MDRO) are present in swine, and likely additional hosts contributing to global AMR. Altogether, our study provides comprehensive genetic support of MDR populations within the gut microbiome of swine.

Background Climate change will result in more frequent droughts that impact soil-inhabiting microbiomes in the agriculturally vital North American perennial grasslands. In this study, we used the combination of culturomics and high-resolution genomic sequencing of microbial consortia isolated from the rhizosphere of a tallgrass prairie foundation grass, Andropogon gerardii . We cultivated the plant host-associated microbes under artificial drought-induced conditions and identified the microbe(s) that might play a significant role in the rhizobiome of Andropogon gerardii under drought conditions. Results Phylogenetic analysis of the non-redundant metagenome-assembled genomes (MAGs) identified the bacterial population of interest – MAG- Pseudomonas . Further metabolic pathway and pangenome analyses detected genes and pathways related to nitrogen transformation and stress responses in MAG- Pseudomonas . Conclusions Our data indicate that the metagenome-assembled MAG- Pseudomonas has the functional potential to contribute to the plant host’s growth during stressful conditions. This study provided insights into optimizing plant productivity under drought conditions.

Environmental change, especially frequent droughts, is predicted to detrimentally impact the North American perennial grasslands. Consistent dry spells will affect plant communities as well as their associated rhizobiomes, possibly altering the plant host performance under environmental stress. Therefore, there is a need to understand the impact of drought on the rhizobiome, and how the rhizobiome may modulate host performance and ameliorate its response to drought stress. In this study, we analyzed bacterial and fungal communities in the rhizospheres of three ecotypes (dry, mesic, and wet) of a dominant prairie grass, Andropogon gerardii . The ecotypes were established in 2010 in a common garden design and grown for a decade under persistent dry conditions at the arid margin of the species’ range in Colby Kansas. The experiment aimed to answer whether and to what extent do the different ecotypes maintain or recruit distinct rhizobiomes after ten years in an arid climate. In order to answer this question, we screened the bacterial and fungal rhizobiome profiles of the ecotypes under the arid conditions of western KS as a surrogate for future climate environmental stress using 16S rRNA and ITS2 metabarcoding sequencing. Under these conditions, bacterial communities differed compositionally among the A. gerardii ecotypes, whereas the fungal communities did not. The ecotypes were instrumental in driving the differences among bacterial rhizobiomes, as the ecotypes maintained distinct bacterial rhizobiomes even after ten years at the edge of the host species range. This study will aid us to optimize plant productivity through the use of different ecotypes under future abiotic environmental stress, especially drought.

An emerging porcine sapelovirus was isolated in a diagnostic specimen from a US swine farm, designated as PSV KS18-01. Full-length genome sequence was obtained through next-generation sequencing. Phylogenetic analysis showed that the virus is more closely related to two Japanese strains but is distantly related to two known US strains. PSV specific diagnostic tools were developed, including the monoclonal antibodies again VP1 and VP2, and a VP1-VP2 antigen-based indirect ELISA. Using this assay, the dynamic response of PSV antibody was investigated in a group of post-weaned pigs that naturally exposed with PSV. The availability of the PSV isolate (KS18-01) and the specific diagnostic reagents and assays provide important tools for PSV control and prevention. Porcine Sapelovirus (PSV), previously named as porcine enterovirus 8, belongs to the genus Sapelovirus in the family Picornaviridae (1). PSV is a non-enveloped, positive-sense singlestranded RNA virus. It has a genome of approximately 7.5 kb, with a typical genomic organization of 5’ untranslated region -Leader-polyprotein (P1-P2-P3)-3’UTR. The polyprotein is synthesized and proteolytically cleaved into seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D) and four structural proteins (VP4, VP2, VP3, VP1) (2). The PSV capsid is icosahedral arrangement with VP1, VP2, and VP3 on the surface, while VP4 embedded in the internal side of the virion. Host antibodies are mainly directed toward the viral capsid proteins, in which VP1 was reported having the ability to stimulate strong antibody response during the infection (2). PSV infection is commonly asymptomatic, but clinical disease of respiratory failure, diarrhea, reproductive disorder, and polioencephlamyelitis have been reported in swine farms from many countries (3-5). Neurotropic strains have been reported from China, Indian, the United Kingdom, and the United States (US). However, little is known about the disease prevalence, morbidity and mortality caused by PSV infection. PSV isolates and diagnostic tools are need for the studies of PSV pathogenesis and epidemiology. In this study, we isolated an emerging PSV in a fecal sample from the US swine farm. The virus isolation was performed on ST cells using the method as described previously (6). The new isolate was designated as PSV KS18-01. The full-length genome sequence was obtained through metagenomic sequencing (7). The genome size of KS18-01 was 7,453 bp, encoding a polyprotein of 2,316 amino acids (aa). Compared to other PSV strains, KS18-01 genome has 79.88-87.91% nucleotide (nt) identity. The 5’-untranslated region (5’-UTR) is the most

A distinct Russian Mammalian orthorubulavirus 5 (PIV5) was detected in cell culture exhibiting cytopathic effect and hypothesized to be contaminated by a scientist with respiratory symptoms. The identification of the divergent strain indicated a lack of knowledge on the diversity of PIV5 strains and calls for surveillance of global PIV5 strains.

Porcine deltacoronavirus (δ-CoV) is the object of extensive research in several countries including the United States. In contrast, the epidemiology of δ-CoVs in wild birds in the US is largely unknown. Our aim was to comparatively assess the prevalence of δ- and γ-CoVs in wild migratory terrestrial and aquatic birds in Arkansas, Illinois, Indiana, Maryland, Mississippi, Missouri, Ohio, Tennessee and Wisconsin. A total of 1236 cloacal/fecal swabs collected during the period 2015–2018 were tested for γ- and δ-CoVs using genus-specific reverse transcription-PCR assays. A total of 61 (4.99%) samples were γ-CoV positive, with up to 29 positive samples per state. In contrast, only 14 samples were positive for δ-CoV (1.14%) with only 1–4 originating from the same state. Thus, unlike previous reports from Asia, γ-CoVs are more prevalent than δ-CoVs in the US, suggesting that δ-CoVs may spread in birds with lower efficiency. This may indicate δ-CoV emerging status and incomplete adaptation to new host species limiting its spread. Phylogenetic analysis of the partial N gene revealed that the newly identified δ-CoV strains were most closely related to the HKU20 (wigeon) strain. Further studies are necessary to investigate the role of aquatic bird δ-CoVs in the epidemiology of δ-CoVs in swine and terrestrial birds.