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Antimicrobial resistance (AMR) is a serious threat to public health globally; it is estimated that AMR bacteria caused 1.27 million deaths in 2019, and this is set to rise to 10 million deaths annually. Agricultural and soil environments act as antimicrobial resistance gene (ARG) reservoirs, operating as a link between different ecosystems and enabling the mixing and dissemination of resistance genes. Due to the close interactions between humans and agricultural environments, these AMR gene reservoirs are a major risk to both human and animal health. In this study, we aimed to identify the resistance gene reservoirs present in four microbiomes: poultry, ruminant, swine gastrointestinal (GI) tracts coupled with those from soil. This large study brings together every poultry, swine, ruminant, and soil shotgun metagenomic sequence available on the NCBI sequence read archive for the first time. We use the ResFinder database to identify acquired antimicrobial resistance genes in over 5,800 metagenomes. ARGs were diverse and widespread within the metagenomes, with 235, 101, 167, and 182 different resistance genes identified in the poultry, ruminant, swine, and soil microbiomes, respectively. The tetracycline resistance genes were the most widespread in the livestock GI microbiomes, including tet (W)_1, tet (Q)_1, tet (O)_1, and tet (44)_1. The tet (W)_1 resistance gene was found in 99% of livestock GI tract microbiomes, while tet (Q)_1 was identified in 93%, tet (O)_1 in 82%, and finally tet (44)_1 in 69%. Metatranscriptomic analysis confirmed these genes were "real" and expressed in one or more of the livestock GI tract microbiomes, with tet (40)_1 and tet (O)_1 expressed in all three livestock microbiomes. In soil, the most abundant ARG was the oleandomycin resistance gene, ole (B)_1. A total of 55 resistance genes were shared by the four microbiomes, with 11 ARGs actively expressed in two or more microbiomes. By using all available metagenomes we were able to mine a large number of samples and describe resistomes in 37 countries. This study provides a global insight into the diverse and abundant antimicrobial resistance gene reservoirs present in both livestock and soil microbiomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lawther, Santos, Oyama, Rubino, Morrison, Creevey, McGrath and Huws.)

Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium Butyrivibrio fibrisolvens . The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes., (Copyright © 2020 Friedersdorff, Kingston-Smith, Pachebat, Cookson, Rooke and Creevey.)

The rumen protozoa, alongside fungi, comprise the eukaryotic portion of the rumen microbiome. Rumen protozoa may account for up to 50% of biomass, yet their role in this ecosystem remains unclear. Early experiments inferred a role in carbohydrate and protein metabolism, but due to their close association with bacteria, definitively attributing these functions to the protozoa was challenging. The advent of 'omic technologies has created opportunities to broaden our understanding of the rumen protozoa. This study aimed to utilize these methods to further our understanding of the role that protozoa play in the rumen in terms of their metabolic capacities, and in doing so, contribute valuable sequence data to reduce the chance of mis or under-representation of the rumen protozoa in meta'omic datasets. Rumen protozoa were isolated and purified using glucose-based sedimentation and differential centrifugation, extracted RNA was Poly(A) fraction enriched and DNase treated before use in a phage-based, cDNA metatranscriptomic library. Biochemical activity testing of the phage library showed 6 putatively positive plaques in response to carboxymethyl cellulose agar (indicative of cellulose activity), and no positive results for tributyrin (indicative of esterase/lipase activity) or egg yolk agar (indicative of proteolysis). Direct sequencing of the cDNA was also conducted using the Illumina HiSeq 2500. The metatranscriptome identified a wealth of carbohydrate-active enzymes which accounted for 8% of total reads. The most highly expressed carbohydrate-active enzymes were glycosyl hydrolases 5 and 11, polysaccharide lyases and deacetylases, xylanases and enzymes active against pectin, mannan and chitin; the latter likely used to digest rumen fungi which contain a chitin-rich cell membrane. Codon usage analysis of expressed genes also showed evidence of horizontal gene transfer, suggesting that many of these enzymes were acquired from the rumen bacteria in an evolutionary response to the carbohydrate-rich environment of the rumen. This study provides evidence of the significant contribution that the protozoa make to carbohydrate breakdown in the rumen, potentially using horizontally acquired genes, and highlights their predatory capacity., (Copyright © 2020 Williams, Thomas, McEwan, Rees Stevens, Creevey and Huws.)

The rumen contains a multi-kingdom, commensal microbiome, including protozoa, bacteria, archaea, fungi and viruses, which enables ruminant herbivores to ferment and utilize plant feedstuffs that would be otherwise indigestible. Within the rumen, virus populations are diverse and highly abundant, often out-numbering the microbial populations that they both predate on and co-exist with. To date the research effort devoted to understanding rumen-associated viral populations has been considerably less than that given to the other microbial populations, yet their contribution to maintaining microbial population balance, intra-ruminal microbial lysis, fiber breakdown, nutrient cycling and genetic transfer may be highly significant. This review follows the technological advances which have contributed to our current understanding of rumen viruses and drawing on knowledge from other environmental and animal-associated microbiomes, describes the known and potential roles and impacts viruses have on rumen function and speculates on the future directions of rumen viral research., (Copyright © 2020 Gilbert, Townsend, Crew, Hitch, Friedersdorff, Creevey, Pope, Ouwerkerk and Jameson.)

This work aimed to gain insight into the transition from milk to solid feeding at weaning combining genomics and metabolomics on rumen contents from goat kids treated with a methanogenic inhibitor (bromochloromethane, BCM). Sixteen goats giving birth to two kids were used. Eight does were treated (D+) with BCM after giving birth and over 2 months. One kid per doe in both groups was treated with BCM (k+) for 3 months while the other was untreated (k-). Rumen samples were collected from kids at weaning (W) and 1 (W + 1) and 4 (W + 4) months after and from does at weaning and subjected to 16S pyrosequencing and metabolomics analyses combining GC/LC-MS. Results from pyrosequencing showed a clear effect of age of kids, with more diverse bacterial community as solid feed becomes more important after weaning. A number of specific OTUs were significantly different as a result of BCM treatment of the kid at W while at W + 1 and W + 4 less OTUs were significantly changed. At W + 1, Prevotella was increased and Butyrivibrio decreased in BCM treated kids. At W + 4 only the effect of treating mothers resulted in significant changes in the abundance of some OTUs: Ruminococcus, Butyrivibrio and Prevotella . The analysis of the OTUs shared by different treatments revealed that kids at weaning had the largest number of unique OTUs compared with kids at W + 1 (137), W + 4 (238), and does (D) (23). D + k+ kids consistently shared more OTUs with mothers than the other three groups at the three sampling times. The metalobomic study identified 473 different metabolites. In does, lipid super pathway included the highest number of metabolites that were modified by BCM, while in kids all super-pathways were evenly affected. The metabolomic profile of samples from kids at W was different in composition as compared to W + 1 and W + 4, which may be directly ascribed to the process of rumen maturation and changes in the solid diet. This study shows the complexity of the bacterial community and metabolome in the rumen before weaning, which clearly differ from that after weaning and highlight the importance of the dam in transmitting the primary bacterial community after birth.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed "CowPI," a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.

Twelve mature (aged 5-16 years) horses and ponies of mixed breed and type were fed restricted (1.25% BM Dry matter) quantities of one of two fiber based diets formulated to be iso-caloric. Diet 1 comprised of 0.8% body mass (BM) of chaff based complete feed plus 0.45% BM low energy grass hay (the same hay used for both diets). Diet 2 comprised 0.1% BM of a nutrient balancer plus 1.15% BM grass hay. Fecal samples were collected at week 10 and week 16. DNA was extracted and the V1-V2 regions of 16SrDNA were 454-pyrosequenced to investigate the bacterial microbiome of the horse. The two most abundant phyla found in both diets and sampling periods were the Firmicutes and Bacteroidetes. There was a clear reduction in Bacteroidetes with a concordant increase in Firmicutes over time. There was a limited degree of stability within the bacterial community of the hindgut of horses, with 65% of bacteria retained, over a 6 week period whilst on a uniform diet. The presence of a core community defined by being present in all samples (each animal/diet combination) included in the study and being present at 0.1% relative abundance (or greater) was identified. In total 65 operational taxonomic units (OTUs) were identified that fit the definition of core making up 21-28% of the total sequences recovered. As with total population the most abundant phyla were the Bacteroidetes followed by the Firmicutes , however there was no obvious shift in phyla due to period. Indeed, when the relative abundance of OTUs was examined across diets and periods there was no significant effect of diet or period alone or in combination on the relative abundance of the core OTUs.