Resistome Analysis of Global Livestock and Soil Microbiomes.

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.

Whole-Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen.

Antimicrobial peptides (AMPs) can efficiently control different microbial pathogens and show the potential to be applied in clinical practice and livestock production. In this work, the aim was to isolate AMP-producing ruminal streptococci and to characterize their genetic features through whole-genome sequencing. We cultured 463 bacterial isolates from the rumen of Nelore bulls, 81 of which were phenotypically classified as being Streptococcaceae. Five isolates with broad-range activity were genome sequenced and confirmed as being Streptococcus lutetiensis. The genetic features linked to their antimicrobial activity or adaptation to the rumen environment were characterized through comparative genomics. The genome of S. lutetiensis UFV80 harbored a putative CRISPR-Cas9 system (Type IIA). Computational tools were used to discover novel biosynthetic clusters linked to the production of bacteriocins. All bacterial genomes harbored genetic clusters related to the biosynthesis of class I and class II bacteriocins. SDS-PAGE confirmed the results obtained in silico and demonstrated that the class II bacteriocins predicted in the genomes of three S. lutetiensis strains had identical molecular mass (5197 Da). These results demonstrate that ruminal bacteria of the Streptococcus bovis/equinus complex represent a promising source of novel antimicrobial peptides.

In silico Screening Unveil the Great Potential of Ruminal Bacteria Synthesizing Lasso Peptides.

Studies of rumen microbial ecology suggest that the capacity to produce antimicrobial peptides could be a useful trait in species competing for ecological niches in the ruminal ecosystem. However, little is known about the synthesis of lasso peptides by ruminal microorganisms. Here we analyzed the distribution and diversity of lasso peptide gene clusters in 425 bacterial genomes from the rumen ecosystem. Genome mining was performed using antiSMASH 5, BAGEL4, and a database of well-known precursor sequences. The genomic context of the biosynthetic clusters was investigated to identify putative lasA genes and protein sequences from enzymes of the biosynthetic machinery were evaluated to identify conserved motifs. Metatranscriptome analysis evaluated the expression of the biosynthetic genes in the rumen microbiome. Several incomplete (n = 23) and complete (n = 11) putative lasso peptide clusters were detected in the genomes of ruminal bacteria. The complete gene clusters were exclusively found within the phylum Firmicutes, mainly (48%) in strains of the genus Butyrivibrio. The analysis of the genetic organization of complete putative lasso peptide clusters revealed the presence of co-occurring genes, including kinases (85%), transcriptional regulators (49%), and glycosyltransferases (36%). Moreover, a conserved pattern of cluster organization was detected between strains of the same genus/species. The maturation enzymes LasB, LasC, and LasD showed regions highly conserved, including the presence of a transglutaminase core in LasB, an asparagine synthetase domain in LasC, and an ABC-type transporter system in LasD. Phylogenetic trees of the essential biosynthetic proteins revealed that sequences split into monophyletic groups according to their shared single common ancestor. Metatranscriptome analyses indicated the expression of the lasso peptides biosynthetic genes within the active rumen microbiota. Overall, our in silico screening allowed the discovery of novel biosynthetic gene clusters in the genomes of ruminal bacteria and revealed several strains with the genetic potential to synthesize lasso peptides, suggesting that the ruminal microbiota represents a potential source of these promising peptides.

Characterization of antibiotic resistance genes in the species of the rumen microbiota.

Infections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

Exploring the rumen fluid metabolome using liquid chromatography-high-resolution mass spectrometry and Molecular Networking.

The rumen primary and secondary metabolite content is intimately related to its community of bacteria, protozoa, fungi, archaea and bacteriophages, ingested feed and the host. Despite the myriad of interactions and novel compounds to be discovered, few studies have explored the rumen metabolome. Here, we present the first study using ultra-high performance liquid chromatography tandem mass-spectrometry and Molecular Networking approach, and various extraction methods on the cell-free rumen fluid of a non-lactating Holstein cow. Putative molecules were annotated based on accurate fragmentation matching the Global Natural Products Social Molecular Networking library, public spectral libraries, or annotated manually. The combination of five extraction methods resulted on 1,882 molecular features observed. Liquid-liquid extraction resulted on the highest molecular features abundance, 1,166 (61.96% of total). Sixty-seven compounds were annotated using Global Natural Products Social Molecular Networking library and public libraries, such as hydrocinnamic and azelaic acid, and monensin. Only 3.56% of molecular features (67) observed had positive match with available libraries, which shows the potential of the rumen as reservoir of novel compounds. The use of untargeted metabolomics in this study provided a snapshot of the rumen fluid metabolome. The complexity of the rumen will remain long unknown, but the use of new tools should be encouraged to foster advances on the rumen metabolome.

Characterization of the rumen lipidome and microbiome of steers fed a diet supplemented with flax and echium oil.

Developing novel strategies for improving the fatty acid composition of ruminant products relies upon increasing our understanding of rumen bacterial lipid metabolism. This study investigated whether flax or echium oil supplementation of steer diets could alter the rumen fatty acids and change the microbiome. Six Hereford × Friesian steers were offered grass silage/sugar beet pulp only (GS), or GS supplemented either with flax oil (GSF) or echium oil (GSE) at 3% kg(-1) silage dry matter in a 3 × 3 replicated Latin square design with 21-day periods with rumen samples taken on day 21 for the analyses of the fatty acids and microbiome. Flax oil supplementation of steer diets increased the intake of polyunsaturated fatty acids, but a substantial degree of rumen biohydrogenation was seen. Likewise, echium oil supplementation of steer diets resulted in increased intake of 18:4n-3, but this was substantially biohydrogenated within the rumen. Microbiome pyrosequences showed that 50% of the bacterial genera were core to all diets (found at least once under each dietary intervention), with 19.10%, 5.460% and 12.02% being unique to the rumen microbiota of steers fed GS, GSF and GSE respectively. Higher 16S rDNA sequence abundance of the genera Butyrivibrio, Howardella, Oribacterium, Pseudobutyrivibrio and Roseburia was seen post flax feeding. Higher 16S rDNA abundance of the genus Succinovibrio and Roseburia was seen post echium feeding. The role of these bacteria in biohydrogenation now requires further study.