Novel integrated computational AMP discovery approaches highlight diversity in the helminth AMP repertoire.

Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described. This study highlights limitations in the homology-based approaches, used to identify putative nematode AMPs, for the characterisation of flatworm AMPs, and reveals that innovative algorithmic AMP prediction approaches provide an alternative strategy for novel helminth AMP discovery. The data presented here: (i) reveal that flatworms do not encode traditional lophotrochozoan AMP groups (Big Defensin, CSαß peptides and Myticalin); (ii) describe a unique integrated computational pipeline for the discovery of novel helminth AMPs; (iii) reveal >16,000 putative AMP-like peptides across 127 helminth species; (iv) highlight that cysteine-rich peptides dominate helminth AMP-like peptide profiles; (v) uncover eight novel helminth AMP-like peptides with diverse antibacterial activities, and (vi) demonstrate the detection of AMP-like peptides from Ascaris suum biofluid. These data represent a significant advance in our understanding of the putative helminth AMP repertoire and underscore a potential untapped source of antimicrobial diversity which may provide opportunities for the discovery of novel antimicrobials. Further, unravelling the role of endogenous worm-derived antimicrobials and their potential to influence host-worm-microbiome interactions may be exploited for the development of unique helminth control approaches.

FI: The Fecobiome Initiative.

Animal husbandry has been key to the sustainability of human societies for millennia. Livestock animals, such as cattle, convert plants to protein biomass due to a compartmentalized gastrointestinal tract (GIT) and the complementary contributions of a diverse GIT microbiota, thereby providing humans with meat and dairy products. Research on cattle gut microbial symbionts has mainly focused on the rumen (which is the primary fermentation compartment) and there is a paucity of functional insight on the intestinal (distal end) microbiota, where most foodborne zoonotic bacteria reside. Here, we present the Fecobiome Initiative (or FI), an international effort that aims at facilitating collaboration on research projects related to the intestinal microbiota, disseminating research results, and increasing public availability of resources. By doing so, the FI can help mitigate foodborne and animal pathogens that threaten livestock and human health, reduce the emergence and spread of antimicrobial resistance in cattle and their proximate environment, and potentially improve the welfare and nutrition of animals. We invite all researchers interested in this type of research to join the FI through our website: www.fecobiome.com.

The rumen eukaryotome is a source of novel antimicrobial peptides with therapeutic potential.

BACKGROUND: The rise of microbial antibiotic resistance is a leading threat to the health of the human population. As such, finding new approaches to tackle these microbes, including development of novel antibiotics is vital. RESULTS: In this study, we mined a rumen eukaryotic metatranscriptomic library for novel Antimicrobial peptides (AMPs) using computational approaches and thereafter characterised the therapeutic potential of the AMPs. We identified a total of 208 potentially novel AMPs from the ruminal eukaryotome, and characterised one of those, namely Lubelisin. Lubelisin (GIVAWFWRLAR) is an α-helical peptide, 11 amino acid long with theoretical molecular weight of 1373.76 D. In the presence of Lubelisin, strains of methicillin-resistant Staphylococcus aureus (MRSA) USA300 and EMRSA-15 were killed within 30 min of exposure with ≥103 and 104 CFU/mL reduction in viable cells respectively. Cytotoxicity of Lubelisin against both human and sheep erythrocytes was low resulting in a therapeutic index of 0.43. Membrane permeabilisation assays using propidium iodide alongside transmission electron microscopy revealed that cytoplasmic membrane damage may contribute to the antimicrobial activities of Lubelisin. CONCLUSIONS: We demonstrate that the rumen eukaryotome is a viable source for the discovery of antimicrobial molecules for the treatment of bacterial infections and further development of these may provide part of the potential solution to the ongoing problem of antimicrobial resistance. The role of these AMPs in the ecological warfare within the rumen is also currently unknown.

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Metaphylogenomic and potential functionality of the limpet Patella pellucida's gastrointestinal tract microbiome.

This study investigated the microbial diversity associated with the digestive tract of the seaweed grazing marine limpet Patella pellucida. Using a modified indirect DNA extraction protocol and performing metagenomic profiling based on specific prokaryotic marker genes, the abundance of bacterial groups was identified from the analyzed metagenome. The members of three significantly abundant phyla of Proteobacteria, Firmicutes and Bacteroidetes were characterized through the literature and their predicted functions towards the host, as well as potential applications in the industrial environment assessed.

- Invited Review - Chemical signalling within the rumen microbiome.

Ruminants possess a specialized four-compartment forestomach, consisting of the reticulum, rumen, omasum, and abomasum. The rumen, the primary fermentative chamber, harbours a dynamic ecosystem comprising bacteria, protozoa, fungi, archaea, and bacteriophages. These microorganisms engage in diverse ecological interactions within the rumen microbiome, primarily benefiting the host animal by deriving energy from plant material breakdown. These interactions encompass symbiosis, such as mutualism and commensalism, as well as parasitism, predation, and competition. These ecological interactions are dependent on many factors, including the production of diverse molecules, such as those involved in quorum sensing (QS). QS is a density-dependent signalling mechanism involving the release of autoinducer (AIs) compounds, when cell density increases AIs bind to receptors causing the altered expression of certain genes. These AIs are classified as mainly being N-acyl-homoserine lactones (AHL; commonly used by Gram-negative bacteria) or autoinducer-2 based systems (AI-2; used by Gram-positive and Gram-negative bacteria); although other less common AI systems exist. Most of our understanding of QS at a gene-level comes from pure culture in vitro studies using bacterial pathogens, with much being unknown on a commensal bacterial and ecosystem level, especially in the context of the rumen microbiome. A small number of studies have explored QS in the rumen using 'omic' technologies, revealing a prevalence of AI-2 QS systems among rumen bacteria. Nevertheless, the implications of these signalling systems on gene regulation, rumen ecology, and ruminant characteristics are largely uncharted territory. Metatranscriptome data tracking the colonization of perennial ryegrass by rumen microbes suggest that these chemicals may influence transitions in bacterial diversity during colonization. The likelihood of undiscovered chemicals within the rumen microbial arsenal is high, with the identified chemicals representing only the tip of the iceberg. A comprehensive grasp of rumen microbial chemical signalling is crucial for addressing the challenges of food security and climate targets.

Climate change as a challenge for pharmaceutical storage and tackling antimicrobial resistance.

The rise of antimicrobial resistance (AMR) remains a pressing global health challenge. Infections that were once easily treatable with first-line antimicrobials are becoming increasingly difficult to manage. This shift directly threatens the wellness of humans, animals, plants, and the environment. While the AMR crisis can be attributed to a myriad of factors, including lack of infection prevention and control measures, over-prescription of antimicrobials, patient non-compliance, and the misuse of antimicrobials, one aspect that has garnered less attention is the role of storage conditions of these medicines. The way medications, particularly antimicrobials, are transported and stored until the point of use can influence their efficacy and, subsequently, may impact the development of resistant microbial strains. This review delves deeper into the often-overlooked domain of climate change (CC) and antimicrobial storage practices and the potential effects. Inappropriate storage conditions, such as exposure to extreme temperatures, humidity or light, can degrade the potency of antimicrobials. When these compromised medicines are administered to patients or animals alike, they may not effectively eradicate the targeted pathogens, leading to partial survival of the pathogens. These surviving pathogens, having been exposed to sub-lethal doses, are more likely to evolve and develop resistance mechanisms. The review discusses the mechanism underlying this and underscores the implications of antimicrobial storage practices in relation to two of the most pressing global health challenges: AMR and CC. The review also presents specific case studies and highlights the importance of monitoring storage practices and supply chain surveillance. Furthermore, the importance of deploying genomic tools to understand the potential impact of storage conditions on the development of AMR is discussed, and antimicrobial storage highlighted as a crucial part of comprehensive strategies in the fight against AMR.

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38.

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris-HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides. Supplementary Information: The online version contains supplementary material available at 10.1007/s10811-023-03136-3.

Exploring the antimicrobial peptidome of nematodes through phylum-spanning in silico analyses highlights novel opportunities for pathogen control.

Antimicrobial Peptides (AMPs) are key constituents of the invertebrate innate immune system and provide critical protection against microbial threat. Nematodes display diverse life strategies where they are exposed to heterogenous, microbe rich, environments highlighting their need for an innate immune system. Within the Ecdysozoa, arthropod AMPs have been well characterised, however nematode-derived AMP knowledge is limited. In this study the distribution and abundance of putative AMP-encoding genes was examined in 134 nematode genomes providing the most comprehensive profile of AMP candidates within phylum Nematoda. Through genome and transcriptome analyses we reveal that phylum Nematoda is a rich source of putative AMP diversity and demonstrate (i) putative AMP group profiles that are influenced by nematode lifestyle where free-living nematodes appear to display enriched putative AMP profiles relative to parasitic species; (ii) major differences in the putative AMP profiles between nematode clades where Clade 9/V and 10/IV species possess expanded putative AMP repertoires; (iii) AMP groups with highly restricted profiles (e.g. Cecropins and Diapausins) and others [e.g. Nemapores and Glycine Rich Secreted Peptides (GRSPs)] which are more widely distributed; (iv) complexity in the distribution and abundance of CSαß subgroup members; and (v) that putative AMPs are expressed in host-facing life stages and biofluids of key nematode parasites. These data indicate that phylum Nematoda displays diversity in putative AMPs and underscores the need for functional characterisation to reveal their role and importance to nematode biology and host-nematode-microbiome interactions.

Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle.

BACKGROUND: Ruminants can utilize urea as a dietary nitrogen source owing to their ability to recycle urea-N back to the rumen where numerous ureolytic bacteria hydrolyze urea into ammonia, which is used by numerous bacteria as their nitrogen source. Rumen ureolytic bacteria are the key microbes making ruminants the only type of animals independent of pre-formed amino acids for survival, thus having attracted much research interest. Sequencing-based studies have helped gain new insights into ruminal ureolytic bacterial diversity, but only a limited number of ureolytic bacteria have been isolated into pure cultures or studied, hindering the understanding of ureolytic bacteria with respect to their metabolism, physiology, and ecology, all of which are required to effectively improve urea-N utilization efficiency. RESULTS: We established and used an integrated approach, which include urease gene (ureC) guided enrichment plus in situ agarose microsphere embedding and cultivation under rumen-simulating conditions, to isolate ureolytic bacteria from the rumen microbiome. We optimized the dilutions of the rumen microbiome during the enrichment, single-cell embedding, and then in situ cultivation of microsphere-embedded bacteria using dialysis bags placed in rumen fluid. Metabonomic analysis revealed that the dialysis bags had a fermentation profile very similar to the simulated rumen fermentation. In total, we isolated 404 unique strains of bacteria, of which 52 strains were selected for genomic sequencing. Genomic analyses revealed that 28 strains, which were classified into 12 species, contained urease genes. All these ureolytic bacteria represent new species ever identified in the rumen and represented the most abundant ureolytic species. Compared to all the previously isolated ruminal ureolytic species combined, the newly isolated ureolytic bacteria increased the number of genotypically and phenotypically characterized ureolytic species by 34.38% and 45.83%, respectively. These isolated strains have unique genes compared to the known ureolytic strains of the same species indicating their new metabolic functions, especially in energy and nitrogen metabolism. All the ureolytic species were ubiquitous in the rumen of six different species of ruminants and were correlated to dietary urea metabolism in the rumen and milk protein production. We discovered five different organizations of urease gene clusters among the new isolates, and they had varied approaches to hydrolyze urea. The key amino acid residues of the UreC protein that potentially plays critical regulatory roles in urease activation were also identified. CONCLUSIONS: We established an integrated methodology for the efficient isolation of ureolytic bacteria, which expanded the biological resource of crucial ureolytic bacteria from the rumen. These isolates play a vital role in the incorporation of dietary nitrogen into bacterial biomass and hence contribute to ruminant growth and productivity. Moreover, this methodology can enable efficient isolation and cultivation of other bacteria of interest in the environment and help bridge the knowledge gap between genotypes and phenotypes of uncultured bacteria. Video abstract.

Beehives possess their own distinct microbiomes.

BACKGROUND: Honeybees use plant material to manufacture their own food. These insect pollinators visit flowers repeatedly to collect nectar and pollen, which are shared with other hive bees to produce honey and beebread. While producing these products, beehives accumulate a considerable number of microbes, including bacteria that derive from plants and different parts of the honeybees' body. Whether bacteria form similar communities amongst beehives, even if located in close proximity, is an ecologically important question that has been addressed in this study. Specific ecological factors such as the surrounding environment and the beekeeping methods used can shape the microbiome of the beehive as a whole, and eventually influence the health of the honeybees and their ecosystem. RESULTS: We conducted 16S rRNA meta-taxonomic analysis on honey and beebread samples that were collected from 15 apiaries in the southeast of England to quantify the bacteria associated with different beehives. We observed that honeybee products carry a significant variety of bacterial groups that comprise bee commensals, environmental bacteria and symbionts and pathogens of plants and animals. Remarkably, this bacterial diversity differs not only amongst apiaries, but also between the beehives of the same apiary. In particular, the levels of the bee commensals varied significantly, and their fluctuations correlated with the presence of different environmental bacteria and various apiculture practices. CONCLUSIONS: Our results show that every hive possesses their own distinct microbiome and that this very defined fingerprint is affected by multiple factors such as the nectar and pollen gathered from local plants, the management of the apiaries and the bacterial communities living around the beehives. Based on our findings, we suggest that the microbiome of beehives could be used as a valuable biosensor informing of the health of the honeybees and their surrounding environment.

Agrifood and net zero.

We consider the science, policy, and implementation (science-policy-society interface) issues around the agrifood system and the UK's transition to net zero. We conclude that agrifood policy should become more targeted, and the marriage of top-down and bottom-up approaches is key to co-create a pathway that is plausible for each stakeholder.

Pre-Weaned Calf Rearing on Northern Irish Dairy Farms-Part 2: The Impact of Hygiene Practice on Bacterial Levels in Dairy Calf Rearing Environments.

Pre-weaned dairy calves are very susceptible to disease in the first months of life due to having a naïve immune system and because of the numerous physiological stressors they face. Hygiene management is a key element in minimizing enteric disease risk in calves by reducing their exposure to pathogens. Samples of milk, concentrate feed and drinking water, boot swabs of bedding and swabs of feed equipment were collected from 66 dairy farms as part of a survey of calf rearing practice and housing design. All the samples were cultured to determine total viable counts (TVC), total coliforms (TCC) and Escherichia coli as indicators of hygiene. Target ranges for levels of TVC, TCC and E. coli were defined from the literature and the sample results compared against them. The TVC targets in milk, MR and water were <4.0 log10 CFU/mL. TCC and E. coli targets of <1.1 log10 CFU/mL (the detection limit) were used for milk, MR, concentrate feed and feeding equipment. For water, the TCC and E. coli targets were <1.0 log10 CFU/100 mL. The targets used for bedding boot swabs were <6.3 log10 TVC CFU/mL and <5.7 log10 TCC or E. coli CFU/mL. Farm management factors were included as fixed effects in a generalized linear mixed model to determine the probability of samples being within each hygiene indicator target range. Milk replacer samples obtained from automatic feeders were more likely to be within the TVC target range (0.63 probability) than those prepared manually (0.34) or milk samples taken from the bulk tank (0.23). Concentrate feed samples taken from buckets in single-calf pens were more likely to have E. coli detected (0.89) than samples taken from group pen troughs (0.97). A very small proportion of water samples were within the indicator targets (TVC 9.8%, TCC 6.0%, E. coli 10.2%). Water from self-fill drinkers had a lower likelihood of being within the TVC target (0.03) than manually filled buckets (0.14), and water samples from single pens were more likely to be within TCC target ranges (0.12) than those from group pens (0.03). However, all self-fill drinkers were located in group pens so these results are likely confounded. Where milk feeders were cleaned after every feed, there was a greater likelihood of being within the TVC target range (0.47, compared with 0.23 when not cleaned after every feed). Detection of coliforms in milk replacer mixing utensils was linked with reduced probability of TVC (0.17, compared with 0.43 when coliforms were not detected) and TCC (0.38, compared with 0.62), which was within target in feeders. Key factors related to increased probability of bedding samples being within TCC target range were use of group calf pens (0.96) rather than single-calf pens (0.80), use of solid floors (0.96, compared with 0.76 for permeable floors) and increased space allowance of calves (0.94 for pens with ≥2 m2/calf, compared with 0.79 for pens with <2 m2/calf). Bedding TVC was more likely to be within the target range in group (0.84) rather than in single pens (0.66). The results show that hygiene levels in the calf rearing environment vary across farms and that management and housing design impact hygiene.

Implications of Organic Dairy Management on Herd Performance and Milk Fatty Acid Profiles and Interactions with Season.

Interest in organic cows' milk has increased due to the perceived superior nutritional quality and improved sustainability and animal welfare. However, there is a lack of simultaneous assessments on the influence of organic dairy practices and dietary and breed drivers on productivity, feed efficiency, health parameters, and nutritional milk quality at the herd level. This work aimed to assess the impact of organic vs. conventional management and month on milk yield and basic composition, herd feed efficiency, health parameters, and milk fatty acid (FA) composition. Milk samples (n = 800) were collected monthly from the bulk tanks of 67 dairy farms (26 organic and 41 conventional) between January and December 2019. Data on breed and feeding practices were gathered via farm questionnaires. The samples were analyzed for their basic composition and FA profile using Fourier transform infrared spectroscopy (FTIR) and gas chromatography (GC), respectively. The data were analyzed using a linear mixed model, repeated measures design and multivariate redundancy analysis (RDA). The conventional farms had higher yields (kg/cow per day) of milk (+7.3 kg), fat (+0.27 kg), and protein (+0.25 kg) and higher contents (g/kg milk) of protein, casein, lactose, and urea. The conventional farms produced more milk (+0.22 kg), fat (+8.6 g), and protein (+8.1 g) per kg offered dry matter (DM). The organic farms produced more milk per kg of offered non-grazing and concentrate DM offered, respectively (+0.5 kg and +1.23 kg), and fat (+20.1 g and +51 g) and protein (+17 g and +42 g). The organic milk had a higher concentration of saturated fatty acid (SFA; +14 g/kg total FA), polyunsaturated fatty acid (PUFA; +2.4 g/kg total FA), and nutritionally beneficial FA alpha linolenic acid (ALNA; +14 g/kg total FA), rumenic acid (RA; +14 g/kg total FA), and eicosapentaenoic acid (EPA; +14 g/kg total FA); the conventional milk had higher concentrations of monounsaturated FA (MUFA; +16 g/kg total FA). Although the conventional farms were more efficient in converting the overall diet into milk, fat, and protein, the organic farms showed better efficiency in converting conserved forages and concentrates into milk, fat, and protein as a result of reduced concentrate feeding. Considering the relatively small differences in the FA profiles between the systems, increased pasture intake can benefit farm sustainability without negatively impacting consumer nutrition and health.

Influence of dietary oils rich in omega-6 or omega-3 fatty acids on rumen microbiome of dairy cows.

The objective of this study was to compare the effect of supplementing dairy cow diets with contrasting sources of omega-6 (soybean oil) and omega-3 (fish oil) PUFA on rumen microbiome. For 63 d, 15 mid-lactating cows were fed a control diet (n = 5 cows; no fat supplement) or control diet supplemented with 2.9% dry matter (DM) of either soybean oil (SO; n = 5 cows) or fish oil (FO; n = 5 cows). Ruminal contents were collected on days 0, 21, 42, and 63 for 16S rRNA gene sequencing. Beta diversity and Shannon, Simpson and Chao1 diversity indices were not affected by dietary treatments. In terms of core microbiome, Succiniclasticum, Prevotella, Rikenellaceae_RC9_gut_group, and NK4A214_group were the most prevalent taxa regardless of treatments. Bifidobacterium was absent in SO diet, Acetitomaculum was absent in FO, and Sharpea was only detected in SO. Overall, results showed that at 2.9% DM supplementation of either SO or FO over 63 days in dairy cow diets does not cause major impact on bacterial community composition and thus is recommended as feeding practice.

Opportunities and challenges of using metagenomic data to bring uncultured microbes into cultivation.

Although there is now an extensive understanding of the diversity of microbial life on earth through culture-independent metagenomic DNA sequence analyses, the isolation and cultivation of microbes remains critical to directly study them and confirm their metabolic and physiological functions, and their ecological roles. The majority of environmental microbes are as yet uncultured however; therefore, bringing these rare or poorly characterized groups into culture is a priority to further understand microbiome functions. Moreover, cultivated isolates may find utility in a range of applications, such as new probiotics, biocontrol agents, and agents for industrial processes. The growing abundance of metagenomic and meta-transcriptomic sequence information from a wide range of environments provides more opportunities to guide the isolation and cultivation of microbes of interest. In this paper, we discuss a range of successful methodologies and applications that have underpinned recent metagenome-guided isolation and cultivation of microbe efforts. These approaches include determining specific culture conditions to enrich for taxa of interest, to more complex strategies that specifically target the capture of microbial species through antibody engineering and genome editing strategies. With the greater degree of genomic information now available from uncultivated members, such as via metagenome-assembled genomes, the theoretical understanding of their cultivation requirements will enable greater possibilities to capture these and ultimately gain a more comprehensive understanding of the microbiomes. Video Abstract.

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.

Performance and milk quality parameters of Jersey crossbreds in low-input dairy systems.

Previous work has demonstrated some benefit from alternative breeds in low-input dairying, although there has been no systematic analysis of the simultaneous effect of Jersey crossbreeding on productivity, health, fertility parameters or milk nutritional quality. This work aimed to understand the effects of, and interactions/interrelations between, dairy cow genotypes (Holstein-Friesian (HF), Holstein-Friesian × Jersey crossbreds (HF × J)) and season (spring, summer, autumn) on milk yield; basic composition; feed efficiency, health, and fertility parameters; and milk fatty acid (FA) profiles. Milk samples (n = 219) and breed/diet data were collected from 74 cows in four UK low-input dairy farms between March and October 2012. HF × J cows produced milk with more fat (+ 3.2 g/kg milk), protein (+ 2.9 g/kg milk) and casein (+ 2.7 g/kg milk); and showed higher feed, fat, and protein efficiency (expressed as milk, fat and protein outputs per kg DMI) than HF cows. Milk from HF × J cows contained more C4:0 (+ 2.6 g/kg FA), C6:0 (+ 1.9 g/kg FA), C8:0 (+ 1.3 g/kg FA), C10:0 (+ 3.0 g/kg FA), C12:0 (+ 3.7 g/kg FA), C14:0 (+ 4.6 g/kg FA) and saturated FA (SFA; + 27.3 g/kg milk) and less monounsaturated FA (MUFA; -23.7 g/kg milk) and polyunsaturated FA (- 22.3 g/kg milk). There was no significant difference for most health and fertility parameters, but HF × J cows had shorter calving interval (by 39 days). The superior feed, fat and protein efficiency of HF × J cows, as well as shorter calving interval can be considered beneficial for the financial sustainability of low-input dairy farms; and using such alternative breeds in crossbreeding schemes may be recommended. Although statistically significant, it is difficult to determine if differences observed between HF and HF × J cows in fat composition are likely to impact human health, considering average population dairy fat intakes and the relatively small difference. Thus, the HF × J cow could be used in low-input dairying to improve efficiency and productivity without impacting milk nutritional properties.

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.

Microbiome-derived antimicrobial peptides offer therapeutic solutions for the treatment of Pseudomonas aeruginosa infections.

Microbiomes are rife for biotechnological exploitation, particularly the rumen microbiome, due to their complexicity and diversity. In this study, antimicrobial peptides (AMPs) from the rumen microbiome (Lynronne 1, 2, 3 and P15s) were assessed for their therapeutic potential against seven clinical strains of Pseudomonas aeruginosa. All AMPs exhibited antimicrobial activity against all strains, with minimum inhibitory concentrations (MICs) ranging from 4-512 µg/mL. Time-kill kinetics of all AMPs at 3× MIC values against strains PAO1 and LES431 showed complete kill within 10 min to 4 h, although P15s was not bactericidal against PAO1. All AMPs significantly inhibited biofilm formation by strains PAO1 and LES431, and induction of resistance assays showed no decrease in activity against these strains. AMP cytotoxicity against human lung cells was also minimal. In terms of mechanism of action, the AMPs showed affinity towards PAO1 and LES431 bacterial membrane lipids, efficiently permeabilising the P. aeruginosa membrane. Transcriptome and metabolome analysis revealed increased catalytic activity at the cell membrane and promotion of ß-oxidation of fatty acids. Finally, tests performed with the Galleria mellonella infection model showed that Lynronne 1 and 2 were efficacious in vivo, with a 100% survival rate following treatment at 32 mg/kg and 128 mg/kg, respectively. This study illustrates the therapeutic potential of microbiome-derived AMPs against P. aeruginosa infections.