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.

- 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.

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.

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.

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.

In silico identification of two peptides with antibacterial activity against multidrug-resistant Staphylococcus aureus.

Here we report two antimicrobial peptides (AMPs), HG2 and HG4 identified from a rumen microbiome metagenomic dataset, with activity against multidrug-resistant (MDR) bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) strains, a major hospital and community-acquired pathogen. We employed the classifier model design to analyse, visualise, and interpret AMP activities. This approach allowed in silico discrimination of promising lead AMP candidates for experimental evaluation. The lead AMPs, HG2 and HG4, are fast-acting and show anti-biofilm and anti-inflammatory activities in vitro and demonstrated little toxicity to human primary cell lines. The peptides were effective in vivo within a Galleria mellonella model of MRSA USA300 infection. In terms of mechanism of action, HG2 and HG4 appear to interact with the cytoplasmic membrane of target cells and may inhibit other cellular processes, whilst preferentially binding to bacterial lipids over human cell lipids. Therefore, these AMPs may offer additional therapeutic templates for MDR bacterial infections.

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation.

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

Gut microbiome colonization and development in neonatal ruminants: Strategies, prospects, and opportunities.

Colonization and development of the gut microbiome is a crucial consideration for optimizing the health and performance of livestock animals. This is mainly attributed to the fact that dietary and management practices greatly influence the gut microbiota, subsequently leading to changes in nutrient utilization and immune response. A favorable microbiome can be implanted through dietary or management interventions of livestock animals, especially during early life. In this review, we explore all the possible factors (for example gestation, colostrum, and milk feeding, drinking water, starter feed, inoculation from healthy animals, prebiotics/probiotics, weaning time, essential oil and transgenesis), which can influence rumen microbiome colonization and development. We discuss the advantages and disadvantages of potential strategies used to manipulate gut development and microbial colonization to improve the production and health of newborn calves at an early age when they are most susceptible to enteric disease. Moreover, we provide insights into possible interventions and their potential effects on rumen development and microbiota establishment. Prospects of latest techniques like transgenesis and host genetics have also been discussed regarding their potential role in modulation of rumen microbiome and subsequent effects on gut development and performance in neonatal ruminants.

Long-Term Effects of Dietary Supplementation with Olive Oil and Hydrogenated Vegetable Oil on the Rumen Microbiome of Dairy Cows.

Dietary lipids increase energy density in dairy cow diets and in some cases can increase beneficial fatty acids (FA) in milk and dairy products. However, the degree of FA saturation may affect the rumen microbiome. The objective of this study was to determine the long-term effects of feeding saturated (hydrogenated vegetable oil; HVO) or unsaturated (olive oil; OO) fatty acid (FA) sources on the rumen microbiome of dairy cows. For 63 days, 15 mid-lactating cows were fed with either a basal diet (no fat supplement), or the basal diet supplemented with 3% dry matter (DM), either HVO or OO. Rumen contents were collected on days 21, 42 and 63 for 16S rRNA gene sequencing using the Illumina MiSeq platform. The results reveal dominance of the phyla Firmicutes (71.5%) and Bacteroidetes (26.2%), and their respective prevalent genera Succiniclasticum (19.4%) and Prevotella (16.6%). Succiniclasticum increased with both treatments at all time points. Prevotella was reduced on day 42 in both diets. Bacterial diversity alpha or beta were not affected by diets. Predicted bacterial functions by CowPI showed changes in energy and protein metabolism. Overall, 3% DM of lipid supplementation over 63 days can be used in dairy cow diets without major impacts on global bacterial community structure.

Freeze-dried Nannochloropsis oceanica biomass protects eicosapentaenoic acid (EPA) from metabolization in the rumen of lambs.

Eicosapentaenoic acid (EPA) from freeze-dried biomass of Nannochloropsis oceanica microalgae resists ruminal biohydrogenation in vitro, but in vivo demonstration is needed. Therefore, the present study was designed to test the rumen protective effects of N. oceanica in lambs. Twenty-eight lambs were assigned to one of four diets: Control (C); and C diets supplemented with: 1.2% Nannochloropsis sp. oil (O); 12.3% spray-dried N. oceanica (SD); or 9.2% N. oceanica (FD), to achieve 3 g EPA /kg dry matter. Lambs were slaughtered after 3 weeks and digestive contents and ruminal wall samples were collected. EPA concentration in the rumen of lambs fed FD was about 50% higher than lambs fed SD or O diets. Nevertheless, the high levels of EPA in cecum and faeces of animals fed N. oceanica biomass, independently of the drying method, suggests that EPA was not completely released and absorbed in the small intestine. Furthermore, supplementation with EPA sources also affected the ruminal biohydrogenation of C18 fatty acids, mitigating the shift from the t10 biohydrogenation pathways to the t11 pathways compared to the Control diet. Overall, our results demonstrate that FD N. oceanica biomass is a natural rumen-protected source of EPA to ruminants.

Microbiomes attached to fresh perennial ryegrass are temporally resilient and adapt to changing ecological niches.

BACKGROUND: Gut microbiomes, such as the rumen, greatly influence host nutrition due to their feed energy-harvesting capacity. We investigated temporal ecological interactions facilitating energy harvesting at the fresh perennial ryegrass (PRG)-biofilm interface in the rumen using an in sacco approach and prokaryotic metatranscriptomic profiling. RESULTS: Network analysis identified two distinct sub-microbiomes primarily representing primary (≤ 4 h) and secondary (≥ 4 h) colonisation phases and the most transcriptionally active bacterial families (i.e Fibrobacteriaceae, Selemondaceae and Methanobacteriaceae) did not interact with either sub-microbiome, indicating non-cooperative behaviour. Conversely, Prevotellaceae had most transcriptional activity within the primary sub-microbiome (focussed on protein metabolism) and Lachnospiraceae within the secondary sub-microbiome (focussed on carbohydrate degradation). Putative keystone taxa, with low transcriptional activity, were identified within both sub-microbiomes, highlighting the important synergistic role of minor bacterial families; however, we hypothesise that they may be 'cheating' in order to capitalise on the energy-harvesting capacity of other microbes. In terms of chemical cues underlying transition from primary to secondary colonisation phases, we suggest that AI-2-based quorum sensing plays a role, based on LuxS gene expression data, coupled with changes in PRG chemistry. CONCLUSIONS: In summary, we show that fresh PRG-attached prokaryotes are resilient and adapt quickly to changing niches. This study provides the first major insight into the complex temporal ecological interactions occurring at the plant-biofilm interface within the rumen. The study also provides valuable insights into potential plant breeding strategies for development of the utopian plant, allowing optimal sustainable production of ruminants. Video Abstract.

Can rumen bacteria communicate to each other?

BACKGROUND: The rumen contains a myriad of microbes whose primary role is to degrade and ferment dietary nutrients, which then provide the host with energy and nutrients. Rumen microbes commonly attach to ingested plant materials and form biofilms for effective plant degradation. Quorum sensing (QS) is a well-recognised form of bacterial communication in most biofilm communities, with homoserine lactone (AHL)-based QS commonly being used by Gram-negative bacteria alone and AI-2 Lux-based QS communication being used to communicate across Gram-negative and Gram-positive bacteria. However, bacterial cell to cell communication in the rumen is poorly understood. In this study, rumen bacterial genomes from the Hungate collection and Genbank were prospected for QS-related genes. To check that the discovered QS genes are actually expressed in the rumen, we investigated expression levels in rumen metatranscriptome datasets. RESULTS: A total of 448 rumen bacterial genomes from the Hungate collection and Genbank, comprised of 311 Gram-positive, 136 Gram-negative and 1 Gram stain variable bacterium, were analysed. Abundance and distribution of AHL and AI-2 signalling genes showed that only one species (Citrobacter sp. NLAE-zl-C269) of a Gram-negative bacteria appeared to possess an AHL synthase gene, while the Lux-based genes (AI-2 QS) were identified in both Gram-positive and Gram-positive bacteria (191 genomes representing 38.2% of total genomes). Of these 192 genomes, 139 are from Gram-positive bactreetteria and 53 from Gram-negative bacteria. We also found that the genera Butyrivibrio, Prevotella, Ruminococcus and Pseudobutyrivibrio, which are well known as the most abundant bacterial genera in the rumen, possessed the most lux-based AI-2 QS genes. Gene expression levels within the metatranscriptome dataset showed that Prevotella, in particular, expressed high levels of LuxS synthase suggesting that this genus plays an important role in QS within the rumen. CONCLUSION: This is the most comprehensive study of QS in the rumen microbiome to date. This study shows that AI-2-based QS is rife in the rumen. These results allow a greater understanding on plant-microbe interactions in the rumen.

Rumen Protozoa Play a Significant Role in Fungal Predation and Plant Carbohydrate Breakdown.

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.