Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

The turkey microbiome is largely understudied, despite its relationship with bird health and growth, and the prevalence of human pathogens such as Campylobacter spp. In this study we investigated the microbiome within the small intestine (SI), caeca (C), large intestine (LI), and cloaca (CL) of turkeys at 6, 10, and 16 weeks of age. Eight turkeys were dissected within each age category and the contents of the SI, C, LI, and CL were harvested. 16S rDNA based QPCR was performed on all samples and samples for the four locations within three birds/age group were sequenced using ion torrent-based sequencing of the 16S rDNA. Sequencing data showed on a genus level, an abundance of Lactobacillus, Streptococcus, and Clostridium XI (38.2, 28.1, and 13.0% respectively) irrespective of location and age. The caeca exhibited the greatest microbiome diversity throughout the development of the turkey. PICRUSt data predicted an array of bacterial function, with most differences being apparent in the caeca of the turkeys as they matured. QPCR revealed that the caeca within 10 week old birds, contained the most Campylobacter spp. Understanding the microbial ecology of the turkey gastrointestinal tract is essential in terms of understanding production efficiency and in order to develop novel strategies for targeting Campylobacter spp.

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.

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.

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Alzheimer’s disease (AD) is a disorder associated with progressive degeneration of memory and cognitive function. Galantamine is a licenced treatment for AD but supplies of the plant alkaloid that it is produced from, galanthamine, are limited. This three-year system study tested the potential to combine Narcissus-derived galanthamine production with grassland-based ruminant production. Replicate plots of permanent pasture were prepared with and without bulbs of Narcissus pseudonarcissus sown as lines into the sward. Two different fertiliser regimes were imposed. The above-ground green biomass of N. pseudonarcissus was harvested in early spring and the galanthamine yield determined. In the second harvest year a split-plot design was implemented with lines of N. pseudonarcissus cut annually and biennially. All plots were subsequently grazed by ewes and lambs and animal performance recorded. Incorporation of N. pseudonarcissus into grazed permanent pasture had no detrimental effects on the health or performance of the sheep which subsequently grazed the pasture. There was no consistency to the effects of fertiliser rates on galanthamine yields. There was no difference in overall galanthamine yield if N. pseudonarcissus was cut biennially (1.64 vs. 1.75 kg galanthamine/ha for annual combined vs biennial cuts respectively; s.e.d = 0.117 kg galanthamine/ha; ns). This study verified the feasibility of a dual cropping approach to producing plant-derived galanthamine.

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, 2 and 3) were effective against numerous bacterial pathogens, including methicillin resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

33 Herein we report the identification and characterisation of two linear antimicrobial peptides 34 (AMPs), HG2 and HG4, with activity against a wide range of multidrug resistant (MDR) 35 bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains, a highly 36 problematic group of Gram-positive bacteria in the hospital and community environment. To 37 identify the novel AMPs presented here, we employed the classifier model design, a feature 38 extraction method using molecular descriptors for amino acids for the analysis, visualization, 39 and interpretation of AMP activities from a rumen metagenomic dataset. This allowed for the in 40 silico discrimination of active and inactive peptides in order to define a small number of 41 promising novel lead AMP test candidates for chemical synthesis and experimental evaluation. In 42 vitro data suggest that the chosen AMPs are fast acting, show strong biofilm inhibition and 43 dispersal activity and are efficacious in an in vivo model of MRSA USA300 infection, whilst 44 showing little toxicity to human erythrocytes and human primary cell lines ex vivo.45 Observations from biophysical AMP-lipid-interactions and electron microscopy suggest that 46 the newly identified peptides interact with the cell membrane and may be involved in the 47 inhibition of other cellular processes. Amphiphilic conformations associated with membrane 48 disruption are also observed in 3D molecular modelling of the peptides. HG2 and HG4 both 49 preferentially bind to MRSA total lipids rather than with human cell lipids indicating that HG4 50 may form superior templates for safer therapeutic candidates for MDR bacterial infections. 52Author Summary 53 We are losing our ability to treat multidrug resistant (MDR) bacteria, otherwise known as 54 superbugs. This poses a serious global threat to human health as bacteria are increasingly 55 acquiring resistance to antibiotics. There is therefore urgent need to intensify our efforts to 56 develop new safer alternative drug candidates. We emphasise the usefulness of complementing 57 wet-lab and in silico techniques for the rapid identification of new drug candidates from 58 environmental samples, especially antimicrobial peptides (AMPs). HG2 and HG4, the AMPs 59 identified in our study show promise as effective therapies for the treatment of methicillin 3 60 resistant Staphylococcus aureus infections both in vitro and in vivo whilst having little 61 cytotoxicity against human primary cells, a step forward in the fight against MDR infections. 4 62 63 The decline in effective treatment strategies for multidrug resistant (MDR) bacterial infections 64 due to the problem of antibacterial resistance threatens our ability to treat infections now and 65 in the future, and calls for an urgent need to explore new safe drug candidates and alternative 66 treatment strategies 1 . The MDR Gram positive bacteria, methicillin resistant Staphylococcus 67 aureus (MRSA), a human opportunistic pathogen, has become a leading causative agent of 68 hospita...