A Synthetic Polymicrobial Community Biofilm Model Demonstrates Spatial Partitioning, Tolerance to Antimicrobial Treatment, Reduced Metabolism, and Small Colony Variants Typical of Chronic Wound Biofilms.

Understanding chronic wound infection is key for successful treatment and requires accurate laboratory models. We describe a modified biofilm flow device that effectively mimics the chronic wound environment, including simulated wound fluid, a collagen-based 3D biofilm matrix, and a five-species mixture of clinically relevant bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Citrobacter freundii). Mixed biofilms were cultured for between 3 and 14 days with consistent numbers of bacteria that exhibited reduced metabolic activity, which increased with a high dose of glucose. S. aureus was recovered from biofilms as a small colony variant, but as a normal colony variant if P. aeruginosa was excluded from the system. Bacteria within the biofilm did not co-aggregate but formed discrete, species-specific clusters. Biofilms demonstrated differential tolerance to the topical antimicrobials Neosporin and HOCl, consistent with protection due to the biofilm lifestyle. The characteristics exhibited within this model match those of real-world wound biofilms, reflecting the clinical scenario and yielding a powerful in vitro tool that is versatile, inexpensive, and pivotal for understanding chronic wound infection.

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.

Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata.

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host-parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host-parasite interaction within the horse host.

Comparative Genomics and Pan-Genomics of the Myxococcaceae, including a Description of Five Novel Species: Myxococcus eversor sp. nov., Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis sp. nov., Myxococcus vastator sp. nov., Pyxidicoccus caerfyrddinensis sp. nov., and Pyxidicoccus trucidator sp. nov.

Members of the predatory Myxococcales (myxobacteria) possess large genomes, undergo multicellular development, and produce diverse secondary metabolites, which are being actively prospected for novel drug discovery. To direct such efforts, it is important to understand the relationships between myxobacterial ecology, evolution, taxonomy, and genomic variation. This study investigated the genomes and pan-genomes of organisms within the Myxococcaceae, including the genera Myxococcus and Corallococcus, the most abundant myxobacteria isolated from soils. Previously, ten species of Corallococcus were known, whereas six species of Myxococcus phylogenetically surrounded a third genus (Pyxidicoccus) composed of a single species. Here, we describe draft genome sequences of five novel species within the Myxococcaceae (Myxococcus eversor, Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis, Myxococcus vastator, Pyxidicoccus caerfyrddinensis, and Pyxidicoccus trucidator) and for the Pyxidicoccus type species strain, Pyxidicoccus fallax DSM 14698T. Genomic and physiological comparisons demonstrated clear differences between the five novel species and every other Myxococcus or Pyxidicoccus spp. type strain. Subsequent analyses of type strain genomes showed that both the Corallococcus pan-genome and the combined Myxococcus and Pyxidicoccus (Myxococcus/Pyxidicoccus) pan-genome are large and open, but with clear differences. Genomes of Corallococcus spp. are generally smaller than those of Myxococcus/Pyxidicoccus spp. but have core genomes three times larger. Myxococcus/Pyxidicoccus spp. genomes are more variable in size, with larger and more unique sets of accessory genes than those of Corallococcus species. In both genera, biosynthetic gene clusters are relatively enriched in the shell pan-genomes, implying they grant a greater evolutionary benefit than other shell genes, presumably by conferring selective advantages during predation.

The Isolation and Genome Sequencing of Five Novel Bacteriophages From the Rumen Active Against Butyrivibrio fibrisolvens.

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

Predatory Organisms with Untapped Biosynthetic Potential: Descriptions of Novel Corallococcus Species C. aberystwythensis sp. nov., C. carmarthensis sp. nov., C. exercitus sp. nov., C. interemptor sp. nov., C. llansteffanensis sp. nov., C. praedator sp. nov., C. sicarius sp. nov., and C. terminator sp. nov.

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously (Corallococcus coralloides and Corallococcus exiguus). A polyphasic approach, including biochemical analysis of fatty acid methyl esters, substrate utilization, and sugar assimilation assays, was taken to characterize eight Corallococcus species strains and the two type strains. The genomes of all strains, including that of C. exiguus DSM 14696T (newly reported here), shared an average nucleotide identity below 95% and digital DNA-DNA hybridization scores of less than 70%, indicating that they belong to distinct species. In addition, we characterized the prey range and antibiotic resistance profile of each strain, illustrating the diversity of antimicrobial activity and, thus, the potential for drug discovery within the Corallococcus genus. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species. The eight candidates are as follows: Corallococcus exercitus sp. nov. (AB043AT= DSM 108849T = NBRC 113887T), Corallococcus interemptor sp. nov. (AB047AT= DSM 108843T = NBRC 113888T), Corallococcus aberystwythensis sp. nov. (AB050AT = DSM 108846T = NBRC 114019T), Corallococcus praedator sp. nov. (CA031BT= DSM 108841T = NBRC 113889T), Corallococcus sicarius sp. nov. (CA040BT= DSM 108850T = NBRC 113890T), Corallococcus carmarthensis sp. nov. (CA043DT= DSM 108842T = NBRC 113891T), Corallococcus llansteffanensis sp. nov. (CA051BT= DSM 108844T = NBRC 114100T), and Corallococcus terminator sp. nov. (CA054AT= DSM 108848T = NBRC 113892T).IMPORTANCECorallococcus is a genus of predators with broad prey ranges, whose genomes contain large numbers of gene clusters for secondary metabolite biosynthesis. The physiology and evolutionary heritage of eight Corallococcus species strains were characterized using a range of analyses and assays. Multiple metrics confirmed that each strain belonged to a novel species within the Corallococcus genus. The strains exhibited distinct patterns of drug resistance and predatory activity, which mirrored their possession of diverse sets of biosynthetic genes. The breadth of antimicrobial activities observed within the Corallococcus genus highlights their potential for drug discovery and suggests a previous underestimation of both their taxonomic diversity and biotechnological potential. Taxonomic assignment of environmental isolates to novel species allows us to begin to characterize the diversity and evolution of members of this bacterial genus with potential biotechnological importance, guiding future bioprospecting efforts for novel biologically active metabolites and antimicrobials.

Genome Analysis, Metabolic Potential, and Predatory Capabilities of Herpetosiphon llansteffanense sp. nov.

Herpetosiphon spp. are ubiquitous, chemoheterotrophic, filamentous gliding bacteria with the ability to prey on other microbes through a "wolf pack" mechanism. The genus currently comprises four known species (H. aurantiacus, H. geysericola, H. giganteus, and H. gulosus), which produce antimicrobial secondary metabolites such as siphonazole. As part of a study isolating myxobacterial wolf pack predators, we serendipitously isolated a novel environmental strain (CA052B) from the edge of a stream at Llansteffan, United Kingdom, which was identified as a member of the Herpetosiphon genus. A lawn culture method was utilized to analyze the predatory activity of CA052B against 10 prey organisms of clinical relevance. CA052B was found to prey on Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, Bacillus subtilis, and Candida albicans Purified CA052B outer membrane vesicles also exhibited killing activity against the prey organisms when tested by flow cytometry. 16S rRNA sequencing of CA052B showed 98 to 99% identity with other Herpetosiphon species members. Comparing the genome of CA052B with the publicly available genomes of H. aurantiacus and H. geysericola revealed average nucleotide identities of only 84% and 91%, respectively, whereas the genome-to-genome distance calculation showed sequence identities of 28.2% and 46.6%, respectively. Biochemical characterization also revealed distinctions between CA052B and both H. gulosus and H. giganteus Thus, strain CA052BT (= DSM 107618T = NBRC 113495T) is proposed to be the type strain of a novel species, Herpetosiphon llansteffanense sp. nov. The genome sequence of CA052B also revealed diverse secondary metabolite biosynthetic clusters, encouraging further exploration of its antibiotic production potential.IMPORTANCE Predatory bacteria are able to kill and consume other microbes and are therefore of interest as potential sources of new antimicrobial substances for applications in the clinic. "Wolf pack" predators kill prey by secreting antimicrobial substances into their surroundings, and those substances can kill prey organisms independently of the predatory cells. The genus Herpetosiphon exhibits wolf pack predation, yet its members are poorly described compared to other wolf pack predators, such as the myxobacteria. By providing a thorough characterization of a novel Herpetosiphon species, including its predatory, biochemical, and genomic features, this study increases our understanding of genomic variation within the Herpetosiphon genus and how that variation affects predatory activity. This will facilitate future rational exploitation of genus members (and other wolf pack predators) as sources of novel antimicrobials.

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

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.

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis.

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Potential sources of high value chemicals from leaves, stems and flowers of Miscanthus sinensis 'Goliath' and Miscanthus sacchariflorus.

Society demands chemicals from sustainable sources. Identification of commercially important chemicals in crops increases value in biorefineries and reduces reliance on petrochemicals. Miscanthus sinensis and Miscanthus sacchariflorus are high-yielding distinct plants, which are sources of high-value chemicals and bioethanol through fermentation. Cinnamates in leaves, stems and flowers were analysed by LC-ESI-MS(n). Free phenols were extracted and separated chromatographically. More than twenty hydroxycinnamates were identified by UV and LC-ESI-MS(n). Several cinnamate hexosides were detected in the M. sinensis flower and in M. sacchariflorus (leaf and stem). Hydroxybenzoic acids and their hexosides were observed in leaf and stem of M. sacchariflorus. Higher concentrations of 3-feruloylquinic acid were observed in M. sacchariflorus stem, suggesting a role in cell-wall biosynthesis. This technique can be used to screen plants in a mapping family to identify genotypes/species with high concentrations of phenols. Plants with low concentrations of antimicrobial phenols may be good feedstocks for fermentation.

Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts.

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford x Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (-1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer (P<0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher (P<0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass (P<0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.