- Volume 1, Issue 1A, 2019

There is a need for novel classes of antimicrobials to be discovered in order to tackle the growing challenges of antimicrobial resistance. Deep-sea sponges are drawing much attention due to the phylogenetically diverse and dense communities of microbes that live within their tissues. Bioprospecting these sponges offers the possibility of exploring a niche environment that could contain novel classes of antimicrobials. To assess the suitability of Pheronema carpenteri (class Hexactinellida, order Amphidiscosida) and Rhabdodictyum sp. (class Hexactinellida, order Lyssacinosida) as a source of antimicrobials, cultivation-dependent strategies were employed. We assess the culturability of sponge-associated bacterial from P. carpenteri (n=3) and Rhabdodictyum sp. (n=2) using8 treatments; 4 temperature incubation treatments (4, 15, 22–25 and 28 °C), nutritional additives (Sponge spicule extract and a low nutrient heterotrophic media additive), and finally a 24 h enrichment stage. Recovered isolates were screen recovered sponge associated-bacteria isolates for bioactivity against Escherichia coli and Micrococcus luteus. Isolates demonstrating high activity were then tested against 7 clinically relevant pathogens; Staphylococcus aurerus 6571, Streptococcus pyogenes, E. coli 1077, Salmonella enterica Serovar Typhimurium LT2, Klebsiella pneumonia 681, Mycoccoccus phlei and Candida albicans. More isolates were recovered from Rhabdodictyum sp. than P. carpenteri (P<0.005). Isolates recovered from P. carpenteri demonstrate high antibacterial activity against both Gram-positive and Gram-negative strains. 112 isolates in total were found to be bioactive against M. luteus, 55 of which were active against both M. luteus and E. coli. The highest potion of bioactive compounds derived from a 15°C treatment and from the inclusion of Sponge Spicule Extract as a nutritional additive. This research presents the first attempts of bioprospecting these two species of deep-sea sponges and thus far has shown promise in their suitability.

Anaerobic rumen fungi (phylum Neocallimastigomycota) occupy the gastrointestinal tract of several herbivorous animals, and by using their powerful hydrolytic enzymes and mechanical forces they degrade plant material in the rumen, essential for rumen efficiency. The rumen microbiome represents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including antimicrobial peptides (AMPs). AMPs are promising drug candidates, and are necessary for targeting the worldwide issue of antimicrobial resistance. Rumen fluid and faecal samples were collected from various large herbivores, and fungal cultures were grown and maintained under anaerobic conditions. After roll tube culture to isolate single-zoospore cultures, sequencing of LSU was undertaken to identify the fungi to species level. Analysis of genomic data from these cultures, alongside published data was undertaken to explore the diversity of AMPs within these fungal genomes. Using functional and computational screening, potentially novel AMPs have been discovered, with isolates showing encouraging activity against some strains of bacteria. Findings indicate that the rumen microbiome may provide alternative antimicrobials for future therapeutic application.

Antimicrobial resistance (AMR) is becoming the biggest substantial threat for public health and societal implications worldwide. Coincidentally, the development of new antibiotics has decreased and downsized by the pharmaceutical companies for the last 40 years. The past decade has witnessed an increasing effort worldwide on exploration of plant-based natural products for new antimicrobial agents. The purpose of this study is to investigate the potential antibacterial activity of traditional herbal medicinal plant against resistant pathogens of public health and economic importance such as methicillin-resistant Staphylococcus aureus (MRSA) and Acine to bacter Baumannii. The broth microdilution method was used to determine the susceptibility of resistant pathogenic strains selected from WHO priority list. A total of 57 plants were chosen based on traditional knowledge and current scientific information that has been claimed to have antimicrobial activity. Preliminary results showed 75 % of screened plants inhibited the growth MRSA (NCTC 12493) some of which exhibited similarantibacterial efficiency compared to the vancomycin (positive antibiotic control), while 21 % shows an inhibitory effect against Acinetobacter Baumannii (NCTC 12156). In addition, significant synergy was observed between some of the plant extracts and vancomycin against MRSA. Further studies are needed for the warrant of these plant candidates to be further developed into therapeutic antimicrobial agents of required efficacy and safety.

The current threat of antimicrobial resistance, the surge in antimicrobial compounds rendered obsolete and the slow emergence of new classes of antibiotics havetriggeredan urgent call for novel alternatives to treat infectious diseases. The vast microbial diversity of unexplored environments and the chemical and structural variety of specialised metabolites within it stand as one of the central points to tackle this challenge. This work focuses on the exploration of the potential for antimicrobial compounds in three new Streptomyces strains – Streptomyces sp. CG885, Streptomyces sp. CG893 and Streptomyces sp. CG926- isolated in the Natural National Park Los Nevados (Colombia) and with proved production of active metabolites against several ESKAPE pathogens. To this purpose, we constructed a 16S rRNA phylogenetic tree and studied the specialised metabolite potential of these isolates using a genome mining approach [1] to predict the presence of putative Biosynthetic Gene Clusters (BGCs). Findings from this bioinformatic prediction were linked with analysis from active extracts obtained through agar plate extraction to inform prioritization of clusters. So far, the computational analysis has predicted between 68 to 93 specialised metabolite BGCs for each strain. Interestingly, most of these compounds show less than 15 % similarity to any known compound in the database and around 69 clusters seem related to previously uncharacterised RiPPs, NRPS and PKS. Further work will focus onthe validation and study of those clusters linked to compounds with potential in clinical development.

The biosynthesis of silver nanoparticles extracellularly using baker’s yeast, Saccharomyces cerevisiae and its antibacterial activity was investigated in this study. Biosynthesised silver nanoparticles were chaterized by using UV-Visible spectroscopy, which showed a distinct observed absorption peak at 429.00 nm that is attributed to the plasmon resonance of silver nanoparticles; X-ray diffraction, which determined the average size of the silver nanoparticles to be approximately 16.07 nm; presence of oval shaped silver nanoparticles determined by scanning electron microscopy; and Fourier-transform infrared, which revealed notable peaks at 3332.2, 2903.6 and 1636.3 cm−1 corresponding to the binding of the silver nanoparticles to active biomolecules, alcohols and phenols, carboxylic acids and aromatic amines respectively. The silver nanoparticles were also found to be stable for ninety days. Antibacterial activity of the silver nanoparticles was also studied. The silver nanoparticles was significantly active (P>0.05) against the test organisms at an extract concentration of 75 µg ml−1. Concentrations less than or equal to 50 µg ml−1 were not as effective as the colony forming units at this concentration, 1.61×106 for methicillin-resistant Staphylococcus aureus and 1.45×106 for Pseudomonas aeruginosa respectively were about the same range a small the colony forming units of the controls. The silver nanoparticles inhibited methicillin-resistant S. aureusmore than they inhibited P. aeruginosa. The LD50 of the synthesized silver nanoparticles after oral administration was seen to be greater than 5000 mg kg−1 body weight and is therefore thought to be safe. This study supports the use of silver nanoparticles as therapeutic agents.

Derivatives of the secondary metabolites of Streptomyces bacteria account for over half of the antibiotics currently used in the clinic. A crucial part of overcoming antimicrobial resistance (AMR) is in the development of new antibiotics that function with a novel mechanism of action, to avoid the rapid acquisition of microbial resistance. We previously reported the isolation of the new species Streptomyces formicae, from African Tetraponera penzigiplant-ants, shown to have potent activity against several strains on the World Health Organisation’s AMR watch list including MRSA. Genetic analysis of this strain revealed one of its 45 biosynthetic gene clusters, a type 2 polyketide synthase, produced all 13 formicamycins, the natural products responsible for S. formicae’s antimicrobial activity. Several regulators of the formicamycin biosynthetic pathway have been identified, including the major repressor (ForJ). Having purified ForJ, DNA binding assays were performed based on previous cappable-RNA and ChIP sequencing experiments to confirm the interaction of ForJ with different putative promoters in the cluster. By optimising gene-reporter fusion assays in this novel strain, it has been possible to characterise some of the promoters in relation to ForJ to determine the effect of its binding upon activity of the gene cluster. Such work is essential in the progression of novel antimicrobials from the laboratory into a clinic as it must be thoroughly understood how a compound is synthesised and how its biosynthetic pathway is regulated before it can be exploited for overproduction.

The discovery of streptomycin by Selman Waksman (1943) brought into focus a new avenue of drugs from natural products, i.e., actinobacterial secondary metabolites. It forms more than 60 % of total bacterial secondary metabolites (mostly from Streptomyces). Interestingly, rare Actinobacteria can produce novel secondary metabolites with unique chemical structures. With the rise of drug resistant microbes, focus on actinobacterial research has shifted towards exploring unusual niches such as those located along land-ocean boundary where freshwater mixes with saltwater. One such ecosystem is the Sundarbans mangrove, located at the apex of Bay of Bengal. The present study aims to identify novel actinobacterial species from Sundarbans which has the ability to produce unique secondary metabolites. Mangrove sediments were collected, overall bacterial diversity and secondary metabolites producing bacterial diversity were elucidated by 16S rRNA and polyketide synthase (PKS) clone library approaches respectively. Fifteen bacterial strains were isolated from the sediment using cultured approach, among which, an isolate I2 was identified based on polyphasic taxonomy. The I2 represents a new species, Myceligenerans indicum sp. nov. This new species also possess PKS genes which indicate ability to produce secondary metabolite(s). Promising antibacterial activity of this new species was found against Escherchia coli XL10, Bacillus subtilis and Vibrio chemaguriensis Iso1 especially for fraction prepared using acetone and dichloromethane (1 : 1). Spectroscopic approaches have revealed the presence of functional groups such as amide, allene, isothiocyanate and ketenimine groups.

The study of biogeography enables an understanding of the distribution patterns of biodiversity across space and time [1]. Therefore, byusing a trait-based approach, such as antibiotic production, it is possible to assess the evolutionary, geographic and ecological variables that affect the Actinobacteria specialized metabolism [2, 3]. This is particularly important as Actinobacteria isolated from marine ecosystems have been shown to be a promising source of new drugs [4, 5]. In this study, a comparative metabolomics approach using molecular networking was applied to understand the role of biogeography on the specialized metabolism of Micrococcus spp. and Pseudonocardia spp. isolated from Arctic and Antarctic marine sediments. The LC-MS/MS analysis showed differences in the specialized metabolism of phylogenetically related strains isolated from different geographic regions. These preliminary results suggest an influence on the microbial chemical space through assessing biogeographic impact. Future work on further marine ecosystems will expand our knowledge on the relationship between the chemistry and ecology of rare Actinobacteria.

The synthesis of hydrogel scaffolds with inherent antimicrobial activity has advantages for their use in tissue engineering. An ultra-short naphthalene lysine conjugated peptide, NapFFK’K’, containing naphthalene (Nap) as a molecule of high aromaticity for gel strength, phenylalanine (F) and epsilon variant lysine (K’) has previously been shown by us to self-assemble forming hydrogels with inherent antimicrobial properties against a limited number of pathogens tested. The aim of this work was to extend the antimicrobial activity studies on NapFFK’K’ including pathogenic bacteria associated with dental infections. NapFFK’K’ was synthesised using the 9-fluorenylmethoxucarbonyl Solid Phase Peptide Synthesis. Peptide purity was analysed by mass spectrometry. Hydrogel formulation was achieved by suspending the peptide in sterile deionized water followed by addition of NaOH and HCl. Hydrogels were tested at peptide concentrations of 1 %, 1.5 % and 2 % w/v against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus, and the Gram-negative bacterium Fusobacterium nucleatum. Bacteria inoculumns were exposed on hydrogel surface for 24 h. Bacterial susceptibility assay, employing the Miles and Misra method, was used to determine antimicrobial activity of hydrogels after 24 h incubation. Our results show that peptide hydrogels exhibit antimicrobial properties against both groups of bacteria but at different peptide concentrations. The 1 % peptide hydrogels was most effective against Gram-positive bacteria whereas the 2 % peptide hydrogel was effective against Fusobacterium nucleatum. Given the efficacy of the self-assembling NapFFK’K’ peptide hydrogels against oral pathogens, they may have potential use in tissue engineering approaches for regenerative endodontic treatments.

This project aims to develop Chaplins, functional amyloid proteins from Streptomyces sp., into a novel nano-thin adhesive material for the defence industry through combining existing protein-based technology with a composite partner. Chaplin proteins were extracted from a range of wild-type strains, while a synthetic promoter system was developed to express and secrete chaplins, which are typically difficult to express and purify. Chaplin proteins were found to have potentially useful adhesive properties and we have been investigating their application in optically transparent adhesives, since adhesives currently used to bond laminated transparent materials are prone to water ingress resulting in degradation of optical clarity and delamination. These adhesives could also be lighter, thinner, and have a lower environmental impact. To determine the potential of this material as a novel synbio-adhesive for bonding glass and polycarbonate we have investigated the bonding ability and transparency of natural and engineered amyloid proteins, both alone and with partner biopolymers and bulking materials. We are now exploring ways to improve the properties of the proteins by modifying the amino acid sequences, incorporating binding domains to the composite partner and via chemical modifications. Our projects has provided proof-of-concept for the use of bio-inspired amyloid protein-based materialscompounds as both adhesives and corrosion resistant metal coatings. Supported by Defence and Security Accelerator (DASA), Defence Science and Technology Laboratory (Dstl) project grants CDE100367 and ACC101824.

The purpose of this research is to further develop existing technology using engineered functional amyloid proteins for corrosion resistance by developing a coating which provides both adhesion to substrate and corrosion resistance. Pathogenic amyloid proteins have been attributed a role in certain diseases such as Alzheimer’s Disease. However, the research undertaken here involves non-pathogenic functional bacterial amyloids, using coelicolor hydrophobic aerial proteins (chaplins or Chp) produced by Streptomyces for improving corrosion resistance. Chaplins have been proven conceptually to provide corrosion resistance properties on metals and, also provide strong adhesive properties in a multi-composite systems. The composition, application and curing of the better bonding chaplin composite will now be optimised for development of a better performing functional coating, which includes inclusion of small metabolites or natural products acting as corrosion inhibitors. This would then allow for benchmarking against existing corrosion-resistant coatings. Upon determining the most relevant formulae, samples will be analysed by using microscopic techniques such as Scanning Electron Microscopy and Atomic Force Microscopy, while corrosion will be tested using immersion tests, weathering via salt-spray and in-situ scanning vibrating electrode.

Biosurfactants (BS) are amphiphilic molecules produced as a secondary metabolite by various bacteria and yeast species and are secreted extracellularly. BS have shown to work in synergy with antibiotics and also demonstrate strong antimicrobial and anti-adhesive characteristics. This coupled with their low toxicity makes them suitable candidates as combination therapies to combat skin infections. In this study, the aim was to investigate the in-vitro antimicrobial and anti-biofilm properties of mannosylerythritol lipids (MELs) produced by Moesziomyces aphidis against Staphylococcus aureus DSM-20231, Streptococcus pyogenes ATCC-19615, Staphylococcus epidermidis DSM – 28319, Pseudomonas aeruginosa DSM-3227, Escherichia coli ATCC – 25 922 and Propionibacerium acnes DSM- 1897. MELs are most predominantly used in skin creams, thus, a rationale was developed to investigate antibiotics used to treat bacterial skin infections, namely, Polymyxin B Sulphate, Neomycin, Mupirocin and Bacitracin. Minimum inhibitory concentration (MIC) values where determined for each antibiotic and BS per bacterium using the broth dilution technique based on CLSI guidelines. BS where extracted by solvent extraction and characterised using Mass Spectrometry – High Performance Liquid Chromatography, standards were quality assured using MALDI-TOF. Flow cytometry determined percentage dead versus alive for each antibiotic, BS and combination of antibiotic and BS. Scanning Electron Microscopy determined the effect of the BS on the bacterial cell walls. This study proves that BS work synergistically with antibiotics to increase the MIC of the antibiotics resulting in a substantial decrease in antibiotic use and at lower concentration. The use of BS combination therapy has the potential to reduce resistant rates and also lengthen the time taken for resistance to develop.

Fusobacterium necrophorum is a strictly anaerobic, non-spore-forming, Gram-negative Bacillus encompassing two subspecies. Fusobacterium necrophorum subsp. necrophorum (FNN) is commonly found in animals, where it is the causative agent of foot rot and abscesses. Fusobacterium necrophorum subsp. fundiliforme (FNF) is found in humans and is the causative agent of Lemierre’s disease, a condition associated with throat infections. Little is currently known about the genomic diversity of the two subspecies. Whole-genome sequences were generated for 18 Fusobacterium necrophorum isolates recovered from recurrent and persistent sort throats in UK patients. Sixty-three whole genome sequences available for Fusobacterium spp. were downloaded from GenBank and compared with the newly sequenced isolates. All-against-all comparisons of average nucleotide identity (ANI) were done to confirm species affiliation. Strains of FNN (n=6) and FNF (n=43) shared ∼97 % ANI with each other, while strains of the same subspecies shared >98 % with one another. All the newly sequenced isolates belonged to FNF. All 49 Fusobacterium necrophorum sensu stricto genomes were subject to comparative and pangenome analyses. The strains fell into two groups, with the majority of strains clustering with the type strain of FNF. Three distinct clusters of strains were identified within the FNF group, and analyses are underway to determine their potential clinical significance. Analyses of virulence-associated leukotoxin genes are also being undertaken. It is envisaged that in-depth analyses of a large collection of Fusobacterium necrophorum genomes, particularly those of FNF, will provide novel insights into pathogenesis of these fastidious anaerobic bacteria.

The human gastrointestinal tract represents a collection of prokaryotic and eukaryotic microorganisms. It has been reported that gut microbiota plays a role in health and disease. Imbalance of gut microbial communities has been implicated in several non-communicable diseases including inflammatory gastrointestinal diseases, Type 2 diabetes, and obesity. However, a role of gut microbiota in adult Southeast Asians remains largely unexplored. Herein, we present gut microbiota data obtained from Thai volunteers living in Chiang Rai Province, Thailand. We use a combination of quantitative PCR, Next Generation Sequencing (NGS), and gas chromatography-mass spectrometry (GC-MS) to investigate microbial communities, individual microbial taxa, and metabolites of NCDs volunteers. We consider prokaryotic taxa and their relationships with body mass index/waist to hip ratio and diet. We find that specific taxa of lower taxonomic levels are associated with the lean and overweight, but not the obese phenotype. We also find that microbial prokaryotic communities differ significantly amongst the different study groups.

Chlamydiae are obligate intracellular bacteria with highly reduced genomes, reflective of their abilities to sequester nutrients from the host. The host range of this phylum far exceeds that described for the Chlamydia genus which encompasses terrestrial animals; however, much of this diversity is uncultivated. Of particular evolutionary significance are members of the earliest diverging family, Candidatus Parilichlamydiaceae, comprising only uncultivated species associated with the fish gill disease, epitheliocystis. Epitheliocystis poses a significant threat to aquaculture industries globally. Due to the lack of culture systems for Ca. Parilichlamydiaceae, little is known about their biology, which also imposes further limitations on answering epidemiological questions. Gill extracts from three Chlamydiales-positive Australian aquaculture species were subject to DNA preparation to deplete host DNA and enrich microbial DNA, prior to metagenome sequencing. We obtained three metagenome-assembled Ca. Parilichlamydiaceae genomes from three different host species, and conducted functional genomics comparisons with diverse members of the phylum. Using these genomes, we developed a novel Ca. Parilichlamydia carangidicola-specific multi locus sequence analysis (MLSA) scheme to investigate genetic diversity in this species Genomic analysis revealed highly reduced genomes (∼0.8 Mbp) that share 342 orthologs with other chlamydial families, with a notable reduction in genes for synthesis of nucleotides and amino acids. MLSA revealed a high level of genetic diversity of Ca. P. carangidicola, with 20 genotypes distributed among 29 samples from four populations within the same aquaculture facility. Culture-independent genomics has provided an unprecedented insight into chlamydial evolution, pathogenicity and epidemiology. This approach could be adapted for further genomic epidemiological investigations.

Diarrhoeal disease remains a major cause of child morbidity, growth faltering and mortality in low and middle income countries (LMICs), with Campylobacter among the most common causes. Previous work has identified the major sources in the UK and US (e.g. contaminated poultry), however little is known about the risk factors and transmission routes in LMICs, where incidence is extremely high (up to 85 % of children infected before 1 year) and suggests a different epidemiology. Risk factors such as household crowding, poor sanitation, consumption of contaminated water and cohabitation with animals, all constitute potential transmission risks but their relative importance is unknown. This is a major concern as frequent or chronic enteric (re)infection is linked to significant morbidity and growth faltering in children. Comparative genomics offers a solution for untangling complex transmission networks. Campylobacter jejuni and C. coli primarily inhabit the gut of birds and mammals and signatures of adaptation can be detected in their genomes. Therefore, by sequencing the genome of human clinical isolates and faecally contaminated environments it is possible to discern its origin. Pilot genomics studies of strains from humans, animals and food in LMICs have identified genomic variation in strains that may indicate differences in source, survival, transmission or virulence (compared to the UK). In particular we have identified globally distributed strains and lineages; country-specific strains; rapid dissemination of accessory genes in specific regions; evidence of within-household spread and strains associated with asymptomatic infection or high levels of antimicrobial resistance.

Exploring and understanding the phylogeny of the Butyrivibrio group is imperative if we are ever to fully understand the consortium of ruminal microbial enzymes that are responsible for the catalysis of multifaceted reactions, such as biohydrogenation. At present, taxonomic classification of the Butyrivibrio group is based primarily on butyrate production. This approach has become antiquated with the development of sequencing technologies and downstream bioinformatics analysis. This study investigated the taxonomic relatedness and functional capacity of the ruminal Butyrivibrio group using 72 genomes. Seventy-one Butyrivibrio group genomes were obtained via JGI (the Hungate 1000 project), and one additional bacterial strain was sequenced by ourselves. A 40 marker phylogenetic tree was constructed and visualised with the interactive Tree Of Life (iTOL), and pangenome analysis conducted using Spine/ClustAGE. Orthologous gene affiliations were identified using OrthAgogue, and glycosyl hydrolase families were identified using dbCAN then aligned with Clustal Omega. Data obtained showed that three primary clades were observed, namely the genus Pseudobutyrivibrio, B. fibrisolvens, and the remaining Butyrivibrio species. Pangenome analysis and orthologous gene affiliations revealed greater diversity within Butyrivibrio than Pseudobutyrivibrio. Butyrivibrio clades consistently showed smaller core genome sizes in comparison to Pseudobutyrivibrio, with core genome percentages as low as 4 %, indicating high levels of variance. Glycosyl hydrolase alignment shows extensive sequence dissimilarity between genes on a nucleotide and amino acid level These findings suggest that the Butyrivibrio group are highly evolved to maintain competitiveness in the rumen and emphasises the need for further research into the biochemical capacity of the Butyrivibrio group.

The issue of genome minimization for a living cell has been the subject of intense study, and raises important theoretical questions and practical opportunities. We have developed an in-silico methodology, based on genome-scale models, to minimize the number of active genes encoding enzymes and transporters in a cell’s metabolic network. In the resultant minimal metabolic networks, all the remaining active genes are essential for keeping the network working to achieve the biomass value predicted by Flux Balance Analysis. We have tested our approach on a set of genome-scale metabolic models of various eukaryotic and prokaryotic organisms, but have focussed on Saccharomyces cerevisiae. The nutrient environments employed have comprised both known and automatically generated sets of nutrients and relative maximum uptake rates. The results generate more than 1000 unique minimal networks for a single condition, demonstrating the complexity of the minimization problem. We then performed a frequency analysis on the affected genes to discover patterns or similarities among the networks and the redundancy of specific pathway-related genes. In addition to this, we also evaluated the networks using a pathway-oriented robustness analysis, to see how the networks respond to random variation in the reaction fluxes. Finally, we have done a cross-species comparison of our algorithm’s results to highlight some of the homologous genes that are retained in the networks of a majority of species. Thus, our work has produced a tool for in silico genome minimization that permits the discovery of mandatory genes in the minimal metabolic networks.