- Volume 1, Issue 1A, 2019

Histomonas meleagridis is a protozoan parasite that causes mortality and morbidity in a wide range of gallinaceous fowl. It most notably affects turkeys, causing 80–100 % mortality in a flock. There is no commercial treatment for this parasite at the moment and attempts at a vaccine have failed. In this study we investigated the potential efficacy of novel rumen and chicken gastrointestinal tract microbiome-derived antimicrobial peptides (AMPs) against H.meleagridis. H. meleagridis was cultivated in growth flasks in Dwyer media. The concentration of the parasite was determined using a Haemocytometer, the cells were counted under the microscope. The AMP challenges were carried out in 96-well plates. The starting concentration of the AMPs were 1024 µg ml−1 down to 2 µg ml−1. The cell densities of H. meleagridis were checked at 24 h. The protozoal cell densities were checked by extracting DNA and performing qPCRs targeted against FeHyD and Rpb1 genes. Fluorescent microscopy was also used to check parasite densities as described above. Five Chicken and three Rumen AMPs resulted in decreases in protozoal cell densities following microscopy, suggesting that the peptides show potential therapeutic application for blackhead disease. Nonetheless, the qPCR primers were non-specific and resulted in bacterial DNA amplification. Consequently, in order to understand the biological function of H. meleagridis and design new qPCR primers allowing quantification, we isolated the macronuclei of a H. meleagridis strain followed by sequencing using the illumine Hi-Seq 2500 and paired ends. Future work will focus on confirming how well the AMPs work agent H. meleagridis by employing electron microscopy to observe how the AMP affect the cells.

Amoebic Gill Disease (AGD) is a major problem in the aquaculture industry, as it is responsible for substantial losses of farmed Atlantic salmon in various worldwide locations. The disease is caused by the usually free-living Paramoeba perurans compromising the gills through the resulting development of hyperplastic lesions and lamellar fusion. These structural changes result in a reduction in the functional surface area of the gill tissues. Recent research has focused on identifying bacteria present within a culture of P. perurans, through performing isolation and identification of bacteria present in the cultures using 16S sequencing. Further NGS sequencing was performed from various culture conditions to provide insight into the changes of the bacterial microbiome during amoeba culture. As attempts to isolate the amoeba from the bacterial contamination has been unsuccessful, consideration into a possible symbiotic relationship between the amoeba and bacteria was considered. A filtering method was used to attempt to identify the genera of bacteria present within the amoeba. The isolation and 16S sequencing identified the presence of various marine bacteria, including those of the Pseudoalteromonas, Halomonas, Cellulophaga and Mesonia genera. The NGS sequencing identified a substantial proportion of sequences to match the Vibrio genus and suggests an association between this genus and the amoeba. If symbiotic relationships between specific bacteria and amoeba can be confirmed, the bacteria could potentially be used as an indicator organism for the risk of AGD outbreak. It may also provide an indirect target for the control and treatment of AGD.

The gastro-intestinal tract hosts a complex microbial ecosystem that helps regulate the physiological, immunological and nutritional functions of the pig so disturbances within this microbiota can have profound effects on porcine health and disease. The gut microbiota is shaped by the environment, immune pressures and diet. The weaning transition represents a critical time-point that can interfere with intestinal development and cause dramatic shifts in the gut microbiota. Previously, in-feed antibiotics were used to counteract the adverse effects of weaning but an EU-wide ban since 2006 has propelled the search for safe and sustainable alternatives within the livestock industry. Studies have indicated that dietary fatty acids are efficient in terms of minimising weaning disorders including elevated incidence of enteric disease and immunodepression and thus represent a promising alternative to antibiotics. Furthermore, previous research has indicated that including fatty acid mixtures in the porcine diet has a positive effect on overall animal performance, increasing growth rate and enhancing feed conversion ratios. It is thought that these health benefits and production gains stem from the ability of fatty acids to mediate the effects of gut microbiota on intestinal immune function through modifying the proportions of microorganisms present. The aim of this research was to use deep sequencing and metagenomic methods to explicitly explore the influence fatty acid supplementation has on shaping the intestinal microbiome during weaning. Results have indicated that fatty acids play a role in modifying the intestinal microflora, establishing an environment that favours the growth of commensal species such as Lactobacillus.

Hantaviruses are a diverse group of single-stranded, negative sense RNA viruses, belonging to the Bunyaviridae family. They are primarily rodent-borne and transmitted into humans through the inhalation of aerosolised excreta of infected animals. Each hantavirus is typically associated with a single reservoir species, resulting in a persistent, yet asymptomatic infection. In humans, however, in addition to asymptomatic infection two disease syndromes are associated with hantavirus infection, hantavirus haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Tatenale Hantavirus (TATV) was first isolated from field voles in NW England in 2013 and has since been detected at other sites in Northern England. However, only two small fragments of the L and S segments have been sequenced, precluding full phylogenetic characterisation of the virus. Field voles were sampled at two sites in Leicestershire (N=104) and Cheshire (N=12), and their lungs subjected to a degenerate pan-hantavirus RT-PCR assay, targeting a section of the polymerase gene. Eleven Cheshire and one Leicestershire sample were TATV positive, and were 94 % homologous across the amplicon. Illumina Hi-Seq sequencing revealed the complete coding regions of the Leicestershire isolate. Analysis of the sequence showed that TATV is most closely related to Khabarovsk virus, with a homology of 91%/88%/89 % (AA) across the L/M/S Segments, respectively. ICTV species demarcation requires an AA difference of >7 % across the S and M, TATV meets the requirement of a novel species. Retrieval of full coding sequence will allow further investigation into TATV, and its potential to infect humans through pseudotyping of the glycoproteins.

Enteric fever is major health issue in developing countries and it is becoming progressively untreatable due to increase of antimicrobial resistance. The causative agent, Salmonella enterica, replicates in host phagocytes in various organs and regulates expression of hundreds of genes in response to host signals. PhoPQ is one of the key regulators and essential for virulence in humans and in a mouse typhoid fever model. The sensor kinase PhoQ responds to diverse stimuli (Mg2+ limitation, low pH, cationic antimicrobial peptides, high osmolarity, and, indirectly, to reducing conditions). However, which signals are predominant in vivo remains unclear. To address this issue, we determined the activity of the PhoPQ regulon using a chromosomally encoded fusion of the PhoP-dependent phoNp promoter to gfp-ova and we quantified single-cell fluorescence levels of Salmonella in spleen of infected mice using flow cytometry. The results show extensive heterogeneity in PhoP-activities in the Salmonella population. Comparison of mutants with sensing defects suggests that, in vivo, PhoQ responds to a combination of antimicrobial peptides, acidic pH and low Mg2+, but not to reducing conditions. Negative feedbacks have also only a minor impact. Single-cell analysis of phoNp and asrp promoters suggests differential environmental pH as a major driver of heterogeneous PhoP activities. A combination of immunohistochemistry and proteomics of Salmonella from infected samples was used to validate this hypothesis. Together, our data show how Salmonella uses the PhoPQ system to integrate various host signals in order to tune expression of virulence factors to the diverse tissue microenvironments that this pathogen inhabits.

Mycobacterium bovis, a member of the M. tuberculosis complex, causes bovine tuberculosis, one of the most important veterinary health problems in the UK. In the absence of improved control the projected economic burden in the UK over the next decade is predicted to be £1bn. Although transmission of M. bovis occurs primarily between infected mammalian hosts, M. bovis has been demonstrated to persist in soil, suggesting an environmental infectious route. M. bovis is likely predated on by environmental amoebae such as Dictyostelium discoideum and as such may have evolved mechanisms to modulate the interaction with amoebae. In this study we have investigated M. bovis interactions in vitro with D. discoideum. We demonstrate that virulent M. bovis evades destruction by D. discoideum. Using a genome-wide M. bovis transposon mutant library, we selected for mutants that failed to escape D. discoideum after 48 h of infection. Mutants of genes encoding the MCE4 transport system, genes involved in sulpholipid synthesis/transport, and genes encoding PPE and PE-PGRS protein families remained associated with D. discoideum. Most strikingly, mutations in 11 genes of the major mycobacterial virulence locus ESX-1, which encodes a T7 secretion system implicated in bacterial transit through host cell membranes, were significantly enriched in D. discoideum. Our data demonstrate that known virulence factors involved in host-pathogen interactions in mammalian hosts also play a role in D. discoideum-M. bovis interactions. Our data further suggest that M. bovis has evolved to actively transit bacteriverous D. discoideum rather that to use it as a replicative niche.

Within a large-scale report commissioned by the Wellcome Trust [1], primary school teachers were described as facing barriers in teaching science. The top barriers described were the lack of budget and resources, lack of time and curricular importance as well as other issues such as a lack of subject knowledge or confidence and concerns relating to space and resource access. Teaching science is just one part of a primary teacher’s complex role and is a subject in which most primary teachers do not have a degree or A level qualification [2,3]. There is little Microbiology content within the National Curriculum, however, schools can introduce additional scientific content within the Primary Key Stages. Within this context, a Microbiology, Genomics and Bioinformatics researcher in association with Key Stage 2 classes in a Norfolk Junior School carried out a joint project for Microbiology-related science in conjunction with Norwich Research Park facilities in May 2017 and the Microbiology Society. Here we report on the findings from teacher and pupil’s perspectives and consider how Microbiology/Hygiene could be presented to this age group in a classroom setting.

1. ‘State of the Nation’ report of UK primary science education. S. Leonardi et al. CFE Research, Leicester LE1 5TE. September 2017.

2. The Royal Society (2010) Science and mathematics education 5–14. A ‘state of the nation’ report. London: the Royal Society

3. ASE Guide to Primary Science Education (Serret and Earle, 2018), reviewed in https://tdtrust.org/cpd-primaryscience

Antibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.

Actinobacteria are ubiquitous in soil and well-known for producing antimicrobial compounds. Increasingly, members of this phylum are found to form symbiotic relationships, for example with plants and insects, and are thought to provide protection against host infection. However, it remains poorly understood how Actinobacteria are recruited to microbiomes and whether secondary metabolites are produced in vivo. Acromyrmex echinatior leaf cutter ants transmit Pseudonocardia bacteria between generations and also recruit Streptomyces to their cuticular microbiome. We show that Pseudonocardia species isolated from the ant cuticle inhibit the fungal nest pathogen Escovopsisweberi and dual RNA-sequencing confirmed that Pseudonocardia secondary metabolite gene clusters are expressed in vivo on the ant cuticle. RNA stable isotope probing showed that ants supply cuticular resources to their microbiome which may fuel interference competition and select for antibiotic-producing bacteria. Similar to leaf cutter ants we also show that plant roots recruit growth-promoting and antibiotic-producing Streptomyces bacteria, but appear not to transmit them via their seeds. Root exudates are hypothesized to play a major role in root microbiome recruitment and DNA stable isotope probing coupled with Illumina sequencing showed that these were actively metabolized by many bacterial genera. However, Streptomyces appeared to be outcompeted by more abundant Proteobacteria, despite the fact that isolates could grow on purified exudates in the absence of competition. Streptomyces root exudate preferences are now being evaluated using comparative metabolomics. Defining the factors that influence the competitiveness of protective bacteria when colonizing microbiomes has implications for the development of more consistent biocontrol strategies and prebiotic techniques.

Streptomyces species are important producers of bioactive compounds such as antibiotics, antitumor and immunosuppressant drugs. Around two-thirds of all known natural antibiotics are produced by these bacteria and antibiotic production is linked to sporulation. The discovery of new bioactive compounds has declined since the 1960s but genome sequencing has revealed the potential to identify many more bioactive compounds. They are many secondary metabolite gene clusters which are inactive (silent) under laboratory conditions. We characterize the highly conserved actinobacterial two component system MtrAB which coordinates sporulation with secondary metabolite production in the two model organisms Streptomyces venezuelae and S. coelicolor. Deletion of the histidine kinase gene mtrB resulted in increased production of the antibiotic chloramphenicol in S. venezuelae and actinorhodin and undecylprodigiosin in S. coelicolor. Chloramphenicol is not usually produced under laboratory condition which suggests that deleting mtrB can activate silent antibiotic clusters. Additionally, we introduced point mutations at the D56 phosphorylation site of MtrA by CRISPR-Cas9 to abolish phosphorylation. This mutant shows the same phenotype as the ΔmtrA strain and chloramphenicol production is increased. Chromatin immunoprecipitation and sequencing (ChIP-seq) was used to identify MtrA targets and revealed that MtrA likely controls secondary metabolite production by binding to the promoter regions of cluster situated regulators in both model organisms. Additionally, MtrA binds upstream of genes involved in DNA replication and cell division. To our knowledge this is the first evidence of the connection of development and secondary metabolite production by a two component system.

Aurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis, has been shown to inhibit the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS). To further assess the utility of this molecule as an anti-virulence compound, a better understanding of its mechanism of action is required. We used whole transcriptome analysis, cell infection and GFP-reporter assays to show that Aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island-which encodes for the T3SS, acting via its master regulator, Ler. We have also observed similar effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). These properties suggest Aurodox may have potential for the treatment of E. coli infections of the gut. Despite the recent interest in the compound, the biosynthesis of Aurodox by Streptomyces goldiniensis is still poorly understood. To gain insight in to this, we have sequenced the whole genome of S. goldiniensis and identified a putative Aurodox biosynthetic gene cluster (BGC) which shares a high level of functional homology with the BGC encoding Kirromycin, a non-methylated Aurodox derivative. In-depth analysis of the BGC supports a model where a unique polyketide synthase pathway involving a combination of both Cis and Trans-Acyltransferases synthesise the Aurodox polyketide backbone, followed by decoration and finally the addition of a methyl group. Future work will include the heterologous expression this BGC to confirm its role in Aurodox biosynthesis, with the ultimate aim to produce novel Aurodox derivatives.

Finding new antimicrobial compounds is vital to combat the growing threat of resistance. Most currently used antibiotics originate from actinomycetes discovered more than half a century ago. We recently reported the new species Streptomyces formicae, isolated from the African fungus-farming plant-ant, Tetraponera penzigi. S. formicae produces a novel family of polyketide antibiotics, the formicamycins, that have potent activity against resistant pathogens including MRSA and vancomycin-resistant Enterococci (VRE). Using CRISPR/Cas9, we have identified and characterised the genes responsible for formicamycin biosynthesis in the native producer. In addition, we used cappable RNA- and ChIP-sequencing to determine the transcriptional organisation of the pathway. We exploited this information to generate multiple mutants of S. formicae that overproduce formicamycins and their biosynthetic intermediates, some of which also have bioactivity. Furthermore, the potential for novel chemistry from S. formicae is not limited to the formicamycin pathway; antiSMASH analysis shows this talented strain contains at least 45 secondary metabolite biosynthetic gene clusters (BGCs). Under standard laboratory conditions, wild-type S. formicae also exhibits antifungal activity against the drug resistant Lamentospora prolificans, and when the formicamycin BGC is deleted, the strain produces even more potent antibacterial activity against MRSA. To identify the biosynthetic pathways for these metabolites, entire BGCs up to 208 kbp were deleted using CRISPR. Overall, this work demonstrates that searching under-explored environments for new species combined with genome editing is a promising route towards finding novel anti-invectives.

Microorganisms have been increasingly exploited for their remarkable ability to produce diverse natural products, which display bioactivities ranging from antimicrobial and anticancer to signalling and developmental. Genome sequencing has revealed that bacteria harbour many more biosynthetic gene clusters (BGCs) for natural products than are currently characterised. Whilst several genome mining tools have been developed to aid discovery, some classes of BGCs remain difficult to identify. One such class is ribosomally-synthesised and post-translationally modified peptides (RiPPs). RiPP BGCs are small with few regions of homology, and the short precursor peptides are rarely annotated in genomes. After developing a targeted genome mining approach for RiPPs, we identified a novel family of BGCs spanning over 200 actinobacterial genomes. The presence of diverse biosynthetic enzymes and sequence variation of the precursor peptides suggest that these BGCs may produce several structurally diverse molecules. We have successfully TAR cloned one such BGC from Streptomycesand expressed it heterologously, and metabolomic analysis led to identification of the pathway product. Gene deletion experiments have also confirmed the involvement of individual biosynthetic enzymes. The compound has been purified and the structure elucidated by NMR. Further work will focus on exploring RiPP BGCs from other species and investigating the biological role of these molecules. The discovery of such a diverse and highly conserved group of BGCs highlights that we are still scratching the surface of the huge biosynthetic capacity of microorganisms, and the use of more sophisticated genome mining tools could help unveil many more important molecules in the future.

The large amount of data from inexpensive sequencing means that the number of putative biosynthetic gene clusters (BGCs) far exceeds our ability to experimentally characterize them. This necessitates the need for development of further tools to analyze putative BGCs to flag those of interest for further characterization. clusterTools implements a framework to aid in the in silico characterization of BGCs by identifying regions of the DNA, containing homologous proteins, or coding sequences containing specific functional domain compositions using user-built HMM rules, in close proximity, reporting results in an easy to visualize manner. clusterTools complements existing software for BGC analysis in two ways. First, by running clusterTools on databases of genomic sequences in an exploratory mode, the user can identify and download regions of interest in the DNA for further processing and annotation in programs such as antiSMASH. Second, if clusterTools is run on databases constructed from putative gene clusters generated by antiSMASH, one can rapidly identify clusters on interest from the group that warrant further analysis and experimental characterization. We demonstrate the use of clusterTools as part of our workflow to identify BGCs of specific classes of natural products that would be difficult to identify with existing methods, particularly clusters containing assembly line domains as components, including those involved in bacterial polyketide alkaloid biosynthesis. clusterTools can also be used to identify novel BGCs by incorporating regulatory and antibiotic resistance elements. Standalone versions of clusterTools are available for Macintosh, Windows, and Linux.

The natural product, prodigiosin, caught researchers’ interest more than a century ago because of its bright red color. Today, interest in this tripyrrolic secondary metabolite remains strong due to its biological effects, including potent antibiotic activity against various Gram-positive bacteria. Further exploration of the potential of this class of molecules requires libraries of analogues. Yet, the total synthesis of prodigiosin-like compounds (prodiginines) proves challenging. This can be overcome by highjacking the bacterial biosynthetic machinery via mutasynthesis. Although a number of different bacteria produce prodigiosin, its biosynthetic pathway is well conserved. The final precursors, MAP and MBC, are always produced independently, before a terminal condensation reaction. Our work focuses on the last step of the formation of MAP: the oxidation of H2MAP. We were able to isolate and characterize HapB, the enzyme catalyzing this reaction in Hahella chejuensis. In addition, we showed that some modifications of alkyl substituents on the C2 and C3 positions of H2MAP did not alter HapB activity significantly. We then fed these analogues of H2MAP into Serratia ΔpigD, a mutant of Serratia ATCC sp.39006 which does not produce any endogenous H2MAP. As expected from the in vitro testing, chain elongation past two carbons on the C2 position could not be accepted whereas all substrates with a modification on the C3 position restored pigmentation, leading to the formation of eight novel prodiginines.

Non-ribosomal peptide synthetases (NRPSs) are responsible for the natural in vivo production of a large number of therapeutically relevant compounds. The non-ribosomal peptides (NRPs) formed by their action are the product of the long modular assembly lines, whilst the terminal step in the biosynthesis is the hydrolytic release, and frequently, macrocyclisation of the aminoacyl-S-thioester by an embedded thioesterase (TE). The surugamide biosynthetic pathway is composed of two NRPS assembly lines. One produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded TE, which led us to question how the peptides are released from the assembly line (and cyclised in the case of surugamide A). We characterised an alpha/beta hydrolase and β-lactamase in vivo and established that the former is a type II TE for surugamide A, but not surugamide F, and that the latter is a trans-acting release factor for both compounds. In vitro substrate utilisation assays unambiguously established that the β-lactamase can produce mature surugamides A and F from N-acetylcysteamine (SNAC) thioester mimics of the cognate terminal biosynthetic intermediates. Using bioinformatics, we estimate that ∼12 % of filamentous Actinobacteria harbor an NRPS system lacking an embedded TE and instead use a trans-acting β-lactamase release strategy. This expands the paradigmatic understanding of how non-ribosomal peptides are released from the terminal NRPS module and adds a new dimension to the synthetic biology toolkit, potentially useful in the search for novel antibiotics.

Microbially-derived antimicrobial compounds are a rich source of clinical antibiotic leads. However, discovery rates have declined over the past 40 years due, in part, to high rediscovery rates of known compounds from traditional soil-based screening approaches. In this study, an ancient hot-spring water source was tested for the presence of antimicrobial-producing bacteria using culture techniques which led to isolation of two organisms capable of inhibiting the growth of multiple bacterial species. Oxford Nanopore whole genome sequencing was used to identify these two isolates as being in one of two genera; Streptomyces and Paenibacillus. Bioinformatic analysis revealed both isolates to have multiple novel secondary metabolite gene clusters. Investigations of the Streptomyces sp. by natural product chemistry techniques showed the organism to produce multiple antimicrobial compounds, these were effective methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. This study underlines the value of investigating non-traditional habitats in the search for novel antibiotic-producing organisms.

Herein we report the identification and characterisation of two linear antimicrobial peptides (AMPs), HG2 and HG4, with activity against a wide range of multi-drug resistant (MDR) bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains, a highly problematic group of Gram-positive bacteria in the hospital and community environment. To identify the novel AMPs presented here, we employed the classifier model design, a feature extraction method using molecular descriptors for amino acids for the analysis, visualization, and interpretation of AMP activitiesfrom a rumen metagenomic dataset. This allowed for the in silicodiscrimination of active and inactive peptides in order to define a small number of promising novel lead AMP test candidates for chemical synthesis and experimental evaluation. In vitrodata suggest that the chosen AMPs are fast acting, show strong biofilm inhibition and dispersal activity and are efficacious in an in vivomodel of MRSA USA 300 infection, whilst showing little toxicity to human erythrocytes and human primary cell lines ex vivo. Observations from biophysical AMP-lipid-interactions and electron microscopy suggest that the newly identified peptides interact with the cell membrane and may be involved in the inhibition of other cellular processes. Amphiphilic conformations associated with membrane disruption are also observed in 3D molecular modelling of the peptides. HG2 and HG4 both preferentially bind to MRSA total lipids rather than with human cell lipids indicating that HG4 may form superior templates for safer therapeutic candidates for MDR bacterial infections.