- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Extremophiles: Living Life at the Edge
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Motilimonas cestriensis sp. nov., isolated from a Cheshire brine spring
More LessThe Cheshire Salt District (UK) is home to a wide range of largely unexplored inland brine springs, whose increased salinity originates from subterranean Triassic salt rock deposits. Our study focused on the Anderton Brine Spring System, a set of pools of varying salinity which are subjected to regular salinity fluctuations depending on drainage and evaporation (recorded salinities have ranged from 1.2% to 13% NaCl). Preliminary investigation of samples obtained from one of these pools (with 4.2 % NaCl) revealed a wealth of novel isolates which were analysed by 16S rRNA gene sequencing. Within these isolated strains, we have detected that strain MKS20 had a 97 % similarity to its closest relative, Motilimonas eburnea, which is currently the only characterised species in a newly discovered genus of the order Alteromonadales. All reported strains within this genus have been isolated from marine environments, namely marine-sediments and the gut of a sea cucumber. MKS20 is the first strain in the genus to be reported from an inland brine spring and thus significantly extends the ecological range of the genus Motilimonas. The distinctiveness of our strain is further supported by preliminary results from polyphasic taxonomic characterisation (e.g. metabolic and enzymatic profiling). Based on these, strain MKS20 represents a putative new species within the genus for which we propose the name Motilimonas cestriensis, referring to Cheshire, the place of isolation.
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Viable metabolisms in a simulated martian chemical environment
There is evidence that water may exist on Mars as brines in the subsurface. The chemistries of these brines will be greatly influenced by the local lithologies, which would impact on the organisms that could potentially live within them. There are multiple metabolisms that are theoretically viable under martian chemical conditions. In order to better establish which of these are capable of supporting persistent growth under martian conditions, we performed a series of enrichments using four geological simulants for Mars: a global composition, an early and unaltered basaltic composition, a sulfur-rich composition, and a haematite-rich composition. Enrichments were inoculated with sediment from Pyefleet mudflats in the Colne estuary (Essex, UK). Mudflat sediment was added to the simulant materials and a brine based on the chemistry of Rocknest. Enrichments were supplied with a H2/CO2 headspace at 1 bar pressure and incubated for 20 days. The enrichments were repeatedly subcultured into fresh simulant material and brines in order to effectively select for a community actively growing in this chemical environment. The enriched community was characterised through the isolation and identification of microbes, microscopy and the amplicon sequencing of 16S rRNA genes amplified from DNA extracted from each stage of the enrichment. Biotic and abiotic experiments were conducted to identify geochemical changes that occur due to the presence of microbial activity. We will present details on the communities enriched on the different martian simulants, metabolisms identified as present within the actively growing community and geochemical changes that were identified.
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Biomineralization of microbes in natural and restored saltmarshes: a missing link in restoration efforts?
More LessSaltmarshes are threatened coastal ecosystems, which are subject to cyclic variation in conditions which create a gradient-rich environment (e.g. salinity, pH). They support very diverse communities of plants and animals, protect the coast from erosion, and play an important role in element cycling and bio-remediation. The importance of saltmarshes has only been recently recognised, which has led to significant efforts in restoring sites that had been previously converted to Agriculture. Such restoration attempts have had limited success in returning these sites to their original biodiversity and biological structure. While not yet fully understood, it is thought that several factors are at play, including persisting differences in soil structure and quality. Coastal environments have been previously shown to harbour significant microbial populations capable of producing CaCO3 biominerals. On the other side, CaCO3 biomineral production is known to affect the texture and overall properties of soil, a property currently used in the improvement of soils for Agriculture. We suggest that these factors could be linked, and that the limitations of saltmarsh restoration efforts might result from differences in biomineral production. Our results provide an overview on differences between samples that we’ve collected in natural and restored sites, based on cultivation and screening efforts. Our insights might provide an important step forward in saltmarsh restoration and contribute to more successful approaches to protect these vital biotopes and increase biodiversity.
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Metabolic profiling and environmental characterisation of salterns in the islands of Cabo Verde
More LessThe Cabo Verde Islands constitute a biodiversity hotspot, encompassing a wide range of different biotopes. Thus far, most surveys and programmes have focused exclusively on the flora and/or fauna of the islands and no effort has been made to systematically assess and preserve locally existing microbial biodiversity. The absence of such studies is especially troubling regarding their traditional salterns, as many of them have been abandoned, and increase of construction in coastal areas has already started to indelibly change some of these unique ecosystems and will likely obliterate some of them. Here we provide an overview of the preliminary data recently collected from salterns in the islands of Sal, Maio, and Boavista and include details on their previously unreported physical-chemical characteristics (salinity, pH, temperature and ionic composition), as well as first results on microbial metabolic profiling. Our data has shown a wide diversity of environmental niches (particularly noticeable in Maio), and showcased differences in substrate use both between and within different salterns. These are the first step of our ongoing efforts in assisting in the survey of several threatened extreme environments in Cabo Verde, characterising their geochemistry and microbiology, and identifying their potential biotechnological applications.
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Production of cross-domain signalling molecules by Halophilic archaea
More LessCell-cell communication through the production of autoinducer molecules has been widely studied in bacteria and found to play a pivotal role in biofilm formation and gene regulation. Quorum Sensing (QS) within the domain Archaea is understudied compared with their bacterial counterparts. The aim of this study was to determine whether archaea are capable of cross-kingdom signalling through the production of QS inducing compounds and QS inhibitory activities. A combination of culture-dependent (crude extracts from archaeal isolates) and culture-independent (genomic mining) techniques were employed to investigate the production of compounds capable of modulating bacterial QS. Crude archaeal extracts were screened for activity using the bioreporter strains Agrobacterium tumefaciens ATCC BAA-2240, Escherichia coli JM109 pSB536, pSB401 and pSB1142, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa MW-1. Active strains were further characterised using initial bio-assay guided fractionation. Preliminary results revealed different strains were capable of eliciting a QS response in the bacterial bio-reporters. Initial characterisation using LC-MS, TLC-overlays, and other biochemical tests, suggests the possible production of Butyryl Homoserine Lactones or homologs of this molecule by at least one of the strains. Conversely, other halophilic archaea were capable of inhibiting the production of QS-controlled pathways, as demonstrated by the reduction in virulence factor production by P. aeruginosa. Further characterisation of these compounds will prove to be an invaluable insight into the poorly understood mechanisms behind archaeal QS and equally will reveal the role of these compounds in cross-kingdom signalling.
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- Fighting Fire with Fire – Deploying Microbes in the Battle Against Disease
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Identifying and inhibiting co-aggregation occurring between bacterial respiratory pathogens and commensal species associated with the altered microbiota of chronic pulmonary disease to identify the effect on the development of multispecies biofilms in vitro
More LessComparisons of pulmonary microbiotas associated with healthy individuals and cystic fibrosis (CF) patients identified a less diverse microbiota among CF patients comprising numerous bacterial species not found among healthy controls. This research aimed to identify if coadhesion occurs between respiratory pathogens P. aeruginosa and S. aureus and some commensal species associated with the microbiota of CF. This was conducted in vitro by spectrophotometric co-aggregation analysis and multi-species biofilm assays in the presence and absence of an inhibitor of lectin-mediated coaggregation, testing the ability of eleven commensal representative species of the CF pulmonary microbiota. Results show that all commensal species selected to represent genera that are associated with the CF pulmonary microbiota are capable of coaggregation in vitro with respiratory pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. S. aureus co-aggregated with a higher affinity to commensal species than P. aeruginosa with a median coaggregation percentage of 62.9 % after 24 h incubation, compared to 41.9 % in P. aeruginosa. When commensal and pathogenic species were cultured in the presence of lactose, biofilm inhibition was observed in 5 of 12 cultures with P. aeruginosa and 7 of 12 with S. aureus. These results demonstrate that some commensals associated with CF participate in coaggregation with respiratory pathogens in vitro which has the ability to alter biofilm production. Identifying coadhesion between commensal and pathogenic species in vitro suggests that coadhesion could be occurring in vivo potentially causing increased susceptibility of the host by enhancing adhesion to the epithelial surface of the respiratory tract, aiding colonization and therefore increased susceptibility to infections.
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Arabian Sea associated bacterial strains a source of novel antibiotics against superbugs
More LessOne of the most severe health threats to both humans and animals is Antimicrobial resistance development in microorganisms AMR is a significant and growing challenge and to combat this issue new antimicrobials are urgently needed Fifteen microbial strains isolated from Arabian Sea were screened for their capacity to produce antimicrobial metabolites of pharmaceutical interest. These strains were associated to the brown seaweed Pelvetia canaliculata (Linnaeus) attached to the rocks of Sonmiani Beach (Karachi, Pakistan). Bacterial strains were isolated from sea weeds attached to rocks of Baluchistan coast line using marine agar 2216 and screened for antibacterial activity by agar well diffusion method and crude extract was made and antimicrobial metabolites were purified using silica gel column and structure of pure compound was elucidated using spectroscopic techniques. Crude extract filtrates from CMG 2180 strain, grew on ZMA medium, showed the most remarkable antimicrobial activity, and thus was chosen for further examination. The identification of CMG 2180 as a probable new type strain of the Actinobacterium Kocuria marina was based on phenotypic aspects and biochemical characteristics (e.g. halotolerant Gram-positive micrococcoid) as well as on the nucleotide sequence analysis of its full-length 16S rRNA gene showing the highest similarity with the type strain KMM 3905 (GenBank accession number EU073966). Interestingly, a unique UV-bioactive compound, for which the name of kocumarin was proposed, was isolated and purified from CMG 2180 strain’s crude extracts by flash silica gel column chromatography and TLC/HPTLC. Using routine methods, kocumarin demonstrated prominent and rapid activities against all tested fungi and pathogenic bacteria including MRSA. Its chemical structure was unraveled by 1D and 2D-NMR spectroscopy as 4-[(Z)−2 phenyl ethenyl] benzoic acid.
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AMR gene removal by conjugative delivery of CRISPR-Cas9
More LessAntimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed.
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Community assembly in the microbiome: ecological insights into infant microbiome development
More LessIn healthy adults, the gastrointestinal tract harbours a diverse community of microbes that play critical roles in health and wellbeing. However, we are not born with this microbiome. Over the first months and years of life the gut microbiome gradually develops, undergoing a process akin to classic primary succession. We here develop ecological theory in order to study the factors that drive these assembly processes. We find that interactions between species can enforce order on microbiome development, with interspecies dependencies driving the predictability of succession. We combine this theory with novel sample processing techniques to interrogate both bacterial and fungal microbiome development in premature infants. We utilize machine learning to infer how members of the microbiome are affected by both one another and clinical interventions. Preliminary results identify specific inter-microbial interactions that may in part drive community dynamics, and identify the impact of antibiotic interventions in perturbing healthy microbiome development. Taken together, these results highlight the importance of disentangling microbial interactions if we are to understand and ultimately manipulate our microbiome communities.
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Isolation of novel bacteriocins active against clinically relevant, pathogenic Clostridioides difficile
More LessThe emergence of antibiotic resistant, hypervirulent strains of the nosocomial pathogen Clostridioides difficile, coupled with increased concerns over the physiological impact of traditional broad-spectrum antibiotics on human health has led to an increased demand for alternative therapies. Bacteriocins are proteinaceous toxins produced by bacteria that kill other bacteria and their antimicrobial activity makes them an attractive potential alternative therapy to antibiotics. This research aims to isolate environmental bacteria from porcine faeces that produce novel bacteriocins with inhibitory activity against C. difficile. At present in this study two distinct organisms with protein mediated inhibitory activity against C. difficile have been isolated and it has been shown that these strains encode various bacteriocins. The current focus of this research aims to attribute the inhibitory activity to one of the encoded bacteriocins or potentially a novel antimicrobial peptide, which could then be exploited as an alternative therapy against C. difficile.
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Moonlighting in myxobacteria: measuring the covariation in GAPDH
More LessMyxobacteria are an order of well-known social predators that able to prey upon a wide range of microbes (including fungi and bacteria). It is currently unknown how they are able to consume such a wide range of prey, although moonlighting proteins, those possessing more than one function, appear to be involved. One moonlighter, is the virtually ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Typically present in the cytoplasm, it has also been found on the outer surface of cells, in the extracellular matrix, and secreted through membrane vesicles. In various pathogenic organisms, GAPDH has been shown to have adhesive properties, binding to several types of human connective tissues. Myxobacterial predation can be viewed as analogous to pathogenic infection, and there is evidence that GAPDH might be involved in attacking prey cells. To complement experimental analyses of the predatory role of GAPDH in myxobacteria, we undertook sequence analysis including multiple sequence alignment (MSA) and covariation analyses. Covariation calculations, using a variety of metrics and matrices, have been used to identify areas and individual residues where structural, functional or phylogenetic correlations occur within the sequence. The MSA data highlighted key conserved residues, likely involved in the catalytic reaction and potentially involved in adhesion. The covariation scores suggest compensatory mutations have taken place in discrete locations along the sequence. These results can be used to selectively mutate residues in order to determine their effect in predation.
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Penicillium spp. strains as a possible weapon to fight microbial infections
More LessBacteria are becoming increasingly resistant to antibiotics, leading to untreatable infections and constituting a major public health hazard (Lewis, 2013). This problem is further increased by the reduction in the number of effective antibiotics to tackle resistant strains (Penesyan et al. 2015), so the search for new compounds is seen as a vital priority. Our study consisted in the analysis of four different environmental strains of Penicillium spp., with screening for their extracellular metabolites for potential antimicrobial activity. Several methods were combined and tested for metabolite production and extraction. The strains were grown on: solid media (I-potato dextrose agar and II-malt extract agar), broth (malt extract broth with III-0 and IV-5% NaCl), and V-tapwater; extractions were performed using three different solvents: A-methanol, B-butanol and C-ethyl acetate. All extracts were tested on three different model organisms: Micrococcus luteus, Escherichia coli and Mycobacterium smegmatis. These are models for Gram-positive and Gram-negative bacteria, and for Tuberculosis. The extracts were compared and analysed in order to determine minimal inhibitory concentrations for each of the microorganisms tested. This research presents preliminary results on the development of potential new chemical compounds to help us circumvent the problem of drug resistance. Lewis, K., 2013. Platforms for antibiotic discovery. Nature reviews Drug discovery, 12(5), pp.371–387. Penesyan, A., Gillings, M. and Paulsen, I.T., 2015. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities. Molecules, 20(4), pp.5286–5298.
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Identification and characterization of bioengineered nisin derivatives that inhibit the opportunistic pathogen Staphylococcus epidermidis
More LessStaphylococcus epidermidis is a major cause of hospital-acquired infections particularly on indwelling medical devices and implants. Infections caused by this pathogen are difficult to treat with standard antimicrobial agents mainly as the bacterium can establish biofilms on artificial surfaces. Novel control methods are needed and one such alternative may be bacteriocins; these are antimicrobial peptides produced by some bacteria that inhibit other specific bacteria. They are potent, safe, and stable and are easy to produce using biotechnological based strategies. Additionally, as they are gene encoded they can be easily modified or engineered to enhance their activity. In the present study, we demonstrate the ability of the bacteriocin nisin to inhibit a collection of clinical S. epidermidis strains under standard laboratory conditions. In addition, a bank of bioengineered nisin derivatives was screened using agar-based deferred antagonism assays and derivatives with enhanced antimicrobial activity compared to the wild-type nisin were identified. These derivative peptides are currently being purified using a combination of chromatography-based approaches and their potency and stability are being examined. Future experiments will focus on examining the ability of the nisin derivatives in inhibiting S. epidermidis biofilms on medical device materials (e.g. stainless steel and polyvinyl chloride) both alone and in combination with conventional antibiotics. It is hoped that one of the nisin derivatives analysed in this study may ultimately be used to control or prevent infections caused by S. epidermidis.
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Endophytic actinobacteria of herbal rhizomes and their pharmaceutical potential to form L-asparaginase
Pharmaceutical treasure of herbs is well known and some of which have long been served as natural medicines or nourishments to enrich human health. It is conceivable that herbs offer unique habitats for endophytic microbes. Interestingly, the metabolic exchange between endophytes and their herbal hosts does not only allow the microbial subsistence but also enable them to form analog bioactive substances to those produced by their hosts. These challenge us to focus on searching for beneficial microbes from herbs and industrialize them as the producers of pharmaceutical products. In this work, we isolated 37 actinobacteria from rhizomes of fingerroot (Boesenbergia rotunda), ginger (Zingiber officinale), galangal (Alpinia galanga) and turmeric (Curcuma longa). These actinobacteria were classified preliminarily based on their morphology to the genus Streptomyces (86 %) and other unknown genera (14 %). To screen for anticancer activity, we found that 68% of all isolated actinobacteria produced L-asparaginase, which is a hydrolytic enzyme that acts as an inhibitor of leukemia. Streptomyces sp. ALP03 was among the active isolates exhibited the highest enzyme activity of L-asparaginase and showed a maximum 97.93 % similarity of its 16S rRNA gene sequence to Streptomyces spongiae Sp080513SC-24T. The cytotoxicity assays against diverse types of cancer cell lines will be carried out to assess the anticancer potential of isolate ALP03 and the others showing distinct L-asparaginase activity. With these findings, we conclude that herbal rhizomes have yet been a promising source for the discovery of useful microbes and their pharmaceutical products.
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Screening bacteriophage activity against E. coli O157:H7 attached to host cells
More LessBacteriophage (phage) therapy, the use of a specific virus to kill infecting bacteria, is often cited as an alternative to antibiotic therapy. But phage treatment could also play an important role in bio-security against the foodborne pathogen E. coli O157:H7. Phage can be relatively easily isolated and shown to have activity against bacterial strains of interest under laboratory conditions. However, to be used successfully for treatment the phage must be active in vivo where the bacteria may be attached to host cells. To overcome this potential hurdle we are using a screen to test phage activity in conditions more realistic to the host environment than standard lab media. As part of this work we have been isolating new phage that are active against E. coli O157:H7 strains. We are currently testing these newly isolated phage against different bacterial strains and under a variety conditions looking for characteristics that will make them good candidates for phage therapy. We will then test the activity of promising candidates against E. coli O157:H7 attached to bovine epithelial (EBL) cells. The aim of this work is to put together a panel of around 20 phage that not only have the appropriate standard characteristics for phage therapy but have also been shown to have activity on attached bacterial cells. The longer term aim of this work is to use these ‘in vivo’ active phage as an intervention on cattle colonized with E. coli O157:H7 thereby reducing the potential of this pathogen entering the food chain.
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Creation and characterisation of probiotic libraries for use in poultry and pigs
More LessProbiotics are defined as live micro-organisms which provide the host with a growth or health advantage. In recent year’s with the emergence of antimicrobial resistance, probiotics, and in particular Lactobacillus, have become increasingly popular as an alternative control strategies for bacterial pathogens. Recent studies have demonstrated that Lactobacillus is not only able to increase the growth rate of animals, an important consideration for commercial farming entities, but is also able to inhibit the colonisation of animals with pathogenic bacteria species such as Salmonella, Campylobacter and Escherichia. This study aimed to isolate and characterise lactobacilli isolates from commercially reared chickens and pigs. Particular attention was paid to the probiotic potential of the isolates. Our approach combined molecular techniques with in vitroscreening to rapidly identify this potential. To date 80 and 105 isolates have been collected from pigs and chickens, respectively. All isolates have been confirmed to belong to the Lactobacillus genus by PCR, speciated by 16S sequencing and determined to be clonally unique using RAPD PCR. Isolates were subsequently tested for their ability to tolerate low pH, bile, aerobic and anaerobic conditions before assessing their AMR profile and determine their ability to inhibit a panel of clinical and type strains of pathogenic bacteria. Isolates identified as potential probiotics are currently undergoing whole genome sequenced as per EFSA guidelines. This study has demonstrated that lactobacilli isolates with suitable probiotic properties can be isolated from commercial poultry and pigs. Furthermore, these isolates may prove useful as control strategies for zoonotic bacterial pathogens.
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Targeting antimicrobial resistance genes in clinical isolates from healthcare-associated infections using CRISPR-Cas9
Of the numerous approaches to counteracting the spread of antimicrobial resistance (AMR), one that is receiving increased attention is the use of CRISPR-Cas-based gene editing technology to remove resistance-conferring sequences from bacteria. Proof-of-principle studies have shown that CRISPR-Cas is able to successfully remove AMR genes from monocultures or very simple bacterial communities in the laboratory, but these constructs and their delivery must be adapted to be used in real-world medical settings. One area where such a technology may be implemented is tackling healthcare-associated infections. Development of a CRISPR-Cas9 cassette that is able to target particular resistance genes would be a powerful tool in addressing these infections. CRISPR-Cas9 will therefore be implemented to target AMR genes in clinical isolates of vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase (CTX-M) or carbapenemase (Oxa-48)-producing Klebsiella pneumoniae and Escherichia coli, by utilising a broad host range conjugative plasmid to deliver this construct via conjugation. Preliminary work using E. coli MG1655 as plasmid donor have shown that all strains can receive the plasmid, and there is significant variation in conjugation frequency between E. coli recipients.
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Investigating soil bacteria for novel antimicrobials which inhibit the opportunistic pathogen, Staphylococcus epidermidis
More LessAccording to the European Center of Disease Control and Prevention, Staphylococcus epidermidis is rapidly becoming a serious concern in hospitals as a cause of resistant infections, particularly in the case of in dwelling and prosthetic medical devices. To overcome this issue, new, potent antimicrobials with novel target pathways need to be uncovered to both reduce morbidity and mortality, and the spread of resistant microbes. 51 bacterial strains with observable antimicrobial activity against S. epidermidis were isolated from samples of soil, collected from various locations around Ireland. The activity was reconfirmed, and the most effective strains were shortlisted using deferred antagonism assays. Characterisation tests, (Gram stains, oxidase and catalase testing and 16S sequencing), were carried out to determine the identities of the bacteria at hand. Presently studies are underway to elucidate the nature of the antimicrobial activity and to govern if the findings are novel. HPLC is being used to purify the compounds, with proteinase K assays evaluating if the antimicrobials are proteinaceous in nature. The future of this study will include the sequencing of any uncovered antimicrobial peptides, MIC determination of antimicrobial compounds, observing the impact on biofilm formation and looking at potential efficacy against other relevant pathogens. With the identification of a novel compounds, this study aims to present an opening into potential new treatment options to help address the current struggle against antimicrobial resistance.
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- Focus on 3Rs – The Growing Role of Organoids and Microbial Models to Understand Human and Animal Diseases
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Exploring the metabolic potential of oleaginous actinomycetes in biodiesel production from cassava wastewater
More LessThe depletion in fossil fuel reserves has instigated the search for renewable sources such as biofuels. Biofuel production provides a sustainable alternative to fossil fuels. However, the progression of biofuel industry has been largely affected by several uncertainties including the sustainability of production processes. Finding an economically viable process and the right substrates have been a source of constant debate. Biodiesel production is one such area which is gaining momentum in the last few decades. We are currently investigating cassava wastewater as a substrate and also as a source of oleaginous bacteria. The production of tri-acyl glycerol from mycolic acid containing actinomycetes predominantly Rhodococcus, has been identified as a potential source. These bacteria which are largely ubiquitous provide a significant amount of triglyceride when cultivated under low cost waste. The growth of oleaginous bacteria for the accumulation of triglycerides on low cost and abundant, cassava waste still remains unexplored, especially in Nigeria which is the largest producer of cassava in the world. We started our initial investigations on the cassava waste water sample following culture dependent and independent approach. We prognosticate that the identification of microflora isolated from different stages in the sample will provide a basis for understanding the nature of the substrate and the potential for the synthesis of biodiesel from cassava waste water.
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Galleria mellonella: a novel infection model for screening potential anti-mycobacterial compounds against members of the Mycobacterium tuberculosis complex
More LessAnimal infection models are vital as drug screens for novel therapeutics to tackle the global tuberculosis (TB) epidemic. However, all pre-existing models have limitations which include ethical constraints. Therefore, efforts to reduce and/or replace conventional animal models in TB research are warranted. Previously, we reported the use of Galleria mellonella (greater wax moth, GM) as a novel infection model for the Mycobacterium tuberculosis (MTB) complex, using Mycobacterium bovis BCG lux, a bioluminescent mutant which allows for the rapid quantification of bacterial burden in vivo. Here we investigated the drug screening potential of GM infected with a lethal dose of BCG lux, treated with first or second-line antimycobacterial drugs over a 96 h period; where drug efficacy was determined every 24 h through bioluminescent measurement of larval homogenates. Improved survival outcome was observed in all larvae treated with antimycobacterials when compared to untreated controls. Furthermore, all drug treatments except pyrazinamide resulted in a significant reduction in bioluminescence of BCG lux in vivo. Isoniazid and rifampicin displayed the highest survival outcome and greatest in vivo drug efficacy, in line with observations reported in mice. However, combined or multiple dosing of either drug showed little to no difference over single dose mono-therapy. Our results demonstrate that GM is a promising infection model for members of the MTB complex, with significant potential for its use in the drug development pipeline as a pre-screening model for novel therapeutics, thereby reducing experimental usage of animals in TB research. Supported by the NC3R’s (NC/R001596/1)
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The Dictyostelium rhomboid proteases and mitochondrial disease
More LessRhomboid membrane proteases are integral to pathogenicity of several microbial eukaryotes and the role of mitochondrial rhomboids is important in pathologies beyond infectious disease. By virtue of their ability to cleave tethered proteins, this relatively recently discovered protein family have been found to activate substrates, release mobile signals, and progress cell regulatory pathways. The Dictyostelium model microbe allows the study of an evolutionarily ancient set of rhomboids, aberrant expression of which affects phagocytosis, response to chemoattractants, phototaxis, growth and cell size, ATP levels, and mitochondrial ultrastructure. We report the particularly mitochondrial focus of rhomboids in this amoeba and consider the relevance of this beyond the model organism, given the central role of the human mitochondrial rhomboid in mitophagy, ageing and neurodegenerative disease.
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Comparison of MRSA skin infection models with HaCaT keratinocytes and a 3D-organotypic skin model
More LessBackground and AimsSkin is a major site for Staphylococcus aureus colonization and invasion. A 3D-organotypic skin model was generated by co-culturing keratinocytes with fibroblasts at air-liquid interface enabling the proliferation, migration and differentiation of the keratinocyte to mimic terminal differentiation of the epidermis.
MethodsThe adherence and internalization of MRSA strain types ST8, ST30, ST59, ST22, ST45 and ST239 were evaluated in the HaCaT keratinocytes and a 3-D organotypic skin model. Acridine orange staining and/or anti-Staphylococcus aureus antibody were used for bacterial localization. TUNEL assay was used to evaluate cell death due to apoptosis.
ResultsMaximum adherence to HaCaT cells were exhibited by both MRSA ST59 and ST8 strain types (P=0.129, Tukey post-hoc test). The maximum percentage of internalization was exhibited by both ST59 and ST30. With ST8, ST30, ST59 and ST239 types, bacteria were present within the cytoplasm whereas localization of ST22 and ST45in the phagosomes of HaCaT keratinocytes were observed. Study of cell death in HaCaT keratinocytes was limited to 24 h due to dislodgement of the keratinocytes with all six strains. The 3-D skin model proved to be better to study MRSA transmigration and cell death which could be monitored for longer time points. ST59 exhibited maximum adhesion and internalization.
ConclusionThe 3-D skin model proves to be a better model to study MRSA skin infections.
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Pigs, PAMs and Pathogens: using primary alveolar macrophages from abattoir acquired porcine lungs to model pulmonary infections
More LessThe similarity between the porcine and human immune system makes pigs an ideal model for studying infectious disease. We have been using alveolar macrophages recovered from abattoir-sourced porcine lungs to create a model to study Streptococcus pneumoniae infections in the lung. Pneumonia is a leading cause of death from infectious disease, with S. pneumoniae the most common cause of bacterial pneumonia. Although much studied, there is still a lot we don’t know about the early stages of infection. Recent research in our lab has found a previously unknown intracellular phase for S. pneumoniae within splenic macrophages Alveolar macrophages were recovered by bronco-alveolar lavage and used in gentamicin protection assays. Macrophages challenged with S. pneumoniae at an MOI of 25 bacteria per cell were found to contain live bacteria up to 5 h after infection. Analysis of samples collected at 45, 90, 180 and 300 min after challenge found less than 10 % of the challenge dose present at 45 min, decreasing to 0.02 % after 5 h. The presence of bacteria inside the macrophages was confirmed by confocal microscopy. Studies are on-going to unpick the processes that allow the bacteria to resist phagocytosis by the macrophages, and to use the model to investigate the interaction of the macrophages with other respiratory pathogens. The use of abattoir-acquired porcine cells contributes to the 3Rs – reducing and replacing laboratory animals, and refinement of method as pigs are more akin to humans than mice. J. McNicholl is sponsored by the Daphne Jackson Trust.
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Using human iPSC derived small intestinal organoids as a model for enteric disease caused by Enterotoxigenic E. coli and Vibrio cholerae
Within the last ten years, iPSC (induced pluripotent stem cells) have been widely shown to have the ability to be re-programmed to produce a wide range of tissues in the presence of certain growth factors. In this project, we re-direct human stem cells derived from fibroblasts into complex 3D small intestinal structures termed organoids. These organoids have been shown to possess all cell types that are present in small intestinal tissue such as, enterocytes, goblet cells, enteroendocrine cells and Paneth cells, as well as possessing microvilli and crypt structures. We demonstrate that it is possible to microinject into the lumen of these small intestinal organoids and to manipulate the conditions for infection of non-invasive bacteria such as Enterotoxigenic E. coli (ETEC) and Vibrio cholerae. Looking at known bacterial virulence factors, we have shown there are differences in patterns of infection among different strains of entero-pathogenic bacteria. In addition, we have shown that the induced human organoids (iHO) elicit a recognisable and measurable host response to bacterial toxin.
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The utilisation of organoids and macrophages derived from Human induced pluripotent stem cells as model systems to investigate host-bacterial interactions
More LessUsing human induced pluripotent stem cell (hiPSC) technology we are developing methods to examine host-bacterial interactions. Due to the fact that undifferentiated human induced pluripotent stem cells are amenable to genetic engineering, can be cultured indefinitely and can further be differentiated into multiple cell types, we are exploiting both organoid and macrophage systems to investigate the interactions between host cells and diarrhoeal pathogens, including enterotoxigenic Escherichia coli and Vibrio cholerae. Utilising both wild type and relevant knockout hiPSC lines we are probing both initial interactions and subsequent utilisation of pathways for the effects of toxins. The further analysis of genetically engineered bacteria extend the usefulness of this model system, and complement the availability of mutant host cells, towards the simultaneous genetic analysis of both pathogen and host.
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Modelling biofilms on infected chronic wounds
More LessChronic wounds, for instance venous, pressure, arterial and diabetic ulcers, are a major health problem throughout the world. Compared with normal wounds, those that take more than four weeks to heal are defined as chronic. Interestingly, the numbers of patients suffering from chronic wounds and the cost for treatment have been increasing during the past two decades. There is increasing evidence that suggests that bacteria infect those chronic wounds and there exist as a biofilm, which affects the wound healing and success of wound treatment. To study biofilms in infected wounds, both in vitro and in vivo biofilm models have been developed. In this project, the colony biofilm assay was used to determine antibiotics effect on removing biofilm. The results of this study so far indicated that mature Staphylococcus aureus biofilms were resistant to vancomycin treatment, which works effectively on killing planktonic cells. However, other antibiotics used topically for healing infected chronic wounds, for example, gentamicin, tetracycline, fusidic acid and mupirocin, were more effective at killing mature biofilms in the colony biofilm model. These sets of experiments were also done with pig skin based on colony biofilm assay. This project aims to build up a new ex vivo dynamic model using pig skin and a 3D print flow chamber to mimic chronic wounds. Hence, the results gathered from the colony biofilm assay will be compared with those obtained for the newly developed model. This model can then be used to study the drug delivery and topical treatment of chronic wounds.
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- From Prokaryotes to Eukaryotes: The Origin and Diversity of Eukaryotes
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Development of antimicrobials against Acanthamoeba
More LessAcanthamoeba is a free-living amoeba widely distributed in the environment which exists as two stages in their life cycle: a motile and trophic replicating trophozoite and a resistant cyst stage. In recent years, the incidence of infections due to Acanthamoeba spp. has shown a remarkable increase. This parasite is the causative agent of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK) and a fatal disease of the central nervous system known as Granulomatous Amebic Encephalitis (GAE) mainly in immunocompromised patients. Although the trophozoite form is much more readily eliminated, at present there are not harmless effective treatments or a single drug that can eliminate both cystic and trophozoite forms. A particular set of compounds known as Minor Grove Binders (MGBs) have the characteristic of binding specifically to minor groove region of double-stranded DNA. The main effects of these MGBs are their ability to interfere with biological functions of DNA such as transcription machinery, also induction of apoptosis, hence cell death. These molecules have received great attention since they can be empirically screened and iteratively refined via chemical synthesis to target various entities such as tumors, bacteria, viruses and parasites. MGBs have been tested in vitrousing a colorimetric alamar Blue viability cell assayagainst Acanthamoeba castellanii Neff strain. To date, 2 hit compounds were able to inhibit trophozoites producing IC50s of 1.56 µM and 12.5 µM.
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- Genetics and Genomics Forum
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Characterization of prophages in Salmonella Typhimurium definitive type 8
More LessBackgroundSalmonella Typhimurium DT8 has been associated with human outbreaks. Anderson phage typing scheme has been used for more than four decades for subtyping of Salmonella Typhimurium, but it has shown some limitations. Here, we characterize prophages among Salmonella Typhimurium DT8 strains to test the potential use of prophage sequence profiles for subtyping.
MethodA total of 54 isolates of Salmonella Typhimurium DT8 were selected for this study which was part of an outbreak study by Ashton et al. (2015)*. Twenty-six isolates were associated with an outbreak in the States of Jersey in 2013 and 28 isolates were non-outbreak-associated isolates. Comparative genomics using a range of bioinformatic tools was carried out including SPAdes for de novo assembly of WGS data. PHASTER was used for characterisation of prophages (http://phaster.ca/).
ResultAll DT8 strains are lysogenic for three prophages (RE-2010, ST64B and Gifsy-2). Moreover, the three prophages showed identical sequence among outbreak and non-outbreak isolates. Interestingly, prophage SSU5 was detected in one non-outbreak isolate. Prophage SSU5 is closely related to cryptic plasmid pHMC2 that infects only rough strains of Salmonella Typhimurium. This study is the first to report the presence of prophage SSU5 in Salmonella Typhimurium DT8.
ConclusionIt is crucial to use accurate, reliable and highly discriminative subtyping methods for epidemiological characterisation and outbreak investigation of Salmonella Typhimurium. Prophage profiling might be unsuitable subtyping method. The emerging genetic analysis should be combined with the conventional method for epidemiological surveillance until WGS-based analysis can be improved and standardized. *https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336196/
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Genome-guided screening of bacterial isolates to identify potential antibiotic producers
More LessThe urgent need for new antibiotics cannot be overemphasized. Bacterial secondary metabolites remain a relatively untapped source of new therapies. The ability to produce these bioactive compounds is however not universal to all bacterial species. Two key indicators are bacterial genome size (>3 Mb), and the presence of antibiotic-encoding biosynthetic gene cluster (BGCs) within the genomes. BGC distribution is largely determined by phylogeny. Another attribute of some antibiotic producers is the ability to withstand nutritional stress. We exploited these attributes to isolate and identify potential antibiotic producers. A minimal substrate medium was used to isolate nutritionally versatile bacterial strains from topsoil collected from the rhizosphere. The genera of isolates were identified by 16S rRNA gene sequence comparison as Pseudomonas, Hafnia and Obesumbacterium. The typical genome size of species in these genera are 6.2 Mb, 4.7 Mb and 5.0 Mb respectively. The antiSMASH database was browsed by phylogeny to determine the distribution pattern of BGCs in these genera. Pseudomonas strains have an average of 7 BGCs within their genomes that may encode antibiotics, whilst Hafnia and Obesumbacterium strains have 2 and 0 respectively. Therefore, the isolated Pseudomonas strain has the greatest potential to biosynthesis antibiotics. However, the biosynthetic potential of other isolates may be understated given the typical genome size of species in their genera, and their ecological origin. Consequently, all isolates are prime candidates for the next stage of the project which involves genome mining for cryptic or silent genes that may encode novel compounds with antibiotic properties. More isolates are also being recovered.
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Characterisation of extended spectrum beta-lactamases (ESBL) resistance in multi-drug resistant Salmonella concord
More LessSalmonellosis in children from Ethiopia is caused mainly by Salmonella Concord, which are highly invasive, multi-drug resistant and extended-spectrum β-lactamase (ESBL) producers. S. Concord infections have been observed in children adopted from Ethiopia, now living in Europe and United States. S. Concord infections are present in parents of these adopted children, posing a significant dilemma for treatment. Data from Salmonella isolates are stored in Public Health England’s in-house Gastro Data Warehouse (GDW) database. Resistance profiles for 37 pure S. Concord isolates were determined using agar incorporation methods using EUCAST guidelines. The minimum inhibitory concentration (MIC) was determined for 18 antimicrobial agents and ESBL resistance was confirmed using Double Disk Synergy and ESBL E-test methods. ESBL resistance was present in 8 isolates with resistance most commonly seen against penicillins and cephalosporins. Isolates 408 and 537, and isolate 527 displayed resistance to 78 % and 56 % of the antibiotics respectively. Drug resistant regions in isolates 408, 527 and 537 that have been previously sequenced by an Illumina HiSeq 2500 were characterised. ESBL genes blaCTXM-15 was present in isolate 527 and 408 and blaSHV-12 was found in isolate 537. Replicons from plasmids, InCI2, InCFIB, InCF2 and IncH2 were located in the assembled sequences of the three previously sequenced isolates. In conclusion, the possible mechanisms causing spread of ESBL resistance in S. Concord is most likely due to acquisition of plasmids through horizontal gene transfer from various Enterobacteriaceae, however further research must be conducted to confirm this to advance antimicrobial research in this area.
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What factors contribute to the long-term persistence of Shigella species as pathogens?
More LessThe genus Shigella is a globally important pathogen responsible for the infectious disease shigellosis which causes an estimated 125 million cases worldwide and up to 100 000 deaths. Shigella species are now recognised as a priority organism by the World Health Organisation due to the increasing antimicrobial resistance (AMR) observed. For adults, treatment of Shigella infections commonly consists of ciprofloxacin, however, recently there have been confirmed cases of ciprofloxacin-resistant strains. Most Shigella is already resistant to ampicillin and trimethoprim so added resistance to ciprofloxacin is a worrying development. Also of note is the role played by toxin-antitoxin systems where they are critical for maintaining large plasmids that typically contain genes of interest such as AMR genes and virulence factors. The Murray collection comprises of several hundred bacterial strains from the pre-antibiotic era (1917–1954) from a range of geographic locations. Within this collection there are many Shigella isolates which are now publicly available for analysis. There are also available datasets for modern collections which have been sequenced for past studies. We will use these data sets to perform bioinformatic analysis and characterise the dynamic changes in the genetic composition across time. The Murray Collection isolates provide a prospective from the pre-antibiotic era and so from the comparisons with modern isolates we will observe the effect of routine antibiotic use has on the evolution of antimicrobial resistance. Through this investigation the hope is to provide unprecedented insight into how AMR and toxin-anti-toxin systems contribute to the long-term persistence and success of Shigella.
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Phase variation of Opa proteins in hypervirulent serogroup W meningococcal isolates
More LessSerotype W, sequence type 11 Neisseria meningitidis are an increasing cause of morbidity and mortality. The mechanisms underpinning the success of this clone remain unclear. Meningococci express up to four Opa proteins, which mediate adhesion to/invasion of host cells. Opa expression undergoes phase variation – a high frequency ON/OFF switch in gene expression due to insertion/deletions of pentameric repeats (5′-CTCTT-3′) in the coding region. NadA functions as an adhesion, and is phase-variable through tetrameric repeats in a regulatory. WGS and PCR-based fragment size analysis of 121 invasive and 51 carriage MenW:ST11 strains was conducted to determine gene expression patterns between invasive and carriage. Inactivating mutations were constructed in opa, pilE and nadA genes of two representative MenW isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 cells. We observe that four opa loci are present in all MenW:cc11 isolates and that OpaB and OpaD share 100 % sequence similarity. Repeat number variability was detected between the ‘original UK’ strain and the novel ‘2013-strain’ of the hypervirulent MenW:cc11 South American sublineage. No significant differences in the patterns of Opa expression were observed between invasive and carriage isolates. In vitro assays with pilE and nadA deletion suggest that NadA has an effect on invasion, while the double mutant shows a reduction in both adhesion and invasion. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains, but may give a niche specific advantage by enhancing phase variation-mediated immune evasion.
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Characterisation of rpoS alleles in UPEC strain CFT073
More LessEscherichia coli is a major cause of urinary tract infections, bacteraemia and sepsis. CFT073 is a well-studied, prototypic urosepsis isolate. This laboratory has previously shown that strain CFT073 is serum-resistant, with resistance being imparted by factors including capsule and extracellular polysaccharides inter alia. It has become apparent that not all CFT073 strains are identical, some harbour a 5 bp insertion in the rpoS gene which results in a truncated, non-functional RpoS. In this study, we compare CFT073 wild-type and an rpoS mutant. The gene sequence of rpoS in each isolate was verified. Next, the sensitivity of the bacteria to hydrogen peroxide was determined. Finally, wild-type and mutant strains were rendered bioluminescent. Bioluminescence permits the real-time detection of viability. The contribution of RpoS to serum resistance was examined by bioluminescence.
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Fitness costs of methicillin resistance in Staphylococcus aureus
More LessStaphylococcus aureus is a Gram-positive bacteria, and a common human pathogen. It is a leading cause of healthcare-associated infections worldwide, causing potentially fatal bacteraemia. Infections can be treated using antibiotics, but many strains have quickly developed resistance to the most common antibiotics. Methicillin resistance in Staphylococcus is acquired through the uptake of the Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec is a large mobile genetic element, which can integrate into the Staphylococcus genome. It carries the mecA gene, which confers resistance to broad-spectrum β-lactam antibiotics, including methicillin. Several different SCCmec elements have been described, some of which can carry additional antibiotic resistance genes, alongside mecA. These can include resistance to common antibiotics or resistance to heavy metals. Smaller SCCmec types do not encode any additional antibiotic resistance genes. These have been shown to confer no fitness cost to the bacteria. Because of this, smaller SCCmec types are becoming increasingly more common, particularly in community-associated MRSA infections.
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Fitting linear mixed models to a highly-structured dataset effectively controls for population structure in bacterial genome-wide association studies
More LessCystic fibrosis is an inherited, autosomal recessive disease that causes an accumulation of viscous mucus within the lung, leading to chronic bacterial infections. Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that can invade the cystic fibrosis lung, and is the most common bacteria isolated from cystic fibrosis patients. In the largest study of its kind, ∼4200 P. aeruginosa isolates were collected from nine cystic fibrosis patients. The isolates were whole-genome sequenced and screened for 14 virulence-related phenotypes. We aim to associate phenotype with genotype by fitting both linear models and linear mixed models association tests. During this study, linear mixed models were found to effectively control for strong population structure signals, allowing relevant associations to be uncovered. When sub-sampling the dataset into unstructured, single-patient groups, fitting linear mixed models were found to associate genotype with phenotype as effectively as fitting linear models. We also found that aggregating non-synonymous SNPs in the same gene to a single knockout mutation increases the power of the association tests to identify relevant associations. The future direction of this study will involve filtering the results to identify any phenotypically-relevant associations. These putative associations will then be experimentally verified.
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Identification of Staphylococcus aureus and methicillin resistant S. aureus in jail populations
More LessStaphylococcus aureus is the leading cause of soft tissue and skin infection, but also causes pneumonia, endocarditis, and bone infection. The same infections can be caused by methicillin resistant S. aureus (MRSA), but are much more difficult to treat because MRSA is highly resistant to antibiotics. In ethnic and socioeconomic minority groups, methicillin resistance in S. aureus is 30 % higher than the general population. As a result, methicillin resistance is likely to have higher prevalence in jails and prisons where the demographic is disproportionately represented by ethnic minorities, individuals with lower average socioeconomic status, and less access to healthcare. Jail inmates are an understudied but very important population because diseases acquired in jails can be easily introduced into the general population and vice versa because cycling into and out of jail is very common. The presence of S. aureus is an important determinant of the development of soft tissue and other infections, making the detection of this bacteria and the identification of antibiotic resistance essential in assessing risk of disease. In this study, we identified S. aureus in samples from a United States county jail through bacterial plating and DNA extraction. Methicillin resistance was identified in S. aureus positive samples through PCR and sequencing. Our analysis has found elevated rates of S. aureus in the jail population being studied. This project will determine if rates of S. aureus and methicillin resistance in S. aureus isolated from this demographic are disproportionate to the general population.
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Causative agents of early childhood caries: challenges and solutions in culturing and sequencing
More LessDental caries, also known as cavities or dental decay, effects children at a rate five times higher than asthma in the United States. This disease is highly preventable but causes extreme pain and costs millions of dollars to treat every year. The presence of Streptococcus mutansand Streptococcus sobrinusplays a major role in the development and progression of tooth decay; therefore, it is important to establish colonization of these bacteria to assess the risk of developing the disease. Streptococcus bacteria are difficult to grow, extract, and sequence due to strict necessary growth conditions. In this study, we evaluated the performance of different storage and culturing protocols and developed strategies for reducing interference by unwanted bacterial and fungal genomes when sequencing extracted samples. We compared storage conditions of samples at various temperatures, and with and without glycerol. We decided the best storage method was at −80 °C in a specialized solution known as Aimes. When sequencing cultures, we encountered various unwanted bacterial and fungal genomes. To reduce this, we modified our culturing methods by including growth in anaerobic conditions and using serial isolation streaking. These modifications have limited the growth of aerobic specimens and increased culture purity before extraction. With this study, we will be able to better understand the oral microbiome and aim to identify virulence factors in S. mutans and S. sobrinus that contribute to the high rates of dental caries in children.
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Evolvability of orthologous genes
More LessThe project aims to generate a library of laboratory orthologous genes for studying evolvability. In this study, we have taken one of histidine biosynthetic genes hisA from Salmonella enterica through alternating rounds of weak selection (simulated by random mutagenesis through error-prone PCR and screens for partial loss of hisA function) followed by strong purifying selection (simulated by additional rounds of random mutagenesis and screens for restored hisA function). We have started from the wild-type hisA gene expressed from relatively weak arabinose inducible promoter ParaBAD. Twenty different single nucleotide polymorphisms (SNPs) that cause slow growth in the absence of histidine were isolated. In twelve of these deleterious mutants the activity was restored to wild-type levels by additional SNP after another round of random mutagenesis. In this study, in some of the compensated his A variants it was difficult to isolate further deleterious mutations, suggesting that these compensating mutations can mask the effects of additional deleterious mutations. By combining the compensating mutations with known deleterious mutations, complete or partial compensation was seen. Orthologs from one of the diverging lineages were also compared for their evolvability toward TrpF activity (ability to synthesize tryptophan) using fluctuation test. Two orthologs in this lineage showed higher evolvability compared to the wild type hisA.
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Genetic regulation of compost and plant degradation in Agaricus bisporus
More LessAgaricus bisporus, the common button mushroom, is a major contributor to the global carbon-nitrogen cycle through its role as a secondary decomposer of plant and leaf litter. It is also an economically significant mushroom with a domestic farm gate value in excess of €120 million. This research aims to elucidate the molecular mechanisms involved in compost and plant matter degradation by A. bisporus. An examination of the entire genome using microarray analysis was carried out on A. bisporusat 48 h intervals over the course of its commercial lifecycle. This data revealed several genes that appear to be involved in the genetic regulation of mushroom production. From this data, genes with potentially critical roles in compost utilisation and plant matter degradation were identified. Five genes of significant interest were selected for further study. These include three carbohydrate degrading genes and an unknown gene which appears to be unique to A. bisporus, all of which had an expression profile that matched the commercial growth pattern. The fifth gene was selected based on its significant down-regulation during peak mushroom growth. To further compliment this data a bioinformatic analysis of known transcription factor binding sites present within each promoter of A. bisporus is also being carried out. The data generated from this study will provide crucial insight into the mechanisms behind the regulation of A. bisporus growth and its ability to degrade plant matter and compost degradation.
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AphA is a master regulator of natural competence in Vibrio cholerae
More LessVibrio cholerae is the etiological agent of cholera, a disease affecting 4.3 million people each year (WHO, 2015). The symptoms of cholera include profuse watery diarrhoea and vomiting, leading to coma and death in serious cases. The El Tor biotype of V. cholerae is globally predominant, and the success of this biotype to cause disease is thought to be due, in part, to the ability of the organism to successfully colonise two different environments; i) marine and brackish coastal waters where V. cholerae forms biofilms on the chitinous surfaces of shellfish, and ii) the human intestine. When bound to chitinous surfaces V. cholerae expresses genes required for biofilm formation, chitin utilisation and natural competence. Conversely, within the human host, V. cholerae switches on the expression of virulence genes. The transcription factor AphA was discovered as a regulator of genes encoding the toxin co-regulated pilus (TCP) of V. cholerae. It is also known that AphA is active when V. cholerae populations have a low cell density. Hence, AphA is generally considered a regulator of virulence and quorum sensing. We have mapped DNA binding by AphA across the entire V. cholerae genome. The majority of AphA target genes encode cell surface and cell envelope proteins. Unexpectedly, we show that AphA also targets key gene clusters within the natural competence regulon of V. cholerae. Using biochemistry and genetics we show that AphA is a master repressor of natural competence and acts to antagonise transcription activation by the cyclic-AMP receptor protein.
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Whole genome analysis of the common pathogens associated with microbial keratitis
BackgroundMicrobial Keratitis is a serious infection of the cornea and a major cause of corneal opacity and vision loss worldwide. Whole-genome sequencing (WGS) of keratitis bacterial isolates has the ability to provide a wealth of clinically-valuable information. The potential of metagenomics has also been evaluated as current techniques for pathogen identification are laborious, time-consuming and associated with poor culture rates, making metagenomics an exciting future prospect.
Methods/ResultsSamples from keratitis patients were collected and isolates grown in agar before DNA was extracted from pure-cultures and WGS data was obtained from a total of 30 isolates, including common causative agents Pseudomonas aeruginosa and Staphylococcus aureus. Libraries were prepared using a miniaturised Nextera XT protocol and sequencing was carried out on Illumina’s NextSeq platform. Genomic analysis was then based around MLST-typing, comparative genomics, and the identification of key genes inferring antibiotic resistance and virulence. Our pathogens exhibited diverse evolutionary lineages and a range of virulence-associated genes, providing insight into pathogenicity. The ability to obtain sufficient DNA quantities for metagenomics sequencing was also assessed using various host depletion and microbiome enrichment techniques.
Discussion/ConclusionThe clinical role of next-generation sequencing is currently limited due to associated costs and required computational resources, however in the future this method could replace current techniques for pathogen identification. WGS provides important insights into the genomes of the common keratitis infectious agents, improving understanding of the aetiology of corneal infections and metagenomics shows future potential to be the centre of culture-independent pathogen identification.
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Properties and directionality of intragenic promoters in E. coli
More LessThe histone-like nucleoid structuring (H-NS) protein targets and binds AT-rich DNA. Oligomerisation of H-NS along AT-rich genes represses transcription from spurious intragenic promoters within the coding sequences of AT-rich genes. In this work, we sought to better understand why promoters occur so frequently within AT-rich DNA. To do this, we compared the properties of (i) canonical promoters (ii) promoters within H-NS bound genes and (iii) promoters generated by random combinations of nucleotides. We have identified and analysed many active intragenic promoters distributed within AT-rich genes in the E. coli genome. We show that these promoters, and promoters from randomly generated AT-rich DNA, differ from canonical promoters in several ways. In particular, spurious promoters are often dependent on AT-tracts upstream of the promoter −10 element. These AT-tracts play a key role by altering DNA curvature and facilitating interactions between the promoter and RNA polymerase sigma factor. Promoter regions inside genes are also often bidirectional.
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Exploring the role of VpsT in Vibrio cholerae lifestyle switching
More LessBiofilm formation is an important stage of the Vibrio cholerae lifecycle. The transcription factor VpsT is a master regulator of biofilm formation that activates the expression of biofilm matrix components in response to elevated intracellular c-di-GMP. VpsT has also been shown to repress the expression of rpoSand genes involved in motility. This suggests VpsT may have a wider role in the transition from a motile to sessile lifestyle. To better understand the role of VpsT in V. cholerae lifestyle switching ChIP-seq (chromatin immunoprecipitation and DNA sequencing) was used to map VpsT binding across the genome. Our data reveals many additional targets, defines the DNA binding motif for VpsT, and expands the VpsT regulon to include genes involved in c-di-GMP metabolism, motility and virulence. The VpsT interaction at target promoters is c-di-GMP dependent and can repress or activate gene expression.
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Genotypic and phenotypic diversity in Candida tropicalis
More LessCandida tropicalis is a human pathogen with significant mortality rates, which is particularly prevalent in tropical regions. The first genome sequence of C. tropicalis was published in 2009. In this study, we sequenced 75 C. tropicalis isolates with the aim of studying the intra-species diversity of this pathogen at a phenotypic and genetic level. These isolates were collected from both clinical and environmental sources, and sequenced using Illumina technology. Phylogenetic analysis of the sequenced isolates using SNP trees and PCA analysis reveals several multi-isolate clusters, which are unrelated to geographical origin. Overall, the genomes of the isolates were stable, with only 4 out of 75 isolates demonstrating any aneuploidy. However, the genomes are highly heterozygous, with one variant, on average, every 136 bases. Variant analysis revealed three highly divergent isolates, with one variant, on average, every 7 bases. Further examination of these isolates revealed that they are the products of hybridisation between two parents; one which is almost identical to the reference strain (>99.9 %), and a second, unidentified parent, which is approximately 4.5 % different in its sequence to the reference strain. Differences in sequence indicate that these hybrids arose from separate hybridisation events. Phenotypic differences are also observed between isolates under certain conditions, particularly in the presence of cell wall stressors, metal stressors and antifungal drugs. Cosine similarity was used to identify correlations between genetic variants and phenotypic variation in all isolates, identifying variants that may be responsible for particular phenotypes. Work is ongoing to confirm these observed correlations.
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Molecular characterization of the activity and requirements of a novel and promiscuous bacteriophage integrase
More LessStx bacteriophages are responsible for the dissemination and production of Shiga toxin genes (stx) across the Shigatoxigenic E. coli (STEC). These toxigenic bacteriophage hosts can cause severe, life-threatening illness, and Shiga toxin (Stx) is responsible for the severe nature of EHEC infection, a subset of pathogenic STEC. At the point of Stx phage infection, the injected phage DNA can direct its integration into the bacterial chromosome becoming a prophage; the host cell is then known as a lysogen. Unusually, our model Stx phage, Φ24B, can integrate into at least four distinct sites within the E. coli genome that shared no easily identifiable recognition sequence pattern. The identification of what are actually required for phage and bacterial DNAs recombination has been tested using both in vitro and in situ recombination assays. These assays enabled the simple manipulation of bacterial attachment site (attB) and phage attachment site (attP) sequences. The aim of the study is to fully characterize the requirements of this promiscuous integrase, carried by the Stx phage Φ24B (IntΦ24B), to drive integration. These assays enabled us to identify the minimal necessary flanking sequences for attB site identified (21 bp and 49 bp from the right and left the cross over region, respectively) and the attP site (200 bp each side). Furthermore, we identified that the Φ24B integrase does not need Integration Host Factor (IHF) to drive integration. Fi
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Investigating the influence of host genetics on the metagenome: the intestinal microbiome of two arid mammalian species
More LessNumerous factors have been shown to influence microbiome composition, including host genetics, diet and environmental variables. Recent studies have explored how host genetics influence human gut microbial communities, and those of pre-clinical models e.g. mice. However to date no studies have determined the influence of host genetics and extreme environments on wild animal microbiomes. Furthermore, when ‘wild’ species have been utilised this has typically involved working on captive individuals, restricted to a single species or limited to single sampling without replicates. Here we present work which takes advantage of a unique opportunity to directly investigate host organism genetic influences and environment on the resident microbiome. Our dataset comprised samples from individuals of two closely related sympatric, independently adapted arid mouse species, subject to the same stresses and diet; Acomys cahirinus and Acomys russatus. These desert dwellers are very highly adapted to the extreme environment they live in, and therefore represent an exciting opportunity to probe key microbiome-environment-host genetic questions. Samples are replicates from wild individuals, separated by a period of four months. Wild individuals were captured on two occasions and faecal samples collected. DNA extracted and subjected to shotgun metagenomic sequencing, generating NGS data. Preliminary analysis allowed us to probe community composition and relative abundance within individuals, and between members of the same species. Utilising Kraken, Centrifuge and Metaphlan we have found that abundant taxa include Lactobacillus and Roseburia. Ongoing analysis will enable us to establish the relative influence of host genetics on the metagenome composition of the two species.
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Accelerated evolution of lager yeast strains for improved flavour profiles
More LessFermented food and beverages have accompanied humans throughout history. Several species of microorganisms can transform raw materials into products with different and improved characteristics, for example alcoholic beverages (wine and beer) or dairy products, such as yogurt. In a world of constant change and competitiveness, brewers have to modulate and create new products to satisfy consumers. Therefore, the aim of my project is to generate lager yeast capable of producing molecules that improve the organoleptic profile of alcoholic beverages and understand the genetic and biochemical changes that increases the production of these aromatic molecules. The initial work has focused on the Ehrlich Pathway and the ester production. To produce changes in the genome, two approaches have been followed: one chemical way, using Radicicol, and one physical way, Heat Shock Thermal Stress (HSTS). Both ways inhibit Heat Shock proteins. Furthermore, this inhibition produces DNA damage, introducing Single sStrand Breaks (SSBs) and Double Strand Breaks (DSBs). The result of this inhbition is damage in the DNA and, furthermore, mutations in the DNA (aneuplidies and INDELS). After the Accelerated evolution, the evolved strains were plated on media containing amino acid analogues. These amino acid analogues select for cells with decreased feed-back inhibition of amino acid biosynthesis. Different mutant strains were generated with these two ways. These strains will be tested by different approaches, as growth curves, genome ploidy using cCGH and CHEF gel and qPCR analysis to measure the flux throughout the fermentation.
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National collection of type cultures: the bacteriophage and plasmid collections and repositories
The National Collection of Type Cultures (NCTC) is the world’s oldest bacterial collection that was specifically established to provide strains globally to support scientific research. In addition to the general catalogue NCTC has a fully curated bacteriophage and plasmid archive that to date has not recently been made available to the wider scientific community. The NCTC bacteriophage collection consists of over 100 bacteriophage and their corresponding bacterial hosts which were originally deposited primarily for their value in bacterial typing. The collection consists of bacteriophage from the following hosts: Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli and Campylobacter. The NCTC Datta plasmid collection is a curated collection of over 500 unique plasmids which were originally curated to examine the biology of plasmid transfer between and within bacterial strains. The NCTC bacteriophage collection is currently being fully characterised using a range of modern methods including genomic sequencing and electron microscopy. The plasmid collection is also being characterised using genomic sequencing and restriction digest profiles. It is intended that once characterised and rebanked both the plasmid and bacteriophage collections will be made available in 2019 to scientists to support research and development. The NCTC bacteriophage and plasmid collections will both be dynamic, representing an active repositories into which microbiologists can deposit phages and plasmids to support accessibility and reproducibility in science.
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Genome dynamics of Streptomyces clavuligerus
More LessStreptomyces clavuligerus is the producer of clavulanic acid; aβ-lactamaseinhibitor that is used in combination with the antibiotic amoxicillin. Genome sequencing has established that S. clavuligerus contains a linear chromosome and four linear plasmids: pSCL1, pSCL2, pSCL3 and pSCL4. Although pSCL4 carries 20 % of the S. clavuligerus coding sequences, none are thought to be essential with the exception of tap and tpgthat encode terminal proteins necessary for priming of the lagging strand at the telomeres. In order to confirm plasmid essentiality and the genomic architecture of S. clavuligerus, we first corroborated the available genome sequences by physically mapping the genome using Pulsed-field Gel Electrophoresis, this confirmed the presence of the replicons pSCL2, pSCL3 and pSCL4 with sizes of 150, 450 and 1800 kilobases respectively. In addition, bioinformatics and physical analyses of the S. clavuligerus genome allowed the identification of similar non-archetypal telomeres in the chromosome and pSCL4 at one end of each replicon. Despite this, the other telomere remains unidentified, which suggests there is a dynamic chromosome-plasmid relationship in S. clavuligerus. Furthermore, in order to study the essentiality of pSCL4 we first introduced a copy of tap-tpg onto the chromosome prior to plasmid curing and/or tap-tpg deletion. Consequently, targeted genome sequence and further physical analyses, especially of the telomeres, will permit us understand the complex dynamic relationship between the megaplasmid and the chromosome in this important industrial microorganism.
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Phylogenetic analysis of integrases of Acinetobacter baumannii genomic islands
Acinetobacter baumannii among other pathogens is capable of acquiring new virulence determinants via mobile genetic elements (MGE). Analysis of the distribution of genomic islands (GIs) has shown in many species that most MGEs cluster tightly with specific lineages on a phylogenetic core genome tree. This study aims to address the questions on the association between GIs distribution among certain clonal lineagesand to test the contribution of G08 and G62 in A. baumannii to metal susceptibility phenotypes. The whole genome phylogenetic single nucleotide polymorphism (SNP) tree was build using the feature frequency profile. As input, the 101 complete A. baumannii genomes deposited in GenBank were used. The matrix was generated using command line blast and the R platform to generate the output. Susceptibility testing of heavy metals was performed on wild-type and GI-deletion mutants using eight different heavy metals. Whole genome phylogenetic SNP tree constructed on all complete deposited A. baumannii genomes showed that the integrase of the metal resistance G08 and G62 are present only in single or very few sequence types (STs). To test the hypothesis that these GI seven if present only in defined lineage are still mobile, G08 and G62 were selected respectively in strains AB0057 and ATCC17978. Phylogenetic analysis of the respective GI integrases confirmed that the G08int and G62int belong to a separate clade within the Acinetobacter tyrosine recombinases. Susceptibility testing in A. baumannii strain ATCC 17978, AB0057 and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility.
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