- Volume 1, Issue 1A, 2019

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.

South American Zika virus (ZIKV) emerged as a novel enzootic pathogen linked with neonatal brain defects and acute neuropathies, its natural history characterised by a sylvatic transmission cycle with arboreal mosquitoes and African primates. Yet infectivity studies in New World primates indigenous to Zika-endemic regions including host range, susceptibility, replication dynamics, tissue tropism and virus persistence are currently lacking. New World species Callithrix jacchus (marmosets) and Saguinus labiatus (red-bellied tamarins) were highly susceptible to sub-cutaneous challenge with South American ZIKVPRVABC59 inducing rapid, high, acute viraemia in each species. Differences in acute phase vRNA in blood were highly statistically significant at day 3 between these New and two Old World species Macaca mulatta (rhesus macaque) and Macaca fascicularis (cynomolgus macaque), Mann-Whitney U testp=0.00058. Comparative quantitative RT-PCR and RNAscope in situ hybridisation analysis of tissue viral copy number and cellular localisation patterns with Old World NHP revealed early, widespread distribution across multiple skin biopsies distant to the inoculation site, lymphoid organs, reproductive sites and the brain. While viraemia was cleared from blood by day 42 in all individuals, ZIKV persistence in most tissues was identified 100 days post-inoculation. Early neuroinvasion and persistence, following acutely resolved infection characterises ZIKV susceptibility in multiple hosts, especially New World species. Establishment of a ZIKV sylvatic cycle in the Americas may provide persistent animal reservoirs for future outbreak resurgence. New World monkeys represent viable models to understand ZIKV pathogenesis, potential therapeutic interventions and vaccine development strategies.

Solventogenic Clostridium spp. have significant potential as a source of renewable biochemicals. Production of solvents such as butanol and acetone from members of this genus is already being commercialised by industry. However, the scale of biological knowledge of these Clostridia lags behind the scale of their application. With the advent of next generation sequencing methods, transposon mutagenesis now provides a large-scale, high-throughput forward approach to understanding the genetics of these species. We are applying transposon directed insertion-site sequencing (TraDIS) to the industrially-relevant solventogenic species Clostridium saccharoperbutylacetonicum. We are utilising this robust TraDIS pipeline to uncover the essential genome of the species under laboratory conditions. Furthermore, we are investigating the species’ tolerance to butanol, a key limiting step in the fermentation process. In collaboration with Green Biologics, we are also examining the essential genome of the species under industrially important fermentation conditions. Understanding the genes that are conditionally essential in these contexts will be key in advancing the biological knowledge of the species as well as providing information that can improve the fermentation process.

The Advanced Biotechnology for Intensive-Freshwater Aquaculture Wastewater Reuse (ABAWARE) project, which is part of the European Commission’s Water Joint Programming Initiative 2016 Joint Call, aims to increase the efficiency and resilience of water use in aquaculture and minimise its negative impact on the environment and human health. This research, which forms one part of the total ABAWARE project, aimed to ascertain the impact of using microbiota and certain plant species, in conjuncture with a more traditional Recirculated Aquaculture System (RAS), as a filtering system had on the arb and G abundance in various samples taken from an aquaculture facility. Sediment and water samples were taken from the inflow, the main fish basin, after the bioactive ponds before filtration, and after filtration. The resistance genes present in these samples were detected using the Wafergen smartchip real-time qPCR system. This system allows for the simultaneous quantification of 348 distinct Antibiotic Resistance Genes for each sample. The samples also underwent microbiome analysis via 16S rRNA metagenomic sequencing. Mothur was used to analyse the sequencing data. This data informs us of the changes in the microbial population changes that are enacted by the various stages within the aquaculture facility.

The increasing demand for rare earth elements (REE) is fuelled by their importance in a green energy future, with the demand for dysprosium predicted to increase by 5 % annually by 2026. Bioleaching approaches are being investigated for the recovery of REE and other precious metals from waste materials, however even 100 % recovery will not be able to meet increasing demand. Therefore, extraction from primary sources will be required. REE do not form high concentration ores, so their extraction can require processing large volumes of material, however REE are frequently associated with other raw materials and REE are sold as by-products of iron mining from Chinese deposits. Bioleaching offers the potential to produce valuable by-products from existing mining operations or to remediate historical mine waste. The diverse nature of REE-bearing minerals means that a variety of established and emerging bioleaching approaches could be applied: organic acid leaching, oxidative leaching of sulphidic ores, and reductive leaching of oxidised ores. We have applied these processes to three bauxites, demonstrating varied responses to bioleaching with each bauxite. The combination of low concentration ores and varied sources provide a challenge to recovery: however, it is one that microbes could take on.

Microbial Se or Te reduction offers a potential route to biorecovery of these elements from solution. Reduction is often efficient and large amounts of these metalloids can be removed from solution, resulting in extensive precipitation around biomass. This is more effective than biomethylation, which can result in only small amounts of removal, and would necessitate a further trapping step to recover volatilized methylated derivatives. In this research, the fungi Aureobasidium pullulans, Mortierella humilis, Trichoderma harzianum, and Phoma glomeratawere used to investigate the formation of selenium- and tellurium-containing nanoparticles during growth on selenium- and tellurium-containing media. Most organisms were able to grow on both selenium- and tellurium-containing media at concentrations of 1 mM and this resulted in extensive precipitation of elemental selenium and tellurium on fungal surfaces observed by the bright red and black colour changes. Red or black deposits were confirmed as elemental selenium and tellurium, respectively, by X-ray powder diffraction. Apart from elemental selenium and tellurium, selenium oxide and tellurium oxide were also found after growth of Trichoderma harzianumin the presence of 1 mM selenite and tellurite together with the formation ofelemental selenium and tellurium. The hyphal matrix provided nucleation sites for metalloid deposition with extracellular protein and extracellular polymeric substances serving to localize the resultant Se or Te nanoparticles. These findings are relevant to remedial treatments for selenium and tellurium contamination, and possible novel approaches for selenium and tellurium biorecovery from liquid matrices.

Mountains of plastic waste consisting of carrier bags and medical packaging are buried in landfill sites dumped in rivers around the world annually. Unfortunately plastics generated by the petrochemical industry are not biodegradable and therefore accumulate in the environment at a rate of over 25 million tones year-1. Therefore there is a huge demand for biodegradable plastics. Polystyrene (PS), polyethylene (PE) and polypropylene (PP) are problematic materials, used for appliance housings, disposable cutlery and general packaging. This study investigates the utilisation of waste PS, PE and PP as a potential additional carbon sources using bacteria to synthesise polyhydroxyalkanoates (PHAs); a value-added material, able to replace some conventional fossil-fuel plastics while being non-toxic, fully biodegradable and biocompatible. Prodegraded waste PS and PP, and thermally treated PEwere used as supplementary carbon sources totryptone soya broth (TSB and BSM) for 48 h fermentations [1, 2, 3]. The bacterial strain Cupriavidus necator H16 was selected as it is non-pathogenic, genetically stable, robust and one of the best natural producers of PHA. The accumulation of PHAs varied from 17 % (wt / wt) of dry biomass in TSB controls to 39–66 % for PS, PE and PP thermally treated samples. The polymers obtained were analysed with nuclear magnetic resonance (NMR) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) to characterise their chemical structure. In conclusion, certain thermal treatment protocols of the waste plastics were shown to be viable for PHA production; with3-hydroxybutyrate and up to 12 mol% of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units formed.

The end of the European milk quotas in 2015 resulted in a steep increase of Irish milk production from 5 to over 7.2 billion litres annually. Dairy processing ads value, but generates up to 10 litres wastewater (WW) per litre processed. Organic pollutants in the WW need to be removed before discharge. Our approach aims to turn this waste into a resource for polyhydroxyalkanoates (PHA), bioplastic production. Bioplastics are promising materials to reduce our dependency on fossil fuels and waste production. The first production step includes an adaption of the dairy processing WW in an anaerobic, hydrolytic reactor, where acidogenic bacteria metabolise the organic fraction of the WW and form volatile fatty acids (VFAs) the building blocks of PHA. The second step comprises the adaption of biomass towards PHA accumulation. The adaption is driven by an aerobic dynamic feeding strategy to increase the formation of the storage molecule PHA. Addition of the WW as substrate is followed by a starvation period, where bacteria capable of PHA storage have a selection advantage. Over time the mixed microbial system is therefore optimised for PHA accumulation. In a final production step the adapted WW and biomass is combined in a fed-batch reactor to produce the end product PHA. The laboratory scale system could be established and is currently optimised. A later scale-up will help to assess the full economic potential of this waste to value approach.

Antimicrobial resistance (AMR) is becoming the biggest problem facing human and animal health care, with a predicted 10 million deaths per year due to antibiotic resistance by 2050 (Review on Antimicrobial resistance, 2014). The focus of many AMR studies is on problems inside clinical settings, such as hospitals and veterinary practices, yet, as this study shows, often the most diverse sources of resistance genes are found in relatively unexplored areas of the environment; in particular dog faeces and dairy cattle manure.

Chromogenic media analysis showed dog faeces had the greatest number and diversity of ESBL-producing bacteria. Multiplex PCR showed inconclusive evidence of what the resistance genes were in both dairy cattle manure and dog faeces. Phylogenetic analysis of the 16S rRNA showed Flavobacteriaceae-like bacteria in dairy cattle manure but the presence of Enterobacteriaceae-like bacteria, most similar to Escherichia coli, in dog faeces.

The major conclusion of this study is that ESBL-producing bacteria are present in high abundance in both dog faeces and dairy cattle manure and that ESBL-producing bacterial species in dog faeces showed higher diversity than first expected while pre-boil wort had no ESBL-producing bacteria.

The results of this study have crucial significance for future studies and the wider community. The statistical, microbiological and phylogenetic data collected provide strong evidence of the role of the environment in the spread of antibiotic resistance. Secondly, the study utilises a holistic visual model to map the spread of resistance and inform the wider community of the issue of AMR in the environment.

To isolate fungal communities from coastal areas of the Red Sea in Saudi Arabia and identify and classify them by molecular techniques. Samples were collected from the seaside of the Red Sea in Jeddah, Saudi Arabia in March 2012 and stored in sterile screw cap bottles for further analysis. Phenotypic and genotypic characterization of fungal isolates was done using standard techniques. Eight fungal genera including Aspergillus, Penicillium, Thielavia, Fusarium, Emericella, Cladosporium, Scytalidium and Alternaria. Most isolated fungi showed significant growth on petroleum media and were thus considered capable of biodegradation of crude oil-based substances. The fungal genera isolated from the Red Sea had 97 –100 % similarity with the related fungi recorded in the GenBank in which they were deposited. The morphological and molecular structure of these marine fungal isolates closely resembles their terrestrial counterparts in the Genbank. The capabilities of these fungal species to utilize petroleum as a source of carbon speaks to future applications in which marine fungi may be utilized in the breakdown of petroleum-based waste in an ecologically efficient manner.

The role of clinical genomics in infectious disease diagnostics and public health microbiology is the topic of discussion during a recent decade. Although much of this work is aimed at describing the structure of outbreak communities, the methodology works equally well to identify pathogens in clinical samples. Clinical genomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The key concept in using clinical genomics methodology is that detection of microbes is independent of culture and is not limited to targets used for in-depth PCR assays. Rather, it is a process of generating large-scale sequence data sets that adequately sample a specimen for microbial content and then of applying computational methods to resolve the sequences into individual species, genes, pathways, or other features.

The strains of Nodularia are known to produce potent hepatotoxic nodularins (NOD) and other bioactive metabolites i.e. nodulopeptins and spumigins. Contemporary, three new nodulopeptins were also isolated from N. spumigena KAC 66 collected from the Baltic Sea. The wide distribution and toxicological effects of N. spumigena on food chain, has caused significant attention towards the effects of the species. In the present investigation the toxicity of NOD and nodulopeptin 901, produced by N. spumigena, was fractionated by Reversed Phase Flash Chromatography (RPFC) and their toxicity was determined by their lethality to water fleas, Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1; eukaryotic threonine phosphatase/protein serine). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. The pure NOD was lethal to D. pulex and D. magna LC50=8.4 µg. ml-1 and 5.0 µg .ml-1, respectively. The newly characterized nodulopeptin 901 was also tested against D. magna (LC50=>100 µg .ml-1). NOD and nodulopeptin 901 inhibited PP1 with weak IC500.038 μg .ml-1 and 25 µg .ml-1, respectively. The anabaenopeptin A (ANA) and anabaenopeptin B (ANAB) were used as reference peptides as they have similarity in structure with newly characterized nodulopeptin 901. The ANA, ANB and linear nodularin (LNOD) inhibited PP1 with IC5070μg .ml-1,100μg .ml-1 and20μg .ml-1, correspondingly. This is the first study that indicates the toxic and inhibitory activities of nodulopeptin 901 and fractions of N. spumigena KAC 66 against daphnids and PP1.

Soft cheese (wara) is an unripened cheese consumed in several parts of West Africa due to its various nutritional qualities. Soft cheese, a coagulated product of raw milk is usually produced from cow milk using Calotropis procera. This study therefore sought to assess the effect of the different coagulants such as Calotropis procera, Carica papaya, lemon juice and steep water from cereals (maize, millet and sorghum) on the amino acid content of soft cheese produced from sheep milk. Raw milk sample was collected from sheep and processed into soft cheese by these coagulants and the amino acid composition of the sample was carried out using standard methods. The result revealed that Calotropis procera coagulated soft cheese has the highest essential amino acid content Leucine (10.21 g/100 g), while steep water from millet coagulated soft cheese has the lowest essential amino acid content methionine (0.72 g/100 g). However, lemon juice coagulated soft cheese has the highest non essential amino acid glutamic acid (16.27 g/100 g) in all the cheese samples. In conclusion, this study revealed that highly nutritious soft cheese can also be gotten from sheep milk other than the commonly used cow milk and other coagulants such as lemon juice can compete favorably well with Calotropis procera in production of highly nutritious soft cheese. It is therefore recommended that soft cheese produced from sheep milk coagulated by lemon juice should be incorporated into daily diet due to its highly nutritional content.

The isolation, physiological and molecular identification of Bacillus spp. for the development of starter culture in Ugba production were carried out. Sixty two bacteria were isolated and 55 Phynotypically characterized as Gram positive, rod shaped, motile, spore-forming and catalase positive typical characteristics of Bacillus spp. Seven isolates were suspected to be Micrococcus and Staphylococcus spp. They were Gram negative, cocci and catalase negative. The genotypic identification of the suspected Bacillus spp. was done using amplification and partial sequencing of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragment analyzed from isolates confirmed the organisms as Bacillus cereus (30), Bacillus subtilis (17) and Bacillus licheniformis (8). Bacillus subtilis was the dominant species in Ugba fermentation as it had the highest recorded number of isolates. This study indicated that molecular characterization of organisms as a good tool for the identification of organisms for the development of starter culture for use in food fermentation.

Corrosion is the main feature of the oil pipelines destruction. Biocorrosion has been detected in various industries, especially in the oil industry. The rusted pipes samples were obtained from the Gandomkar petroleum pipeline station, Iran. For screening the corrosion producing bacteria, the samples were cultured in the selective culture medium, manganese agar and Iron oxidizing agar incubated at 30 °C for 18 h. The purified individual colonies were subjected to macroscopic, microscopic and molecular examinations, respectively. The cultivation of corrosion based material on manganese agar isolated cream-coloured colonies, convex with the surrounding smooth. The microscopic examinations showed Gram-negative coccobacilli. Based on macroscopic, microscopic and molecular examinations the bacterial isolate was identified as Stenotrophomonas maltophilia strains PBM-IAUF-4 with the accession number of KU145280.1 in Gene Bank. The cultivation of corrosion based material on Iron oxidizing agar isolated cream-coloured colonies cream-coloured colonies that had swarming, convex. The results showed Gram-negative bacilli. Based on macroscopic, microscopic and molecular examinations the bacterial isolate was identified as Kluyvera intermedia strains PBM-IAUF-1 with the accession number of KU145277.1 in Gene Bank. This is the first report of isolation and identification of corrosion-producing bacteria from, Gandomkar, Iran. The first isolated bacterium was identified as Stenotrophomonas maltophilia. The second isolated bacterium was identified Kluyvera intermedia. Both bacteria were isolated for the first time in the world from pipeline corrosion samples. This study confirmed the role of bacteria in the corrosion of oil pipelines.

Reliability and reproducibility of transcriptomics-based studies are dependent on RNA integrity. Microfluidics-based techniques targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of RT-Q-PCR amplicons of the glutamine-synthetase A (glnA) transcript. We showed, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure byamplicon sequencing of the 16S rRNA, amoA and glnA transcripts. We successful sequenced transcripts for all three targets even from highly-degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcripts profiles. Since both RT-Q-PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

Provision of clean drinking water is regarded as being the most significant positive intervention in human health and it plays a significant role in supporting global health. Population growth, economic development and climate change all drive urbanisation and increase demand for clean water and the infrastructure for its delivery. As demand for clean water increases, so will the pressure to ensure its safety. Indwelling sensor networks offer real-time, long-term and intelligent monitoring system, and would enable optimisation of networks for quality. Electrochemical sensors are inexpensive and simple to construct and operate. However, prolonged exposure of the sensors to water causes biofouling which compromise their performance even in weeks or days, making early detection of performance failure critical. By using a combination of electroanalytical methods (cyclic voltammetry, chronoamperometry, electrochemical impedance spectroscopy and hydrodynamic voltammetry) we can quantify the mass transport and kinetics effects of the first six days old biofilm layer to electrode reactions. We used agarose hydrogels and cellulose acetate layer as model biofilms to provide rapid and simplified characterisation of early fouling effects which then compared with lab grown biofilm of Pseudomonas fluorescens. We present a protocol forin situdiagnosis of electrochemical sensors fouling during early stages of biofouling. We also demonstrate that the behaviour of lab grown biofilm can be approached by using a simple model made of hydrogels (i.e. agarose) or a dried layer of an organic solution (i.e. cellulose acetate) which provides a potential application in other fields beyond environmental sensors development such as infectious disease and novel antibiotic researches.

Root associated microbiomes (RAMs) are complex communities which provide benefits to host plants via disease suppression, abiotic stress relief and increased nutrient bioavailability. Most RAM studies have focussed on bacteria and fungi, archaea have largely been overlooked as many studies fail to utilize archaea-specific 16S primers. However, there are reports of archaea being detected and isolated from the rhizosphere and endosphere of crop species, and one report of a plant-growth promoting (PGP) ammonia oxidising archaeon (AOA). Here, we aimed to assess the role of AOA within the wheat (Triticum aestivum) RAM. We applied archaea-specific primers and 16S amplicon sequencing to profile the archaeal community associated with wheat roots grown in agricultural soil. To assess PGP capacity we treated wheat seeds with a concentrated inoculum of model AOA Candidatus Nitrosocosmicus franklandus C13. In contrast to prior reports this had no impact on plant biomass, indicating N. franklandus may be a passive member of the wheat RAM. Stable isotope probing (SIP) experiments have confirmed that bacterial species metabolise Arabidopsis thaliana root exudates. Fractions are being examined to assess whether archaeal species can do the same, and a similar SIP experiment will be performed in wheat. An enrichment culture experiment using root exudates will also be applied to identify and isolate archaea capable of metabolising wheat root exudates. Here we show that AOA are present within the wheat RAM; to understand the niche occupied by these microbes we must further probe how they interact with host metabolites, and whether they contribute to host fitness.

The ability to selectively sequester bacteria from a mixed population is a desirable aim across a range of fields. Already some technologies exist to attempt to meet these aims spanning microfluidics and automated flow cytometry to antibody conjugated magnetic nanoparticle precipitation. However, these technologies are generally limited to the confinement of small populations in defined locations. Furthermore they may not be able to differentiate morphologically similar but phenotypically divergent cells. We aim to produce a novel technology to isolate bacteria from mixed populations by utilising polymeric carbohydrate ligands which bind to inducible bacterial adhesion proteins. To achieve this aim an inducible mutant of the fim operon in Escherichia coli has be constructed, thus allowing for switchable production of the mannose binding organelle, Type 1 fimbriae. Furthermore, we have previously observed that mannosylated polymers selectively bind to Type 1 fimbriated E. coli and to our induced mutant. Novel mannose functionalised polymers are being synthesised via a reversible addition-fragmentation chain transfer (RAFT) scheme. These polymers contain a catechol terminus which may be conjugated to magnetic Fe3O4 nanoparticles thus facilitating selective bacterial sequestration by magnetic separation. The successful development of this polymer-based bacterial sequestration platform could potentially enable the equivalent of immunoprecipitation in large-scale fermentation processes or the precise manipulation of living cells in laboratory scale procedures.

Brown seaweeds such as Laminaria species are a rich source of polysaccharides such as laminarin and fucoidan which have a variety of functional food and animal feed applications, as well as alginates with demonstrated biological and pharmacological activities. Macroalgal surfaces are rich in carbon-based constituents which provide a suitable environment for growth and colonization by diverse bacterial communities. Several environmental and non-environmental factors can influence the composition and abundance of epibacterial communities associated with seaweeds. In addition to the biological, physical and chemical properties of the macroalgal surface, seasonal variations have been found to play a significant role in the structure of the associated microbial communities. Variations in macroalgal epibacterial communities have also been observed within different parts of the host algal species. However, to date, in-depth studies on bacterial communities associated with macroalgal species, their ecological role and interactions with the algae are still scarce. To gain an insight into the diversity and composition of the microbial communities associated with the brown alga Laminaria digitata, the communities derived from different parts of the alga including the blade, meristem, stipe and holdfast; were investigated using metagenomic Illumina sequencing of 16S rRNA gene amplicons. Seasonal variations in the microbial populations were found in samples taken from the Irish coast in different seasons between 2017 and 2018. This metagenomic-based investigation provides a detailed view of the seasonal variations in the bacterial populations associated with Laminaria digitata and helps provide further insights into potential interaction between this macroalga and its epiphytic bacterial communities.

Extended-spectrum β-lactamase (ESBL)/AmpC producing bacteria are one of the critical priority resistant bacteria that contributes to treatment failure and increased death rates. In this work we aimed to study the role of wastewater treatment plants (WWTPs) as reservoirs of ESBL/AmpC producing faecal coliforms. The effluent samples were collected from two WWTPs and faecal coliforms were isolated from all samples using the membrane filtration method. Bacterial isolates were subjected to antimicrobial susceptibility testing toward cefotaxime and ceftazidime. The isolates that showed a resistance phenotype to these antibiotics were considered as putative ESBL/AmpC producing bacteria. These bacteria were subjected to the AmpC test using a protocol with phenylboronic acid. The AmpC negative strains in the AmpC test served as samples for multiplex PCR containing primers specific for blaTEM, blaSHV and blaCTX-M. In total, 498 faecal coliforms were isolated from WWTP effluent samples. For the antibiotic susceptibility testing 99 isolates were considered as ESBL/AmpC producing bacteria. Among them, 26 isolates were found to be positive in the AmpC test. The PCR results revealed that 49 isolates carried blaTEM, 6 bla SHV12, 1 blaCTX-M1 and 5 blaCTX-M15. The ESBL/AmpC producing faecal coliforms in WWTP effluent are discharged to the receiving water environment. These data need to be considered when analysing the risk of WWTP effluent to the environment and to human health, as many of the bacteria identified are not analysed in assessment of risk of pollution from WWTPs globally.

Poor soil conditions limit the building of new infrastructure, which is needed for an ageing and expanding population. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination, therefore there is demand for a sustainable approach to this process. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate (CaCO₃), which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase mechanical properties. Commonly used bacteria include Sporosarcina pasteurii and Bacillus subtilis. A range of factors influences MICCP which presents challenges with process optimisation. These factors need to be optimised in the laboratory before they can be applied for engineering purposes. The overall aims of my research are to optimise urease production in S. pasteurii and B. subtilis and to investigate the distribution and binding of these bacteria with various sand particles, by means of syringe and glass column set ups. These bacteria will be compared with engineered bacteria which can overproduce urease to investigate the impact on precipitation efficiency. Factors to control bacteria biofilm formation to influence the morphology of CaCO₃ will be investigated to determine the impact of various crystal shapes on soil properties. Ultimately, raw data generated from the project will be used for predicting biocementing at a lab scale for building computational models.

With an annual death toll of 7 million, the effect of air pollution on human morbidity and mortality is a major global problem. Air pollution associated with infectious respiratory disease is responsible for a mortality rate of 1.5 million annually. However, the impact of air pollution on bacterial behaviour is largely unknown. Our work has shown for the first time that black carbon (BC), a major component of air pollution, increases dissemination of colonising Streptococcus pneumoniae and Staphylococcus aureusin in-vivo infection models, and also alters biofilm structure, composition, and function. However, the biological mechanisms responsible for dissemination in the host and biofilm alterations by BC are unknown. BC is known to elicit an oxidative stress in eukaryotic tissues, therefore we hypothesise that the S. aureus oxidative stress response may play an important role in the bacterial response to BC. Our data shows that exposure to BC is toxic to S. aureusand that oxidative stress response of mutant strains show increased sensitivity to BC than the wildtype strain. Transcriptional analysis demonstrated that the expression of key oxidative stress genes in the oxidative stress response pathway are induced in the presence of BC. These findings demonstrate that BC has a metabolic effect on S. aureus and that the oxidative stress response is required for bacterial survival to BC. Furthermore, the induction of the S. aureus oxidative stress response may be important for increased dissemination in the host through adaptation of bacterial cells to the host immune response.

Manure spreading onto land is an important agricultural process. Manure is recycled as organic fertiliser; however it can introduce manure-derived antibiotic resistant bacteria into the environment. Grassland consists of approximately 70 % of global agricultural land and is a vital source of food for livestock. Despite the important role grassland plays in food security, the impact of manure application on its resistome and microbiome is relatively unknown. Antibiotic resistance is a multifactorial issue, involving an intertwining relationship between animals, humans and the environment. Therefore, it is critical to fully understand all potential routes of antimicrobial resistance (AMR) transmission. As the microbiome of grassland is an under-researched area, it is a possible source of AMR transmission to animals which may enter the food chain. A pot trial mesocosm experiment was carried out to investigate the impact of manure application on the microbiome of the phyllosphere of perennial ryegrass (Lolium perenne). Pig slurry was applied to six pots of L. perenne and grass and soil samples were taken two weeks following manure application. Following sonication, viable bacteria were isolated from the soil, manure and grass by plating on selective agars supplemented with antibiotics. Isolates were screened for antibiotic resistant bacteria by antibiotic susceptibility testing. DNA was extracted from the soil, grass and manure and underwent microbial community compositional analysis by 16S rRNA sequencing on the Illumina Miseq platform. The results from this mesocosm experiment will contribute to a further field trial to investigate the impact various manure types have on the microbiome and resistome of grassland.

Geoactive fungi such as Aspergillus niger play a significant role in bioweathering processes and element cycling. These organisms are able to secrete a range of organic acids, such as oxalic acid, into their microenvironment. This enables them to mediate mineral dissolution, leading to metal solubilization and precipitation in the form of secondary biominerals. In this investigation, such biotransformation processes were explored as a means of cobalt bioprocessing, an E-tech element identified as being of key strategic importance, in addition to other mineralogically related metals. A range of Co-bearing mineral phases were investigated, including a Co-bearing lithiophorite [(Al,Li)MnO2(OH)2] and erythrite (Co3(AsO4)2·8H2O), in addition to seafloor ferromanganese nodules. Bioleaching and bioprecipitation studies were carried out to investigate the ability of A. nigerto leach cobalt and related metals from Co-bearing minerals, and to precipitate them in biomineral form as a means of cobalt biorecovery. The objective of the work is to investigate the natural biotransformation of cobalt-bearing minerals, to investigate the factors that influence cobalt bioprocessing and to optimise the maximal yield of cobalt biominerals.

The adaptive radiation of Pseudomonas fluorescens SBW25 populations in static liquid microcosms results in the appearance of the biofilm–forming Wrinkly Spreader. This adaptive mutant is able to colonise the high O2-rich region at the top of the liquid column established by the metabolic activity of earlier wild-type colonists, and by doing so, enjoys a significant fitness advantage over non-biofilm–forming competitors including the ancestral Pf. SBW25. Although the underlying molecular biology and evolutionary ecology of the Wrinkly Spreader is well understood, we have recently questioned the need for expensive biofilm–formation to colonise the air-liquid (A-L) interface, as O2-directed flagella-mediated swimming (aerotaxis) should be sufficient to maintain cells in this region. Our investigations show that swimming can overcome displacement by Brownian diffusion and microcurrents within the liquid column. However, it is not sufficient to explain the high levels of enrichment at the A-Linterface shown by Wrinkly Spreader cells. A comparison of the liquid surface tension of wild-type and Wrinkly Spreader cultures, supernatants, and washed cells, suggests that the Wrinkly Spreader produces a surface-active compound weakly associated with the cell which helps penetration of the A-L interface and allows cells to remain in the high-O2 region without further expenditure of energy. Our results suggest that this penetration is key to the following biofilm–formation which supports higher populations at the A-L interface and that this explains the adaptive advantage of the Wrinkly Spreader.

In this work, geoactive fungi including Aspergillus niger, Beauveria caledonica and Paecilomyces javanicus, were used to investigate their biocorrosion and deteriorative effects on copper metal to gain an understanding of the roles that fungi may play in biodeterioration of such a material in the built environment. It was clearly demonstrated that the test fungi possessed a high tolerance to copper metal. New biominerals resulted from fungal interactions with copper metal mainly arising from organic acid excretion. Copper oxalate was formed by oxalate excretion from the fungi and different patterns of bioweathering and biomineralization were generated on the copper surfaces. In addition, copper could be dissolved by certain fungi, result in significant biodeterortive effects such as etching and pitting. These results provide compelling evidence for deteriortion of copper metal by fungi and that organic acids, particularly oxalate, play an important role in this process. Such properties of metal biocorrosion and deterioration indicate the potential significance of fungi in biodeterioration of metal substrates and the importance of considering methods of protection and preservation in the built environment.

The medical importance of honey has been extensively demonstrated. Although high osmolarity, acidity and hydrogen peroxide (H2O2) proved to be the most prevalent factors in honey’s activity, the underlying antimicrobial mechanism remains obscure. Our aim is to provide insight into the physiological changes and genetic responses in honey-treated bacteria, thus improving our understanding of this natural product as a potential novel antimicrobial. A model honey composed of sugars, gluconic acid, and H2O2as they are accumulated in honey after enzymatic reaction happens, was used the investigation of honey’s activity. The bactericidal action of the model was tested on E. coli K-12 strain MG1655. Flow cytometry (FC) and Atomic Force Microscopy (AFM) identified physiological changes such as membrane potential, blebbing, and cell lysis. Reactive Oxygen Species (ROS) accumulation was observed in individual cells by FC. Transposon Directed Insertion Sequencing (TraDIS) identified mutants’ fitness over a time course of E. coli treatment by model honey. The loss of selenocysteine (selAB) and formate dehydrogenase (fdhDE) mutants, proved the redox- balancing activity as essential for the repression of ROS in stressed cells. High susceptibility of energy metabolism (atpABD) and peptidoglycan synthesis (prc) mutants, indicated the strain unable to maintain the reductive cell environment necessary for cellular activities, post honey exposure. Our findings identified some of the honey’s targets when acting as an antimicrobial. The synergies observed support the use of honey as an antimicrobial; however, the identification of mutations that led to enhanced resistance to honey is an important finding that needs further study.

The MELiSSA (Micro Ecological Life Support System Alternative) project aims to create a closed loop system, capable of providing all the necessary food, water and oxygen for astronauts on long Space missions. The MELiSSA loop is comprised of four compartments, with compartment IVA containing photoautotrophic bacteria, such as Arthrospira platensis, a good source of oxygen, edible biomass and micronutrients. One important example of the latter is cobalamin (B12) deficiency of which can lead to pernicious anaemia and neurological systems, and thus would be detrimental to astronauts on long Space missions. The molecule is not made by plants or fungi, so the ultimate source of B12 in the environment is prokaryotes, and its synthesis requires over 20 enzymatic reactions. Many eukaryotic algae also require cobalamin for growth, and some species have been shown to accumulate the vitamin when grown in coculture with cobalamin synthesising bacteria. The aim of this project is to extend existing knowledge of algal-bacterial mutualisms involving cobalamin. Eukaryotic species such as Haematococcus pluvialis and Chlorella vulgaris, both certified as safe for human consumption, along with A. platensis will be investigated with different bacterial partners to maximise cobalamin accumulation. Not only will this research help provide adequate nutrition on long Space missions, it will also support nutritional supply on Earth with the rise of veganism, as well as, aiding in understanding the dynamics of algal-bacterial mutualisms, which is of interest in terms of nutrient cycling in the environment.

Drinking Water Distribution Systems (DWDS) are diverse ecosystems where the majority of microorganisms live forming biofilms, which can alter the water quality if they are mobilised to the bulk water. Biofilm communities can be affected by the increase of temperature due to climate change, thus compromise the distribution of safe water. To understand the effect of temperature on biofilms in DWDS, biofilm was developed for 30 days at 16 °C and 24 °C using a full-scale experimental DWDS facility. Samples were collected at the end of the experiment from removable coupons inserted into the pipes. DNA was extracted and the 16S rRNA and ITS rRNA genes were sequenced and analysed, for the bacterial and fungal diversity respectively. Differences in bacterial and fungal diversity at both temperatures were observed at family level. At 16 °C bacterial community was dominated by Comamonadaceae (21.48 %), Pseudomonadaceae (16.41 %) and Sphingobacteriaceae (12.99 %). However, at 24 °C the most abundant family was Pseudomonadaceae (50.60 %) followed by Sphingomonadaceae (9.59 %) and Sinobacteraceae (7.82 %). Fungal diversity showed that at 16 °C the most abundant family was Nectriaceae (68.9 %), followed by Helotiales (24.5 %) and Filobasidiales (1.5 %). However, at 24 °C the community was dominated by Nectriaceae (98.15 %) and the following families showed a low relative abundance, Rhizopodaceae (0.95 %) and Cryptomycota (0.24 %). Temperature is a key factor for microbial growth in DWDS and affect the composition of the microbial communities. Temperature increase leads changes and a loss in complexity in bacterial and fungal communities of biofilms, which can affect the water quality.

Lanthanum is an important member of the rare earth elements (REE) that are a global strategic resource and have many technological applications ranging from microelectronics manufacturing to the production of clean and renewable energy. Aspergillus niger is widely used in industrial fermentations due to its production of multiple secondary metabolites including citric and oxalic acids. Since it is a ubiquitous soil inhabitant and produces geoactive agents, it can play a role in the biotransformation of metal-containing minerals. Previous studies found that A. niger is capable of mineral solubilization and secondary mineral formation, many metals being precipitated as oxalates. However, there is limited knowledge about the biotransformation of La mediated by fungi. The aim of this project was to explore the mechanisms and factors determining the interactions between A. niger and La. In this study, fungal growth on La-supplemented solid media was carried out and it was discovered that crystalline deposits were formed around fungal colonies in the presence of LaCl₃. These biogenic crystals were recovered and subjected to examination for their elemental composition, morphological features and mineral phases using energy dispersive X-ray analysis (EDXA), scanning electron microscopy (SEM) and X-ray diffraction (XRD) respectively. These confirmed the biotransformation of lanthanum and identified the products as lanthanum oxalate [La₂(C₂O₄)₃·10H₂O], which was further transformed into La₂O₃ by thermogravimetric (TG) treatment. Geochemical modelling also supported these results. Our findings provide a new aspect for the bioprecipitation and biorecovery of REE from solution using fungal culture systems.

The biogenic production of CaCO₃ via Microbially Induced Calcite Precipitation (MICP) has been reported in many microbial strains. This process has a wide variety of current and potential applications, namely in civil engineering, agriculture and bio-remediation. The number of species used in such applications remains limited and seems to be biased towards ureolytic strains. Urea degradation is the best documented process leading to MICP, however, there are several alternative processes, possibly more relevant, which are mostly overlooked. In general, and despite being widely reported, the MICP process is still poorly understood, and has been chronically understudied from a genomic perspective. Here we report on the genus-wide analysis of MICP capability, centred on genomic-based analysis, and focusing on the genus Bacillus. This genus harbours several species capable of MICP and is the most widely used regarding its biotechnological application. The very high number of species within this genus, and availability of whole genome sequence data for several makes it an ideal target for this analysis. Our preliminary results uncover a diverse range of MICP-associated genes, identifies similar genomic profiles within phylogenetic subgroups, and questions the importance of urease activity for CaCO₃ production in the genus Bacillus. This study is the first of its kind and provides key insights into the genomic basis of MICP, while testing the feasibility of a genomic-based prediction method for fast identification of new strains with such capabilities, which would be applicable to other genera and be particularly useful for downstream applications.

Actinobacteria have provided a rich source of novel antimicrobial compounds throughout the antibiotic era. Thermophilic Actinobacteria growing at higher temperatures (>50 °C), have not been extensively studied, despite producing important antibiotics such as thermomycin and anthramycin. We are testing the hypothesis that thermophilic Actinobacteria produce new and unusual antimicrobials at higher temperatures, potentially leading to the discovery of novel heat stable compounds; especially those active against life-threatening fungal infections in humans such as invasive aspergillosis caused by Aspergillus fumigatus, which has developed resistance to current treatments. Compost is a rich source of thermophilic Actinobacteria responsible for generating the heat required for decomposition and yet this niche has been overlooked in terms of natural product discovery. A. fumigatusis a fungus that also lives in compost and also contributes to the composting process and we reasoned that Actinobacteria living in this environment might display activity against pathogenic strains of the fungus. Samples from a series of ‘windrows’ at a commercial green waste processing facility yielded 13 thermophilic Actinobacteria, and strains of Aspergillus fumigatus. The phylogeny and species identity of the bacterial strains were determined by 16S rRNA sequencing. Candidate strains were screened for the ability to inhibit ESKAPE pathogens as well as A. fumigatus, using agar overlays and MIC assays. Selected strains were analysed by whole genome DNA sequencing and likely antimicrobials predicted. Compound identification using mass spectrometry and metabolic profiling has been undertaken on strains that display antibiotic activity, providing a path for the development of new antimicrobials for clinical use.

Environmental microbiome research shows that microbial communities help to shape complex ecological processes that influence human and environmental health. Researchers are currently investigating the potential health-inducing interactions between humans and the environmental microbiome, particularly in urban areas. However, not only are there inherent technical issues to overcome in implementing the findings of this research, but there are also complex social, political and economic factors that affect the design, construction and management phases. Drawing on a recent paper discussing opportunities for Microbiome-Inspired Green Infrastructure (MIGI) (Robinson et al. 2018), we set out criteria for an ‘Environmental Microbiome Toolkit for Urban Designers’ that could be used by planners, architects, landscape architects and civil engineers. We provide a worked example of this toolkit to design a public space in Sheffield, demonstrating practical design techniques to consider the environmental microbiome and its role in human and ecosystem health. The Landscape Institute (LI) is the professional body that regulates and represents landscape architects, providing guidance across all spheres of the profession. One of the key functions of the LI is to develop and maintain the LI Plan of Work, regulating the work that landscape architects undertake at each stage of a project, from landscape assessment through to conceptual and detailed design, contract administration and landscape management. We apply these industry-standards to show how the environmental microbiome should be considered in landscape assessment, design and management, bridging the gap between research and practice and providing a common reference point for future policy development and industry regulation.

Selenium and tellurium are two metalloids essential for future green energy technologies due to their associated photovoltaic and photoconductive properties. In addition, selenium and tellurium oxyanions can be toxic in the environment and can potentially affect human health. This work aims to examine some geochemical influences on Se/Te reduction carried out by selected yeast strains to identify what limitations there are to the process, and their importance. Several yeast strains, capable of selenite or tellurite reduction, were isolated from environmental soil samples on solid media containing selenite or tellurite, reduction being detected by the colour change of colonies to red (Se) or black (Te). Such reduction resulted in the formation of nanoparticles of elemental Se0 or Te0. Growth was assessed in the presence of selenite or tellurite and minimum inhibitory concentrations determined. Rates of selenite and tellurite depletion were determined in different growth conditions and the production of elemental Se0 or Te0 was analysed using energy dispersion X-ray analysis (EDXA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). This work furthers understanding of selenium and tellurium transformation by yeasts also suggests potential routes for Se/Te biorecovery by the formation of Se/Te nanoparticles.

Forty-one aerotolerant subsurface bacterial colonies were isolated from terrestrial subsurface environment at various depth from 5 feet to 50 feet at 5 feet intervals. Out of 41 colonies, 32 morphologically distinct bacterial colonies were selected for gram staining reaction, various sugar utilization pattern and scanning electron microscopic studies. Most of the bacterial colonies were gram-positive rods; some are gram-negative rods, gram-positive cocci and coccobacilli, and gram-negative coccobacilli respectively. Seven bacterial colonies NTN33, NTN34, NTN35, NTN36, NTN01, NTN02 and NTN03 isolated from 40 to 50 feet depth were neither gram positive nor gram negative, and no distinct morphological shape but gigantic structure have identified in that bacteria. The bacterial size were identified on the bacteria NTN36, NTN35, NTN01, NTN30, NTN34, NTN02 as 4.5 µm, 4.1 µm, 3.5 µm, 3.4 µm, 3.2 µm and 2.2 µm. Gigantic rod surrounded by capsular sheath and gigantic rod with footprint like appearance were identified in the bacteria NTN02 and NTN36; those are isolated from 45 and 50 feet depth. Intracellular granules were also found in bacteria NTN34, NTN35 and NTN01. In total, 24 different sugars had used to analyse the sugar utilization pattern of that 32 bacteria. Most of the bacteria have utilized only 03 different types of sugars either maltose, galactose and glycerol or lactose, trehalose and mannose. However, the bacteria NTN16, NTN12, NTN06, NTN24, NTN27, NTN28, NTN34 and NTN02 had utilized 14 different sugars. Nevertheless, very few bacteria have utilized the l-arabinose, α-methyl-d-gluconate and inulin. None of the bacteria had utilized the sugar sodium gluconate, salicin, glucosamine and dulcitol.

Laboratory experiments were conducted to determine the effect of temperature (20 °C and 30 °C) on the rate of tissue decomposition in organic compost for a period of 28 days. Porcine tissue was used as a substitute for human body parts in burial microenvironment. Measurements of the decay include porcine tissue mass loss, soil pH, the metabolic activity of soil microbes, viable count and Gram stain of associated microflora. Incubation temperature had a significant impact on the post-mortem decay rates and stages at 30 °C in comparison to the samples incubated at 20 °C. Bacterial enumeration demonstrated microbial burden to be higher at higher temperatures, suggesting accelerated decomposition. Gram staining identified mostly Gram-negative bacteria, decomposers that might have originated from the organic meat as opposed to soil bacteria. There was a significant difference in soil pH levels during harvest times and at the end of the experiments in both microenvironments. It is an indication that measured pH of the depository may reveal whether the body parts were recently buried. A fluorescein diacetate (FDA) method that measured metabolic activity in compost demonstrated the highest release of fluorescein during harvests at 30 °C. A non-parametric Friedman test of differences among repeated measures rendered significant results (α=0.00) showing differences in microbial activity according to the temperature level. The results suggest that depositional microenvironment would be significantly modified by the decaying organic matter with the rise of temperature. This way, the temperature would make an impact on the decay of buried remains.

Poison frogs secrete alkaloid toxins in their skin as defence mechanisms against predators. Numerous studies have shown that the origin of alkaloid toxins in the skin is through ‘sequestration from diet’, i.e. uptake and storage of toxins or their chemical precursors, mostly from consumed arthropods. There exists the intriguing possibility that the gut microbiome of these frogs may play a role in this process. We address this question by looking at the organism together with its associated microbial communities, an effective symbiotic relationship between host and microbiome that could have allowed phenotypic adaptation of the host to a toxic diet. We sequenced the Bacterial and Archaeal 16S rRNA regions of the gut microbiome of 7 Poison frog species and 9 outgroup frog species caught in the rainforest of Eastern Peru. Frog species were selected based on sharing similar microhabitats and comparable individual sizes. A comparative analysis of the microbiome composition across all our samples allowed us to identify a core group of abundant symbiotic microbes unique to poison frogs in spite of intrinsic variation within species. We speculate this group could be associated to their ability to sequester toxins and we carried further metagenomic sequencing to allow us to determine possible functions that may be involved in toxin processing in these frogs.

As biogas from anaerobic digestion becomes an increasingly attractive biofuel, the need to improve the quality of biogas has come to the fore. Biological upgrading focuses on adding enough hydrogen to an anaerobic biogas reactor to allow methanation of the remaining carbon dioxide by methanogenic Archaea (in situ upgrading). Alternatively, biogas and hydrogen can be mixed in the absence of feedstock, in a reactor operated exclusively to facilitate methanogenesis (ex situ upgrading). This novel technology can encounter inhibition at high loading rates of hydrogen: however, in contrast to anaerobic digestion, the dynamics of this thermophilic functional microbial community are sparsely characterised. High-resolution 16S rDNA community profiles from four anaerobic biogas upgrading reactors were constructed to determine how feedstock, hydrogen, and CO₂ influence biomethanation. Presence/absence of a feedstock led to large differences between in situ and ex situ communities, determining the dominant methanogen genera, and encouraging distinct populations of hydrolysing and fermenting Firmicutes. Although high hydrogen flow rates (∼37 L/day) caused a collapse in methanogenic Methanothermobacter populations in situ, ex situ hydrogen rates greatly exceeded these levels (∼400 L/day) without collapse of Methanobacterium, despite some observed instability and proliferation of likely homoacetogens. Subsequent reduction of hydrogen rates ex situ (259 L/day) appeared to create a niche for hydrogen production, indicated by increased abundance of various syntrophic fermenters known to supply biogenic hydrogen. In either upgrading setup, instability due to increased hydrogen levels manifested as a disruption of fermenting and hydrolysing populations prior to disruption of methanogens.

Recently scientific and industrial interest has grown in relation to antimicrobial surfaces. This interest is mainly due to the persistent and prevalent microbial contamination of industrial and medical surfaces. A critical issue is the dissemination of bacterial colonies across biotic and abiotic surfaces, from surface contact-contact interaction. This ability to colonise materials presents a major problem for cross-contamination and pathogenic bacterial proliferation, resulting in wide-spread distribution and mutation, presenting increased risk to human health. There are two approaches to prevent bacterial spread – disinfection and antimicrobial surfaces. The use of disinfectants presents pollution to the surrounding environment, and the increased development of resistant microbial strains. The beneficial design of antimicrobial polymers enables contact-killing, without the release of biocides into the environment. This project aimed at synthesising sulfur polymeric materials through inverse vulcanisation, with the aim of producing functional antimicrobial materials. As of yet, there is no publication evaluating the antimicrobial effect of diisopropenyl benzene (DIB) and dicyclopentadiene (DCPD) co-polymers as bulk solids, even though elemental sulfur is known to exhibit antimicrobial effects. There is growing demand for materials with antimicrobial capabilities, especially in medical environments, where the epidemiology of hospital acquired infections is of great research interest. The aim of this study was to evaluate the antimicrobial properties of both S-DIB and S. DCPD using Escherichia coli (DSM 1576) and Staphylococcus aureus (DSM 346) against an internationally recognised standard (ISO 22196). To gain further in-depth analysis, confocal microscopy was employed to access the surface impact on bacterial cells.

It is acknowledged that the environment facilitates antibiotic resistance development and spread but not much research has been undertaken to understand the mechanisms involved. This study focuses on antibiotic resistance transfer by enterococci, normal inhabitants of the mammalian gut and important healthcare associated pathogens, under a variety of environmental conditions. These are novel environmental strains, isolated from a farm in Monaghan, Ireland. The primary aim of this project is to quantify factors controlling the horizontal gene transfer (HGT) of antibiotic resistance genes via pheromone-induced conjugation. The main objectives include; identifying enterococcal isolates capable of transferring antibiotic resistance genes under laboratory conditions; using whole genome sequencing (WGS) to characterise genetic events involved in conjugal transfer; developing markers of transfer efficiency; and developing environmentally relevant models to measure enterococcal HGT. The transfer of vancomycin resistance genes from a donor to a recipient cell have been demonstrated and transfer efficiencies range from 1.09×10−1 to 9.74×10−5. This data shows that some donors are better at donating vancomycin resistance genes than other donors and some recipients are better than other recipients at receiving them. Preliminary data also shows the transfer of trimethoprim, tetracycline and erythromycin and we can see that within a localised environment Enterococcus faecalis can spread resistance genes very differently. In conclusion this project hopes to further our knowledge of the mechanism of pheromone-induced conjugation in environmental Enterococci and to show that environmental enterococcal strains can become multi-drug resistant just as easily as clinical isolates.

Antibiotics are extensively fed to beef cattle as they act as rumen modulators, improving animal efficiency and decreasing methane emissions. However, current recommendations by health agencies to limit/ban antibiotic use in animal production call for alternatives such as natural additives. In this study, we aimed to evaluate the efficacy of natural additives in methane mitigation. Bulls (½Angus and ½Nellore) 16±2.2 months old, with average body weight of 385±20.7 kg were fed a basal diet (70 % concentrate, 30 % corn silage) offered ad libitum for 62 days in a feedlot and randomised on five treatments (8 bulls/treatment): control treatment, and addition of 1.5, 3.0, 4.5, or 6.0 g/day/animal of a blend of natural additives containing 37.5 % each of clove essential oil, the commercial blend containing vanillin, eugenol and thymol, 12.5 % and 12.5 % of castor and cashew oils). Methane production from rumen fluid was estimated based on the theoretical fermentation balance for observed molar distribution of VFAs in the rumen. DNA extracted from rumen fluid were sequenced and analysed for methane genes within the MG-RAST database. The natural additives linearly reduced methane production (76 %, P<0.02). Evaluation of Archaeaabundance showed a reduction (79 %, P<0.05) in the major methane producing genera: Halorhabdus, Ferroplasma, Methanoplanus, Picrophilus. A reduction of Fibrobacter and Lactobacillus (71 %), the greatest producers of acetate releasing hydrogen for methane formation was also observed. Our findings suggest that natural additives such as essential oils may be useful in the mitigation of greenhouse gases such as methane in animal production.

Conventional microbial sampling of air in hospitals is usually carried out using settle plates or impaction air samples. This provides little information about intermittent contamination events and is unhelpful for source attribution. Direct continuous bioaerosol sampling is an established technology used to characterise ambient external air. Portable instruments such as the Wideband Integrated Bioaerosol Sensor (WIBS) combine laser particle size and shape detection with signals of biological origin (fluorescence from amino acids and NAD(P)H) characteristic of viable bioaerosols. Monitoring is continuous for weeks at a time and data collected remotely over the internet. We present evidence of the utility of WIBS analysis in characterising air in hospitals in three different environments: operating theatres (plenum ventilated and ultraclean), a respiratory ward, and a specialist cystic fibrosis outpatients. The airborne particle profile was quantitatively and qualitatively different in each environment. Plumes of biologically-relevant airborne particles were detected and source investigation of failing conventional counts in an operating theatre aided by the continuous record. Nebulised drugs contributed a detectable effect on airborne particles which lasted for several hours on the ward despite air changes. A significant effect of plasma air treatment on airborne particles in the ward was detected by WIBS and not conventional cultures. Continuous monitoring may in future allow objective standard setting for airborne particles in different hospital environments and facilitate rapid detection of airborne infection risks.

The Polar Regions have excellent potential for bioprospecting because the microorganisms that live there have adapted to extreme environmental conditions; and these adaptations can be harnessed in a variety of biotechnological applications. In this study, we describe the community structure of a range of different cryospheric habitats to identify environments appropriate for specific bioprospecting activities. Samples were collected from Midtre Lovénbreen glacier and surrounds in Spitzbergen, Svalbard in late June to early July 2017. Many cryospheric environment types were sampled including air, snow, slush, meltwater, cryoconite, proglacial water, soil and seawater. DNA was extracted and prepared for 16S amplicon sequencing. Amplicon sequence variants (ASVs) were assigned using DADA2 and contaminants were removed using the Decontam package in R. We searched the scientific literature for biotechnological applications of abundant community members in each environment. Each environment type displayed a unique community structure, with some physically linked environment types, like glacial snow, slush and meltwater showing continuity through space, and synchronised changes over time. The snow, slush and meltwater habitats were low in biomass and dominated by Gammaproteobacteria, with Cyanobacteria increasing in abundance as melt progressed. Cryoconite was dominated by Cyanobacteria, while sea water was dominated by Bacteriodetes, Gammaproteobacteria and Cyanobacteria. Soil was by far the most biodiverse habitat, with many phyla represented, and a large number of Actinobacteria suitable for antimicrobial discovery. Many of the current EPS, cold-active enzymes, fatty acids, antioxidants and antifreeze proteins are sourced from Proteobacteria and Cyanobacteria, the most abundant phyla in this study.

The conditions of the deep subsurface, combined by perturbations caused by geological disposal of radioactive waste create multiple extreme conditions (limited space availability, high pH, high temperature etc.) for microbial communities. Microbial activity has the potential to cause corrosion of steel and alteration of bentonite clays used in geological disposal facilities. To understand the limits on microbial growth, and the potential for microbial activity to affect the swelling behaviour of the bentonite and metal corrosion, a suite of laboratory experiments is being conducted. In situ repository conditions have been replicated in the MIND (Horizon2020) project. Preliminary results show evidence of corrosion in all experiments, an increase in the basal spacings of smectites in the zone immediately surrounding the steel and inoculated samples had evidence of calcite crystal formation, accompanied by differences in the iron phases. These experiments simulate the in situ conditions well, but the complex nature of this experimental design (high pressure and flow) reduces the practicality of varying the environmental conditions. To complement these investigations, a low-tech solution has been implemented with unpressurised, hydrated bentonite batch experiments. The simpler nature of this set-up allows for investigation of more parameters. Microcosms with artificial groundwater used in the MIND set-up are being compared to the MIND groundwater composition, modelled to represent permafrost conditions. The effect of incubation temperature is also being investigated. Combined, these experiments will help to understand the influence of microbes under the extremes of geological disposal facilities and how their behaviour may change by external parameters.

The 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.

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.

Saltmarshes 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.

The 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.

Cell-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.

Comparisons 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.

One 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.

Antimicrobial 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.

In 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.

The 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.

Myxobacteria 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.

Bacteria 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.

Staphylococcus 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.

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.

Bacteriophage (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.

Probiotics 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.

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.

According 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.

The 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.

Animal 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)

Rhomboid 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.

The 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.

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.

Using 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.

Chronic 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.

Acanthamoeba 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.

The 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.

Salmonellosis 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.

The 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.

Serotype 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.

Escherichia 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.

Staphylococcus 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.

Cystic 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.

Staphylococcus 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.

Dental 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.

The 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.

Agaricus 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.

Vibrio 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.

The 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.

Biofilm 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.

Candida 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.

Stx 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

Numerous 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.

Fermented 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.

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.

Streptomyces 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.

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.