- 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.