bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophotonics: multimodal sensors for bacteria identification and phenotyping
Authors: Thorpe Steve , Krauss Thomas , Thomas Gavin , Johnson Steven
Published: April 2019

A comprehensive understanding of bacteria phenotypes requires tools that are able to characterise structure and function across multiple length scales, from communities and individual cells down to single molecules. Multimodal sensing combines multiple transduction technologies in parallel to probe different properties simultaneously, and thereby increase the range of measurable interactions, the amount of information that can be extracted, and improve detection accuracy. Electrophotonics is a multimodal sensing approach that combines electrochemical and photonic techniques in a single, integrated device that provides enhanced quantitative measurements of chemical reactions. We present a new electrophotonic device based on a Si3N4 guided mode resonant (GMR) structure with an integrated indium tin oxide (ITO) electrode. The GMR structure is sensitive to refractive index changes at the sensor surface, enabling label free, real time detection of biomolecules, microorganisms and imaging of molecular interactions with micrometre-scale resolution to provide spatial information about surface binding interactions. The ITO electrode has been shown to be compatible with voltammetry-based techniques for interrogating redox behaviour along with electrochemical impedance spectroscopy. We demonstrate the wide range of microbiological applications of electrophotonic technology including the characterisation of redox active protein electron transfer and surface adsorption, bacterial adherence and growth on chemically functionalised surfaces, and label free parallel detection of clinically relevant biomarkers. We believe that the multimodal measurements with this novel technology can provide new approaches to investigate and understand microbial biology.
Shining a light on microbial dark matter: a role for the forgotten B vitamins in marine algal communities?
Authors: Absolon Dominic , Smith Alison , Helliwell Katherine
Published: April 2019

The ocean is home to a huge diversity of life that perform a wide range of functions key to earth systems. Marine algae communities contribute a significant proportion of net carbon fixation of the globe as well as forming the basis of the food webs for every trophic level above their own. Despite their importance, many of the taxa that make up these microalgal communities are relatively unknown and understudied, due to their recalcitrance to lab culturing. This concept is known as microbial dark matter and applies in particular to many non-photosynthetic lineages which have been long overlooked by the research community. To investigate whether dependencies of these taxa on certain B vitamins may play a role in this unculturability, data sets produced form the Tara oceans expedition will be analysed. The Stramenopile lineage will be analysed in greater detail for their metabolic potential to synthesise the B vitamins, and/or whether they may dependent on an external source. Both Single-Amplified Genomes (SAG) and metatrascriptomic data sets will be analysed to determine the complement of genes present in different Stramenopile lineages. This will then be correlated with the global distribution data attainable from analysing Tara data sets. By this means, we hope to understand more about the metabolic contribution of this taxonomic group to the community, how this supports the community as a whole and whether dependence on B vitamins is a contributing factor to unculturability.
Investigating the impact of Herpes simplex virus type 1 latency-associated non-coding RNAs on apoptosis in human neuronal cells
Authors: Jacobs Amy , Efstathiou Stacey , Nicoll Michael
Published: April 2019

Herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.
Characterization the role of key DNA sensors in herpes simplex virus replication
Authors: Moshrif Hanan , O’Hare Peter
Published: April 2019

DNA sensors including cGAS, STING and IFI16 are key components of the innate immune response to infection. However, the precise mechanisms of action, in particular the relative importance of direct suppression of replication versus paracrine signalling of an antiviral state to susceptible cells remains unclear. We examined the kinetics of herpes simplex virus infection and spread in a relevant cell type, human keratinocytes, lacking one or other of these DNA sensors using time-lapse microscopy. We also examine transcriptional induction of interferon from the native locus, at single cell and single molecule level using highly sensitive RNA FISH. Our results reveal distinct aspects of the roles of these factors and reveal outcomes not appreciated by other methods. Cells lacking either of these factors showed increased susceptibility to initial infection (prior to any downstream paracrine signalling) but with quite different outcomes. Lack of cGAS resulted in increased cellular migration and cell density at the infection focus. On the other hand, cells lacking STING showed lower cell density and significantly increased cytopathic effect likely curtailing virus yield. Initial results demonstrate that we can analyse interferon transcription at single cell level with exquisite sensitivity down to a few transcripts per cell and reveal profound spatial heterogeneity in responses to induction by PAMP ligands. Altogether, our results reveal new insight into the spatial landscape of the initiation and spread of HSV and key cellular responses which likely integrate pathways including innate immunity, apoptosis and cell migration.
Why does a heterotrophic marine protist produce carotenoids? Genetic approaches to investigate the ecophysiology of the thraustochytrid Aurantiochytrium limacinum
Authors: Rius Mariana , Rest Joshua , Collier Jackie
Published: April 2019

Thraustochytrids are abundant and ubiquitous osmoheterotrophic marine protists (labyrinthulomycetes, stramenopiles) thought to function ecologically as fungus-like decomposers. Some thraustochytrids have the ability to synthesize carotenoids, including carotenes (e.g. beta-carotene) and xanthophylls (e.g. astaxanthin), which is uncommon among heterotrophic eukaryotes. Carotenogenic thraustochytrids appear to have acquired carotenoid biosynthetic enzymes by horizontal gene transfer from bacteria. Heterotrophic production of carotenoids is typically associated with protection against oxidative stress, and in thraustochytrids may be particularly associated with protecting large amounts of essential omega-3 polyunsaturated fatty acids stored in lipid droplets. To gain better understanding of carotenoid function in thraustochytrids, and thus new insight into the ecophysiology of these organisms, we have produced mutants of the thraustochytrid Aurantiochytrium limacinum in which the trifunctional gene Aurli_150841, encoding the first three carotenogenesis-specific reactions (phytoene synthase, phytoene desaturase, lycopene cyclase), has been interrupted by double homologous recombination with a construct containing a zeocin resistance (BleoR, shble) expression cassette. As predicted, the Aurli_150841 knockout mutants lack the carotenoid pigmentation found in the wild-type. Complementation with the wild-type Aurli_150841 to confirm that this phenotype is due to the knockout is in progress. Differences between the wild-type and Aurli_150841 knockout mutants in features such as growth rate and biomass yield, lipid content, survival in stationary phase and response to oxidative stress are being evaluated under growth conditions that induce different amounts of carotenoid accumulation in the wild-type.
Uncovering the molecular basis of viable but non culturable (VBNC) cells
Authors: Wagley Sariqa , Titball Richard , Butler Clive
Published: April 2019

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.
Developing genetic manipulation platforms for Naegleria gruberi
Authors: Kazana Eleanna , von der Haar Tobias , Tsaousis Anastasios D.
Published: April 2019

Naegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.
Clinical management of low-level hepatitis B surface antigen in haemodialysis patients with a recent history of HBV vaccination – the results of a UK-wide survey
Authors: Goddard Megan , Timms Judith
Published: April 2019

Identifying transient low-level hepatitis B (HBV) surface antigen (sAg) post-vaccination is a well-established phenomenon in haemodialysis patients. These results create a clinical conundrum; should patients be deemed potentially infectious, requiring further testing and heightened infection control precautions, or should negligible risk be assumed? National guidance covering this scenario is lacking; therefore we conducted a survey of the clinical practice of UK virology departments. Thirty-six laboratories were contacted and 17 responses received (47 %). Eleven responders had observed sAg positivity post-vaccination, for a maximum duration of 3 weeks. Clinical management was highly variable. Only 3 hospitals had specific written policies, with others following an internal consensus or a standard protocol for sAg positive patients without recent vaccine. Further testing consisted of HBV serology (3/11), HBV PCR (1/11) or PCR and serology (7/11). Three departments were highly risk averse, recommending universal isolation with dedicated dialysis machines. Two departments did not advise additional precautions and 5 based decisions on risk factors e.g. foreign travel. Heightened precautions were stopped on the basis of negative serology (1/11), PCR (5/11) or serology and PCR (2/11). To summarise, vaccine-derived sAg positivity is commonly encountered on haemodialysis units. Recommendations on the interval between vaccination and testing would alleviate the issue, with this study suggesting a minimum of 3 weeks. Infection control precautions varied considerably. In most cases, patients are risk assessed for isolation as a minimum; however this may depend on local expertise and the availability of isolation facilities. It is clear that national guidance would standardise patient care.
Genome dynamics of Streptomyces clavuligerus
Authors: Algora Lis , Herron Paul
Published: April 2019

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.
Functional and structural studies of Chikungunya Virus nsP1 Protein
Authors: Loveday Kate , Levitt Emily , Nguyen Trang , Edwards Thomas , Tuplin Andrew
Published: April 2019

Chikungunya virus (CHIKV) is a single-stranded, positive-sense RNA virus of the Alphavirus genus. CHIKV is an arbovirus, whose spread is mediated by Aedes species mosquitos and is associated with debilitating joint pain and febrile symptoms in infected humans. A lack of vaccine or specific antivirals, combined with increasing global spread, has facilitated the re-emergence of CHIKV in recent years. Our research focuses on the CHIKV encoded non-structural protein-1 (nsP1), which has methyltransferase activity and is essential for virus genome replication. Through a combination of structural, biochemical and reverse genetic approaches, we aim to investigate the relationship between the molecular structure and both canonical and non-canonical functions of nsP1, at different stages of CHIKV replication. We demonstrated that substitution of an in-frame methionine (M24), towards the N-terminal of nsP1, severely inhibits CHIKV replication in a host cell-dependent manner. Specifically, we demonstrated that an M24>A substitution had no significant effect on sub-genomic replicon replication but blocks production of infectious CHIKV virions – suggesting a role in later stages of virus replication, such as packaging or egress, rather than genome replication or translation. Utilising such a reverse genetic approach, analysis of a panel of M24 substitutions has improved our understanding on non-canonical yet essential functions of nsP1 during CHIKV replication. In order to further elucidate both the structural and biochemical basis for the observed mutant phenotypes, we developed a system for bacterial expression and purification of recombinant CHIKV nsP1 and have established crystallography trails, prior to analysis of the molecular structure by X-ray crystallography.
Investigation into the physicochemical interactions of silica nanoparticles and EPS biomolecules within the biofilm matrix of Pseudomonas spp.
Authors: Hiebner Dishon , Barros Caio , Devlin Henry , Fulaz Stephanie , Quinn Laura , Vitale Stefania , Casey Eoin
Published: April 2019

Difficulties in the removal of bacterial biofilms in the industrial and biomedical sectors have driven the development of new technologies. Although numerous studies have highlighted the use of nanoparticles (NPs) as antibiofilm agents, the fundamental physicochemical interactions between NPs and the biofilm matrix is still poorly understood 1. The development of ‘smart nanoparticles’ for biofilm removal requires an in-depth understanding of the complex interactions between NPs and biomolecules within the extracellular polymeric substances (EPS) of the biofilm matrix. These interactions are highly dependent on the physical and chemical properties of the NPs 2. In order to identify and characterize the specificity of binding and the direct interaction between silica NPs (SiNPs) and EPS matrix components of Pseudomonas spp. biofilms, a range of experiments were carried out. Biofilms were exposed to SiNPs of different sizes, charges and surface functionalization while biomolecules such as proteins, polysaccharides, and eDNA were fluorescently labelled and their distribution, relative abundance and their colocalization with SiNPs within the biofilm was quantitatively assessed using CLSM microscopy. Changes to the SiNPs size and surface-chemistry dramatically affected their interactions with biomolecules in the biofilm matrix. This includes the increased affinity (or interaction) of SiNPs to preferentially bind to proteins and beta-linked polysaccharides and also lead to changes in the degree to which aggregation of SiNPs occurs within and on the surface of the biofilm.

1. Ikuma K et al. (2015). Front. Microbiol. 6, 591

2. Bewersdorff, et al. (2017). Int. J. Nanomed. 12, 2001–2019
Multi-scale variability analysis of Arctic soil microbial communities
Authors: Malard Lucie , Pearce David
Published: April 2019

Understanding distribution patterns at various spatial scales is a central issue in microbial ecology. Beyond the lone identification of biogeographical patterns, understanding the environmental drivers behind community diversity and structure is key. While many studies identify pH as a major parameter structuring microbial communities at large spatial scales, many other variables impact distribution patterns on smaller scales. Here, we investigated the biogeographical patterns of Arctic soil microbial communities from 1 m to 500 m, within Adventdalen, Svalbard, using 16S sequencing, gravimetric measurements and X-ray fluorescence. Multivariate analyses identified key environmental variables shaping microbial communities and revealed the importance of soil moisture, organic carbon and elements such as aluminium, calcium and potassium in structuring distribution patterns. The indicator species analyses identified key associations between environmental variables and OTUs. Using geostatistical kriging, we mapped the biodiversity and distribution of key OTUs across the landscape. Overall, our results highlight the spatial heterogeneity in Arctic soils and identifies the sampling scale needed to characterize microbial communities within an area of interest with seemingly homogeneous landscape.
Genomic mining of thermophilic actinobacteria from compost for novel antimicrobials
Authors: Halket Gillian , Munro Carol , Herron Paul
Published: April 2019

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.
Proteomic analysis reveals vector-virus interactions between Zika virus and Aedes aegypti mosquito cells
Authors: Gestuveo Rommel , Varjak Margus , Kohl Alain
Published: April 2019

Zika virus (ZIKV) is an arbovirus (family Flaviviridae) mainly transmitted by Aedes mosquitoes causing febrile illness and Zika congenital syndrome in infants if mothers were infected during pregnancy. ZIKV manipulates its host’s cellular machinery in order to facilitate infection and evade antiviral responses. The identification of host and vector proteins involved in these processes may lead to novel antiviral strategies. In this study, Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C) or anchored capsid (AC) proteins were developed to investigate virus-vector protein interactions. To identify interaction partners, immunoprecipitation (IP) of V5-tagged C or AC was performed and subjected to proteomic analyses using nLC-MS/MS under label-free quantification conditions. A total of 148 and 53 mosquito protein interactors unique to C and AC were identified, respectively. Protein network and gene ontology analyses showed biological processes possibly important for ZIKV infection. To investigate further the role of these proteins during infection, 25 were chosen for dsRNA-based knockdown screen and infection with reporter virus (ZIKV-Nluc) in AF5 cells. Significant reduction in reporter virus signal was observed during knockdown of 6 interactors suggesting a pro-viral role for these proteins during infection. This was corroborated by conducting the same knockdown experiments but infecting with a clinical isolate of ZIKV (PE243), which showed reduced virus RNA levels and titre. Interestingly, three of the six proteins are part of the ubiquitin-proteasome pathway (UPP). Currently, functional experiments are underway to investigate the role of UPP during ZIKV infection.
D-serine: trick or treat?
Authors: Turner Natasha
Published: April 2019

D-serine is an amino acid that has become a focus in recent years due to its unique role in many biological processes. It is a host metabolite in humans with diverse roles in neurotransmission and signalling. Previous work in our group showed that D-serine can play a critical role in controlling expression of pathogenic virulence factors in bacteria, specifically Escherichia coli, as well as impacting microbial community composition through niche specificity. In enterohaemorrhagic E. coli (EHEC), the presence of D-serine results in down regulation of the type 3 secretion system (T3SS), resulting in inability to colonise. In contrast, uropathogenic E. coli (UPEC) can catabolise this metabolite and colonise the bladder where D-serine is present in high concentrations, leading to a urinary tract infections (UTI). Hence, in different pathotypes, D-serine can act as a positive (treat) or negative (trick) environmental stimulus. UPEC and neonatal meningitis E. coli (NMEC) strains often carry the DsdXCA operon which allows for the metabolism of D-serine. This locus is responsible for the detoxification of D-serine, allowing for tolerance of D-serine as a carbon source. My work has focused on understanding the role of D-serine as a signal for gene expression in UPEC and NMEC through the action of the regulator, DsdC. Using global approaches I have characterised the binding sites of DsdC across the chromosome revealing new insights into how this protein contributes to UPEC and NMEC pathogenesis. The work is important as it helps us understand how specific pathogens sense their environment and cause disease.
Assessment of novel disinfection technologies, and bacterial contamination in the healthcare setting
Authors: Murray Jason , Ternan Nigel , Gill Chris , McMullan Geoff , Farren David , Scott Michael
Published: April 2019

Worldwide, hundreds of thousands of healthcare acquired infections (HAIs) are reported each year. Contamination of hospitals is a source of, and allows dissemination of HAIs. In healthcare settings one of the major vectors of contamination is healthcare workers’ uniforms. As surfaces become contaminated, bacteria can then be contacted by patients or staff who may indirectly spread bacteria to patients. Direct and indirect spread of bacteria could result in infection of patients and increased infection rates. Further consequences include increased levels of antibiotic use and costs. A pilot study was conducted at Antrim Area Hospital, Northern Health and Social Care Trust. 100 pre-shift and 100 post-shift healthcare workers’ uniforms were assessed for Staphylococcus aureus and Enterococcus spp. isolates. We found increased levels of antibiotic resistant S. aureus and Enterococcus spp. contamination on post-shift uniforms compared to zero to minimal contamination of pre-shift uniforms. A biobank of isolates was subsequently characterised for antibiotic sensitivity using European Union Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines – 51 % of S. aureus isolates were classed multi-drug resistant. Genomic diversity was assessed using Random Amplification of Polymorphic DNA (RAPD) – high levels of similarity was found amongst isolates. As one means of reducing uniform bioburden, we conducted analysis of a novel surface-active organisilane disinfectant named Goldshield (GS). GS was marketed as a long lasting antimicrobial prevent (re)contamination. GS technology displayed bactericidal, sporicidal and anti-biofilm properties in laboratory testing providing rationale for an intervention where GS could be incorporated into hospital laundry and assessed for potential use in infection control.
Dissemination of a phage-encoded virulence factor in a pandemic S. Typhimurium
Authors: Tassinari Eleonora , Bawn Matt , Duffy Geraldine , Kingsley Robert A.
Published: April 2019

Salmonella Typhimurium is the second most common cause of foodborne salmonellosis. Phage-mediated horizontal gene transfer contributes to the virulence of S. Typhimurium. An example of a phage-encoded virulence gene is sopE, a T3SS effector, found rarely in Typhimurium and associated with epidemics. The current pandemic and multi-drug resistant monophasic variant of S. Typhimurium (S. 4,[5],12:i:-) acquired sopE in multiple events following the lysogeny of a previously undescribed bacteriophage, mTmV. The current study aimed to further investigate the association of the virulence gene with S. 4,[5],12:i:- and assess its epidemiological impact. To this end, a large collection of clinical S. Typhimurium isolates from the UK have been analysed for the sopE gene and mTmV presence using a phylogenomic approach. While a large proportion of S. 4,[5],12:i:- (41 %) carried the sopE gene, few isolates outside the epidemic clade harboured it. Notably, the mTmV bacteriophage was identified only in S. 4,[5],12:i:-, although laboratory experiments demonstrated that the phage host range is not restricted to it. Nonetheless, we identified the phage in other S. enterica serovars circulating in the same ecological niche of S. 4,[5],12:i:-. In addition, a genomic characterisation of mTmV was performed revealing an unexpected level of phage variation. Finally, we identified a novel phage-like element harbouring the gene. The study revealed the large dissemination and selection of the virulence gene in the current epidemic, which is mobilised by multiple and distinct mobile genetic elements.
Pneumococcal invasive disease preceded by intracellular replication within splenic macrophages
Authors: Carreno David , Wanford Joseph , Jasiunaite Zydrune , Hames Ryan , Restrepo Marcos I. , Orihuela Carlos J. , Andrew Peter W. , Moxon E. Richard , Oggioni Marco R.
Published: April 2019

During bacteremic pneumonia, the prevailing dogma is that bacteria seed from the lungs into the blood. Recently, we have shown that experimental murine sepsis is preceded by intracellular replication within splenic macrophages (Ercoli Nat Microbiol 2018), which shed into the bloodstream initiating invasive disease. Here we aimed to investigate a role for the spleen in the pathogenesis of bacteraemia following pneumonia. We analysed by confocal microscopy the fate of pneumococci during ex vivo human spleen perfusions (REC reference: 18/EM/0057), in spleens during pneumonia in non-human primates (Reyes PLOS one 2016) and mice. During ex vivohumanspleenperfusion, clusters of pneumococci were observed within macrophages and the size of bacterial clusters increased over time. To associate these infectious foci to invasive pneumococcal disease during pneumonia, we analysed spleens in a baboon pneumonia model, and detected pneumococcal clusters in splenic macrophages. To test the functional relevance of these data, we treated intranasally-challenged mice with a single, non-therapeutic sub-MIC dose of azithromycin, known to concentrate inside macrophages. Data showed that bacterial lung-counts were identical in treated and untreated mice. Untreated mice showed signs of disease, had high blood and spleen-counts, whereas mice treated with the non-therapeutic dose showed no signs of disease, had low spleen-counts and no bacteraemia. Thus, the number of pneumococci in the spleen, not the lung, correlates to blood-counts during bacterial pneumonia. We hypothesise that after initial control of invasive infection by the spleen, bacteraemia associated with pneumonia arises from a sub-set of splenic macrophages that are permissive for bacterial replication.
Comparison of temperate bacteriophages of Pseudomonas aeruginosa from the lungs of chronically infected non-cystic fibrosis bronchiectasis patients over a period 10 years
Authors: Duignan Libby , Tairq Adnan , Hamel Jeremie , Rheault Jean-Guillaume Emond , de Soyza Anthony , Perry Audrey , Levesque Roger , Smith Darren
Published: April 2019

The accessory genome of Pseudomonas aeruginosa (PA), is frequently perceived insignificant compared to the core genome, however typically contains temperate bacteriophage (phage) genomes that effect the bacterial host. PA presence in chronically infected lungs correlates with loss of lung function particularly in cystic fibrosis (CF) and non-CF bronchiectasis (nCFBR). This study focuses on isolates from chronically infected nCFBR patients isolated over a decade, shedding light on how temperate phages change over the course of a chronic infection with antibiotic treatment. As temperate phages insert themselves into the bacterial genome they also have the ability to cause genetic diversity to their host’s genome, which drives evolution at an increased rate. By analysing the PA genomes isolated from the chronically infected lungs of patients over a period of 10 years. It was possible to predict the temperate phages in the genomes as well as induce the phages from the bacteria and sequence them. This then identifies which phages are rooted within the genome and which are inducible and therefore may transfer, granting horizontal gene transfer between strains in the lungs. The aim of this study is to ascertain whether by comparing the phages longitudinally and horizontally (when multiple strains were seen within a sample) it is possible to determine if these phage have a role in the PA infection within the lungs becoming chronic. This may also give an idea to why these PA infections are chronic and are so hard to clear from the lung, which is yet unknown.
Elucidating conserved interactions between viral glycoproteins and cellular factors
Authors: Rix Thomas , Kelly James , Bailey Dalan
Published: April 2019

Viral glycoproteins are found on the surface of all enveloped viruses, mediating binding to host receptors and the initiation of entry events. In addition, numerous vaccines employ the same viral glycoproteins as immunogens, either vectored or recombinant in nature. During infection viral glycoproteins are thought to interact with various host-factors, facilitating their trafficking to the cell surface. However, these interactions are not currently well understood or characterised. Using a human gene expression microarray, the cellular response to expression of various viral glycoproteins (Ebola, Nipah, VSV and Measles) was assessed in vitro. Specifically, we found a number of genes with a fold change greater than 2 displaying significantly altered expression across all four glycoprotein transfections. A subset of these genes were selected for validation by qPCR and extended to RSV (respiratory syncytial virus) fusion protein (F) transfection. The expression of these genes was then further investigated in Measles and RSV infected cells. Our data has identified host genes with altered expression in response to a diverse panel of viral glycoproteins, potentially elucidating a conserved set of pathways important for viral glycoprotein activity. Greater understanding of the proteins and pathways involved in glycoprotein expression has the potential to identify mechanisms underpinning host susceptibility to disease as well as improving the yield of vaccine producing cells.
From Antarctic DNA to stress tolerant crop plants – exploiting the why protein domain
Authors: Mertens Jasmin , Anderson Dominique , Ferreras Eloy , Cowan Don
Published: April 2019

Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress conditions [1]. A phylogenetic analysis of multiple WHy homologues from different species suggested that the ancestral origin of this protein gene lies within the ancient archaea [1]. Our previous studies have shown that this bacterial protein elicits significant protection against freeze and cold stress in recombinant E. coli [2]. Expression of the WHy gene in Arabidopsis resulted in a wide range of statistically significant stress-tolerant phenotypic properties. These included an increase of up to 6-fold higher germination efficiency of transgenic recombinant seeds compared to the WT, and a 100 % survival rate of WHy gene-expressing plants compared to 0 % survival of adult WT plants after freeze shock. Similar improvements in survival rates were observed for recombinant plants in drought stress experiments.

References

Mertens J, Aliyu H, Cowan DA (2018). Applied and environmental microbiology, AEM-00539.

Anderson D, Ferreras E, Trindade M, Cowan D (2015). FEMS Microbiology Letters, 362(15):fnv110.
Clostridium difficile: cell surface biogenesis
Authors: O’Beirne Shauna , Kirk Joseph A. , Fagan Robert P.
Published: April 2019

On the C. difficile cell surface is a proteinaceous paracrystalline array, known as the S-layer. The S-layer of C. difficile is composed of two proteins: the high molecular weight (HMW) and the low molecular weight (LMW) S-layer proteins, derived from the pre-protein SlpA. PS-II, an anionic polymer found in all C. difficile strains examined to date, has been identified as the ligand responsible for the attachment of S-layer and associated cell wall proteins. Early efforts to knock out slpA proved unsuccessful and the genes thought to encode the PS-II synthesis pathway were also thought to be essential. However, by using bacteriocins that specifically target the S-layer, we recently isolated a mutant which had no evident S-layer due to a mutation in the slpA gene. As the S-layer was previously thought to be essential, it now brings into question whether PS-II is also essential. In the strain lacking an S-layer, we have now created a deletion mutant in the putative PS-II polymerase and we are attempting to generate additional mutations in the polysaccharide synthesis pathway. Analysis of these mutants will provide insights into the mechanism of PS-II synthesis and shed light on its function in cell morphogenesis.
Characterisation of bovine dendritic cells following FMDV infection
Authors: Yasmin Amina , Moffat Katy , Reid Elizabeth , Charleston Bryan , Milling Simon , Seago Julian
Published: April 2019

Dendritic cells (DCs) are the sentinels of the immune system, responsible for recognising invading pathogens and priming the adaptive immune system for appropriate responses. Hence they are considered potential targets for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Little is known of the events of FMDV replication in bovine moDCs. Present work therefore sought to characterize FMDV and its immune complex (IC) replication in bovine moDCs in vitro. A chimeric heparin sulphate FMDV (O1M) was used in this study. Immuno-fluorescence microscopy (IFM) and quantitative RT-PCR was used to analyse viral replication at 0–6, 8, 16 and 24 hpi. Plaque assays were used to investigate the yields of live virus produced in moDCs at 0, 4, 8 and 24 hpi. FMDV and IC FMDV could infect moDC. In moDC infected with FMDV alone, or with immune-complexed (IC) FMDV, replication was observed by IFM between 2–4 and 1–16 hpi. In contrast, for both FMDV and FMDV IC infections RT-PCR analyses showed viral replication peaked at 4 hpi and then decreased between 8 to 24 hpi. Plaque assays using supernatants of the infected moDC showed no evidence of an increase in viral titre at 24 hpi. The detection of viral nsp (3AB and derivatives) suggests replication of FMDV persists for longer in moDCs when entry is mediated by IC. However, the lack of increase in virus yield suggests replication is abortive. One possible explanation for this difference could be that bovine moDCs are able to recognise non-immune complexed FMDV more rapidly.
Assessing metabolite biogeography of Micrococcus spp. and Pseudonocardia spp. isolated from marine environments
Authors: Parra Jonathan , Duncan Katherine R.
Published: April 2019

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

References

1. Lomolino, M. V., Riddle, B. R. and Whittaker, R. J. Biogeography. (Sinauer, 2016).

2. Morlon, H. et al. The biogeography of putative microbial antibiotic production. PLoS One 10, 1–15 (2015).

3. Krause, S. et al. Trait-based approaches for understanding microbial biodiversity and ecosystem functioning. Front Microbiol 5, 1–10 (2014).

4. Fenical, W. and Jensen, P. R. Developing a new resource for drug discovery: Marine actinomycete bacteria. Nat Chem Biol 2, 666–673 (2006).

5. Hug, J. et al. Concepts and Methods to Access Novel Antibiotics from Actinomycetes. Antibiotics 7, 44 (2018).
The environmental microbiome toolkit for urban designers
Authors: Watkins Harry , Robinson Jake
Published: April 2019

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.
Characterisation of new environmental bacteriophages targeting the Escherichia coli LamB outer membrane porin
Authors: Zeng Ziyue
Published: April 2019

Bacteriophages are viral parasites of bacteria. A successful infection starts with the adsorption of the bacteriophage to a specific receptor on the host cell surface. Most bacteriophages are thought to have a narrow host range but this can be extended in certain cases. One strategy forextending host range is to first express a known functional bacteriophage receptor protein in bacteria previously non-susceptible to the bacteriophage, thereby enabling adsorption and potential infection by viruses that target the specific receptor. To investigate the feasibility of this approach, a plasmid (pMUT13) encoding the Escherichia coli LamB porin, the receptor for bacteriophage Lambda, was transferred into three different enterobacterial genera, namely Citrobacter, Yersinia, and Serratia. Over 100 environmental bacteriophages were isolated that infected these pMUT13-containing strains, and some bacteriophages were shown to infect their respective hosts in a LamB-dependent way. The host ranges of the environmental bacteriophages were cross-tested across the heterologous genera and surface adsorption kinetics investigated. Unlike bacteriophage Lambda, which is a member of the Siphoviridae, these newly-isolated LamB-dependent bacteriophages were more commonly members of the Myoviridae, based on transmission electron microscopy and whole genome sequences. Furthermore, an interesting selection of evolved bacteriophage mutants with broader host range were isolated, and the key mutations involved in their evolution to adapt to new hosts were investigated by genome analysis.
Selenium and tellurium oxyanion reduction by yeasts
Authors: Codognotto Lara , Gadd Geoffrey Michael
Published: April 2019

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.