bluuebunny/crossref_metadata_2025 huggingface dataset:
An exploration of antigen expression of hepatitis C entry receptors on equine cells in relation to equine hepacivirus A
Authors: Sohrmann Mascha , Daly Janet M. , Scott Julia , Tarr Alexander W. , Kydd Julia H.
Published: April 2019

Equine hepacivirus A (EqHV) belongs to the family Flaviviridae and has been identified as the hepacivirus phylogenetically most closely related to Hepatitis C virus (HCV). Like HCV, EqHV is a hepatotropic virus that has been reported in over fourteen countries from six continents. It has a high prevalence in some populations, in particular the Thoroughbred breed. However, much is still unknown about EqHV, such as its pathogenesis and mode of transmission. The main four receptors used by HCV for viral entry to human hepatocytes are Cluster of Differentiation-81 (CD-81), occludin (OCLN), claudin-1 (CLDN-1) and Scavenger Receptor Class B Member 1 (SCAR-B1). This study investigated the distribution and expression of these entry receptors on equine cells by flow cytometry, immunocytochemistry and immunohistochemistry. Using a human liver cell line (Huh-7) as a positive control, antibodies against HCV receptors appeared to cross-react with antigens on equine cells. The proportion of fetal horse kidney cells that expressed CD-81, OCLN, and CLDN-1 was 37.2 %, 16.0 %, and 7.0 %, respectively, whereas the equine dermal cells (E.Derms) expressed CD-81 (96.0 %), OCLN (1.2 %) and CLDN-1 (1.7 %). CD-81, OCLN and CLDN-1 were also expressed on E.Derms and Huh7 cells, as detected by immunocytochemistry and on equine liver cells and the allantochorionic region of Thoroughbred placenta by immunohistochemistry. These findings form the basis for further comparative investigation into the entry receptors used by EqHV to infect equine and human cells. Such information may inform future studies on EqHV pathogenesis and mode(s) of transmission.
A synthetic biology approach for African horse sickness vaccine platforms
Authors: Stewart Meredith , Lopez Alejandro , Herder Vanessa , Ortego Javier , Palmerini Massimo
Published: April 2019

African horse sickness virus (AHSV) is the causative agent of African horse sickness (AHS), a highly fatal disease of equids. Currently, live attenuated vaccines are used to control AHS especially in South Africa, but they are in general subject to restrictions. We took advantage of previously published AHSV structural and antigenic data to simplify AHSV vaccine development using an AHSV-4 reverse genetics approach. We systematically substituted the tip and central domains of AHSV-4 outer capsid protein VP2 with the corresponding region from other serotypes to generate a chimeric protein. AHSV S2 chimeric and mono-reassortant viruses for all 9 serotypes were recovered by reverse genetic using AHSV-4 as backbone and used to immunise guinea pigs. Presence of neutralising antibody titres were determined using a fluorescence-based neutralisation assay. The exchange of the tip and central domain of VP2 switched the serotype specificity of the rescued chimeric viruses, however, sera from AHSV-4VP2DTip only neutralised the homologous AHSV-4 reference virus. Mono-reassortants, but not recombinant viruses containing chimeric VP2, induced antibodies with low levels of cross-neutralisation between phylogenetically related serotypes. Interestingly, most of the sera raised were able to neutralise AHSV-4, indicating the presence of other neutralising epitopes within the virus. These results raise the possibly of generating a single virus that affords protection against multiple serotypes. Our research highlights the ability to manipulate the AHSV genome to rapidly generate ‘synthetic’ viruses using a single platform approach for vaccine development.
The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence
Authors: Jin Yi , Bakshi Siddharth , Caporale Marco , Taggart Aislynn , Stewart Meredith , Palmarini Massimo
Published: April 2019

African horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (a, b and g). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVDNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVDNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVDNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVDNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.
Human papillomavirus E6 regulates the trafficking of gap junction protein Cx43
Authors: Dong Li , Johnstone Scott , Graham Sheila
Published: April 2019

High-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.
Modification of the ADP-ribose-1"-monophosphatase domain in recombinant infectious bronchitis virus affects viral replication in vitro and attenuates the virus in vivo
Authors: Dowgier Giulia , Keep Sarah , Britton Paul , Bickerton Erica
Published: April 2019

The gammacoronavirus infectious bronchitis virus (IBV) is responsible for an acute respiratory disease in domestic fowl, which has high economic impact and welfare implications in the poultry industry. The IBV non-structural protein, nsp3, is a multifunctional protein containing several putative domains, including an ADP-ribose-1’-monophosphatase (ADRP) domain conserved among coronaviruses. Inactivation of the ADRP domain in alpha- and betacoronaviruses is associated with reduced pathogenicity in vivo and altered interferon response and cytokine profiles in the host, without affecting viral replication in vitro. Therefore, recombinant viruses lacking ADRP functions have been proposed as ideal candidates for live attenuated vaccines. A recombinant IBV (rIBV) was generated in the backbone of the pathogenic M41-K strain containing a mutation in the ADRP domain catalytic core, known to abolish ADRP function in other coronaviruses. The ADRP-defective rIBV was characterised in vitro and in vivo; conversely to previously described ADRP-defective coronaviruses, in vitro analysis showed a reduction of viral replication, and the rIBV displayed a distinctive plaque phenotype. No reversion of the mutation occurred after serial passages of the virus in primary avian cell culture, nor in ex vivotracheal organ cultures which wereutilisedas a surrogate for in vivo stability testing. Pathogenicity experiments conducted in vivo resulted in a reduction in clinical signs in comparison to M41-K-infected birds, and tracheal ciliary activity, a marker for pathogenicity, was comparable to mock infected birds. These data support the role of ADRP as a pathogenic determinant and demonstrate the potential of ADRP-defective rIBV as a promising candidate vaccine.
Marine Streptomyces spp. isolates with synthetic polyesters-degrading activity
Authors: Leao de Almeida Eduardo , Felipe Carrillo Rincón Andrés , Nevalainen Seija Elizabeth , Jackson Stephen , O’Leary Niall , Dobson Alan
Published: April 2019

The rapid expansion of global plastic production in the last number of decades (>355 million tonnes in 2017), coupled with poor waste management, has resulted in an estimated 5–12 million metric tonnes of plastic waste entering our oceans. Packaging applications account for much of the current waste production, and commonly include polyethylene- and polyethylene terephthalate (PET)-based materials that are resistant to natural degradation processes, particularly in marine environments. In response to this global marine pollution issue and the continuing demand for effective treatment of such plastics in terrestrial environments (e.g. 27.1 million tons of annual, post-consumer plastic waste in Europe alone), researchers have focused on addressing the biodegradation of recalcitrant plastic waste such as PET. To this end, we screened 20 Streptomyces spp. strains isolated from marine sponges for polycaprolactone (PCL)-degrading activity, which is considered a model substrate for PET. Although the Streptomyces genus is commonly explored for natural products discovery, little is still known about its potential for polymer degradation. Genomic analysis of two of the Streptomyces isolates which screened positive for PCL-degrading activity were found to have PET-hydrolase gene homologs that shared 41 % identity to the well-characterised PETase from Ideonella sakaiensis 201 F6. One of these genes was subsequently heterologous expressed in E. coli in order to further characterise the enzymatic activity and other biochemical properties of the enzyme.
SNP based transmission study of badgers infected with Mycobacterium bovis in the edge risk area of England
Authors: O’Cathail Colman , Barron Elsa Sandoval , Swift Ben , Bennett Malcolm , Emes Richard
Published: April 2019

Mycobacterium bovisis the causative agent of bovine tuberculosis (bTB), one of the most costly and persistent agricultural infectious diseases still widespread in England today. However, not all parts of England are created equally with respect to the incidence of bTB. In 2012 the Department for the Environment, Food and Rural Affairs (DEFRA) divided the country into three risk areas; the high, low and edge risk areas. The perpetuation of the bTB epidemic is often blamed on the known wildlife host of M. bovis, the European badger (Meles meles). Despite this, no large scale studies examining both the prevalence and transmission patterns of M. bovis in this species has been undertaken. Here we describe the results of a major survey of 600 road-kill badgers from 6 counties within the edge risk area of England. Mycobacterium like colonies were isolated from over 80 carcasses of which, 65 were confirmed to be tuberculosis complex positive by PCR. These isolates were spoligotyped, VNTR typed and then subsequently whole-genome sequenced. We describe a SNP based transmission analysis of the sequenced isolates that provides a higher degree of resolution between the badgers compared to the described molecular typing methods.
Of microscopes and microbes; novel applications of optical microscopy to microbiology
Authors: Rooney Liam , Hoskisson Paul , McConnell Gail
Published: April 2019

The development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume >100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.
Discovery of novel highly divergent RNA viruses in European rodents and rabbits
Authors: Tsoleridis Theocharis , Chappell Joseph , Onianwa Okechukwu , Monchatre-Leroy Elodie , Umhang Gérald , Shi Mang , Tarlinton Rachael , McClure Patrick , Holmes Edward , Ball Jonathan
Published: April 2019

Although RNA viruses are likely to exist in every species of cellular life, our knowledge of their biodiversity and evolution has been focused on those that can cause disease in humans and in economically important animals and plants. Recently published studies, however, have shown that every healthy organism can carry viruses. In this study, we present the unbiased discovery of highly divergent RNA viruses in European rodents and rabbits using Next Generation Sequencing (NGS). Tissue samples from different organs were collected from UK rodents and French rabbits and were initially screened for viruses with PCR. Following up on preliminary data, the positive samples were sent for NGS to acquire full genomes and perform unbiased virus discovery. Our findings encompass a number of novel viruses including astrovirus, rotavirus A, hantavirus, picornavirus, coronavirus, paramyxovirus etc. Among the novel viruses, the picornavirus interestingly showed 50 % and 34 % similarity to the closest relative for the non-structural and the structural protein respectively at amino acid level, suggesting a potentially novel genus within the family. This study shows the presence of highly divergent RNA viruses in European rodents and rabbits enriching thus our current knowledge of their origin and evolution. Finally, this study shows that these animal species can be the reservoirs of RNA viruses notorious for cross-species transmission, increasing the risk of a spill-over to humans or livestock.
Understanding the pathogenic process of uropathogenic Escherichia coli ST127 using proteomics on uroepithelial co-culture samples
Authors: Saeedi Nizar , Sharma Vikram , Jackson Simon , Upton Mathew
Published: April 2019

Background

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). Strains of sequence type (ST) 127 exhibit the highest virulence potential of most UPEC strains, but little is known about pathogenicity during infection. We sought to investigate this using a quantitative proteomics approach.

Material/Methods

Three strains of UPEC ST127 (EC18, EC41 and SA189), in addition to non-pathogenic strain E. coli K12, were analysed in co-culture with the uroepithelial cell-line HT1197 for 5 h. We analyzed the bacterial and uroepithelial proteome along with the secreted proteins in the medium (secretome). The digested proteins and peptides from all fractions were separated on a Dionex Ultimate 3000 RSLC nano flow system and analyzed in an Orbitrap Velos Pro FTMS. Data were processed using Persues software.

Results

Label free quantitative proteomics revealed different proteomic profiles of the co-cultured strains. Gene Ontology enrichmentanalysis showed upregulation in the pentose phosphate pathway and glycolysis/glycogenesis in EC18 (an O-antigen deficient mutant). These two pathways could be important routes of carbon flux through the central metabolic pathways during growth in urine. Co-culture of SA189 with HT1197 cells leads to apparent cytotoxic effects in HT1197 cells not seen with other UPEC strains. Analysis of the SA189 secretome revealed highly abundant bacterial proteins, some of which (e.g. aromatic-amino-acid aminotransferase) were uniquely found during co-culture conditions.

Conclusion

Proteomics is crucial towards increasing our understanding of the pathogenic potential of UPEC ST127 strains and may facilitate identification of novel diagnostic or therapeutic targets to reduce UTI.
Identification and characterisation of the key virulence determinants of asymptomatic pathogenic Escherichia coli
Authors: Hallam Jennifer
Published: April 2019

Diarrhoeal disease is a major public health concern, with over 500 000 childhood deaths recorded every year, (www.who.int). The leading cause of infantile diarrhoea is pathogenic Escherichia coli (EPEC), characterised by watery diarrhoea which varies in levels of severity. In recent studies a growing incidence of asymptomatic EPEC carriage has been recognised in Asia and the Middle East, suggesting the possibility that communities may act as a reservoir for this microorganism, (Kader, 2009; Alikhani, Mirsalehian and Aslani, 2018). Using a combination of molecular biology and bioinformatic analysis, we aim to characterise asymptomatic strains from these regions, determine their key virulence factors and find new ways of combatting this pathotype. At present we have developed a diagnostic multiplex PCR which incorporates known virulence genes of diarrhoeagenic E. coli pathotypes for quick and simple detection of EPEC. Additionally, we have optimised an infection assay using HeLa cells to phenotypically characterise typical and atypical colonisation and also identified bacteriocins capable of killing EPEC.
Characterisation of the microbiome for two hexactinellid sponges and purification of associated antimicrobial agents from their resident microbes
Authors: Koch Matthew , Best Poppy , Farnham Garry , Kiernan Michele , Bishop Alistair , Warburton Philip , Howell Kerry , Upton Mathew
Published: April 2019

The imminent threat of antimicrobial resistance has necessitated that the search for novel antimicrobials be widened to lesser-explored environments. Marine and freshwater sponges have emerged as the most prolific source of such compounds over the last decade, representing the most widely sampled phyla in the hunt for novel biologics over the last 45 years. Most of the work however has focused on sponges from shallow waters, with the deep-sea sponge microbiome highlighted as a major source of untapped antimicrobial potential. Optimisation of bacterial recovery was carried out for two previously unstudied species of deep-sea Hexactinellid sponge species (Pheronema carpenteri and Rhabdodictyum delicatum recovered from the Rockall Trough, North Atlantic), using a variety of culture media, supplementation and environmental conditions. This optimisation was carried out in parallel with 16S rDNA metagenomic sequencing in order to determine community composition for both sponge species (IonTorrent, Life Technologies). All recovered isolates were assayed for antimicrobial activity, forming a panel of ‘active’ organisms. Two isolates (Ph16-28; A11) were selected for downstream purification and characterisation of the responsible antimicrobial agent via column chromatography. Isolate identities are currently being confirmed via draft whole-genome sequencing (MinION, Oxford Nanopore), and are suspected to be members of the Bacillus and Streptomyces genera. Current data provides a working axiom for the cultivation of deep-sea sponge microbes and suggests the deep-sea sponge microbiome to be a promising source for novel antimicrobials.
Waste not, want not: enhancing the ability of yeast to utilise its own leftovers from the brewing industry to fuel the transport industry with ethanol
Authors: Beaton Ainsley , Tucker Nicholas , Escalettes Franck , Magneschi Leonardo
Published: April 2019

The global brewing industry produces a large amount of waste, 85 % of this is composed of spent brewers’ grain. One use for this waste product is in the bioethanol industry where the yeast, S. cerevisiae uses the spent grain as a feedstock. Due to the nature of the feedstock, there is a lack of utilisable carbon for S. cerevisiae. To obtain optimum utilisation of the waste product in conjunction with high process efficiency, enhanced carbon metabolism of the production strain is required. As well as expanded nutrient utilisation there is also a requirement to maintain high ethanol production and ethanol tolerance that industrial strains have acquired in a preferred growth medium. We are using high-throughput phenotypic arrays to rapidly identify strains best able to grow in a wide range of conditions, including various carbon and nitrogen sources and multiple stress inducing conditions. This method has shown small but measurable differences between production strains in industrially relevant growth conditions. In collaboration with an industrial partner, both targeted and random chromosomal integration of transgenes have been made to multiple candidate production strains to improve recycled feedstock utilisation and process efficiency. In addition, whole genome sequencing is being utilised to interrogate the genetic basis for phenotypic differences between production strains. It has been found that some important null phenotypes are at the transcription level, this information is now in use to drive future rounds of genetic manipulation.
Induction and characterisation of a 25-hydroxycholesterol associated immune response to Gram positive and negative bacteria in a whole blood model of sepsis
Authors: Chick Heather , Griffiths William J. , Wang Yuqin , Humphrey Tom J. , Wilkinson Thomas S.
Published: April 2019

Early diagnosis and treatment of sepsis is one of the biggest challenges to ICU clinicians. Globally, 19 million cases occur annually and it is the third biggest cause of death in the UK. Sepsis is characterised by an uncontrollable, non-specific immune response to an infection, and as a result is difficult to diagnose. Recent research has found that 25-hydroxycholesterol (25-HC) plays a crucial role in the immune response to viral infection. Less is known about the role of sepsis-associated bacteria in this response. To identify novel biomarkers in bacterial sepsis a whole blood model was used and the cellular and molecular responses measured to well-characterised bacteria (Escherichia coli K12 and Staphylococcus epidermidis RP62A) using flow cytometry, ELISA and high performance liquid chromatography-mass spectrometry (LC-MS). Following bacterial infection, mononuclear cells and granulocytes decrease rapidly in response to both K12 and RP62A. This corresponds to a concomitant increase in total CD45 and CD19 expression and the concentration of the proinflammatory cytokines IL-6, CCL3 and CCL20. Proinflammatory responses were significantly more pronounced in K12 infection. There were significant increases in 25-HC in response to K12 infection, and this effect was partially blocked through inhibition of TLR2 or TLR4. Our results suggest the importance of using both cellular and humoral screening to identify unique pathways induced by sepsis causing bacteria. In addition, the current study provides some of the first evidence that 25-HC may be involved in a bacterial driven immune response. This study has importance when designing novel biomarkers to predict sepsis.
Transcriptomic analysis indicates the mode of action of the novel antibiotic MGB-BP-3 against Staphylococcus aureus
Authors: Nieminen Leena , Lemonidis Kimon , Browning Douglas , Hunter Iain , Suckling Colin , Tucker Nicholas
Published: April 2019

MGB-BP-3 (MGB) is a novel synthetic antibiotic inspired by Distamycin – a natural product that is capable of binding to the minor groove of DNA. MGB has a high bactericidal activity against a broad range of Gram-positive bacteria without the toxicity associated with the natural products that it was inspired by. Its oral formulation, developedfor the treatment of Clostridium difficile infections, is currently progressing through a phase 2 clinical trial. This study investigatesthe mode of action of this novel antibiotic. To allow better understanding of MGB’s mode of action, RNA-Seq analysis was undertaken on S. aureus following challenge with 0.5 x MIC (0.1 µg ml−1) MGB-BP-3. Triplicate samples of RNA were extracted at 10 min after challenge. RNA-Seq analysis identified 698 transcripts showing significant changes in expression profile, which were confirmed by quantitative RT-PCR. Amongst these, 62 essential genes showed transcriptional arrest. Glycolysis, pentose phosphate pathway and the TCA cycle were affected. In addition, biosynthesis of nucleotides and certain amino acids were altered and Biolog phenotype arrays were performed in the presence of MGB to confirm this. DNA binding assays demonstrated MGB binding to intergenic regions upstream of strongly down-regulated essential genes (mraY and dnaD). Attempts to evolve resistance to MGB have so far been unsuccessful unlike with the rifampicin control. In conclusion our findings are consistent with a bactericidal mode of action of MGB at the transcriptional level of multiple essential genes.
Understanding the evolution and metabolic capabilities of the Butyrivibrio group
Authors: Pidcock Sara , Skvortsov Timofey , Santos Fernanda , Ting Karen Siu , Creevey Chris , Huws Sharon
Published: April 2019

Exploring and understanding the phylogeny of the Butyrivibrio group is imperative if we are ever to fully understand the consortium of ruminal microbial enzymes that are responsible for the catalysis of multifaceted reactions, such as biohydrogenation. At present, taxonomic classification of the Butyrivibrio group is based primarily on butyrate production. This approach has become antiquated with the development of sequencing technologies and downstream bioinformatics analysis. This study investigated the taxonomic relatedness and functional capacity of the ruminal Butyrivibrio group using 72 genomes. Seventy-one Butyrivibrio group genomes were obtained via JGI (the Hungate 1000 project), and one additional bacterial strain was sequenced by ourselves. A 40 marker phylogenetic tree was constructed and visualised with the interactive Tree Of Life (iTOL), and pangenome analysis conducted using Spine/ClustAGE. Orthologous gene affiliations were identified using OrthAgogue, and glycosyl hydrolase families were identified using dbCAN then aligned with Clustal Omega. Data obtained showed that three primary clades were observed, namely the genus Pseudobutyrivibrio, B. fibrisolvens, and the remaining Butyrivibrio species. Pangenome analysis and orthologous gene affiliations revealed greater diversity within Butyrivibrio than Pseudobutyrivibrio. Butyrivibrio clades consistently showed smaller core genome sizes in comparison to Pseudobutyrivibrio, with core genome percentages as low as 4 %, indicating high levels of variance. Glycosyl hydrolase alignment shows extensive sequence dissimilarity between genes on a nucleotide and amino acid level These findings suggest that the Butyrivibrio group are highly evolved to maintain competitiveness in the rumen and emphasises the need for further research into the biochemical capacity of the Butyrivibrio group.
Rational design of TFDs as novel oligonucleotide antimicrobials to treat Gram-negative infections
Authors: Bahia Sandeep , Morris Christopher , Laurence Louis , McArthur Michael
Published: April 2019

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.
Macrolide-resistant Mycoplasma genitalium in male and female patients attending a Northern Irish Genitourinary Medicine Clinic
Authors: Cox Ciara , McKenna James , McCaughey Conall , Watt Alison
Published: April 2019

Background

Mycoplasma genitalium (MG) can cause urethritis in men and pelvic inflammatory disease in women. Despite the increasing rate of MG resistance to first-line macrolide treatment due to 23S rRNA gene mutations, the availability of testing remains limited in UK laboratories. This study obtained preliminary data on the rate of macrolide resistant strains circulating in N. Ireland.

Methods

Clinical specimens from 1052 patients attending a Genitourinary Medicine (GUM) clinic in Belfast from April 2017 to April 2018 were screened using an in-house MG PCR assay. MG positive samples were subsequently tested using Resistance Plus™ MG assay (Speedx, Australia) to detect 23S rRNA gene mutations associated with macrolide resistance.

Results

Amongst all study samples, 3.9 % (n=41) tested positive for MG. Out of these 41.5 % (n=17) had a 23S rRNA mutation. The rate of MG was highest in rectal swabs (8.13 %) followed by vaginal swabs (4.96 %) and then male urines (2.85 %). The rate of 23 S rRNA mutations associated with macrolide resistance in positive vaginal swabs was 21.4 % whereas the rate amongst urine and rectal swabs was at least 50 %.

Conclusion

Data from this study adds to the evidence base to perform MG testing in risk groups in N. Ireland to improve patient outcomes and reduce antimicrobial resistance.
Molecular typing of human enteroviruses: reanalysis of previously untypable EV isolates and the rapid detection of EV-D68
Authors: Howson-Wells Hannah , McClure Patrick , Clark Gemma , Irving William , Kidd I. Michael
Published: April 2019

Molecular diagnostics have superseded cell culture and neutralisation as the gold standard for enterovirus (EV) diagnosis and serotyping in resource rich countries. EV positive isolates from Nottingham University Hospitals NHS Trust are referred to the Enteric Virus Unit (EVU) for typing; performed through amplification and sequencing of viral capsid protein-1 (VP1). An audit of local EVU typing data (2013–2017 showed a significant (P<0.005) increase in untypable EV strains isolated from clinical specimens. Following a literature review of published EV typing assays and in silico analyses of 2201 EV genomes, an end point RT-snPCR VP1-typing assay was derived from a widely cited protocol and successfully employed to produce a simple, reliable and cost-effective typing assay. Twenty-three previously untypable isolates were identified, with phylogenetic analyses of the VP1 region showing broad distribution across EV-A and B species. This assay detected serotypes across EV species A-D, including EV-D68, responsible for severe neurological and respiratory disease. Following an increase in severe EV infections, including six cases of suspected acute flaccid myelitis, this RT-snPCR was employed locally as a rapid typing assay. In combination with 5’UTR PCR and an EV-D68 specific PCR, designed to amplify the complete VP1 region, EV-D68 was detected in respiratory and neurological specimens, including the CSF of a patient with brainstem encephalitis.
Exploring the molecular inter-relationship of SRPK1 and SRSFs during Human Rhinovirus (HRV)-infection and their subsequent effects on viral replication
Authors: Rozario Christoforos , Graham Sheila , Stevenson Andrew , Maciewicz Rose
Published: April 2019

Introduction

HRV infections cause asthma exacerbations, while HRV-induced wheezing in pre-school children is associated with asthma development in later life. Molecular mechanisms underlying this pathophysiology remain elusive, while HRV-C is increasingly associated with disease severity. Transcriptomic analysis of human respiratory (nasal) epithelia from an in vivo HRV-C infection model revealed changes in expression of cellular Serine/Arginine Protein Kinase 1 (SRPK1) and its substrates, the Serine/Arginine Splicing Factors (SRSFs) suggesting that HRV infection targets pre-mRNA splicing and/or its regulating factors.

Hypothesis

1) HRV splicing modulation results in host transcriptome changes, leading to impaired anti-viral responses and/or immunoregulation; 2) HRV-induced changes to SR proteins result in enhanced HRV-translation and replication.

Methods

HRV-A RNA was synthesised by in-vitro transcription from pRV16.11. HRV replication was measured by RT-qPCR and titration in HeLa cells. Protein expression was monitored by western blotting and confocal microscopy. SPRIN340 was used as a specific SRPK1 inhibitor. Alternatively, SRPK1 was depleted via siRNA interference.

Results

1) SRPK1, SRSF1-6 and SRSF9 are expressed in both A549 and HeLa cells; levels were greater in the latter. 2) SRPIN340 and siSRPK1 treatments decrease the levels of SRPK1 leading to decreased expression of SRSFs in HeLa and A549 cells. 3) HRV16 infection decreased SRPK1, SRSF1-6 and SRSF9 expression in HeLa and A549 cells.

Conclusion

Our initial data suggest that HRV-infection specifically alters molecules involved in pre-mRNA splicing, providing us with insights into the molecular mechanisms underlying the distinct pathology of HRV infection, as well as the aetiology of HRV-associated asthma.
Biofilm production and other virulence factors in Streptococcus spp. isolated from clinical cases of bovine mastitis in Poland
Authors: Kaczorek-Łukowska Edyta , Małaczewska Joanna , Wójcik Roma , Siwicki Andrzej
Published: April 2019

Mastitis is a common disease in dairy cattle. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland. Most of the isolates analyzed demonstrated an ability to produce biofilm (over 70 %). Virulence genes were searched for in S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33 %), cylE (78 %), bca (37 %), and cfb (100 %). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes were Ia (n=8) and II (n=8), in addition to some strains that were not classified in any of the groups (n=6). Out of the eight selected genes for S. uberis only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76 % and 86 % of the strains, respectively. The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.
Determining the function of proteins degraded by Human Cytomegalovirus
Authors: Fletcher-Etherington Alice , Nightingale Katie , Lin Kai-Min , Ravenhill Benjamin , Nobre Luis , Weekes Michael
Published: April 2019

Human Cytomegalovirus (HCMV) is associated with significant morbidity and mortality in the immunocompromised, and is a leading cause of congenital infection. Only three drugs are available for treatment, all with significant toxicities. New therapies and a vaccine are urgently required. Susceptibility to viral infection and disease is determined in part by antiviral restriction factors (ARFs) and the viral proteins that have evolved to degrade them. Small-molecule disruption of the interaction between an ARF and a viral antagonist can inhibit viral replication and may be utilised for antiviral therapies. To identify novel restriction factors against HCMV, we previously developed a multiplexed proteomic approach to identify proteins that are actively degraded early during HCMV infection. We reasoned that these would be enriched in known and novel ARFs that the virus must degrade in order to replicate. 35 proteins were shown to be degraded according to stringent statistical criteria, which included the known anti-HCMV restriction factors Sp100 and MORC3, and a novel ARF, HLTF. Here, we present preliminary results from a combination of viral replication assays to identify other novel ARFs. These include a new ‘two-colour’ approach to characterise HCMV restriction, which aims to eliminate variability in cell density by mixing populations of cells in a single cell culture well. Preliminary studies have identified a number of proteins that inhibit virus replication, as well as a novel dependency factor which the virus may degrade in order to attenuate cellular immune signalling or help establish latency.
Enterobacteriaceae-selective modification of the intestinal microbiota using oligonucleotide antimicrobials
Authors: Wong Nichola , McArthur Michael
Published: April 2019

The human gut microbiota is complex, dense, and hugely influential to health. Imbalances in the microbiota have been associated with numerous disease states, in many cases due to the overgrowth of Enterobacteriaceae, such as Escherichia coli. We describe an oligonucleotide antimicrobial that selectively reduces levels of Enterobacteriaceae in vitro, in a model of the human colonic microbiota, and in vivo in a murine study, whilst leaving the core microbiota intact. The antimicrobials are Transcription Factor Decoys (TFDs) that bind to and competitively inhibit an identified transcription factor necessary for growth in the intestine. This is highly conserved amongst Enterobacteriaceae and controls anaerobic respiration and response to nitrosative stress caused by the innate immune response of the host. A nanoparticulate formulation delivers the TFDs to the cytoplasm of E. coli, as visualized by confocal microscopy, and rapidly kills the bacteria under microaerobic conditions. When applied to anin vitro model of the human intestinal microbiota the TFD produced a decrease up to log10 6 c.f.u. ml−1 in coliforms within the Enterobacteriaceae family while other families remain intact. When delivered orally to the intestines of mice similar results were seen: Enterobacteriaceae were decreased or cleared from the wild-type intestinal microbiota while the remaining bacteria were unaffected. This demonstrates that TFDs can be used to make precise changes to the microbiota and has utility in testing associations between dysbiosis and disease and developing microbiota targeted therapeutics.
Evaluating the epizootic risk to poultry of a novel Chinese H7N9 virus variant with increased pathogenicity in turkeys
Authors: James Joe , Thomas Saumya , Mahmood Sahar , Seekings Amanda , Slomka Marek , Brown Ian , Brookes Sharon
Published: April 2019

Previously we successful infected turkeys with the China-origin H7N9 low pathogenicity avian influenza virus (LPAIV, A/Anhui/1/13, referred to as ‘wild-type’ (Wt)) which successfully transmitted to contact turkeys with virulent outcomes, highly unusual for LPAIV infection, particularly as the LPAIV cleavage site remained unchanged in all experiments. Sequencing of progeny viruses revealed consistent emergence of the L226Q polymorphism in the HA gene, termed the ‘turkey-adapted’ (ty-ad) virus. Ty-ad and Wt were used to compare the epizootic risk posed by both H7N9 LPAIVs in turkeys and to explore the mechanisms which underpins any differences. The Wt and ty-ad viruses robustly infected inoculated and contact turkeys, producing similar shedding titres. However, the ty-ad virus was more pathogenic than the Wt virus in directly-infected and contact turkeys, causing 100 % (Wt) compared to 16 % (ty-ad) survival. The ty-ad virus was detected in broader range of turkey organs, and at higher titre, compared to the Wt variant. This contrasted with pathogenicity and tissue-tropism observations for both viruses in chickens. The wt and ty-ad viruses did not replicate without trypsin in vitro, affirming a typical LPAIV phenotype. The L226Q polymorphism is known to alter receptor binding, with key differences in receptor distribution between turkey and chicken tissues observed. Replication kinetics differences in a range of avian cells will be reported for both viruses. Consequently, if this ty-ad variant were to arise more frequently in nature, it would pose an increased virulent risk to turkeys. It is therefore important to maintain surveillance and understanding of China-origin H7N9 viruses.
Antimicrobial activity of naphthalene lysine conjugated peptide hydrogels
Authors: Afami Marina Ellie , Karim Ikhlas El , Laverty Garry , Lundy Fionnuala
Published: April 2019

The synthesis of hydrogel scaffolds with inherent antimicrobial activity has advantages for their use in tissue engineering. An ultra-short naphthalene lysine conjugated peptide, NapFFK’K’, containing naphthalene (Nap) as a molecule of high aromaticity for gel strength, phenylalanine (F) and epsilon variant lysine (K’) has previously been shown by us to self-assemble forming hydrogels with inherent antimicrobial properties against a limited number of pathogens tested. The aim of this work was to extend the antimicrobial activity studies on NapFFK’K’ including pathogenic bacteria associated with dental infections. NapFFK’K’ was synthesised using the 9-fluorenylmethoxucarbonyl Solid Phase Peptide Synthesis. Peptide purity was analysed by mass spectrometry. Hydrogel formulation was achieved by suspending the peptide in sterile deionized water followed by addition of NaOH and HCl. Hydrogels were tested at peptide concentrations of 1 %, 1.5 % and 2 % w/v against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus, and the Gram-negative bacterium Fusobacterium nucleatum. Bacteria inoculumns were exposed on hydrogel surface for 24 h. Bacterial susceptibility assay, employing the Miles and Misra method, was used to determine antimicrobial activity of hydrogels after 24 h incubation. Our results show that peptide hydrogels exhibit antimicrobial properties against both groups of bacteria but at different peptide concentrations. The 1 % peptide hydrogels was most effective against Gram-positive bacteria whereas the 2 % peptide hydrogel was effective against Fusobacterium nucleatum. Given the efficacy of the self-assembling NapFFK’K’ peptide hydrogels against oral pathogens, they may have potential use in tissue engineering approaches for regenerative endodontic treatments.
The identification of novel vaccine antigens and their potential role in preventing VTEC infections in children
Authors: Quinn Conor , Frost Susanna , McClean Siobhán
Published: April 2019

VTEC are a group of strains of E. coli, which cause severe bloody diarrhoea. VTEC infections can lead to the development of haemolytic uremic syndrome (HUS), which is the main cause of kidney failure among children. HUS can cause other life-long complications including seizures, bowel perforation and blindness. Ireland currently has the highest incidence of VTEC infections compared to any other European country. There are no vaccines available to protect children and immunocompromised adults against VTEC infections. Due to the risk of patients experiencing severe symptoms and complications, there is an urgent need for a vaccine against this infection. Bacterial proteins involved in host cell attachment have previously been shown to be efficacious prophylactic vaccine antigens for other infections. We have shown that an O157 strain, NCTC12900 has 1.3-fold higher binding to HT29 cells than the commensal strain, HS (P=0.0162). We have used a proteomic approach to identify the bacterial proteins involved in attachment to two human gastrointestinal epithelial cell lines, HT29 and Caco-2. We have identified seven host cell attachment proteins in VTEC, strain NCTC12900, that are not found in commensal strain, HS. These antigens will be examined for their ability to protect mice from VTEC challenge.