bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrated phenotypic and genomics analysis to elucidate differences in stress resistance and virulence of Listeria monocytogenes strains
Authors: Stratakos Alexandros , Linton Mark , Ward Patrick , Ijaz Umer , Scates Pam , McBride Jane , Gundogdu Ozan , Corcionivoschi Nicolae
Published: April 2019

Listeriosis is an important food-borne disease responsible for high rates of morbidity and mortality. L. monocytogenes has been the cause of several food-borne outbreaks and product recalls throughout the world. It can adapt and survive in a wide range of stress conditions which makes it difficult for food producers to eradicate. The goal of this study was to use phenotypic assays and whole genome sequencing to elucidate possible links between food related stress resistance and virulence phenotypes in L. monocytogenes strains originating from different sources. Four L. monocytogenes isolates from sweetcorn and one isolate from a food processing environment (control) were sequenced and evaluated for the ability to survive in acid (pH 3.5, 15 min), in the presence of a commercial antimicrobial mixture (2 % v/v, 90 min), heat (60 °C, 5 min) and hydrogen peroxide (420 mM, 15 min). Results showed that the strains had different resistance levels to the above stressors with the environmental strain being more susceptible to heat and the commercial antimicrobial. Also, results showed that the four sweetcorn isolates were more virulent than the environmental isolate as they had significantly higher attachment and invasion capacity onto HCT-8 cells (P Pan-genome analysis revealed that the four isolates fall within a class associated with recent outbreak strains. Single Nucleotide Polymorphisms (SNPs) analysis was performed on the five genome sequences and subsequent cluster analyses on the resulting whole genome SNP matrix revealed differences between the strains.
Disruption of the mce operon from Streptomyces affects spore resistance and results in precocious germination
Authors: Addington Emily , Uguru Gabriel , Clark Laura
Published: April 2019

Streptomyces coelicolor is a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosis as an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Heat kill assays show that the deletion of the mce operon result in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. The spores of a Δmce mutant also exhibit a precocious germination phenotype seen on SEM images confirmed with germination assays.
Distance-decay patterns overshadow effects of long-term fertilization and tillage on microbial community structure in agricultural soils
Authors: Brown Claire , Helgason Thorunn , Dytham Calvin , Moir James
Published: April 2019

Community profiling is one of the most utilised tools in microbial ecology today. The relationships between microbial communities and their environment affect ecosystem function in fields spanning from medicine to agriculture; and understanding the community dynamics of a microbial community is key to understanding the complex effects of human intervention. In this study, we look at the effects of long-term tillage and fertilization regimes in soils from an agricultural block-designed field trial set up in 2001. By studying the microbial community composition, absolute microbial abundance and diversity of denitrification functional genes in the context of environmental data, we were able to address the question of how specific land management histories affect the diversity and distribution of bacteria and denitrification genes within agricultural soils. It was found that microbial communities appear to be largely unaffected by land management history, and cluster predominantly by spatial location within the field, despite lack of significant environmental variation. In this well-established agricultural field trial, Euclidean distance is the major identifiable determinant of microbial community dissimilarity (as well as dissimilarity in microbial abundance). That ecological drift, rather than physicochemical factors can be the major determinant of genetic potential may have consequences for attempts to understand nutrient availability in agricultural systems. Additionally, the overwhelming variation caused by spatial distance indicates that block designed experiments may not always have sufficient statistical power to identify any effects of human treatment.
Metabolic recuperation in valine production by mutant strain
Authors: Jamil Misbah
Published: April 2019

The contemplation of present research work to get over scantiness of amino acid by amelioration ofmicroorganism. As a matter of fact the use of chemical and physical factors symbolize a new era in the excerption of microorganism for proficient of producing a desired product. In this workbiosynthetic pathway of valine and their regulation was observed in Bacillus cereus, Corynebacterium and Pseudomonas fluoresence. The contrivance of operation of ems mutants was analyze in different fermentation media at 24, 48, 72 and 96 h incubation under optimum condition by acidic ninhydrin method. Optical density was recorded by spectrophotometer 470 nm for Valine. The effects of carbon and nitrogen sources and growth factors on the production of valine were studied. In this studies mutant evince that mutation confabulate upon the bacteria the ability to produce valine many times more than its parental strain within the same span of incubation period. The objective of this work to step up headway to search unconventional sources to overcome nutritional exhortation of valine.
Sustainable natural production of vitamins for human consumption in long space missions using synthetic ecology approaches
Authors: Harrison Ellen
Published: April 2019

The MELiSSA (Micro Ecological Life Support System Alternative) project aims to create a closed loop system, capable of providing all the necessary food, water and oxygen for astronauts on long Space missions. The MELiSSA loop is comprised of four compartments, with compartment IVA containing photoautotrophic bacteria, such as Arthrospira platensis, a good source of oxygen, edible biomass and micronutrients. One important example of the latter is cobalamin (B12) deficiency of which can lead to pernicious anaemia and neurological systems, and thus would be detrimental to astronauts on long Space missions. The molecule is not made by plants or fungi, so the ultimate source of B12 in the environment is prokaryotes, and its synthesis requires over 20 enzymatic reactions. Many eukaryotic algae also require cobalamin for growth, and some species have been shown to accumulate the vitamin when grown in coculture with cobalamin synthesising bacteria. The aim of this project is to extend existing knowledge of algal-bacterial mutualisms involving cobalamin. Eukaryotic species such as Haematococcus pluvialis and Chlorella vulgaris, both certified as safe for human consumption, along with A. platensis will be investigated with different bacterial partners to maximise cobalamin accumulation. Not only will this research help provide adequate nutrition on long Space missions, it will also support nutritional supply on Earth with the rise of veganism, as well as, aiding in understanding the dynamics of algal-bacterial mutualisms, which is of interest in terms of nutrient cycling in the environment.
Enhancing the unexplored chemical diversity of Streptomyces sp. to produce new antibiotics active against multidrug resistant Acinetobacter baumannii
Authors: Burns Joshua , Law Samantha , Edwards Christine , Lawton Linda
Published: April 2019

Streptomyces species are a major source of antibiotics but are often grown under restrictive conditions that limit biosynthetic gene expression. As a result, the vast majority of secondary metabolites within a single species remain unexpressed in the lab along with the huge variety in chemical structures and bioactivities. The overarching aim of this project was to identify conditions that produce novel antibiotics, specifically against Gram-negative pathogens. The culture collection NCIMB contains hundreds of Streptomyces isolated from around the world. Strains were selected from the collection guided by preliminary bioactivity studies and available literature. These were then grown in an extensive variety of conditions designed to stimulate production of a wide variety of secondary metabolites, detected by UPLC-MS and analysed using the freely available metabolomic tools MZmine, MetaboAnalyst, and GNPS. Conditions included various carbon and nitrogen sources, temperatures, stresses, epigenetic inhibitors, and other microbes. Compounds active against the Gram negative multidrug resistant pathogen Acinetobacter baumannii were detected in the scaleup culture supernatant fractions. Metabolite identification through GNPS did not detect any previously discovered compounds active against A. baumannii, indicating a potentially novel antibiotic against one of the WHO’s priority pathogens.
Role of oxalate in fungal precipitation and biorecovery of lanthanum
Authors: Kang Xia , Gadd Geoffrey
Published: April 2019

Lanthanum is an important member of the rare earth elements (REE) that are a global strategic resource and have many technological applications ranging from microelectronics manufacturing to the production of clean and renewable energy. Aspergillus niger is widely used in industrial fermentations due to its production of multiple secondary metabolites including citric and oxalic acids. Since it is a ubiquitous soil inhabitant and produces geoactive agents, it can play a role in the biotransformation of metal-containing minerals. Previous studies found that A. niger is capable of mineral solubilization and secondary mineral formation, many metals being precipitated as oxalates. However, there is limited knowledge about the biotransformation of La mediated by fungi. The aim of this project was to explore the mechanisms and factors determining the interactions between A. niger and La. In this study, fungal growth on La-supplemented solid media was carried out and it was discovered that crystalline deposits were formed around fungal colonies in the presence of LaCl3. These biogenic crystals were recovered and subjected to examination for their elemental composition, morphological features and mineral phases using energy dispersive X-ray analysis (EDXA), scanning electron microscopy (SEM) and X-ray diffraction (XRD) respectively. These confirmed the biotransformation of lanthanum and identified the products as lanthanum oxalate [La2(C2O4)3·10H2O], which was further transformed into La2O3 by thermogravimetric (TG) treatment. Geochemical modelling also supported these results. Our findings provide a new aspect for the bioprecipitation and biorecovery of REE from solution using fungal culture systems.
Phylogenetic analysis of integrases of Acinetobacter baumannii genomic islands
Authors: Al-Jabri Zaaima , Zamudio Roxana , Horvath-Papp Eva , Ralph Joseph D. , Al-Muharrami Zakariya , Oggioni Marco , Rajakumar Kumar
Published: April 2019

Acinetobacter baumannii among other pathogens is capable of acquiring new virulence determinants via mobile genetic elements (MGE). Analysis of the distribution of genomic islands (GIs) has shown in many species that most MGEs cluster tightly with specific lineages on a phylogenetic core genome tree. This study aims to address the questions on the association between GIs distribution among certain clonal lineagesand to test the contribution of G08 and G62 in A. baumannii to metal susceptibility phenotypes. The whole genome phylogenetic single nucleotide polymorphism (SNP) tree was build using the feature frequency profile. As input, the 101 complete A. baumannii genomes deposited in GenBank were used. The matrix was generated using command line blast and the R platform to generate the output. Susceptibility testing of heavy metals was performed on wild-type and GI-deletion mutants using eight different heavy metals. Whole genome phylogenetic SNP tree constructed on all complete deposited A. baumannii genomes showed that the integrase of the metal resistance G08 and G62 are present only in single or very few sequence types (STs). To test the hypothesis that these GI seven if present only in defined lineage are still mobile, G08 and G62 were selected respectively in strains AB0057 and ATCC17978. Phylogenetic analysis of the respective GI integrases confirmed that the G08int and G62int belong to a separate clade within the Acinetobacter tyrosine recombinases. Susceptibility testing in A. baumannii strain ATCC 17978, AB0057 and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility.
The biocide triclosan triggers multiple regulatory systems in Staphylococcus aureus to induce antibiotic tolerance
Authors: Walsh Dean , Aylott Jonathan , Hardie Kim
Published: April 2019

The biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.
Efficacy of novel eugenol tosylate congeners as antifungal compounds in combination with fluconazole against Candida albicans
Authors: Molepo Julitha
Published: April 2019

The incidences of Candida albicans infections and their changing drug resistance patterns have drastically increased in recent years. Therefore, new drugs and alternative treatment strategies are promptly required. Combination therapy and the use of natural products have been extensively studied as alternative treatment. In this study, we synthesized Eugenol Tosylate Congeners (ETCs 1–6) and evaluated their antifungal activity profile alone and in combination with fluconazole (FLC) against four FLC susceptible and three FLC resistant clinical isolates of Candida albicans isolates according to CLSI guidelines. For insight mechanism of antifungal action of ETCs, activity of plasma membrane H+-ATPase pump of these C. albicans isolates was determined by monitoring the pH of the external medium. ETC 1 and ETC 4 were the most active congeners against the resistant isolates with the MIC ranging from 125 to 250 µg ml−1. The MFC of ETCs ranged from 1000 µg ml−1 to 2000 µg ml−1. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms showed 36 % of synergy, 29 % of additive, 33 % of indifferent and 2 % of antagonistic interactions. These compounds also inhibit H+efflux activity of H+-ATPase pump at varying degrees. Our results suggest that these ETCs may be directly binding to this pump and thereby inhibiting H+-efflux in Candida cells. These results advocate the potential of these compounds in developing new antifungal drugs; however, further studies are required to understand the other mechanisms involved and in vivo efficacy and toxicity of these compounds.
Deciphering the regulatory mechanisms of formicamycin biosynthesis in Streptomyces formicae, a novel antibiotic against MRSA
Authors: Noble Katherine , Devine Rebecca , Som Nicolle , Hutchings Matthew
Published: April 2019

Derivatives of the secondary metabolites of Streptomyces bacteria account for over half of the antibiotics currently used in the clinic. A crucial part of overcoming antimicrobial resistance (AMR) is in the development of new antibiotics that function with a novel mechanism of action, to avoid the rapid acquisition of microbial resistance. We previously reported the isolation of the new species Streptomyces formicae, from African Tetraponera penzigiplant-ants, shown to have potent activity against several strains on the World Health Organisation’s AMR watch list including MRSA. Genetic analysis of this strain revealed one of its 45 biosynthetic gene clusters, a type 2 polyketide synthase, produced all 13 formicamycins, the natural products responsible for S. formicae’s antimicrobial activity. Several regulators of the formicamycin biosynthetic pathway have been identified, including the major repressor (ForJ). Having purified ForJ, DNA binding assays were performed based on previous cappable-RNA and ChIP sequencing experiments to confirm the interaction of ForJ with different putative promoters in the cluster. By optimising gene-reporter fusion assays in this novel strain, it has been possible to characterise some of the promoters in relation to ForJ to determine the effect of its binding upon activity of the gene cluster. Such work is essential in the progression of novel antimicrobials from the laboratory into a clinic as it must be thoroughly understood how a compound is synthesised and how its biosynthetic pathway is regulated before it can be exploited for overproduction.
The ‘missing’ gastric microbe; the impact of gastric cancer-associated microbiota on Helicobacter pylori growth in vitro and its implications in gastric carcinogenesis
Authors: Alfawaz Dana , Katsande Paidamoyo , Jordan Helen , Kuehne Sarah , Ferrero Richard , Rossiter Amanda
Published: April 2019

In recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pylori is the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O2) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori
in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.
Examining receptor usage by the morbilliviruses: who, or what, is really at risk?
Authors: Conceicao Carina , Willet Brian , Bailey Dalan
Published: April 2019

There are seven ‘established’ morbilliviruses: rinderpest (RPV), measles (MeV), small ruminant (PPRV), cetacean (CeMV), phocine (PDV), canine (CDV) and feline (FmoV) morbillivirus, the majority of which are thought to have narrow host ranges. The exception is CDV, which has been reported in almost all families of the order Carnivora, as well as non-human primates. Morbilliviruses enter cells through one of two proteinaceous receptors: signalling lymphocyte activation molecule F1 (SLAMF1), present on the surface of many immune cells; or Nectin-4, expressed on the basolateral side of various polarised epithelial cells. Given the close similarity of these viruses and their universal use of related receptors, there are realistic fears morbilliviruses may spill-over into naïve hosts, i.e. following eradication of RPV in cattle. This spill-over is not entirely unprecedented; PDV and MeV are thought to share common ancestors with CDV and RPV, respectively. To examine morbillivirus receptor usage and predict future spill-over and zoonotic transmission events, we have developed a high-throughput, quantifiable, cell-cell fusion assay. Effector cells, expressing the viral F and H glycoproteins as well as a split-luciferase reporter, are co-cultured with target cells expressing SLAMF1 or Nectin-4 receptors and the remaining half of the reporter. Upon functional receptor engagement the effector and target cells fuse, allowing cytoplasmic mixing and reconstitution of the reporter, a process which can be quantified. Using these assays we are investigating whether the receptor usage profile of morbilliviruses represents an important factor in spill-over transmission into new hosts.
Candida albicans TLOs and fitness: phenotypic analysis of a TLO null strain of C. albicans generated via CRISPR-Cas9 mutagenesis
Authors: Fletcher Jessica , Flanagan Peter , Sullivan Derek , Moran Gary
Published: April 2019

Background

Candida albicansis a fungus that is both a commensal and opportunistic pathogenic in humans. Genomic analysis has highlighted the expansion of the telomere associated ORF (TLO) gene family as unique in C. albicans. In C. albicans there are 15 different members of the TLO family present, compared to only two in its closest relative, C. dubliniensis. Here we show that deleting all TLOs from C. albicans reduces fitness and effects multiple phenotypes.

Method

A guide RNA (gRNA) with sequence with homology to the TLOgenes was introduced to C. albicans AHY940 (aLEU2/Δleu2), along with the other components of the CRISPR-Cas9 (Nguyen et al. 2017).

Results

The Δtlo strain was found to be generally pseudohyphal in morphology, compared to the wild type. It also showed defective growth in nutrient rich YEPD and YEP-Galactose. Growth on YEPD agar produced colonies similar in appearance to WT, however hyphal induction via growth on Spider agar was greatly reduced in the mutant strain compared to WT. Resistance to oxidative stress was examined and the Δtlostrain was more susceptible to stress induced by both H2O2 and tBOOH. In tests to determine the resistance of the Δtlostrain to cell wall perturbating compounds it was seen that this strain is much less resistant to Congo Red and Calcofluor white than WT. Biofilm formation on plastic surfaces also was reduced in the Δtlo strain compared to that of the WT.

Conclusions

Deletion of the TLO genes in C. albicans greatly impacts phenotypes associated with virulence, and generally results in a less fit strain of C. albicans.
Screening bacteriophage activity against E. coli O157:H7 attached to host cells
Authors: Low Alison , Gally David
Published: April 2019

Bacteriophage (phage) therapy, the use of a specific virus to kill infecting bacteria, is often cited as an alternative to antibiotic therapy. But phage treatment could also play an important role in bio-security against the foodborne pathogen E. coli O157:H7. Phage can be relatively easily isolated and shown to have activity against bacterial strains of interest under laboratory conditions. However, to be used successfully for treatment the phage must be active in vivo where the bacteria may be attached to host cells. To overcome this potential hurdle we are using a screen to test phage activity in conditions more realistic to the host environment than standard lab media. As part of this work we have been isolating new phage that are active against E. coli O157:H7 strains. We are currently testing these newly isolated phage against different bacterial strains and under a variety conditions looking for characteristics that will make them good candidates for phage therapy. We will then test the activity of promising candidates against E. coli O157:H7 attached to bovine epithelial (EBL) cells. The aim of this work is to put together a panel of around 20 phage that not only have the appropriate standard characteristics for phage therapy but have also been shown to have activity on attached bacterial cells. The longer term aim of this work is to use these ‘in vivo’ active phage as an intervention on cattle colonized with E. coli O157:H7 thereby reducing the potential of this pathogen entering the food chain.
The role of SARM in the control of immune response driven by Klebsiella pneumonia infection
Authors: Feriotti Claudia , Bengoechea Jose
Published: April 2019

Introduction

Klebsiella pneumonia is a Gram-negative, capsulated bacteria, which is an important cause of community-acquired and nosocomial pneumonia. Klebsiella co-opts cellular functions dedicated to control immune balance to limit the activation of inflammatory responses. SARM (Sterile α-and armadillo-motif containing protein), the fifth identified member of the TIR (Toll-interleukin 1 receptor (1LR)) adaptor family, negatively regulates IRF and NF-kB activation by affecting TLR4 and TLR3 TRIF-dependent signalling. It is currently unclear the role, if any, of SARM in bacterial infections. Here, we aim to dissect the contribution of SARM in Klebsiella infections, and, specifically, to investigate whether Klebsiella may exploit SARM as part of the pathogen’s portfolio immune evasion strategies.

Results

SARM contributed to Klebsiella anti-inflammation strategies in macrophages through by increasing AKT phosphorylation (to limit phagosome-lysosome fusion), and preventing the activation of NF-kB (to control inflammatory responses). SARM also negatively regulated type I IFN regulatory factor (IRF3) by decreasing IRF3 phosphorylation. Notably, SARM played a role as inflammasome inhibitor, as observed by increased IL-1β secretion in the supernatants of infected sarm-/-. SARM also inhibited ASC oligomerization in Klebsiella-infected macrophages as seen by the increased ASC monomers release by sarm-/- BMDMs. SARM was also required for pyroptosis following Klebsiella infection. Interestingly, Klebsiella induced the expression of SARM in a TLR4-TRAM-TRIF-IRF3-IFNAR dependent manner, demonstrating that Klebsiella exploits type I IFN to trigger SARM to control inflammasome activation, and the activation of inflammatory responses.

Conclusions

These findings have uncovered how Klebsiella manipulates the TLR adaptor SARM to dampen the activation of host defences.
The study of rotavirus phylogenetic diversity, re-assortment and interspecies transmission and associated virome in Northern Irish livestock and wildlife
Authors: Tregaskis Paula Lagan , Mooney Mark , Lemon Ken
Published: April 2019

Background

Rotavirus (RV) is a highly infectious pathogen of livestock causing diarrhoea and dehydration, and substantial economic loss. RV is an RNA virus with a segmented genome that has evolved significantly creating diverse strains. RV is believed to be endemic within livestock and the genotyping of positive isolates will allow the diversity of the rotavirus to be phylogenetically mapped for evidence of re-assortment and interspecies transmission. Rotavirus positive samples and rotavirus negative samples were examined using a metagenomics approach through next generation sequencing (NGS) to study the virome of symptomatic animals.

Methods

RV in symptomatic livestock, and a small subset of wildlife and exotic animal faeces samples (n=326), originating from Northern Ireland, were screened by RT PCR for the RV VP6 gene. NGS libraries using a de novo metagenomics approach were run on the MiSeq reagent V3 600 cycle as pairend reads. Data was quality checked by Fast QC, assembled in SPAdes, processed through NCBI blast (n) and then MEGAN to display the taxonomical content.

Results

The prevalence of RV VP6 gene was n=108 (33 %). Initial Sanger sequencing results showed porcine G3, G4 and G5 and bovine P7 and P13 strains. Preliminary metagenomic results indicates re-assortment and interspecies transmission with a positive bovine sample showing RV acquired re-assortment from human and equine RV strains. Analysis shows there is a large viral community present in both RV positive and RV negative animals. The metagenomic profiling through NGS data set will give a better understanding of the symptomatic virome in livestock.
Identification of putative packaging signals in the RNA of foot-and-mouth disease virus (FMDV)
Authors: Neil Chris , Logan Grace , Newman Joseph , Ward Joseph , Rowlands David , Stonehouse Nic , Tuthill Toby
Published: April 2019

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.
Development of anti-virulence polymers targeting mycobacteria
Authors: Madduri Bala , Creese Oliver , Francisco Fernandez-Trillo Dr , Alderwick Luke
Published: April 2019

Modern medicine is under the excruciating pressure of drug resistant bacterial strains which are ever advancing with the introduction of every new class of antibiotics. Traditional bactericidal and bacteriostatic drugs, while effective in eliminating the susceptible bacterial strains, also impose a selective pressure on bacteria which often leads to the emergence of antimicrobial resistance. An alternative approach is the development of anti-virulence therapies, which aims reduce bacterial pathogenesis while avoiding the selective pressure of classical antimicrobial inhibitors, thus rendering bacteria harmless and potentiating natural elimination from the host by innate immunity defence mechanisms. We have synthesised a selection of functional polymers of poly(acryloyl hydrazide) using a panel of aldehyde functionalisation groups and evaluated their anti-virulence properties on both Mycobacterium bovis BCG and Mycobacterium smegmatis mc2 155, two surrogate organisms to study Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis. Using a combination of microscopy and in vitro studies, we have shown the effectiveness of anti-virulence polymers in reducing mycobacterial phagocytosis in J774 macrophages with minimal antimicrobial activity.
The persistence and dynamics of commensal poultry gut flora within a broiler rearing house
Authors: Brown Helen , Healy Sara , Pursley Isabella , Parker Daniel , Hayes Grant , Horton Daniel , Ragione Roberto La , Neilson David
Published: April 2019

Commercial poultry rearing systems often house successive flocks of birds with limited between-flock cleaning of the poultry houses. Previous research focused on opportunities for successive flocks to become colonised with pathogenic bacterial species. However, there is a paucity of information regarding the transfer and persistence of commensal bacterial between flocks, and if this might confer health benefits on subsequent flocks. The work presented here utilised 16S community sequencing to characterise the development of the microbial flora of commercially reared broiler chickens and turkeys to determine microbial environmental persistence. DNA was isolated from caecal contents, faeces, and various bedding samples collected from nine sites over a nine month period. Samples were taken from houses used for rearing chickens or turkeys or where alternating chicken and turkey flocks were reared. Measures of alpha diversity for the different samples suggested that both chickens and turkeys had a similar microbiota. Unsurprisingly, members of the microflora could also be found in the environmental samples tested, although survival was dependent on the phyla and bedding material. Further analysis of the samples is currently underway, in order to determine the extent, if any, of microbial transfer between flocks, with a particular focus on potential poultry microbiota species differences. This study demonstrated how commensal microbes are able to persist within poultry rearing sheds and if this transfer impacts on subsequent flock performance. Alongside increasing understanding of microbial environmental persistence, the work also shows how effective current biosecurity methods are in controlling the transfer of all microbes, including pathogens.
A SLIC answer to a continuing problem
Authors: Hammond Robert
Published: April 2019

Background

The burden of Anti-Microbial Resistance (AMR) is a growing problem globally. Here we present a device that determines susceptibility rapidly from primary human or animal samples and could turn the tide of AMR. SLIC (Scattered Light Integrating Collector) is a sensitive device for the detection of microbes based on the scattering of laser light.

Methods

Proof of concept studies were carried out initially to establish the lower limit of detection. This was found to be 10–50 c.f.u. ml−1. This exquisite sensitivity allowed us to commence work establishing rapid MICs. Starting with an inoculum of 105/ml bacteria and using a relevant range of antibiotic concentrations the MIC can be established in less than one microbial doubling period.

Results

The rapid and sensitive detection SLIC affords allows for fast growing organisms such as E. coli and S. aureus to have their MICs established in less than 10 min, for any antibiotic. For slow growing organisms such as M. bovis we are able to establish an MIC in H. influenzae and Mycoplasma spp.

Conclusion

As bacterial quantification is continuously monitored we are able to see the action of antibiotics in real time. Using this facility, we can readily distinguish between lytic antibiotics and bactericidal but non-lytic antibiotics. This provides the opportunity to gain new insights into the mechanisms of action and the effect antibiotics have on microbes in a new way in a novel Point-of-Care device.
Enigma of gastric microbiome: diversity, genomic and transcriptomic insights into Ochrobactrum spp. (a non H. pylori, urease positive bacterium)
Authors: Gohil Kushal , Dharne Mahesh
Published: April 2019

The dominance and pathogenicity of Helicobacter pylori in the gastric niche is eminent. Urease test is used to diagnose the infection of H. pylori. However, nowadays several reports indicated the emergence of organisms other than H. pylori (Non H. pylori bacteria– NHPs). We have reported urease positive, Ochrobactrum intermedium from the non-ulcer dyspeptic individual. Therefore such urease positive bacteria raise a question on the reliability of urease test. Since 2005, nearly 35 cases are reported for the presence of Ochrobactrum spp. from clinical specimens. Their high level of resistance to antibiotics and phylogenetic relationship to anthropozoonotic pathogen, Brucella makes them a probable human pathogen. Therefore, it is necessary to study their prevalence, survival mechanism and function in the human stomach. To check their pervasiveness in the Indian population, 218 urease positive gastric biopsy samples were processed and 62 Ochrobactrum spp. isolates were identified with and without H. pylori. Population study of Ochrobactrum spp. was also done using multilocus sequence typing (MLST) which revealed 45 sequence type and clonal population. As the prevalence of Ochrobactrum spp. is approx. 30 %, their survival mechanism was studied under in vitro gastric conditions (acidic pH, urea, microaerophilic environment), using microarray and RNA seq methods. The differential gene expression analysis showed 2 different acid resistance mechanism, i.e. Urease dependent acid resistance mechanism like H. pylori, and Amino acid dependent acid resistance mechanism. Our finding necessitates further detailed investigation of such NHPs in gastric environment and role of such bacteria in gastric niche and warrants further refinement of urease based diagnosis.
Study of the regulation of human IFIT1 stability
Authors: Leong Xin Yun , Sweeney Trevor
Published: April 2019

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are produced in both interferon-dependent or interferon-independent manners after pathogen-associated molecular pattern recognition. Four IFITs (IFIT1, IFIT2, IFIT3 and IFIT5) have been characterized in humans. They are cytoplasmic proteins with repetitive tetratricopeptide repeats, protein motifs well characterized to mediate protein-protein interactions. IFITs play several functions in cells and their antiviral roles are well established. IFIT1 principally binds non-self cap0 mRNAs and inhibit their translation. On its own, IFIT1 overexpresses poorly in cells. Co-expression with IFIT3 enhances IFIT1 protein levels. Likewise, downregulation of endogenous IFIT3 decreases the expression of IFIT1 at the protein level. The stabilization is dependent on the interaction of the C terminus of IFIT3 with IFIT1 via a specific motif in both proteins, disruption of which greatly reduced IFIT1 stability. It is currently unclear why efficient IFIT1 expression is regulated by its interaction with IFIT3 in this manner. To address this, we have begun to investigate the process of IFIT1 degradation. We have used a range of inhibitors to disrupt specific cellular protein degradation pathways. Surprisingly, inhibition of proteasome and lysosome pathways showed protection of IFIT1 from active degradation, suggesting the protein may be degraded via various routes. We will discuss our current efforts to generate stabilized IFIT1 mutants using random and insertional mutagenesis to identify motifs that may contribute to IFIT1 turnover. These findings will contribute to a deeper understanding of IFITs role during the immune response and may identify methods by which their function can be manipulated.
Modified time-temperature combinations reduce beef carcass contamination but will this benefit be seen thoughout the food chain?
Authors: Koolman Leonard , McSharry Siobhan , Whyte Paul , Bolton Declan
Published: April 2019

The overall value of Irelands beef exports is worth approximately €2.5bn which is an annual increase of 5 % in 2017. However, the beef industry faces many challenges to export products to distant markets including a short shelf life and other economic losses that are mainly caused by microbial contamination. One of the simplest approaches to limit this contamination on the surfaces of beef carcasses is to use alternative carcass chill regimes. Carcasses underwent an industry standard chill process (10 °C for 10 h followed by 0 °C for 38 h) and were compared with carcasses that underwent a more rapidly chilled process (0 °C for 5 h and −2 °C for 3 h). Bacterial concentrations (mesophilic and psychrophilic total viable counts, total Enterobacteriaceae counts, Lactic Acid Bacteria, Pseudomonas spp., Brochothrix thermosphacta, Clostridium spp.), physiochemical (pH, temperature, water activity (aw)) and organoleptic (colour, odour, texture) changes were monitored throughout the entire beef food chain (carcass → primal → retail steak) until end of shelf life. Rapidly chilled carcasses had significantly (P<0.05) less bacterial surface contamination compared to conventionally chilled carcasses. There was also significantly (P<0.05) less evaporative loss on carcasses and primals which will have a positive economic impact on the beef industry. This longitudinal study is one of the largest trials ever performed on beef shelf life extension.
The bacterial microbiome of in vitro cultures of Paramoeba perurans
Authors: MacPhail David , Koppenstein Rhea , Longshaw Matt , Henriquez Fiona
Published: April 2019

Amoebic Gill Disease (AGD) is a major problem in the aquaculture industry, as it is responsible for substantial losses of farmed Atlantic salmon in various worldwide locations. The disease is caused by the usually free-living Paramoeba perurans compromising the gills through the resulting development of hyperplastic lesions and lamellar fusion. These structural changes result in a reduction in the functional surface area of the gill tissues. Recent research has focused on identifying bacteria present within a culture of P. perurans, through performing isolation and identification of bacteria present in the cultures using 16S sequencing. Further NGS sequencing was performed from various culture conditions to provide insight into the changes of the bacterial microbiome during amoeba culture. As attempts to isolate the amoeba from the bacterial contamination has been unsuccessful, consideration into a possible symbiotic relationship between the amoeba and bacteria was considered. A filtering method was used to attempt to identify the genera of bacteria present within the amoeba. The isolation and 16S sequencing identified the presence of various marine bacteria, including those of the Pseudoalteromonas, Halomonas, Cellulophaga and Mesonia genera. The NGS sequencing identified a substantial proportion of sequences to match the Vibrio genus and suggests an association between this genus and the amoeba. If symbiotic relationships between specific bacteria and amoeba can be confirmed, the bacteria could potentially be used as an indicator organism for the risk of AGD outbreak. It may also provide an indirect target for the control and treatment of AGD.
Retrospective point-prevalence study of Enterovirus D68 (EV-D68) detection in the symptomatic paediatric population presenting to the Children’s Hospital Emergency Department, RBHSC, Northern Ireland
Authors: Balgos Annamarie , Cox Ciara , McKenna James , Feeney Susan
Published: April 2019

Enterovirus D68 (EV-D68) detection has recently been associated with severe neurological symptoms in adults and children in European countries. The spectrum of disease by EV-D68 ranges from asymptomatic to acute respiratory symptoms, hospitalisation, and sporadically to neurological symptoms, including acute flaccid paralysis (AFP) and acute flaccid myelitis (AFM), and death. This has led to increased vigilance for detection of enterovirus D68, especially in cases that present with the more severe clinical syndromes. In normal cases, enterovirus is transmitted by faecal-oral routes and/or respiratory routes, however in the case of EV-D68 it is almost exclusively passed through respiratory transmission. An infection of EV-D68 is generally distinguished by its rapid onset of disease. The incubation period of the disease between 3 to 5 days, which is unlike the other enteroviruses which usually have an incubation period of around 10 days. This study describes a retrospective analysis of 150 non-invasive respiratory specimens (nose/throat or throat swabs) collected from paediatric outpatients presenting to Childrens Hospital Emergency Department with respiratory and/or CNS symptoms including fever and seizure. Specimens have been collected from November 2017- November 2018. All specimens tested by four RT-PCR assays for Pan-Enterovirus, EV-D68 specific, human Rhinovirus and RNaseP (quality control). Results on prevalence of EV-D68, as single or co-infection, are presented. Results indicate low prevalence of EV-D68 in the Northern Ireland symptomatic paediatric population. As EV-D68 is an emerging infection it is critical to remain vigilant particularly in the case of neurological presentation.
Investigating the effect of alkaline stress on biofilm formation by Salmonella enteritidis
Authors: Papakonstantinou Katerina , Efthimiou Georgios
Published: April 2019

Bacterial biofilm formation is an important survival strategy in multiple environments. It is affected by the attachment surface, the bacterial strain and the surrounding environment. In Salmonella enteritidis, a biofilm-forming foodborne pathogen, the molecular biofilm regulators act as on-off controls between the sessile and the planktonic population, while the exact underlying formation mechanism still remains unclear. The aim of this project is to study the effect of alkaline environment on the formation of Salmonella biofilms and to examine the architecture of biofilm produced under alkaline conditions, by use confocal microscopy. Neutral pH was found to be the optimal pH for Salmonella biofilm formation, while pH 10 significantly reduces it (P-value=0.015). However, cell viability remains high at pH 10, which suggests that the pathogen can easily survive the alkaline stress. Biofilm morphology at pH 7 is characterized by thick cell clusters, whereas at pH 10 it is characterised by thin layers of individual cells. These findings can help us understand how Salmonella enteritidis survives under highly alkaline conditions, potentially leading to the design of new and more effective disinfection strategies involving highly alkaline detergents.