bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication of the Chikungunya virus genome requires cellular chloride channels
Authors: Müller Marietta , Jones Natalie , Todd Eleanor , Khalid Henna , Merits Andres , Mankouri Jamel , Tuplin Andrew
Published: April 2019

Chikungunya virus (CHIKV) causes fever and debilitating joint pain, with frequent long-term health implications and cumulating fatalities worldwide. There are no specific antivirals and vaccines, therefore understanding CHIKV replication is essential to establish treatments and preventative measures. Cellular ion channels are druggable targets and are known to facilitate replication of RNA viruses. To determine if the activities of cellular chloride channels (Cl--channels) are required during CHIKV replication, we applied broad-ranging inhibitors and siRNA to mammalian and invertebrate cells. The Cl--channel inhibitors DIDS, 9-ACA and NPPB significantly reduced the titre of released CHIKV progeny at 12 h post-infection in a dose-dependent manner suggesting that Cl--channels are pro-viral factors. Analysis of viral protein expression and time-of-inhibitor-addition studies indicated that CHIKV requires Cl--channels at post-entry and pre-egress stages. Replication of a sub-genomic replicon was restricted and genome copy numbers reduced by Cl--channel inhibition, implying that Cl--channels are involved in genome replication. siRNA knock-down identified the chloride intracellular channels (CLIC) 1 and 4 to be required for the CHIKV infectious cycle with CLIC1 interacting with the viral protein nsP3. We hypothesise that the channels play a role in formation or maintenance of the membranous, viral replication-complexes and that this important role is conservt amongst the mammalian and invertebrate hosts. These findings advance our understanding of CHIKV replication in the two host environments and help to identify drugs/druggable targets for treatment and prevention of CHIKV disease.
Clinical evaluation of the novel molecular bacterial load assay for real-time monitoring of tuberculosis treatment response
Authors: Mtafya Bariki , Sabiiti Wilber , Sabi Issa , Msuya Yahya , Ntinginya Nyanda
Published: April 2019

Background

Monitoring the treatment of tuberculosis (TB) relies on less sensitive smear microscopy (SM) and culture methods which are very slow. We evaluated the novel Molecular bacterial load assay (MBLA) for implimentability and real-time monitoring of TB treatment in a clinical setting.

Methods

Therapy naive (Xpert MTB/RIF confirmed) TB positive patients were enrolled in Mbeya, Tanzania. Sputum samples were collected at baseline and thereafter at week 2, month 2, 5 and 6 of treatment. Samples were analysed for M. tuberculosis (M.tb) by MBLA and compared to SM, culture and clinical monitoring.

Results

59 TB patients were enrolled for the study. Median age, 37 (18-65) years, 62.7 % (37/59) male, 45.6 % (27/59) HIV positive and 8.47 % (5/59) were re-treatment. Mean BL (± SD) at baseline was 5.48 ± 1.3 declining to 3.42 ± 0.7  at month 2 and 3.51 ± 0.62 log10CFU/ml at month 6 of treatment. This corresponds MBLA positivity of 92.98, 65.5 and 7.84 % at baseline, month 2 and 6 respectively. In contrast, positivity of SM and culture were 78.95, 9.62 and 0 %, and 85.96, 25 and 3.39 % at baseline, month 2 and 6 respectively. Decline in test positivity reflected resolution of clinical signs. While night sweat, and chest pain resolved earlier on in treatment, resolution of cough was slow and consistent with MBLA. Furthermore, the turn-around-time for MBLA results was 24  h compared to median (range) of 14.83 (4.33–42)  days for liquid culture.

Conclusion

MBLA exhibited higher sensitivity and shorter turn-around-time than standard tests and clinical signs. This demonstrates the potential of MBLA to offer real-time results for clinical decision making.
Comparative analysis of the composition and change of the microbiome of diabetic foot ulcers from patients on different therapies
Authors: Walker Thomas , Spencer Janice , Dancer Stephanie , Craft John
Published: April 2019

Diabetic foot ulcers (DFUs) are slow healing wounds which arise from co-morbidities associated with diabetes. Often these ulcers become infected leading to gangrene, osteomyelitis and sepsis. Current treatment options include debridement and a topical irrigant which have limited success. Understanding the effects that these treatments have on microbiome of DFUs and on wound healing is poorly understood. This study compared the efficacy of two irrigant solutions (Prontosan and Electrolysed water –E.W.) on their impact on the DFU microbiome, their role in DFU healing and their effect on biofilm viability. Sequential samples taken from 7 patients undergoing treatment with either one of the irrigants, over a 4 week period revealed commonly observed genera present included Staphylococci (96 %), Propionibacterium (96 %) and Finegoldia (89 %). A unique composition and diversity was observed in the microbiome of each individual DFU. Increasing microbial diversity within the DFUs was correlated with an elevated percentage abundance of anaerobic and Gram negative genera whilst inversely correlated with facultative anaerobic and Gram positive genera. No significant reduction in diversity or species richness of the DFU microbiomes was observed after treatment with either irrigant. Both Prontosan and E.W. had similar effects upon S. aureus biofilms reducing viability by 82.013 % and 86.89 % respectively however E.W. efficacy was strain specific. In addition, E.W. was ineffective at preventing biofilm formation in 6/8 (75 %) S. aureus strains. Better understanding of the DFU microbiome and investigations into novel therapies is paramount to aid our ability to improve the quality of life for diabetic patients.
Different oral niches give rise to varying levels of genetic diversity in Streptococcus mitis
Authors: Davison Charlotte , Beleza Sandra
Published: April 2019

Background

The commensal bacterium, Streptococcus mitis is a major coloniser of the oral cavity. This close cousin of Streptococcus pneumoniae has been identified to occupy a range of oral niches independent of pH, epithelial surface and redox state. We have previously observed a high level of genetic diversity in S. mitis with an open pan-genome and only 46 % of the genome identified as core.

Aim

We aim to identify the driving force behind the high levels of genetic diversity identified in S. mitis and determine if this diversity can be attributed to oral location.

Methods

Samples were collected from the buccal mucosa and tongue dorsum. A total of 75 S. mitis isolates were whole genome and Ion Torrent sequenced and subject to bioinformatic analysis.

Results

In contrast to S. pneumoniae, genetic diversity in S. mitis is predominantly driven by mutation with recombination playing only a minor role. It was also observed that isolates collected from the tongue dorsum were significantly more diverse than those collected from the buccal mucosa. Due to the mutational nature of S. mitis diversity, this suggests varying pressures on S. mitis in these two environments. Study is now underway to establish the genetic differences between S. mitis from these oral niches and determine how this will impact future study and study design of this significant oral microbiome component.
Characterising Birnaviridae replication and reassortment in vitro: virus factories derived from distinct input viruses form in the cytoplasm of co-infected cells and coalesce over time
Authors: Campbell Elle , Wells Joanna , Gray Alice , Broadbent Andrew
Published: April 2019

The Birnaviridae family is comprised of non-enveloped viruses with a double-stranded RNA genome that is divided into two segments, A and B. Birnaviruses are responsible for major economic losses to the poultry and aquaculture industries, and reassortment complicates their epidemiology and control. However, little is known about the nature of theirreplication in cells, or the molecular mechanism underpinning reassortment. In order to address this, we rescued two recombinant infectious bursal disease (IBD) viruses, with either a GFP11 or Tetracysteine (TC) tag at the 3’ end of segment B (IBDV-GFP11 and IBDV-TC, respectively). DF-1 cells were either transfected with GFP1-10 prior to IBDV-GFP11 infection, or stained with ReAsH following IBDV-TC infection, which led to the apprearance of green or red foci in the cytoplasm, respectively. Foci co-localised with VP3 and dsRNA, suggesting these were virus factories (VFs). The average number of VFs significantly decreased from 60 to 5 per cell between 10 and 24 h post infection (P<0.01), while the average area significantly increased from 1.24 µm2 to 45.01 µm2 (P<0.01), suggesting VFs coalesce in the cytoplasm over time. Red, green and yellow foci were observed in the cytoplasm of co-infected cells, suggesting that VFs are initially derived from distinct input viruses prior to coalescence. Live cell imaging revealed that larger VFs were more static while smaller VFs were more mobile, and fusion events were observed. We speculate that VF coalescence is required for birnavirus reassortment, and current work is aimed at determining the cellular factors that drive coalescence.
Characterising the human intestinal mycobiome during healthy ageing
Authors: James Steve , Phillips Sarah , Baker David , Hall Lindsay , Carding Simon
Published: April 2019

Although fungi are fundamental to the human microbiome, the diversity and dynamics of the mycobiome is poorly understood, particularly in considering their association with infectious disease, autoimmune disorders and atopy that affect immunocompromised individuals and infants. Characterising the human mycobiome faces several challenges relating to their low abundance and lack of standardized procedures for sample collection and isolation of viable cells and/or quality genetic material for culture-dependent and independent taxonomic and functional characterisation. To address these issues, we have developed a mycobiome analysis pipeline employing both culture-dependent and independent methods to identify as well as isolate, where possible, the fungal taxa populating the human intestinal tract. In a proof-of-concept study this pipeline has been used to identify fungal populations in faecal samples obtained from a small cohort of young infants, aged 2 years or younger. All were born prematurely, and severely immunocompromised and at risk from invasive and potentially lethal microbial infections, including those caused by fungal overgrowth. We have used this combined approach successfully to identify the fungi present in each individual infant, and to recover viable isolates. To date, Candida albicans and C. parapsilosis are the most frequently isolated fungi. While both are major opportunistic human fungal pathogens, C. parapsilosis is particularly problematic to preterm babies, due to its innate ability to form biofilms. Detailed characterisation of these isolates is currently underway. Two large-scale longitudinal microbiome studies have started at the Quadram Institute, and our validated analysis pipeline will be incorporated to define the fungal component of each study participant.
Bacterial lipocalins protect bacteria from antibiotic-induced membrane lipid peroxidation
Authors: Naguib Marwa , Valvano Miguel
Published: April 2019

Burkholderia cenocepacia, an opportunistic bacterium causing chronic respiratory infections in patients with cystic fibrosis, produces the bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures antibiotics outside bacterial cells, providing a novel extracellular mechanism of antimicrobial resistance. Sublethal concentrations of bactericidal antibiotics (e.g. polymyxin B, norfloxacin) and superoxide ion stress inducers (e.g. paraquat) stimulate transcription of bcnA, suggesting BcnA may also play a role in oxidative stress responses. bcnA gene homologues can be genetically linked to an upstream gene encoding a predicted membrane cytochrome b-561 protein, which we have named bcoA (bcnA cytochrome oxidase-associated gene). RT-PCR showed that both bcnA and bcoA are co-transcribed in B. cenocepacia. We hypothesize that BcnA and BcoA may be components of an unrecognized pathway to protect B. cenocepacia and other bacteria from membrane lipid-derived peroxidation damage. Here, we show that bcnA and bcoA deletion mutants display enhanced membrane lipid peroxidation and fail to survive under conditions that stimulate peroxidative stress (e.g. cold stress and salt stress). Compared to wild type, the levels of the lipid peroxidation biomarker malondialdehyde are also increased in the mutants upon exposure to near-lethal concentrations of polymyxin B and norfloxacin. No differences in bacterial survival were detected in parental and mutants under anaerobiosis. These findings indicate that BcnA and BcoA participate in a previously unrecognised peroxidation detoxification pathway that likely protects the outer membrane lipid bilayer under extreme stress conditions such as antibiotic killing.
Genetic regulation of compost and plant degradation in Agaricus bisporus
Authors: Dunne Keith , O`Donoghue Martin , Grogan Helen , Burton Kerry , Heneghan Mary
Published: April 2019

Agaricus bisporus, the common button mushroom, is a major contributor to the global carbon-nitrogen cycle through its role as a secondary decomposer of plant and leaf litter. It is also an economically significant mushroom with a domestic farm gate value in excess of €120 million. This research aims to elucidate the molecular mechanisms involved in compost and plant matter degradation by A. bisporus. An examination of the entire genome using microarray analysis was carried out on A. bisporusat 48 h intervals over the course of its commercial lifecycle. This data revealed several genes that appear to be involved in the genetic regulation of mushroom production. From this data, genes with potentially critical roles in compost utilisation and plant matter degradation were identified. Five genes of significant interest were selected for further study. These include three carbohydrate degrading genes and an unknown gene which appears to be unique to A. bisporus, all of which had an expression profile that matched the commercial growth pattern. The fifth gene was selected based on its significant down-regulation during peak mushroom growth. To further compliment this data a bioinformatic analysis of known transcription factor binding sites present within each promoter of A. bisporus is also being carried out. The data generated from this study will provide crucial insight into the mechanisms behind the regulation of A. bisporus growth and its ability to degrade plant matter and compost degradation.
A micro luminescence-based assay to measure serum susceptibility in Escherichia coli
Authors: Hartnett Aileen , Smith Stephen , Knowles Susan , Lydon Sara
Published: April 2019

Neonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.
A pilot study to determine the relationship between bacterial populations in the cloaca and the caecum of broiler chickens
Authors: Andreani Nadia , Goddard Matthew
Published: April 2019

The study of animal digestive tracts reveals important information on the host’s health status. For livestock, being able to predict the effect of different treatments on the gut microbiome has important implications for increased sustainability, enhanced animal welfare and increased food safety. However, gut contents can be investigated only after the slaughter of the animal, but cloaca/rectal samples may be collected from live animals and reduce the number of animals killed for experimental purposes. The aim of this study is comparing the microbial communities of caecum and cloaca associated with eight poultry broiler flocks from two English farms. 16S amplicon libraries were run on a MiSeq with a 250 bp PE read metric. The data were evaluated with in qiime1 and qiime2. Comparisons of bacterial communities of cecum and cloaca revealed they are significantly different in terms of the number and types of bacterial species, as well as their abundance (P-value Indicator species analysis of cecum samples showed the class Bacilli were enriched, while Clostridia had greater prevalence in cloaca. Finally, no pathogenic bacterial species of poultry were identified in the analysed animals. Despite the fact sampling cloaca content could be a method to reduce cost and suffering for research purposes, this study reveals the limit of the use of cloacal microbiomes to provide a window into poultry alimentary canal microbiomes.
Prevalence of microbial parasites in captive animals across wildlife parks
Authors: Betts Emma , Gentekaki Eleni , Carpenter Angus , Harland Adrian , Tsaousis Anastasios
Published: April 2019

Microbial eukaryotes (parasites/protists) are widely distributed and are common inhabitants of the gastro-intestinal (GI) tract of humans and animals. Some species, including Giardia, Entamoeba and Cryptosporidium are associated with symptomatic gastro-intestinal illness. However, others, for example Blastocystis, have questionable pathogenicity as they can be found in symptomatic and asymptomatic individuals. The aim of this study is to investigate selected protists which present health concerns to humans or animals. To date, approximately 180 faecal samples from 33 mammalian species, four bird species and one reptile across two wildlife parks in the Southeast England have been collected. A combination of cell culturing techniques, microscopy and molecular biology have been carried out to positively identify different protists including Blastocystis, Cryptosporidium, Eimeria, Entamoeba, Giardia and Isospora. Preliminary data show over fifty percent of the animals are sequence positive for at least one species, with approximately thirty percent exhibiting co-habitation with two or more different species. This study provides one of the first thorough investigations into distribution and prevalence of GI tract protists in wildlife parks in the UK. As a result, it has enhanced our awareness regarding what may constitute a normal eukaryotic component of the gut microbiome, in addition to aiding conservation efforts by examining the impact captivity has on an animal’s microbiome and potential implications this may have on their release.
AphA is a master regulator of natural competence in Vibrio cholerae
Authors: Haycocks James , Warren Gemma , Grainger David
Published: April 2019

Vibrio cholerae is the etiological agent of cholera, a disease affecting 4.3 million people each year (WHO, 2015). The symptoms of cholera include profuse watery diarrhoea and vomiting, leading to coma and death in serious cases. The El Tor biotype of V. cholerae is globally predominant, and the success of this biotype to cause disease is thought to be due, in part, to the ability of the organism to successfully colonise two different environments; i) marine and brackish coastal waters where V. cholerae forms biofilms on the chitinous surfaces of shellfish, and ii) the human intestine. When bound to chitinous surfaces V. cholerae expresses genes required for biofilm formation, chitin utilisation and natural competence. Conversely, within the human host, V. cholerae switches on the expression of virulence genes. The transcription factor AphA was discovered as a regulator of genes encoding the toxin co-regulated pilus (TCP) of V. cholerae. It is also known that AphA is active when V. cholerae populations have a low cell density. Hence, AphA is generally considered a regulator of virulence and quorum sensing. We have mapped DNA binding by AphA across the entire V. cholerae genome. The majority of AphA target genes encode cell surface and cell envelope proteins. Unexpectedly, we show that AphA also targets key gene clusters within the natural competence regulon of V. cholerae. Using biochemistry and genetics we show that AphA is a master repressor of natural competence and acts to antagonise transcription activation by the cyclic-AMP receptor protein.
The selective sequestration of bacterial populations using polymeric glycomaterials
Authors: Petch Joshua , Heeb Stephan , Camara Miguel , Mantovani Giuseppe
Published: April 2019

The ability to selectively sequester bacteria from a mixed population is a desirable aim across a range of fields. Already some technologies exist to attempt to meet these aims spanning microfluidics and automated flow cytometry to antibody conjugated magnetic nanoparticle precipitation. However, these technologies are generally limited to the confinement of small populations in defined locations. Furthermore they may not be able to differentiate morphologically similar but phenotypically divergent cells. We aim to produce a novel technology to isolate bacteria from mixed populations by utilising polymeric carbohydrate ligands which bind to inducible bacterial adhesion proteins. To achieve this aim an inducible mutant of the fim operon in Escherichia coli has be constructed, thus allowing for switchable production of the mannose binding organelle, Type 1 fimbriae. Furthermore, we have previously observed that mannosylated polymers selectively bind to Type 1 fimbriated E. coli and to our induced mutant. Novel mannose functionalised polymers are being synthesised via a reversible addition-fragmentation chain transfer (RAFT) scheme. These polymers contain a catechol terminus which may be conjugated to magnetic Fe3O4 nanoparticles thus facilitating selective bacterial sequestration by magnetic separation. The successful development of this polymer-based bacterial sequestration platform could potentially enable the equivalent of immunoprecipitation in large-scale fermentation processes or the precise manipulation of living cells in laboratory scale procedures.
Analysis of bacterial competition using imaging mass spectrometry
Authors: Castaño Laia , Gilmore Ian , Johnston Blair , Chrubasik Michael , Duncan Katherine
Published: April 2019

The bacterial order actinomycetales are responsible for the production of 65–70 % of microbially produced specialised metabolites with diverse biological activities, with some actinomycetale strains containing over 30 Biosynthetic Gene Clusters encoding for these metabolites. However, only approximately 10 % of these genes are typically transcribed in a mono-culture setting. Furthermore, it has been observed that microbial interactions may induce these cryptic gene clusters providing an ecological advantage to the producer strains. To understand the chemical exchange between strains isolated from the marine environment, microbial interactions were assessed using 49 actinomycetale strains, two Pseudomonas and one Bacillus strain. In total, 72 tri-cultures (three strains) were analysed resulting in 29 strains that showed an altered phenotypes as a result of the interaction. These were then evaluated in a one-to-one culture (two strains) followed by bioactivity screening. Using this data, nine tri-cultures and 27 one-to-one cultures were evaluated using tandem Mass Spectometry, enabling chemically interesting interactions to be prioritized for Time of Flight Secondary Ionisation Mass Spectometry (ToF-SIMS) analysis. ToF-SIMS enables the spatial distribution of parent ions within a sample, in this case, two bacterial strains interacting in a Petri dish. The results that will be presented demonstrate that microbial interactions induce the production of metabolites and ToF-SIMS represents an exciting strategy to study bacterial chemical ecology.
Identification of sites of Infectious Bronchitis Virus RNA synthesis
Authors: Doyle Nicole , Rayon Selma , Simpson Jennifer , Hawes Pippa , Maier Helena J
Published: April 2019

Infectious bronchitis virus (IBV), an avian gammacoronavirus, is an important pathogen causing significant animal welfare problems and economic losses to the global poultry industry. Positive-strand RNA viruses, including coronaviruses, induce cellular membrane rearrangements during replication forming replication organelles, which are thought to support efficient viral RNA synthesis. IBV replication has been shown to induce the formation of double membrane vesicles (DMVs), zippered ER and tethered vesicles, known as spherules. Although these are proposed to be the site of viral RNA synthesis, this is as yet unconfirmed and is therefore the focus of these studies. Historically, dsRNA has been used as a marker for sites of coronavirus RNA synthesis, however IBV-associated dsRNA and nsp12 (the viral RNA-dependent RNA polymerase) do not colocalise in infected cells. We have determined the cellular location of the viral genome using Fluorescence In Situ Hybridisation (FISH). By comparing the immunofluorescence labelling of cells permeabilised with different detergents, we have demonstrated that dsRNA can be found within membrane-protected compartments, while nsp12 is not, indicating that the virus could be isolating the dsRNA in DMVs or spherules, affording protection from the host immune response. By incorporating uridine analogues over the course of infection with IBV, we have visualised sites of nascent viral RNA synthesis using super-resolution microscopy. This has shown that dsRNA appears to colocalise more strongly with nascent RNA than nsp12. Using these methods as well as looking at the ultrastructural level we are able to begin to discover the location of sites of IBV RNA synthesis.
Evolutionary genomics of the host-restricted pathogen Staphylococcus aureus subsp. anaerobius reveals extensive genome decay and signatures of adaptation
Authors: Yebra Gonzalo , Neamah Maan M , Wee Bryan A , Richardson Emily J , de la Fuente Ricardo , Ross Fitzgerald J , Penadés José R
Published: April 2019

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.
Characterization of a bacteriophage from avian Staphylococcus aureus associated with innate immune evasion
Authors: Parr Emily , Richardson Emily , Hume David , Fitzgerald J. Ross
Published: April 2019

Staphylococcus aureus is an important human and livestock pathogen. An S. aureus phage (φAvβ) inserted into the chromosome at the beta toxin gene (beta-converting phage) is present in approx. 90 % of human strains and contributes to human-specific innate immune evasion. Comparative genomic analysis of S. aureus isolates from infected poultry has revealed an avian-specific subfamily of beta-converting phages represented by multiple variants with distinct integrase gene alleles. To investigate the role of the avian beta-converting phages in host-pathogen interactions, a φAvβ-deficient strain with non-functional beta-toxinand a φAvβ-deficient strain with restored beta-toxin were constructed by allele replacement in an avian pathogenic S. aureus strain. Compared to the wild type, both φAvβ-deficient strains have reduced net extracellular growth in vitro in chicken bone-marrow derived macrophages. Further investigation using GFP-tagged bacteria has revealed that both φAvβ-deficient strains show reduced initial phagocytosis and intracellular survival compared to the wild type. Absence of φAvβ is also associated with decreased killing of the chicken bone-marrow derived macrophages. We are currently investigating the mechanism underlying this phenotype using deletion mutants of the candidate phage effector genes. Ongoing work also involves using RNAseq to investigate differential host transcriptional response of the macrophages to S. aureus in presence/absence ofφAvβ. Overall, these data will contribute new information relating to the evolution of avian S. aureus and mechanisms of bacterial host-adaptation.
Decaying Ascophyllum nodosum as a source of algal cell wall degrading enzymes with potential utility in enzyme-assisted extraction technologies
Authors: Ihua Maureen , Mohammed Halimah , Guihéneuf Freddy , Margassery Lekha , Jackson Stephen , Clarke David , Dobson Alan
Published: April 2019

Seaweeds are of huge interest in the food, pharmaceutical and agricultural industries due to their high nutritional content and the prevalence of useful bioactive compounds. Current extraction methods of macroalgal-derived metabolites are however problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular as they allow the development of more efficient and eco-friendly processes. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25°C and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina Miseq sequencing and culture-dependent approaches, including the novel iChip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay with a substantial decrease in the relative abundances of certain phyla including Planctomycetes and Verrucomicrobia observed during the decay period. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species from the phylum Bacteroidetes together with a number of Vibrio species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculous and were shown to be equally efficient as commercial enzymes in their extraction efficiencies.
Development of streptomyces to utilise sustainable feedstock in fermentations
Authors: Birke Anna , Kendrew Steve , Huckle Benjamin , Hunter Iain , Hoskisson Paul
Published: April 2019

The genus Streptomyces is comprised of soil-dwelling Gram-positive Actinobacteria that are widely used for the industrial production of antibiotics. S. clavuligerus is used for the industrial production of clavulanic acid, which is a potent b-lactamase inhibitor, and is, therefore, able to restore the sensitivity of b-lactamase-producing bacteria penicillins and cephalosporins. In fermentations, the carbon sources available for utilisation by the producing organism have profoundly impact central carbon and specialised metabolic pathways. We have a long-term goal of using carbon sources from food waste to produce clavulanic acid with a view to developing more sustainable fermentations. To achieve this, the carbon utilisation profile of S. clavuligerus has to be diversified. Wildtype S. clavuligerus is a natural glucose auxotroph and has adapted to utilise glycerol most efficiently. It has been shown that the lack of glucose utilisation by S. clavuligerus is due to the insufficient expression of genes whose products are required for glucose uptake (glcP) and phosphorylation (glk). To enable glucose utilisation by S. clavuligerus strains, we have constructed strains for heterologous expression of either glcP or glk from different Streptomyces species. Further, the range of utilisable carbon sources for growth and clavulanic acid production has been investigated. Growth on solid media has revealed interplay between carbon and nitrogen metabolism, with extracellular protease production being regulated in a carbon source-dependent manner. Therefore, the role of protease secretion and its relationship with clavulanic acid production has been examined, revealing a complex role between carbon catabolite repression, protease production and clavulanic acid biosynthesis.
Whole genome analysis of the common pathogens associated with microbial keratitis
Authors: Glenn Michael , Kaye Stephen , Neal Timothy , Roberts Paul , Winstanley Craig , Fairley Derek , Simpson David
Published: April 2019

Background

Microbial Keratitis is a serious infection of the cornea and a major cause of corneal opacity and vision loss worldwide. Whole-genome sequencing (WGS) of keratitis bacterial isolates has the ability to provide a wealth of clinically-valuable information. The potential of metagenomics has also been evaluated as current techniques for pathogen identification are laborious, time-consuming and associated with poor culture rates, making metagenomics an exciting future prospect.

Methods/Results

Samples from keratitis patients were collected and isolates grown in agar before DNA was extracted from pure-cultures and WGS data was obtained from a total of 30 isolates, including common causative agents Pseudomonas aeruginosa and Staphylococcus aureus. Libraries were prepared using a miniaturised Nextera XT protocol and sequencing was carried out on Illumina’s NextSeq platform. Genomic analysis was then based around MLST-typing, comparative genomics, and the identification of key genes inferring antibiotic resistance and virulence. Our pathogens exhibited diverse evolutionary lineages and a range of virulence-associated genes, providing insight into pathogenicity. The ability to obtain sufficient DNA quantities for metagenomics sequencing was also assessed using various host depletion and microbiome enrichment techniques.

Discussion/Conclusion

The clinical role of next-generation sequencing is currently limited due to associated costs and required computational resources, however in the future this method could replace current techniques for pathogen identification. WGS provides important insights into the genomes of the common keratitis infectious agents, improving understanding of the aetiology of corneal infections and metagenomics shows future potential to be the centre of culture-independent pathogen identification.
Genome mining of ant-associated bacteria to identify novel secondary metabolites
Authors: Hamilton Jacob , Holmes Neil , Worsley Sarah , Patrick Elaine , Pierce Naomi , Mueller Ulrich , Boomsma Jacobus , Wilkinson Barrie , Hutchings Matthew
Published: April 2019

Antibiotic discovery has stagnated since the end of the 1960s, despite an increasing level of antimicrobial resistance in the clinic. However, recently there has been a renewed interest in searching for novel antibiotics, particularly in under-explored niches. Symbiotic relationships between bacteria and eukaryotes, for example plants and insects, can prove to be a fruitful source of novel secondary metabolites. Many ant species form mutualistic relationships with antibiotic-producing bacteria to protect their colonies against parasitic invasion. However, the antibiotic-producing potential of bacteria associated with different ant species has rarely been explored in detail. We isolated 20 bacteria from the colonies of ant species in the genera Acromrymex, Cyphomyrmex and Mycocepurus, which are all fungus-farming ants, as well as Allomerus and Tetraponera genera which predate on other insects. High quality genome sequences were generated for each bacterial strain using PacBio sequencing. AntiSMASH analysis showed that many of the strains had the potential to produce many secondary metabolites, including a modified candicidin-like compound and several other clusters with low percentage homology to known antimicrobial compounds. Under standard lab conditions less than 20 % of strains show bioactivity against bacteria Bacillus subtillis and fungi Candida albicans however their genomes suggest a much higher potential. Pleiotropic methods such as growth media variations and co-culture with other microorganisms are now being used to switch on cryptic clusters in these novel isolates. Genome mining of symbiotic strains could make a valuable contribution to antibiotic discovery since such strains are constantly having to evolve in response to their host.
The effect of soil pH and phosphorus interactions on nitrous oxide emissions and microbial communities involved in nitrogen cycling
Authors: Grau Butinyac Meritxell , O’Flaherty Vincent , Richards Karl , Wall David , Brennan Fiona
Published: April 2019

Excess application of Nitrogen (N) to agricultural soils can lead to environmental pollution. As nitrous oxide (N2O) is a potent greenhouse gas, it is of critical importance to reduce its emission for climate change mitigation. Availability of nitrogen can facilitate denitrification, an anaerobic respiratory pathway carried out by microbial communities in which N2O is an intermediate product. An understanding of soil, climatic and edaphic factors influencing microbial communities and their activity is key to reducing N2O emissions. Soil pH strongly impacts microbial community structure, with a direct effect on NosZ, the pH-sensitive enzyme catalysing N2O reduction. We thus expect that microbial communities in acidic soils have a reduced capacity to mitigate N2O emissions. It is likely that other management factors, like phosphorus application, interact with pH; causing changes to chemical nutrient availability and direct effects on microbial composition. The complexity linking N2O emissions and microbial activity (impacted by soil pH), and the interacting role phosphorus availability plays in this relationship, is not yet understood. In this study, the capacity of microbial communities to denitrify, as well as the functional microbial community impacted by soil pH, were analysed by potential denitrification assays, measuring N2O fluxes and N2O/(N2O+N2) ratios; and qPCR analysis of denitrification genes. This was investigated across two soil types with a pH gradient and range of phosphorus application rates. Understanding the link between the microbial communities and N2O production can be applied in agricultural management to reduce emissions from fields.
Production and characterization of antibodies neutralizing H9N2 avian influenza virus
Authors: Lukosaityte Deimante , Sadeyen Jean-Remy , Iqbal Munir , Digard Paul
Published: April 2019

Monoclonal antibodies (mAbs) that neutralize influenza A virus have been shown to be potent therapeutic reagents if used pre- or post – exposure to the pathogen. Whilst the majority of this research has focused on human antibody therapeutics, there is an urgent need for generation of antibodies able to neutralize avian influenza viruses (AIV). Several mAbs against H9N2 virus have previously been generated using mouse hybridomas. To facilitate development of passive immunization strategies, the variable chains of these antibodies were characterized in the context of isotype and species specific Fc fragments. Replacement of the IgG2 Fc region with an IgG1 Fc region enhanced neutralizing activity for one of the tested antibodies. Additionally, chicken chimeric antibodies were generated which showed comparable neutralization titres to those generated by the original hybridomas. To identify if antibodies could function as a single chain variable fragment antibodies (scFvs) these were produced in insect S2 cells. This confirmed that H9N2 virus can be neutralized by scFvs. However, an example with high HI titres but lost detectable neutralizing activity was found, possibly due to the loss of bivalent interaction between antigen and antibody. Antibodies showing superior neutralization activity in vitro will be subsequently tested for their potency in in vivo infection. We propose that passive immunization can reduce the impact of AIV in poultry by inducing immediate protection and bypassing immunocompromised individuals.
An analysis of the role of statins in reducing the virulence of Candida albicans
Authors: Ajdidi Ahmad , Kavanagh Kevin
Published: April 2019

The yeast Candida albicans has the ability to induce several systematic and superficial diseases in the immunocompromised or immunosuppressed host. Statins are widely used drugs for the control of cholesterol biosynthesis in the body and are therefore used in treatment of hypercholesterolemia. There is increasing evidence for the potential use of statins in preventing and treating fungal infections. The aim of this study was to assess the effect of statins on C. albicans and to characterize the proteomic alterations occurring in C. albicans in response to statin. Pre-growth of C. albicans in the presence of statin lead to lower levels of ergosterol, reduced adherence to buccal epithelial cells and decreased virulence in Galleria mellonella larvae. Cells also showed increased permeability as measured by elevated amino acid and protein leakage. Proteomic analysis of C. albicans exposed to statin revealed differential abundance of proteins related to ergosterol biosynthesis such as squalene monooxygenase (4.52 fold increase), and lanosterol synthase (2.84 fold increase). Proteins involved in oxidative stress response such as small heat shock protein 21 (6.33 fold decrease) and glutathione peroxidase (2.05 fold decrease) and adherence related proteins such as yeast-form wall protein 1 (6.26 fold decrease) and Ecm33p protein (2.06 fold decrease) were also altered in abundance. These results indicate that statins have the ability to reduce the growth of C. albicans and induce an oxidative stress response. Statins may have potential to control fungal infections in vivo if used alone or in combination with conventional antifungal agents.
Detection and quantification of Staphylococcus aureus heterogeneity to identify antibiotic-induced persistence
Authors: Hassan Marwa , Butler Mark , Ranzoni Andrea , Cooper Matthew
Published: April 2019

Persister cells are characterised as being viable but non-culturable, a state that preserves their metabolic energy to survive the environmental stress, which allows for recurrent infections. Detection of persisters is, therefore, not possible with standard culture-dependent methods. Furthermore, the effect of antibiotics on the induction of persisters has not been assessed. This study aimed to identify antibiotic-induced persistence and determine the percentage of heterogeneity. Vancomycin, daptomycin and dalbavancin were assessed by standard MIC methods against selected Staphylococcus aureus strains. Replicates of MIC assays were stained with propidium iodide to quantify live/dead and a reactive oxygen species (ROS) dye to detect and quantify persisters using culture-independent single-cell sorting, independently. A comparative analysis was then performed. Dalbavancin showed the lowest MIC values against tested S. aureus strains followed by daptomycin and vancomycin. Cell sorting of vancomycin-, daptomycin- and dalbavancin-treated S. aureus strains showed a range of 1.9–10.2 %, 17.7–62.9 % and 7.5–77.6 % live cells based on the strain, respectively, in which daptomycin, in particular, was a strong inducer of a persister population. Persisters represented 3.7–16 % of the bacterial population. The culture-independent identification of antibiotic-induced persistence through studying at the single-cell level showed different efficacy of antibiotics than standard MIC. Vancomycin was the most effective antibiotic against tested strains followed by dalbavancin then daptomycin as assessed by cell sorting. Therefore, re-evaluation of standard MIC methods may be required to assess the efficacy of antibiotics. Additionally, the detection of daptomycin-associated persisters may provide an elucidation to the reported rapid resistance development in vivo.
Applying transposon-directed insertion site sequencing to industrially important, solventogenic species Clostridium saccharoperbutylacetonicum
Authors: de Lussy-Kubisa Laurence , Chaudhuri Roy , Fagan Robert
Published: April 2019

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