bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogenotrophic methanogenesis dominates at high pH
Authors: Wormald Richard , Humphreys Paul
Published: April 2019

One potential design for a geological disposal facility (GDF) for intermediate level radioactive waste (ILW) involves the use of a cement base grout which will establish a highly alkaline environment for extended time periods [1]. Methane generation by colonising microbes could impact the long-term performance of the facility by influencing gas pressures and potentially leading to the migration of 14C to the biosphere [1]. Sediments acquired from a wide-range of anthropogenic alkaline sites in the UK were used to develop acetoclastic and hydrogenotrophic methanogen enrichment cultures over a broad range of pH values (7.0–12.0). The generation of methane from hydrogen and acetate was assessed to determine the dominant methanogenic pathways. Archaeal community analysis via Illumina MiSeq was employed to describe the populations involved and the acetoclastic inhibitor methyl fluoride was utilised to confirm the lack of acetate-dependent methane generation under alkaline conditions. High pH (pH>9.0) microcosms employing alkaline sediments were dominated by hydrogen-consuming methanogens of the orders Methanobacteriales and Methanomicrobiales, with no acetate consumption detected under these conditions. In contrast, neutral pH microcosms employing control sediments were dominated by acetoclastic methanogens of the order Methanosarcinales and demonstrated high acetate consumption rates. The rate of acetate consumption and proportion of acetoclastic methanogens decreased in a linear fashion as the pH within cultures was increased, however hydrogen consumption rates remained stable up to pH 11.0. The data shown suggests hydrogenotrophic methanogenesis is the dominant methanogenic pathway at high pH which could have important consequences on gas pressures within a GDF.
Investigating the role of nitric oxide in plant root colonisation by Streptomyces spp.
Authors: Newitt Jake , Munnoch John , Worsley Sarah , Hutchings Matt
Published: April 2019

Streptomyces is a genus of soil dwelling actinomycetes that play an important role in plant health through association with plant roots. They provide an array of benefits to the plant host such as infectious disease prevention and plant growth promotion. We investigate the role of nitric oxide (NO), a ubiquitous signalling molecule used by plants and bacteria alike, in root colonisation by Streptomyces coelicolor. Plating studies were conducted for Arabidopsis thaliana and Triticum aestivum. Relative colonisation was determined by comparing selective recovery of marked mutant strains, alongside a marked control. The effect of increased endogenous NO was interrogated with a deletion mutant of nsrR-hmpA – genes responsible for NO detoxification in Streptomyces. Strains were also engineered to express recombinant NO synthase genes, to investigate the impact of NO production by the bacteria. We show that S. coelicolor ΔnsrR-hmpA is significantly more competent at colonising T. aestivum rhizosphere compared to the control (P<0.005). Endosphere colonisation is sporadic for both mutant and control, this observation is supported by fCLSM imaging. Preliminary data indicates that the Streptomyces strains engineered to express NO synthase at high levels, colonise poorly. This suggests that NO is a dynamic and finely tuned signalling molecule. We are excited to present promising new evidence to support an as yet undescribed link between NO and plant root colonisation by Streptomyces coelicolor. Understanding the mechanisms that underpin this process is the first step in exploiting these interactions for agricultural technology.
Surfactants from the sea: rhamnolipid production by marine bacteria
Authors: Twigg Matthew , Tripathi Lakshmi , Zompra Katerina , Salek Karina , Irorere Victor , Gutierrez Tony , Spyroulias Georgios , Marchant Roger , Banat Ibrahim
Published: April 2019

Biosurfactants produced from microbial sources are increasingly viewed by industry as more sustainable and less toxic alternatives to their chemically derived counterparts. One major class of biosurfactant that has the potential for commercial exploitation are the rhamnolipids. Rhamnolipids are composed of one or two rhamnose monosaccharides covalently bonded to fatty acid chains of varying molecular weights. The major microbial producer of rhamnolipid is Pseudomonas aeruginosa, however as this is a known human pathogen many industries are reluctant to utilise rhamnolipids synthesised by this bacterium. In order to avoid this problem a consortium of both academic and industrial partners have been screening marine bacteria for their ability to synthesis biosurfactants in a project called MARISURF. Here we report our findings of rhamnolipid production by two marine bacterial strains. Rhamnolipid production by these strains was first identified via the phenotypic screening of surface tension reduction. Rhamnolipid synthesis was then confirmed and characterised via HPLC-MS and NMR. Both 16S rDNA and subsequent genomic sequencing revealed these strains to be Marinobacter sp. and Pseudomonas mendocina, both species where rhamnolipid production was previously un-reported. Finally, both strains were assessed for potential pathogenicity using the Galleria mellonella model. Importantly for commercial exploitation, neither strain was shown to be harmful to G. mellonella over a 72 h infection period. Confirmed identification of rhamnolipid production in bacterial strains isolated from the marine environment highlights global oceans as an untapped resource in the ocean for the discovery of novel sources of biosurfactants.
Biofertilizer/biopesticide potentiality of zinc solubilizing Pseudomonas aeruginosa FA-9 and Enterobacter sp. FA-11 isolated from the wheat rhizosphere grown in arid zone
Authors: Ullah Muhammad Abaid , Yusuf Fauzia
Published: April 2019

Bacterial strains were obtained from wheat rhizosphere and screened for zinc solubilization on agar plates. Two strains FA-9 and FA-11 were found efficient for Zn solubilizing activity and were identified as Pseudomonas aeruginosa and Enterobacter sp. by 16S rRNA and gyrase (gyrB) genes analysis, respectively. The strain FA-9 produced a clear zone diameter of 63 mm, 60 mm and 51 mm on agar plate amended with different zinc ores. The strain FA-11 produced a zone diameter of 17 mm with zinc carbonate and 20 mm with zinc oxide while no zone was observed with zinc phosphate. Both strains showed no visible activity with ZnS ore. Similarly, FA-9 and FA-11 increased maximum soluble zinc content (102 µg ml−1 and 45 µg ml−1) from zinc carbonate ore as compared to zinc oxide ore (102 µg ml−1 and 45 µg ml−1) in liquid broth. It was noted that both strains exhibited less potential (7 µg ml−1 and 0.57 µg ml−1) to solubilize ZnS ore in liquid broth. A comparison between agar plate assay and liquid broth quantification shows that agar plate assay does not present the solubilizing potential of ZSB precisely. The strains FA-9 and FA-11 produced auxin with l-tryptophan (3.25 µg ml−1 and 2.86 µg ml−1) and without l-tryptophan (1.23 µg ml−1 and 1.02 µg ml−1). Both strains expressed exo-polysaccharides (EPS) and siderophores activity along with phosphate (P) solubilization, ACC deaminase and antifungal activities. The ACC deaminase and N-fixation activity was confirmed by the amplification of acdS and nifH genes respectively.
Occurrence of extended-spectrum β-lactamase (ESBL)/AmpC producing bacteria in wastewater treatment plant effluent
Authors: Thuy Do Thi , O’Flaherty Aidan , Walsh Fiona
Published: April 2019

Extended-spectrum β-lactamase (ESBL)/AmpC producing bacteria are one of the critical priority resistant bacteria that contributes to treatment failure and increased death rates. In this work we aimed to study the role of wastewater treatment plants (WWTPs) as reservoirs of ESBL/AmpC producing faecal coliforms. The effluent samples were collected from two WWTPs and faecal coliforms were isolated from all samples using the membrane filtration method. Bacterial isolates were subjected to antimicrobial susceptibility testing toward cefotaxime and ceftazidime. The isolates that showed a resistance phenotype to these antibiotics were considered as putative ESBL/AmpC producing bacteria. These bacteria were subjected to the AmpC test using a protocol with phenylboronic acid. The AmpC negative strains in the AmpC test served as samples for multiplex PCR containing primers specific for blaTEM, blaSHV and blaCTX-M. In total, 498 faecal coliforms were isolated from WWTP effluent samples. For the antibiotic susceptibility testing 99 isolates were considered as ESBL/AmpC producing bacteria. Among them, 26 isolates were found to be positive in the AmpC test. The PCR results revealed that 49 isolates carried blaTEM, 6 bla SHV12, 1 blaCTX-M1 and 5 blaCTX-M15. The ESBL/AmpC producing faecal coliforms in WWTP effluent are discharged to the receiving water environment. These data need to be considered when analysing the risk of WWTP effluent to the environment and to human health, as many of the bacteria identified are not analysed in assessment of risk of pollution from WWTPs globally.
Extraction and identification of components of the biofilm matrix in Pseudomonas species biofilms
Authors: Quinn Laura , Barros Caio , Vitale Stefania , Casey Eoin
Published: April 2019

The surface associated communities of microorganism in biofilms are encased in a matrix of extracellular polymeric substances (EPS). The EPS is made up of mainly polysaccharides, proteins, nucleic acids and lipids and plays an important role in maintaining the integrity of the biofilm (1). Although the general composition of the EPS is known, it can be highly variable among strains and among different growth conditions for the same strain. Due to the large variety of biopolymers in nature and the difficulty in their analysis, EPS has been called ‘the dark matter of biofilms’ (2). In order to develop a comprehensive understanding of the matrix of the biofilm, EPS was extracted from four Pseudomonas spp., mCherry-expressing Pseudomonas fluorescens, GFP-expressing Pseudomonas putida and the wild types of Pseudomonas fluorescens and Pseudomonas putida. The extractions were carried out on biofilms grown on glass slides using the cation exchange resin (CER) method. Colorimetric methods were used to quantify the sugars and proteins present in the EPS. These colorimetric assays showed that there was a larger amount of proteins present compared to sugars. The proteins present in all four biofilms of Pseudomonas spp. were identified by LC-MS/MS while NMR and HPLC were used to identify the sugars present. The knowledge gained by these results have the potential to aid in the development of biofilm eradication methods through the targeting of specific components of the EPS. 1. Starkey, M., et al. (2004), American Society of Microbiology: 174–191.2. Flemming, H.-C. and J. Wingender (2010). Nature Reviews Microbiology 8 : 623.
Investigating the role of the bacterial mechanosensitive channel YnaI in Salmonella pathogenesis
Authors: Asogwa Mimi , Miller Samantha , Spano Stefania , Stevens Mark
Published: April 2019

Mechanosensitive channels are required for bacteria cells to survive hypoosmotic shock (transition from high to low osmolarity environment). YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. Previous studies have suggested that S. Typhimurium YnaI may be implicated in host colonization during infection of farmed animals. Disruption of ynaI impaired intestinal colonization in pigs, cattle and chicken. To investigate S. Typhimurium YnaI structure and function, the S.Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the S. Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. To further understand the role of YnaI in S. Typhimurium pathogenesis, S. Typhimurium ynaI was deleted and the ability of mutant to survive and replicate in host cells was investigated. The S. Typhimurium YnaI channel has unique characteristics when expressed in E. coli: S. Typhimurium YnaI channel conferred almost complete protection against 0.3 M NaCl hypoosmotic shock when overexpressed, but interestingly, high level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media. Deletion of ynaI from S. Typhimurium led to increased internalization in macrophages and epithelial cells. Data derived from this study reveals novel characteristics of S. Typhimurium YnaI which may provide insights into other functions of the S. Typhimurium YnaI channel.
Using a metabolic model of Acetobacterium woodii for insights into its utility for biotechnological purposes
Authors: Mesfin Noah , Fell David , Mesfin Noah
Published: April 2019

Acetogens are microbes which produce acetate as a fermentation by-product. They have diverse phylogeny but a metabolic feature in common called the Woods-Ljungdahl Pathway (WLP), which confers the ability to fix carbon dioxide via a non-photosynthetic route. Electrons for this process are derived from diverse substrates including molecular hydrogen and carbon monoxide. The ability of acetogens to utilise components of syngas (H2, CO, CO2) make them an attractive target for metabolic engineering for industrially relevant products. We have previously reported the construction of a genome-scale metabolic model of the model acetogen Acetobacterium woodii using a sequenced and annotated genome of strain DSM1030. The model consists of 836 metabolites, 909 reactions and 84 transporters and can account for growth on diverse substrates reported in the literature. We identified the reactions used to catabolise fifteen single substrates and 121 substrate pair combinations, and used this to construct a sub-model representing a core set of energy producing catabolic pathways. We then introduced heterologous reactions to allow for the production of chemical of interest. Elementary modes analysis of this extended sub-model was applied to further decompose it into unique sets of the smallest functioning sub-networks. With CO2 and H2 as substrates, we find routes for the production of several chemicals where small amounts of excess ATP are produced simultaneously. Repeated analysis with alternative renewable feedstocks such as methanol and formate, indicate a wider potential in producing compounds of interest while also maintaining energy generation and co-factor conservation.
Evolutionary strategies of Bdellovibrio bacteriovorus predators and prey
Authors: Summers Kim , Kreft Jan-Ulrich
Published: April 2019

Antibiotic resistance is a serious threat to human health and new treatments for bacterial infections are urgently needed. Bacteriophages, first used at the beginning of the 20th century, and the predatory bacterium Bdellovibrio bacteriovorus (discovered in 1962) are potential alternatives to antibiotics. We developed a mathematical predator prey model to explore the effects of Bdellovibrio and bacteriophage on prey bacterial numbers. Our system has an abiotic resource that is consumed by the E. coli prey following Monod kinetics and up to two predator species with Holling type I or type II functional responses. As Bdellovibrio spends considerable time in the periplasm of its prey as a ‘bdelloplast’, this stage is also modelled, giving a delay between prey removal and ‘birth’ of predators. We used the model to examine the effects of Bdellovibrio and a bacteriophage on prey populations and found a distinct difference in effectiveness between Bdellovibrio and bacteriophages. We also looked at how various biological factors change predation effeciency. We found that there is an optimal predator:prey ratio for the predator. We also discovered that there is an optimal attack rate and an optimal mortality for the predator.
A novel deep-sea sponge bacterium producing two promising antimicrobial candidates
Authors: Aldarhami Abdu , Felek Arif , Farnham Garry , Desriac Florie , Howell Kerry , Upton Mathew
Published: April 2019

Background

Natural product screening methods are arguably the most efficient way to identify novel antibiotics. Exploiting obscure, hard to reach environments, implementing the latest high-throughput next generation sequencing techniques, performing in silico analysis and synthesis/recombinant expression of promising candidates may increase the discovery of unique agents.

Materials/methods

Deep-sea sponges were collected (∼1000 m depth) in the North Atlantic Ocean and bacteria were recovered. One strain (EU4) was selected for detailed analysis. Strain EU4 produced an inhibitor into liquid media. This compound was purified using liquid chromatography andmatrix-assisted laser desorption/ionization (MALDI) analysis was performed. In parallel, the draft genome was obtained and analysed using BAGEL-3 and anti SMASH-4.0mining tools. Successful synthetic production was obtained for a candidate identified using antiSMASH.

Results

Analysis ofthedraft genome (5.8Mbp) indicates that strain EU4is a novel member of the Bacillaceae. To date, the produced compound showed activity towards Micrococcus luteus only, while the synthetic compound displays a broad spectrum of activity towards Gram positive and negative bacteria. In addition, based on MALDI analysis, the synthetic and the naturally produced compounds possess different molecular weights, being approximately 4 kDa and 1.7 kDa, respectively.

Conclusions

Bacteria recovered from deep-sea sponges couldpotentially be a rich source for novel compounds. In silico analysis of producer genomes has provided a means of identifying cryptic compounds, not produced in culture. Further study of both compounds, which showed diverse activity spectra, may lead to promising new candidates for development into clinically relevant therapeutics.
Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays and ELISA
Authors: Mayora-Neto Martin , Bentley Emma , Wright Edward , Temperton Nigel , Ploemen Ivo , Soema Peter , ten Have Rimko , Masterson Stuart , Michaelis Martin , Wass Mark , Scott Simon
Published: April 2019

The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck hindering research efforts. A safe alternative to working with native viruses are pseudotyped viruses (PV) which are non-replicating particles bearing surface glycoprotein(s) that can be used for antibody detection. The aim of this study was to create a diagnostic tool to distinguish between genera and species of pathogenic filoviruses (e.g. neutralization tests and ELISA), avoiding the cross reactivity currently seen. High titre PVs bearing the receptor glycoprotein (GP) of different filovirus species, plus specific epitope chimeras, were successfully generated. Next, lyophilisation studies to assess particle stability/degradation transportation and long-term storage were conducted. Filoviruses maintained their titres for at least 1.5 years after lyophilisation when kept in temperatures of up to 4 °C, with all filovirus genera following a similar trend. At higher temperatures, PVs degraded to unworkable titres. Reconstituted PVs also performed well in neutralisation assays. A chimeric cuevavirus GP bearing ebolavirus (Zaire sp.) epitopes KZ52 and 1 H3 retained infectivity, with average titres of approximately 1×10 7 RLU ml−1, similar to wild type, indicating its structure was not compromised. These chimeras are now being assessed in neutralisation tests using specific monoclonal antibodies and incorporated into ELISA with PVs as antigens. The data suggests lyophilised PVs are amenable to long-term storage, and their GPs can be modified to create artificial antigens for diagnostics and serosurveillance.
Cross-sectional study of respiratory Aspergillus spp. colonization or infection in patients with various stages of chronic obstructive pulmonary disease (COPD) using culture vs non-culture based technique
Authors: Waqas Sarmad , Dunne Katie , Talento Alida Fe , Wilson Graham , Martin-Loeches Ignacio , Keane Joseph , Rogers Thomas R
Published: April 2019

Background

COPD patients are now recognized to be at increased risk of colonization by Aspergillus spp. which may progress to invasive pulmonary aspergillosis (IA). Published data on the frequency of Aspergillus detection in COPD are limited.

Methods

A cross-sectional study was undertaken to determine Aspergillus colonization or infection in COPD patients undergoing bronchoscopy for any indication. Culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL).

Results

One hundred and fifty patients were included (44.7 % female, mean age 68.2 years). 21.3 % were inpatients, 74.7 % outpatients and 4 % were ICU patients. Investigation of lung masses was the most common indication (43.3 %) for bronchoscopy. Most patients (81.3 %) were either GOLD stage 1 or 2 COPD. Cancer was the most frequent co-morbidity (60.48 %). 12 % and 48.7 % were on systemic and inhaled steroids respectively. Lung mass was the most common (28.43 %) CT imaging finding. Seventeen patients (11.3 %) had a positive result for Aspergillus (Culture+Galactomannan+PCR). 76.4 % out of these seventeen were in the early stages (GOLD stage 1 or 2) of COPD.

Conclusion

Aspergillus sp. was detected in 3.3 % of patients by culture, which increased to 11.3 % if culture was combined with either a positive GM or PCR result. Overall the frequency of Aspergillus detection in this population of COPD patients was low which may reflect the predominance of Gold stages 1 and 2 among the study population.
Combating and detecting Histomonas meleagridis, the causative agent of black head disease, through the administration of novel antimicrobial peptides derived from microbiomes
Authors: Pickup James , Oyama Linda , Nixey Cliff , Huws Sharon
Published: April 2019

Histomonas meleagridis is a protozoan parasite that causes mortality and morbidity in a wide range of gallinaceous fowl. It most notably affects turkeys, causing 80–100 % mortality in a flock. There is no commercial treatment for this parasite at the moment and attempts at a vaccine have failed. In this study we investigated the potential efficacy of novel rumen and chicken gastrointestinal tract microbiome-derived antimicrobial peptides (AMPs) against H.meleagridis. H. meleagridis was cultivated in growth flasks in Dwyer media. The concentration of the parasite was determined using a Haemocytometer, the cells were counted under the microscope. The AMP challenges were carried out in 96-well plates. The starting concentration of the AMPs were 1024 µg ml−1 down to 2 µg ml−1. The cell densities of H. meleagridis were checked at 24 h. The protozoal cell densities were checked by extracting DNA and performing qPCRs targeted against FeHyD and Rpb1 genes. Fluorescent microscopy was also used to check parasite densities as described above. Five Chicken and three Rumen AMPs resulted in decreases in protozoal cell densities following microscopy, suggesting that the peptides show potential therapeutic application for blackhead disease. Nonetheless, the qPCR primers were non-specific and resulted in bacterial DNA amplification. Consequently, in order to understand the biological function of H. meleagridis and design new qPCR primers allowing quantification, we isolated the macronuclei of a H. meleagridis strain followed by sequencing using the illumine Hi-Seq 2500 and paired ends. Future work will focus on confirming how well the AMPs work agent H. meleagridis by employing electron microscopy to observe how the AMP affect the cells.
Novel insights into human cytomegalovirus gene function from a multiplexed proteomic screen of multiple block deletion viruses
Authors: Nightingale Katie , Fielding Ceri , Davies Colin , Wang Eddie , Antrobus Robin , Davison Andrew , Wilkinson Gavin , Stanton Richard , Tomasec Peter , Weekes Michael
Published: April 2019

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.
Oligonucleotide transcription factor decoys as tools to control bacterial transcription
Authors: Laurence Louis , McArthur Michael , Wong Nichola
Published: April 2019

Transcription Factor Decoys (TFDs) are short synthetic oligonucleotides that contain the binding site for specific bacterial transcription factors. When translocated into bacterial cytoplasm they can rapidly kill cells when targeted against essential bacterial pathways. Translocation is currently achieved by combination of the TFD with a proprietary lipidic delivery agent, CM2, to form nanoparticles. These interact with highly conserved anionic phospholipids, such as Cardiolipin, to effect delivery to both Gram-positive and Gram-negative bacteria. Simplifying translocation would be an advance that would allow serial screening of large libraries of TFDs to delineate genetic regulatory networks in numerous types of bacteria, including emerging strains. To achieve this we have combined key chemical moieties of the CM2 delivery molecule to the oligonucleotide conjugate by Click chemistry and show that these discrete conjugates are capable of translocation. Confocal laser scanning microscopy was used to monitor the uptake of the TFD-conjugates to E. coli and in parallel their effect on the targeted genetic pathways was confirmed with reporter strains and plating under selective conditions. Hence, it was confirmed that these conjugates can be used as tools to efficiently and specifically modify gene expression by inhibition of selected transcription factors.
Characterizing avian pathogenic Escherichia coli from diagnostic cases in Georgia, USA – comparison of gene profiles with tissue of isolation
Authors: Logue Catherine , Newman Darby , Nolan Lisa , Barbieri Nicolle
Published: April 2019

Colibacillosis caused by Avian Pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality and carcass condemnation to the poultry industry worldwide resulting in significant economic losses. We assessed a multiplex PCR for classifying diagnostic APEC and characterized these isolates using gene profile analysis.48 E. coli isolates collected between August and October 2018 through the Poultry Disease Research Center (PDRC) Diagnostic Laboratory were analyzed. Isolates were screened using multiplex PCR targeting genes associated with APEC chromosomal and plasmid virulence. Isolates were assessed for relationship between gene profile and host tissue of origin. Overall, isolates met the criteria for definition as well-developed pathogens with more than 90 % of isolates positive for the genes iroN, ompTp and hlyF; 78 % were positive for aerJ and 67 % for iss. A significantly lower prevalence was observed for cvaC, etsB, ireA and papC (range 5–36 %). When overall gene prevalence was examined for tissue of isolation, we found that APEC from the ovary, bone marrow, pericardium and lung had higher average numbers of genes compared to isolates recovered from skin and yolk sac. Genes associated with the ColV virulence plasmid (iss, iroN, hlyF and ompTp) were detected in 43 of 48 isolates (89.5%) further confirming the ColV plasmid is the defining trait of the APEC subpathotype. The use of a multiplex panel to screen for APEC has shown good correlation with pathogenesis, and tissue source and correlates well with invasive strains. Path panel diagnostics is available through PDRC, providing significant value to APEC screening.
Use of Nano- SIMS at the single cell-level to evaluate drug penetration into mycobacterial biofilms
Authors: Akwani Winifred , Hingley-Wilson Suzie , Chambers Mark , Rakowska Paulina , McMahon Greg
Published: April 2019

Non-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause high mortality and morbidity amongst patients who are immunocompromised or have chronic lung diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate and are associated with the development of increased antimicrobial resistance (AMR). Treatment for M. abscessus and chimaera infections often requires 2–3 antibiotics over 2 years. The question we want to address is whether the increased AMR and treatment time in NTM biofilm formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms (M. abscessus and M. chimaera). Nano-SIMS maps the relative abundance of different ions down to the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce treatment duration and AMR development.
Deciphering adaptation through mobile element pangenome composition
Authors: Waters Nicholas , Holmees Ashleigh , Abram Florence , Pritchard Leighton , Brennan Fiona
Published: April 2019

Pangenome analysis can help reveal functional and physiological differences between organisms by linking genes present in their genomes with phenotypic characteristics. While this works well for closely-related organisms and large numbers of isolates, analysis of species with a large pangenome may be problematic as optimal sample size increases with intraspecies diversity. Organisms such as E. coli exhibit high intraspecies diversity, resulting in an exceptionally large pangenome, pushing the limits of what constitutes a species. However, the phylogeny of the mobile elements can differ strongly from the phylogeny of the rest of the genome, reflecting how rapidly these elements can be gained or lost. We suggest when studying adaptations on a short genetic time-scale (ie, within species or strain), the mobile regions of the genome may be more informative than the more stable parts of the genome. To this end, we developed a pipeline, Horizontally Acquired Partial Pangenome of Inserted Elements (or ‘happie’), which allows researchers to study the mobile pangenome. Thus, happie could be used as a proxy to detect genes associated with a particular trait amidst the noise of a diverse genetic background. We aim to use happie to compare a panel of known soil-persistent E. coli isolates and a curated panel of non-soil-persistent isolates. This will allow us to determine whether specific mobile elements (or genes carried on those elements) are associated with soil persistence, which would indicate that the cost of harbouring the mobile element was less than the advantage conferred.
Zika virus: out of sight but not out of the mind. Viral persistence and host responses within the central and peripheral nervous systems
Authors: Ferguson Debbie , Hall Jo , Ham Claire , Jenkins Adrian , Giles Elaine , Kempster Sarah , Berry Neil , Almond Neil
Published: April 2019

Following the rapid spread of Zika virus infection through the Caribbean and Americas, model systems of Zika virus infection have been established to determine disease pathology and potential health impacts for those infected with this neurotropic flavivirus. NIBSC has established both Old World (rhesus and cynomolgus macaques) and New World (red-bellied tamarins) non-human primate disease models of human Zika virus infection. Animals were infected sub-cutaneously with the Caribbean Zika isolate PRVABC59 and pairs terminated during primary viremia (3 dpc) or post clearance of plasma viremia (42 and 101 dpc). FFPE sections from multiple tissues, including brain and peripheral nerves were analysed for Zika virus RNA (RNAscope), viral proteins and host responses (immunohistochemistry). All animals became infected and rapidly cleared high levels of peripheral viremia. Zika virus RNA was detected in multiple tissues from 3 dpc with low levels of viral RNA and viral NS1 protein remaining detectable through to 101 dpc. Viral RNA was associated with both glial and neuronal cells throughout the brain and schwann cell nuclei in peripheral nerves. Neuroinflammation (astrogliosis, microgliosis, peri-vascular cuffing) and increased levels of CD3+, CD8+ or CD45+ cells were present from 3 dpc through to 101 dpc. Disruption of MAP2 neuronal dendrite staining and peripheral nerve myelin basic protein staining was also observed. The continued detection of Zika virus within tissues 3 months post infection and in the absence of a detectable peripheral viremia raises questions regarding potential long term effects of this virus and associated inflammatory responses within the nervous systems.
Investigation of molecular mechanisms of polymerase I (Pol-l) inhibitor PMR-116 using Isothermal Titration Calorimetry (ITC)
Authors: Panov Konstantin , Alsahafi Sameerh
Published: April 2019

Cancer has been identified as a group of diseases characterized by abnormal cell growth. In eukaryotic cells, the nucleolus is the region of the nucleus that constructs ribosomal subunits. Polymerase 1 transcription site has been used as the marker to particular aggressive tumours seen when the nucleoli increases in size and number. The nucleolar size correlates with the level of rRNA synthesis, which is transcribed by RNA polymerase 1 (RNAP1) during the initial stage of ribosome biogenesis. In recent years the rRNA transcription has emerged as novel target for anti-cancer therapy and specific RNAP1 transcription inhibitor are currently undergoing clinical trials for anti-cancer therapy. Thus, the proposed therapeutic strategies for solid tumour growth or inhibition of cancer cell proliferation is the selective inhibitor RNAP1 transcription. The currently available inhibitors are characterised by a different mechanism and different levels of genotoxicity. Two drugs, (CX-5461 and PMR-116) are small molecules and selective inhibitor of RNAP1 transcription which have moderate effect on transcription by other nucleolar polymerases and protein translation. CX-5461 inhibits transcription by displacing essential promoter recognition factor SL1 thus preventing an initiation of transcription. PMR-116 demonstrated a great potential as RNAP1 inhibitor, is characterized by low cytotoxity and very high anti-cancer effect. However, the exact molecular mechanisms of RNAP1 inhibition is unknown. Therefore, this experiment aims to identify the stage of the transcription cycle affected by PMR-116 by using a combination in vitro and vivo based assays. Also, to determine the drug target into the molecular mechanism of PMR-116 by using biochemical methods including Isothermal Titration Calorimetry (ITC).
The long isoform of ZAP widely restricts Paramyxoviruses
Authors: Thibault Patricia A. , Ryan Andrew P. , Hung Chuan-tien , Daugherty Matthew , Lee Benhur
Published: April 2019

Paramyxoviruses (PVs) are negative-sense RNA viruses that are important in human and animal health, and can cause severe zoonotic diseases. We screened a wide range of animal and human PV matrix proteins for host interactions, and found that the long (L) isoform of the host protein ZAP (zinc-finger antiviral protein) interacted with all the tested viral matrixes. ZAP-L is constitutively expressed, has a prenlyation motif for membrane-localization, and primarily mediates antiviral activity by binding and degrading viral RNAs through the exosome complex. However, ZAP restriction of PVs has not been demonstrated in the literature. We found that knockdown of ZAP-L results in increased replication of a panel of five human and animal PVs. Overexpression of ZAP-L (but not ZAP-short) restricts replication of PVs in a reciprocal pattern – with the notable exception of Sendai virus (SeV) – and as with other viruses, mutating the prenylation motif of ZAP-L abolishes restriction. RT-qPCR of PV RNAs and pulled-down RNAs does not indicate specific targeting of a viral transcript by ZAP-L, although overall genome abundance is reduced. Finally, immunoprecipitation of ZAP shows an additional RNA-independent interaction between ZAP-L and SeV-nucleocapsid not found with HPIV3-nucleocapsid, a closely-related PV. Thus, we have observed that ZAP-L interacts with the matrix of, and restricts replication of, a wide range of paramyxoviruses. A PV that is not restricted (SeV), has an additional interaction with ZAP via its nucleocapsid protein that may ameliorate ZAP restriction. Investigating ZAP-related restriction differences between closely-related PVs may shed light on anti-viral mechanisms of ZAP.
Effect of antibacterial lipids on biofilm formation by Streptococcus mutans
Authors: Harvey Veritia , Brady Damien , Kealey Carmel
Published: April 2019

Streptococcus mutans is the major cariogenic organism associated with Dental Caries, a widespread chronic disease of the oral cavity (Muras et al. 2018). It is associated with oral biofilm formation, production of organic acids, and has the capacity to out-compete non-cariogenic commensal species (Lemos et al. 2013). Recent studies carried out on fatty acids demonstrated effective antimicrobial activity against S. mutans (Hughes, 2014). This study will evaluate the activity of lipids on S. mutans biofilm formation and proposes that dietary constituents may be used as a natural therapy to maintain oral hygiene. Clinical isolate S. mutans 3014 D5929 was exposed to various concentrations of lipid for 24 h. Crystal violet assay was performed for quantification of biofilm biomass. Fluorescent microscopy using SYTO® 9 and Alexa Fluor® 647-labelled dextran conjugate was performed to visualise biofilm formation pre- and post-exposure to MCO. Biofilm biomass was reduced for all lipid concentrations. Fluorescent microscopy indicated a significant reduction in bacterial cell number and a lack of structural biofilm upon exposure to fatty acid mixtures when compared to control. The research demonstrated that lipid does have S. mutans based antimicrobial and antibiofilm capabilities.
Understanding the ecology and evolution of polymicrobial wound infections
Authors: da Silva Ana , Sloan Tim , Williams Paul , Diggle Steve
Published: April 2019

Chronic wounds (CW) are a common complication of diabetic ulcers (DUs), which are a major burden to health care systems worldwide and can result in lower limb amputation due to the intractability of the infection. In DUs there is a high probability of the infecting bacteria evolving considerable phenotypic and genetic diversity, as has previously been shown for chronic lung infections. However, it is not known whether this is also the case for chronic DUs, and whether diversity impacts on virulence and antibiotic resistance. To study this, bacterial populations were isolated from different samples from patients with DUs. Phenotypic diversity was investigated in P. aeruginosa populations through the analysis of phenotypes traditionally associated with pathogenicity, and through a whole genome study. Phenotypic variation in P. aeruginosa isolates taken from different patients was observed, but little variation within the same CW (with exception of one patient with a leg ulcer). Antibiotic resistance was found to increase during the course of infection, and it became apparent that P. aeruginosa colonisation in DUs is via a single strain per ulcer, and potentially per patient, even though some sample-specific phenotypic profiles were found to arise from a homogenous population. For this case, a detailed genomic analysis between bone and blood isolates was done, including a comparison of their transcriptomes using RNAseq. The results suggest that the loss of flagellum facilitated evasion of the innate immune system, which allowed bacteria to go undetected and spread systemically causing the rapid decline in the patient’s health.
Aedes aegypti(Aag2)-derived clonal mosquito cell lines reveal impact of pre-existing persistent co-infection with the insect-specific Bunyavirus Phasi Charoen-like virus on arbovirus replication
Authors: Wallace Louisa , Fredericks Anthony , Bernal-Rubio Dabeiba , Davidson Andrew , Fernandez-Sesma Ana , Maringer Kevin
Published: April 2019

Aedes aegyptiis a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes, however the impact of persistent insect-specific virus infections that more accurately mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. We generated single cell-derived clonal cell lines from the commonly used Ae. aegypticell line Aag2. Two of the fourteen Aag2-derived clonal cell lines studied in detail harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus or the rhabdovirus vesicular stomatitis virus. Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide well-characterised Aag2-derived clonal cell lines as a valuable resource to the arbovirus research community.
Cancer control is a key functionality underlying evolution of extended lifespan in mammals
Authors: Kowalczyk Amanda , Partha Raghavendran , Clark Nathan , Chikina Maria
Published: April 2019

The biological origin of life expectancy remains a fundamental and unanswered scientific question with important ramifications for human health, especially as the bulk of burden of human healthcare shifts from infectious to age-related diseases. The striking variability in life-span among animals occupying similar ecological niches1and the numerous mutations that have been shown to increase lifespan in model organisms2–5point to a considerable genetic contribution. Using mammalian comparative genomics, we correlate lifespan phenotypes with relative evolutionary rates, a measure of evolutionary selective pressure6. Our analysis demonstrates that many genes and pathways are under increased evolutionary constraint in both Long-Lived Large-bodied mammals (3L) and mammals Exceptionally Long-Lived given their size (ELL), suggesting that these genes and pathways contribute to the maintenance of both traits. For 3L species, we find strong evolutionary constraint on multiple pathways involved in controlling carcinogenesis, including cell cycle, apoptosis, and immune pathways. These findings provide additional perspective on the well-known Peto’s Paradox that large animals with large numbers of cells do not get cancer at higher rates than smaller animals with fewer cells7. For the ELL phenotype, our analysis strongly implicates pathways involved in DNA repair, further supporting the importance of DNA repair processes in aging8–12. Moreover, these correlations with lifespan phenotypes are consistent across the entire mammalian phylogeny, suggesting that additional constraint on these pathways is a universal requirement for long lifespan.
Estrogen accelerates heart regeneration by promoting inflammatory responses in zebrafish
Authors: Xu Shisan , Xie Fangjing , Fallah Samane , Babaei Fatemeh , Zhu Lina , Wong Kin Fung , Liang Yimin , Ramalingam Rajkumar , Sun Lei , Wang Xin , Lam Yun Wah , Cheng Shuk Han
Published: April 2019

Sexual differences are observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we report that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and suppressed by estrogen-antagonist tamoxifen. Injuries to the heart, but not other tissues, increased plasma estrogen level and expression of estrogen receptors, especiallyesr2a, in zebrafish hearts. The resulting endocrine disruption induces the expression of female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered more pronounced immune and inflammatory responses in females. These responses, previously shown to enhance heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in female. Furthermore, a brief exposure to estrogen could precondition zebrafish for an accelerated heart regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to heart injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provides a new model system for the study of sexual differences in human cardiac repair.