bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessing acute and chronic Staphylococcus aureus growth and virulence in an ex vivo model of cystic fibrosis lung infection
Authors: Crealock-Ashurst Branagh , Harrison Freya , Sweeney Esther 
Published: July 2020

Staphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable.
There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation.
Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.
Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Authors: De Ste Croix Megan , Neelam Dave , Oldfield Neil , Lucidarme Jay , Turner David , Borrow Ray , Sheppard Samuel , Martinez-Pomares Luisa , Bayliss Christopher 
Published: July 2020

Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates.
We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states.
Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.
What are the implications of genetic variation for the control of Japanese encephalitis virus?
Authors: Bish John , Rose Nicola , Daly Janet 
Published: July 2020

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. Annually it causes between 13,000 and 20,000 deaths mainly in South-East Asia. Vaccination programmes have drastically reduced the number of JEV cases however outbreaks do still occur. Recently G1 has overtaken G3 as the dominant genotype in most endemic countries and as all currently licensed vaccines are based on G3 it is necessary to evaluate the potential of emerging genotypes to break through current control measures.
This project seeks to generate a virus-like-particle (VLP) neutralisation assay in order to investigate the potential for emergent or divergent genotypes of JEV to infect vaccinated individuals. Using this assay, we will evaluate the ability of vaccinee sera to neutralise emergent genotypes as well as to introduce a variety of SNPs that have been shown to enhance virulence and investigate whether any combination of these are able to invalidate vaccine protection.
There is also concern that recombination between wild-type and live attenuated vaccine strains may occur in the vaccine setting which could result in rescue of the virulent potential of the attenuated virus. Evidence of this is being sought in silico using publicly available data and existing techniques.
These data will provide a clearer picture of the nature of JEV in endemic areas and whether the current strategy of vaccination is sufficient to prevent naturally acquired infection in the long term or if other strategies must now be considered.
A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus
Authors: D. Drumm Shauna , Owens Rebecca , Mitchell Jennifer , M. Keane Orla 
Published: July 2020

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins.
Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes.
Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages.
These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.
Investigating the effect of Herpes Simplex Virus 1 Latency-Associated Non-Coding RNAs on the Human Neuronal Transcriptome
Authors: Jacobs Amy , Mate Ryan , Fritzsche Martin , O’Hare Peter , Efstathiou Stacey 
Published: July 2020

Herpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons.
This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia.
Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.
Identification of Rab GTPases involved in restricting Salmonella Typhi growth in mouse macrophages
Authors: Solano-Collado Virtu , Baldassarre Massimiliano , SpanÒ Stefania 
Published: July 2020

Salmonella enterica serovar Typhi (S. Typhi) is a human adapted pathogen and the causative agent of typhoid fever, a life-threatening infection that kills hundred of thousand people every year, being particularly devastating in developing countries. S. Typhi host-restriction is partly due to the Rab32-dependent antimicrobial pathway, which is crucial to prevent the growth of S. Typhi in mouse macrophages. However, the exact mechanisms used by macrophages to kill S. Typhias well as the molecular basis of these mechanisms in the adaptation to the human host are unknown.
In order to identify host genes required to kill S. Typhi, we have performed a targeted short-hairpin RNA (shRNA) screen in primary mouse macrophages, aiming to i) optimize the conditions to perform silencing screenings in primary mouse macrophages and ii) identify novel Rab GTPases involved in S. Typhi host-restriction. For this, pooled shRNAs are used to knockdown gene expression in macrophages using lentiviral-based transduction system. After infection with a fluorescently-labelled S. Typhi strain, macrophages containing different numbers of intracellular bacteria are sorted by flow cytometry and targeted genes identified by next-generation sequencing.
This small-scale screen allowed us to optimize the screening conditions to perform genome-wide screenings in primary macrophages. More importantly, we have identified other Rab GTPases required in mouse macrophages to control S. Typhi survival confirming that this approach can be used to identify genes that macrophages use to control S. Typhi infection and extending our knowledge of the immunity mechanisms controlling the growth of intracellular pathogens.
UV irradiation of HSV-1 virions induces the differential recruitment of host intrinsic and innate immune regulators to infecting viral genomes
Authors: Iliev Victor , Orr Anne , McFarlane Steven , Boutell Chris 
Published: July 2020

Recognition of virus-derived nucleic acids is a key process in the activation of immune defences to viral infection. However, the spatiotemporal recruitment of host immune regulators to infecting viral DNA (vDNA) genomes remains poorly defined. Here we utilize 5-Ethynyl-2’-deoxycytidine (EdC) nucleotide labelling of WT or UV-irradiated HSV-1 genomes in combination with click chemistry and high-resolution confocal microscopy imaging to investigate the recruitment and proximity of key intracellular immune regulators to infecting vDNA during HSV-1 infection.
We report that UV irradiation of HSV-1 virions restricts vDNA decompaction upon delivery to infected cells. UV treatment reduced the frequency in PML-NB recruitment to nuclear genomes relative to non-irradiated vDNA. These data demonstrate an important role of genome decompaction in the recruitment of intrinsic host factors to nuclear infecting vDNA. Additionally, UV-irradiated HSV-1 genomes favoured a premature cytosolic deposition phenotype, due to ineffective association with microtubules as shown by nocodazole treatment. The cytosolic UV irradiated vDNA showed enhanced association with both cGAS and STING. Interestingly, high-resolution Airyscan imaging identified a consistent unreported 3D interaction between both cGAS and STING, the strength of association of which was enhanced in the context of UV infection. This enhanced recruitment correlated with elevated levels of IFN-β transcription relative to non-irradiated vDNA, despite the presence of non-irradiated cytosolic genomes.
Our data demonstrates that UV irradiation of herpesviruses, a well-established procedure for virus inactivation, has multiple effects on genome structure and sub-cellular localisation that differentially influence the spatiotemporal recruitment of intrinsic and innate host immune regulators to infecting genomes.
Toxigenic Corynebacterium ulcerans: re-emergence of a zoonotic infection
Authors: Enefer Alice , Rose Samuel , Litt David , Gower Charlotte , Foster Geoffrey , Groves Natalie , Said Bengü , Russell Katherine , Chand Meera , Amirthalingam Gayatri , Fry Norman 
Published: July 2020

Diphtheria is a potentially life-threatening infection in humans. The three species capable of causing diphtheria are: Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Although UK cases are rare, recently there has been an increase in reported toxigenic C. ulceransassociated with companion animals. Potentially toxigenic corynebacteria are sent to the National Reference Laboratory, Public Health England (PHE), London, UK for species confirmation, determination of presence of the diphtheria toxin gene by real-time PCR, and confirmation of toxin expression by the Elek test. We reviewed submissions of C. ulcerans between January 2006 and November 2019.
Fifty-three isolates of toxigenic C. ulcerans were received, 29 from humans and 24 from animals. The most common animal hosts were dogs (15) and cats (7), but isolates were also received from a horse and a captive rhinoceros. Multi-locus sequence typing (MLST) data were derived from whole genome sequencing. In three human cases, C. ulcerans was isolated from companion animals and in all three, typing data supported an epidemiological link between human and animal hosts. The sequence types (STs) for these linked isolates were ST331 (human and dog, human and cat) and ST551 (human and cat).
Although MLST data are limited, the finding of the same ST (ST331) from canine and feline sources indicates that strains can be carried by both hosts.
Management of diphtheria cases is an important public health issue. Typing data can support epidemiological linkage and identify possible sources, allowing the potential intervention and treatment of animals thus avoiding onward transmission.
Use of probiotic bacteria to selectively modulate Candida albicans virulence in biofilms
Authors: Cummins Thomas , Hughes Megan , Marchesi Julian , Morse Daniel 
Published: July 2020

Candida albicans is an opportunistic fungal pathogen present in the oral cavities of up to two-thirds of people. Despite typically existing as a commensal microorganism, it has pathogenic potential, particularly in older, immunocompromised individuals. A common Candida-associated infection is denture-associated stomatitis (DS), which presents clinically as areas of erythema on the palatal mucosa, and discomfort for the denture-wearer. In vitro, previous work has shown that the expression of C. albicans virulence factors varies according to its interactions with other oral microorganisms.
Mature single- and mixed-species biofilms (with Candida and several strains of common oral bacteria) were grown on poly(methyl methacrylate) (PMMA) coupons, representing dentures. Additionally, to some coupons, individual probiotic strains were added. Total RNA was extracted, reverse transcribed and putative virulence gene expression was determined by RT-qPCR relative to ACT1, a housekeeping gene. Biofilm-infection assays of FADU and TR146 epithelial cell lines were also performed by pre-culturing cells, then adding single- or mixed-species inocula overnight. Quantification of cell damage determined by lactate dehydrogenase assay.
Biofilm co-culture with the addition of certain probiotic strains downregulated C. albicans virulence genes in both short-term and long-term mixed-species biofilms.
With an increasing aged population that is heavily reliant on the use of antibiotics that can negatively affect the microbiota of patients, there is a requirement to look at the benefits of prophylactics, from both an economic and patient well-being viewpoint. The results show the realistic possibility of using probiotics to prevent or restrict development of Candida-associated oral diseases.
The Missing Link: Developing a pipeline for accelerated antibiotic discovery from Streptomyces through linking ‘omics data
Authors: Scobie Darren , Hjorleifsson Grimur , Herron Paul , Rogers Simon , Duncan Katherine 
Published: July 2020

The genus Streptomyces has proven to be a rich reservoir of specialized metabolites, accounting for 80% of all microbially produced antibiotics including chloramphenicol and nystatin from S. venezuelae and S. noursei respectively. However, the discovery of novel microbial chemistry is still greatly needed to combat antimicrobial resistance. Comparative metabolomics, using platforms such as Global Natural Products Social Molecular Networking (GNPS), as well as tools such as antiSMASH and BiGSCAPE have aided the mining of biosynthetic gene clusters (BGC’s) across datasets but comparing the chemistry to the encoding biosynthetic gene clusters is a significant bottleneck.
In this study, ten Streptomyces strains were selected, based on phylogeny and availability of genome sequence. The strains were cultured on 6 types of Actinomycete-specific media to maximise metabolite diversity. Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was used to obtain spectral data from crude metabolite extracts enabling comparative metabolomics analysis via the GNPS platform. As the genome sequences were publicly available, genome mining of BGC’s was achieved using antiSMASH resulting in 260 BGC’s across the ten strains. This revealed 53 gene cluster families when analysed using BiGSCAPE, the largest encoding for 8 metabolites.
In future, both biosynthetic (BGC’s) and chemistry (parent ions) datasets will be computationally linked based on strain presence/absence. The development of standardised datasets that enable cross-‘omics comparison will aid prioritisation of novel antibiotics, especially when combined with bioactivity data.
An image-based screening system for the study of Mycobacterium avium persistence and drug discovery
Authors: Nagalekshmi Karthika , O’Kane Cecilia , Schroeder Gunnar 
Published: July 2020

Mycobacterium avium infects human macrophages causing opportunistic infections. A steady increase of these infections over the past four decades and resistance to common anti-mycobacterial drugs, create an urgent need for new treatments; however, drug discovery is held back by a lack of knowledge about how M. avium replicates or persists in host cells.
We implemented an image-based assay using a fluorescence dilution (FD) system to measure M. avium replication and persistence. M. avium strain 104 carrying a plasmid encoding GFP and TurboFP635 under constitutive and inducible promoters, respectively, is induced prior to infection of THP1 macrophages and the fluorescent signals are tracked over time in the absence of the inducer. Loss of the TurboFP635 signal while GFP signal is maintained, identifies replicating and retention of both signals non-replicating bacteria.
In the absence of inducer, the M. avium 104 FD strain replicated in the macrophages, leading to increasing numbers of GFP-expressing intracellular bacteria and concomitant loss of TurboFP635 signal in >90% of the infected cells after 24 hours. Upon re-induction, these bacteria expressed TurboFP635, suggesting they are metabolically active and alive. We observed the presence of a small non replicating population that persisted over 96 hours pi. We applied our assay to compare the effect of a panel of anti-mycobacterial drugs, revealing different effects on killing, intracellular replication and induction of persisting, non-replicating bacteria, illustrating the power of this system to facilitate the dissection of the biology of persistence and anti-mycobacterial drug discovery in the future.
Investigation of Pseudomonas aeruginosa biofilm formation within washing machines using novel nanosensor technology
Authors: Blunk Birte , Camara Miguel , Williams Paul , Aylott Jonathan , Hardie Kim 
Published: July 2020

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infections. Our investigation is focussed on a current and growing concern: the formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures (30°C to 40°C) rather than at higher temperatures above 60°C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa, one of the predominant bacteria found in washing machines.
Little is known about environmental microniches present in biofilms. Here, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilms. We use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to pH variation. Using time lapse imaging, pH changes were tracked in real time at a single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms. We also look at the isolation and identification of P. aeruginosa from household washing machines. Whole genome analysis was performed to identify different genomic features relevant to antimicrobial resistance (AMR) and biofilm formation. Furthermore, testing of different washing detergent formulations revealed a range of abilities to disrupt biofilm formation or kill P. aeruginosa, which will facilitate the development of more effective washing agents to limit the emergence of AMR within biofilms.