bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uncovering the molecular mechanisms of the Pseudomonas aeruginosa PA14 response to myxobacterial predation
Authors: Sydney Natashia , Whitworth David , Tucker Nicholas 
Published: July 2020

Myxobacteria are Gram-negative bacteria, notable for their predatory and antimicrobial activities, which dictate the outcomes of their interactions with neighbouring organisms. They are abundant and widespread in nature, and can significantly affect the microbiome of an environment.
We hypothesise that there are underlying molecular mechanisms in prey specieswhich govern the prey’s susceptibility/resistance to the antimicrobial activity of myxobacteria. In this work we attempt to define the mechanisms by which Pseudomonas aeruginosa PA14 resists predation bythe model myxobacterium Myxococcus xanthus.

Pseudomonas aeruginosa is an opportunistic pathogen of humans and plants. With the rise in antibiotic resistant organisms, Pseudomonas spp. are categorised as World Health Organisation priority 1 antibiotic-resistant bacteria and are our prey of choice in this study.
In collaboration with Dr N. Tucker (Strathclyde), and using a strain of M. xanthus expressing mCherry (courtesy of E. Hoiczyk, Sheffield), we developed 96-well plate assays of predation which measured the optical density of both predator and prey and the florescence of predator at different point intervals. Predation was assayed againsta library of approximately 5700 PA14 mutants to identify strains with increased/decreased susceptibility to predation. Responses of PA14 mutants varied with time and between mutants, allowing us to create a shortlist of candidate genes involved in the prey response to predation.
We are currently performing a preliminary analysis of the data using the Integrated Genomic Viewer and Circos plots to assess the genomic organisation of the prey genes that influence susceptibility and resistance to predation.
Use of a laboratory model hospital sink system to investigate fluctuation of Gram-negative bacteria in sink waste traps
Authors: Ferguson Harry , Aranega Bou Paz , Moore Ginny 
Published: July 2020

Hospital sinks in the UK have recently been under scrutiny as possible reservoirs for Gram-negative bacteria, especially carbapenem resistant Enterobacterales (CRE). These strains have been found in intensive care wards across the country and can re-enter the clinical environment, representing a risk to vulnerable patients.
Two sink waste traps known to be colonized with CRE were collected from a hospital and fitted to a vertical-draining and rear-draining handwash sink installed within a laboratory model sink system. Sinks were automatically flushed four times a day and, as per usual in the model, TSB was provided once daily to maintain microbial populations. Gram-negative bacteria were regularly monitored using selective culture, MALDI-TOF and antibiotic disk diffusion. The short-term effect of adding simulated IV fluids (5% glucose or 0.9% NaCl) and the impact of sink design on Gram-negative proliferation were investigated.
Communities included Enterobacter asburiae; Klebsiella oxytoca; Pseudomonas aeruginosa and Citrobacter freundii, among others, including CRE. The addition of simulated IV fluids did not induce Gram-negative bacterial proliferation in the time frame of the experiment. Differences were observed in the fluctuation of Gram-negative levels after flushing between the different sink designs. Gram-negative numbers in vertical-draining sinks decreased immediately after the tap was flushed and subsequently increased between flushes. However, in rear-draining sinks, little fluctuation was observed.
Hospital sink waste traps can harbour Gram-negative bacteria resistant to antibiotics. In our experimental conditions, the type of sink was the determining factor in the magnitude of fluctuation in Gram-negative populations while simulated IV fluids had little effect.
DAAs treatment is associated with up-regulation of CD81 on peripheral B-lymphocytes
Authors: Hassuna Noha A , Abdel-Malek Mohamed Ahmed Yehya , Nasif Mohamed Khalid Ali , Medhat Mohamed , Partridge Lynda J 
Published: July 2020

Cellular surface expression of CD81 (an essential co-receptor for HCV) is critical for successful HCV infection. Furthermore, CD81 cross-linking with HCV-E2 protein impedes activation signaling pathways in different lymphocytes (T-cells, B -cells and NK cells). The expression of CD81 on peripheral lymphocytes is known to be downregulated following successful dual anti-HCV therapy. On the other hands, no reports are yet available regarding its expression levels following the newly used treatment regimen in Egypt; direct-acting antivirals (DAAs): Sofosubvir & daclatsvir for three months. Thus, the aim of the current study was to evaluate the expression levels of CD81 on T and B lymphocytes in HCV-infected patients before and after successful treatment with DAAs. Cellular CD81 expression was measured on CD3+ (T lymphocytes) and CD19+ (B Lymphocytes) lymphocytes by flow cytometry from 19 patients with chronic HCV infection.
All the HCV viruses were of genotype 4. We found no correlation between CD81 expression on either CD3+ or CD19+ lymphocytes and viral load. The expression of CD81 on CD19+ lymphocytes was markedly reduced at the end of the treatment. On the contrary, CD81 was significantly increased on CD3+ lymphocytes following successful treatment.
Our data indicate that successful treatment of HCV infection is associated with a reduction in surface CD81 expression on B lymphocytes with a concomitant increase on T lymphocytes.
How do temperate bacteriophages affect the fitness of Pseudomonas aeruginosa?
Authors: Plahe Grace , E. Alison Heather , Goodhead Ian , James Chloe 
Published: July 2020

The Liverpool Epidemic Strain(LES) of Pseudomonas aeruginosa is a key opportunistic bacterium and a major cause of mortality and morbidity in cystic fibrosis (CF) patients. It has been established to carry distinctive prophages within its genome, providing the host bacterium with advantages through horizontal gene transfer.
Several well-known partnerships between prophage and their bacterial hosts have been characterised, however, very little is known about other phage-host systems. This project explores the dynamics between Pseudomonas aeruginosa PAO1 and its phage, determining whether they confer an advantage in the CF lung.
Evidence that Chlamydia abortus vaccine strain 1B produces indistinguishable placental lesions to wild type strains
Authors: Gaston Caspe Sergio , Frew David , Milne Elspeth , Chianini Francesca , McNeilly Tom , Sargison Neil , Longbottom David 
Published: July 2020

Chlamydia abortus is the most commonly diagnosed cause of abortion in small ruminants around the world [1]. Control of chlamydial abortion is achieved in several European countries using an attenuated live 1B C. abortus vaccine strain, which can be distinguished from virulent wild-type strains by PCR-RFLP analysis [2]. Application of this method has provided molecular evidence that the 1B strain can cause abortion in ewes [3, 4]. The objective of this study was to define the distribution of lesions and bacterial load in cotyledons from ewes vaccinated with the 1B strain compared to normal wild-type infections.
A Chlamydia-free flock of 75 multiparous adult ewes were vaccinated twice, two years apart, each prior to mating, with the commercial 1B vaccine. In the second lambing season following the last vaccination, placentae (n=116) were collected and analysed by C. abortus real-time qPCR [3]. Only two of the placentae, both from the same ewe, were found to be positive. Viable organisms were isolated from these placentae and confirmed by RFLP-PCR [3] to be vaccine-type. All cotyledons from these placentae were analysed by histopathology and immunohistochemistry [5], and compared with those from wild-type infected placentae. The lesions in the vaccine-type infected placentae were indistinguishable from the wild-type infected placentae in terms of their severity, load and distribution.
These results suggest that the 1B vaccine strain of C. abortus can cause chlamydial abortion in ewes producing typical placental lesions to wild-type infected animals, and could be circulating with the potential to cause natural infection and disease.
3D co-cultures of epithelial cells with immune cells as a model of HSV and oncolytic HSV infections
Authors: Epifano Ilaria , Conner Joe , McNeish Iain A. , V. Graham Sheila 
Published: July 2020

Virus infectivity is commonly investigated with in vitro monolayer cell cultures or in vivo animal models. Ease of growth and manipulation and low cost characterise standard cell culture. Animal models allow investigation of infectivity in the context of tissue structure and environment but are costly and can be limited by species variations. Neither approach can recapitulate the human context, the tissue microenvironment and human immune components. Three-dimensional organotypic raft tissue models can provide most of the advantages of in vitro and in vivo models.
We successfully established HSV and oncolytic HSV (HSV1716) infection in 3D raft cultures of epithelial non-tumour (HaCaT) and tumour (SiHa, OVCAR3 and TOV21G) cell lines. Our 3D models allowed the evaluation and quantification of virus replication and the recovery of the virus both in culture media and tissues.
We developed a complex 3D co-culture of epithelial cells with human immune cells in order to mimic the tissue microenvironment. This innovation allowed us to study the effect of immune cells in cell killing by HSV1716 in the in vitro tissues. In HSV1716-infected co-culture tissues, immune cells were identified throughout the tissue and some migrated to the areas of infection. The immune activity was identified through increased IL-8 release. Moreover, combining infection with immune cell infiltration increased tumour cell killing in the 3D co-culture model. This new co-culture model could be further developed to identify the role of immune cells in oncolytic viroimmunotherapy and to dissect the involvement of specific single immune cell subpopulations.
Artificial Intelligence and deep learning turns to antibiotic discovery
Authors: Crossman Lisa 
Published: July 2020

Deep learning (DL) is a subset of Artificial Intelligence employing neural networks that require the use of a training set and are modelled on circuit pathways in the human brain. Whilst AI is a burgeoning field today, its roots are in the 1950s. DL algorithms use multiple layers to progressively extract higher level features from the raw input. Many different architectures of neural network exist, and for the most part are involved in applications with image recognition. Some recent uses of DL include self-driving cars; there are also creative projects using DL to create fake faces or human poses for use as models, composing music, creating novel art from different styles of art and writing fake news.

Streptomyces spp. are well-known as important producers of bioactive compounds such as antibiotics. These bioactive compounds are often encoded as secondary metabolites in the organisms by large gene clusters such as non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). The NRPS and PKS assemble peptides using enzymatic units arranged in modules that can function in an iterative or sequential fashion independent of messenger RNA. Each NRPS or PKS is capable of assembling one type of peptide. Fusions of the two also exist.
Here we have trained deep learning neural networks to provide us with simulated “fake” secondary metabolite sequences. We examine the characteristics of these sequences and how they could be used to guide us with antibiotic discovery.
Assessing the ability of Tigecycline and Meropenem to clear intra-macrophage Klebsiella pneumoniae
Authors: Asim Kinza , J. Wanford Joseph , Oggioni Marco 
Published: July 2020

The emergence of hypervirulent Klebsiella pneumoniae of serotype K1 and K2 are a major cause of life-threatening, community-acquired infections. Recent studies demonstrated that Kp can persist for long periods of time within the spleen and liver and survive within macrophages. We aimed to explore whether two clinically relevant antimicrobials differed in their capacity to clear within-macrophage Kp.
The mouse monocyte cell line J774a were used to model Kp macrophage infection (cultured in RPMI, 10% Fetal-bovine-serum, 37oC, 5%CO2). Cells were harvested and seeded in a 96-well plate at 2x106 cells/mL and incubated overnight. The following day, cells were infected with hypervirulent, K1 Kp (NTUH-K2044) for one hour at an MOI of 10. A 1-hour treatment with gentamicin and polymyxin-Bwas used to kill extracellular Kp. Cells were then washed, and incubated with serial 2-fold dilutions of meropenem and tigecycline for 6 hours. In parallel, a killing assay was performed with antibiotic, and Kp in cell culture media alone to compare the intracellular and extracellular activity of each antibiotic.
We show that whilst the majority of the inoculum was resistant to phagocytosis, a small fraction of Kp were able to adhere to macrophages, enter the cell, and persist for up to 6h. Furthermore, we demonstrate that lower concentrations of tigecycline were required to inhibit intracellular Kp, compared with meropenem, which required concentrations in excess of the planktonic MIC to clear the intracellular niche.
These data indicate that there is reason to re-examine the antimicrobial treatment regimens for hypervirulent Kp infection with a focus on intracellularly active drugs.
Unravelling the requirement for host cell chloride channels during HRSV infection
Authors: Pearson Hayley , Barr John , Mankouri Jamel 
Published: July 2020

Ion channels are a diverse class of transmembrane proteins that selectively allow ions across membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is emerging as an important host-pathogen interaction that regulates critical stages of the virus multiplication cycle including entry, replication and egress.
Human respiratory syncytial virus (HRSV) causes severe respiratory tract infections globally and is one of the most lethal respiratory pathogens for infants in developing countries, with frequent development of bronchiolitis. Furthermore, it is the most significant cause of hospitalisation of infants in the UK. Evidence also indicates that severe childhood HRSV infection contributes towards the increased incidence of adult asthma. No HRSV vaccine is available, and currently the only treatment is immunoprophylaxis which is prohibitively expensive and only moderately effective; thus new treatment options are required.
Utilising GFP-expressing HRSV in combination with an extensive panel of channel specific pharmacological inhibitors, we have identified an important role of cellular chloride (Cl-) channels during HRSV infection. Interestingly, pharmacological inhibition of specific Cl- channel families has ruled out involvement of the CFTR and instead highlighted a critical requirement for calcium-activated Cl- channels (CaCCs). Time of addition studies using CaCC blockers have indicated that these channels play a post-entry role during HRSV infection. Using genetic knockdown techniques we have isolated a single channel of interest and are now further investigating its role in facilitating HRSV multiplication, as well as assessing the importance of Cl- channels in replication cycles of other negative sense RNA viruses.
Modulation of cAMP levels by a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria
Authors: Thomson Michael , Larrouy-Maumus Gerald 
Published: July 2020

Antimicrobial tolerance is the gateway to the development of antimicrobial resistance and is therefore a major issue that needs to be tackled.
The second messenger, cyclic-AMP (cAMP) is conserved across all taxa of life. It is involved in propagating the signal from environmental stimuli and converting it into a response. In bacteria such as M. tuberculosis (Mtb), P. aeruginosa, V. cholerae and B. pertussis, cAMP has been implicated in virulence, regulation of metabolism and gene expression. Cyclic AMP signalling in mycobacteria is especially complex – with 16 enzymes that produce cAMP in Mtb alone.
By discovery of a novel, actinobacteria conserved enzyme that degrades cAMP, we have developed a tool to modulate cAMP levels in mycobacteria. By using a combination of metabolomics, bioenergetics and time-to-kill assays, we show that when this enzyme is overexpressed in the model organism M. smegmatis, there is a 3.3 -fold decrease in intracellular cAMP levels. This was concomitant with 7-fold increased ATP. The unbalanced ATP/cAMP ratio consequently altered cell envelope permeability, compromised bioenergetics and most importantly, led to a decrease in the tolerance to various frontline antimicrobials.
Taken together, this work provides clear evidence that cAMP is involved in antimicrobial tolerance in mycobacteria and that this may represent a promising new target for antimicrobial development.
H5N8 highly pathogenic avian influenza virus introduction risk routes in a high biosecurity floor reared poultry setting
Authors: Warren Caroline , Slomka Marek , Nunez Alejandro , Thomas Saumya , Mahmood Sahar , Byrne Alexander , Banyard Ashley , James Joe , Watson Samantha , Brookes Sharon 
Published: July 2020

The European H5N8 highly pathogenic avian influenza virus (HPAIV) epizootic during 2016-2017 resulted in both wild bird and poultry deaths throughout the EU. This in vivo study investigated the potential for indirect infection of naïve birds through contaminated drinking water or feed, to assess potential disease incursion into a high biosecurity commercial floor housed setting.
Three-week-old Ross 308 chickens were exposed to H5N8 A/wigeon/Wales/52833/2016 (H5N8-2016) virus at a high (1 x 106 EID50/ml) or low (1 x 104 EID50/ml) dose, in either drinking water or feed for a 24 hour period. Chickens directly-infected with a high dose of H5N8-2016 (intra-nasal) acted as positive controls. Viral shedding, environmental contamination and clinical signs were monitored for ten days post infection (dpi).
All directly-infected birds shed virus and were humanely terminated at 3 dpi. Immunohistochemical analysis of nasal epithelium and caecal tonsil lymphoid tissue, obtained at post mortem from directly-infected chickens (2 dpi), showed the presence of influenza antigen in both tissues. Only birds exposed to high dose virus in drinking water, shed virus and showed clinical disease presentation (67% mortality). Interestingly low levels of antigen were detected in the nasal epithelium, whereas higher levels were detected in the caecal tonsil.
All surviving chickens from each group, remained uninfected and did not seroconvert. Our findings suggest virus bio availability in different substrates is variable (feed and water) and possible routes of viral contamination leading to disease ingress at poultry premises may have different outcomes including disease presentation.
In vitro reassortment and adaptation of influenza A viruses circulating in swine
Authors: Ferrando VerÓnica , Börgeling Yvonne , Mellmann Alexander , Gandhi Shrey , Brunotte Linda , Stoll Monika , Harder Timm , Beer Martin , Ludwig Stephan 
Published: July 2020

Since the last influenza pandemic in 2009, H1N1pdm has been introduced into the swine population in Europe where, in combination with swine influenza A virus (IAV) lineages, it started to generate a variety of reassortant viruses of unknown zoonotic risk for humans. To study these reassortment events, we isolated a wild swine lung cell clone (C22) susceptible to IAV infection. We established conditions for co-infection and passaging of H1N1pdm and swine avian-like H1N1. After 7 passages, we plaque-purified C22-adapted strains, characterized their genome composition by next-generation sequencing and analysed replication abilities in swine and human lung cell lines as well as in human lung tissue ex vivo.
Among C22-adapted viruses isolated from co-infection, we revealed reassortants carrying PB1/PA/NA or only PB1/PA from H1N1pdm. We also detected exclusively swine H1N1-derived strains. All isolates carried distinct mutations. As expected, adapted viruses reached higher titers compared to both parental strains in swine lung cells. Furthermore, all C22-adapted viruses were able to replicate in human lung A549 cells without any prior adaptation to the human host. Strikingly, all reassortants were able to infect and efficiently replicate in human lung tissue ex vivo, indicating that these viruses might pose a zoonotic risk.
To summarize, we successfully established an in vitro swine-like model to study reassortment and adaptation of IAVs currently circulating in swine. Our results indicate that our model might be a useful tool to prospectively evaluate the compatibility of different IAV strains to generate reassortants, which might represent a threat to the human population.
Phenotypic characterization and ecological succession of microorganisms during the fermentation of Cassava and Maize
Authors: Ukwuru Michael , Kalu Achi Ome , Nnambuihe Ekwenye Uchechi 
Published: July 2020

Fermented foods are consumed by a very large population in Africa but the products have many drawbacks ranging from shelf life instability to contamination and toxicity. These foods therefore require an upgrade through improved fermentation processes. This work determined the phenotypic characteristics of the fermenting microorganisms and microbial ecological succession during fermentation of cassava and maize to determine the predominant fermenting microorganisms. Cassava roots and maize grains were fermented using the traditional method of processing them into fufu and ogi for 72 h and 48 h respectively. Samples were drawn every 12 h for analysis. Enumeration and characterization of lactic acid bacteria were carried out on MRS medium with subsequent microscopic examination, physiological, biochemical reaction tests and API 50 CH gallery. Yeast isolates were identified by their morphological characteristics. Thirteen lactic acid bacteria were isolated from the fermenting cassava and 6 from the fermenting maize. The Isolates were Gram positive and catalase negative. Lactobacillus plantarum, L. fermentum and L. pentusus predominated in both fermentations while Candida tropicalis, C. krusei and Saccharomyces cerevisae also predominated in both fermentations. Candida inconspicuo was found only in cassava fermentation. The results of this work revealed the microbial ecology of fermented cassava and maize which is a prerequisite to the understanding needed to develop a multifunctional starter culture for these fermentations for their upgrade.
Keywords: Cassava, Maize, Fermentation, lactic acid bacteria, Yeasts.