bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Characterization of bacteriocin related genes discovered in a novel probiotic isolate Enterococcus faecium W1
Authors: Aziz Fauzia , Khan Nasim , Ahmed Safia , Jones Ian , Andrews Simon
Published: July 2020

Aims: To isolate and characterize the bacteriocin activity identified by the genomic analysis of a novel enterococcal isolate, Enterococcus faecium W1

Methods and Results: Culture fermentates of a novel E. faecium isolate, W1 showed high levels of antimicrobial activity against Listeria monocytogenesisand a variety of other Gram+ve and Gram-ve bacteria. Ammonium sulphate precipitation of the fermentates followed by hydrophobic interaction chromatography showed bactericidal activity correlated with fractions containing only low molecular weight proteins. The same fractions had activity on human cancer cell line HT-29 measured by cytotoxicity assay (MTT) and apoptosis induction. Whole-genome sequencing of E. faecium W1 revealed five bacteriocin related gene clusters, a lack of virulence factors and lack of antibiotic resistance genes. To confirm that antimicrobial activity was correlated with the bacteriocin genes identified, selected bacteriocin genes were cloned and expressed as His-tagged proteins in E. coli. Bactericidal activity against L. monocytogenes was associated with protein purified by metal affinity chromatography. A structure-function analysis to investigate the role of key residues in activity is ongoing.

Conclusion:
E. faecium W1 is a promising candidate for use as a probiotic supplement or in food preservation. The bacteriocin related genes are functional and could be improved for future use as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: There is a need for new approaches to the growing problem of antibiotic resistance. Expression of bacteriocins identified in E. faecium W1 could contribute to this endeavour in addition to its use as a probiotic organism.
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.
Convergence of virulence and drug efflux traits in a siderophore ABC transporter on mobile genetic elements in Gram negative pathogens
Authors: Farzand Robeena , Rajakumar Kumar , Mukamolova Galina , Bare Mike , O’Hare Helen
Published: July 2020

The accessory genome of the human pathogen Klebsiella pneumoniae is large, variable and highly mobile. This reservoir of genes leads to the emergences of hospital outbreak strains with enhanced virulence and multidrug resistance, such as ST258, and acts as a source for transfer of these traits to other Gram negative pathogens. One such mobile genetic element, ICEKp, is prevalent in isolates of invasive disease where it enhances iron acquisition by the siderophore yersiniabactin. Yersiniabactin is also a virulence factor in pathogenic Escherichia coli and Yersinia species. Similarities between siderophore transporters and drug efflux pumps led us to postulate that the yersiniabactin transport proteins could contribute to antimicrobial resistance. We determined the effect of loss and gain of ICEKp, or the transporters alone, on iron acquisition and drug sensitivity of K. pneumoniae and Escherichia coli. Deletion of ICEKp impaired iron acquisition of clinical isolate K. pneumoniae HS11286 due to reduced siderophore secretion and reduced ability to acquire iron from siderophores. A simultaneous increase in sensitivity to a broad range of antimicrobials could be complemented by reintroduction of the ybtPQ ABC transporter. Furthermore, transfer of ICEKp to E. coli occurred efficiently by conjugation and conferred a similar decrease in sensitivity to antimicrobials.
Exploring potentials of indigenous yeasts in fermentation of Chambourcin Grapes
Authors: Tang Chun , Wee Josephine
Published: July 2020

Background: Indigenous yeasts present on grape berries have been shown to impact the winemaking process and final wine quality. In this study, we used Chambourcin, a hybrid grape cultivar, to isolate indigenous yeasts for potential use in winemaking. Hybrid grapes are of particularly interest due to higher resistance to cold temperature and fungal diseases.

Methods: Yeasts were isolated from spontaneous fermentation of crushed grapes by plating on Dichloran Rose Bengal Chloramphenicol agar and identified by Sanger sequencing. Yeast candidates were selected for their ability to grow in Yeast Extract-Peptone-Dextrose broth supplemented with varying ethanol concentration. Candidate yeasts were chosen for mock fermentation using sterile Chambourcin juice. Volatile and non-volatile compounds that predict wine quality (flavor and aroma) were measured by UPLC and GC-MS.

Results:
Hanseniaspora uvarum, Starmerella bacillaris, Candida californica, and Zygoascus meyerae were predominantly isolated from Chambourcin. S. bacillaris isolate 180002 and C. californica isolate 180004 were able to tolerate up to 10% ethanol compared to S. cerevisiae (ethanol tolerance up to 12%). Our results demonstrate that isolate 180002 grown in ethanol supplemented medium is comparable to the commercial S. cerevisiae. Various aroma profiles of three main chemical families – esters, higher alcohol and volatile acids in products fermented by S. bacillaris and other isolates were observed.

Conclusion:
Starmerella bacillaris isolate 180002 demonstrates potential for use in winemaking with hybrid grapes based on ethanol tolerance and production of wine related chemical compounds. This study further supports the application of indigenous grape-associated yeasts in creating flavor and aroma diversity of wine.
Real-time WGS monitoring identifies L. monocytogenes outbreaks in The Netherlands and contributes to a rapid detection of the source
Authors: Castelijn Greetje , van Overbeeke Lennert , Lee Seungeun , Wullings Bart , te Loeke Nathalie , Franz Eelco , Friesema Ingrid , van der Voort Menno
Published: July 2020

Whole genome sequencing (WGS) is increasingly used by food regulatory agencies and public health institutes. It is a powerful tool to identify the source of a foodborne outbreak. Real-time WGS analysis helps to act fast during a foodborne outbreak, and with that the impact of an outbreak can be significantly decreased. In The Netherlands real-time WGS analysis is performed for L. monocytogenes originating from humans and from the food chain, and WGS data is shared between the food regulatory agencies (WFSR and NVWA) and the public health institute (RIVM). Consequently, by molecular typing and cluster analysis probable infection sources of L. monocytogenes are identified. These analysis already identified 18 clusters of human L. monocytogenes isolates related to food isolates, and also several clusters that might suggest persistence of L. monocytogenes in different production environments. Real-time WGS analysis for example contributed to the fast identification of the source of a ST-6 L. monocytogenes outbreak originating from ready-to-eat meat products, and the subsequent termination of this outbreak. The timeframe of human cases (n=19) and strains isolated from the RTE meat products, together with the genetic relatedness of the strains suggest that the source of the outbreak was a L. monocytogenes strain which persisted in the production environment. This shows the effectiveness of real-time WGS analysis in solving foodborne outbreaks in the Netherlands and its potential for the food industry in the prevention of these outbreaks in the future.
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.
Identification of isoprene-degrading bacteria in phyllosphere and soil communities from high isoprene-emitting oil palm trees by DNA-stable isotope probing
Authors: Carrión Ornella , Gibson Lisa , M.O. Elias Dafydd , P. McNamara Niall , A. van Alen Theo , J.M. Op den Camp Huub , Vimala Supramaniam Christina , Colin Murrell J.
Published: July 2020

Isoprene is the most abundant biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those from methane. Due to its volatile nature and high reactivity, isoprene plays a complex role in atmospheric chemistry and hence, climate. However, very little is known about its biological degradation in the environment. The vast majority of isoprene (500 Tg ·y-1) is produced by terrestrial plants and oil palm is considered one of the highest isoprene-producing trees, with estimated emissions of 175 μg·g-1 dry leaves ·h-1. Oil palm is also a heavily cultivated crop since it is the source of 30% of the vegetable oil in the world and in countries such as Malaysia represents >85% of total agricultural land. The vast expansion of a single crop that emits such high amounts of isoprene have raised serious concerns about its impact on air quality and climate change. We performed DNA Stable Isotope Probing (DNA-SIP) to study the isoprene-degrading community of oil palm trees in a Malaysian plantation and identified novel genera of isoprene-utilising bacteria in both oil palm soils and leaves. isoA amplicon sequencing data also confirmed that oil palm trees harbour a novel diversity of isoA genes, which encode the alpha subunit of the isoprene monooxygenase, a key enzyme in isoprene metabolism. In addition, metagenome assembled genomes (MAGs) were reconstructed from metagenomes from oil palm soil and leaf incubations and analysed to identify isoprene degradation gene clusters in these microorganisms. Finally, analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere.
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.
Characterisation of a new megaplasmid family associated with the spread of multidrug resistance in Pseudomonas aeruginosa
Authors: Cazares Adrian , P.J. Hall James , L. Wright Laura , Grimes Macauley , Emond-Rhéault Jean-Guillaume , Pongchaikul Pisut , Santanirand Pitak , C. Levesque Roger , L. Fothergill Jo , Winstanley Craig
Published: July 2020

Unlike in other important pathogens, the role of plasmids in the emergence of antimicrobial resistance (AMR) in Pseudomonas aeruginosa (Pa) has remained largely unaddressed. Previous work on AMR in Pa has mostly used genome sequencing methods that are limited because of the difficulty of using short-read data to detect and reconstruct complex plasmids. Here, using superior long-read sequencing, comprehensive bioinformatics analyses, and experimental characterization, we uncover an emerging family of important Pseudomonas megaplasmids and report its contribution to dissemination of multidrug resistance (MDR) on a global scale. Firstly, we identified large plasmids with a key role in the spread of MDR in a hospital in Thailand, and characterised their resistance regions revealing evidence of duplication, recombination and shared repeats, indicative of dynamic adaptation. Applying phylogenomics and pangenomics approaches we linked related megaplasmids and defined a core and pangenome for the family, exposing wide variations in AMR genes carriage. We then surveyed thousands of publicly available genomes, leading to discovery of dozens of megaplasmid relatives overlooked in multiple datasets, including already published studies. By integrating all this information and looking beyond the pathogenic species we gained valuable insights into the evolution of the megaplasmid family and revealed its widespread and multispecies distribution. We also showed that members of this family are stable in the absence of antibiotic pressure, bear no fitness cost to their host, and can be readily transferred between different Pseudomonas species. Our findings expand the bacterial plasmidome and provide insights on how MDR plasmids emerge from environmental reservoirs.
Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance
Authors: Jengarn Juggragarn , Daly Janet , Maina Meshach , Van Diemen Pauline , Scott Simon , Temperton Nigel
Published: July 2020

Influenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses.
The Type VI Secretion System of Pseudomonas aeruginosa: a gun loaded with antimicrobial bullets
Authors: Howard Sophie , Furniss Chris , Bonini Dora , Amin Himani , Paracuellos-Torrecilla Patricia , Costa Tiago , Mavridou Despoina , Filloux Alain
Published: July 2020

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe respiratory infections in people who are immunocompromised. P. aeruginosa possesses the Type VI Secretion System (T6SS), a bacterial weapon that injects effectors into neighbouring prokaryotes and eukaryotes. The T6SS is crucial for bacterial warfare, allowing P. aeruginosa to kill its competitors, which promotes its dominance in mixed microbial environments. P. aeruginosa has three T6SSs, H1/2/3-T6SS, these are structural homologs but deliver unique effectors. Effectors are delivered via the secreted component, a Hcp tube topped with a VgrG and PAAR spike. Only the first three identified effectors are delivered by Hcp1. Since then, there has been a bias in identification of VgrG or PAAR delivered effectors, mostly as these are encoded next to the spike proteins. Some P. aeruginosa effectors not only kill bacteria but have a dual role in pathogenesis. Our aim was to identify a comprehensive set of Hcp-delivered effectors for all three systems. Using Hcp1/2/3, systematic pull-down screens were performed to identify novel interaction partners. After confirming interaction, antibacterial toxicity was evaluated, identifying new Hcp delivered T6SS effectors for Hcp2 and Hcp3, which are toxic in the bacterial cytoplasm. These new anti-bacterial effectors may kill bacteria in novel ways, which could lead to novel antibiotics. Additionally, a toxin fusion proved too large for secretion and blocked the T6SS, revealing a Hcp-delivered effector size limit. Future work will focus on fully characterising these new toxins, as well as to look into the potential eukaryotic role of other interaction partners.
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.
Structural characterization of genomic RNA-coat protein contacts in single-stranded RNA viruses by high-resolution cryo-EM
Authors: Mata Carlos , Chandler-Bostock Rebecca , Scott Andrew , Lulla Valeria , Firth Andrew , Bingham Richard , Dykeman Eric , Rowlands David , Ranson Neil , Twarock Reidun , Stockley Peter
Published: July 2020

Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.
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.
Ultra- structure of Toombak; smokeless tobacco of Sudan and its effects on oral and systemic health
Authors: Sami Amel , Stanton Catherine , Ross Paul , Ryan Tony , Elimairi Imad
Published: July 2020

Introduction: Toombak is a smokeless tobacco used by the Sudanese. Nicotiana Rustica leaves are fermented, and sodium bicarbonate is added, increasing Ph. This causes high absorption spikes of unprotonated nicotine through mucosal epithelium, and into the blood, distinguishing Toombak as an addictive product. Furthermore,Toombak hashighmicrobial contamination due to its open field, rural production.

Materials and methods: 21 tobacco samples were collected from 3 main towns in the capital Khartoum, Sudan. Analysis included microbiome (16S rRNA sequencing), metabolome (Liquid and Gas Chromotography- volatile organic compound method), heavy metal content and scanning electron microscopy (SEM).

Results: Compared to limit of detection values (LOD), Tobacco specific nitrosamines (TSNA’s), in particular, 4(methylnitrosamino)-1-(3-pyridyl)–butanone (NNK) were markedly increased [NNK; 1.2-4.7 mg/g; LOD 0.02]. Free nicotine ranged from 16-31 mg/g; [LOD 0.01]. High choline and carnitine, volatile aldehydes; and benzyl alcohol were also detected. 8 cations, of which iron [1465 mg/kg] and copper [3.76 mg/kg] were strikingly observed, compared to average levels from other products; [Iron 20-60mg/kg, Copper 1.3mg/kg]. 8 Phyla, including but not limited to; Actinobacteria; cornyebacterium(contain nitrate reductase genes) and Firmicutes; staphylococcus and facklamia, were sequenced. SEM highlighted a non-homogenous product with elevated sodium spectrum.

Conclusions: TSNA’s were potent in Toombak due to high starting nicotine and rich nitrate reductase bacteria. High choline and carnitine can promote cardiovascular disease through their conversion to trimethylamine-N-oxide. Diphtheria and infective endocarditis are associated with cornyebacterium and facklamia respectively while staphylococcus can lead to numerous systemic opportunistic diseases.
Identification of genes that contribute to fitness of African and Global clades of Salmonella Enteritidis during infection of macrophages
Authors: Yee Fong Wai , Canals Rocío , Predeus Alex , Perez-Sepulveda Blanca , A. Feasey Nicholas
Published: July 2020

Non-typhoidal Salmonella (NTS) usually cause gastroenteritis in humans, but in recent years NTS have begun to cause epidemics of bloodstream infections in Africa. Salmonella Enteritidis is the second most common serovar associated with this invasive form of NTS disease (iNTS) in Africa. To establish a systemic infection, Salmonella must survive and replicate within host cells, with macrophages being a primary target. Genomic characterisation of S. Enteritidis isolates from human bloodstream has identified two new clades that are unique to Africa and distinct from the Global Epidemic clade. The African S. Enteritidis clades exhibit genomic degradation, and possess a distinct prophage repertoire and are multi-drug resistant. However, little is known about the virulence factors that allow African S. Enteritidis to cause systemic infection in susceptible hosts. We screened libraries of random insertion mutants of African and Global S. Enteritidis by transposon insertion sequencing (TIS), and identified about 280 genes belonging to each clade that contribute to bacterial survival and replication in murine macrophages. The genes were associated with 5 pathogenicity-islands, or encoded the global regulators PhoPQ and OmpR-EnvZ. Experiments are ongoing to investigate the role in intra-macrophage replication of genes that are uniquely identified in African Salmonella. It is hoped that our findings will contribute to a greater understanding of African Salmonella infection biology, and that some of the virulence-associated genes could be potential targets for novel therapeutics.
Non-human primates in the Gambia harbour human-associated pathogenic Escherichia coli strains
Authors: Foster-Nyarko Ebenezer , Alikhan Nabil-Fareed , Ravi Anuradha , Thilliez Gaëtan , Thomson Nicholas , Baker David , Kay Gemma , D. Cramer Jennifer , O’Grady Justin , Antonio Martin , Pallen Mark
Published: July 2020

Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between different host species. We have investigated genetic diversity and antimicrobial resistance in Escherichia coli isolates from a range of non-human primates dispersed across the Gambia: patas monkey (n=1), western colobus monkey (n=6), green monkey (n=14) and guinea baboon (n=22). From 43 stools, we recovered 99 isolates. We performed Illumina whole-genome shotgun sequencing on all isolates and nanopore long-read sequencing on isolates with antimicrobial resistance genes. We inferred the evolution of E. coli in this population using the EnteroBase software environment. We identified 43 sequence types (ten of them novel), spanning five of the eight known phylogroups of E. coli. Many of the observed sequence types and phylotypes from non-human primates have been associated with human extra-intestinal infection and carry virulence characteristics associated with disease in humans, particularly ST73, ST217 and ST681. However, we found a low prevalence of antimicrobial resistance genes in isolates from non-human primates. Hierarchical clustering showed that ST442 and ST349 from non-human primates are closely related to isolates from human infections, suggesting recent exchange of bacteria between humans and monkeys. Our results are of public health importance, considering the increasing contact between humans and wild primates.
RNA-seq provides molecular insights into interaction modification in simplified microbial community
Authors: De Boever Frederik , Potin Philippe , Green David
Published: July 2020

A major challenge in microbial ecology is to understand the stability of interspecies interactions when progressing from pairs of interacting species to multispecies interaction networks. A lack of direct evidence, and a conceptual framework to explore how direct and indirect effects shape cellular responses in species-rich networks has hindered progress in our understanding of these combined effects. Here we aimed to investigate whether higher-order interactions shape community dynamics and transcriptional profiles of all interacting partners in a simplified microbial community that includes a primary producer (Nannochloropsis oceanica CCAP849/10) and two heterotrophic bacteria (Marinobacter sp. FDB33 and Alteromonas sp. FDB36). By combining co-cultivation assays, quantification of absolute abundances, nutrient analysis, and simultaneous RNA-sequencing, we reveal genome-wide transcriptional responses in all binary co-cultivation partners and show that the third partner can profoundly alter binary interactions at the phenotypic and transcription level. Our study demonstrates the context-dependency of binary interactions, whereby environmental conditions and the presence of specific organisms can affect the cellular physiology of the interacting partners and ultimately the stability of the community. Furthermore, our approach provides a powerful tool for probing the molecular basis of emergent properties in more complex systems.
Developing global whole cell standards for the microbiome field
Authors: Sergaki Chrysi , Anwar Saba , Logan Alastair , Rijpkema Sjoerd , C.A. Amos Gregory
Published: July 2020

The microbiome field has developed rapidly over the last decade, however there is no effective standardisation of protocols in place, with no accredited or certified reference materials available to the wider community. As part of the NIBSC Microbiome program, we developed whole cell standards for microbiome research which can act as global working standards and will be put forward for consideration as the first whole cell World Health Organization International Reference Reagents for microbiome analysis. The developed reagents consist of common bacteria found in human gut and have been tested using different commercial DNA extraction kits in order to detect biases introduced through different DNA extraction processes. Multiple replicates of DNA extracted the whole cell standard using different DNA extraction kits were assessed using Next Generation Sequencing and evaluated using the number of input cells in the standards, as measured by microscopy and flow cytometry. These NIBSC whole cell standards are aimed to standardise the DNA extraction steps in microbiome analytical pipelines and serve as a tool to more accurately capture the representation of each microbe in the gut microbiome. This could be achieved by setting a specific threshold of accepted error levels by the microbiome community and we aim to set this through a large collaborative study in 2020-21. This work is part of a larger NIBSC microbiome program that is developing site specific DNA, whole cell and matrix spike-in reagents for use in microbiome research.
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.
Isolation and characterization of Anoxygenic phototrophs from shrimp ponds in Thailand
Authors: Ramirez Xelimar , Luamanu Imeleta , Michael Ceballos Ruben , Padilla Crespo Elizabeth
Published: July 2020

Anoxygenic phototrophic purple bacteria are ubiquitous in aquatic and terrestrial environments and demonstrate broad phenotypic diversity. Purple bacteriaderive energy from light under anaerobic conditions via anoxygenic photosynthesis, a process in which water is not the electron donor. It has been suggested that these bacteria are useful for a variety of applications, including: wastewater treatment; heavy metal remediation; nitrogen fixation; and, control of CH4 emissions. In this study, the goal was to isolate and characterize PNSB from shrimp ponds in Thailand. Surface water and sediment were collected. Enrichment cultures were prepared using Pfenning’s mineral media. As indicated by development of reddish color and turbidity, anoxygenic phototrophic growth was observed within two days of incubation. Cultures in liquid media and on solid plates exhibited a deep red or purple color ten weeks post-inoculation. Under light microscopy, enrichments consist of communities dominated by thin, elongated gram-negative cells with granules of elemental sulfur, which are characteristic of purple bacteria. Molecular methods confirm the presence of pufLM, a genetic biomarker for purple bacteria (e.g., Thiohalocapsa marina, Allochromatium vinosum, Roseovarius tolerans). Initial sequencing of key genes (i.e., pufLM) indicate that these environmental samples contain novel isolates or “geographic variants” that have not been previously described. We have developed a few pure cultures of multiple species from these environmental samples. Since shrimp farming is a key industry in southern Thailand, the characterization of the microbial communities in these ecosystems, including anoxygenic phototrophs, will provide insights into how to maintain water quality in these food production systems.