bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro antimicrobial efficacy of Cnidoscolus aconitifolius leaf extract and honey on Staphylococcus epidermidis clinical isolate
Authors: Ogbu Hanson , Igboanusi Emeka , Ezenobi Nkechi
Published: April 2019

The search for alternative, potent, cost-effective treatment of ailments caused by resistant microorganisms and the role of plants and their products as essential sources of medicinal agents is receiving increasing attention. Several of these natural products are reported to have capacity to produce natural compounds of high structural diversity that serve as defense agents against invading microorganisms. Cnidoscolus aconitifolius also known as tree spinach is an indigenous tropical tree that has gained lot of importance in its nutritive value and traditional use. This study evaluates the possible effect of Cnidoscolus aconitifolius leaf extract alone, extract/honey combination on Staphylococcus epidermidis clinical isolate. Phytochemical analysis and antimicrobial efficacy of the extract were performed using standard methods. The results of phytochemical analysis reveal the presence of carbohydrate, tannins, alkaloids, steroids, flavonoids, anthraquinone, saponins and carotenoids. Antimicrobial activities showed inhibition zone values of 8.0±0.1 mm for aqueous extract alone, 9.0±0.1 mm for aqueous extract/honey combination. The finding suggests that C. aconitifolius might be a good source of compounds that can be used to inhibit the growth of Staphylococcus epidermidis pathogen and further supports its popular and wide traditional applications in the treatment of various illnesses. Hence the need for further research to exploit the full potential of C. aconitifolius tree in order to influence their extensive consumption, storage, improvement and production.
HSV-1 genome decompaction plays an important role in viral DNA sensing by PML-NB intrinsic antiviral regulators
Authors: Iliev Victor , Orr Anne , Boutell Chris
Published: April 2019

Constituent proteins that reside within promyelocytic leukaemia nuclear bodies (PML-NBs) are known to play an important role in cellular restriction of herpesvirus gene expression as a component of the intrinsic antiviral immune response to DNA virus infection. However, the precise mechanism(s) of PML-NB genome detection, entrapment, and silencing remain unknown. We have recently reported a sensitive method of viral genome detection that enables the visualisation of infecting EdC (5-Ethynyl-2’-deoxycytidine) labelled HSV-1 genomes (HSV-1EdC). We report that ultraviolet light (UV) irradiation of HSV-1EdC virions inhibits the nuclear recognition of viral DNA (vDNA) by core PML-NB constituent proteins (PML, Sp100, and Daxx). This impairment of recruitment was independent of UV source, but dependent on dose and time course of UV irradiation that correlated with a loss in viral genome decompaction upon nuclear entry. Moreover, UV treatment promoted premature uncoating of viral genomes within the cytoplasm of infected cells in a dose-dependent manner. Our data highlights the importance of genome decompaction in PML-NB sensing of infecting viral genomes. Additionally, it uncovers a previously undescribed mechanism of action for the induction of innate immune defences during herpesvirus infection that have historically utilised UV inactivation to promote the activation of cellular pattern recognition receptors (PPRs) and induction of interferon stimulated gene (ISG) expression.
The role of RNA-RNA interactions in the assembly and reassortment of influenza A viruses
Authors: Knight Michael , Dadonaite Bernadeta , Gilbertson Brad , Trifkovic Sanja , Rockman Steven , Laederach Alain , Brown Lorena , Fodor Ervin , Bauer David LV
Published: April 2019

Influenza A virus has a genome consisting of 8 segments of negative sense RNA. When two influenza A virus strains infect the same cell, there is potential for the progeny to package segments from both strains. This process is termed reassortment and can lead to rapid genetic shifts that have previously generated strains of influenza responsible for pandemic events. Recent evidence suggests that assembly of the eight influenza genomic segments for packaging into a virion is mediated by RNA-RNA interactions between the segments. These interactions are likely to contribute to the varying compatibilities for reassortment observed between segments from different strains of influenza. We have captured complete RNA-RNA interaction maps for several influenza A viruses using a high-throughput sequencing approach and identify extensive, redundant, networks of RNA-RNA interactions between the genomic viral RNA segments. We extended this analysis to H1N1 and H3N2 reassortants, and found that by manipulating these interactions, we can drive preferential co-segregation of segments during reassortment. This work provides the first direct evidence that RNA-RNA interactions between the influenza virus genomic segments are a key factor in driving reassortment between viral strains.
The great escape: dissecting the interactions between Mycobacterium bovis and the soil amoeba Dictyostelium discoideum
Authors: Butler Rachel , Smith Alex , Mendum Tom , Chandran Aneesh , Wu Huihai , Lefrancois Louise , Soldati Thierry , Stewart Graham
Published: April 2019

Mycobacterium bovis, a member of the M. tuberculosis complex, causes bovine tuberculosis, one of the most important veterinary health problems in the UK. In the absence of improved control the projected economic burden in the UK over the next decade is predicted to be £1bn. Although transmission of M. bovis occurs primarily between infected mammalian hosts, M. bovis has been demonstrated to persist in soil, suggesting an environmental infectious route. M. bovis is likely predated on by environmental amoebae such as Dictyostelium discoideum and as such may have evolved mechanisms to modulate the interaction with amoebae. In this study we have investigated M. bovis interactions in vitro with D. discoideum. We demonstrate that virulent M. bovis evades destruction by D. discoideum. Using a genome-wide M. bovis transposon mutant library, we selected for mutants that failed to escape D. discoideum after 48 h of infection. Mutants of genes encoding the MCE4 transport system, genes involved in sulpholipid synthesis/transport, and genes encoding PPE and PE-PGRS protein families remained associated with D. discoideum. Most strikingly, mutations in 11 genes of the major mycobacterial virulence locus ESX-1, which encodes a T7 secretion system implicated in bacterial transit through host cell membranes, were significantly enriched in D. discoideum. Our data demonstrate that known virulence factors involved in host-pathogen interactions in mammalian hosts also play a role in D. discoideum-M. bovis interactions. Our data further suggest that M. bovis has evolved to actively transit bacteriverous D. discoideum rather that to use it as a replicative niche.
Antibacterial activity of traditional herbal medicine
Authors: He Qiqi
Published: April 2019

Antimicrobial resistance (AMR) is becoming the biggest substantial threat for public health and societal implications worldwide. Coincidentally, the development of new antibiotics has decreased and downsized by the pharmaceutical companies for the last 40 years. The past decade has witnessed an increasing effort worldwide on exploration of plant-based natural products for new antimicrobial agents. The purpose of this study is to investigate the potential antibacterial activity of traditional herbal medicinal plant against resistant pathogens of public health and economic importance such as methicillin-resistant Staphylococcus aureus (MRSA) and Acine to bacter Baumannii. The broth microdilution method was used to determine the susceptibility of resistant pathogenic strains selected from WHO priority list. A total of 57 plants were chosen based on traditional knowledge and current scientific information that has been claimed to have antimicrobial activity. Preliminary results showed 75 % of screened plants inhibited the growth MRSA (NCTC 12493) some of which exhibited similarantibacterial efficiency compared to the vancomycin (positive antibiotic control), while 21 % shows an inhibitory effect against Acinetobacter Baumannii (NCTC 12156). In addition, significant synergy was observed between some of the plant extracts and vancomycin against MRSA. Further studies are needed for the warrant of these plant candidates to be further developed into therapeutic antimicrobial agents of required efficacy and safety.
The Dictyostelium rhomboid proteases and mitochondrial disease
Authors: Thompson Elinor , Rafiq Mehak
Published: April 2019

Rhomboid membrane proteases are integral to pathogenicity of several microbial eukaryotes and the role of mitochondrial rhomboids is important in pathologies beyond infectious disease. By virtue of their ability to cleave tethered proteins, this relatively recently discovered protein family have been found to activate substrates, release mobile signals, and progress cell regulatory pathways. The Dictyostelium model microbe allows the study of an evolutionarily ancient set of rhomboids, aberrant expression of which affects phagocytosis, response to chemoattractants, phototaxis, growth and cell size, ATP levels, and mitochondrial ultrastructure. We report the particularly mitochondrial focus of rhomboids in this amoeba and consider the relevance of this beyond the model organism, given the central role of the human mitochondrial rhomboid in mitophagy, ageing and neurodegenerative disease.
Investigating the mechanisms of BK polyomavirus egress and virus-host interactions
Authors: Ho Sophia , Caller Laura , Beale Rupert , Crump Colin
Published: April 2019

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that can establish a lifelong, silently persistent infection in the kidney and is estimated to infect 70–90 % of the world’s population. In immunocompromised individuals, particularly bone marrow and kidney transplant patients, increases in BKPyV replication can result in significant pathological conditions. In the case of kidney transplant patients, this can result in nephropathy, which in severe cases can result in the deterioration of allograft function and loss of the transplanted organ. There are currently no antiviral treatments with strong evidence of clinical efficacy against BKPyV. Though little is known about BKPyV egress from infected cells, we have evidence showing that BKPyV can be released in a non-lytic manner by an unconventional cellular secretory pathway that bypasses the Golgi apparatus. Here, we investigate the mechanisms behind BKPyV non-lytic egress through studying the effects of knocking out candidate host proteins involved in unconventional secretory pathways on BKPyV release, examining the effects of BKPyV infection on host cell protein secretion, and ascertaining which host proteins are essential for the BKPyV life cycle via a whole-genome CRISPR screen. These experiments are uncovering novel virus-host interactions that, when targeted, could lead to antiviral effects.
Type I interferon activity promotes a cellular environment that supports the establishment of latency by human cytomegalovirus
Authors: Olaizola-Rebe Paula , Onyerindu Chinedu , Kiesworo Kevin , Lodha Manivel , Stamminger Thomas , Peters Nicholas , Murray Matthew , Reeves Matthew
Published: April 2019

Type I interferons (IFN) are potent inducers of an anti-viral state in response to infection and have been demonstrated to inhibit cytomegalovirus (CMV) replication both in vitro and in vivo. CMV, like all herpes viruses, has the capacity to establish lifelong infections of host through the establishment of latency. As the very early stages of viral entry can trigger IFN responses we investigated the impact of IFN on the establishment of latent human CMV (HCMV) in myeloid progenitor cells. Here we show that priming of myeloid THP1 cells with type I IFN prior to infection skews infection towards a more efficient establishment of latency. This is evidenced by detection of reduced lytic gene expression, increased latent gene expression, and increased levels of reactivation following differentiation. Blockade of IFN signalling with neutralising antibodies antagonised the latent phenotype suggesting that endogenous IFN production upon infection contributed to the effect observed. Intriguingly, whilst both IFNα2 and IFNβ can drive latent infection individually, their effects were dose-dependent and demonstrated a biphasic impact on the establishment of latency, with the highest doses of IFN preventing both lytic and latent infection. These data demonstrate that the HCMV derives an unexpected benefit from IFN production. They support a hypothesis that, although anti-viral in nature, concentration-specific effects of IFN may be evident in the cells which can modulate different outcomes post infection in persistent viruses such as HCMV. Future work is identifying the IFN concentration-specific effects responsible for a cellular environment that favours the establishment of latency.
Accelerated evolution of lager yeast strains for improved flavour profiles
Authors: de la Cerda Roberto , Bond Ursula
Published: April 2019

Fermented food and beverages have accompanied humans throughout history. Several species of microorganisms can transform raw materials into products with different and improved characteristics, for example alcoholic beverages (wine and beer) or dairy products, such as yogurt. In a world of constant change and competitiveness, brewers have to modulate and create new products to satisfy consumers. Therefore, the aim of my project is to generate lager yeast capable of producing molecules that improve the organoleptic profile of alcoholic beverages and understand the genetic and biochemical changes that increases the production of these aromatic molecules. The initial work has focused on the Ehrlich Pathway and the ester production. To produce changes in the genome, two approaches have been followed: one chemical way, using Radicicol, and one physical way, Heat Shock Thermal Stress (HSTS). Both ways inhibit Heat Shock proteins. Furthermore, this inhibition produces DNA damage, introducing Single sStrand Breaks (SSBs) and Double Strand Breaks (DSBs). The result of this inhbition is damage in the DNA and, furthermore, mutations in the DNA (aneuplidies and INDELS). After the Accelerated evolution, the evolved strains were plated on media containing amino acid analogues. These amino acid analogues select for cells with decreased feed-back inhibition of amino acid biosynthesis. Different mutant strains were generated with these two ways. These strains will be tested by different approaches, as growth curves, genome ploidy using cCGH and CHEF gel and qPCR analysis to measure the flux throughout the fermentation.
National collection of type cultures: the bacteriophage and plasmid collections and repositories
Authors: Fazal Mohammed-Abbas , Agendia Juandem , Yokoya Kazutomo , Hannah Matthew , Russell Julie , Alexander Sarah
Published: April 2019

The National Collection of Type Cultures (NCTC) is the world’s oldest bacterial collection that was specifically established to provide strains globally to support scientific research. In addition to the general catalogue NCTC has a fully curated bacteriophage and plasmid archive that to date has not recently been made available to the wider scientific community. The NCTC bacteriophage collection consists of over 100 bacteriophage and their corresponding bacterial hosts which were originally deposited primarily for their value in bacterial typing. The collection consists of bacteriophage from the following hosts: Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli and Campylobacter. The NCTC Datta plasmid collection is a curated collection of over 500 unique plasmids which were originally curated to examine the biology of plasmid transfer between and within bacterial strains. The NCTC bacteriophage collection is currently being fully characterised using a range of modern methods including genomic sequencing and electron microscopy. The plasmid collection is also being characterised using genomic sequencing and restriction digest profiles. It is intended that once characterised and rebanked both the plasmid and bacteriophage collections will be made available in 2019 to scientists to support research and development. The NCTC bacteriophage and plasmid collections will both be dynamic, representing an active repositories into which microbiologists can deposit phages and plasmids to support accessibility and reproducibility in science.
The genomic (re)definition of EPEC
Authors: Heinz Eva , Mather Alison , Petty Nicola , Kallonen Teemu , Semmler Torsten , Wieler Lothar , Elias Waldir , Regua-Mangia Adriana , Corander Jukka , Gomez Tânia , Jenkins Claire , Frankel Gad , Thomson Nicholas
Published: April 2019

Enteropathogenic E. coli (EPEC) were the first E. coli strains linked to human disease (1945), and pose a serious health threat. The burden for public health changed over the past century as it did for other diarrheal diseases; whilst it is still endemic in large parts of South America, cases in Europe are commonly associated with recent travel. Despite its long-standing importance for global health and its significant impact on child mortality in Low- and Middle Income Countries (LMICs), we still have very limited understanding of what defines an EPEC beyond the diagnostic LEE island, or indeed if there are any shared characteristics, as most in-depth analyses of their pathogenic determinants are confined to few model strains. We present a study analysing ∼1300 whole-genome sequences combining published and newly sequenced datasets of EPEC and non-EPEC strains to map the evolutionary history and molecular determinants. Importantly, we have expanded the published data with whole-genome sequences of 300 historical and contemporary EPEC strains from a large collection of clinical isolates, mainly from Brazil and England, which enables us to compare the epidemiology in a high-income with a LMIC over an extended time frame. We furthermore present a molecular definition of EPEC, including mapping of phage islands and de-novo prediction of effectors in this large-scale dataset, as well as investigating the patterns of adhesins and other secretion systems, thus characterising different EPEC lineages which have emerged numerous times during the evolution of E. coli.
The present & the past: a review of newly accessioned bacterial strains into the UK’s National Collection of Type Cultures
Authors: Turnbull Jake David , Gurung Sunita , Russell Julie E. , Alexander Sarah
Published: April 2019

The National Collection of Type Cultures (NCTC) is a bacterial culture collection which is taxonomically and biologically diverse. NCTC holds approximately 6000 different strains from over 900 different species; among them strains originally isolated in the 19th century, strains for use as controls as stipulated EUCAST and ISO guidelines, type strains on which the description of bacterial species are based and other strains from a variety of backgrounds. The remit of NCTC is to provide authentic bacterial cultures of medical and veterinary interest to the scientific community, to support and enhance the reproducibility of scientific research and to improve global public health. To fulfil this remit, remain scientifically relevant and to preserve the legacy of contemporary medical bacteriology for future scientists, NCTC accessions strains of clinical significance: such as recently circulating and outbreak strains, diagnostic escape mutants and strains with novel antimicrobial resistance profiles. In 2018, 166 bacterial strains were accessioned into the NCTC and made available to the scientific community. These include NCTC 14052: a reference strain for emergent hyper-virulent K. pneumoniae, 4 type strains of newly described bacterial species, 82 strains accessioned from the Murray Collection of pre-antibiotic era Enterobacteriaceae and 8 strains with antimicrobial resistance mechanisms previously unrepresented in the collection, including NCTC 14208: a N. gonorrhoeae isolated from an instance of combined ceftriaxone and azithromycin treatment failure. Through literature review we have highlighted their value to the scientific community, both in their own right and in the context of bacterial strains already held by the NCTC.
Mastitis and animal husbandry – high-throughput sequencing as a support tool
Authors: Blake Daniel , Haynes Edward , Kyriazakis Ilias
Published: April 2019

Mastitis is a disease of the mammary gland which affects most mammals and is one of the costliest ongoing challenges in modern dairy farming. The most significant challenges in mastitis management are the speed and accuracy of diagnosis and the use of antibiotics. Current mastitis diagnosis in veterinary practices in the United Kingdom utilises culture-based techniques. However, this approach has some limitations including; processing time, species selection biases and culture failure. This ongoing PhD project seeks to assess the utility of high-throughput sequencing technology in addressing the current challenges in mastitis diagnosis. Samples were obtained from cattle from farms in North Yorkshire, United Kingdom. Cattle were selected from monthly somatic cell count records as mastitis positive cases with a cell count between 250 000 and 550 000 cells ml−1 or control animals with a count below 200 000 cells. Mastitis positive cases were further split into bacteriology positive and negative groups. During sampling a cow-side somatic cell test (California Milk Test) was performed to confirm the monthly database readings. All farms sampled were conventional dairy farms with comparable management systems. Samples were collected by trained veterinarians following a pre-defined protocol designed to limit sample contamination. Microbial community diversity, richness and composition will be compared between sample groups to survey for variations which may explain why culture fails in culture negative subclinical cases. 131 sample animals have been assigned to study groups with samples collected, DNA extracted and prepared for sequencing on the Illumina MiSeq platform with results expected in January 2019.
Comparison of MRSA skin infection models with HaCaT keratinocytes and a 3D-organotypic skin model
Authors: Barua Nilakshi
Published: April 2019

Background and Aims

Skin is a major site for Staphylococcus aureus colonization and invasion. A 3D-organotypic skin model was generated by co-culturing keratinocytes with fibroblasts at air-liquid interface enabling the proliferation, migration and differentiation of the keratinocyte to mimic terminal differentiation of the epidermis.

Methods

The adherence and internalization of MRSA strain types ST8, ST30, ST59, ST22, ST45 and ST239 were evaluated in the HaCaT keratinocytes and a 3-D organotypic skin model. Acridine orange staining and/or anti-Staphylococcus aureus antibody were used for bacterial localization. TUNEL assay was used to evaluate cell death due to apoptosis.

Results

Maximum adherence to HaCaT cells were exhibited by both MRSA ST59 and ST8 strain types (P=0.129, Tukey post-hoc test). The maximum percentage of internalization was exhibited by both ST59 and ST30. With ST8, ST30, ST59 and ST239 types, bacteria were present within the cytoplasm whereas localization of ST22 and ST45in the phagosomes of HaCaT keratinocytes were observed. Study of cell death in HaCaT keratinocytes was limited to 24 h due to dislodgement of the keratinocytes with all six strains. The 3-D skin model proved to be better to study MRSA transmigration and cell death which could be monitored for longer time points. ST59 exhibited maximum adhesion and internalization.

Conclusion

The 3-D skin model proves to be a better model to study MRSA skin infections.
Generation of a recombinant GFP-tagged infectious bronchitis virus (IBV)
Authors: Webb Isobel , Keep Sarah , Stuart Jamie , Oade Michael , Britton Paul , Bickertron Erica
Published: April 2019

Infectious Bronchitis Virus (IBV) is a highly contagious gammacoronavirus which infects poultry. Using reverse genetics, enhanced green fluorescence protein (eGFP) has been inserted into a pathogenic strain of IBV, M41, in place of a newly identified open reading frame (ORF), ORF7. ORF7 has been identified in several IBV strains including M41 and apathogenic strain Beau-R. ORF7 is located immediately downstream of the N gene, preceding the 3’ UTR. This region has been chosen because we are interested in investigating whether a protein can be transcribed from the associated transcription regulatory sequence (TRS-B). M41 has a deletion at the 3’ end of the genome and does not encode all of ORF7, suggesting it is not essential for the viral replication and is not required for a pathogenic phenotype. The TRS-B, TAACA for ORF7 in Beau-R, is non-canonical and is located after the stop codon for the N gene. In M41 only part of the TRS-B is present, TAA, so nucleotides CA were added alongside the eGFP sequence. A number of viruses were successfully rescued. Growth kinetics in primary Chicken Kidney cells (CK) were comparable to the parent virus M41-K. The stability of the eGFP sequence has been assessed in CK cells and in embryonated eggs. The newly constructed TRS-B however was not utilised suggesting that it is not solely the TRS-B that controls the transcription of ORF 7.
Discovering the biology behind the organism while developing genetic tools for Corallochytrium limacisporum
Authors: Kozyczkowska Aleksandra , Najle Sebastian , Ruiz-Trillo Iñaki , Casacuberta Elena
Published: April 2019

To address biological questions that cannot be answered by current model organisms, we need to develop genetic tools in the specific taxa that can provide the best answers. Such is the case of the origin of animals in which, genetic tools need to be developed among the closest unicellular relatives of animals. To fill this gap, we are developing genetic tools in Corallochytrium limacisporum, a close unicellular relative of animals that also has a fascinating biology. Corallochytrium is a marine free-living walled saprotroph that develops through a choenocyte. Moreover, because of the basal phylogenetic position of Corallochrytria together with Ichthyosporea, this lineage is especially informative to fill the void of information between yeast and metazoans. We have successfully developed transient and a stably transfection protocols by introducing the resistance gene to puromycin, allowing us to select individual transformants. Deep characterization of the established transformed lines has revealed important biological features of this organism such as the plasticity of its genome, the mode of plasmid integration, and some differences between the two known strains (Hawaii and India). Currently we are better characterizing these features and, in parallel, developing genome-editing technologies. Progress and the potential implications of our research will be presented and further discussed.
Investigating virus diversity in humans and non-human animals in Vietnam
Authors: Ashworth Jordan , Lu Lu , Woolhouse Mark ,   on behalf of the VIZIONS Consortium
Published: April 2019

Despite ∼60 % of human pathogens being of zoonotic origin, the diversity of viruses in non-human hosts remains understudied. To investigate the viral diversity present in Vietnam, the Vietnam Initiative on Zoonotic Infections (VIZIONS) collected 2100 rectal swabs and faecal samples from several non-human hosts (including pigs, rats and bats) as well enteric hospital patients and individuals with high animal contact. Samples underwent metagenomic sequencing (Illumina HiSeq), assembly (MetaSPAdes) and screening for viral sequences (Blastn and DIAMOND Blastp). The majority (87.5%) of viral sequences identified in humans shared high nucleotide identity (>95 %) to previously described viral species, with the exception of sequences assigned to Anelloviridae and Picobirnaviridae. Approximately half (46.5%) of viral sequences identified in non-human hosts shared between 60 to 80% nucleotide identity to its closest match in GenBank. For all hosts other from rats the family with the highest frequency of low identity hits was Picobirnaviridae. Sources of diversity at a viral family level were mostly host specific, with pigs having diverse astrovirus and smacovirus sequences, rats for rotavirus and adenovirus and bats for coronavirus and parvovirus. These results highlight the historic bias of sequencing viruses from human hosts and the need to shift focus to non-human hosts. Pig, bats and rats in Vietnam harbor a large diversity of potentially novel viruses that may be threats to animal and human health.
Investigation of a hospital Enterobacter cloacae NDM-1 outbreak using whole genome sequencing
Authors: Fairley Derek , Protaschik Yuri , Finn Ruth , Turton Jane , Doumith Michel , Elbaz Wesam , Hedderwick Sara , Bradley Declan
Published: April 2019

Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.
The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James’s Hospital, Dublin
Authors: Griffin Auveen , Dunne Katie , Talento Alida , Rogers Tom
Published: April 2019

Triazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes – previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. VIPcheck™ plates provide a simple agar based screening method. Each 4-well plate contains a growth control (GC) well and 3 wells containing itraconazole (4 mg l−1), voriconazole (2 mg l−1) and posaconazole (0.5 mg l−1). Briefly, 25 µl of a 0.5-2 McF suspension AF is inoculated into each well and plates are read after 48 h incubation at 37 °C. Any growth in a triazole containing well is suggestive of resistance. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints. We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples. A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=18 resistant to ≥1 triazole drug as previously determined by AFST and/or molecular methods). The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells. Our results suggest that the VIPcheck™ plate is a reliable screening method for triazole resistance.
Creation and characterisation of probiotic libraries for use in poultry and pigs
Authors: Brown Helen , Pursley Isabella , Horton Daniel , Ragione Roberto La
Published: April 2019

Probiotics are defined as live micro-organisms which provide the host with a growth or health advantage. In recent year’s with the emergence of antimicrobial resistance, probiotics, and in particular Lactobacillus, have become increasingly popular as an alternative control strategies for bacterial pathogens. Recent studies have demonstrated that Lactobacillus is not only able to increase the growth rate of animals, an important consideration for commercial farming entities, but is also able to inhibit the colonisation of animals with pathogenic bacteria species such as Salmonella, Campylobacter and Escherichia. This study aimed to isolate and characterise lactobacilli isolates from commercially reared chickens and pigs. Particular attention was paid to the probiotic potential of the isolates. Our approach combined molecular techniques with in vitroscreening to rapidly identify this potential. To date 80 and 105 isolates have been collected from pigs and chickens, respectively. All isolates have been confirmed to belong to the Lactobacillus genus by PCR, speciated by 16S sequencing and determined to be clonally unique using RAPD PCR. Isolates were subsequently tested for their ability to tolerate low pH, bile, aerobic and anaerobic conditions before assessing their AMR profile and determine their ability to inhibit a panel of clinical and type strains of pathogenic bacteria. Isolates identified as potential probiotics are currently undergoing whole genome sequenced as per EFSA guidelines. This study has demonstrated that lactobacilli isolates with suitable probiotic properties can be isolated from commercial poultry and pigs. Furthermore, these isolates may prove useful as control strategies for zoonotic bacterial pathogens.
Influence of gestational and developmental age on human airway epithelial innate immune responses to Respiratory Syncytial Virus (RSV) in early life
Authors: Groves Helen , Broadbent Lindsay , Guo-Parke Hong , Shields Mike , Power Ultan
Published: April 2019

Background

RSV is a major respiratory pathogen in infants, with preterm and young infants being at increased risk of severe disease. Airway epithelial cell (AEC) innate immune responses represent an important first line of defence. Development of these responses in early life is poorly understood.

Methods

Well differentiated cultures were generated using nasal AECs harvested from term and preterm infants at birth and from the same infants at one-year old. Cultures were infected with RSV and resulting cytopathology and innate immune responses studied.

Results

RSV growth kinetics were similar between preterm and term newborn cohorts at birth and in the one-year-old repeat cohort. Secretion of interferon lambda-1 (IFN-l1), CXCL10 (IP-10), CCL5 (RANTES) and CXCL8 (IL-8) were similar between RSV-infected preterm and term newborn AECs. Following RSV infection, significantly higher secretion of IFN-l1 (P=0.0034), CXCL10 (P<0.05) and CCL5 (P<0.05) was noted in one-year-derived AECs compared to newborn AECs. We also identified differential expression of a novel endogenously expressed anti-RSV protein, pleiotrophin (PTN), which interacts with nucleolin, a cofactor for RSV entry. PTN expression/secretion was significantly decreased in preterm versus term AECs (P=0.014) and in newborn compared to one-year WD-PNECs (P=0.0008).

Conclusion

These exciting findings represent the first description of age- and prematurity-related differences in AEC innate immune responses, demonstrating greater RSV-induced expression of pro-inflammatory chemokines with increasing age and developmental differences in expression of the novel anti-RSV protein PTN. These findings may, in part, explain the increased susceptibility of preterm and very young infants to severe RSV disease.
St. Abb’s Head phlebovirus – a separate virus species or a strain of Uukuniemi phlebovirus?
Authors: Szemiel Agnieszka M , Willett Brian J
Published: April 2019

St. Abb’s Head virus (SAHV), a member of the genus Phlebovirus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments. SAHV was isolated from a pool of seabird ticks (Ixodes uriae) collected at a seabird colony in St. Abb’s Head National Nature Reserve, Berwickshire, Scotland in 1979. Antigenically, SAHV appeared to be related to the Uukuniemi serogroup of phleboviruses. Similarly, the proteins of SAHV shared similar biochemical properties to Uukuniemi phlebovirus. Here, we describe an in depth molecular characterisation of SAHV. Using next generation sequencing technology, we demonstrate that SAHV is very closely related to the Uukuniemi phlebovirus (UUKV). We examine the growth of SAHV in mammalian, avian and tick celllines and define its target cell tropism.
Do uropathogenic E. coli require changes before it can spread into the bloodstream?
Authors: Goswami Cosmika , Fox Stephen , Leanord Alistair , Evans Thomas
Published: April 2019

Background

Uropathogenic Eschericha coli are the leading cause of urinary tract infections (UTIs). The microbe can spread from bladder to kidney and finally to blood. We wished to determine whether genetic changes accompany the passage of these infections from urine to blood.

Material/methods

12 paired urine and blood samples were collected from patients in Greater Glasgow and Clyde; the interval between sample collection time between pairs was less than 48 h. Whole genomic sequencing of these paired samples was performed using the Illumina MiSeq platform. De novoassembly of reads was carried out using Shovill assembler; whereas SNPs were identified using SMALT, VarScan and Gubbins tools.

Results

Urine and blood samples in each pair had the same MLST type. Surprisingly, however, there were multiple differences in the presence of plasmid genes, phage elements and insertion sequences within pairs, as well as numerous SNPs. For example, the mercury reductase merAgene and mercuric transport protein merPhave been acquired in a plasmid of a blood isolate compared to the contemporaneous urine sample. We also identified missense mutations in genes involved in several metabolic pathways in bloodstream isolates. Several of the observed gene deletions/insertions and SNPs were found in more than one of the paired blood and urine isolates.

Conclusions

The observed sequence differences between contemporaneous blood and urine isolates suggests that genomic differences accumulate within the urinary bladder prior to blood stream invasion. The observed blood stream variants may thus possess a selective advantage in invasion and/or survival within blood.
The chitin attenuator: the Ca2+/calcineurin pathway maintains the viability of Candida albicans cells with supra-normal chitin levels
Authors: da Silva Dantas Alessandra , Gow Neil , Walker Louise
Published: April 2019

Chitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.
The Candida albicans arginase family encodes enzymes with diverse catabolic activities that differentially influence host–fungus interactions
Authors: Schaefer Katja , Bates Steven , Ames Ryan , Christou Stella , Gow Neil
Published: April 2019

In the blood stream, arginine is an essential amino acid that is required by phagocytes to synthesize iNOS. Previously we showed that the fungus Candida albicans induces host arginase production that diverts arginine from the pathway that leads to the production of nitrite oxide. We therefore investigated whether C. albicans arginase activity also contributed to the protection of the fungus by competing for arginine during infections. Three C. albicans genes had been annotated as putative arginase encoding genes. Heterologous expression of these genes suggested all three had some arginase activity and one gene product (Car1) encoded a bone fide arginase that was required for growth on arginine. However, single and double mutations in the two other genes (AGM1 and GBU1) did not affect growth on arginine as a single nitrogen source and were found instead to encode agmatinase and guanidinobutyrase respectively that participate in two other pathways related to arginine metabolism. This family of three enzymes therefore exhibits mixed biochemical activities and collectively participate in the catabolism of exogenous and endogenous sources of arginine. Virulence of the triple mutant lacking all three genes was reduced in a Galleria infection model, but single or double mutants were fully virulent. None of the single or multiple mutants affected host NO production suggesting they do not influence the oxidative burst of phagocytes. In addition, CAR1 expression was required for hyphal growth. This family of enzymes therefore represent a novel enzyme set that is essential for growth in vivo and indirectly for fungal virulence.
The effect of dietary fatty acid supplementation on gut microbiome development in weaning piglets
Authors: McKenna Robyn , McAdam Paul , Smyth Victoria , Robinson Mark , Cosby Louise
Published: April 2019

The gastro-intestinal tract hosts a complex microbial ecosystem that helps regulate the physiological, immunological and nutritional functions of the pig so disturbances within this microbiota can have profound effects on porcine health and disease. The gut microbiota is shaped by the environment, immune pressures and diet. The weaning transition represents a critical time-point that can interfere with intestinal development and cause dramatic shifts in the gut microbiota. Previously, in-feed antibiotics were used to counteract the adverse effects of weaning but an EU-wide ban since 2006 has propelled the search for safe and sustainable alternatives within the livestock industry. Studies have indicated that dietary fatty acids are efficient in terms of minimising weaning disorders including elevated incidence of enteric disease and immunodepression and thus represent a promising alternative to antibiotics. Furthermore, previous research has indicated that including fatty acid mixtures in the porcine diet has a positive effect on overall animal performance, increasing growth rate and enhancing feed conversion ratios. It is thought that these health benefits and production gains stem from the ability of fatty acids to mediate the effects of gut microbiota on intestinal immune function through modifying the proportions of microorganisms present. The aim of this research was to use deep sequencing and metagenomic methods to explicitly explore the influence fatty acid supplementation has on shaping the intestinal microbiome during weaning. Results have indicated that fatty acids play a role in modifying the intestinal microflora, establishing an environment that favours the growth of commensal species such as Lactobacillus.
An investigation of the molecular and cellular mechanisms of in vitro phage therapy in human cells
Authors: Møller-Olsen Christian , Ho Siu , Ross Toby , Sagona Antonia
Published: April 2019

As the consequences of increasing bacterial resistance to traditional antibiotics become evident, the importance of research and development of therapeutic alternatives is apparent. Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host. However, whether and how bacteriophages can kill intracellular bacteria in a human cell environment remains elusive. E. coli strains displaying the K1 polysaccharide capsule virulence factor, are nosocomial pathogens responsible for urinary tract infections (UTIs), neonatal meningitis and potentialprecursors for septicaemia. These different types of infections were modelled in vitro by infecting human bladder epithelial cells (T24 HTB-4) and humancerebral endothelial cells (hCMEC/D3) with E. coli EV36, a strain displaying the K1 capsule. The infected human cells then received in vitro phage therapy using bacteriophage K1F that specifically targets E. coli strains displaying the K1 capsule. The rate of bacterial infection and the molecular and cellular mechanisms of in vitro phage therapy was analysed by means of flow cytometry, confocal and live microscopy. We show that rfp-tagged E. coli EV36-RFP and gfp-tagged bacteriophage K1F-GFP, enter the human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP can efficiently kill intracellular E. coli EV36-RFP in human urinary bladder epithelial cells and humancerebral endothelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy. Collectively this data contribute evidence-based knowledge for the ongoing development of phage therapy.