bluuebunny/crossref_metadata_2025 huggingface dataset:
The MtrAB-LpqB two component system links development with secondary metabolite production in Streptomyces species and can be manipulated to switch on silent secondary metabolite clusters
Authors: Som Nicolle F. , Heine Daniel , Holmes Neil A. , Munnoch John T. , Knowles Felicity , Chandra Govind , Seipke Ryan F. , Hoskisson Paul A. , Wilkinson Barrie , Hutchings Matthew I.
Published: April 2019

Streptomyces species are important producers of bioactive compounds such as antibiotics, antitumor and immunosuppressant drugs. Around two-thirds of all known natural antibiotics are produced by these bacteria and antibiotic production is linked to sporulation. The discovery of new bioactive compounds has declined since the 1960s but genome sequencing has revealed the potential to identify many more bioactive compounds. They are many secondary metabolite gene clusters which are inactive (silent) under laboratory conditions. We characterize the highly conserved actinobacterial two component system MtrAB which coordinates sporulation with secondary metabolite production in the two model organisms Streptomyces venezuelae and S. coelicolor. Deletion of the histidine kinase gene mtrB resulted in increased production of the antibiotic chloramphenicol in S. venezuelae and actinorhodin and undecylprodigiosin in S. coelicolor. Chloramphenicol is not usually produced under laboratory condition which suggests that deleting mtrB can activate silent antibiotic clusters. Additionally, we introduced point mutations at the D56 phosphorylation site of MtrA by CRISPR-Cas9 to abolish phosphorylation. This mutant shows the same phenotype as the ΔmtrA strain and chloramphenicol production is increased. Chromatin immunoprecipitation and sequencing (ChIP-seq) was used to identify MtrA targets and revealed that MtrA likely controls secondary metabolite production by binding to the promoter regions of cluster situated regulators in both model organisms. Additionally, MtrA binds upstream of genes involved in DNA replication and cell division. To our knowledge this is the first evidence of the connection of development and secondary metabolite production by a two component system.
Marine fungal dark matter in the global ocean
Authors: Chrismas Nathan , Cunliffe Michael
Published: April 2019

Marine fungi are a major part of ‘microbial dark matter’, with most organisms known from sequence data and currently not in culture. Interest in marine fungi has substantially increased over the past decade, and studies using culture independent methods have indicated that fungal diversity in the oceans may be greater than previously estimates based on cultivation alone. There remains much to learn about the true diversity of marine fungi in the global oceans and the ecological roles that they could play. The Tara Oceans expedition has allowed for significant advancements in our understanding of the global diversity of planktonic microorganisms. Interrogation of the Tara Oceans 18S rRNA gene dataset for fungal sequences shows that fungi are found throughout the global oceans, appearing in all marine regions covered by the Tara Oceans expedition. In the survey, Ascomycota and Basidiomycota were common, while some locations had increased abundances of Chytridiomycota. Differences in community composition were observed between oceanic regions and, although clear signals were not apparent due to the nature of the sampling, there was some indication of community variation between upwelling, coastal, shelf and gyral provinces. Different size sampling fractions appeared to capture different portions of the pelagic fungal community. These findings highlight a number of ecological questions: How important are oceanic currents in determining fungal biogeography? What is the relationship between marine fungi and biogeochemical processes? What is actually there? By targeting these questions directly, we will be able to bring the dark matter of marine fungi into the light.
Stage-specific gene regulation in Perkinsus olseni parasites
Authors: Zielinski Jana , Einarsson Elin , Lassadi Imen , Waller Ross
Published: April 2019

Perkinsus species are important marine parasites of molluscs including mussels and oysters with substantial commercial and environmental impact. Perkinsus forms a sister lineage to the apicomplexan parasites (e.g. malaria agent, Plasmodium) and share similar invasion-related structures with these better studied pathogens. However, much less is known of the transmission and invasion biology of Perkinsus spp. We are developing genetic tools to study these parasites, and Perkinsus is emerging as an experimental model for marine parasites. The life cycle of Perkinsus species start with motile zoospores that are ingested by the host via filtration of sea water. After infection of the mollusc, these cells develop into a replicating non-motile vegetative form of the parasite, the trophozoite. Upon host death or morbidity, the parasites are released back into the water column where they differentiate into zoosporangia that release up to 100 motile zoospores. In vitro, Perkinsus species can be maintained as trophozoites in an axenic sea water-based media, and this is the best studied form of the parasite. However, Perkinsus olseni, can be triggered to sporulate in vitro using Ray’s fluid thioglycollate media, and we seek to better study this phase of the lifecycle that is essential for disease spread. To do this, we are determining stage-specific gene expression, during the transformation from trophozoites to zoospores, by RNA-Seq. This will both identify genes that are specific to this growth stage, and also identify zoospore-specific promoters that can be used in experimental manipulation and study of this poorly known cell stage.
Understanding aurodox: A Type III secretion system inhibitor from Streptomyces goldiniensis
Authors: McHugh Rebecca Elizabeth
Published: April 2019

Aurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis, has been shown to inhibit the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS). To further assess the utility of this molecule as an anti-virulence compound, a better understanding of its mechanism of action is required. We used whole transcriptome analysis, cell infection and GFP-reporter assays to show that Aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island-which encodes for the T3SS, acting via its master regulator, Ler. We have also observed similar effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). These properties suggest Aurodox may have potential for the treatment of E. coli infections of the gut. Despite the recent interest in the compound, the biosynthesis of Aurodox by Streptomyces goldiniensis is still poorly understood. To gain insight in to this, we have sequenced the whole genome of S. goldiniensis and identified a putative Aurodox biosynthetic gene cluster (BGC) which shares a high level of functional homology with the BGC encoding Kirromycin, a non-methylated Aurodox derivative. In-depth analysis of the BGC supports a model where a unique polyketide synthase pathway involving a combination of both Cis and Trans-Acyltransferases synthesise the Aurodox polyketide backbone, followed by decoration and finally the addition of a methyl group. Future work will include the heterologous expression this BGC to confirm its role in Aurodox biosynthesis, with the ultimate aim to produce novel Aurodox derivatives.
Investigating the antimicrobial efficacy of MSCs as a potential novel therapy for Mycobacterium avium pulmonary infection
Authors: Shaw Timothy , Schroeder Gunnar , Krasnodembskaya Anna , O’Kane Cecilia
Published: April 2019

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) has rapidly increased in global prevalence over the last two decades. NTM-PD occurs mainly in patients with pre-existing structural lung disease and current treatment strategies are often ineffective and poorly tolerated. Outside of the cystic fibrosis population, the most common NTM isolates are from Mycobacterium avium complex (MAC). Mesenchymal stromal cells (MSCs) have potent antimicrobial and immunomodulatory properties including direct microbial killing and enhancement of phagocytic function. Their effect on MAC species is unknown. Human MSCs were infected with M. avium at a multiplicity of infection (MOI) of 2. Human monocyte-derived macrophages (MDMs) were also infected with a clinical isolate of M. avium at MOI of 2. After 4 h, MSCs were added at a ratio of 1 MSC:3 MDMs. After 24 and 74 h, colony counts were performed on supernatants and cell lysates. MSCs reduced total bacterial counts of M. avium by 24 % at 24 h (from 295×103/ml to 225×103/ml, P<0.05) and 40 % at 72 h (from 403×103/ml to 243×103/ml, P<0.05). MSCs reduced total bacterial counts of M. avium in infected MDMs by >40 % (from 381×103/ml to 209×103/ml, P<0.05) after 24 h and >70 % after 72 h (from 1050×103/ml to 314×103/ml, P<0.05). MSCs have modest direct antimicrobial effect against MAC, but potently enhance their killing by macrophages. Mechanistic studies are required to understand the mechanisms of the antimicrobial effect, with the aim of exploiting these therapeutically in pulmonary MAC disease.
Identification of novel host factors influencing human cytomegalovirus replication using a two-step siRNA screen
Authors: Lee Chen-Hsuin , Griffiths Samantha , Digard Paul , Pham Nhan , Auer Manfred , Haas Juergen , Grey Finn
Published: April 2019

As an obligate intracellular parasite, human cytomegalovirus (HCMV) completely relies on host machinery to replicate. Understanding which host factors are required for virus replication contributes to our understanding of virus biology and cell biology, identification of potential targets for antiviral therapy. High-throughput small interfering RNA (siRNA) screens are a powerful approach to identify novel host-virus interactions. Conventional screens often use reporter genes as a proxy for virus replication, rather than measuring production of infectious virus. We developed a two-step siRNA screen that independently measured primary replication and virus production. Screening with a library targeting almost 7000 genes, we identified 37 genes involved in early stages of HCMV replication and 15 genes specifically involved in later aspects, such as late gene expression, assembly and egress. These include factors in ubiquitin-dependent protein degradation pathway, and components of the mediator complex. Furthermore, we showed that the induction of SIN3A, a transcriptional regulator that forms a repressor complex with histone deacetylase 1 and 2, is essential for late gene expression and virus production. This study demonstrates a powerful two-step high throughput approach which identifies key host factors underpinning HCMV replication and informs our understanding of how the virus interacts with its host.
Illuminating the molecular choreography of multi-segmented RNA genomes
Authors: Borodavka Alex , Strauss Sebastian , Coria Aaztli , Dykeman Eric , Jungmann Ralf , Lamb Don
Published: April 2019

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.
Identifying the role of complement receptor 2 (CR2) on follicular dendritic cells (FDCs) in the persistence of foot and mouth disease virus (FMDV)
Authors: Gordon Lucy , Mabbott Neil , Charleston Bryan , Perez Eva
Published: April 2019

More than 50 % of cattle (regardless of foot and mouth disease (FMD) vaccination) become persistently infected for long periods of time (years) after exposure to FMD virus (FMDV). The mechanisms associated with establishment of persistent infections are still poorly understood. I am testing the hypothesis that complement receptor 2 (CR2) on follicular dendritic cells (FDCs) located in the germinal centers of the lymphoid tissue, are involved in the trapping and long-term persistence of FMDV and that this persistence is essential for the maintenance of long-term antibody responses to FMDV. The aim of this study was to assess FMDV antigen retention and the generation of the specific immune response in vivo, in a mouse model. Groups of mice were treated with an anti-CR2 monoclonal antibody, named 4B2, which has been described previously to block CR2 long-term in vivo, blocking for 6 weeks after a single injection of 2 mg (Kulik et al., 2015). After treatment with 4B2, animals were infected with FMDV and lymphoid tissues and serum samples evaluated for the presence of antigen and the humoral immune response, respectively. The ability of 4B2 to block the binding of FMDV and PAP immune complexes (ICs) to FDCs in vitro as well as results about the role of FDC in trapping FMDV via CR2 in vivo is currently being investigated and will be discussed.
Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens identifies isogenic strains in multiple outbreaks and novel virulence-related features
Authors: Kiu Raymond , Caim Shabhonam , Pickard Derek , Dougan Gordon , Painset Anais , Swift Craig , Mather Alison , Amar Corinne , Hall Lindsay
Published: April 2019

Background

Clostridium perfringensis a major enteric pathogen known to cause gastroenteritis in humans. Although major outbreak cases are frequently reported, to date no Whole Genome Sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of C. perfringens-associated outbreak strains.

Methods

We have applied both genomic and phylogenetic analyses on a sub-set of 109 newly-sequenced C. perfringens strains isolated from gastroenteritis-associated disease cases (including food-poisoning and care-home diarrhoea) from England and Wales between 2011 – 2017 to probe virulence profiles including toxin and AMR genes, plasmid features and genomic epidemiology of these case isolates.

Results

Our data identified that highly-similar C. perfringens strains were associated with 9 care home-associated individual outbreaks over a 5 year interval, indicating potential common sources linked to these distinct outbreaks. Enterotoxin gene cpe was encoded in all but 4 isolates (96.4 % type-F strains), and it was further determined that virulence plasmids encoding cpe were extensively distributed in the isolates (97 % care-home isolates carry pCPF5603 plasmid; 60 % food-poisoning isolates carry pCPF4969 plasmid). Further virulence factors, such as β2-toxin, were enriched in these isolates (46.7 %). Phage proteins were also commonly identified, with additional analysis indicating phages may contribute to spread of virulence determinants.

Conclusion

This study highlights the genotypic and epidemiological relatedness of a large collection of C. perfringens strains isolated from gastroenteritis-associated cases from across the UK and Wales. Key points revealed include the potential circulation of disease-associated strains, and impact of cpe-encoding-plasmid disseminations, linked to outbreak cases.
Phylogenetic and genomic characterisation of narnaviruses: a diverse group of non-encapsidated RNA viruses
Authors: Dinan Adam , Olendraite Ingrida , Brown Katherine , Firth Andrew
Published: April 2019

Narnaviruses are a group of single-stranded, positive-sense RNA viruses, which are non-encapsidated and hence normally transmitted vertically. Narnaviral genomes encode a single protein: the RNA-dependent RNA polymerase (RdRp), which catalyses viral replication. Currently, there are just two recognised species within the genus Narnavirus, both of which are known to infect Saccharomyces yeast. Here, we systematically identify and characterise narnaviral genomes in public sequence databases, using a combination of in silicoapproaches. We identify two major clades of narnaviruses, and propose the establishment of a taxonomic framework based upon their molecular characteristics. Codon usage bias across both clades was analysed and compared with those of potential host taxa from across the eukaryotic domain of life. In one clade, we demonstrate the widespread presence of a long reverse-strand open reading frame (rORF), which typically occupies >90 % of the full-length genomic RNA. Comparative analysis shows that the putative rORF-encoded proteins are highly divergent in amino acid composition, with a central region of increased conservation. These findings shed new light on one of the most divergent clades of eukaryote-infecting viruses.
Understanding the adaptive response of Streptomyces coelicolor to the glycopeptide antibiotic teicoplanin
Authors: Connelly Samuel , Hong Hee-Jeon
Published: April 2019

Glycopeptide antibiotics are clinically important as a second-line therapy for the treatment for nosocomial infections caused by Gram-positive pathogens. A universal mode of action for glycopeptide antibiotics is to target the terminal residues, d-Alanyl-d-Alanine on the cell wall peptidoglycan intermediate lipid II, arresting the later stages of peptidoglycan biosynthesis. This weakens the cell wall, making it susceptible to rupture. A general resistance mechanism for glycopeptide antibiotics requires the core genes, vanRSHAX, that detect the presence of a glycopeptide (VanS) and upregulate genes (VanR) which orchestrate the remodelling of d-Ala-d-Ala on lipid II to d-Ala-d-Lactate (VanHAX). Glycopeptide affinity for lipid II is reduced by 1000-fold and biological activity impaired. Our previous study has shown that altering the termini of peptidoglycan precursors by VanHAX action was not sufficient for the resistance of the glycopeptide teicoplanin in S. coelicolor, which is instead mediated mainly by VanJ. Using RNA-seq, this study is designed to understand how the adaptive response in an S. coelicolor wild type (M600) and a vanJ mutant strain differ after exposure to teicoplanin. We have also compared these data with available data on the cell wall targeting antibiotics vancomycin, bacitracin and moenoymycin. By doing so, we aim to gain insight into the molecular basis of the improved activity of teicoplanin over vancomycin as well as identify novel cellular targets of teicoplanin which can help inform the design of future glycopeptides with desirable therapeutic properties.
Intracellular survival of Enterobacter cloacae complex in human macrophages
Authors: Parau Georgiana , Bisaro Fabiana , Marshall Helina , Valvano Miguel A.
Published: April 2019

The Enterobacter cloacae complex (Ecc) is a group of enteric Gram-negative bacteria that also exist as commensals in nature. Ecc bacteria are also responsible for nosocomial outbreaks, targeting primarily immunocompromised patients and causing a wide range of systemic infections. Infection is often fatal as management is complicated by the high-level multidrug resistance expressed by Ecc isolates. Indeed, the Enterobacter species represent the last ‘E’ of the ESKAPE pathogens, which are the global leading causes of nosocomial infections. Despite the clinical relevance of Ecc, little is known about their virulence-associated properties and pathogenicity. Bridging this gap in knowledge is fundamental to provide a blueprint to better tackle Ecc infections. The aim of this study is to explore how E. cloacae interact with human macrophages and identify the bacterial and host cell factors involved in this process. Differentiated, human THP-1 monocytic macrophages were infected with fluorescent E. cloacae for various lengths of time. Macrophage infection was analysed by confocal microscopy and images processed on ImageJ and LAS X. Data suggests that approximately 90 % of intracellular E. cloacae are killed by 5 h post-infection, but the remaining 10 % of the bacterial population survives and persists up to 48 h post-infection. We have also observed E. cloacae colocalising with early and late endosomes as well as lysosomes. Our current results suggest that E. cloacae can subvert normal phagocytic trafficking and possibly adapt to survive inside the acidic lysosomal environment.
Development of a genetic modification system for the dinoflagellate Amphidinium carterae
Authors: Nimmo Isabel , Barbrook Adrian , Nisbet Ellen , Howe Chris
Published: April 2019

Dinoflagellates are important marine algae, being an essential symbiont in corals. Loss of the dinoflagellates causes coral bleaching, and ultimately death of coral reefs. Current efforts to study dinoflagellates are greatly hindered by the lack of a reliable method of genetic transformation. The chloroplast genome of dinoflagellates is fragmented into multiple plasmid-like minicircles, each carrying at most a few genes. We have used ‘artificial’ minicircles, based on fusions between endogenous minicircles and E. coli plasmids to establish a transformation system and optimize parameters for it. We have determined the sensitivity of wild type dinoflagellate strains to possible selective agents. We have compared the performance of a number of transformation methods, including electroporation and particle bombardment, and find that electroporation is not effective for transformation, whereas particle bombardment (‘biolistics’) is. We have assessed different parameters for biolistics and subsequent selection. We have shown the successful maintenance of sequence from an artificial minicircle within a dividing cell population over a period of six months, as well as evidence (using RT-PCR) of transcription and the expected phenotype for the inserted gene. This represents a significant step forward in developing the genetic modification of Amphidinium carterae. We are currently testing the protocol with other dinoflagellates.
Understanding virus resistance due to ISG15-loss-of-function
Authors: Vasou Andri , Holthaus David , Andrejeva Jelena , Bamford Connor , McLauchlan John , Randall Richard , Hughes David
Published: April 2019

Viral infections induce profound cellular responses resulting the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, while others regulate the cellular response. For most viruses, the specific antiviral ISG(s) is not known, which has potential consequences for the quest for new therapeutics. The ubiquitin-like protein ISG15 is a major regulator of antiviral response and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where patients exhibit elevated ISG expression in the absence of infection. Using CRISPR/Cas9 knockout technology, we have recapitulated these effects in cultured cells, confirming ‘free’ ISG15’s role as a central regulator of type-I IFN antiviral response. We also show that during an antiviral response, ISG15-deficiency leads to significant physiological defects (inhibition of translation and proliferation) and resistance to parainfluenza viruses. We asked if virus resistance was due to the direct antiviral activity of ISGs, or whether cells were non-permissive due to physiological defects. We took advantage of the knowledge that IFIT1 is the principle antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in ISG15-deficient cells, confirming that resistance was due to the antiviral response and not due to physiological state related to ISG15-deficiency. We also compared infections with related viruses where IFIT1 has known intermediate antiviral activity (PIV2) and low activity (PIV3); restoration of replication with these viruses reflected their sensitivity to IFIT1 restriction. Based on the observations in IFIT1-knockdown cells, we propose a novel platform for the identification of antiviral ISGs based on recovery of virus infection.
Of mice or men? Developing an ex vivo model of Staphylococcus aureus infection in the cystic fibrosis lung
Authors: Sweeney Esther , Harrison Freya , Guzman Katerina , Tormo-Mas M. Angeles
Published: April 2019

Staphylococcus aureus is one of the dominant organisms isolated from the airways of cystic fibrosis (CF) patients, particularly early in life, and is usually regarded as pathogenic. However, there remains significant gaps in our understanding of the role of S. aureus in the progression of pulmonary infection and lung disease in CF. Mouse models of S. aureus lung infection, even in CF animals, frequently demonstrate pneumonia and abscesses of the lung, a phenomenon very rarely observed in people with CF. Furthermore, live host models are associated with high costs and are limited in duration and sample size for ethical reasons. Most in vitro models fail to consider the influence of host tissue interaction or spatial structure on the development and persistence of infection. We have previously described an ex vivo pig lung model (EVPL) of cystic fibrosis for Pseudomonas aeruginosa lung infection. Here we show the progression of this model to support the growth of Staphylococcus aureus. Our data suggests that, in our model, S. aureus cells may preferentially aggregate in artificial sputum rather than adhere to lung tissue. In the context of historical case reports, this result potentially reflects the clinical situation in cystic fibrosis more accurately than mouse models and could have substantial clinical significance.
Co-selection of antibiotic resistance caused by a legacy of PTE pollution in Gram-negative bacteria
Authors: Tonner Rebecca , Peshkur Tanya , Rodgers Kiri , MacLellan Ian , Williams Roderick , Hursthouse Andrew , Henriquez Fiona , Knapp Charles
Published: April 2019

Antimicrobial resistant bacteria can become harboured in sediments of post-industrial estuaries. Subsequently, their resistance traits could be enriched by pollutants deposited in the sediments. Recent evidence strongly suggests this may pose hazards that not only affects the health care sector, but could also impact tourism and the aquaculture industries. The River Clyde, UK was chosen for this study due to its extensive industrial history, and three sites were chosen to sample from representing different levels and types of industrial activities—two highly polluted and one relatively ‘pristine’ site. We extracted and analysed for metal pollutants (or ‘potentially toxic elements’, PTE), and other geochemical characteristics for all sediment cores. Gram-negative, enteric bacteria were isolated from all sediment cores from the three sites. Their susceptibilities to antibiotics and metals were assayed—determining minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The results indicate that co-selection of PTEs and antibiotic resistance does occur, and this impacts bacteria that are potential human pathogens. Higher concentrations of metals in the environment correlated to antibiotic resistance and higher MICs to metals than among bacteria found in less polluted sites. To continue to protect human health, the interactions between environmental and human health must be fully understood. This study provides critical information behind the specific causes of antibiotic resistance due to a legacy of pollution.
Occurrence and evaluation of antimicrobial susceptibility of Staphylococcus aureus isolated from chicken eggs, Eastern Ethiopia
Authors: Birmeka Jelalu Kemal , Beji Wakene , Tesfamariam Gebregeorgis
Published: April 2019

Staphylococcus aureus is responsible for a variety of infections in humans and animals that particularly causes staphylococcal food poisoning when it present in foods. This study was aimed to isolate Staphylococcus aureus present on the shell surfaces and in the contents of chicken eggs, and determine antimicrobial susceptibility patterns. A total of 335 egg samples were obtained from open market (n=174) and poultry farm (n=161). A sterile cotton swab was used to sample the surface of eggs. After sterilizing the shells, the egg contents were sampled. Isolation of Staphylococcus aureus was done based on ISO. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of the total 335 eggs sample examined, 93 (27.8 %) samples yielded S. aureus. Out of these, 28 (17.4 %) were from poultry farm while 65 (37.4 %) were obtained from open market. Similarly, 63 (18.8 %) were from the shell while 30 (8.9 %) were from the content. The level of S. aureus in egg contents was significantly higher in the open market (CI=0.0962–0.6085; P=0.003). All 76 S. aureus isolates were resistant to at least one of the antimicrobials tested with overall 3.9–92.0 % level of resistance pattern showing higher resistant to penicillin (92 %), and ampicillin (89.5 %). Multiple drug resistance was detected in 86.8 % of the total S. aureus isolates. The study showed high level of S. aureus with considerable antimicrobial resistant pattern. Further study is needed to better define bacterial resistance to antimicrobial agents with emphasis of multiple drug resistant.
Assembly of a portal-like structure in feline calicivirus following receptor engagement
Authors: Conley Michaela , McElwee Marion , Goodfellow Ian , Bhella David
Published: April 2019

The mechanism by which non-enveloped RNA viruses, such as the caliciviruses, escape the endosome, is poorly understood. The Caliciviridae are a family of viruses which include many important human and animal pathogens, most notably norovirus which causes winter vomiting disease. We used cryoEM and asymmetric three-dimensional reconstruction to investigate structural changes in the capsid of feline calicivirus (FCV) that occur upon virus binding to its cellular receptor; feline junctional adhesion molecule-A (fJAM-A). We discovered that following receptor engagement substantial conformational changes in the FCV capsid lead to the assembly of a portal-like structure at a unique three-fold symmetry axis. Atomic models of the major capsid protein, VP1, in the presence and absence of fJAM-A were calculated, revealing the conformational changes induced by the interaction. In the course of this analysis we discovered a large portal-like structure which assembles at a unique three-fold axis. The portal-like complex comprises 12 copies of the minor capsid protein VP2. We calculated an atomic model of VP2 and revealed structural changes in VP1 that lead to the formation of a pore in the capsid shell at the portal vertex. VP2 is encoded by all caliciviruses although despite being critical for the production of infectious virus, its function and structure were, until now, undetermined. We hypothesise that the VP2 portal-like complex is the method by which the virus escapes the endosome during virus entry, allowing delivery of the viral genome into the cytoplasm for replication to then ensue.
Antagonism of mosquito innate immunity by the chikungunya virus nsP3 protein
Authors: Guo Jinchao , Gao Yanni , Harris Mark
Published: April 2019

Chikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitos and causing fever, rash and chronic arthralgia. There are no vaccines or antiviral agents available for CHIKV therefore it is important to understand the molecular details of virus replication. To address this, we previously conducted a mutagenic analysis of the central alphavirus unique domain (AUD) of the CHIKV non-structural protein 3 (nsP3), testing replication of a subgenomic replicon in a variety of mammalian and mosquito (Aedes albopictus) cell lines. One mutant (M219A) exhibited a different phenotype in two Aedes albopictus cell lines (U4.4 and C6/36): replicating as wildtype in C6/36 but was blocked in U4.4. As U4.4 cells have an intact RNAi response whereas C6/36 have a frameshift mutation in the Dicer-2 (Dcr2) gene and express an inactive Dcr2 protein, we proposed that the replication of M219A was suppressed by the RNAi antiviral response in U4.4 cells, while wild type nsP3 was able to counteract this response. To further investigate this hypothesis we have extended the mutagenic analysis to screen other residues in proximity with M219 within the AUD. All of these mutants retain wildtype levels of replication in mammalian and C6/36 cells, except for W220A which replicates poorly. Evaluation of the replication of these mutants in U4.4 is ongoing. We are also pursuing a CRISPR/Cas9 approach to ablate expression of Dcr2 in U4.4 cells, and generating stable C6/36 cells expressing Dcr2 to confirm our hypothesis. Our studies will shed light on how CHIKV nsP3 can antagonise mosquito innate immunity.
Isolation of novel bacteriocins active against clinically relevant, pathogenic Clostridioides difficile
Authors: McLaughlin Yasmin
Published: April 2019

The emergence of antibiotic resistant, hypervirulent strains of the nosocomial pathogen Clostridioides difficile, coupled with increased concerns over the physiological impact of traditional broad-spectrum antibiotics on human health has led to an increased demand for alternative therapies. Bacteriocins are proteinaceous toxins produced by bacteria that kill other bacteria and their antimicrobial activity makes them an attractive potential alternative therapy to antibiotics. This research aims to isolate environmental bacteria from porcine faeces that produce novel bacteriocins with inhibitory activity against C. difficile. At present in this study two distinct organisms with protein mediated inhibitory activity against C. difficile have been isolated and it has been shown that these strains encode various bacteriocins. The current focus of this research aims to attribute the inhibitory activity to one of the encoded bacteriocins or potentially a novel antimicrobial peptide, which could then be exploited as an alternative therapy against C. difficile.
Exploring the metabolic potential of oleaginous actinomycetes in biodiesel production from cassava wastewater
Authors: Awoniyi Marcus
Published: April 2019

The depletion in fossil fuel reserves has instigated the search for renewable sources such as biofuels. Biofuel production provides a sustainable alternative to fossil fuels. However, the progression of biofuel industry has been largely affected by several uncertainties including the sustainability of production processes. Finding an economically viable process and the right substrates have been a source of constant debate. Biodiesel production is one such area which is gaining momentum in the last few decades. We are currently investigating cassava wastewater as a substrate and also as a source of oleaginous bacteria. The production of tri-acyl glycerol from mycolic acid containing actinomycetes predominantly Rhodococcus, has been identified as a potential source. These bacteria which are largely ubiquitous provide a significant amount of triglyceride when cultivated under low cost waste. The growth of oleaginous bacteria for the accumulation of triglycerides on low cost and abundant, cassava waste still remains unexplored, especially in Nigeria which is the largest producer of cassava in the world. We started our initial investigations on the cassava waste water sample following culture dependent and independent approach. We prognosticate that the identification of microflora isolated from different stages in the sample will provide a basis for understanding the nature of the substrate and the potential for the synthesis of biodiesel from cassava waste water.
Phase variation of Opa proteins in hypervirulent serogroup W meningococcal isolates
Authors: Dave Neelam , Green Luke , Lucidarme Jay , Oldfield Neil , Bayliss Christopher
Published: April 2019

Serotype W, sequence type 11 Neisseria meningitidis are an increasing cause of morbidity and mortality. The mechanisms underpinning the success of this clone remain unclear. Meningococci express up to four Opa proteins, which mediate adhesion to/invasion of host cells. Opa expression undergoes phase variation – a high frequency ON/OFF switch in gene expression due to insertion/deletions of pentameric repeats (5′-CTCTT-3′) in the coding region. NadA functions as an adhesion, and is phase-variable through tetrameric repeats in a regulatory. WGS and PCR-based fragment size analysis of 121 invasive and 51 carriage MenW:ST11 strains was conducted to determine gene expression patterns between invasive and carriage. Inactivating mutations were constructed in opa, pilE and nadA genes of two representative MenW isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 cells. We observe that four opa loci are present in all MenW:cc11 isolates and that OpaB and OpaD share 100 % sequence similarity. Repeat number variability was detected between the ‘original UK’ strain and the novel ‘2013-strain’ of the hypervirulent MenW:cc11 South American sublineage. No significant differences in the patterns of Opa expression were observed between invasive and carriage isolates. In vitro assays with pilE and nadA deletion suggest that NadA has an effect on invasion, while the double mutant shows a reduction in both adhesion and invasion. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains, but may give a niche specific advantage by enhancing phase variation-mediated immune evasion.
BactiVac, a network to support the study, development and implementation of bacterial vaccines
Authors: Cunningham Adam , Dean Johanna , Pope Susan , Balandyte Evelina , MacLennan Calman A.
Published: April 2019

Infections account for >20 % of all deaths worldwide, and are particularly problematic in low-middle income countries (LMICs), with bacterial infections killing approximately 5 million people annually. The crisis of antimicrobial resistance means our options for controlling infections are narrowing. Vaccines are a cost-effective approach to prevent infectious disease. However, there are many bacterial infections against which we lack any licensed vaccine. The BactiVac Network (birmingham.ac.uk/bactivac), was launched in August 2017 and is led by Profs Cal MacLennan and Adam Cunningham. BactiVac is a global bacterial vaccinology network established to accelerate the development of vaccines against bacterial infections, particularly those relevant to LMICs. BactiVac brings together academic, industrial and other partners involved in vaccine research against human and animal bacterial infections from the UK and LMICs. The Network fosters partnership and provides catalyst pump-priming project and training funding to encourage cross-collaboration across our membership. Full details are available on our website. BactiVac has >600 members across >60 countries with 39 % based in LMICs and 10 % in industry. Membership is free – join us at bit.ly/apply BactiVac Benefits of membership include: Access to catalyst funding – third round for pump-priming projects closes 5 May 2019, open call for training awards Invitation to subsidised Annual Network Meetings–next meeting will be held in March 2020 Access to Members’ Directory – to develop collaborations and access distinct expertise. We encourage you to become a member, be involved in this initiative, and to be part of its growing success now and in the future.
Characterisation of rpoS alleles in UPEC strain CFT073
Authors: McGarry Naoise , Smith Stephen
Published: April 2019

Escherichia coli is a major cause of urinary tract infections, bacteraemia and sepsis. CFT073 is a well-studied, prototypic urosepsis isolate. This laboratory has previously shown that strain CFT073 is serum-resistant, with resistance being imparted by factors including capsule and extracellular polysaccharides inter alia. It has become apparent that not all CFT073 strains are identical, some harbour a 5 bp insertion in the rpoS gene which results in a truncated, non-functional RpoS. In this study, we compare CFT073 wild-type and an rpoS mutant. The gene sequence of rpoS in each isolate was verified. Next, the sensitivity of the bacteria to hydrogen peroxide was determined. Finally, wild-type and mutant strains were rendered bioluminescent. Bioluminescence permits the real-time detection of viability. The contribution of RpoS to serum resistance was examined by bioluminescence.
Moonlighting in myxobacteria: measuring the covariation in GAPDH
Authors: Zwarycz Allison , Whitworth David
Published: April 2019

Myxobacteria are an order of well-known social predators that able to prey upon a wide range of microbes (including fungi and bacteria). It is currently unknown how they are able to consume such a wide range of prey, although moonlighting proteins, those possessing more than one function, appear to be involved. One moonlighter, is the virtually ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Typically present in the cytoplasm, it has also been found on the outer surface of cells, in the extracellular matrix, and secreted through membrane vesicles. In various pathogenic organisms, GAPDH has been shown to have adhesive properties, binding to several types of human connective tissues. Myxobacterial predation can be viewed as analogous to pathogenic infection, and there is evidence that GAPDH might be involved in attacking prey cells. To complement experimental analyses of the predatory role of GAPDH in myxobacteria, we undertook sequence analysis including multiple sequence alignment (MSA) and covariation analyses. Covariation calculations, using a variety of metrics and matrices, have been used to identify areas and individual residues where structural, functional or phylogenetic correlations occur within the sequence. The MSA data highlighted key conserved residues, likely involved in the catalytic reaction and potentially involved in adhesion. The covariation scores suggest compensatory mutations have taken place in discrete locations along the sequence. These results can be used to selectively mutate residues in order to determine their effect in predation.
Identification of Staphylococcus aureus and methicillin resistant S. aureus in jail populations
Authors: Whealy Ryann , Erickson Daryn , Cocking Jill , Hepp Crystal , Fofanov Viacheslav
Published: April 2019

Staphylococcus aureus is the leading cause of soft tissue and skin infection, but also causes pneumonia, endocarditis, and bone infection. The same infections can be caused by methicillin resistant S. aureus (MRSA), but are much more difficult to treat because MRSA is highly resistant to antibiotics. In ethnic and socioeconomic minority groups, methicillin resistance in S. aureus is 30 % higher than the general population. As a result, methicillin resistance is likely to have higher prevalence in jails and prisons where the demographic is disproportionately represented by ethnic minorities, individuals with lower average socioeconomic status, and less access to healthcare. Jail inmates are an understudied but very important population because diseases acquired in jails can be easily introduced into the general population and vice versa because cycling into and out of jail is very common. The presence of S. aureus is an important determinant of the development of soft tissue and other infections, making the detection of this bacteria and the identification of antibiotic resistance essential in assessing risk of disease. In this study, we identified S. aureus in samples from a United States county jail through bacterial plating and DNA extraction. Methicillin resistance was identified in S. aureus positive samples through PCR and sequencing. Our analysis has found elevated rates of S. aureus in the jail population being studied. This project will determine if rates of S. aureus and methicillin resistance in S. aureus isolated from this demographic are disproportionate to the general population.
Characterising the inhibition profile of an antimicrobial produced by Enterococcus
Authors: Cole Nathaniel , Walsh Fiona
Published: April 2019

Introduction

Antibiotic resistance is one of the greatest problems facing the 21st century with few new classes of antibiotics being discovered. The forefront of antibiotic discovery has been the soil microbiome and it is still a valuable resource for identifying microbes with possible antibiotic producing capabilities leading to novel classes of antibiotics. This justifies continuing investigation into the soil microbiome for antibiotic producing bacteria, to help tackle the growing trend in antibiotic resistance. A bacterial soil isolate was found to inhibit Enterococcus faecalis ATCC 29212 and the aim of this work was to further characterise the inhibition profile of the antibacterial.

Methods

Bacterial plug and supernatant assays were used to access the inhibition ability of a soil bacterial isolate against the WHO ‘priority pathogens’. Identification of the bacteria was carried out using 16S rRNA and whole genome sequencing. Synergy tests were carried out using broth dilution assays.

Results

The soil isolate (N5) was identified as Enterococcus and showed antibacterial activity against Staphylococcus aureus MRSA and E. faecalis VanA. This antibacterial is secreted into the supernatant which still showed inhibitory activity against MRSA and VRE. Synergy with N5 and ciprofloxacin (0.2  µg ml−1) against E. faecalis ATCC 29212 was also observed.

Conclusion

Both VRE and MRSA are important players in nosocomial infections and are displaying high levels of resistance. Further study of this antibacterial could lead to the development of a new compound to help overcome resistance mechanisms or a novel antimicrobial.
Augmenting Staphylococcal infection: the importance of timing
Authors: Carnell Oliver , Sutton Joshua , Foster Simon
Published: April 2019

Staphylococcus aureus (S. aureus) acts as a commensal in the microbiome of the skin and nasopharynx. However, on gaining access to the bloodstream it can cause an array of pathogenic outcomes. S. aureus can crowdsource the microflora to assist in becoming an opportunistic pathogen as our lab has recently published findings that co-inoculation of S. aureus with commensals, acting as pro-infectious agents, leads to a much more robust, virulent infection. This benefits S. aureus at doses where it would otherwise be cleared by the immune system. Pro-infectious agents do not need to be live commensals as isolated cell wall peptidoglycan also augments infection. This work aimed to assess the effects of inoculation with pro-infectious agents before and after infection with S. aureus. It was found that pro-infectious agents needed to be co-administered in order to fully augment infection. This gives mechanistic insight where S. aureus and the pro-infectious agents need to be in the same local environment or phagocyte to augment infection.