- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Oral Abstract
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- Non-human Pathogens
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Discovery of novel highly divergent RNA viruses in European rodents and rabbits
Although RNA viruses are likely to exist in every species of cellular life, our knowledge of their biodiversity and evolution has been focused on those that can cause disease in humans and in economically important animals and plants. Recently published studies, however, have shown that every healthy organism can carry viruses. In this study, we present the unbiased discovery of highly divergent RNA viruses in European rodents and rabbits using Next Generation Sequencing (NGS). Tissue samples from different organs were collected from UK rodents and French rabbits and were initially screened for viruses with PCR. Following up on preliminary data, the positive samples were sent for NGS to acquire full genomes and perform unbiased virus discovery. Our findings encompass a number of novel viruses including astrovirus, rotavirus A, hantavirus, picornavirus, coronavirus, paramyxovirus etc. Among the novel viruses, the picornavirus interestingly showed 50 % and 34 % similarity to the closest relative for the non-structural and the structural protein respectively at amino acid level, suggesting a potentially novel genus within the family. This study shows the presence of highly divergent RNA viruses in European rodents and rabbits enriching thus our current knowledge of their origin and evolution. Finally, this study shows that these animal species can be the reservoirs of RNA viruses notorious for cross-species transmission, increasing the risk of a spill-over to humans or livestock.
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Combating and detecting Histomonas meleagridis, the causative agent of black head disease, through the administration of novel antimicrobial peptides derived from microbiomes
More LessHistomonas meleagridis is a protozoan parasite that causes mortality and morbidity in a wide range of gallinaceous fowl. It most notably affects turkeys, causing 80–100 % mortality in a flock. There is no commercial treatment for this parasite at the moment and attempts at a vaccine have failed. In this study we investigated the potential efficacy of novel rumen and chicken gastrointestinal tract microbiome-derived antimicrobial peptides (AMPs) against H.meleagridis. H. meleagridis was cultivated in growth flasks in Dwyer media. The concentration of the parasite was determined using a Haemocytometer, the cells were counted under the microscope. The AMP challenges were carried out in 96-well plates. The starting concentration of the AMPs were 1024 µg ml−1 down to 2 µg ml−1. The cell densities of H. meleagridis were checked at 24 h. The protozoal cell densities were checked by extracting DNA and performing qPCRs targeted against FeHyD and Rpb1 genes. Fluorescent microscopy was also used to check parasite densities as described above. Five Chicken and three Rumen AMPs resulted in decreases in protozoal cell densities following microscopy, suggesting that the peptides show potential therapeutic application for blackhead disease. Nonetheless, the qPCR primers were non-specific and resulted in bacterial DNA amplification. Consequently, in order to understand the biological function of H. meleagridis and design new qPCR primers allowing quantification, we isolated the macronuclei of a H. meleagridis strain followed by sequencing using the illumine Hi-Seq 2500 and paired ends. Future work will focus on confirming how well the AMPs work agent H. meleagridis by employing electron microscopy to observe how the AMP affect the cells.
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The bacterial microbiome of in vitro cultures of Paramoeba perurans
More LessAmoebic Gill Disease (AGD) is a major problem in the aquaculture industry, as it is responsible for substantial losses of farmed Atlantic salmon in various worldwide locations. The disease is caused by the usually free-living Paramoeba perurans compromising the gills through the resulting development of hyperplastic lesions and lamellar fusion. These structural changes result in a reduction in the functional surface area of the gill tissues. Recent research has focused on identifying bacteria present within a culture of P. perurans, through performing isolation and identification of bacteria present in the cultures using 16S sequencing. Further NGS sequencing was performed from various culture conditions to provide insight into the changes of the bacterial microbiome during amoeba culture. As attempts to isolate the amoeba from the bacterial contamination has been unsuccessful, consideration into a possible symbiotic relationship between the amoeba and bacteria was considered. A filtering method was used to attempt to identify the genera of bacteria present within the amoeba. The isolation and 16S sequencing identified the presence of various marine bacteria, including those of the Pseudoalteromonas, Halomonas, Cellulophaga and Mesonia genera. The NGS sequencing identified a substantial proportion of sequences to match the Vibrio genus and suggests an association between this genus and the amoeba. If symbiotic relationships between specific bacteria and amoeba can be confirmed, the bacteria could potentially be used as an indicator organism for the risk of AGD outbreak. It may also provide an indirect target for the control and treatment of AGD.
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The effect of dietary fatty acid supplementation on gut microbiome development in weaning piglets
More LessThe 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.
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Investigation and genome characterisation of tatenale hantavirus in wild rodent populations in the United Kingdom
Hantaviruses are a diverse group of single-stranded, negative sense RNA viruses, belonging to the Bunyaviridae family. They are primarily rodent-borne and transmitted into humans through the inhalation of aerosolised excreta of infected animals. Each hantavirus is typically associated with a single reservoir species, resulting in a persistent, yet asymptomatic infection. In humans, however, in addition to asymptomatic infection two disease syndromes are associated with hantavirus infection, hantavirus haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Tatenale Hantavirus (TATV) was first isolated from field voles in NW England in 2013 and has since been detected at other sites in Northern England. However, only two small fragments of the L and S segments have been sequenced, precluding full phylogenetic characterisation of the virus. Field voles were sampled at two sites in Leicestershire (N=104) and Cheshire (N=12), and their lungs subjected to a degenerate pan-hantavirus RT-PCR assay, targeting a section of the polymerase gene. Eleven Cheshire and one Leicestershire sample were TATV positive, and were 94 % homologous across the amplicon. Illumina Hi-Seq sequencing revealed the complete coding regions of the Leicestershire isolate. Analysis of the sequence showed that TATV is most closely related to Khabarovsk virus, with a homology of 91%/88%/89 % (AA) across the L/M/S Segments, respectively. ICTV species demarcation requires an AA difference of >7 % across the S and M, TATV meets the requirement of a novel species. Retrieval of full coding sequence will allow further investigation into TATV, and its potential to infect humans through pseudotyping of the glycoproteins.
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- Offence and Defence
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Signal-integration through PhoPQ enables Salmonella to adapt to heterogeneous tissue microenvironments
More LessEnteric fever is major health issue in developing countries and it is becoming progressively untreatable due to increase of antimicrobial resistance. The causative agent, Salmonella enterica, replicates in host phagocytes in various organs and regulates expression of hundreds of genes in response to host signals. PhoPQ is one of the key regulators and essential for virulence in humans and in a mouse typhoid fever model. The sensor kinase PhoQ responds to diverse stimuli (Mg2+ limitation, low pH, cationic antimicrobial peptides, high osmolarity, and, indirectly, to reducing conditions). However, which signals are predominant in vivo remains unclear. To address this issue, we determined the activity of the PhoPQ regulon using a chromosomally encoded fusion of the PhoP-dependent phoNp promoter to gfp-ova and we quantified single-cell fluorescence levels of Salmonella in spleen of infected mice using flow cytometry. The results show extensive heterogeneity in PhoP-activities in the Salmonella population. Comparison of mutants with sensing defects suggests that, in vivo, PhoQ responds to a combination of antimicrobial peptides, acidic pH and low Mg2+, but not to reducing conditions. Negative feedbacks have also only a minor impact. Single-cell analysis of phoNp and asrp promoters suggests differential environmental pH as a major driver of heterogeneous PhoP activities. A combination of immunohistochemistry and proteomics of Salmonella from infected samples was used to validate this hypothesis. Together, our data show how Salmonella uses the PhoPQ system to integrate various host signals in order to tune expression of virulence factors to the diverse tissue microenvironments that this pathogen inhabits.
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The great escape: dissecting the interactions between Mycobacterium bovis and the soil amoeba Dictyostelium discoideum
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.
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- Teaching Microbiology in Higher Education Symposium
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Microbiology in primary school teaching
More LessWithin a large-scale report commissioned by the Wellcome Trust [1], primary school teachers were described as facing barriers in teaching science. The top barriers described were the lack of budget and resources, lack of time and curricular importance as well as other issues such as a lack of subject knowledge or confidence and concerns relating to space and resource access. Teaching science is just one part of a primary teacher’s complex role and is a subject in which most primary teachers do not have a degree or A level qualification [2,3]. There is little Microbiology content within the National Curriculum, however, schools can introduce additional scientific content within the Primary Key Stages. Within this context, a Microbiology, Genomics and Bioinformatics researcher in association with Key Stage 2 classes in a Norfolk Junior School carried out a joint project for Microbiology-related science in conjunction with Norwich Research Park facilities in May 2017 and the Microbiology Society. Here we report on the findings from teacher and pupil’s perspectives and consider how Microbiology/Hygiene could be presented to this age group in a classroom setting.
1. ‘State of the Nation’ report of UK primary science education. S. Leonardi et al. CFE Research, Leicester LE1 5TE. September 2017.
2. The Royal Society (2010) Science and mathematics education 5–14. A ‘state of the nation’ report. London: the Royal Society
3. ASE Guide to Primary Science Education (Serret and Earle, 2018), reviewed in https://tdtrust.org/cpd-primaryscience
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- The Biological and Chemical Tales of the Antibiotic Makers
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Regulation of exploratory growth and antibiotic production in Streptomyces venezuelae by the two-component system CutRS
More LessAntibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.
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The chemical ecology of protective microbiomes in plant roots and leafcutter ants
More LessActinobacteria are ubiquitous in soil and well-known for producing antimicrobial compounds. Increasingly, members of this phylum are found to form symbiotic relationships, for example with plants and insects, and are thought to provide protection against host infection. However, it remains poorly understood how Actinobacteria are recruited to microbiomes and whether secondary metabolites are produced in vivo. Acromyrmex echinatior leaf cutter ants transmit Pseudonocardia bacteria between generations and also recruit Streptomyces to their cuticular microbiome. We show that Pseudonocardia species isolated from the ant cuticle inhibit the fungal nest pathogen Escovopsisweberi and dual RNA-sequencing confirmed that Pseudonocardia secondary metabolite gene clusters are expressed in vivo on the ant cuticle. RNA stable isotope probing showed that ants supply cuticular resources to their microbiome which may fuel interference competition and select for antibiotic-producing bacteria. Similar to leaf cutter ants we also show that plant roots recruit growth-promoting and antibiotic-producing Streptomyces bacteria, but appear not to transmit them via their seeds. Root exudates are hypothesized to play a major role in root microbiome recruitment and DNA stable isotope probing coupled with Illumina sequencing showed that these were actively metabolized by many bacterial genera. However, Streptomyces appeared to be outcompeted by more abundant Proteobacteria, despite the fact that isolates could grow on purified exudates in the absence of competition. Streptomyces root exudate preferences are now being evaluated using comparative metabolomics. Defining the factors that influence the competitiveness of protective bacteria when colonizing microbiomes has implications for the development of more consistent biocontrol strategies and prebiotic techniques.
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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
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.
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Understanding aurodox: A Type III secretion system inhibitor from Streptomyces goldiniensis
More LessAurodox, 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.
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Discovering novel antimicrobials from Streptomyces formicae, a symbiont of fungus farming plant ants, using CRISPR/Cas9 genome editing
More LessFinding new antimicrobial compounds is vital to combat the growing threat of resistance. Most currently used antibiotics originate from actinomycetes discovered more than half a century ago. We recently reported the new species Streptomyces formicae, isolated from the African fungus-farming plant-ant, Tetraponera penzigi. S. formicae produces a novel family of polyketide antibiotics, the formicamycins, that have potent activity against resistant pathogens including MRSA and vancomycin-resistant Enterococci (VRE). Using CRISPR/Cas9, we have identified and characterised the genes responsible for formicamycin biosynthesis in the native producer. In addition, we used cappable RNA- and ChIP-sequencing to determine the transcriptional organisation of the pathway. We exploited this information to generate multiple mutants of S. formicae that overproduce formicamycins and their biosynthetic intermediates, some of which also have bioactivity. Furthermore, the potential for novel chemistry from S. formicae is not limited to the formicamycin pathway; antiSMASH analysis shows this talented strain contains at least 45 secondary metabolite biosynthetic gene clusters (BGCs). Under standard laboratory conditions, wild-type S. formicae also exhibits antifungal activity against the drug resistant Lamentospora prolificans, and when the formicamycin BGC is deleted, the strain produces even more potent antibacterial activity against MRSA. To identify the biosynthetic pathways for these metabolites, entire BGCs up to 208 kbp were deleted using CRISPR. Overall, this work demonstrates that searching under-explored environments for new species combined with genome editing is a promising route towards finding novel anti-invectives.
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Genome-led discovery of novel microbial natural products
More LessMicroorganisms have been increasingly exploited for their remarkable ability to produce diverse natural products, which display bioactivities ranging from antimicrobial and anticancer to signalling and developmental. Genome sequencing has revealed that bacteria harbour many more biosynthetic gene clusters (BGCs) for natural products than are currently characterised. Whilst several genome mining tools have been developed to aid discovery, some classes of BGCs remain difficult to identify. One such class is ribosomally-synthesised and post-translationally modified peptides (RiPPs). RiPP BGCs are small with few regions of homology, and the short precursor peptides are rarely annotated in genomes. After developing a targeted genome mining approach for RiPPs, we identified a novel family of BGCs spanning over 200 actinobacterial genomes. The presence of diverse biosynthetic enzymes and sequence variation of the precursor peptides suggest that these BGCs may produce several structurally diverse molecules. We have successfully TAR cloned one such BGC from Streptomycesand expressed it heterologously, and metabolomic analysis led to identification of the pathway product. Gene deletion experiments have also confirmed the involvement of individual biosynthetic enzymes. The compound has been purified and the structure elucidated by NMR. Further work will focus on exploring RiPP BGCs from other species and investigating the biological role of these molecules. The discovery of such a diverse and highly conserved group of BGCs highlights that we are still scratching the surface of the huge biosynthetic capacity of microorganisms, and the use of more sophisticated genome mining tools could help unveil many more important molecules in the future.
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clusterTools: functional element identification for the in silico prioritization of biosynthetic gene clusters
More LessThe large amount of data from inexpensive sequencing means that the number of putative biosynthetic gene clusters (BGCs) far exceeds our ability to experimentally characterize them. This necessitates the need for development of further tools to analyze putative BGCs to flag those of interest for further characterization. clusterTools implements a framework to aid in the in silico characterization of BGCs by identifying regions of the DNA, containing homologous proteins, or coding sequences containing specific functional domain compositions using user-built HMM rules, in close proximity, reporting results in an easy to visualize manner. clusterTools complements existing software for BGC analysis in two ways. First, by running clusterTools on databases of genomic sequences in an exploratory mode, the user can identify and download regions of interest in the DNA for further processing and annotation in programs such as antiSMASH. Second, if clusterTools is run on databases constructed from putative gene clusters generated by antiSMASH, one can rapidly identify clusters on interest from the group that warrant further analysis and experimental characterization. We demonstrate the use of clusterTools as part of our workflow to identify BGCs of specific classes of natural products that would be difficult to identify with existing methods, particularly clusters containing assembly line domains as components, including those involved in bacterial polyketide alkaloid biosynthesis. clusterTools can also be used to identify novel BGCs by incorporating regulatory and antibiotic resistance elements. Standalone versions of clusterTools are available for Macintosh, Windows, and Linux.
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Mutasynthesis of novel prodiginines derived from the antibiotic prodigiosin by exploiting substrate specificity of H2MAP oxidases PigB and HapB
More LessThe natural product, prodigiosin, caught researchers’ interest more than a century ago because of its bright red color. Today, interest in this tripyrrolic secondary metabolite remains strong due to its biological effects, including potent antibiotic activity against various Gram-positive bacteria. Further exploration of the potential of this class of molecules requires libraries of analogues. Yet, the total synthesis of prodigiosin-like compounds (prodiginines) proves challenging. This can be overcome by highjacking the bacterial biosynthetic machinery via mutasynthesis. Although a number of different bacteria produce prodigiosin, its biosynthetic pathway is well conserved. The final precursors, MAP and MBC, are always produced independently, before a terminal condensation reaction. Our work focuses on the last step of the formation of MAP: the oxidation of H2MAP. We were able to isolate and characterize HapB, the enzyme catalyzing this reaction in Hahella chejuensis. In addition, we showed that some modifications of alkyl substituents on the C2 and C3 positions of H2MAP did not alter HapB activity significantly. We then fed these analogues of H2MAP into Serratia ΔpigD, a mutant of Serratia ATCC sp.39006 which does not produce any endogenous H2MAP. As expected from the in vitro testing, chain elongation past two carbons on the C2 position could not be accepted whereas all substrates with a modification on the C3 position restored pigmentation, leading to the formation of eight novel prodiginines.
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Chain termination of structurally disparate non-ribosomal peptides by a trans-acting β-lactamase
More LessNon-ribosomal peptide synthetases (NRPSs) are responsible for the natural in vivo production of a large number of therapeutically relevant compounds. The non-ribosomal peptides (NRPs) formed by their action are the product of the long modular assembly lines, whilst the terminal step in the biosynthesis is the hydrolytic release, and frequently, macrocyclisation of the aminoacyl-S-thioester by an embedded thioesterase (TE). The surugamide biosynthetic pathway is composed of two NRPS assembly lines. One produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded TE, which led us to question how the peptides are released from the assembly line (and cyclised in the case of surugamide A). We characterised an alpha/beta hydrolase and β-lactamase in vivo and established that the former is a type II TE for surugamide A, but not surugamide F, and that the latter is a trans-acting release factor for both compounds. In vitro substrate utilisation assays unambiguously established that the β-lactamase can produce mature surugamides A and F from N-acetylcysteamine (SNAC) thioester mimics of the cognate terminal biosynthetic intermediates. Using bioinformatics, we estimate that ∼12 % of filamentous Actinobacteria harbor an NRPS system lacking an embedded TE and instead use a trans-acting β-lactamase release strategy. This expands the paradigmatic understanding of how non-ribosomal peptides are released from the terminal NRPS module and adds a new dimension to the synthetic biology toolkit, potentially useful in the search for novel antibiotics.
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Identification of novel antimicrobial-producing bacteria from an ancient water source by Oxford Nanopore Whole Genome Sequencing and Natural Product Chemistry
More LessMicrobially-derived antimicrobial compounds are a rich source of clinical antibiotic leads. However, discovery rates have declined over the past 40 years due, in part, to high rediscovery rates of known compounds from traditional soil-based screening approaches. In this study, an ancient hot-spring water source was tested for the presence of antimicrobial-producing bacteria using culture techniques which led to isolation of two organisms capable of inhibiting the growth of multiple bacterial species. Oxford Nanopore whole genome sequencing was used to identify these two isolates as being in one of two genera; Streptomyces and Paenibacillus. Bioinformatic analysis revealed both isolates to have multiple novel secondary metabolite gene clusters. Investigations of the Streptomyces sp. by natural product chemistry techniques showed the organism to produce multiple antimicrobial compounds, these were effective methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. This study underlines the value of investigating non-traditional habitats in the search for novel antibiotic-producing organisms.
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Genomic analysis of Burkholderia ambifaria identifies key specialised metabolites for biopesticidal applications
BackgroundBurkholderia bacteria are renowned for the biosynthesis of an extensive repertoire of specialised metabolites encoded by biosynthetic gene clusters (BGCs). Burkholderia ambifaria is a historical biopesticide and produces multiple antimicrobials with activity against nematodes, fungi and bacteria. We evaluated the distribution of antimicrobial activity and BGCs across the species which, despite their biopesticidal potency, is poorly understood.
ResultsGenomic analysis of 64-B. ambifaria whole-genomes revealed considerable diversity. Pathway prediction identified 1272 BGCs across 64 strains that were de-replicated to 38 distinct BGCs. Known BGCs accounted for 13 of the BGCs; 7 of which corresponded to known antimicrobials, and included enacyloxins and bactobolins. The antimicrobial activity of the strains was assessed in a classical overlay assay against animal and plant pathogens. The combined genome mining and in vitro activity screens identified a potent anti-Gram-positive and anti-Oomycete compound, cepacin. A damping-off disease model with the pathogen Pythium ultimum and legume Pisum sativum (garden peas) highlighted cepacin as a key specialised metabolite in crop protection. B. ambifaria strain persistence in a mouse inhalation infection model was reduced by removal of the third replicon, whilst retaining the biopesticidal properties of the B. ambifaria strain.
ConclusionThis work highlights the diversity and prevalence of specialised metabolites in B. ambifaria, and the importance of the potent antimicrobial cepacin in protection against damping-off disease in agriculture. Construction of a B. ambifaria strain that retained biopesticidal properties yet was attenuated for persistence within a murine infection model provides a potential route towards developing safe biopesticidal agent.
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In silico identification of two novel antimicrobial peptides with antibacterial activity against multi-drug resistant Staphylococcus aureus
Linda Oyama, Hamza Olleik, Ana Carolina Nery Teixeira, Matheus M Guidini, James A Pickup, Alan R Cookson, Hannah Vallin, Toby Wilkinson, Denise Bazzolli, Jennifer Richards, Mandy Wootton, Ralf Mikut, Kai Hilpert, Marc Maresca, Josette Perrier, Matthias Hess, Hilario C Mantovani, Narcis Fernandez-Fuentes, Christopher J Creevey and Sharon A HuwsHerein we report the identification and characterisation of two linear antimicrobial peptides (AMPs), HG2 and HG4, with activity against a wide range of multi-drug resistant (MDR) bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains, a highly problematic group of Gram-positive bacteria in the hospital and community environment. To identify the novel AMPs presented here, we employed the classifier model design, a feature extraction method using molecular descriptors for amino acids for the analysis, visualization, and interpretation of AMP activitiesfrom a rumen metagenomic dataset. This allowed for the in silicodiscrimination of active and inactive peptides in order to define a small number of promising novel lead AMP test candidates for chemical synthesis and experimental evaluation. In vitrodata suggest that the chosen AMPs are fast acting, show strong biofilm inhibition and dispersal activity and are efficacious in an in vivomodel of MRSA USA 300 infection, whilst showing little toxicity to human erythrocytes and human primary cell lines ex vivo. Observations from biophysical AMP-lipid-interactions and electron microscopy suggest that the newly identified peptides interact with the cell membrane and may be involved in the inhibition of other cellular processes. Amphiphilic conformations associated with membrane disruption are also observed in 3D molecular modelling of the peptides. HG2 and HG4 both preferentially bind to MRSA total lipids rather than with human cell lipids indicating that HG4 may form superior templates for safer therapeutic candidates for MDR bacterial infections.
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Enhancing the unexplored chemical diversity of Streptomyces sp. to produce new antibiotics active against multidrug resistant Acinetobacter baumannii
More LessStreptomyces species are a major source of antibiotics but are often grown under restrictive conditions that limit biosynthetic gene expression. As a result, the vast majority of secondary metabolites within a single species remain unexpressed in the lab along with the huge variety in chemical structures and bioactivities. The overarching aim of this project was to identify conditions that produce novel antibiotics, specifically against Gram-negative pathogens. The culture collection NCIMB contains hundreds of Streptomyces isolated from around the world. Strains were selected from the collection guided by preliminary bioactivity studies and available literature. These were then grown in an extensive variety of conditions designed to stimulate production of a wide variety of secondary metabolites, detected by UPLC-MS and analysed using the freely available metabolomic tools MZmine, MetaboAnalyst, and GNPS. Conditions included various carbon and nitrogen sources, temperatures, stresses, epigenetic inhibitors, and other microbes. Compounds active against the Gram negative multidrug resistant pathogen Acinetobacter baumannii were detected in the scaleup culture supernatant fractions. Metabolite identification through GNPS did not detect any previously discovered compounds active against A. baumannii, indicating a potentially novel antibiotic against one of the WHO’s priority pathogens.
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Direct proteolytic control of an extracytoplasmic function RNA polymerase sigma factor
More LessStreptomyces species are well known for harbouring a large number of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors; nearly all of regulated genes required for morphological differentiation and/or response to environmental stress. The activity of ECF σ factors is typically modulated by a cognate anti-σ factor protein co-encoded at the same locus. In previous work, we identified σAntA as a cluster-situated regulator of starter unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is controlled by a cognate anti-σ factor, σAntA is an orphan, which raises intriguing questions about how its activity is controlled. Inspection of the σAntA amino acid sequence revealed a carboxyterminal di-alanine, which is the only motif of the Clp-protease recognition signal obligately required for proteolysis. Here, we show by immunoblotting that the abundance of 3xFLAG-σAntA in vivo is enhanced by alteration of the C-terminal Ala-Ala to Asp-Asp and that abundance of both variants is elevated in the absence of genes encoding the Clp-protease and its regulatory subunits ClpX and ClpA. We also show that (His)6-SUMO-σAntA, but not a variant lacking the C-terminal di-alanine motif, is degraded by the ClpXP protease in vitro. These data unambiguously establish direct proteolysis as an alternative control strategy for ECF RNA polymerase σ factors and expands the paradigmatic understanding microbial signal transduction regulation.
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Use of Nano- SIMS at the single cell-level to evaluate drug penetration into mycobacterial biofilms
More LessNon-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause high mortality and morbidity amongst patients who are immunocompromised or have chronic lung diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate and are associated with the development of increased antimicrobial resistance (AMR). Treatment for M. abscessus and chimaera infections often requires 2–3 antibiotics over 2 years. The question we want to address is whether the increased AMR and treatment time in NTM biofilm formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms (M. abscessus and M. chimaera). Nano-SIMS maps the relative abundance of different ions down to the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce treatment duration and AMR development.
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The Savage Dawn Peptide: an antibiotic woven from 12th century Welsh poetry
More LessBacterial antibiotic resistance is widely regarded to be one of the most pressing threats facing humanity. Finding new antibiotics is a vital research area and can now be supported by a vast reservoir of readily available ‘omic data on the back of the explosion of low cost sequencing technologies. Antimicrobial Peptides (AMPs): endogenous peptides that provide a fast and effective means of defence against pathogens as part of the innate immune response. The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists. Large reservoirs of existing sequences exist and are well annotated and understood. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides cheaply available from a wide array of third party companies. A well organised screening program can screen 100 s of prospects a day against model bacterial organisms to test for activity and is one of the few areas of biological science that can scale to meet the data output from computational prediction toolkits. AMPLY, an in-house tool designed at Aberystwyth University, supported by Life Science Wales and working in collaboration with Tika Diagnostics at St. George’s Hospital (London) and Queen’s University (Belfast) is part of a next wave of computational drug discovery platforms and is already uncovering a treasure trove of novel AMPs in diverse microbial environments. However, AMPLY’s abilities extend beyond the analysis of ‘omic data alone and its predictive modelling has extracted a therapeutically viable novel AMP built from a string of ancient Welsh poetry.
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- The Microbial Pangenome
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Genomic evolution of Klebsiella pneumoniae clones: the good, the bad and the ugly
Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.
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The genomic (re)definition of EPEC
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.
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The co-occurrence and co-exclusion of evolving objects in prokaryotes
More LessThroughout evolution, evolving objects (domains, genes, operons etc.) have continuously combined, forming new proteins, gene clusters, and genomes. Horizontal gene transfer, particularly among prokaryotes, has facilitated this combinatorial process. Thus, evolving objects that interact positively or synergistically with each other are expected to co-occur more often than by chance; conversely, evolving objects may avoid co-occurrence, indicating an antagonistic or redundant functionality between objects. In this work, we use methods adapted from graph theory to understand patterns of co-occurrence and exclusion in prokaryotes. We have implemented multi-level graph models in which each node (vertex) is a gene or species connected by an edge (relationship) to another node to display these coincidence relationships. Our method incorporates the phylogenetic distribution and synthenic distances of these genes, and we demonstrate how these concepts can be used to identify conserved clusters of vertical and horizontally inherited units of selection. We apply these multi-level graph models to a variety of datasets including prokaryotic pangenomes, and metagenomic sequencing datasets from human-associated microbial communities. We find evidence for genes that significantly co-occur with each other within each of these datasets; these genetic clusters include objects from characterized biological pathways but also include genes with unknown functions. Further, we identify genes that exclude each other, indicating evolving objects with antagonistic or redundant biological functions. This work represents a different approach to understanding the evolution of prokaryotes and allows us to draw novel hypotheses as to the potential role of these genetic clusters in prokaryote biology.
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Deciphering adaptation through mobile element pangenome composition
More LessPangenome analysis can help reveal functional and physiological differences between organisms by linking genes present in their genomes with phenotypic characteristics. While this works well for closely-related organisms and large numbers of isolates, analysis of species with a large pangenome may be problematic as optimal sample size increases with intraspecies diversity. Organisms such as E. coli exhibit high intraspecies diversity, resulting in an exceptionally large pangenome, pushing the limits of what constitutes a species. However, the phylogeny of the mobile elements can differ strongly from the phylogeny of the rest of the genome, reflecting how rapidly these elements can be gained or lost. We suggest when studying adaptations on a short genetic time-scale (ie, within species or strain), the mobile regions of the genome may be more informative than the more stable parts of the genome. To this end, we developed a pipeline, Horizontally Acquired Partial Pangenome of Inserted Elements (or ‘happie’), which allows researchers to study the mobile pangenome. Thus, happie could be used as a proxy to detect genes associated with a particular trait amidst the noise of a diverse genetic background. We aim to use happie to compare a panel of known soil-persistent E. coli isolates and a curated panel of non-soil-persistent isolates. This will allow us to determine whether specific mobile elements (or genes carried on those elements) are associated with soil persistence, which would indicate that the cost of harbouring the mobile element was less than the advantage conferred.
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- Vaccines Against Bacterial Pathogens
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Mathematical modelling to characterise the in vivo dynamics of Salmonella in the naïve and immunised host
More LessSystemic salmonellosis encompasses typhoid and paratyphoid fever, and invasive non-typhoidal salmonellosis, with high mortality and morbidity amongst children and the immunocompromised in low-resource settings. Immunisation efforts remain hampered by the unavailability of safe vaccines with cross-protectivity against causative Salmonella strains. Characterisation of the within-host Salmonella dynamics in the naïve and immunised host can elucidate the mechanisms by which different vaccine types exert their protective effect, and help in vaccine selection and design. Experimental data tracking the changes in bacterial population composition in the different tissues of the host at different timepoints can be coupled with mechanistic mathematical models to estimate the parameters governing the processes of bacterial replication, killing and inter-organ migration. Using a recently described minimisation-divergence estimation approach, we extend a three-compartmental mechanistic model and re-analyse existing datasets to better characterise the bacterial migratory processes between the blood, liver and spleen in the early stages of infection, and the overall Salmonella dynamics in the later phases in the naïve host. We apply the same model to published data from mice immunised with either a live-attenuated or killed whole-cell vaccine to identify their in vivo differential impacts on Salmonella migration, replication and death. Finally, we identify alternative experimental designs to improve the statistical qualities of the mathematical model and allow better inference of parameters governing the unobserved processes of bacterial dynamics.
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BactiVac, a network to support the study, development and implementation of bacterial vaccines
More LessInfections 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.
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Bactericidal fully-human monoclonal antibodies can be cloned from patients convalescing from invasive meningococcal disease
More LessReverse vaccinology 2.0 (RV 2.0), in which the cloning and recombinant expression of antigen-specific antibodies is followed by determination of their functional activity, is a valuable approach that can unravel novel vaccine antigens, or reinforce the vaccine candidacy of already known antigens. Invasive meningococcal disease (IMD) remains a serious source of concern even with the availability of vaccines. Incomplete strain coverage is a limitation of current vaccines, hence efforts to identify candidate antigens that will compose supplementary or replacement vaccines are necessitated. In this proof-of-principle study, we sought to assess the applicability of RV 2.0 to anti-meningococcal vaccine antigen discovery. Antibody-secreting cells (ASCs) obtained from a patient convalescing from serogroup B (MenB) IMD, were isolated and sorted singly using FACS. The specificity and functionality of each antibody produced by individual ASCs were assessed in ELISA and bactericidal assays, respectively. Eight cross-reactive anti-meningococcal antibodies were successfully cloned; three of these mediated complement-dependent killing of antigenically-heterologous MenB strains. Western blot data shows binding of these three bactericidal antibodies to a ∼35 kDa antigen. None of the three bactericidal antibodies were reactive with a target in current vaccine formulations strongly suggesting that the ∼35 kDa antigen does not compose available vaccines. Unequivocal determination of the identity of the ∼35 kDa antigen is ongoing. Given the need for antigens that would compose improved or novel anti-meningococcal vaccines, this study shows that the RV 2.0 approach has the potential to be a powerful tool in the identification of functionally-immunogenic anti-meningococcal antigens.
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Development of in vitro functional antibody assays to identify and assess vaccine targets against E. coli bacteraemia
More LessExtra-intestinal pathogenic E. coli (ExPEC) is one of the leading causes of bacteraemia and urinary tract infections (UTI) worldwide. At present, there is no licensed vaccine available and with the increasing incidence of multidrug resistance (MDR) the demand for developing alternatives to antibiotics is paramount. We have developed in vitro functional antibody assays to fast track screening of patient convalescent sera from ExPEC infections to identify potential vaccine antigens. A functional immunological assay will also facilitate the assessment of vaccine candidates against the many disease-causing serotypes circulating in the population. Outer membrane vesicles (OMVs) purified from a mutant (ΔlpxM) MDR O25 (ST131) strain were used to immunise mice to raise antiserum against the bacterial strain. Immunised mice were protected in a mouse model of E. coli bacteraemia. The antiserum from protected animals was used to develop an antibody-mediated bacterial killing assay and a complement deposition assay (CDA). The antiserum mediated killing with human IgG and IgM-depleted plasma as the complement source, and also showed deposition of C3b and C5b-9 onto the bacterial surface determined by flow cytometry. This may be a useful screening tool for mining for new antigens and in assessing the antisera raised against the candidates for cross serotype protection.
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Genomics for rational autogenous vaccine design to control Campylobacter infection in poultry
More LessCampylobacter is the leading cause of food-borne gastroenteritis worldwide and the most common cause of infection in humans have been linked to the consumption of contaminated poultry meat. Previous poultry vaccines have provided short term reduction in Campylobacter intestinal load in chickens but have limited commercial efficacy because of high diversity and rapid evolution of strains. Genome-wide association study (GWAS) of isolates from humans and chicken have identified genes associated with survival of Campylobacter through the poultry processing chain. These genes represent targets for vaccine design that would be selectively neutral within the chicken gut and therefore could be sustained in the population. We designed an autogenous vaccine based on isolates with survival-associated genes and monitored the efficacy in the chicken and through processing. First, we sampled for Campylobacter across five broiler farms feeding into one abattoir in Norfolk and characterized genomic diversity using next generation sequencing (NGS), including the identification of survival genes. These data were then used in a predictive model to identify the minimum number of strains to be used in vaccine design based on known immunogenicity of the strains. Second, the vaccine was developed and given at two time-points to a whole farm of breeder chickens. Third, vaccine efficacy was monitored at three time points post-vaccination in vaccinated breeders and their progeny. The cfu/g reduction in the ceacum and neck skin were determined. This study demonstrates the potential for NGS in autogenous vaccine design to reduce harmful strains of bacteria that are carried commensally in livestock.
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- Virology Workshop: Antivirals and Vaccines
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In-ovo antiviral assay of methanolic leaf extract of Cymbopogon citratus (Lemon grass) on Newcastle disease virus
This study determined the in-ovo antiviral effect of crude Methanolic leaf extract of Cymbopogon citratus on Newcastle disease virus. Cold extraction was carried out using analytical grade methanol. Phytochemical screening of the crude extract was carried out using standard procedures. Antiviral assay was carried out in nine-day old specific pathogen free embryonated hens’ eggs in three designs made of five eggs per group (Virus with extract, virus only and un-inoculated groups) with concentrations ranging from 12.5 to 100 mg ml−1. Egg toxicity of the extract was determined for concentrations of 12.5, 25, 50, 100, 200, 300 and 400 mg ml−1. Inoculated eggs were incubated at 37 °C and observed daily for 96 h for embryo survival and mortality. Spot haemagglutination was carried out on bacteria-free allantoic fluid from the embryonated eggs to detect the presence of the virus. Phytochemical assay revealed the presence of saponins, flavonoids, steroids, terpenes, phlebotannins and terpenoids. Mild toxicity was observed at concentrations of 100 mg ml−1 and above. There was no haemagglutination of fluid from the eggs inoculated with a combination of Virus and extract at concentrations of 50 and 100 mg ml−1. The current findings demonstrated that leaf extract of Cymbopogon citratus has potential medicinal value as well as antiviral activity against Newcastle disease virus in-vivo. The specific mechanism of action remains to be studied to further elucidate on its potential as a therapeutic product for the treatment of Newcastle Disease.
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Study on seroprevalence of IgG antibody of Varicella–Zoster virus in pregnant mothers, neonates and children of six of months old
More LessThis was a cross sectional and observational study, conducted in the Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet. The study period was from July 2010 to June 2011 in objective to explore the Varicella-Zoster virus immune status (VZV-IgG) in pregnant mothers and neonates up to six months post birth. For this purpose, 60 pregnant women, 60 new born babies and 60 infants aged six months were selected. The mean age of the pregnant woman was 28.8 (SD±4.7) years. The sex of new born babies and six months aged infants was identical [29 (48.3 %) male vs 31 (51.7 %) male; P=0.715]. Seroprevalence rate of IgG antibody of Varicella-Zoster Virus in Pregnant, mothers 81.7%, in new born babies 78.3 % and in infants aged six months were 10.0 %. The seropositivity of VZV IgG level in both pregnant mothers and newborn babies were almost similar but the infant aged six months were significantly lower than that of new born babies (p This showed that a significant proportion of Bangladeshi pregnant mother is susceptible to varicella and infant aged six months is highly susceptible to varicella. Any vaccination strategy must have to take into account these epidemiological variability of the country.
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Rabies virus: defining antigenic requirements for pan-lyssavirus neutralisation
More LessThe lyssavirus genus is a diverse group of viruses all capable of causing an invariably fatal disease known as rabies, most commonly caused by the prototype species rabies virus (RABV). Alongside RABV the lyssavirus genus currently contains 15 other viruses capable of causing rabies. These viruses are broadly categorised into phylogroups according to the predicted level of vaccine protection, with protection from current vaccines and therapeutics afforded against phylogroup I but not II or III. Current evidence suggests that for a protective neutralising antibody response against RABV a neutralising antibody titre of 0.5 IU ml−1 is sufficient. This arbitrary value has been developed and promoted as a serological cut-off based on the reactivity of defined sera with a standardised dose of RABV. Studies using cross protection assays, have suggested that for protection against more divergent members of the genus, even those in phylogroup I, 10-fold or greater than the 0.5 IU ml−1 antibody titres are required. The continued discovery of novel lyssaviruses globally warrants an in-depth assessment of the protective titre required to protect against all the lyssaviruses to inform occupationally high-risk groups (e.g., scientists, bat workers and speleologists). Based on live virus neutralization assays, a minimum of 7 distinct lyssavirus glycoprotein antigens would have to be included in any pan-lyssavirus vaccine. Certainly, representative immunogens from all lyssavirus species characterized in phylogroups II and III are required to stimulate a pan-lyssavirus response.
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The application of CRISPR/Cas9 system in the generation of viral vectored avian influenza vaccines
More LessAvian influenza is highly contagious poultry disease and the mortality can reach 100 %, leading to huge economic burden and threat to food security. Vaccination is the most effective strategy for prevention and control of influenza. Recombinant vector vaccines are effective promising vaccines capable of immunizing against multiple pathogens. Traditional methods for constructing recombinant vaccines involve homologous recombination or bacterial artificial chromosomes but these methods can be time-consuming and labour-intensive. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a recently developed gene editing technology which has proven beneficial to gene modification and offers an alternative for constructing recombinant vaccines. There are mainly two methods used for gene insertion; error-prone non-homologous end joining (NHEJ) and the high-fidelity homology-directed repair (HDR) pathway. Owing to its high fidelity, most studies focus on using HDR for vaccine development but NHEJ offers some attractive advantages through its high efficiency. In our study, both HDR and NHEJ dependent CRISPR/Cas9 systems were explored for the rapid generation of recombinant influenza vaccines using duck enteritis virus and herpesvirus of turkeys as vectors.
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The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus replication
Arbidol (arb, umifenovir) is used clinically in several countries as an anti-influenza virus drug. We have previously shown that arb inhibits many viruses including hepatitis C virus, Ebola and Zika, and that the primary mode of action appears to be via inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. We have also shown that arb is a good inhibitor of (non-enveloped) poliovirus types 1 and 3. Here, we evaluate the antiviral potential of arb against another picornavirus, foot-and-mouth disease virus (FMDV), an important veterinary pathogen. Sub-cytotoxic concentrations of arb inhibited the replication of FMDV replicon RNA. arb inhibition of FMDV RNA replication was not a result of generalised inhibition of uptake of cargo (e.g. plasmid DNA or RNA), nor did arb inhibit FMDV replication when added at 4 h post-transfection of FMDV replicon RNA. FMDV replication was blocked by the replication inhibitor guanidium hydrochloride (GuHCl). Upon GuHCl removal, FMDV replication was restored, and arb suppressed this recovery of virus replication. For other picornaviruses, recovery of virus replication upon GuHCl removal has been shown to require translation. However, arb did not suppress cap- or internal ribosome entry site (IRES)-dependent translation. arb also inhibited infectious equine rhinitis associated virus (ERAV), a close relative of FMDV. Testing of arb against infectious FMDV is in progress. Collectively, the data demonstrate that arb inhibits certain picornaviruses by a mechanism that appears to be independent of effects on virus entry but involves inhibition of genome replication.
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Development of low bio-containment assays to characterise the antibody responses in pigs to Nipah virus vaccine candidates
Nipah virus (NiV) is a zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. Since 1998, NiV outbreaks have occurred in Malaysia, Bangladesh and India. NiV poses a significant pandemic threat due to its broad host range and the widespread distribution of its natural host, the Pteropus fruit bat. Despite this, there are currently no licenced therapeutics or vaccines for use in either humans or livestock. Aiming to develop a safe and inexpensive vaccine to protect livestock in future outbreaks and to prevent onward transmission to humans, we are evaluating the immunogenicity and efficacy of three candidate vaccines in pigs. These vaccines are based on the NiV G or F surface glycoproteins, proteins which are essential for mediating virus-cell or cell-cell entry. The candidate vaccines include: (i) a recombinant soluble NiV G protein subunit, (ii) a recombinant molecular clamp stabilised NiV F protein subunit and (iii) a replication deficient, adenoviral vectored NiV G protein. We have developed low biocontainment assays to characterise the antibody responses to NiV and to aid identification of immune correlates of protection through quantification of both antigen-specific and neutralising antibody responses to our vaccine candidates. This includes anti-NiV F/G indirect ELISAs, a microneutralisation test using pseudotyped particles, and a microfusion inhibition test using a quantifiable cell-cell fusion assay. Using these assays we have demonstrated that all three of our novel vaccines are immunogenic in pigs and capable of generating a robust antibody response, with evidence for neutralising antibodies.
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HSV-1 genome decompaction plays an important role in viral DNA sensing by PML-NB intrinsic antiviral regulators
More LessConstituent 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.
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Flavivirus membrane (M) proteins as potential ion channel antiviral targets
The Flavivirus small integral membrane (M) protein resides within mature infectious particles yet, unlike the envelope (E) protein which mediates membrane fusion upon encountering low pH within the acidifying endosome, the function of M within this context is unknown. We are investigating whether Zika virus (ZIKV) M protein exhibits channel activity, acting as a viroporin playing a role mediating virus entry and uncoating. Importantly, ZIKV entry was blocked in a dose-dependent fashion by the prototypic channel blocker rimantadine and the drug also prevented virus spread in mouse models of ZIKV infection. Molecular dynamics simulations supported that M protein is able to oligomerise into a hexameric viroporin channel, opening of which is promoted in an acidified environment via protonation of a conserved histidine residue. Rimantadine is predicted to bind onto this structure in silico at a lumenal binding site, against which we are currently designing improved small molecule inhibitors that could form the basis of novel M protein targeted drug discovery. Importantly, drugs targeting M might either prevent or reduce the severity of ZIKV infections, including those crossing the placenta, and could also be translated for use against other Flaviviruses.
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Development of Bacteroides thetaiotaomicron outer membrane vesicles as a universal, mucosal vaccine for Influenza A virus
More LessInfluenza A Virus (IAV) is a zoonotic pathogen which causes seasonal epidemics worldwide, resulting in significant morbidity and mortality. Due to rapid virus evolution, available vaccines/antiviral drugs must be re-formulated and re-administered in most years. There is therefore an unmet need for a ‘universal’ vaccine to provide long-lasting, adaptive immunity to multiple strains of IAV. The aim of this study was to assess outer membrane vesicles (OMVs) produced by Bacteroides thetaiotaomicron (Bt) containing IAV antigens as a candidate universal mucosal vaccine. First, using intranasally-delivered fluorescently-labelled OMVs and FACS we demonstrated good uptake of OMVs by dendritic cells in both secondary and tertiary lymphoid tissue, showing OMVs are efficiently trafficked from the mucosa to the lymphatic system. Immunohistology confirmed this and showed expansion of lymphoid tissues and lymphoid follicles after OMV delivery. The stem/stalk of IAV HA is less variable and will generate antibodies that are cross-protective. Thus, we produced OMVs (termed H5F) that contained the stalk region of HA from strain A/VietNam/1203/04 (H5N1). We inoculated mice intranasally with H5F expressing OMVs or wild type OMVs and assessed antibody production and protection from challenge using a lethal dose of a different strain of influenza (PR8; H1N1). OMV-H5F-inoculted mice produced IgG and IgA in the respiratory tract that recognised both H5 HA and H1 HA. OMV-H5F inoculated mice were also protected from lethal challenge and supported lower virus titres. Our results indicate a strong potential for this approach to generate a universal IAV vaccine.
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Use of Bacteroides-derived microvesicles for mucosal vaccines
The vast majority of infectious agents enter the body via mucosal sites yet there are very few licensed mucosal vaccines able to generate protective immunity at the sites of pathogen entry. A major obstacle in developing mucosal vaccine is delivering biologically active vaccine antigens (Ag) to mucosa-associated lymphoid tissues to prime protective immune responses. To address these issues we have developed a drug delivery technology platform to deliver intact antigens to the respiratory and gastrointestinal tract using outer membrane vesicles (OMV) naturally produced by Bacteroides thetaiotaomicron (Bt), a non-pathogenic human commensal gut bacterium. We have developed the capability of engineering Bt to express antigens of interest in their OMVs which we have shown are stable for long periods of time across a wide temperatures range. They also have inherent adjuvanticity as shown by the ability of native OMVs to elicit the formation of organised lymphoid follicles comprising dendritic cells and T and B cells in both the upper and lower respiratory tract after intranasal administration. The intransal administration of Bt OMVs expressing the pre-fusion headless hemagglutinin mini-stem protein of influenza type A virus (IAV) subtype, H5N1, induced high titre antigen-specific antibodies in the respiratory mucosa (IgA) and serum (IgG) that conferred heterotypic protection to infection by a H1N1 IAV. Collectively, our data demonstrates the feasibility of using Bt OMVs in mucosal vaccine formulations for respiratory infections.
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- Virology Workshop: Clinical Virology
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Development of serologic tools for diagnosis and surveillance of Chikungunya
More LessChikungunya (CHIKV), an arbovirus that belongs to the Alphavirus genus of the Togaviridae family, causes a disease characterized by acute onset of fever accompanied by arthralgia. CHIKV also has been associated with cases of meningoencephalitis (primarily in neonates), Guillain–Barré syndrome and hemorrhagic disease. The clinical similarities, cross-reactivity and cocirculation of arboviruses in Brazil have complicated their differentiation, highlighting the need for new diagnostic tools. A serologic test can be useful for acute detections as well as for surveillance and epidemiological studies. Point of care tests currently available to detect CHIKV infections have been associated with low accuracy. The aim of this work was to design a synthetic gene and generate a recombinant protein to be used as antigens in diagnostic assays. Computational methods were used to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKV) was synthetized and the protein was purified by affinity chromatography. Antigenicity of the protein was initially confirmed by western-blot using sera from CHIKV infected mice. Additionally, the seroreactivity of r-CHIKV protein was evaluated using a panel of sera samples from human patients, CHIKV seropositive or not, by indirect IgG ELISA. The r-CHIKV protein showed sensitivity of 95 % and specificity of 96 %. No cross-reactivity was found against sera of Zika and Dengue positive patients. These results indicate that this proteins maybe useful antigen to detect CHIKV infections in ELISA assays. Our next step includes the development of a rapid test kit.
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Clinical management of low-level hepatitis B surface antigen in haemodialysis patients with a recent history of HBV vaccination – the results of a UK-wide survey
More LessIdentifying transient low-level hepatitis B (HBV) surface antigen (sAg) post-vaccination is a well-established phenomenon in haemodialysis patients. These results create a clinical conundrum; should patients be deemed potentially infectious, requiring further testing and heightened infection control precautions, or should negligible risk be assumed? National guidance covering this scenario is lacking; therefore we conducted a survey of the clinical practice of UK virology departments. Thirty-six laboratories were contacted and 17 responses received (47 %). Eleven responders had observed sAg positivity post-vaccination, for a maximum duration of 3 weeks. Clinical management was highly variable. Only 3 hospitals had specific written policies, with others following an internal consensus or a standard protocol for sAg positive patients without recent vaccine. Further testing consisted of HBV serology (3/11), HBV PCR (1/11) or PCR and serology (7/11). Three departments were highly risk averse, recommending universal isolation with dedicated dialysis machines. Two departments did not advise additional precautions and 5 based decisions on risk factors e.g. foreign travel. Heightened precautions were stopped on the basis of negative serology (1/11), PCR (5/11) or serology and PCR (2/11). To summarise, vaccine-derived sAg positivity is commonly encountered on haemodialysis units. Recommendations on the interval between vaccination and testing would alleviate the issue, with this study suggesting a minimum of 3 weeks. Infection control precautions varied considerably. In most cases, patients are risk assessed for isolation as a minimum; however this may depend on local expertise and the availability of isolation facilities. It is clear that national guidance would standardise patient care.
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Molecular typing of human enteroviruses: reanalysis of previously untypable EV isolates and the rapid detection of EV-D68
More LessMolecular diagnostics have superseded cell culture and neutralisation as the gold standard for enterovirus (EV) diagnosis and serotyping in resource rich countries. EV positive isolates from Nottingham University Hospitals NHS Trust are referred to the Enteric Virus Unit (EVU) for typing; performed through amplification and sequencing of viral capsid protein-1 (VP1). An audit of local EVU typing data (2013–2017 showed a significant (P<0.005) increase in untypable EV strains isolated from clinical specimens. Following a literature review of published EV typing assays and in silico analyses of 2201 EV genomes, an end point RT-snPCR VP1-typing assay was derived from a widely cited protocol and successfully employed to produce a simple, reliable and cost-effective typing assay. Twenty-three previously untypable isolates were identified, with phylogenetic analyses of the VP1 region showing broad distribution across EV-A and B species. This assay detected serotypes across EV species A-D, including EV-D68, responsible for severe neurological and respiratory disease. Following an increase in severe EV infections, including six cases of suspected acute flaccid myelitis, this RT-snPCR was employed locally as a rapid typing assay. In combination with 5’UTR PCR and an EV-D68 specific PCR, designed to amplify the complete VP1 region, EV-D68 was detected in respiratory and neurological specimens, including the CSF of a patient with brainstem encephalitis.
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Wombling surplus diagnostic nucleic acid for novel pathogenesis and genetic epidemiology of viral infections
Nottingham University Hospitals Trust receives circa 13 000 samples for diagnosis of respiratory and neurological viruses per annum, however positive results are achieved in approximately 50 % of respiratory and 20 % of neurological investigations. We therefore aimed to retrospectively extend the diagnostic spectrum for these samples by applying a battery of degenerate PCR assays to surplus diagnostic nucleic acids.218 previously negative respiratory specimens collected in 2016 from children under 5 years old, identified positivity of 11 % for Human Bocavirus, 5 % for Human Enteroviruses (including 3 cases of Enterovirus D68), 4 % for Human Coronaviruses and one individual positive for Trichodysplasia Spinulosa Polyomavirus. Complementary investigation of 1730 previously negative specimens from children and adults with neurological symptoms yielded positive results for Hepatitis E, BK Polyomavirus and Astroviruses in addition to Entero- and Parechoviruses apparently missed by standard diagnostic assays. Our extensive archive further allowed us to investigate relatively rare viral infections in significant numbers. Therefore we also studied the genetic and clinical epidemiology of the human Rubulavirus pathogens Parainfluenza 2 and 4 in 121 and 237 patients respectively between 2013 and 2017. This indicated co-circulation of three clusters of Parainfluenza 4 in Nottingham with greater presentation of subtype 4b than 4a and 2 clades of Parainfluenza 2. 5 fatalities were recorded in the Parainfluenza 2 cohort. In summary, surplus nucleic acid from viral diagnostic laboratories represents a valuable resource for both service development and clinical research. Coronavirus, Bocavirus and Enterovirus testing have since been implemented in the routine diagnostic panel for respiratory investigations.
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Clinical evaluation of herPHEgen®: a herpesvirus genotype-to-phenotype antiviral resistance database and testing service
Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infections may be life-threatening in immunocompromised patients. Antiviral treatment is available but resistance can develop. The gold standard for resistance testing is cell culture-based phenotyping which is slow (3–4 weeks) and has a high (50 %) failure rate. Faster (<10 working days) genotypic testing of viral thymidine kinase (TK) and DNA polymerase (DNApol) can be used with >95 % success rate. However, a reference database for interpreting drug susceptibility from genetic data is lacking. We developed an HSV genotypic test and genotype-to-phenotype drug resistance database. We evaluated the service using 325 clinical samples, previously characterised by phenotypic susceptibility testing, from 248 treatment-experienced patients. The median age was 42.5 years [IQR30.0–51.0] and 50.8 % (n=126) were female. Clinical details were as follows: 42.3 % (n=105) haemato-oncology patients; 12.1 % (n=30) HIV-infected; 4.4 % (n=11) unspecified immunosuppression; 2.4 % (n=6) congenital infection; 1.2 % (n=3) solid organ transplant; 37.5 % (n=93) unknown. HSV-1 was identified in 58.2 % (n=189) samples. Phenotypic testing identified resistance in 63.7 % (n=207) samples. Genotypic testing identified a resistance-associated mutation (RAM) in TK and/or DNApol in 200/207 samples, a positive percent agreement of 96.6 %, whereas a RAM was detected in 1/118 susceptible samples, a negative percent agreement of 99.2 %. Most RAM occurred in TK (n=195; 97.0 %) with few in DNApol (n=32; 15.9 %). In summary, through herPHEgen we have developed a robust HSV resistance testing service, providing clinicians with timely and accurate results. This will improve clinical decision-making, optimising treatment efficacy and minimising toxicity in immunocompromised patients with HSV.
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Low prevalence of resistance mutations to integrase inhibitors (INI) in patients living with HIV-1 at baseline using next generation sequencing (NGS)
More LessCurrent guidelines indicate that baseline INI resistance testing is not required; however, increased prescribing may lead to increased resistance mutation prevalence. NGS allows the detection of minority variants (MVs) not be detected by conventional methods. Barts Health NHS Trust patient records were reviewed to identify INI naïve patients prescribed INI between March 2014 and March 2017. Baseline plasma samples were extracted for nucleic acid and amplified in a single RT-PCR. Libraries were prepared using the Nextera XT library preparation kit and sequencing performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences and resistances determined using the Stanford HIV resistance database (HIVdb). MVs were variants <20 % of the overall population. Of 248 patients prescribed INI, 128 patients were excluded from the study due to an undetectable viral load or lack of sample. Of the remaining 121 patients, integrase sequences were successfully obtained from 91. No major INI mutations were detected. Six patients were identified with an HIVdb INI score ≥10: E157Q (4 patients) and T97A (2 patients). Only 1 MV accessory mutations was observed (A128T). Of patients with resistance mutations, 5 received raltegravir- and 2 received dolutegravir-based regimes. No treatment failures were observed. Prevalence of baseline INI mutations increased in recent years: 0/12 (0 %) patients in 2014–2015, 2/32 (6.3 %) patients in 2015–2016 and 5/47 (10.6 %) patients in 2016–2017. Resistance MVs to INI were extremely rare. Although this study supports current baseline INI resistance testing guidelines, increasing mutation prevalence’s may require future revision of guidelines.
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Applying Next Generation Sequencing Technology to Hepatitis C genotyping in a routine diagnostic laboratory
More LessBackgroundTo meet the target of Hepatitis C elimination in the UK, a rapid and high throughput method of genotyping is desirable. Current methods target the NS5B region with a nested PCR followed by Sanger sequencing. Drawbacks include insufficient information to guide second line treatment in the event of failure and poor sensitivity compared to assays targeting the 5’UTR.
MethodPreviously tested HCV positive patient samples were selected and RNA extracted using an ultra-centrifugation manual extraction method. A two-step reverse transcription long range PCR generated amplicons which were quantified using the Qubit fluorometer and libraries prepared using the Nextera XT library preparation kit. Sequencing was performed using an Illumina Miseq and genomes were assembled using a Linex-based bioinformatics pipeline.
ResultsA total of 150 samples were tested, with full HCV genomes obtained for genotypes 1–6 and the sensitivity of the assay corresponded to around 1000 IU ml−1. Where previous sequence data for resistance associated variants (RAVs) was available, mutations were consistent. Furthermore, the assay identified a 2 k/1b recombinant and allowed the typing of previously unassigned genotypes, demonstrating its utility in the diverse local population.
ConclusionThis study displayed the utility of a whole genome sequencing approach to HCV. A major benefit of whole genome analysis was the inclusion of regions targeted by antiviral therapy such as NS5A and NS3. This allowed the identification of RAVs in patients experiencing treatment failure. Its application to dried blood spots is currently being investigated as part of a wider screening programme.
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