- Volume 2, Issue 7A, 2020
Volume 2, Issue 7A, 2020
- Abstracts from Annual Conference 2020
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- Poster Presentation
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Comparison of human gut microbial profiles of matched faecal and biopsy samples from healthy individuals using culture dependent and independent methods
Faecal samples have often been used to characterise the gut microbiota in health and disease. There is significant debate whether faecal bacterial communities accurately reflect the mucosa associated bacterial populations, which are considered critical in the aetiopathogenesis of several gastrointestinal diseases. We simultaneously assessed faecal and mucosal microbiota from healthy volunteers to unravel the degree of concordance between the two profiles. Paired fresh rectal biopsies and faecal samples were obtained from ten healthy volunteers and processed under stringent anaerobic conditions. Composition and diversity of the microbiota were studied using next generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene and culturomics. Bacterial richness and diversity were comparable between mucosal and faecal samples with no significant statistical differences. The relative abundance of Oxalobacteraceae, Propionibacteriaceae, Campylobacteraceae and Corynebacteriaceae were significantly increased (Corncob analysis; FDR=0.00027, 0.000046, 0.011 and 0.025 respectively) in biopsy compared to faecal samples at the family level. Conversely, there was increased abundance from the family Ruminococcaceae and Clostridiaceae (Corncob analysis; FDR=0.025 and 0.025 respectively) in faecal samples. Principal Coordinates Analysis of a Bray Curtis distance matrix generated from sequence variant tables did not show distinct clustering of biopsy and faecal samples (PERMANOVA; p=0.991). A total of 528 bacteria were isolated from a subset of 6 volunteer samples (biopsy and faeces) out of which there were 97 unique and 39 novel species identified. Our study showed good concordance between faecal and gut mucosal microbial profile, corroborating that faecal samples can act as a convenient surrogate to study gut microbiota.
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Enhancing the natural toxicity of Salmonella Typhimurium towards tumours
More LessBackground and objective: Salmonella is the underlying cause of foodborne diseases and poses a major public health problem worldwide. Research has developed a method to efficiently treat cancer using some of the same bacteria behind food poisoning, one of these bacteria is Salmonella which targets and penetrats tumours specifically by being attracted to the compounds produced by tumour cells and accumulating at the tumour site and inducing inflammation. In this project we aim to investigate the mechanism of Salmonella Typhimurium which has a tremendous ability to invade, replicate and compete to survive inside the cells by virtue of effector proteins such as: sipA, sipB, and AvrA which it possesses.
Method: The S.Typhimurium strains used are wild-type SL1344 ( ΔsipA, ΔsipB, ΔavrA and VV341) and attenuated strain of SL7207. Bacteria were cultured in LB broth in a 37 °C shaker overnight to reach a stationary phase before using it to infect B16F10 (mouse melanoma).
Results: The initial results show that the infection of B16F10 with wild-type SL1344 had a high level of invasion compared to the low number of bacteria with the deletion of sipB which impaired its entry into the cell. Similarly, the mutant strains ΔsipA and ΔavrA show an increasing number of intracellular bacteria, like the wild-type strain. We will be investigating further on the innate mechanisms of Salmonella in disrupting tumour growth and progression, that might help maximize the potential of using these bacteria in monotherapy or in tandem with other useful therapies.
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Characterising the role of Proline Tyrosine Kinase 2 during Adherent-invasive Escherichia coli infection of macrophages
More LessStudies have implicated that Adherent-invasive Escherichia coli (AIEC) in the aetiology of Crohn’s Disease (CD), a chronic inflammatory bowel disease (IBD). CD-associated AIEC are characterised by an ability to adhere to and invade intestinal epithelial cells and replicate intracellularly in macrophages. This occurs as AIEC form vacuoles within phagolysosomes, preventing macrophage-mediated killing. However, little is known about the interaction of macrophage proteins with AIEC.
In this study, we used an in vitro infection model to identify macrophage proteins associated with AIEC intracellular replication. We identified the importance of proline-rich tyrosine kinase 2 (PYK2) in AIEC infection. PYK2 is widely expressed in epithelial cells and hematopoietic cells and plays essential roles in tumorigenesis and tumour progression. PYK2 is also overexpressed in patients with intestinal and colorectal cancer (CRC). CRC is one of the major long-term complications of IBD, especially in patients with long disease duration, a large extent of colitis, and uncontrolled inflammation. Moreover, PYK2 has been identified as an IBD risk loci via large scale genome-wide association studies.
Our in vitro models have demonstrated that the addition of PYK2 inhibitor PF-431396 during infection results in a significant decrease in intracellular replication of AIEC. This has been quantified using fluorescence immunostaining and imaging flow cytometry. These results suggest that PYK2 may play a role in intracellular bacteria survive and replication. Our future work aims to further understand the relationship between PYK2, AIEC and CD.
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Prevalence of Tinea unguium (Onychomycosis) in Toe Nails of boot wearing group (The Footballers) in Owerri, Imo State. Nigeria
More LessFoot mycoses are a frequent disease that represents a public health problem worldwide. This study aims to evaluate the epidemiology of foot mycoses among footballers in Owerri, Imo State, in order to determine the fungal etiological agents and to identify possible risk factors. To investigate the treatment and preventive measures for the susceptible groups. A total of 485 samples were collected; tinea unguium were confirmed in 88.2% of cases. A prospective study of fifty footballers was undertaken during one year (2018-2019). A complete mycological diagnosis was carried out on all footballers. The results obtained showed that out of the 50 toe nails samples of footballers examined, 28(56.0%) had positive cases of the infection on direct microscopy which served as the screening test. The causative pathogens of Onychomycosis isolated from fungal culture are dermatophytes , and the most frequent pathogen was Trichophyton rubrum 15(30.0%), yeast Candida albicans 9(18.0%). Non-dermatophyte molds were observed in 8(16.0%) cases and Fusarium sp. was the frequent genus 14(28.0%)and Aspergillus sp. 4(8.0%). The main predisposing factors of fungal foot infections were practicing ritual washing (56.6%) and frequentation of communal showers (50.5%). Confirmatory test such as germ-tube test was done on the Candida albicans isolate for proper identification. The age group 31- 35 years had the highest prevalence of onychomycosis 12(92.31%) and age group 16-20 years presented the lowest prevalence of onychomycosis 4(28.57%). Proper care of toe nails and boots is necessary to prevent the increasing rate of this infection.
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Metagenomic mining of novel/innovative commercial glycoenzymes
More LessIn biological science, metagenomics has revolutionised the process of drug discovery. This is because metagenomics serves as a tool for in depth characterisation by examining, sequencing, replicating and identifying the whole DNA/genome collected from any mixed population (Mahapatra et al., 2019). Metagenomics is used to study Carbohydrate Active Enzymes (CAZymes) containing microbial communities due to molecular-based culture-independent methods (Kunath et al., 2017). CAZymes are made of multiple domains; each domain serves as the key mediator for a specific function for the protein and structure. In the Carbohydrate-Active enZYmes (CAZy) database 20% of all protein domains are identified as domains of unknown function (DUFs). DUFs are mostly ignored due to not being the most abundant in many genomes. However, in a paper by Goodacre 2014, DUFs are shown to be essential and likely related to the organism survival function due to their relation to essential proteins(Goodacre et al., 2014). In the CAZy database multiple DUFs are associated with catalytic CAZyme domains, which could result in them having a similar function or increase their catalytic activity. We present DUFs that are considered to have the capability to help CAZymes break down polysaccharide chains and tools/methods we used to achieve these results.
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The selection and co-selection of antimicrobial resistance by non-antibiotic drugs and plant protection products
More LessNon-antibiotic compounds including metals and biocides co-select for clinically relevant antimicrobial resistance (AMR) genes. Presently, there is little research looking at the effects of plant protection products (PPPS) such as herbicides and insecticides on the development and spread of resistance. Agricultural activities require the direct application of PPPs in large quantities and at high concentrations to soil. These chemicals are applied alongside antibiotics and manures which may contain antibiotic residues, other selective agents and resistant bacteria. The selection pressure exerted by this mixture of chemicals may result in enrichment of resistant bacteria and/or the exchange of resistance genes between environmental and clinically relevant bacteria. The impact of these chemicals on AMR and microbial diversity in terrestrial environments is poorly understood.
PPPs from different substance groups were tested for antimicrobial activity using the SELECT method developed by Murray et al. (2019; under review). In this method, bacterial communities were exposed to PPPs and selective concentrations were identified by a significant reduction in community growth. Results from these experiments determined the concentration at which long term evolution experiments were carried out in soil microcosms. Selection for AMR was investigated using metagenomic analysis and targeted real-time PCR.
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A comparative approach to understanding selection on extracellular secretion
More LessMany bacterial genes encode proteins that are secreted extracellularly. These proteins can be considered cooperative because all surrounding cells can benefit from their production. Therefore, it has been hypothesized that these cooperative genes would more frequently lie on mobile elements, such as plasmids, which can transfer to other cells. This could stabilise cooperation, leading to the prediction that plasmids should carry proportionally more cooperative genes than the less mobile chromosome. However, it is unknown whether this prediction holds across the bacterial tree of life. To address this, we analysed the gene content of the chromosome and plasmid(s) of 1620 genomes comprising 51 diverse bacterial species. We find that across species analysed, plasmids do not carry proportionally more cooperative genes than the chromosome. Contrary to prediction, the role of mobile elements in promoting cooperative behaviour is highly variable across bacterial species.
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A potential new paradigm of denitrification in pathogenic Neisseria
More LessMetals are bacterial nutrients. Upon infection by microorganisms, the animal host innate immune system typically reduces the availability of metals. In response, bacterial pathogens can activate pathways for metal uptaketo avoid metal starvation. This competition for metals at the host-pathogen interface is termed “nutritional immunity”.
We are interested in how the obligate human pathogen Neisseria gonorrhoeae acquires nutrient copper (Cu). This Gram-negative bacterium expresses several respiratory cuproenzymes that are required for growth and metabolism in both aerobic and anaerobic conditions. These cuproenzymes include the putative nitrous oxide reductase (NosZ). NosZ contains 12 Cu atoms per functional dimer and it catalyses the reduction of nitrous oxide (N2O) to dinitrogen (N2). This reduction is an intermediate step in the denitrification pathway, in which nitrite (NO2) is used as the terminal electron acceptor for respiration instead of O2. In this project, we will determine whether NosZ is expressed as a functional enzyme in N. gonorrhoeae. We will then examine how this enzyme acquires nutrient Cu and subsequently assembles its active site.
Given the rise in antibiotic resistance and the worldwide recognition of multidrug-resistant N. gonorrhoeae as a major threat to public health, we hope that a fundamental understanding of the physiology and metabolism of this organism will yield new strategies for anti-infectives, for instance by manipulating Cu availability at the site of infection.
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Influence of phosphate dosing on biofilm development on Drinking Water Distribution Systems infrastructure surfaces
Phosphate is added to drinking water by UK water companies as a treatment to prevent the corrosion and metal leaching, like lead, in pipes. However, phosphate is a nutrient for microorganisms, and it can favour biofilm formation in Drinking Water Distribution Systems (DWDS), which can alter the water quality and safety. This study analyses the effect of phosphate addition on biofilm formation over different materials and its consequences for drinking water quality by i) using controlled experimental pipeline facility representative of a real-scale DWDS with high-density polyethylene coupons and ii) using a small-scale DWDS biofilm reactors with lead coupons. Biofilms developed over one month were exposed to the effect of different phosphate dosing and compare with UK normal water phosphate concentrations. During the experiment, physico-chemical analysis of water and microbial analysis of biofilms was carried out. Sequencing analysis of the 16s rRNA gene, from extracted DNA obtained from biofilms, provided information on any bacterial changes, and Scanning Electron Microscopy gave information about the biofilm organization. The results indicate that microorganisms find more difficult to establish and develop biofilms under high phosphate dosing, resulting in biofilms with less cells. Also, some physico-chemical parameter seems to be affected by phosphate dosing, like chlorine and lead. It is expected that differences in the biofilm community will be found depending on phosphate dosing. This study will provide information on the effect of phosphate on biofilm development in different pipes materials, which will facilitate to adjust an optimal phosphate dose to prevent plumbosolvency in DWDS.
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Combination of Rifampicin and Colistin against antibiotic resistant Enterobacteriaceae
More LessAntimicrobial resistance is one of the greatest challenges for humanity. Patients, especially, admitted to the intensive care unit have been exposed to higher risk of healthcare associated infections mostly caused by antibiotic resistance since the imprudent use of antibiotics over the years. The discovery and development of new antibiotics are facing difficulties with the continuous evolution of drug resistance in bacteria, and the reduced investment in R&D for antibiotics from pharmaceutical companies. Novel strategies are urgently needed to control this pandemic threat of antibiotic resistance. Antibiotic combination with two or more drugs may be useful in targeting resistant gram-negative bacteria by producing synergistic effect and enhanced bactericidal activates. In this study, we determined the combination of rifampicin and colistin against Extended Spectrum Beta Lactamase (ESBL), carbapenemase producing and colistin resistant Enterobacteriaceae using chequerboard method and time kill curves. We measured the combination effects based on Fractional Inhibitory Concentration index (FICI) and the efficacy of bacterial reduction comparing to that of single antibiotic. Interestingly, we found that the combination of rifampicin and colistin showed synergistic activities against the tested bacteria, indicating FIC index ≤0.5. The time kill curve shows that the two drugs combination exhibits 99% kill whilst the single antibiotic had no activities. Thus, the combination of rifampicin and colistin demonstrated synergistic activity with reduced MIC and the increased rate of killing against both ESBL and carbapenemsase producing and colistin resistant Enterobacteriaceae.
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Molecular identification of ticks and tick borne pathogens in Nigerian Dogs: A public health threat
More LessGlobal warming has changed the range of tick species and the tick borne diseases (TBDs) they carry; there is an increasing need to assess the risk and impact of TBDs. This study focused on TBDs of Dogs in Nigeria, where despite a large burden of ticks and TBDs there is little data on the prevalence of either for veterinarians and human health professionals to make informed decisions regarding treatment and control.
Most existing studies on cattle have indicated that Anaplasma, Babesia, Theileria, Ehrlichia, Hepatozoon and Candidatus Neoehrlichia species are present in Nigeria. However, dogs kept in much closer contact with their owners, may represent a higher risk of zoonotic TBDs transmission. This study used a combination of morphological and molecular identification of ticks and tick borne pathogens, and IDEXX 4Dx kits for pathogen antibody detection from canine blood, we examined 93 dogs to date.
Rhipicephalus sanguineus was the most abundant tick found on Nigerian dogs. Babesia species were the most abundant tick borne pathogen, followed by Ehrlichia species. This study serves as the first report of Babesia microti like species, Theileria parva, Babesia bovis, Anaplasma phagocytophilium and Ehrlichia chaffeensis in Nigerian dogs. There was no evidence of Borrelia Burgdorferi sensu lato nor Dirofilaria species in Nigerian dogs. The presence of Babesia microti and Anaplasma phagocytophilium is a direct public threat. Further testing of additional 200 dog samples and multivariate modelling of demographic risk factors, will enable us develop treatment and control guidelines for TBDs for Nigerian Veterinarians and Dog owners.
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Investigating selection for antimicrobial resistance by non-antibiotic drugs (NADs)
A recent study screening over 1000 drugs has shown that non-antibiotic drugs (NADs), including human targeted drugs, have been shown to have antimicrobial effects on representative strains of human gut bacteria. NADs are present in both wastewater effluent and freshwater systems, where they may only be partially metabolised. Concentrations of antibiotics found in the environment have been shown to select for resistance in complex microbial communities, and it is thought that concentrations of NADs in the environment may also select for resistance. This project aims to determine if NADs select for antimicrobial resistance, by investigating effects in a complex bacterial community. Pharmaceuticals from 7 different drug classes have been screened for antimicrobial activity. The most potent compounds will be used in exposure experiments to identify any existing antimicrobial resistance genes that confer cross-resistance to both antibiotics and NADs using a metagenomics approach. In addition, changes in community composition will be investigated to determine if there is enrichment for opportunistic pathogens after exposure to NADs. This project ultimately aims to inform on environmental water quality standards and environmental risk assessment of wastewater treatment effluent in order to mitigate the growing problem of environmental antimicrobial resistance.
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Next generation sequencing and in vivo pathogenicity study of multidrug-resistance efflux pump genesoqxAB-encoding plasmids in Escherichia coli
More LessBackground: Multidrug-resistant efflux pump genes oqxAB-encoding plasmids are wildly disseminated in livestock, and also identified in human samples. Giving the high presence in environment and multidrug-resistant nature of oqxAB, deeper understanding of oqxAB-encoding plasmids is necessary.
Objective: To investigate the prevalence, virulence, and phylogenetic of oqxAB-encoding plasmids.
Method: A total of 43 oqxAB carrying Escherichia coli were isolated from human and livestock from China and Hong Kong, these isolates were characterised by disc-diffusion susceptibility test and PCR. The 43 samples were sequenced using PacBio or Illumina platforms, and analysied with five oqxAB-encoding plasmid sequences extracted from Genbank. in vivo pathogenicity study performed by injecting E. coli J53 transconjugants containing oqxAB plasmid into Galleria mellonella larvae.
Result: Among the 48 oqxAB-encoding plasmids, 27 were IncFII F18 plasmids, 12 F16 plasmids, six F33 plasmids, two F24 plasmids, and one F14 plasmid. Virulence regions in plasmids with same Inc group encoded similar set of virulence factors and antibiotic resistance genes. Co-existence of blaCTX-M, fosA3, and oqxAB genes was identified in all F33 plasmids and one F18 plasmid. in vivo pathogenicity study suggested oqxAB plasmids caused significant health deterioration in larvae but no significant increase in death.
Conclusion: Our data shows similar sets of pathogenic factors are frequently co-carried by oqxAB plasmids of same Inc group, suggesting high correlation between plasmids identified from China and Hong Kong. Co-existence of fosA3, blaCTX-M, and oqxAB suggests resistance to most clinical antibiotics. in vivo test indicates oqxAB plasmid can increase pathogenicity of the host bacteria.
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Risk factors associated with the mortality of Acinetobacter baumannii
More LessBackground: Acinetobacter baumannii (AB) was declared an antibiotic-resistant “Priority 1 pathogen” by WHO. We sought to investigate the predisposing risk factors to this pathogen.
Methods: In a retrospective study, adults who were admitted to Sohar hospital during 2016-2017 and had a positive laboratory-confirmed culture of AB were studied.We classified patients into 2 groups based on 30-day, all-cause mortality and compared the characteristics. Exploratory classification and regression tree (CART) analysis was performed to explore risk factors for mortality to include to a logistic regression model.
Results: A total of 321 patients were included, age was (Mean±SD) 57.42±20.22, male gender was 180(56.07%), mortality was 140(44%). Survivors vs deceased had; length of stay 38.25±88.74 vs 51.31±79.19 (p=0.002),multi-drug resistantisolates 134(51.34%) vs 127(48.66%) p=<0.001, critical care admission 35(38.04%) vs 57(61.96%) p=<0.001, comorbidities 114(47.50%) vs 126(52.50%) p=<0.001 and history of invasive procedures 82(59.85%) vs 55(40.15%) p=0.27. Logistic regression revealed that the odds of dying increase by a factor of 1.044 for every additional year of age, 1.844 times higher for male compared to female, 4.412 times higher for patients admitted into critical care units compared to general wards, 3.138 times higher for patients admitted with a diagnosis of infection, 2.356 times higher for patients with hospital-acquired AB infection compared to community-acquired.
Conclusion: Both modifiable and non-modifiable risk factors are associated with mortality and overall health status may contribute to infection outcome. Stabilization of comorbidities and effective antimicrobial treatment could be the mainstay of successful prevention.
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Machine learning models to identify patient and microbial genetic factors associated with carbapenem-resistant Klebsiella pneumoniae infection
More LessThe World Health Organization considers carbapenem-resistant Enterobacteriaceae (CRE) an urgent public health threat due to global prevalence, limited treatment options, and high mortality rates. Carbapenem-resistant Klebsiella pneumoniae (CRKP), the most common CRE species, is especially prevalent in long-term acute care hospitals (LTACHs) in the United States. Among patients colonized with CRKP, only a subset develop clinical infection. We used CRKP whole-genome sequences and associated clinical metadata from unique patient samples from 20 LTACHs (n=366) to (1) identify both clinical and microbial features associated with infection compared to colonization, and (2) evaluate the contribution of microbial features in infection prediction. Modifiable clinical features associated with infection could inform clinical practice, while microbial features could help predict clinical infection. We performed L2 regularized logistic regression 100 times for each feature set. Clinical predictors of infection include having a central line or gastrostomy tube. Genomic predictors of infection include iron scavenging genes (known to be associated with invasive infections in healthy hosts) and the number of antibiotic resistance genes. Furthermore, we found that clinical and genomic features (separate or combined) have similar predictive power (AUROC IQRs: clinical=0.56-0.64, genomic=0.54-0.63, combined=0.59-0.67). This suggests that genomic features may be associated with certain clinical features. These results provide insight into potential clinical and microbial drivers of CRKP infection in LTACH patients and provide a starting point for investigating the biological basis of infection, identifying patients at high risk of infection, and devising targeted strategies to prevent infection.
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Fatty acids as novel treatment options for Pseudomonad and Staphylococcal infection
More LessPseudomonas aeruginosa and Staphylococcus aureus are bacteria pathogens that cause a myriad of infections affecting various sites in the body including the eyes, ears, lungs, skin, heart, bones, and blood amongst others. These bacteria can be disseminated via the blood to other parts of the body away from the primary site of infection and consequences vary from mild to severe with death occurring in certain instances. Both bacterial infections can occur individually, as well as in co-infection resulting in even worse outcomes. P. aeruginosa and S. aureus exhibit multidrug resistance against current antibiotic treatment regimens, which accentuates the challenge in managing the infections caused by these bacteria. To prevent the looming era of untreatable bacterial infections, alternative treatment regimens that are cost effective and accessible are needed. To explore novel treatment options, twenty-five organic compounds comprising fatty acids and their derivatives were screened for antibacterial activity in broth microdilution assay to determine the minimum inhibitory concentration and minimum bactericidal concentration against both P. aeruginosa and S. aureus. Five candidates (N–nonanoic acid, butyric acid, heptanoic acid, palmitoleic acid, and isopropyl myristate) were effective against P. aeruginosa. Seven candidates (N–nonanoic acid, palmitoleic acid, tridecanoic acid, sebaic acid, undecanoic acid, monolaurin, and monocaprin) were effective against S. aureus. Candidates such as N–nonanoic acid and palmitoleic acid were effective against both P. aeruginosa and S. aureus, demonstrating that the same fatty acids show potential to be used against both Gram negative and Gram positive bacterial infections.
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Use of a laboratory model hospital sink system to investigate fluctuation of Gram-negative bacteria in sink waste traps
More LessHospital sinks in the UK have recently been under scrutiny as possible reservoirs for Gram-negative bacteria, especially carbapenem resistant Enterobacterales (CRE). These strains have been found in intensive care wards across the country and can re-enter the clinical environment, representing a risk to vulnerable patients.
Two sink waste traps known to be colonized with CRE were collected from a hospital and fitted to a vertical-draining and rear-draining handwash sink installed within a laboratory model sink system. Sinks were automatically flushed four times a day and, as per usual in the model, TSB was provided once daily to maintain microbial populations. Gram-negative bacteria were regularly monitored using selective culture, MALDI-TOF and antibiotic disk diffusion. The short-term effect of adding simulated IV fluids (5% glucose or 0.9% NaCl) and the impact of sink design on Gram-negative proliferation were investigated. Communities included Enterobacter asburiae; Klebsiella oxytoca; Pseudomonas aeruginosa and Citrobacter freundii, among others, including CRE. The addition of simulated IV fluids did not induce Gram-negative bacterial proliferation in the time frame of the experiment. Differences were observed in the fluctuation of Gram-negative levels after flushing between the different sink designs. Gram-negative numbers in vertical-draining sinks decreased immediately after the tap was flushed and subsequently increased between flushes. However, in rear-draining sinks, little fluctuation was observed. Hospital sink waste traps can harbour Gram-negative bacteria resistant to antibiotics. In our experimental conditions, the type of sink was the determining factor in the magnitude of fluctuation in Gram-negative populations while simulated IV fluids had little effect.
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Characterization of bacteriocin related genes discovered in a novel probiotic isolate Enterococcus faecium W1
More LessAims: To isolate and characterize the bacteriocin activity identified by the genomic analysis of a novel enterococcal isolate, Enterococcus faecium W1
Methods and Results: Culture fermentates of a novel E. faecium isolate, W1 showed high levels of antimicrobial activity against Listeria monocytogenesisand a variety of other Gram+ve and Gram-ve bacteria. Ammonium sulphate precipitation of the fermentates followed by hydrophobic interaction chromatography showed bactericidal activity correlated with fractions containing only low molecular weight proteins. The same fractions had activity on human cancer cell line HT-29 measured by cytotoxicity assay (MTT) and apoptosis induction. Whole-genome sequencing of E. faecium W1 revealed five bacteriocin related gene clusters, a lack of virulence factors and lack of antibiotic resistance genes. To confirm that antimicrobial activity was correlated with the bacteriocin genes identified, selected bacteriocin genes were cloned and expressed as His-tagged proteins in E. coli. Bactericidal activity against L. monocytogenes was associated with protein purified by metal affinity chromatography. A structure-function analysis to investigate the role of key residues in activity is ongoing.
Conclusion: E. faecium W1 is a promising candidate for use as a probiotic supplement or in food preservation. The bacteriocin related genes are functional and could be improved for future use as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: There is a need for new approaches to the growing problem of antibiotic resistance. Expression of bacteriocins identified in E. faecium W1 could contribute to this endeavour in addition to its use as a probiotic organism.
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Foot-and-mouth disease virus non-structural protein 3A modulates cellular innate immune responses to influence host tropism
More LessThe picornavirus foot-and-mouth disease virus (FMDV) is responsible for one of the most significant diseases of livestock, leading to large economic losses due to reduced productivity and trade embargoes for areas not certified as disease-free. The picornavirus non-structural protein 3A is involved in replication of the viral RNA genome and is implicated in host tropism of several picornaviruses. Deletions in the C-terminus of 3A have been observed in FMDV outbreaks specific for swine and such viruses are non-pathogenic in cattle. The mechanism for species specific attenuation of FMDV is unknown.
We have shown that FMDV containing a C-terminal deletion in 3A is attenuated in bovine cell culture and that the attenuated phenotype can be reversed by the JAK1/2 inhibitor Ruxolitinib (Rux), identifying a role for the induction of interferon stimulated genes (ISGs) in the restricted bovine tropism of the 3A-deleted virus.
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Generation of Equine Herpesvirus type 1 glycoprotein pseudotyped lentiviral particles for use as a tool for tropism and diagnostic studies
Equine herpesviruses (EHVs) are enveloped DNA viruses infecting mainly members of the Equidae family and also members of other taxa. EHVs primarily causing respiratory disease, however EHV type 1 (EHV-1) can produce cases of a neurological disease, abortion and neonatal death, sometimes as regional outbreaks. Thus these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 presents a complex array of 12 glycoproteins on its surface envelope, but it is unclear which ones are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a perfect study tool. In 2016, Rogalin & Heldwein successfully generated the first functional herpesvirus pseudotype, bearing the four glycoproteins gB, gD, gH and gL from human Herpes simplex 1. Our study is the first to attempt pseudotyping of EHV-1. We have employed homologous glycoproteins of EHV-1 in lentivirus PV generation, using different mammalian cells (e.g. epithelial, dermal, CNS) as transduction targets. The glycoprotein sequences obtained from an EHV-1 strain isolated from organs of aborted foetus during a significant outbreak in Normandy (France) in 2010. Future work will focus on the development of a PV assay for detection of neutralising antibodies in naturally infected horses for diagnostics and for vaccine evaluation.
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A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus
More LessIn Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins.
Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.
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Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa
More LessPseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.
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Generation of a bacterial clone for assessing the impact of climatic stress conditions on microbial proliferation
More LessDocumented increases in atmospheric Carbon Dioxide (CO2) concentrations have contributed to a rise in average global temperatures. Environmental variation due to climate change is expected to affect the growth of microorganisms. Hence, there is a need to assess the induced adaptations of microorganisms, which are common biological contaminants, to environmental changes. Therefore, an enhanced green fluorescent protein (eGFP) expressing Escherichia coli BL21(DE3) clone was generated. Plasmid pAP1698-4 was used as the donor for the eGFP gene and pD454-MBP as the recipient plasmid to produce pD454-MBPeGFP. Expression of eGFP in the clone was confirmed using confocal microscopy. The growth of the clone was characterised by plate counting technique. Variation in the length of the lag phase, λ, and growth rate, μmax, kinetic parameters of the clone was observed, compared to the wildtype BL21(DE3). A live/dead kinetic assay, using eGFP for the quantification of live cells and propidium iodide (PI) as a stain for dead cells, was optimised using a microplate reader with controlled temperature and CO2 conditions. Full growth curves were collected when culture media was inoculated with 4 to 6 Log10CFU.mL-1. The optimal PI concentration was 150 nM; higher concentrations inhibited growth, and lower concentrations gave no signal difference compared to the blank. The growth kinetics of the clone under different environmental conditions; between 400 ppm to 2500 ppm CO2, combined with 37°C to 42°C, were evaluated using the live/dead kinetic assay, allowing assessment of response to induced environmental stress.
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Analysis of β-lactam, azithromycin and fosfomycin resistance in non-typhoidal Salmonella: Characterisation of an S. Infantis plasmid
More LessNon-typhoidal Salmonella (NTS)infections are associated with high morbidity and mortality. β-lactams are used as first-line treatment but resistance to these has increased considerably in recent years. Azithromycin and fosfomycin are used as alternatives; however, the incidence of resistance in these drugs is also increasing. Epidemiological surveillance on 35,372 NTS received by Public Health England was conducted for analysis of demographics, including global travel. Genomic typing and antimicrobial resistance data for Salmonellaisolates were used to determine the prevalence of β-lactam, azithromycin and fosfomycin resistance in NTSover a four year period. No isolates were resistant to β-lactams, azithromycin or fosfomycin alone but all isolates were resistant to multiple antimicrobial classes. IncHI2, IncY and IncN plasmids were predominantly found in the most multi-drug resistant isolates. Multi-drug resistance (MDR) was particularly a concern in the S. Infantis population. Therefore, long read sequencing was used to characterise an MDR S. Infantis isolate. Three drug regions were identified in a IncFIB, a mega plasmid identified in this isolate. The resistance determinants fosA, arsA, arsD and blaCTXM65,were discovered on the same drug region. Analysis of IncFIB in this S.Infantis isolate revealed 99% similarity to a IncFIB plasmid in S. Infantis isolated from chickens in the USA. Thishas not been reported before, warranting efforts for enhanced surveillance programmes to identify sources of emerging resistance, which will aid in establishing control measures for prevention of spread of resistance.
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Convergence of virulence and drug efflux traits in a siderophore ABC transporter on mobile genetic elements in Gram negative pathogens
More LessThe accessory genome of the human pathogen Klebsiella pneumoniae is large, variable and highly mobile. This reservoir of genes leads to the emergences of hospital outbreak strains with enhanced virulence and multidrug resistance, such as ST258, and acts as a source for transfer of these traits to other Gram negative pathogens. One such mobile genetic element, ICEKp, is prevalent in isolates of invasive disease where it enhances iron acquisition by the siderophore yersiniabactin. Yersiniabactin is also a virulence factor in pathogenic Escherichia coli and Yersinia species. Similarities between siderophore transporters and drug efflux pumps led us to postulate that the yersiniabactin transport proteins could contribute to antimicrobial resistance. We determined the effect of loss and gain of ICEKp, or the transporters alone, on iron acquisition and drug sensitivity of K. pneumoniae and Escherichia coli. Deletion of ICEKp impaired iron acquisition of clinical isolate K. pneumoniae HS11286 due to reduced siderophore secretion and reduced ability to acquire iron from siderophores. A simultaneous increase in sensitivity to a broad range of antimicrobials could be complemented by reintroduction of the ybtPQ ABC transporter. Furthermore, transfer of ICEKp to E. coli occurred efficiently by conjugation and conferred a similar decrease in sensitivity to antimicrobials.
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Exploring potentials of indigenous yeasts in fermentation of Chambourcin Grapes
More LessBackground: Indigenous yeasts present on grape berries have been shown to impact the winemaking process and final wine quality. In this study, we used Chambourcin, a hybrid grape cultivar, to isolate indigenous yeasts for potential use in winemaking. Hybrid grapes are of particularly interest due to higher resistance to cold temperature and fungal diseases.
Methods: Yeasts were isolated from spontaneous fermentation of crushed grapes by plating on Dichloran Rose Bengal Chloramphenicol agar and identified by Sanger sequencing. Yeast candidates were selected for their ability to grow in Yeast Extract-Peptone-Dextrose broth supplemented with varying ethanol concentration. Candidate yeasts were chosen for mock fermentation using sterile Chambourcin juice. Volatile and non-volatile compounds that predict wine quality (flavor and aroma) were measured by UPLC and GC-MS.
Results: Hanseniaspora uvarum, Starmerella bacillaris, Candida californica, and Zygoascus meyerae were predominantly isolated from Chambourcin. S. bacillaris isolate 180002 and C. californica isolate 180004 were able to tolerate up to 10% ethanol compared to S. cerevisiae (ethanol tolerance up to 12%). Our results demonstrate that isolate 180002 grown in ethanol supplemented medium is comparable to the commercial S. cerevisiae. Various aroma profiles of three main chemical families – esters, higher alcohol and volatile acids in products fermented by S. bacillaris and other isolates were observed.
Conclusion: Starmerella bacillaris isolate 180002 demonstrates potential for use in winemaking with hybrid grapes based on ethanol tolerance and production of wine related chemical compounds. This study further supports the application of indigenous grape-associated yeasts in creating flavor and aroma diversity of wine.
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The role of flagella in Pseudomonas-Pedobacter social motility across a surface
More LessBacteria often reside in multi-species communities where many behaviors result from interspecies relationships. In a two-species community, we show that co-culture of Pseudomonas fluorescens and Pedobacter sp. permits motility across a hard agar surface where neither species moves alone. Pseudomonas species engage in surface motility, including swimming and swarming, but these require moist environments. We are exploring the role of the Pseudomonas flagella in social motility. We deleted genes related to flagellar structure and function to study the importance of flagellar elements on the social phenotype. Using microscopy and swimming assays, we evaluate the effects of gene deletions on the presence and function of flagella in the resulting mutants, and evaluate the effect on social motility by observing the phenotype on both hard (2% w/v) and soft agar (1% w/v). Removal of the flagellar filament abolishes social motility, indicating a requirement for flagella in social motility. Removal of the flagellar motor also abolishes social motility, demonstrating that flagella must be functional. However, removal of membrane-spanning structural components, or part of the type III secretion system, results in mutants that lack flagella, but participate in a similar motile behavior with Pedobacter. Here we describe a role for flagella in motility of a two-species consortium across a hard agar surface, an environment considered non-permissive for flagellar motility. The requirement for both bacterial species indicates we are observing motility as a social phenotype, with a contribution from Pedobacter that enables the Pseudomonas flagella to function under conditions relevant in the natural soil environment.
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Real-time WGS monitoring identifies L. monocytogenes outbreaks in The Netherlands and contributes to a rapid detection of the source
Whole genome sequencing (WGS) is increasingly used by food regulatory agencies and public health institutes. It is a powerful tool to identify the source of a foodborne outbreak. Real-time WGS analysis helps to act fast during a foodborne outbreak, and with that the impact of an outbreak can be significantly decreased. In The Netherlands real-time WGS analysis is performed for L. monocytogenes originating from humans and from the food chain, and WGS data is shared between the food regulatory agencies (WFSR and NVWA) and the public health institute (RIVM). Consequently, by molecular typing and cluster analysis probable infection sources of L. monocytogenes are identified. These analysis already identified 18 clusters of human L. monocytogenes isolates related to food isolates, and also several clusters that might suggest persistence of L. monocytogenes in different production environments. Real-time WGS analysis for example contributed to the fast identification of the source of a ST-6 L. monocytogenes outbreak originating from ready-to-eat meat products, and the subsequent termination of this outbreak. The timeframe of human cases (n=19) and strains isolated from the RTE meat products, together with the genetic relatedness of the strains suggest that the source of the outbreak was a L. monocytogenes strain which persisted in the production environment. This shows the effectiveness of real-time WGS analysis in solving foodborne outbreaks in the Netherlands and its potential for the food industry in the prevention of these outbreaks in the future.
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How do temperate bacteriophages affect the fitness of Pseudomonas aeruginosa?
More LessThe Liverpool Epidemic Strain(LES) of Pseudomonas aeruginosa is a key opportunistic bacterium and a major cause of mortality and morbidity in cystic fibrosis (CF) patients. It has been established to carry distinctive prophages within its genome, providing the host bacterium with advantages through horizontal gene transfer.
Several well-known partnerships between prophage and their bacterial hosts have been characterised, however, very little is known about other phage-host systems. This project explores the dynamics between Pseudomonas aeruginosa PAO1 and its phage, determining whether they confer an advantage in the CF lung.
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Molecular detection of Mycoplasma amphoriforme and Ureaplasma spp. from patient samples previously investigated for Mycoplasma pneumoniae infection
More LessM. amphorifrome(MAM), is a novel pathogen associated with chronic respiratory tract infections in immunocompromised patients, but prevalence in immunocompetent patients is not established. Ureaplasma spp. are a known cause of hyperammonaemia in lung transplant patients. To date, their prevalence has been well documented in neonatal lungs but observations in adults is lacking.
161 samples were obtained from Public Health England, submitted previously for M. pnuemoniae (MPN) investigation, which were screened for MAM using quantitative PCR. MAM positive samples were then screened for macrolide resistance using 23s rRNA (domain V) PCR amplification and Sanger-sequencing. The 161 samples were also screened for Ureaplasma spp. using conventional PCR involving different primer sets for initial screening and subsequent differentiation between Ureaplasma spp. by targeting a variable region.
10 of 161 samples were found positive for MAM (6.2%), all of which were found to be genotypically macrolide susceptible and 9/10 positives were isolated from males. Three of the total positives were previously identified to be MPN positives. None of the samples taken during the summer months had any MAM DNA detected. Five of the 161 samples tested positive for U. parvum (3.1%), 3/5 of which were from isolated from males.
These data suggest that MAM maybe an emerging pathogen that can cause persistent respiratory infections. The lungs may represent a possible reservoir for U. parvum and source for donor-derived lung infections. Further investigation is required into the prevalence of these organisms.
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Comparing the innate and humoral immune responses of different chicken lines to Infectious Bronchitis Virus (IBV) infection
More LessInfectious Bronchitis Virus (IBV) is a gammacoronavirus that is prevalent in commercial chicken flocks, resulting in characteristic clinical signs including snicking, rales, decreased tracheal ciliary activity, reduced weight gain and reduced egg production. Preliminary results indicate that there is a different clinical response to IBV infection in different chicken lines. Therefore, we aim to determine whether there is a differential innate or humoral immune response to IBV between chicken lines.
A series of in vivo experiments were conducted comparing brown leghorns (Rhode Island Red, RIR (Roslin)) to white leghorns (from Valo and Ovagen). Trachea and bursa were collected from infected and control birds at four-, six- and fourteen-days post infection (dpi). There was a difference in snick rate and rales between the RIRs and the white leghorns (both lines). However, no difference was observed in ciliary activity. Viral load was determined by absolute quantification using qRT-PCR. The viral load in the trachea of RIRs was significantly lower (p<0.05) at 6 dpi compared to 4 dpi, unlike in Ovagen birds where there was no significant difference between the timepoints. Relative gene expression of IFN-α, IFN-β, IFN-γ, IL-6 and IL-1β in these tissues will be measuredby qRT-PCR. Serum was processed from whole blood collected at zero and ten dpi for use in IBV specific ELISAs which will measure antibody responses in the chicken lines. This project aims to explore immune responses against IBV as well as identifying the causes of variability in experimentation using chickens to investigate IBV infection.
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Identification of Rab GTPases involved in restricting Salmonella Typhi growth in mouse macrophages
More LessSalmonella enterica serovar Typhi (S. Typhi) is a human adapted pathogen and the causative agent of typhoid fever, a life-threatening infection that kills hundred of thousand people every year, being particularly devastating in developing countries. S. Typhi host-restriction is partly due to the Rab32-dependent antimicrobial pathway, which is crucial to prevent the growth of S. Typhi in mouse macrophages. However, the exact mechanisms used by macrophages to kill S. Typhias well as the molecular basis of these mechanisms in the adaptation to the human host are unknown.
In order to identify host genes required to kill S. Typhi, we have performed a targeted short-hairpin RNA (shRNA) screen in primary mouse macrophages, aiming to i) optimize the conditions to perform silencing screenings in primary mouse macrophages and ii) identify novel Rab GTPases involved in S. Typhi host-restriction. For this, pooled shRNAs are used to knockdown gene expression in macrophages using lentiviral-based transduction system. After infection with a fluorescently-labelled S. Typhi strain, macrophages containing different numbers of intracellular bacteria are sorted by flow cytometry and targeted genes identified by next-generation sequencing.
This small-scale screen allowed us to optimize the screening conditions to perform genome-wide screenings in primary macrophages. More importantly, we have identified other Rab GTPases required in mouse macrophages to control S. Typhi survival confirming that this approach can be used to identify genes that macrophages use to control S. Typhi infection and extending our knowledge of the immunity mechanisms controlling the growth of intracellular pathogens.
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Microwaves can reverse the tumour phenotype of human papillomavirus type 16 (HPV16)-positive keratinocytes in 3D cell culture models: a novel therapy for HPV-associated disease?
More LessHigh-risk human papillomavirus (HPV) is the causative agent of benign, precancerous and cancerous lesions, in both anogenital and oropharyngeal sites. Increased expression of the viral oncoproteins E6 and E7 are responsible for tumour progression. The treatment of these precancerous and cancerous lesions is invasive, painful and with long-term side effects. Localised microwaves have been used successfully in the clinic for the treatment of verrucas, which are caused by low-risk HPV genotypes (>75% success rate versus >33% for cryotherapy). Moreover, local hyperthermia is known to have anti-tumour effects.
Ten-second microwave treatment of 3D in vitro-grown cervical tumour tissues (HPV16-positive SiHa cell) resulted in cell death in the treated zone while the tissue integrity was disrupted in the adjacent area. Microwaves induced apoptosis (induction of cleaved caspase 3) and autophagy (induction of LC3) and inhibited cell proliferation (loss of Ki67 and MCM2) in the entire tissue. Furthermore, HPV16 E6 and E7 expression was reduced in cells in the treated and transition zones, with subsequent induction of expression of the apoptosis-regulator, p53 over a 24 hour period following microwave treatment. Thermal stress, identified with the Heat Shock Protein 70 (HSP70) and translational stress identified by G3BP expression, was observed in the transition zone.
In conclusion, we demonstrate that the microwave treatment induces cell stress pathways and inhibits HPV oncoprotein expression that causes tumour progression. Induction of apoptosis and reduced cell proliferation suggest a reversal of the cervical tumour phenotype in the 3D tissues.
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Characterisation of surfactant-expressing bacteria and their potential bioremediation properties from hydrocarbon-contaminated and uncontaminated soils
More LessWe are investigating characteristics associated with oil degradation amongst bacteria isolated from clean and hydrocarbon contaminated soils from Nigeria and the UK.Our focus has been to identify bacteria expressing surfactants following isolation on Pseudomonas selective (PSA-CFC) and non-selective nutrient media and investigate the nature of surfactants, heavy metal resistance and hydrocarbon-degrading enzymes expressed by the bacteria. Of five sites sampled, a total of 1460 colonies were tested using the drop collapse assay, and 110 were found to express surfactants reducing liquid surface tensions as assessed by quantitative tensiometry to between 24.7 and 26.7 mN.m-1 (Tukey-Kramer HSD, α=0.05). We undertook a range of growth and behaviour-based assays on 60 selected strain which, when investigated by Hierarchical cluster analysis (HCA) demonstrated that this collection showed considerable phenotypic diversity. Eight out of the 60 strains could grow at a high temperature (50 °C), 35 of the 60 strains utilized diesel as a sole carbon source, and most of the strains could tolerate high concentrations (up to 20 mM) of heavy metals. Identification by 16S rDNA sequencing revealed that some of the strains belong to Pseudomonas, Bacillus, and Stenotrophomonas genera. We found using bioinformatics analysis of eight-selected draft genome sequences (AntiSMASH and RAST) NRPS-like (probable surfactants), cytochrome P450, catechol-1,2/2,3-dioxygenase, lipase, and heavy metal resistance gene sequences. We intend to use the information provided in this research to select strains for potential applications in in-situor ex-situ bioremediation of hydrocarbon-contaminated soils.
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Identification of interferon-stimulated genes with anti-HCMV activity
As a first line of defence, the interferon-mediated innate immune response is pivotal to protect cells against invading pathogens. At the heart of it are hundreds of interferon-stimulated genes (ISGs) upregulated substantially shortly after viral infection. Some of these ISGs act indirectly to interfere with virus life cycle via regulation of interferon signalling pathways whereas other ISG can act directly to inhibit viral replication via interaction with viral components. Although many of the ISGs have been identified decades ago, only a handful of them have been characterized about their antiviral activity. Here, we used an arrayed expression lentivirus library of more than 400 ISGs to identify the ISGs with anti-HCMV activity. We performed parallel screens using wild type fibroblast cells and IRF3 KO fibroblast cells generated by CRISPR/Cas9 editing to identify ISGs more likely to directly inhibit HCMV, as opposed to activation of IFN signalling. The IRF3-independent ISGs identified in the screen include those that signal through IRF3 independent pathways such as IRF7 or known to inhibit HCMV directly such as IDO, RIPK2 and AIM2 validating the screening approach. Interestingly, we also identified novel IRF3-independent anti-HCMV ISGs, indicating they may play a role in directly inhibiting the virus.
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A novel in vitro urethra model to demonstrate bacterial displacement during urinary catheter insertion
Background: There is currently no standard established in vitro model to test the efficacy of intermittent catheters to prevent or control introduction/movement of bacteria into the urethra during device insertion. This study aimed to address this issue by developing a reproducible agar based in vitro urethral model.
Method: A novel in vitro model and testing method was developed to quantify the displacement of bacterial growth after intermittent catheter insertion.The urethral model consists primarily of a preformed channel within a specifically formulated agar based matrix. The urethra model was inoculated at one side of the channel to act as the urethral meatus, a catheter was then inserted. After incubation the bacteria within the urethra channel was quantified.
Results: Once optimised, the model produced reliable and reproducible results with both E. coli and S. aureus (P≥0.265). The model was used to test three different intermittent catheter types. When compared to the growth control there was a significant difference in bacterial distribution when inserting an uncoated (P≤0.001) or hydrophilic coated (P≤0.009) catheter; there was no significant difference when a prototype catheter was inserted with either bacterial species used (P≥0.423).
Conclusion: These findings support the hypothesis that a single catheter insertion can initiate a catheter-associated urinary tract infection. The in vitro urethra model and associated methodology provide a new research tool for the development and validation of emerging technologies in urological healthcare.
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Design of stable Enterovirus 71 virus like particles (VLPs) as a potential approach to vaccine development
More LessWe have previously described the design of stable and immunogenic polio virus-like particles (VLPs) (Fox, et al. 2017) as an alternative approach to vaccine production. Unlike current polio vaccines, recombinantly-expressed VLP vaccines are non-infectious so would pose no risk of accidental escape from production plants, threatening eradication. To do this we devised a pipeline for the identification of stabilising mutations which could then be combined in a single construct to produce suitable particles; this strategy may have applications for other enterovirus vaccines.
Enterovirus 71 (EV71) is one of causative agents of hand, foot and mouth disease which is usually mild but in some cases neurological and systemic complications may occur. Recently there have been several outbreaks with significant mortality in South East Asia as well as increasing numbers of reports of outbreaks in Europe. VLP vaccines might be a useful alternative to inactivated vaccines currently in use or development.
EV71, like poliovirus, produces empty particles that are antigenically different from the virion. If, like poliovirus, these empty particles are less immunogenic than the virion, it would be necessary to stabilise them in the native conformation. We are attempting to do this (1) by incorporating modifications that proved successful in the context of poliovirus and (2) by identifying new candidate mutations using an analogous pipeline. Here we will report the characterisation of a range of different modifications that have stabilising and de-stabilising effects on EV71 particles as well as unexpected effects on morphogenesis.
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How environment shapes horizontal gene transfer
More LessThe evolutionary fate of a horizontal gene transfer (HGT) event is determined by its fitness on the recipient cell, i.e., whether it is beneficial, neutral or deleterious. The distribution of fitness effects (DFE), thus is a fundamental predictor of the outcome of an HGT event.
The environment plays a considerable role in determining the fitness cost of a horizontally transferred gene. We have studied the fitness effects of genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli in six environments, that potentially represent the conditions experienced by the two species. The data suggests high variability of genes in different environments. Genes, whose fitness varies substantially between environments, may be able to persist in populations while being deleterious in one environment, they may be neutral or even beneficial in another environment, suggesting that environmental fluctuations may increase the likelihood of HGT.
In addition to the in vitro environments, we are also looking at, how changes in the intrinsic environment of a cell, after an HGT event, could affect fitness. An increase in protein dosage due to functional similarity of the horizontally transferred gene to the endogenous gene can cause an imbalance in the cell, thereby leading to a negative fitness effect. By comparing the growth rates of each ortholog gene with the wild type strain, we can elucidate when gene dosage acts as a barrier to HGT.
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UV irradiation of HSV-1 virions induces the differential recruitment of host intrinsic and innate immune regulators to infecting viral genomes
More LessRecognition of virus-derived nucleic acids is a key process in the activation of immune defences to viral infection. However, the spatiotemporal recruitment of host immune regulators to infecting viral DNA (vDNA) genomes remains poorly defined. Here we utilize 5-Ethynyl-2’-deoxycytidine (EdC) nucleotide labelling of WT or UV-irradiated HSV-1 genomes in combination with click chemistry and high-resolution confocal microscopy imaging to investigate the recruitment and proximity of key intracellular immune regulators to infecting vDNA during HSV-1 infection.
We report that UV irradiation of HSV-1 virions restricts vDNA decompaction upon delivery to infected cells. UV treatment reduced the frequency in PML-NB recruitment to nuclear genomes relative to non-irradiated vDNA. These data demonstrate an important role of genome decompaction in the recruitment of intrinsic host factors to nuclear infecting vDNA. Additionally, UV-irradiated HSV-1 genomes favoured a premature cytosolic deposition phenotype, due to ineffective association with microtubules as shown by nocodazole treatment. The cytosolic UV irradiated vDNA showed enhanced association with both cGAS and STING. Interestingly, high-resolution Airyscan imaging identified a consistent unreported 3D interaction between both cGAS and STING, the strength of association of which was enhanced in the context of UV infection. This enhanced recruitment correlated with elevated levels of IFN-β transcription relative to non-irradiated vDNA, despite the presence of non-irradiated cytosolic genomes.
Our data demonstrates that UV irradiation of herpesviruses, a well-established procedure for virus inactivation, has multiple effects on genome structure and sub-cellular localisation that differentially influence the spatiotemporal recruitment of intrinsic and innate host immune regulators to infecting genomes.
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Assessing the ability of Tigecycline and Meropenem to clear intra-macrophage Klebsiella pneumoniae
More LessThe emergence of hypervirulent Klebsiella pneumoniae of serotype K1 and K2 are a major cause of life-threatening, community-acquired infections. Recent studies demonstrated that Kp can persist for long periods of time within the spleen and liver and survive within macrophages. We aimed to explore whether two clinically relevant antimicrobials differed in their capacity to clear within-macrophage Kp.
The mouse monocyte cell line J774a were used to model Kp macrophage infection (cultured in RPMI, 10% Fetal-bovine-serum, 37oC, 5%CO2). Cells were harvested and seeded in a 96-well plate at 2x106 cells/mL and incubated overnight. The following day, cells were infected with hypervirulent, K1 Kp (NTUH-K2044) for one hour at an MOI of 10. A 1-hour treatment with gentamicin and polymyxin-Bwas used to kill extracellular Kp. Cells were then washed, and incubated with serial 2-fold dilutions of meropenem and tigecycline for 6 hours. In parallel, a killing assay was performed with antibiotic, and Kp in cell culture media alone to compare the intracellular and extracellular activity of each antibiotic.
We show that whilst the majority of the inoculum was resistant to phagocytosis, a small fraction of Kp were able to adhere to macrophages, enter the cell, and persist for up to 6h. Furthermore, we demonstrate that lower concentrations of tigecycline were required to inhibit intracellular Kp, compared with meropenem, which required concentrations in excess of the planktonic MIC to clear the intracellular niche.
These data indicate that there is reason to re-examine the antimicrobial treatment regimens for hypervirulent Kp infection with a focus on intracellularly active drugs.
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Characterisation of a new megaplasmid family associated with the spread of multidrug resistance in Pseudomonas aeruginosa
Unlike in other important pathogens, the role of plasmids in the emergence of antimicrobial resistance (AMR) in Pseudomonas aeruginosa (Pa) has remained largely unaddressed. Previous work on AMR in Pa has mostly used genome sequencing methods that are limited because of the difficulty of using short-read data to detect and reconstruct complex plasmids. Here, using superior long-read sequencing, comprehensive bioinformatics analyses, and experimental characterization, we uncover an emerging family of important Pseudomonas megaplasmids and report its contribution to dissemination of multidrug resistance (MDR) on a global scale. Firstly, we identified large plasmids with a key role in the spread of MDR in a hospital in Thailand, and characterised their resistance regions revealing evidence of duplication, recombination and shared repeats, indicative of dynamic adaptation. Applying phylogenomics and pangenomics approaches we linked related megaplasmids and defined a core and pangenome for the family, exposing wide variations in AMR genes carriage. We then surveyed thousands of publicly available genomes, leading to discovery of dozens of megaplasmid relatives overlooked in multiple datasets, including already published studies. By integrating all this information and looking beyond the pathogenic species we gained valuable insights into the evolution of the megaplasmid family and revealed its widespread and multispecies distribution. We also showed that members of this family are stable in the absence of antibiotic pressure, bear no fitness cost to their host, and can be readily transferred between different Pseudomonas species. Our findings expand the bacterial plasmidome and provide insights on how MDR plasmids emerge from environmental reservoirs.
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Investigating the coding capacity of rotaviruses using a newly developed reverse genetics system
More LessBovine rotavirus (RV) infection causes severe diarrhoea in young dairy calves and has a significant economic impact on livestock production as a result of high morbidity and mortality caused. Development of technologies to engineer infectious RV using an entirely plasmid-based reverse genetics (RG) system has proven challenging. A breakthrough was made when Kanaiand co-authors (PNAS, 2017)developed a plasmid-only-based RG system for the simian RV strain SA11.We are currently developing an analogous RG system for the bovine RF RV strain. Having parallel systems for different RV strains will help to validate phenotypic changes induced by site-directed mutagenesis (SDM) within the RV genome.
The coding capacity of the 11-segmented dsRNA RV genome has been largely unexplored. Using bioinformatic analyses, we have identified four segments with up to five putative alternative initiation codons which are in moderate or strong Kozak context. Furthermore, some occur in segments for which the canonical start codon occurs within 15 nucleotides of the start of the segment, further suggesting the possibility of alternative translation start sites to generate coding diversity. We are now applying our RG systems to investigate RV coding capacity using TnT transcription assays, radiolabelling and SDM.
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Toxigenic Corynebacterium ulcerans: re-emergence of a zoonotic infection
Diphtheria is a potentially life-threatening infection in humans. The three species capable of causing diphtheria are: Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Although UK cases are rare, recently there has been an increase in reported toxigenic C. ulceransassociated with companion animals. Potentially toxigenic corynebacteria are sent to the National Reference Laboratory, Public Health England (PHE), London, UK for species confirmation, determination of presence of the diphtheria toxin gene by real-time PCR, and confirmation of toxin expression by the Elek test. We reviewed submissions of C. ulcerans between January 2006 and November 2019.
Fifty-three isolates of toxigenic C. ulcerans were received, 29 from humans and 24 from animals. The most common animal hosts were dogs (15) and cats (7), but isolates were also received from a horse and a captive rhinoceros. Multi-locus sequence typing (MLST) data were derived from whole genome sequencing. In three human cases, C. ulcerans was isolated from companion animals and in all three, typing data supported an epidemiological link between human and animal hosts. The sequence types (STs) for these linked isolates were ST331 (human and dog, human and cat) and ST551 (human and cat).
Although MLST data are limited, the finding of the same ST (ST331) from canine and feline sources indicates that strains can be carried by both hosts.
Management of diphtheria cases is an important public health issue. Typing data can support epidemiological linkage and identify possible sources, allowing the potential intervention and treatment of animals thus avoiding onward transmission.
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Effects of trimethylamine N-oxide on the growth and metabolism of gut bacteria
More LessTrimethylamine N-oxide (TMAO) is an osmolyte that enters the bloodstream directly through consumption of fish, or through microbial metabolism of it and other dietary methylamines such as carnitine and choline, producing trimethylamine (TMA). TMA produced by microbes enters the blood and is transported to the liver, where it is converted back to TMAO by flavin-containing monooxygenases before being excreted in the urine. TMAO has been shown to potentially have beneficial effects on metabolic health and the gut-brain axis at normal physiological levels, while elevated serum levels are associated with cardiometabolic disease in Western patients. Bacteria in the family Enterobacteriaceae exhibit changes in their growth and metabolome in the presence of TMAO but why this occurs is not known. Growth experiments show that a caecal isolate of Klebsiella pneumoniae, a member of the Enterobacteriaceae, experience a more rapid growth rate, when grown anaerobically in the presence of TMAO, but may require oxygen to be present to produce TMA. K. pneumoniae will be subjected to differing concentrations of TMAO and oxygen to examine the effects on the metabolites produced, the genes expressed, and the rate of growth. This will be done by analysing spent media using GC-MS, qPCR targeting genes involved in anaerobic respiration, and measuring the turbidity of cultures over time. By understanding how bacteria in the gut interact with TMAO new insights can be gained as to how gut bacteria and their metabolites influence human health.
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Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance
More LessInfluenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses.
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Understanding the evolution of Mycobacterium tuberculosis lineages using an integrated genomics and metabolomics approach
Despite being the number one cause of death from an infectious disease, little is known of the 7 phylogenetic lineages of Mycobacterium tuberculosis (Mtb). These lineages are thought to have adapted differently to their human hosts, as they are geographically localised. As a result, they show variation at the phenotype level, such as virulence and the ability to develop antibiotic resistance, and at a genomic level, such as in single nucleotide polymorphisms (SNPs). We have linked the differences in SNPs between lineages to differences in metabolites (i.e. what is ultimately produced by a cell). Through multi-omic integration of these datasets we have discovered lineage-specific metabolomic changes, potentially as a result of genomic adaptation. The differences between lineages will provide insight into new biological pathways to target and manipulate in future research.
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Use of probiotic bacteria to selectively modulate Candida albicans virulence in biofilms
More LessCandida albicans is an opportunistic fungal pathogen present in the oral cavities of up to two-thirds of people. Despite typically existing as a commensal microorganism, it has pathogenic potential, particularly in older, immunocompromised individuals. A common Candida-associated infection is denture-associated stomatitis (DS), which presents clinically as areas of erythema on the palatal mucosa, and discomfort for the denture-wearer. In vitro, previous work has shown that the expression of C. albicans virulence factors varies according to its interactions with other oral microorganisms.
Mature single- and mixed-species biofilms (with Candida and several strains of common oral bacteria) were grown on poly(methyl methacrylate) (PMMA) coupons, representing dentures. Additionally, to some coupons, individual probiotic strains were added. Total RNA was extracted, reverse transcribed and putative virulence gene expression was determined by RT-qPCR relative to ACT1, a housekeeping gene. Biofilm-infection assays of FADU and TR146 epithelial cell lines were also performed by pre-culturing cells, then adding single- or mixed-species inocula overnight. Quantification of cell damage determined by lactate dehydrogenase assay.
Biofilm co-culture with the addition of certain probiotic strains downregulated C. albicans virulence genes in both short-term and long-term mixed-species biofilms. With an increasing aged population that is heavily reliant on the use of antibiotics that can negatively affect the microbiota of patients, there is a requirement to look at the benefits of prophylactics, from both an economic and patient well-being viewpoint. The results show the realistic possibility of using probiotics to prevent or restrict development of Candida-associated oral diseases.
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Gene transfer agents: Another step towards a mechanistic understanding of expression
More LessGene transfer agents (GTAs) are small viruses that package and transfer random pieces of the producing cell’s genome but are unable to transfer all the genes required for their own production. GTAs are able to spread any DNA in the host cell and so their potential impact upon bacterial evolution and antimicrobial resistance is immense.
Our discovery that the product of gene rcc01865 is a specific GTA activation factor (GafA) for the model Rhodobacter capsulatus GTA (RcGTA) and that GafA is essential for RcGTA production, has provided the link between GTA production and host regulatory pathways. However, while GafA has significantly improved our understanding of GTA regulation the complete mechanism is unclear. Our goal was to investigate the GafA mechanism of action in more detail. We demonstrate direct protein-protein interaction between GafA and the RNA polymerase omega subunit (RNAP Ω) using bacterial-two-hybrid and pull down assays. Further evidence for the interaction has come from random and site directed mutagenesis of gafA and targeted truncations. GafA mutants were also tested to assess their impact on RcGTA production. RNAP Ω is thought to recruit alternative sigma factors to the RNAP holoenzyme. Regions of GafA also share sequence homology with known sigma factor proteins, and we propose that GafA acts as an alternative sigma factor to co-ordinate expression of disparate RcGTA genes. Our results advance our understanding of this fascinating mode of horizontal gene transfer, not only in the model species but also in other potential GTA producing species that contain gafA homologues.
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Impact of air pollution on buff-tailed bumblebees (Bombus terrestris) and their gut microbiome
More LessBumblebees play a major role in global pollination. Consequently, their health is of high importance for food security worldwide. Yet, recent population estimates show that their numbers are declining. This decline has been attributed to habitat loss, infection and use of pesticides. An important factor for bee health that contributes to population survival is the gut microbiome composition. The bee gut microbiome provides protection from pathogens, is specific to the host and helps break down food. Without a balanced gut microbiome, the health of the bee is threatened through increased infection and mortality. The bee gut microbiome is relatively simple, being dominated by 8 core bacterial species providing a convenient study system. Previous published data shows that air pollution has an impact on bacterial behaviour. Therefore, our hypothesis is exposure to air pollution causes an imbalance in the bee gut microbiome. To test this, we exposed bees to black carbon (BC), a major component of air pollution particulate matter. We assessed the effects on bee behaviour, microbiome composition and gut bacteria treated in vitro. Bees treated with BC showed a significant increase in viable bacterial cells in their faecal community. Independent culture of gut commensals showed that BC significantly alters the structure of their biofilms, which are important for colonisation in vivo. This supports the hypothesis that air pollution can cause an imbalance in the bee gut microbiome, and may adversely influence bee health and pollinator populations.
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Interreg 2 seas Project: Health For Dairy Cows, H4DC
Cryptosporidium spp. are microbial parasites that infect the gastrointestinal tract of humans and many animals, causing cryptosporidiosis, a disease characterised by acute watery diarrhoea. In dairy farms, C. parvum is the most common species in calves, leading to high mortality rate, stunted growth, and consequently high economic losses. Trade between farms and breeding centres is a major risk factor in the spread of such parasites, posing a threat to other farms worldwide as well as to human health. This problem is aggravated by the lack of good breeding practices, efficient detection tools, and lack of effective anti-cryptosporidial drugs.
To address cryptosporidiosis in dairy farms, we have established the ‘Health For Dairy Cows (H4DC)’ consortium in order to tackle some of the aforementioned issues.
Herein, we will present preliminary data from a 3-step strategy:
1)Dissemination of pilot farms in France, Belgium, The Netherlands and England. This collaboration will serve to test the effect of new husbandry practices in the occurrence of Cryptosporidium, which will aim to decrease Cryptosporidium incidence and ultimately decrease the economic burden of cryptosporidiosis.
2)These pilot farms will later be used as testing-grounds of the low-cost and easy-to-use in-situ C. parvum detection tool that will be developed during this project.
3)Development of a cell-based drug-screening system which will be used to screen various drugs and compounds for anti-Cryptosporidium activity.
Finally, data from these findings will be used to establish model and strategies in order to transfer the developed technologies to both farmers and biotech/pharmaceutical companies.
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The Missing Link: Developing a pipeline for accelerated antibiotic discovery from Streptomyces through linking ‘omics data
More LessThe genus Streptomyces has proven to be a rich reservoir of specialized metabolites, accounting for 80% of all microbially produced antibiotics including chloramphenicol and nystatin from S. venezuelae and S. noursei respectively. However, the discovery of novel microbial chemistry is still greatly needed to combat antimicrobial resistance. Comparative metabolomics, using platforms such as Global Natural Products Social Molecular Networking (GNPS), as well as tools such as antiSMASH and BiGSCAPE have aided the mining of biosynthetic gene clusters (BGC’s) across datasets but comparing the chemistry to the encoding biosynthetic gene clusters is a significant bottleneck.
In this study, ten Streptomyces strains were selected, based on phylogeny and availability of genome sequence. The strains were cultured on 6 types of Actinomycete-specific media to maximise metabolite diversity. Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was used to obtain spectral data from crude metabolite extracts enabling comparative metabolomics analysis via the GNPS platform. As the genome sequences were publicly available, genome mining of BGC’s was achieved using antiSMASH resulting in 260 BGC’s across the ten strains. This revealed 53 gene cluster families when analysed using BiGSCAPE, the largest encoding for 8 metabolites.
In future, both biosynthetic (BGC’s) and chemistry (parent ions) datasets will be computationally linked based on strain presence/absence. The development of standardised datasets that enable cross-‘omics comparison will aid prioritisation of novel antibiotics, especially when combined with bioactivity data.
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The complete sequence, annotation and comparative analysis of the Escherichia coli IncFII family plasmid pMccB17, encoding biosynthetic and immunity functions for the antimicrobial peptide microcin B17
More LessMicrocin B17 (Mcb17) is a ribosomally synthesized and post-translationally modified peptide (RiPP), produced by Escherichia coli, that inhibits bacterial DNA gyrase in a similar way to quinolones. The Mcb17 operon, consisting of seven genes encoding biosynthetic and immunity/export functions, was originally found on a plasmid, pMccB17. This circular plasmid, previously known as pRYC17, was originally found in Escherichia coli strain LP17, isolated from the intestinal tract of a healthy newborn at Hospital La Paz, Spain and was transferred by conjugation to E. coli K-12 [Baquero et al. (1978) J. Bacteriol. 135: 342]. pMccB17 is a low copy number IncFII plasmid in the same incompatibility group as R100 and R1. Not much is known about this plasmid aside from the facts that it carries the Mcb17 operon, does not possess any conventional antibiotic resistance markers and its size was estimated to be approximately 70 kb.
We extracted the plasmid from E. coli K-12 strain RYC1000 [pMccB17] and sequenced it twice using an Illumina short-read method, firstly together with the host bacterial chromosome, then plasmid DNA was purified and sequenced separately. PCR primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete sequence has 83 predicted genes, initially identified by Prokka and subsequently manually reannotated using BLAST. Comparison to other IncFII plasmids shows a large proportion of shared genes, especially in the conjugative plasmid backbone. However, pMccB17 which is a MOBF12 plasmid lacks transposable elements and in addition to the Mcb17 operon, this plasmid carries 25 genes of unknown function.
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MORF: An online tool for exploring microbial cell responses using multi-omics analysis
More LessWith continuing improvements and reducing costs of high-throughput technologies, microbiologists are increasingly collecting multi-omics datasets. However, the tools and techniques used to analyse these kinds of data are often highly specialised and require bioinformatics, statistics and often coding experience. Many studies also tend to report on a single aspect of the data whilst overlooking other potentially interesting phenomena. Consequently, many of these multi-omics data sets are not being used to their full potential. MORF was created as a solution to these problems by providing access to multi-omics datasets through an online interface which presents the data in a user-friendly and accessible way. No coding experience or specialist statistical knowledge is required, and users are free to explore the data using interactive graphics and simple analysis tools.
Here we demonstrate MORF using multi-omics datasets from two experiments using bacteria in industrial fermentation processes. First, Escherichia coli engineered to produce styrene, a valuable chemical used in the manufacture of polymers, and secondly a Clostridium which produces the biofuel butanol. A key outcome was the identification of targets believed to be involved in responding to membrane stress, which we identified using MORF’s differential gene and protein analysis tools. Work is underway to further characterise and engineer these targets to improve product yields. In conclusion, MORF provides a framework for omics analysis that can be applied to any organism or set of experimental conditions, and will help researchers and collaborators to make the most of their data.
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Modulation of cAMP levels by a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria
More LessAntimicrobial tolerance is the gateway to the development of antimicrobial resistance and is therefore a major issue that needs to be tackled.
The second messenger, cyclic-AMP (cAMP) is conserved across all taxa of life. It is involved in propagating the signal from environmental stimuli and converting it into a response. In bacteria such as M. tuberculosis (Mtb), P. aeruginosa, V. cholerae and B. pertussis, cAMP has been implicated in virulence, regulation of metabolism and gene expression. Cyclic AMP signalling in mycobacteria is especially complex – with 16 enzymes that produce cAMP in Mtb alone.
By discovery of a novel, actinobacteria conserved enzyme that degrades cAMP, we have developed a tool to modulate cAMP levels in mycobacteria. By using a combination of metabolomics, bioenergetics and time-to-kill assays, we show that when this enzyme is overexpressed in the model organism M. smegmatis, there is a 3.3 -fold decrease in intracellular cAMP levels. This was concomitant with 7-fold increased ATP. The unbalanced ATP/cAMP ratio consequently altered cell envelope permeability, compromised bioenergetics and most importantly, led to a decrease in the tolerance to various frontline antimicrobials.
Taken together, this work provides clear evidence that cAMP is involved in antimicrobial tolerance in mycobacteria and that this may represent a promising new target for antimicrobial development.
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Studying the surface-tethered AaaA as a virulence factor and anti-P. aeruginosa drug target
More LessP. aeruginosa is a leading cause of bacterial wound infections, and is associated with a disproportionally high level of mortality in burn patients especially. Because of its wide arsenal of virulence factors and biofilm formation ability, infections with P. aeruginosa are often chronic and extremely difficult to treat. This study uses an ex-vivo skin model to study the role of the virulence factor AaaA (PA0328) in chronic wound infections. AaaA, or the Arginine-specific aminopeptidase of Pseudomonas aeruginosa A, is a surface-tethered autotransporter which cleaves N-terminal arginine from peptides. In the oxygen and nutrient-limited environments of chronic wounds, this free arginine could serve as a nutrient source for P. aeruginosa via alternative metabolic pathways. Changes in local arginine concentrations may also alter host iNOS and Arginase immune responses which influence inflammation and wound healing, in order to favour a chronic infection. Being surface-tethered and immunogenic, AaaA is also of interest as a potential antimicrobial drug or vaccine target. This study aims to probe the role of AaaA on both pathogen survival and host response in the wound context, using a combination of in situ transcriptional reporters, immunofluorescence and laser-scanning microscopy, and RT-qPCR to localise and quantify P. aeruginosa survival and aaaA expression, as well as resident immune cell invasion, and expression of immune factor genes, in wounds over time. Here, we present preliminary data examining AaaA expression and function in P. aeruginosa biofilm cultures and ex-vivo skin wound infections, as well as the results of preliminary inhibitor screens.
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Structural characterization of genomic RNA-coat protein contacts in single-stranded RNA viruses by high-resolution cryo-EM
Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.
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An image-based screening system for the study of Mycobacterium avium persistence and drug discovery
More LessMycobacterium avium infects human macrophages causing opportunistic infections. A steady increase of these infections over the past four decades and resistance to common anti-mycobacterial drugs, create an urgent need for new treatments; however, drug discovery is held back by a lack of knowledge about how M. avium replicates or persists in host cells.
We implemented an image-based assay using a fluorescence dilution (FD) system to measure M. avium replication and persistence. M. avium strain 104 carrying a plasmid encoding GFP and TurboFP635 under constitutive and inducible promoters, respectively, is induced prior to infection of THP1 macrophages and the fluorescent signals are tracked over time in the absence of the inducer. Loss of the TurboFP635 signal while GFP signal is maintained, identifies replicating and retention of both signals non-replicating bacteria.
In the absence of inducer, the M. avium 104 FD strain replicated in the macrophages, leading to increasing numbers of GFP-expressing intracellular bacteria and concomitant loss of TurboFP635 signal in >90% of the infected cells after 24 hours. Upon re-induction, these bacteria expressed TurboFP635, suggesting they are metabolically active and alive. We observed the presence of a small non replicating population that persisted over 96 hours pi. We applied our assay to compare the effect of a panel of anti-mycobacterial drugs, revealing different effects on killing, intracellular replication and induction of persisting, non-replicating bacteria, illustrating the power of this system to facilitate the dissection of the biology of persistence and anti-mycobacterial drug discovery in the future.
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Taking charge: Using electrochemical impedance spectroscopy to quantify biofilm formation in Pseudomonas spp.
The opportunistic pathogen, Pseudomonas aeruginosa, is infamous for its ability to rapidly form biofilms (<24 h) in inhospitable environments and the development of antimicrobial resistance (AMR). It has been seen to have resistance to nearly all known antibiotics, including last-line antibiotics colistin and carbapenems. AMR is currently considered one of the biggest threats to human health, causing 700,000 deaths annually, expected to rise to 10 million deaths per year by 2050. P. aeruginosa, alongside other opportunistic pathogens, has been implicated in infections following various surgical procedures. Such infections compromise patient recovery and, when a medical implant is present a biofilm can develop that will ultimately require a complex revision surgery to remove the infection.
In this study, impedance spectroscopy and differential pulse voltammetry were carried out in parallel to measure electrochemical and impedance properties of bacteria, allowing for identification and quantification of pyoverdine and pyocyanin; bacterial metabolites. Three Pseudomonas spp. (P. aeruginosa, P. fluorescens and P. putida) were assayed in liquid culture at OD600. The sensor was standardised with pyoverdine and pyocyanin, with an electrochemical reading taken every 30 minutes up to 4 hours. This assay was repeated with Pseudomonas spp. growing in biofilms in LB broth, with a screen-printed electrode as the solid surface. Readings were then used to correlate metabolite production to biofilm production in each Pseudomonas sp. Pyoverdine correlated with biofilm formation for all three assayed Pseudomonas, with variation in the quantity of metabolite produced between species. This allows the two metabolites to be used as indicators of biofilm mass on devices and surfaces.
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Incidence of malaria parasites in human immuno-deficiency virus clients attending General Hospital Awo-Omamma, Imo State Nigeria
More LessThe incidence of malaria parasite in Human Immuno-deficiency Virus clients attending Awo-omamma General Hospital, Owerri Imo state Nigeria was studied. A total of 200 blood samples were collected; 150 samples were collected from sero-positive HIV clients while 50 samples were collected from sero-negative HIV clients which served as control samples. Out of this 200 clients 85(42.5%) were males while 65(32.5%) were females. The blood samples were analyzed using Malaria Rapid Test Kit for the presence of Plasmodium falciparum, using standard medical laboratory procedure. The result revealed an overall prevalence of 43 (28.7%) for HIV positive participants that tested positive to malaria parasite, 15 (17.6%) were male while 28 (43.1%) were female. Analysis based on age revealed that the highest prevalence was among those within the age group 30-39 years having 20 (10%) while those with the least prevalence were observed among those within the age group 20-29 years having 32 (16%). Analysis of malaria parasite based on CD4+ cell count among HIV clients revealed that 51(34%) had CD4+ cell count above 200cell/μl while 23 (15.3%) had CD4+cell count below 200cell\μl. This study has shown that there is a low prevalence of malaria parasite (Plasmodium falciparum) among HIV/AIDs clients with high CD4+ attending HIV clinic in Awo-omamma General Hospital, Imo state. It is recommended that more efforts be made to eradicate malaria completely as this will go a long way in reducing the rate of mortality among HIV clients.
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Increasing production of the antibiotic-abyssomicin C in Micromonospora maris
More LessAbyssomicin C is a polyketide antibiotic produced by Micromonospora maris AB-18-032. Previous work on abyssomicin C indicated that it inhibits growth of infectious pathogens such as Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). It does this by suppressing para-aminobenzoic acid (pABA) synthesis, which is required for folic acid biosynthesis in bacteria. This makes abyssomicin C an appealing antibiotic drug, as it is specific only to bacteria. However, its yield in chemical synthesis (4 %) and biosynthesis (60.0 mg/L) are low. Ribosome engineering, through the selection of streptomycin-resistant and rifampin-resistant mutants, of M. maris may result in strains with a higher titer of production. After screening by bioassay and sequencing, the mutant genes in six Ochi mutants were identified. Of these mutants, four of them are able to produce a higher titer of abyssomicin C. The other two strains were detected to produce the other secondary metabolite.
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Ultra- structure of Toombak; smokeless tobacco of Sudan and its effects on oral and systemic health
More LessIntroduction: Toombak is a smokeless tobacco used by the Sudanese. Nicotiana Rustica leaves are fermented, and sodium bicarbonate is added, increasing Ph. This causes high absorption spikes of unprotonated nicotine through mucosal epithelium, and into the blood, distinguishing Toombak as an addictive product. Furthermore,Toombak hashighmicrobial contamination due to its open field, rural production.
Materials and methods: 21 tobacco samples were collected from 3 main towns in the capital Khartoum, Sudan. Analysis included microbiome (16S rRNA sequencing), metabolome (Liquid and Gas Chromotography- volatile organic compound method), heavy metal content and scanning electron microscopy (SEM).
Results: Compared to limit of detection values (LOD), Tobacco specific nitrosamines (TSNA’s), in particular, 4(methylnitrosamino)-1-(3-pyridyl)–butanone (NNK) were markedly increased [NNK; 1.2-4.7 mg/g; LOD 0.02]. Free nicotine ranged from 16-31 mg/g; [LOD 0.01]. High choline and carnitine, volatile aldehydes; and benzyl alcohol were also detected. 8 cations, of which iron [1465 mg/kg] and copper [3.76 mg/kg] were strikingly observed, compared to average levels from other products; [Iron 20-60mg/kg, Copper 1.3mg/kg]. 8 Phyla, including but not limited to; Actinobacteria; cornyebacterium(contain nitrate reductase genes) and Firmicutes; staphylococcus and facklamia, were sequenced. SEM highlighted a non-homogenous product with elevated sodium spectrum.
Conclusions: TSNA’s were potent in Toombak due to high starting nicotine and rich nitrate reductase bacteria. High choline and carnitine can promote cardiovascular disease through their conversion to trimethylamine-N-oxide. Diphtheria and infective endocarditis are associated with cornyebacterium and facklamia respectively while staphylococcus can lead to numerous systemic opportunistic diseases.
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AMR Escherichia coli and its temporal and spatial variability within the aquatic environment
More LessPhenotypic and genotypic identification methods have been used to determine the temporal and spatial dynamics of AMR-Escherichia coli in a mainly rural watercourse that receives WWTP-effluent compared to a parallel river which does not. We aimed to investigate the incidence of plasmid-mediated mcr-1and β-lactamase-genes in E. coli recovered from both water and Asellus aquaticus samples throughout two-calendar-years.
Samples of the water and A. aquaticus were recovered from the relevant locations each month. CHROMagar ESBL agar was used throughout to isolate and identify ESBL-E. coli. The presence of AMR-genes was confirmed using the ‘BSAC’ antibiotic-disk-synergy method and PCR analysis to confirm the presence of mcr-1and ESBL-genes. The CHROMagar ESBL agar was found to be 99.7% (n=578) accurate when confirmed with a PCR analysis of the ESBL-genes. Seventy-six-point-six percent (n=449) of the isolated ESBL-E. coli were correctly identified as ESBL-producing organisms using the ‘BSAC’ method. Interestingly 61.9% (n=358) of the ESBL-E. coli were also found to carry the mcr-1 gene.
Our data shows that AMR levels were highest at the WWTP-effluent throughout the two-years for both water and A. aquaticus samples. The incidence of AMR-E. coli 1km downstream of the effluent discharge was equivalent to the parallel river sites, suggesting that the dispersal of AMR from the WWTP-effluent is limited, although AMR-E. coli were found in relatively high numbers at the WWTP-effluent. We argue that the presence of AMR in the freshwater invertebrate A. aquaticus could represent an important route by which AMR can spread from the aquatic environment to the terrestrial environment.
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Identification of genes that contribute to fitness of African and Global clades of Salmonella Enteritidis during infection of macrophages
More LessNon-typhoidal Salmonella (NTS) usually cause gastroenteritis in humans, but in recent years NTS have begun to cause epidemics of bloodstream infections in Africa. Salmonella Enteritidis is the second most common serovar associated with this invasive form of NTS disease (iNTS) in Africa. To establish a systemic infection, Salmonella must survive and replicate within host cells, with macrophages being a primary target. Genomic characterisation of S. Enteritidis isolates from human bloodstream has identified two new clades that are unique to Africa and distinct from the Global Epidemic clade. The African S. Enteritidis clades exhibit genomic degradation, and possess a distinct prophage repertoire and are multi-drug resistant. However, little is known about the virulence factors that allow African S. Enteritidis to cause systemic infection in susceptible hosts. We screened libraries of random insertion mutants of African and Global S. Enteritidis by transposon insertion sequencing (TIS), and identified about 280 genes belonging to each clade that contribute to bacterial survival and replication in murine macrophages. The genes were associated with 5 pathogenicity-islands, or encoded the global regulators PhoPQ and OmpR-EnvZ. Experiments are ongoing to investigate the role in intra-macrophage replication of genes that are uniquely identified in African Salmonella. It is hoped that our findings will contribute to a greater understanding of African Salmonella infection biology, and that some of the virulence-associated genes could be potential targets for novel therapeutics.
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Bifidobacterium adolescentis shows potential to strengthen host defence against gastrointestinal infection via inhibition of the opportunistic pathogen Candida albicans and stimulation of human-isolated macrophages killing capacity in vitro
The human gut microbiota enhances the host’s resistance to enteric pathogens via colonisation resistance, a phenomenon that is driven by multiple mechanisms, such as production of antimicrobial metabolites and activation of host immune responses. However, there is limited information on how individual gut bacterial species, particularly many of the dominant anaerobes, might impact the host’s defence.
This study investigated the potential of specific human gut isolates to bolster the host’s resistance to infection. First, by antagonising the opportunistic fungal pathogen Candida albicans, and secondly, by modulating the killing capacity of human-isolated macrophages in vitro.
Co-culturing C. albicans with faecal microbiota from different healthy individuals revealed varying levels of fungal inhibition. In vitro assays with a panel of representative human gut anaerobes confirmed that culture supernatants from certain bacterial isolates, in particular of Bifidobacterium adolescentis, significantly inhibited C. albicans growth. Mechanistic studies revealed that microbial fermentation acids including acetate and lactate, in combination with the associated decrease in pH, were strong drivers of this inhibitory activity.
In the second in vitro assay, human-isolated macrophages were exposed to bacterial supernatants, and subsequently tested for their capacity to eliminate adherent-invasive Escherichia coli. Among the gut anaerobes tested, B. adolescentis was revealed to exert the strongest immunostimulatory and killing effect when compared to the unstimulated macrophages control.
B. adolescentis is known to be stimulated by dietary consumption of resistant starch andmay therefore represent an attractive target for the development of probiotic and prebiotic interventions tailored to enhancethe host’s natural defences against infection.
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A horizontally gene transferred copper resistance locus enables survival of community acquired methicillin resistant Staphylococcus aureus USA300 in host cells
The spread of community acquired, methicillin resistant Staphylococcus aureus (CA-MRSA) is an increasing problem seen outside the healthcare setting. One such strain, CA-MRSA USA300, is epidemic in the United States. USA300 shows a heightened resistance to the innate immune system, in particular to macrophage engulfment. Two horizontally acquired genes, encoding an efflux pump (CopX) and lipoprotein (CopL), were discovered in 2 different lineages of USA300, representing CA-MRSA epidemics in North and South America. Removal of either of these genes resulted in elevated copper concentrations in the cytoplasm of S. aureus, implying a function in copper hyper-resistance. While copper is an essential part of metabolic machinery, it is toxic at high concentrations and is utilised by macrophages to kill bacteria in the phagosome. Supporting this, USA300 with functional copXL genes showed increased survival in macrophages compared to their copXL negative counterparts. Although the role of CopX as an efflux pump explains the rise in intracellular copper concentration upon its mutation, the role of the CopL lipoprotein is still unknown. Therefore, to better understand the function of CopL and how it might influence S. aureus host interaction, transcriptomic analysis is underway to identify downstream targets. This has the potential to uncover an exciting mechanism linking metal resistance to host virulence.
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Non-human primates in the Gambia harbour human-associated pathogenic Escherichia coli strains
Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between different host species. We have investigated genetic diversity and antimicrobial resistance in Escherichia coli isolates from a range of non-human primates dispersed across the Gambia: patas monkey (n=1), western colobus monkey (n=6), green monkey (n=14) and guinea baboon (n=22). From 43 stools, we recovered 99 isolates. We performed Illumina whole-genome shotgun sequencing on all isolates and nanopore long-read sequencing on isolates with antimicrobial resistance genes. We inferred the evolution of E. coli in this population using the EnteroBase software environment. We identified 43 sequence types (ten of them novel), spanning five of the eight known phylogroups of E. coli. Many of the observed sequence types and phylotypes from non-human primates have been associated with human extra-intestinal infection and carry virulence characteristics associated with disease in humans, particularly ST73, ST217 and ST681. However, we found a low prevalence of antimicrobial resistance genes in isolates from non-human primates. Hierarchical clustering showed that ST442 and ST349 from non-human primates are closely related to isolates from human infections, suggesting recent exchange of bacteria between humans and monkeys. Our results are of public health importance, considering the increasing contact between humans and wild primates.
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Replication kinetics of a CHIKV Asian strain with 76 aa duplication in the nsP3 HVD, in comparison to the wild-type Asian and ECSA strains
Introduction: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes fever, rash and polyarthralgia. CHIKV has expanded its circulating regions to the Indian Ocean islands, Europe, Americas and Southeast Asia. Two CHIKV lineages, the ASIAN and ECSA are circulating in Malaysia. In 2009, a CHIKV strain with a 76 amino acid (aa) duplication in its nsP3 hypervariable domain (HVD), identified as CHIKvASIAN09MUM-Dup+ was isolated from a patient co-infected with DENV-2. Indels and duplication have been found in many other alphaviruses, and suggested to play a role in the survivality of the viruses.
Objectives: We aim to compare and relate the replication kinetics and virulency in-vitro of CHIKvASIAN09MUM-Dup+ with the wild-type Asian and ECSA strains.
Methods & Results: Genotypic analysis was conducted on three CHIKV strains in Malaysia, the CHIKvASIAN06UM-Dup-, CHIKvECSA08UM-Dup- and CHIKvASIAN09MUM-Dup+. We found that CHIKvASIAN09MUM-Dup+ has significant low replication rates in Vero, C6/36 and Rhabdosarcoma cells as compared to the wild-type strains. The highest titers were reached by CHIKvASIAN09MUM-Dup+ in all cells are 6.5 to 6.75 log10 TCID50/mL, which is 100 fold lower compared to the wild-type strains.
Conclusion: The significantly low replication rate of Dup+ strain in all the cells, maybe suggestive to be due to co-infection and co-existence with DENV, where the aa duplication may play a role in overcoming competitive suppression. This preliminary finding agrees with reported events, where alphaviruses use insertion, deletion and duplication of amino acid in nsP3 HVD as strategies to influence replication in host, viral virulency, pathogenesis and survivality for evolution adaptation.
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Developing global whole cell standards for the microbiome field
More LessThe microbiome field has developed rapidly over the last decade, however there is no effective standardisation of protocols in place, with no accredited or certified reference materials available to the wider community. As part of the NIBSC Microbiome program, we developed whole cell standards for microbiome research which can act as global working standards and will be put forward for consideration as the first whole cell World Health Organization International Reference Reagents for microbiome analysis. The developed reagents consist of common bacteria found in human gut and have been tested using different commercial DNA extraction kits in order to detect biases introduced through different DNA extraction processes. Multiple replicates of DNA extracted the whole cell standard using different DNA extraction kits were assessed using Next Generation Sequencing and evaluated using the number of input cells in the standards, as measured by microscopy and flow cytometry. These NIBSC whole cell standards are aimed to standardise the DNA extraction steps in microbiome analytical pipelines and serve as a tool to more accurately capture the representation of each microbe in the gut microbiome. This could be achieved by setting a specific threshold of accepted error levels by the microbiome community and we aim to set this through a large collaborative study in 2020-21. This work is part of a larger NIBSC microbiome program that is developing site specific DNA, whole cell and matrix spike-in reagents for use in microbiome research.
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Regulation of Rhodobacter capsulatus gene transfer agent production
More LessHorizontal gene transfer (HGT) enables the spread of antimicrobial resistance, virulence, metabolic and other genes conferring an advantage to the organism. HGT is enhanced in biofilms because of increased cell-cell contact (conjugation), and eDNA in the biofilm matrix causing development of competence and providing material for transformation. Production of the Rhodobacter capsulatus gene transfer agent (RcGTA), another mechanism of HGT, could also increase in biofilm as high cell density increases the proportion of GTA particles produced that encounter a target cell. RcGTA is a phage-like particle that packages ∼4.5 kb pieces of random DNA from the producing cell’s genome and transfers it to a recipient cell. Five loci comprise the RcGTA genome: a 15kb cluster containing most of the RcGTA structural genes, a cell lysis locus, two structural loci encoding head spikes and tail fibres, and a maturation/regulation locus that includes that master regulator, gafA.
I assayed RcGTA production using gene transfer bioassays and biofilm using a 96 well plate assay. I will present data showing that deletion of four GTA-related genes, including gafA itself, all lead to reduced biofilm production. All four gene knock-outs also strongly reduce GTA-mediated gene transfer, suggesting GTA production and biofilm are co-regulated. I will also present work to characterize GTA production in biofilms, for example by monitoring the transfer of fluorescent protein genes through confocal microscopy, and assessing how specific regulators control this process. Biofilms are ubiquitous in the environment so studying the spread of antimicrobial resistance genes by GTAs is important.
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Identifying drivers of microbial community composition associated with aquatic primary producers: metagenomic analysis of cyanobacterial cultures
More LessThe energy derived from aquatic primary production is fundamental to driving Earth’s life support systems – but they don’t achieve all this by themselves. Heterotrophic bacteria found in the photic zones of aquatic environments have a fundamental role in this too. Our group’s interest is in understanding how heterotrophs help autotrophs: who in these communities are important, and how and why they are important. Answering this is important in both natural and manmade environments so we can model these environments, and as appropriate, manipulate them, such as applying designer microbiomes to aid industrialisation of algal cultivation. Metagenomic analysis of 31 marine and freshwater cyanobacterial cultures from the Culture Collection of Algae & Protozoa resulted in assembly of >400 bacterial metagenomes (MAGs) with ca. 14 unique MAGs per culture. Community composition was clearly partitioned by salinity as a driver but collectively niche accounted for most community taxonomic variation. No universal core microbiome was identified, but taxonomic composition of marine cultures bore notable similarities to marine eukaryotic algal communities and to a natural cyanobacterial mat community found next to a northern Chilean geyser. Stable taxonomic associations imply that these taxa may have functional importance to their algal host. Functional analysis of the MAGs is underway and we will test whether the relative taxonomic variability contrasts with low functional variation between communities. If true, this implies that primary producers drive community assembly in a functionally predictable way, and that function, not taxonomy, is the more important parameter to understand.
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The investigation of virus-driven intrahepatic cholangiocarcinoma through dysregulated hepatic differentiation
More LessIntrahepatic Cholangiocarcinoma (iCCA), bile duct cancer, is increasing in incidence worldwide. Infection with hepatitis C virus (HCV) is a known risk factor for developing iCCA. Recent studies of HCV associated hepatocellular carcinoma (HCC) have demonstrated that HCV infection induces oncogenic gene expression patterns resulting from altered epigenetic profiles. Importantly, whilst ∼90% of patients treated with new direct acting antivirals (DAAs) attain a sustained virological response, the risk of HCC and other malignancies is not reduced by the same extent as previous interferon therapies. We, and others, have shown that this may be related to an imprinted gene expression pattern persisting post-treatment.
The cellular origin of primary liver cancers can vary due to liver cell plasticity. However, the incidence of mixed iCCA-HCC tumour phenotypes and the dual iCCA/HCC risk associated with HCV infection led us to hypothesise that virus-infected hepatic progenitor cells (HPC) might be the source of virus-driven malignancies. Accordingly, we demonstrated that adult HPCs were susceptible to HCV infection ex vivo. Moreover, models of hepatic differentiation revealed that HCV disrupts this process via hijacking the HIPPO signalling pathway with oncogenic hallmarks persisting following viral cure.
To explore HCV-induced alterations during cholangiocyte-specific differentiation, we have chosen to exploit the immortalised non-transformed HPC line, HepaRG and induced pluripotent stem cell derived HPCs. We will describe endeavours to develop a robust model of HCV-mediated perturbation of cholangiocyte-specific differentiation in order to identify new treatments that complement DAA therapy in order to eliminate the risk of malignancy.
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Gambian poultry isolates from hyperendemic group of AMR Escherichia coli strains in sub-Saharan Africa
Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from ten chickens and nine guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (four of them novel), of which ST155 was the most common (22/68, 32%). These strains span four of the eight main phylogroups of E. coli, with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64%) encoded resistance to ≥3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates sit within a tight genomic cluster (100 alleles difference) of strains from poultry and livestock in sub-Saharan Africa (the Gambia, Uganda and Kenya). Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted.
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Investigating the impact of M1 protein from Group A Streptococcus on fibrin clot formation, structure and fibrinolytic potential
Background: Fibrin formation is an essential part of innate immunity, sealing off infections to limit bacterial spreading.The surface-anchored M1 protein is a major virulence determinant of Group A streptococcus (GAS). During early infection M1 is cleaved from the cell surface by SpeB, a streptococcal protease regulated by CovR/S. M1 forms a supramolecular complex with fibrinogen however the impact on fibrin formation is not known.
Methods: The effects of recombinant M1 (rM1) were assessed in fibrin clots made from plasma or purified fibrinogen incubated with thrombin, by confocal microscopy and scanning electron microscopy. Clotting and lysis profiles (with plasminogen activators and plasminogen) were investigated kinetically using thromboelastography (ROTEM).
Results: rM1 (0.47-60 μg/ml) increased clotting rates to produce heterogeneous clots with irregular fibre bundles and compacted fibrin. Formation of the protective fibrin biofilm was also disrupted by rM1. Furthermore, mechanical strength of fibrin clots was reduced with increasing rM1 concentrations and was undetectable above 15.5 μg/ml. Purified and plasma clots formed with rM1 were more susceptible to lysis by plasmin, with a 1.4 – 2-fold reduction in lysis times.
Conclusions: At sites of GAS infection, cleaved M1 may bind to fibrinogen generating fibrin clots which: lack the protective film at the clot surface; are mechanically weaker; and are less resistant to lysis by plasmin. GAS strains of M1-type are commonly associated with invasive infections; the impact of M1 on fibrin structure could contribute to the severity of GAS infection by compromising the fibrin barrier that limits bacterial proliferation and migration.
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Isolation of thermophilic Actinobacteria from compost and identification of bioactive compounds with antimicrobial properties
More LessTo combat the problem of antimicrobial resistance, we are testing the hypothesis that thermophilic Actinobacteria produce novel antimicrobials at higher temperatures, with potential activity against life-threatening infections like invasive aspergillosis caused by the fungus Aspergillus fumigatus. Samples from “windrows” at a green waste processing facility yielded 36 potential thermophilic Actinobacterial strains isolated at 50oC, as well as strains of A. fumigatus. The phylogeny and identities of the bacterial strains were determined by 16S rDNA sequencing. Three strains - DJT 15 Streptomyces thermoviolaceus subsp. apingens, DJT 32 Saccharomonospora viridis and DJT 36 Saccharomonospora glauca - have shown inhibitory activity in bioassays against the ESKAPE pathogens, two of which (DJT 32 and 36) also inhibited the growth of the fungal pathogen Aspergillus fumigatus isolated from the same compost. Strain DJT 32 has also been shown to have an inhibitory effect against azole resistant human pathogenic strains of A. fumigatus. Whole genome Sequencing data of DJT 15 and 32 have been used to identify possible biosynthetic gene clusters for antimicrobial compounds (novel or otherwise) through AntiSmash analysis. Alongside this, bioactive compounds have been extracted from broth cultures of each strain using HP-20 Resin method, and the metabolites will be identified using LC:MS combined with metabolic profiling. Extraction and identification of novel metabolites will provide a path for the development of new antimicrobials for clinical use. This study has shown that thermophilic Actinobacteria produce antimicrobial compounds at higher temperatures, against Staphylococcus aureus and against the highly pathogenic fungus, A. fumigatus.
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Anoxygenic phototroph pufLM gene sequences derived from tropical aquatic sampling sites: Diversity, distribution, and phylogenetics
More LessPurple sulfur bacteria (PSB) and purple non-sulfur bacteria (PNSB) are characterized by their ability to perform anoxygenic photosynthesis. PSB and PNSB are ubiquitously found in coastal waters, enclosed lagoons, stagnant water, mangrove soils, estuaries, and similar environments. In this study, we examine microbial diversity in PSB enrichments derived from a variety of tropical sampling sites (e.g., Thailand, Puerto Rico) associated with shrimp ponds, coastal mangroves, fresh water ponds, and Nymphaeaceae (i.e., water lily) plant tissue. Since 16S rRNA-based analyses are inadequate to describe the diversity of phototrophic bacteria, other biomarkers (e.g., pufLM) are used to construct phylogenies and elucidate biogeography. Our samples indicate that the majority of sequences associated with freshwater pond PSB were related to known marine, halophilic, or salt-tolerant PSB (e.g., Marichromatium, Allochromatium, Thiococcus, and Thiohalocapsa). Phylotypes not closely-associated with known species of PSB (or PNSB) were also found. PNSB gene sequences, which appear to be related to Rhodopseudomonas and Rhodoplanes, were mostly found in freshwater samples and from Nymphaeaceae plant tissues, suggesting a difference in the ecology and distribution of these two broader bacterial groups. This difference is likely due to differences in habitat such as physical (e.g., temperature) and chemical parameters (e.g., salinity). Our preliminary analyses demonstrate a rich diversity of anoxygenic phototrophic bacteria from tropical sampling sites. Few studies have described the diversity of purple bacteria in tropical environments using full pufLM gene sequences. Employing next-generation sequencing (NGS) appears to provide greater resolution towards a deeper understanding of the global diversity and distribution of these anoxygenic phototrophs.
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Isolation and characterization of Anoxygenic phototrophs from shrimp ponds in Thailand
More LessAnoxygenic phototrophic purple bacteria are ubiquitous in aquatic and terrestrial environments and demonstrate broad phenotypic diversity. Purple bacteriaderive energy from light under anaerobic conditions via anoxygenic photosynthesis, a process in which water is not the electron donor. It has been suggested that these bacteria are useful for a variety of applications, including: wastewater treatment; heavy metal remediation; nitrogen fixation; and, control of CH4 emissions. In this study, the goal was to isolate and characterize PNSB from shrimp ponds in Thailand. Surface water and sediment were collected. Enrichment cultures were prepared using Pfenning’s mineral media. As indicated by development of reddish color and turbidity, anoxygenic phototrophic growth was observed within two days of incubation. Cultures in liquid media and on solid plates exhibited a deep red or purple color ten weeks post-inoculation. Under light microscopy, enrichments consist of communities dominated by thin, elongated gram-negative cells with granules of elemental sulfur, which are characteristic of purple bacteria. Molecular methods confirm the presence of pufLM, a genetic biomarker for purple bacteria (e.g., Thiohalocapsa marina, Allochromatium vinosum, Roseovarius tolerans). Initial sequencing of key genes (i.e., pufLM) indicate that these environmental samples contain novel isolates or “geographic variants” that have not been previously described. We have developed a few pure cultures of multiple species from these environmental samples. Since shrimp farming is a key industry in southern Thailand, the characterization of the microbial communities in these ecosystems, including anoxygenic phototrophs, will provide insights into how to maintain water quality in these food production systems.
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Investigating the functional relationship between streptokinase variants from Group A Streptococcus, and associated M-like proteins
More LessBackground: Streptokinase (SK) from Group A streptococcus (GAS) activates human plasminogen to generate plasmin, which degrades fibrin clots tofacilitate bacterial dissemination. Sequence variants of SK from diverse GAS strains form distinct evolutionary clusters.Unlike cluster 1 SK, cluster 2 variants have very little activity in solution and depend on co-factors (e.g. fibrin(ogen)).Cluster 2 SK variants also appear to correlate with cell-surface M-like proteins: SK2a with M1 (fibrinogen-binding) and SK2b with PAM (plasminogen-binding).
Methods: Plasminogen activation by recombinant SKs (rSK2a, rSK2b) was investigated by chromogenic assay; nickel-coated microtiter plates were used to immobilise recombinant M proteins (rPAM and rM1) via a C–terminal His tag to mimic cell surface plasmin generation.
Results: Plasminogen activation by rSK2b is stimulated ∼18-fold by rPAM in solution; when rPAM is immobilised stimulation exceeds 100-fold. Fibrin is the most potent stimulator of rSK2a activity (7-fold increase) compared to fibrinogen (4-fold); when rM1 was included, either in solution or immobilised, there was no further stimulation of rSK2a activity with fibrinogen.
Discussion: Stimulation ofSK2b activity by plasminogen bound to immobilised PAM suggests an important role for cell-surface plasmin generation. SK2a activity appears to be independent of M1, targeting fibrin directly. SK variants are commonly associated with distinct disease manifestations with SK2b commonly expressed by invasive skin-tropic strains of GAS and SK2a by nasopharynx-tropic strains. An improved understanding of the molecular mechanism of action by GAS SK variants may help to identify potential novel therapeutic targets for the treatment of invasive GAS diseases.
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Synergistic degradation of a biodegradable plastic film by a marine microbial community
More LessBackground: The need for replacing conventional plastics has led to an increase of the use biodegradable plastics. Most biodegradable plastic materials are certified for compostability, and their degradation mechanisms by marine bacterial communities, is still largely unknown.
Methods: Bacterial communities that degrade a PBAT-based biodegradable film (PF) were enriched from marine samples collected from the Mediterranean (Greece and Italy) and North Sea (Germany). DNA, RNA and proteins were extracted simultaneously from cultures that were starved and induced with biodegradable films, for metagenomic, transcriptomic and proteomic analyses. Mineralization of the films was assessed by CO2 evolution and film disintegration was physicochemically assessed.
Results: Within the enriched marine communities, Alphaproteobacteria and Gammaproteobacteria were the most abundant classes. Among these groups, Marinobacter was the most predominant genus on the PF film. Hydrolases similar to PETases, MHETases and terephthalic acid dioxygenases, the enzymes needed for polyethylene terephthalate (PET) degradation, were expressed when communities were exposed to the biodegradable PF film. The PETases-like hydrolases (Ple) belonged to Marinobacter, MHETases-like hydrolases (Mle) to Marinobacter and Pseudooceanicola species, and the putative terephtalate dioxygenases (Tph) to Saccharospirillum and to a α-proteobacterium candidate. Ple proteins were mainly abundant and upregulated on the PF film while Mle and Tph were abundant in the free-living fraction. Around 60% of the tested biodegradable plastic was converted to CO2 and no traces of the film were detected after the mineralization was complete.
Conclusion: Biodegradable plastics degradation is achieved synergistically by labour division among specialized film-attached and free-living bacteria. Ultimately, their complex interaction leads to the complete mineralization of a biodegradable plastic.
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Impact of Amoxicillin treatment on E. coli in dairy cow faeces
More LessIntroduction: There has been a global drive for increased antimicrobial stewardship. In the UK, this drive has focused on decreased usage of antimicrobials, specifically HP-CIAs. It is well known that antimicrobials are selection drivers for antimicrobial resistance. How a given dose will impact the prevalence of resistance in a microbiome, and therefore the associated risk of a resistant infection is less understood. Questions also remain around the impact of antimicrobial therapy on resistance across a herd, if selection occurs within an animal receiving treatment, what is the potential risk of this resistance spreading throughout other animals?
Methods: E. COLI ISOLATION Tryptone Bile X-Glucuronide (TBX) media was used for selective growth of non – toxigenic E. coli from dairy cow faeces. SUSCEPTIBILITY TESTING EUCAST Disk Diffusion testing guidelines were followed to determine susceptibility of faecal isolates to a panel of 8 antimicrobials from 5 different classes. ISOLATE FINGERPRINTING To determine clonality of faecal isolates ERIC-PCR was used as an efficient method to provide a genomic fingerprint.
Results This work is ongoing. Current work suggests that low frequency Amoxicillin treatment has no significant selection for resistance. However, there appears to be some instances of co-selection for Streptomycin, Tetracycline and Sulphonamide resistance, and several multi drug resistance isolates have been identified. More work needs to be done to confirm the impact of low frequency Amoxicillin treatment on E. coli resistance and identify the mechanism behind suspected co-selection and multi drug resistance.
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Human host cell entry restriction of Lassa and other arenaviruses
More LessArenaviruses are the largest family of viral haemorrhagic fever causing viruses. They have worldwide distribution and are divided into Old World (OW) and New World (NW) viruses based on their phylogeny, geographical distribution and serological cross-reactivity. Endemic to West Africa and South America, these emerging RNA viruses jump the species barrier from their natural rodent hosts to humans, resulting in illnesses ranging from mild flu-like syndromes to severe and highly fatal haemorrhagic zoonoses. Recent increased frequency of outbreaks and associated high fatality rates of the most common arenavirus, Lassa, in Nigeria has emphasised that these viruses should no longer be treated as causes of sporadic epidemics. The immense impact of these outbreaks on human health is further exacerbated by the lack of vaccines and effective treatments and makes it imperative to understand the molecular basis of viral pathogenesis and immune evasion.
Virus entry is a key determinant of viral host range, cellular tropism and disease outcome, hence, targeting this step of the arenavirus lifecycle could have significant impact on the control of viral infection. Our data demonstrate for the first time a synergistic restriction activity against arenavirus entry by two cellular host factors known for their control of enveloped virus infections. This co-operative restriction activity appears to conserved and we have evidence that arenaviruses may have evolved strategies to escape inhibition, through entry receptor switching, thus alluding to an understanding of the dynamics of arenavirus infection and adaptations that the viruses have made to escape host restriction pressures.
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Structural investigation of cyclic nucleotide binding proteins from Trypanosoma cruzi
More LessFora targeted therapy of Trypanosomiasis, new antiparasitic drugs should be specifically directed against essential pathways in the parasite life cycle. Among these potential targets are signal transduction pathways, which have remained largely unexplored in Trypanosoma species. Of special interest is cAMP-mediated signaling, since cAMP has been shown to play critical roles in the life cycle of T. cruzi and in host cell during invasion. The presented research focuses on the identification and characterisation of novel cAMP response proteins (CARPs) in T. cruzi by using a multi disciplinary approach involving the parasitology group of Dr Martin Edreira (University of Buenos Aires, Argentina) and the structural biology group of Dr Ivan Campeotto (University of Leicester, UK). The aim of the project is not only to increase our knowledge about T. cruzi biology but also to target CARPs for the design and development of novel therapeutic agents against Chagas disease. To date, protein crystals of one of the members of the CARP family have been obtained, paving the way for structure determination and for a structure-based drug design approach.
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Diverse mutational routes to the restoration of motility in a soil bacterium
More LessRestoration of a phenotype following insertion of a crippling mutation gives us insights into the adaptability of an organism. Mutational routes to the restoration of phenotypes can be diverse. Deletion of the master regulator (fleQ) for flagellar synthesis genes renders Pseudomonas fluorescens immotile, but after being put under strong selection to swim, it reliably re-evolves motility.
In order to uncover how the immotile bacteria regain their ability to swim they are inoculated into swimming agar plates and sampled from the leading edge of the motile zone. These samples are sequenced at a range single locus sites, predicted to be where causative mutations for the restoration of motility will be found (as indicated from previous research).
In the engineered immotile strain Pf0-2x, a huge variety of mutations, consisting of SNPs, multi-codon deletions, and frameshifts are observed across all candidate loci. In a complex environment (LB) 50% of initial restorative mutations occur in glnA, a glutamine synthetase gene. In a minimal media environment (M9) 45% of these mutations occur in ntrB, a gene for a kinase that forms part of a two component system. These findings contrast strongly with similar work in the related P. fluorescens strain SBW25, where the same SNP is repeatedly found across all environments. Interestingly, this SNP has never been seen in Pf0-2x.
These results highlight how preferential mutational routes can vary across environments, and that strain-to-strain differences can have a major impact types of mutations that are uncovered and selected for by evolution.
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Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon
More LessCampylobacter is the leading bacterial cause of gastroenteritis worldwide and its incidence is especially high in low- and middle-income countries (LMIC). Disease epidemiology in LMICs is different compared to high income countries like the USA or in Europe. Children in LMICs commonly have repeated and chronic infections even in the absence of symptoms, which can lead to deficits in early childhood development. In this study, we sequenced and characterized C. jejuni (n=62) from a longitudinal cohort study of children under the age of 5 with and without diarrheal symptoms, and contextualized them within a global C. jejuni genome collection. Epidemiological differences in disease presentation were reflected in the genomes, specifically by the absence of some of the most common global disease-causing lineages. As in many other countries, poultry-associated strains were a major source of human infection but almost half of local disease cases (15 of 31) were attributable to genotypes that are rare outside of Peru. Asymptomatic infection was not limited to a single (or few) human adapted lineages but resulted from phylogenetically divergent strains suggesting an important role for host factors in the cryptic epidemiology of campylobacteriosis in LMICs. Preprint: https://www.medrxiv.org/content/10.1101/2020.04.26.20075689v1
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Antimicrobial resistance: Transdisciplinary research on humans, antimicrobials and microbes
More LessCalls for action on antimicrobial resistance (AMR) have existed almost as early as the discovery of penicillin and the sulpha drugs. Since then solutions to AMR have circled around the development of new antimicrobials and the rationalisation of their use via various configurations of regulation of access and distribution, promotion of diagnostics and education of prescribers and consumers. Research by historians and social scientists (HSS) are increasingly demonstrating the various limitations and unintended consequences of many of these approaches, while also seeking to propose not only different ways to study AMR as a problem, but also address it. Part of this, involves serious engagement with microbiological insights (i.e. related to the microbiome) and methods to move beyond the impasses of outdated concepts (e.g. germ theory), methodological reductionism and disciplinary boundaries. Based on our empirical research on AMR and human microbiome science, we demonstrate how AMR is a transdisciplinary problem requiring contributions from HSS’s research and expertise in order to devise socially meaningful and microbiologically effective solutions. We have identified four areas where such contributions would be beneficial: (1) policies (e.g. AMR policies still assume germ theory and operate within silos); (2) AMR solutions are human centred (i.e. neglect of the microbiome and pay limited attention to other nonhumans) vs one health; (3) epidemiological variables and microbiological discourse (i.e. often employ outdated anthropological and philosophical concepts, such as westernised, modern, traditional); (4) Rhetorics and lexicon (i.e. can be morally and conceptually simplistic, like ‘war’, ‘sweets’, ‘good’/’bad’ bugs, ‘irrational’).
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Molecular identification of antibiotic resistance genes of bacteria isolates from ready to eat food and drinking water sold in Imo State University
More LessFood and water are fundamental need of human as its quality is of great concern to the generality of consumers, despite quality control measures during their production, unacceptable microbiological load often makes them unsafe. In this study, we analyzed various ready to eat food and water for presence of coliform bacteria, fungi count, antibiotic resistance gene and plasmid DNA, Samples were found to be positive for various antibiotic resistance genes namely, Sul1, Sul2, Tet A and TetB, interestingly, considerable total coliform bacteria were found from isolates and this result was further confirmed using illumina sequencing of the 16SrRNA gene. All Samples were negative for plasmid DNA, These finding deserve attention as the presence of coliform bacterial and antibiotic resistance genes potentiate health risk to members of university community and as such calls for strigent supervision and saftety and implementation of food safety regulations.
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Characterization of a multi-modular bacterial enzyme with binding and hydrolysis activities on fungal cell wall components: Insights into bacterial-fungal interactions in the soil
More LessBacteria from the phylum Bacteroidetes is known as good degraders because of their abilities in secreting glycoside hydrolases (GHs). As a member in Bacteroidetes, Chitinophaga pinensis is capable to hydrolyze several glycans (McKee et al., 2019). We found that a multi-modular CAZyme produced by C. pinensis contains not only two GHs but also two uncharacterized domains. Although it is common for CAZmes to have non-catalytic modules like carbohydrate-binding domains (CBMs), the domains in this protein are uncommon, and their functions unknown.
Our enzyme contains five domains – a GH5_46 enzyme, a GH18 enzyme, two non-identical uncharacterized domains, and a Por C-terminal secretion domain. I have shown that the GH5 can specifically degrade pustulan (β-1,6-glucan), and is the only β-1,6-glucanase found in its sub-family. The GH18 is a chitinase that reacts with β-chitin. Interestingly, the two uncharacterized domains both bind polysaccharides, and improve enzyme stability. However, they do not necessarily bind the same glycan as their GH partners. Our result demonstrates how two distinct polysaccharide-degrading modules within one protein can cooperate. We also discuss the impact of the binding domains on the GH activity.
We anticipate our research can stimulate the exploration of β-1,6-glucanase activity in family GH5, and expand the database of carbohydrate specificities in bacteria. The two unusual binding domains we have characterized suggest an unexplored aspect of bacterial physiology in the soil.
References:
McKee, L.S., et al., 2019. Focused metabolism of β-Glucans by the soil Bacteroidetes species Chitinophaga pinensis. Appl. Environ. Microbiol., 85(2), pp.e02231-18.
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Using structural biology of methanogen enzymes to aid our understanding of methane formation
More LessMethane is a potent greenhouse gas (28-fold more potent than carbon dioxide) and is a significant gas contributing to global climate change. Approximately a billion tons of methane are produced each year by methanogenic archaea in ruminants. These archaea possess a number of unusual traits such as isoprenoid-based lipids, unusual cell wall chemistry and a unique energy metabolism (methanogenesis) that requires six methanogen-specific cofactors. Many of the enzymes involved in these processes have no direct analogues in the host animal. To gain insights into the fundamental biology of rumen methanogens we have determined crystal structures of key enzymes with archaeal-specific traits. Over 600 enzymes were targeted for structure determination which produced approximately 200 purified soluble enzymes for crystallographic screening. More than 50 different enzymes have produced crystals and 30 structures have been solved for individual enzymes to date. The results have helped illuminate our understanding of methane formation at this critical juncture in the world’s history.
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Can m6A-modified RNA be used as an Anti-Viral Target?
More LessKaposi’s sarcoma-associated herpesvirus (KSHV) is a DNA virus associated with several HIV-associated malignancies.
Like all herpesviruses, KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND-1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).
Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner. Taking this into consideration, novel ligands have been assessed as effective anti-viral reagents. The importance of A versus m6A within the lytic phase of KSHV’s lifecycle will be investigated by combining in silicoscreening with biophysical techniques and cell based assays.
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Identification of potential key genetic factors in the long-term success of Shigella as pathogens
More LessBacterial of the genus Shigella are a major contributor to the global diarrhoea burden causing 100,000 deaths per annum globally. Further to this, increasing antibiotic resistance in Shigella and the lack of a licenced vaccine has led WHO to recognise Shigella as a priority organism for the development of new antibiotics. Understanding what drives the long-term persistence and success of this pathogen will thus aid the global management of shigellosis and may identify targets relevant to other enteric bacteria. To identify key genetic drivers of Shigella evolution over the past 100 years, we have used the historical Murray collection; comprising several hundred pre-antibiotic era (1917 – 1954) Enterobacteriaceae from diverse geographical locations. We employed genome-wide association studies to sequences from over 100 Shigella isolates from the Murray collection alongsidemore modern (i.e. 1950s – 2018) isolates to identify genetic factors (SNPs and genes) significantly associated with time as a continuous variable. GWAS hits (e.g. a putative resistance protein) then underwent variation, functional and phenotypic analysis to examine the plausibility of their role in shaping Shigella populations and as potential targets for managing this pathogen.
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Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates.
We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states.
Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.
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Digital Outreach: Promoting a global understanding of antimicrobial resistance (AMR)
More LessTechnology allows educators to reach a global audience without amassing a large carbon footprint. The Skype in the Classroom programme from Microsoft enables me to spark curiosity and engage a wide range of students in dialogue to explore global issues, in particular, the threat of antimicrobial resistance, while supporting me to develop my skills as an educator. Through a multi-disciplinary approach, I employ Skype in the Classroom as a future-proof, powerful tool in our arsenal against the evolving microbial threat. We are not only responsible for teaching the next generation of scientists, but also the next generation of thought-leaders and professionals, who will work collaboratively towards our common goals.
Antimicrobial resistance is one of the grand challenges of the 21st century, and global connectivity is one of the best solutions to promoting better understanding of this threat. The Skype in the Classroom programme has allowed me to teach sessions to students in Norway, India, Brazil, Miami, Florida and Puerto Rico, with students ranging from 10 to 18 years old. I use skype and Teams to deliver online lessons in an accessible way, starting with the scope of the problem, covering the technical detail of how resistance emerges and is spread, and looking at some of the newest approaches to tackling AMR. Using a ‘challenge-based-learning’ approach, we discuss how different professionals and roles within society can contribute to the issue. This makes the class applicable to students with a wide range of backgrounds, including those without a keen interest in science.
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