- 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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