bluuebunny/crossref_metadata_2025 huggingface dataset:
Galleria mellonella: a novel infection model for screening potential anti-mycobacterial compounds against members of the Mycobacterium tuberculosis complex
Authors: Asai Masanori , Li Yanwen , Singh Khara Jasmeet , Robertson Brian , Langford Paul , Newton Sandra
Published: April 2019

Animal infection models are vital as drug screens for novel therapeutics to tackle the global tuberculosis (TB) epidemic. However, all pre-existing models have limitations which include ethical constraints. Therefore, efforts to reduce and/or replace conventional animal models in TB research are warranted. Previously, we reported the use of Galleria mellonella (greater wax moth, GM) as a novel infection model for the Mycobacterium tuberculosis (MTB) complex, using Mycobacterium bovis BCG lux, a bioluminescent mutant which allows for the rapid quantification of bacterial burden in vivo. Here we investigated the drug screening potential of GM infected with a lethal dose of BCG lux, treated with first or second-line antimycobacterial drugs over a 96 h period; where drug efficacy was determined every 24 h through bioluminescent measurement of larval homogenates. Improved survival outcome was observed in all larvae treated with antimycobacterials when compared to untreated controls. Furthermore, all drug treatments except pyrazinamide resulted in a significant reduction in bioluminescence of BCG lux in vivo. Isoniazid and rifampicin displayed the highest survival outcome and greatest in vivo drug efficacy, in line with observations reported in mice. However, combined or multiple dosing of either drug showed little to no difference over single dose mono-therapy. Our results demonstrate that GM is a promising infection model for members of the MTB complex, with significant potential for its use in the drug development pipeline as a pre-screening model for novel therapeutics, thereby reducing experimental usage of animals in TB research. Supported by the NC3R’s (NC/R001596/1)
Metagenomic analysis reveals significant seasonal variations in the epiphytic bacterial communities associated with different parts of the brown seaweed Laminaria digitata
Authors: Ihua Maureen , Dobson Alan , Stengel Dagmar
Published: April 2019

Brown seaweeds such as Laminaria species are a rich source of polysaccharides such as laminarin and fucoidan which have a variety of functional food and animal feed applications, as well as alginates with demonstrated biological and pharmacological activities. Macroalgal surfaces are rich in carbon-based constituents which provide a suitable environment for growth and colonization by diverse bacterial communities. Several environmental and non-environmental factors can influence the composition and abundance of epibacterial communities associated with seaweeds. In addition to the biological, physical and chemical properties of the macroalgal surface, seasonal variations have been found to play a significant role in the structure of the associated microbial communities. Variations in macroalgal epibacterial communities have also been observed within different parts of the host algal species. However, to date, in-depth studies on bacterial communities associated with macroalgal species, their ecological role and interactions with the algae are still scarce. To gain an insight into the diversity and composition of the microbial communities associated with the brown alga Laminaria digitata, the communities derived from different parts of the alga including the blade, meristem, stipe and holdfast; were investigated using metagenomic Illumina sequencing of 16S rRNA gene amplicons. Seasonal variations in the microbial populations were found in samples taken from the Irish coast in different seasons between 2017 and 2018. This metagenomic-based investigation provides a detailed view of the seasonal variations in the bacterial populations associated with Laminaria digitata and helps provide further insights into potential interaction between this macroalga and its epiphytic bacterial communities.
Carbon, Nitrogen and Phosphorous assimilation in V. cholerae – a case of imperfect nutrient sensing
Authors: Paranjape Shridhar , R Shashidhar
Published: April 2019

Vibrio cholerae experiences frequent feast and famine conditions. In the absence of all the three nutrient components (Carbon, Nitrogen, and Phosphorous), the cells could manage starvation and survive up to 6 months with only 1 log reduction in viability. Addition of carbon or nitrogen to the starvation media resulted in 3 log reduction in cell number. Simultaneous addition of carbon and nitrogen (Phosphorous starvation) reduced the cell viability to a below detectable level. The suboptimal growth conditions; non-metabolizable source of carbon and nitrogen; variable C: N ratio; inhibition of metabolism and cell division, increased the cell survival under phosphate starvation. However, when all the three components are limited, the cells did not initiate active metabolism and could conserve the energy for long-term survival. This observation suggests that integration of carbon, nitrogen and phosphorus sensing is imperfect in V. cholerae and it cannot down-regulate the metabolism during phosphorous limitation. The carbon and nitrogen could prime the cells to accelerate the rate of metabolism, irrespective of the presence of phosphorous, thereby creating an energetically unfavorable situation. The lack of crucial component phosphorus fails to activate a stringent response that results in increased futile cycling of nutrients, loss of ATP and cell death. The Vibrio genus was found to be less efficient in surviving phosphate starvation than E. coli and S. Typhimurium. The two-component system CreC (a response regulator of phosphorous) is absent in V. cholerae and may be responsible for the lack of stringent response to phosphorus starvation.
Generation of recombinant avian coronaviruses indicates the S gene is a factor in pathogenicity
Authors: Stevenson-Leggett Phoebe , Keep Sarah , Oade Michael , Britton Paul , Bickerton Erica
Published: April 2019

The avian coronavirus infectious bronchitis virus (IBV) is the most economically important disease of chickens in the UK, causing significant losses as a result of poor weight gain and reduced egg quality in infected birds. IBV expresses a large spike (S) glycoprotein on the surface of the virion which is responsible for attachment to host cells and is the main antigenic target for neutralising antibodies during infection. Previous work has also demonstrated that the S protein determines cell tropism in vitro. In order to investigate the involvement of the S gene in IBV pathogenesis and explore the potential for vaccine propagation in cell culture, recombinant viruses were generated using vaccinia virus based reverse genetics. Two isolates of the pathogenic M41 strain were mutated to include the S gene from a non-pathogenic lab strain with extended tropism (Beau-R) or a heterologous pathogenic field strain with restricted tropism (4/91), resulting in two recombinant IBVs termed M41K-BeauR(S) and M41K-4/91(S), respectively. These viruses were characterised in vitro and in vivo to determine the involvement of the S gene in IBV replication and pathogenicity. M41K-BeauR(S) was attenuated in vivo but exhibited the extended host tropism of the S donor strain. M41K-4/91(S) remained pathogenic and also adopted the restricted in vitro tropism of 4/91. This indicates that the S gene not only determines the cellular tropism of the virus but also plays a key role during in vivo infections, and that replacing the ectodomain of IBV S can significantly alter the pathogenicity of the resulting virus.
Attenuation of E. coli O157:H7 virulence by a combination of natural plant extracts and organic acids before and after refrigerated storage
Authors: Stratakos Alexandros , Linton Mark , Ward Patrick , Sima Filip , Kelly Carmel , Pinkerton Laurette , Gundogdu Ozan , Corcionivoschi Nicolae
Published: April 2019

Anti-virulence strategies are an alternative approach to combat zoonotic bacterial pathogens. Although control measures are in place against E. coli O157, it still remains a global health concern with cattle being the most important reservoir. Different feed constituents have varying effects on the virulence of E. coli O157 present in the gut which then are transferred to meat processing surfaces and meat during slaughter. This study explored the anti-virulence properties of a mixture of natural plant extracts and organic acids (citrus, grape and oregano extracts, lactic and citric acid) before and after refrigerated storage as means of reducing the risk from E. coli O157 by reducing its virulence. Assessment of the effects of sub-inhibitory concentrations (0.1 and 0.5 %, v/v) of the antimicrobial mixture before refrigerated storage showed that the pathogen’s motility and adhesion onto HCT-8 cells was significantly reduced (P<0.05) in a dose-dependent manner. Shiga-toxin 2 production was also significantly reduced. Real-time PCR analysis revealed that the mixture of natural antimicrobials repressed expression of adhesion (eae) and shiga-toxin 2 (stx2) genes, which was consistent with the observed reduction in adhesion and toxin production. The same virulence factors were investigated after simulated storage of E. coli O157 in meat simulation medium. Results revealed that after exposure to the antimicrobial mixture, the virulence was significantly lower compared to non-treated control after refrigerated storage. The present work shows the potential of the antimicrobial mixture in reducing virulence and thus risk from E. coli O157 by applying it as a feed additive.
The optimization of microbial induced calcium carbonate precipitation in soil improvement using engineered bacteria
Authors: Haystead Jamie
Published: April 2019

Poor soil conditions limit the building of new infrastructure, which is needed for an ageing and expanding population. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination, therefore there is demand for a sustainable approach to this process. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate (CaCO3), which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase mechanical properties. Commonly used bacteria include Sporosarcina pasteurii and Bacillus subtilis. A range of factors influences MICCP which presents challenges with process optimisation. These factors need to be optimised in the laboratory before they can be applied for engineering purposes. The overall aims of my research are to optimise urease production in S. pasteurii and B. subtilis and to investigate the distribution and binding of these bacteria with various sand particles, by means of syringe and glass column set ups. These bacteria will be compared with engineered bacteria which can overproduce urease to investigate the impact on precipitation efficiency. Factors to control bacteria biofilm formation to influence the morphology of CaCO3 will be investigated to determine the impact of various crystal shapes on soil properties. Ultimately, raw data generated from the project will be used for predicting biocementing at a lab scale for building computational models.
The microbial diversity of a sulfur-rich and saline cold pool in the Canadian high Arctic
Authors: Christopher Macey Michael , Stephens Ben , Fox-Powell Mark , Schwenzer Susanne P. , Pearson Victoria K. , Cousins Claire R. , Olsson-Francis Karen
Published: April 2019

Currently, the surface of Mars cannot sustain liquid water, but there is evidence suggesting that water was present in the Noachian. Although water might exist in the subsurface of Mars, it could not sustain in the present day unless it was highly saline. Thus, saline springs in polar desert environments are analogues with which to investigate martian conditions. An example of this is Axel Heiberg Island, located in a region of continuous permafrost in the Canadian High Arctic, which hosts sulfidic and highly saline springs. In this study, cultivation-dependant and independent techniques were used to investigate the microbial diversity of a sediment sample collected from a saline cold (3–8 °C) pool at Colour Peak Springs on Axel Heiberg. Both DNA and RNA were extracted from the samples, and the microbial community was characterised using the 16S rRNA gene from the extracted nucleic acids. The metabolic profile was characterised by screening DNA and cDNA for functional genes relating to the cycling of carbon (coxL, xoxF, cbbL), nitrogen (nifH, nosZ, nod) and sulfur (dsrB, soxB). The community profiles were used to inform enrichment strategies, allowing for the isolation and characterisation of several halophilic isolates including strains of Marinobacter, Halomonas, Halanaerobium and Loktanella. Through this work we have been able to develop an in-depth characterisation of the metabolic and phylogenetic diversity that is present and viable within this analogue site. This allows us to start building an understanding of the underlying mechanisms and strategies that enable organisms to persist in these environments.
The antimicrobial activity of Micromonospora sp.
Authors: Mark David , DeWald Jan , Tucker Nicholas , Herron Paul
Published: April 2019

With antimicrobial resistance becoming an increasingly severe issue in both the developed and developing regions of the world, new strategies need to be employed to identify and characterise novel antimicrobial activity. Pseudomonas aeruginosa is a Gram negative pathogen and a major cause of opportunistic infections in burn victims and cystic fibrosis patients. Due to a wide repertoire of antibiotic resistance mechanisms, these infections are difficult to cure and thus there is a need to develop novel antimicrobial treatments. Members of the Actinobacterial genus Micromonospora produce a broad range of bioactive secondary metabolites, a number of which possess antimicrobial properties. The goal of this research is to examine the antimicrobial properties of an actinomycete strain – identified by 16S sequencing as Micromonospora –originally isolated from the Atacama desert, Chile, with a focus on identifying and characterising anti-pseudomonad activity. Bioactivity was screened for against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii and Enterococcus faecium. AntiSMASH analysis of assembled Illumina sequencing data was used to identify putative biosynthetic gene clusters. Antimicrobial bioactivity was observed, and potential antimicrobial biosynthetic gene clusters were identified through genome mining. This work has identified antimicrobial activity from a region of underexplored ecology, and highlights the importance of sampling and examining areas which have previously been considered too hostile to support life.
Streptomyces coelicolor M145 MIP-like proteins: to identify their role in a non-pathogenic organism
Authors: Thomas Amy , Fernández-Martínez Lorena
Published: April 2019

Understanding the role of virulence loci within pathogenic organisms can be vital in exploring the evolution of disease. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find macrophage infectivity potentiator-like proteins (MIPs) (Clark et al., 2013). MIPs are a subset of immunophilins associated with virulence in a range of micro-organisms (Norville et al., 2011). It is unknown the role they possess in non-pathogenic strains such as Streptomyces coelicolor M145. This project will identify the role of MIPs in a non-pathogenic strain through cloning, overexpression and knock out of three genes encoding putative MIP-like proteins (SCO1638, SCO1639 and SCO2620). The phenotypes will then be characterised by growth under contrasting conditions. Antibiotic production will also be measured and compared to the wild-type M145 strain. The overexpression and mutant strains will also be tested for the ability to infect amoeba using amoeba infection assays compared to a wild-type control. The results from this study will contribute to the understanding of the role of MIPs and therefore the evolution of virulence within Streptomyces species.
Genomic analysis of Burkholderia ambifaria identifies key specialised metabolites for biopesticidal applications
Authors: Mullins Alex , Murray James , Bull Matthew , Jenner Matthew , Jones Cerith , Webster Gordon , Green Angharad , Neill Daniel , Connor Thomas , Parkhill Julian , Challis Gregory , Mahenthiralingam Eshwar
Published: April 2019

Background

Burkholderia bacteria are renowned for the biosynthesis of an extensive repertoire of specialised metabolites encoded by biosynthetic gene clusters (BGCs). Burkholderia ambifaria is a historical biopesticide and produces multiple antimicrobials with activity against nematodes, fungi and bacteria. We evaluated the distribution of antimicrobial activity and BGCs across the species which, despite their biopesticidal potency, is poorly understood.

Results

Genomic analysis of 64-B. ambifaria whole-genomes revealed considerable diversity. Pathway prediction identified 1272 BGCs across 64 strains that were de-replicated to 38 distinct BGCs. Known BGCs accounted for 13 of the BGCs; 7 of which corresponded to known antimicrobials, and included enacyloxins and bactobolins. The antimicrobial activity of the strains was assessed in a classical overlay assay against animal and plant pathogens. The combined genome mining and in vitro activity screens identified a potent anti-Gram-positive and anti-Oomycete compound, cepacin. A damping-off disease model with the pathogen Pythium ultimum and legume Pisum sativum (garden peas) highlighted cepacin as a key specialised metabolite in crop protection. B. ambifaria strain persistence in a mouse inhalation infection model was reduced by removal of the third replicon, whilst retaining the biopesticidal properties of the B. ambifaria strain.

Conclusion

This work highlights the diversity and prevalence of specialised metabolites in B. ambifaria, and the importance of the potent antimicrobial cepacin in protection against damping-off disease in agriculture. Construction of a B. ambifaria strain that retained biopesticidal properties yet was attenuated for persistence within a murine infection model provides a potential route towards developing safe biopesticidal agent.
Identifying novel antimicrobials from anaerobic rumen fungi
Authors: Evans Rebeca , Griffith Gareth , Callaghan Tony , Thomas Ben , Huws Sharon
Published: April 2019

Anaerobic rumen fungi (phylum Neocallimastigomycota) occupy the gastrointestinal tract of several herbivorous animals, and by using their powerful hydrolytic enzymes and mechanical forces they degrade plant material in the rumen, essential for rumen efficiency. The rumen microbiome represents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including antimicrobial peptides (AMPs). AMPs are promising drug candidates, and are necessary for targeting the worldwide issue of antimicrobial resistance. Rumen fluid and faecal samples were collected from various large herbivores, and fungal cultures were grown and maintained under anaerobic conditions. After roll tube culture to isolate single-zoospore cultures, sequencing of LSU was undertaken to identify the fungi to species level. Analysis of genomic data from these cultures, alongside published data was undertaken to explore the diversity of AMPs within these fungal genomes. Using functional and computational screening, potentially novel AMPs have been discovered, with isolates showing encouraging activity against some strains of bacteria. Findings indicate that the rumen microbiome may provide alternative antimicrobials for future therapeutic application.
In vivo and ex vivo models of infectious bursal disease virus (IBDV) in inbred chicken lines differing in their resistance to the disease
Authors: Asfor Amin , Dulwich Kate , Broadbent Andrew
Published: April 2019

Infectious bursal disease virus (IBDV) targets B cells in the bursa of Fabricius (BF), causing immunosuppression in chickens and mortality. Susceptibility differs between inbred chickens, with 0 % mortality in ‘resistant’ lines and up to 80 % mortality in ‘susceptible’ lines. However, the mechanism of disease resistance is not understood. In order to address this, chickens (n=18) from three ‘resistant’ lines (15, C and O) and one ‘susceptible’ line (W) were infected with the very virulent IBDV strain, UK661. Clinical scores were recorded and tissues harvested at necropsy on day one, two and three post-infection for RNA extraction and virus titration, compared to non-infected controls. Interestingly, within a given line, we observed a range of symptoms, with some individuals experiencing more severe disease than others, despite no difference in viral replication. Line 15 was the least susceptible to disease based on the average clinical scores (3.2 (15), 5.7 (C), 4.8 (O) and 4.7 (W)) and the percentage of birds with a clinical score of 2 or above (17 % (15), 100 % (C), 83 % (O) and 83 % (W)). The average peak virus replication was also significantly lower in line 15 birds (6.3 log10 fold change) compared to lines C or O (7.0 and 6.8 log10 fold change) (P<0.01). RNA-sequence analysis will be performed using BF samples to understand the biological pathways that confer IBDV resistance. Moreover, primary bursal cells harvested from resistant and susceptible lines were infected with IBDV ex vivo and ongoing work aims to quantify differentially expressed genes in these cells.
Application of furanone compounds for the modulation of biofilm formation in common wound pathogens
Authors: Proctor Chris , Ternan Nigel , McCarron Paul
Published: April 2019

Chronic wounds are a significant issue in healthcare, presenting a considerable economic burden to the NHS and a serious health risk to patients. The majority of non-healing wounds have been shown to contain a biofilm which prolongs the inflammatory stage of wound healing and significantly delays wound healing. This often causes a normal wound to progress and become chronic, presenting further problems for patients including increased risk of secondary infection, further deterioration of the wound and an increase in treatment intensity. This project aims to assess the efficacy of several compounds in modulating the formation of biofilms in a number of clinically relevant pathogens when used at sub-inhibitory concentrations. The organisms used in this project include Pseudomonas aeruginosa and Staphylococcus aureus. We aim to test the efficacy of three plant derived compounds including 4-hydroxy-2,5-dimethyl-3(2 h) furanone (HDMF), 2-methyltetrahydrofuran-3-one (MTHF) and l-ascorbic acid. Future work will characterise the efficacy of these compounds when delivered to a biofilm from a hydrogel based delivery system. At sub-inhibitory concentrations in pure solution, candidate molecules tested to date showed no ability to reduce biofilm formation. Indeed, treatment with HDMF resulted in greater production of biofilm in P. aeruginosa and treatment with all compounds showed no difference in biofilm formation by S. aureus. To characterise the impact of hydrogel based delivery on compound efficacy all candidate molecules were loaded into a hydrogel and shown to be effectively released from it. Experiments to characterise the modulatory potential of these compounds when released from a hydrogel are currently underway.
The effect of local release antibiotic beads on in-vitro bacterial growth from tissue taken from infected diabetic foot ulcers
Authors: Fletcher Julie , Porter Robert , Aiken Sean , Delury Craig , Michell Stephen
Published: April 2019

Diabetic foot infection is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. Recent figures published by Public Health England show that there are more than 163 diabetes related amputations in England every week. This study investigates the effect of antibiotic loaded calcium sulfate (Stimulan® Rapid Cure) beads on in-vitro bacterial growth from tissue taken from diabetic foot infections. Patients were recruited from the Macleod Diabetes and Endocrine Centre at the Royal Devon and Exeter Hospital. Inclusion in the study was based on clinical recognition of an infected foot ulcer requiring wound debridement. Debrided tissue was homogenised and 50 µl spread over the surface of Columbia blood agar and fastidious anaerobe agar. Three replicate calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar. Each bead contained approximately 3.4 mg and 1.6 mg of vancomycin and gentamicin respectively. Plates were incubated aerobically or anaerobically as appropriate. Zones of inhibition were recorded at 1 and 4 days. Calcium sulfate beads containing vancomycin and gentamicin were able to inhibit bacterial growth in all tissue homogenates tested with zone diameters ranging from 16 to 40 mm. Local release of antibiotics could have the benefit of achieving high local concentrations within poorly vascularised tissue which may inhibit bacterial growth at the wound site. By improving treatment of diabetic foot infections, it may be possible to prevent amputation, maintain mobility and conserve quality of life.
Identification and characterization of bioengineered nisin derivatives that inhibit the opportunistic pathogen Staphylococcus epidermidis
Authors: O’ Shea Clíona , Twomey Ellen , Field Des , Begley Máire
Published: April 2019

Staphylococcus epidermidis is a major cause of hospital-acquired infections particularly on indwelling medical devices and implants. Infections caused by this pathogen are difficult to treat with standard antimicrobial agents mainly as the bacterium can establish biofilms on artificial surfaces. Novel control methods are needed and one such alternative may be bacteriocins; these are antimicrobial peptides produced by some bacteria that inhibit other specific bacteria. They are potent, safe, and stable and are easy to produce using biotechnological based strategies. Additionally, as they are gene encoded they can be easily modified or engineered to enhance their activity. In the present study, we demonstrate the ability of the bacteriocin nisin to inhibit a collection of clinical S. epidermidis strains under standard laboratory conditions. In addition, a bank of bioengineered nisin derivatives was screened using agar-based deferred antagonism assays and derivatives with enhanced antimicrobial activity compared to the wild-type nisin were identified. These derivative peptides are currently being purified using a combination of chromatography-based approaches and their potency and stability are being examined. Future experiments will focus on examining the ability of the nisin derivatives in inhibiting S. epidermidis biofilms on medical device materials (e.g. stainless steel and polyvinyl chloride) both alone and in combination with conventional antibiotics. It is hoped that one of the nisin derivatives analysed in this study may ultimately be used to control or prevent infections caused by S. epidermidis.
Shining a light on antibiotic selection: optimised live/dead fluorescence spectrometry for rapid antimicrobial susceptibility testing
Authors: Julia Robertson , Ou Fang , McGoverin Cushla , Vanholsbeeck Frederique , Swift Simon
Published: April 2019

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We propose to use live/dead spectrometry as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We have developed a spectroscopic device (Optrode) to measure fluorescence from SYTO 9 and propidium iodide stained cells that can be used to enumerate the bacterial load. We propose a procedure using the Optrode that will take bacteria in a clinical sample, challenge with a panel of antibiotics, and measure live/dead ratios to determine the best bactericidal choice. Using calibration data we optimised the live/dead spectrometry protocol outlined in the kit instructions, improving upon media selection for growth and staining, and analytical parameters. We applied the optimised methodology to detect live and dead Escherichia coli in populations challenged with ampicillin. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI. Following on from the promising results generated with ampicillin, live/dead spectrometry of ampicillin challenged cells was characterised in terms of antibiotic concentration, growth phase, and susceptibility to treatment for each treatment time. The generated data demonstrated that reliable detection of E. coli knockdown by ampicillin using live/dead spectrometry requires log phase cells challenged with a suitable concentration for a particular treatment time.
Fundamental nanoparticle interactions with biofilms of Pseudomonas species
Authors: Devlin Henry , Barros Caio , Fulaz Stephanie , Hiebner Dishon , Quinn Laura , Vitale Stefania , Casey Eoin
Published: April 2019

The use of engineered nanoparticles (NPs) as a technique for antimicrobial delivery aimed at biofilm treatment is an emerging field of research. There have been numerous studies involving a wide range of NPs showing varying results regarding anti-bacterial effects (1). However, research focusing on specific interactions between functionalized nanoparticles and the extracellular polymeric substances (EPS) of biofilms are limited. The complexity of the biofilm matrix may be hindering the understanding of the fundamentals which govern biofilm – nanoparticle interactions. There are a wide range of physicochemical properties which influence the uptake and retention of nanoparticles within the matrix including NP size and charge properties, biofilm topography and porosity and EPS composition (2). These aspects must be considered when studying biofilm – nanoparticle interactions. In order to identify these specific interactions, a series of experiments were carried out using mCherry-expressing Pseudomonas fluorescens and GFP-expressing Pseudomonas putida biofilms. Using high throughput fluorescent intensity measurements and confocal microscopy, it was possible to investigate the uptake of surface functionalized silica NPs by the two biofilms and obtain valuable information regarding biofilm – nanoparticle interactions. The results suggest that specific NP surface functionalization has a major role in guiding the interaction and binding of EPS components, possibly due to electrostatic interactions between NPs and the EPS. The findings of this research will help with the future design of nanoparticles with specific modes of action towards components in the EPS.1. Ramos M., et al. (2018). International Journal of Nanomedicine13 : 1179–1213.2. Nevius BA., et al.(2012). Ecotoxicology21 : 2205–2213.
Global gene expression profiling of a virulent Klebsiella pneumoniae strain during pulmonary infection
Authors: Kidd Timothy , Dumigan Amy , Forde Brian , Phan Minh-Duy , Canals Rocio , Hinton Jay , Schembri Mark , Bengoechea Jose
Published: April 2019

Background

Klebsiella pneumoniae (Kpn) is an important respiratory pathogen associated with significant mortality, fierce inflammatory responses and high rates of antimicrobial resistance. The increasing incidence of multidrug resistant Kpn has significantly narrowed the therapeutic options available; as such, there is an urgent need to better understand Kpn pathophysiology to identify novel therapeutic targets. Here we performed an in vivo transcriptomic analysis of Kpn isolated from a mammalian host with pulmonary infection.

Methods

C57BL/6 mice were intranasally inoculated with the virulent Kp52.145 strain (serotype O1:K2); with lungs extracted, homogenised and pooled (n=3; in duplicate) at 32 h post-infection for bacterial RNA purification and RNA-Seq (Illumina). Differential gene expression (analysed using Degust [Voom/Limma]; FDR cut-off =0.01, abs log-FC=2) was assessed in comparison with mid-log phase growth in Lennox broth.

Results

Overall, we identified >900 differentially expressed genes (DEGs), comprising ∼17 % of the combined chromosomal and plasmid coding sequence repertoire. 52 % of the DEGs were upregulated during infection, including several siderophore-independent iron-, manganese- and zinc-uptake systems (e.g. hmuRSTUV, sitABCD, mntH and znuACB). We also observed a marked in vivo oxidative stress signal, with several Kpn oxidative stress response genes (e.g. oxyR, katE, katG) upregulated during infection. In contrast, expression of mgrB, a negative-regulator of the PhoPQ two-component system associated with resistance to host antimicrobial peptides was downregulated.

Conclusion

This study provides a novel insight into Kpn gene expression during pulmonary infection. Overall, our data suggest that adaptation to metal starvation, oxidative stress and innate immune defenses are critical for the success of Kpn lung infection.
Genomic evolution of Klebsiella pneumoniae clones: the good, the bad and the ugly
Authors: Wyres Kelly , Wick Ryan , Judd Louise , Froumine Roni , Tokolyi Alex , Gorrie Claire , Lam Margaret , Duchêne Sebastián , Jenney Adam , Holt Kathryn
Published: April 2019

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.
Biosynthesis of silver nanoparticles using baker’s yeast, Saccharomyces cerevisiae and its antibacterial activities
Authors: Olobayotan Ifeyomi , Akin-Osanaiye Bukola
Published: April 2019

The biosynthesis of silver nanoparticles extracellularly using baker’s yeast, Saccharomyces cerevisiae and its antibacterial activity was investigated in this study. Biosynthesised silver nanoparticles were chaterized by using UV-Visible spectroscopy, which showed a distinct observed absorption peak at 429.00 nm that is attributed to the plasmon resonance of silver nanoparticles; X-ray diffraction, which determined the average size of the silver nanoparticles to be approximately 16.07 nm; presence of oval shaped silver nanoparticles determined by scanning electron microscopy; and Fourier-transform infrared, which revealed notable peaks at 3332.2, 2903.6 and 1636.3 cm−1 corresponding to the binding of the silver nanoparticles to active biomolecules, alcohols and phenols, carboxylic acids and aromatic amines respectively. The silver nanoparticles were also found to be stable for ninety days. Antibacterial activity of the silver nanoparticles was also studied. The silver nanoparticles was significantly active (P>0.05) against the test organisms at an extract concentration of 75 µg ml−1. Concentrations less than or equal to 50 µg ml−1 were not as effective as the colony forming units at this concentration, 1.61×106 for methicillin-resistant Staphylococcus aureus and 1.45×106 for Pseudomonas aeruginosa respectively were about the same range a small the colony forming units of the controls. The silver nanoparticles inhibited methicillin-resistant S. aureusmore than they inhibited P. aeruginosa. The LD50 of the synthesized silver nanoparticles after oral administration was seen to be greater than 5000 mg kg−1 body weight and is therefore thought to be safe. This study supports the use of silver nanoparticles as therapeutic agents.
Pseudomonas aeruginosa antibiogram profiles are poor indicators of genetic relatedness
Authors: McKenna James , Fairley Derek , Neill Lesley , Crawford Lyndsey
Published: April 2019

Pseudomonas aeruginosa is a significant nosocomial pathogen responsible for severe and life threatening infections particularly in immunocompromised patients. This organism is ubiquitous in healthcare environments particularly water systems which act as a reservoir of infection. Recognition of a potential outbreak and having the ability to quickly identify and mitigate sources of exposure is critical for effective infection control. Historically analysis of P. aeruginosa antibiogram profiles represents a convenient and frequently used ‘first line’ indicator of strain relatedness. Reported here is a comparison of P. aeruginosa antibiogram profiles with those obtained using rapid Variable Number Tandem Repeat (VNTR) for patient and environmental isolates in in three separate local nosocomial outbreaks. The results demonstrate that antibiogram profiles from P. aeruginosa should not be employed as presumptive indicators of relatedness, doing so can falsely re-assure clinicians. Use of rapid molecular typing method VNTR allows genotypically identical strains to be unambiguously identified within 48 h.
Diversity of eukaryotic gut microbiota of northern Thai populations
Authors: Yowang Amara , Jinatham Vasana , Tsaousis Anastasios , Kullawong Niwed , Popluechai Siam , Gentekaki Eleni
Published: April 2019

Gut eukaryome refers to the collection of fungi and protists in the gut. Until recently, all eukaryotic gut microbes were considered parasites and subject to elimination. Though this is true in some instances, critical evaluation of the literature reveals that most microbial eukaryotes are harmless often colonizing the human gut for long periods of time. Evidence is accumulating that the eukaryome plays important ecological roles in gut communities, as well as, in health and disease of the host. Nonetheless, systematic examination aiming to obtain baseline information of their prevalence and diversity in human populations is lagging. To address this knowledge gap, we collected fecal samples from a population of adults residing in north Thailand (n=211), who showed no gastrointestinal (GI) symptoms and had no history of GI diseases. We then examined the prevalence and diversity of two commonly found eukaryotic genera: the stramenopile Blastocystis (Blastocystis spp.) and the yeast Candida (Candida tropicalis and Candida albicans). Twenty three percent of individuals were positive for Blastocystis. Their sequences grouped in six of the nine clades that colonize humans. Twelve percent of the study population was positive for Candida, 4 % for C. albicans and 8 % for C. tropicalis, while concurrent colonization was also noted in some individuals. Eukaryotic bacterial interactions, as well as, interplay with diet and body mass index are also discussed. This is the first study providing data on the eukaryome of Thai populations and evidence that microeukaryotes traditionally considered as pathogenic asymptomatically colonize the gut of healthy humans.
Structural and phenotypic analysis of Chikungunya Virus RNA structures during viral genome replication and translation
Authors: Kendall Catherine , Khalid Henna , Mueller Marietta , Kohl Alain , Merits Andres , Stonehouse Nicola J , Tuplin Andrew
Published: April 2019

Chikungunya virus (CHIKV) is a pathogenic, positive-sense RNA virus of the Alphavirus genus, which causes fever and debilitating joint pain in humans. CHIKV is transmitted by Aedes mosquitoes and is currently re-emerging in the absence of approved vaccines or antiviral therapies. One approach to vaccine production is to attenuate CHIKV replication through disruption of genomic RNA secondary structures. We have mapped the RNA structure of the 5’ region of the CHIKV genome using selective 2’-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. We have identified five highly-conserved RNA structures within the nsP1-coding region of ORF-1 and, using a reverse genetics approach, determined their impact on virus replication in infectious virus and luciferase-reporter subgenomic replicon systems. Our results suggest that RNA structures within the nsP1-coding region are required for efficient CHIKV genome replication in mammalian and mosquito cells, potentially via vertebrate/invertebrate host-specific mechanisms. For example, disruption of one of the stem-loops inhibits CHIKV replication in mammalian cell lines, while having no significant effect in mosquito cells. Restoration of the structure via compensatory silent mutations restores replication – indicating RNA structure-dependent enhancement of CHIKV replication. Conversely, disruption of an adjacent stem-loop inhibits CHIKV replication in mosquito cells but not in mammalian cell lines. Our structural data also suggests that higher-order interactions within this region impact CHIKV replication. As arboviruses continue to re-emerge, it is critical that we improve our understanding of their replication cycles. RNA structures may constitute novel targets for vaccine attenuation or drug design.
Phylogenetics and vector competence of a bovine ephemeral fever virus strain from Israel
Authors: Fernandez de Marco Mar , Hernandez-Triana Luis , Dorey-Robinson Daniel , McElhinney Lorraine , Sanders Chris , Carpenter Simon , Fooks Anthony , Zalensky Olga , Gelman Boris , Erster Oran , Johnson Nicholas
Published: April 2019

Bovine ephemeral fever virus (BEFV) [Family Rhabdoviridae: genus Ephemerovirus] causes a transient febrile illness in cattle that results in economic losses and morbidity, with occasional mortality. Epidemics of the disease have been particularly costly in countries of the Middle East, including Israel. The virus is considered a vector-borne pathogen although the exact relationship with a number of blood-feeding arthropods has not been established. In order to improve developments in diagnostic detection, phylogeographic investigations and virus-vector relationships of BEFV we have derived the first genome of this virus from an isolate from Israel and used this to assess recent outbreaks of disease. We have also investigated the vector competence of BEFV with a number of target arthropod species. The complete genome sequence of BEFV (Israel strain 1) is 14 850 base pairs in length and shows over 95 % identity with the only other Middle Eastern genome for BEFV from Turkey. A wider phylogeny shows that these viruses form a clade, previously described as cluster II, which is clearly distinct from BEFV strains derived from China, Japan and Australia. However, there is a distinct separation of those viruses from Israel to others in the Middle East. Preliminary vector competence studies suggest that at least three species of mosquito that are potential transmission vectors of the virus are incapable of infection with, or transmission of, BEFV. Current studies are ongoing to assess other blood-feeding insect species as potential vectors.
Investigating the nuclear localisation and proteolytic activity of the meningococcal App and MspA autotransporters
Authors: Dutta Shoma , Oldfield Neil , Wooldridge Karl
Published: April 2019

Autotransporter proteins are major secreted virulence factors of Gram-negative bacteria. They are translocated across the inner membrane via the Sec machinery and the outer membrane via the Bam complex and a series of periplasmic chaperones, respectively. The passenger domain may then be proteolytically cleaved and released into the external milieu. The meningococcal autotransporters Adhesion and penetration protein (App) and Meningococcal serine protease A (MspA) are secreted S6-peptidase family autotransporters. Our previous work has shown that FITC-labelled recombinant App or MspA can be taken up by host cells and translocated into the nucleus. App and MspA can also bind to, and cleave recombinant host histones. We furthered investigate the ability of App and MspA (and their inactive derivatives) to cleave recombinant and host-derived histones. Our data demonstrate proteolytic activity of App and MspA on recombinant H3 and Hep-2 cell-derived H3 (which may undergo post-translational modifications that are not applied to the recombinant protein); no cleavage was observed when the histone proteins were treated with proteolytically inactive mutants of the autotransporter proteins. We have also further investigated the nuclear localisation of App and MspA by deleting areas of interest within the meningococcal autotransporters and assessing the impact on nuclear localisation in order to identify the autotransporter motifs required to direct App and MspA to the nuclear compartment. In summary, our results confirm that App and MspA can reach the nuclear compartment of the host cell and clip host-derived histone H3.
Leveraging structural relationships as a novel mode of viral classification
Authors: Magill Damian , Skvortsov Timofey , O’Flaherty Vincent , McGrath John
Published: April 2019

100 years have passed since the independent discovery of the humble bacteriophage (phage) by Frederick Twort and Felix d’Herelle in 1915 and 1917 respectively, and since then, it has become commonly accepted that phages represent the most abundant biological entities on Earth. Despite this fact, viral taxonomy lies in extremely treacherous waters, ever changing to accommodate the next series of phylogenetic mysteries. The utilisation of genes such as the terminase large sub-unit can in some cases provide a robust taxonomic marker, but this is often found to fail at higher taxonomic levels. In addition, the rapid evolutionary dynamics and highly modular nature of phages provide yet more phylogenetic roadblocks, necessitating additional and multifaceted approaches as a means of resolution. Here, we describe a novel approach towards the taxonomic classification of phage systems. Tools for accurately predicting the three dimensional structure of proteins are improving at an unprecedented rate due to the fact that the number of protein sequences far exceeds the number of experimentally determined structures. Our approach leverages these methods through a pipeline which compares models of phage marker genes in order to permit the inference of phylogenetic relationships based on cross model superimposition. We hope this method will supplement other approaches in providing a more holistic approach to viral classification.