- Volume 2, Issue 7A, 2020

Pseudomonas 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.

Documented 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.

Non-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.

The 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.

Background: 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.

Bacteria 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.

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.

The 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.

M. 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.

Infectious 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.

Salmonella 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.

High-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.

We 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.

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.

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.

We 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.

The 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.

Recognition 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.

The 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.