- Volume 1, Issue 1A, 2019

Modern medicine is under the excruciating pressure of drug resistant bacterial strains which are ever advancing with the introduction of every new class of antibiotics. Traditional bactericidal and bacteriostatic drugs, while effective in eliminating the susceptible bacterial strains, also impose a selective pressure on bacteria which often leads to the emergence of antimicrobial resistance. An alternative approach is the development of anti-virulence therapies, which aims reduce bacterial pathogenesis while avoiding the selective pressure of classical antimicrobial inhibitors, thus rendering bacteria harmless and potentiating natural elimination from the host by innate immunity defence mechanisms. We have synthesised a selection of functional polymers of poly(acryloyl hydrazide) using a panel of aldehyde functionalisation groups and evaluated their anti-virulence properties on both Mycobacterium bovis BCG and Mycobacterium smegmatis mc2 155, two surrogate organisms to study Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis. Using a combination of microscopy and in vitro studies, we have shown the effectiveness of anti-virulence polymers in reducing mycobacterial phagocytosis in J774 macrophages with minimal antimicrobial activity.

Triazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes – previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. VIPcheck™ plates provide a simple agar based screening method. Each 4-well plate contains a growth control (GC) well and 3 wells containing itraconazole (4 mg l−1), voriconazole (2 mg l−1) and posaconazole (0.5 mg l−1). Briefly, 25 µl of a 0.5-2 McF suspension AF is inoculated into each well and plates are read after 48 h incubation at 37 °C. Any growth in a triazole containing well is suggestive of resistance. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints. We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples. A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=18 resistant to ≥1 triazole drug as previously determined by AFST and/or molecular methods). The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells. Our results suggest that the VIPcheck™ plate is a reliable screening method for triazole resistance.

Antimicrobial resistance (AMR) poses an ever-increasing threat to public health; the prevalence of resistant bacterial strains has reduced the clinical efficacy of many existing therapeutics and is therefore contributing to rising mortality rates due to difficult to treat bacterial infections. Two key approaches used to mitigate the threat of AMR are the discovery of novel therapeutics with activity against these resistant strains, and educating the wider public about the impact of AMR, and steps that can be taken to reduce the development of resistance. We are combining both approaches to enhance the impact of our public engagement activities. During a recent event at the University of Plymouth, a member of the public isolated the bacterial strain ‘36A’ from the button of a lift control panel. Simultaneous antagonistic screening identified antimicrobial activity against a range of both Gram-positive and Gram-negative bacteria. 36A was then subjected to draft genome sequence determination via the MinION platform (Oxford Nanopore). Growth media were optimised to enhance antimicrobial activity, with fermentation in LB broth and subsequent purification of the culture supernatant via multi-stage column chromatography resulting in the isolation of four putative antimicrobial compounds. Initial characterisation has shown that each compound has a peptidic component, all showing stability and potency at a relatively low concentration against MRSA, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Structural characterisation has been carried out using mass spectrometry, with further characterisation and cell toxicity studies ongoing. The producing strain has been identified as a member of the Paenibacillus genus.

Early diagnosis and treatment of sepsis is one of the biggest challenges to ICU clinicians. Globally, 19 million cases occur annually and it is the third biggest cause of death in the UK. Sepsis is characterised by an uncontrollable, non-specific immune response to an infection, and as a result is difficult to diagnose. Recent research has found that 25-hydroxycholesterol (25-HC) plays a crucial role in the immune response to viral infection. Less is known about the role of sepsis-associated bacteria in this response. To identify novel biomarkers in bacterial sepsis a whole blood model was used and the cellular and molecular responses measured to well-characterised bacteria (Escherichia coli K12 and Staphylococcus epidermidis RP62A) using flow cytometry, ELISA and high performance liquid chromatography-mass spectrometry (LC-MS). Following bacterial infection, mononuclear cells and granulocytes decrease rapidly in response to both K12 and RP62A. This corresponds to a concomitant increase in total CD45 and CD19 expression and the concentration of the proinflammatory cytokines IL-6, CCL3 and CCL20. Proinflammatory responses were significantly more pronounced in K12 infection. There were significant increases in 25-HC in response to K12 infection, and this effect was partially blocked through inhibition of TLR2 or TLR4. Our results suggest the importance of using both cellular and humoral screening to identify unique pathways induced by sepsis causing bacteria. In addition, the current study provides some of the first evidence that 25-HC may be involved in a bacterial driven immune response. This study has importance when designing novel biomarkers to predict sepsis.

Ventilator-associated pneumonia (VAP) is amongst the most common healthcare-associated infections worldwide. Current understanding of the underlying mechanisms has focussed on either the microbiological or physiological elements but host-microbe interactions, which are instrumental in pathogenesis, have received less research focus. This work aims to explore clinically relevant and reproducible models to investigate these interactions in the context of VAP. A clinical isolate of Pseudomonas aeruginosa, a pathogen common in VAP, was investigated in systems of increasing complexity. Sensitivity to key antibiotics (LVX, MEM, and TZP) used for treatment of VAP was unaffected by the presence of cytokines (IL-1β, IL-6, and TNFα) in vitro. Larvae of Galleria mellonella, an in vivo insect model with a rudimentary immune system, was used to test virulence of P. aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. P. aeruginosa killed 100 % of larvae within 24 h. S. aureus and K. pneumoniae killed 43.5 % and 50 % of larvae respectively, within 8 days. An ex vivo mammalian model was developed, which demonstrated abundant P. aeruginosa proliferation on lung tissue. After validating inactivation of host lung tissue, we identified changes in the expression of P. aeruginosa quorum sensing genes LasI and RhlI, specifically induced by host interaction. Our results suggest that host factors may influence bacterial growth and gene expression. We will use these early data to validate and expand our models prior to investigation of clinical samples from VAP patients. We will report our most recent findings in the development of clinically relevant models to investigate VAP.

Melioidosis is caused by the Gram-negative, intracellular bacterium Burkholderia pseudomallei. The epidemiology of the disease is unclear- it is limited to tropical regions, where monitoring can be sparse and further complicated by the multifaceted nature of the condition. The most common indication of acute melioidosis is pneumonia (51 %) that can progress to acute fulminant sepsis with multifocal infiltrates with high rates of mortality. Meliodosis has been estimated to cause 89 000 deaths annually with an estimated mortality rate of approximately 50 %. Due to ease of the dissemination of the pathogen, its ability to form biofilms, the high degree of antimicrobial resistance and paucity of effective treatments, controlling the infection is a major challenge. Our approach is to develop nanoparticles capable of effectively delivering antimicrobials to biofilms formed by Burkholderia species. Formulations of a proprietary lipidic delivery agent, CM2, have been tested against two species: B. cepacia UCB717 that has no capsule and B. thailandensis (that is used as a surrogate for B. pseudomallei), including strains with and without capsules both of which readily form biofilms. Confocal laser scanning microscopy was used to monitor the uptake of the nanoparticles and the MIC and MBEC of CM2 alone (and analogues), in combination with a panel of antimicrobials and a novel oligonucleotide antimicrobial, termed a Transcription Factor Decoy (TFD), were measured. The most efficacious combinations will be formulated for delivery by inhalation prior to testing in animal models.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis with over 550 million cases reported yearly. The World Health Organization has listed C. jejuni as one of 12 microorganisms on a global priority list for antibiotic resistance due to a rapid increase in the number of strains resistant to fluoroquinolone antibiotics. This fluoroquinolone resistance is conferred through a single point mutation in the QRDR region within the gyrA gene which is also involved in DNA supercoiling homeostasis. We recently revealed that changes in DNA topology play a major role in the regulation of virulence in C. jejuni with relaxation of DNA supercoiling associated with increased attachment to and invasion of human epithelial cells. The aim of this study was to investigate whether fluoroquinolone resistant strains of C. jejuni displayed altered supercoiling associated phenotypes. A panel of mutants were derived against nalidixic acid and ciprofloxacin and shown to have a greater ability to form viable biofilms under aerobic conditions and that this phenotype was associated with changes in DNA supercoiling levels. These mutants were also shown to have an increased ability to attach to and invade epithelial cells in vitro and conferred an increase in the killing efficiency of Galleria mellonella. We report for the first time that fluoroquinolone resistance in C. jejuni is associated with an increase in virulence and the ability to form viable biofilms in oxygen rich environments. These altered phenotypes may play a critical role in the continued increase in fluoroquinolone resistance observed for this important pathogen.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen with a high mortality and morbidity rate. It has many mechanisms of resistance to antibiotics, which makes it hard to treat. Pyocin is a protein that is produced by P.aeruginosa that kills related strain bacteria. pyocin type S bind to ferrisiderophore receptors that uses the TonB system to translocate into the bacterial cell. The aim of this study was to express and purify receptor and translocation (R+T) domains of pyocin S1, S2 and S3 plus the TonB1 receptor and ToLAIII receptor of P.aeruginosa to determine if pyocins S uses these to transverse the inner membrane of target cells. IPTG was used to induce the protein in the expression and analyzed by SDS-PAGE gel, giving fragment size of 11 kDa (ToLAIII), 23 kDa (TonB1), 45 kDa (S2 R+T) and 79 kDa (S3 R+T). Protein purification was cried on the four proteins using affinity chromatography technique by His-tag in C-terminal of S2 and S3 R+T domain, N-terminal of TonB1 and ToLAIII proteins. In addition, Gel Filtration chromatography was used to further purify the proteins so that the interaction between them could be tested. This study confirmed that the proteins used is expressed and purified, so in the future study the gel filtration would be carried out for R+T domains of the other S pyocins and TonB1 protein so that the interaction would be tested between the proteins in the study.

The microbiome-gut-brain (MGB) axis is a bi-directional route of communication that exists between the brain and the microbes that reside in the gut. The MGB axis is becoming of increasing importance as significant alterations in the gut microbiota are now linked to numerous neurological conditions, however, little is currently known about the microbiome derived mediators of communication. Here we used mass spectrometry imaging (MSI), a label free imaging technique, to identify bacterial products that cross the blood brain barrier in specific pathogen free (SPF) mice. We identified two bacterial molecules abundant in white matter regions of the murine that were absent in the brain and gut in germ free (GF) mice. We have identified the primary gut microbial producers of these metabolites to be members of the Lachnospiraceae family. Both molecules were found to be structurally similar to carnitine and localise with carnitine in the SPF mouse brain. Using a primary murine cell culture model of the central nervous system white matter we show that these molecules are capable of significantly impairing mitochondrial basal respiration. Given their systemic presence in the mouse and their presence in human biological samples, these metabolites may have significant implications for diseases associated with mitochondrial dysfunction and an altered gut microbiota. These results are the first to describe a direct molecular inter-kingdom communication between prokaryotes and the mammalian brain that can facilitate functional inhibition in mammalian brain cells.

The 2013–16 epidemic of Ebola virus disease underscored the shortcomings of the international community to both respond to outbreaks and conduct critical research in complex humanitarian crises. To address these concerns, the UK Government has formed the UK Public Health Rapid Support Team (UK- PHRST). The UK-PHRST is a collaboration between Public Health England and the London School of Hygiene and Tropical Medicine with the University of Oxford and Kings College London as academic partners. The UK-PHRST has a novel triple mandate to work in low- and middle-income countries (LMICs) to:

• Respond to outbreaks
• Conduct innovative operational research during and between outbreaks to generate evidence on best practices
• Build LMIC and regional capacity for outbreak response

Antimicrobial resistance is a major global healthcare challenge. Beyond the discovery of novel antimicrobial agents, the development of novel formulations for enhancing current antibiotics is a promising strategy to reduce the rate of treatment failure. Drug delivery nano-carriers can achieve high local concentration of antimicrobial agents, reduce toxicity, and improve biodistribution and pharmacokinetics. We have taken two approaches to enhance antibiotic delivery and effectiveness. Firstly, we used a bespoke targeted liposomal system for intracellular antibiotic delivery to phagocytic cells. This enables treatment of an intracellular Gram-negative infection with a cell-impermeable antibiotic. Targeted liposomes were found to significantly enhance uptake compared to uncoated liposome control formulations both in in vitro and in vivo (zebrafish model). Secondly, liposomal nanoformulations were utilised to deliver peptide antibiotics, where liposomes protect peptides from degradation, and allow potential co-delivery of combination therapeutics. We investigated different liposomal formulations and drug combinations against E. coli and S. aureus. We show that peptide-loaded liposomes are more efficient compared to free drug in inhibiting the growth of both Gram-negative and Gram-positive bacteria. These initial results suggest that liposome-mediated delivery can be utilised for the repositioning and repurposing of existing antibiotics, potentially allowing for the treatment of diverse infections in a more effective manner.

Antibiotic resistance is a major global health problem. Preservation of antibiotics, underpinned by the availability of novel antimicrobials that avoid the emergence of antimicrobial resistance and antibiotic cross-resistance is an important societal goal. We have developed a novel biocidal complex (iodo-thiocyanate complex or ITC), drawing the inspiration from naturally occurring peroxidase-catalysed systems. This study was aimed to reveal the potential of ITC for induction of resistance and cross-resistance, and, thus, different aspects of resistance were explored. We show that the repeated exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus to sub-inhibitory levels of ITC during serial passage of batch cultures did not generate ITC resistance. By comparison, E. coli and S. aureus developed low-level and high-level resistance to levofloxacin (LVX), respectively. Further, we attempted to generate de novo resistance in antimicrobial-sensitive E. coli during 20 days of continuous culturing when exposed to gradually increasing concentrations of ITC and LVX. The exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based analysis of ITC-challenged bacterial populations. By contrast, the resistance to LVX was rapidly induced, selected for high-level and enriched with a distinctly characteristic genome mutational pattern. WGS of LVX-challenged population revealed that the majority of mutations appeared in the genes of LVX target proteins and drug influx. This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.

Streptococcus pneumoniae (S.p.) and Respiratory Syncytial Virus (RSV) are two major pathogens commonly found to coexist in respiratory secretions in patients with acute respiratory infection. Though there is increasing evidence of a synergistic interplay between these two pathobionts, the exact mechanisms remain obscure. The aim of our study was to decipher how coinfection with RSV alter pneumococcal growth dynamics and host immune response and how this impact on the colonisation and invasive properties of S.p. Using in vivo mouse model, we made the key observation that upon coinfection with RSV, the density of pneumococcal colonisation in the nasopharynx and dissemination to the lower respiratory tract were significantly higher in mice previously colonised with S.p. These mice also presented more severe weight loss and delayed recovery compared to mono-infected animals as well as significantly heightened pro-inflammatory cytokine profiles. Measurement of in vitro transepithelial electrical resistance (TEER) showed that, upon RSV coinfection, S.p. transmigrate through the epithelial barrier without altering epithelial integrity suggesting a transcellular mechanism rather than paracellular migration. Moreover, RSV-pneumococcal coinfection of human primary nasal epithelial cell demonstrated major changes in host protein expression involved in the catalytic activity, ubiquitination, cytoskeletal organisation, and endocytosis. Simultaneously, significant upregulation observed in bacterial proteins involved in the ribosomal activity, streptococcus-induced tissue inflammation, DNA supercoiling, and bacterial viability during oxidative stress, affecting both the survival and the virulence of S.p. Our results explain the complex interactions between pneumococci, RSV and host and help towards further understanding the significance of viral-bacterial co-infection in clinical settings.

Asymptomatic Bacterial Vaginosis (BV) damages vaginal epithelium increasing risk of sexually-transmitted infections. Gardnerella vaginalis, Atopobium vaginae, Prevotella amnii, P. bivia and Candida albicans are associated with BV. Presence of Lactobacillus spp. is indicative of a healthy microbiota. Symptomatic BV is treated with metronidazole. The role of microbiota and metronidazole treatment in the recurrence and persistence of asymptomatic BV remains to be elucidated. This study uses whole genome sequencing (WGS) to determine the microbiota changes with metronidazole treatment. DNA from 20 vaginal swabs was obtained at four time points over 12 months from five African American women and was subjected to WGS. The first time point is the untreated baseline. All subjects were tested every 4 months and received a course of metronidazole for each episode of BV during the 12 months period. Nugent scores were used to classify BV status. The microbial profiles were analyzed along with the sociodemographic metadata. Despite treatment, the participants did not recover from BV — two participants experienced persistent BV, and the rest had recurrent BV. WGS analyses show that G. vaginalis was the most abundant organism as compared to Lactobacillus species. The metronidazole treatment resulted in the loss of Lactobacillus and Prevotella species. One participant scored healthy based on Nugent score at one time point, during when Lactobacillus species dominated the microbiome. Based on this pilot longitudinal study, metronidazole may not be an effective treatment for asymptomatic BV. Studies with larger cohorts can lead to statistically significant conclusions to develop alternative interventions for asymptomatic BV.

Chronic wounds affect approximately 2 % of the worldwide population and incur healthcare costs in the billions. Key to their persistence is the formation of microbial biofilms, which are accounted for in nearly 80 % of all non-healing wounds. The smart dressing presented herein aims to detect a range of volatile infection protagonists, with a striking colour change that can be visualised with the naked eye, providing 24/7, non-invasive monitoring of infection development and antimicrobial treatment efficacy. A range of coloured indicator films housing dyes responsive to volatile analytes in the wound headspace were developed and tested against porcine skin inoculated with Pseudomonas aeruginosa. Digital images of the indicator film were captured at regular time intervals and the resulting images were aligned and split into red, green and blue (RGB) colour channels to yield semi-quantitative data. vAPCI-MS was exploited to identify additional volatiles for incorporation into the smart dressing design. A CO₂-sensing film comprising xylenol blue dye underwent a marked colour change from blue to yellow within 12 h of inoculation with PAO1, whilst indicators monitoring uninoculated control skin remained blue (no colour change). In addition, vAPCI-MS identified putrescine as an additional volatile of interest, and responsive indicator films were developed for its detection. The marked colour change exhibited by each indicator film is easily visualised by eye and can be digitally analysed to provide semi-quantitative data. This early warning, point-of-care technology is a promising candidate in combatting biofilm development in wounds.

Bacterial infection and corrosion are the two of the most significant causes of metallic implant failure. In our study, we innovated a facile two-step approach to synthesising a TiO2-PTFE nanocomposite coating on stainless steel, which endows the implant surface with both antibacterial and anticorrosion properties. By harnessing the adhesion and reactivity of bioinspired polydopamine, the TiO2-PTFE coating was uniformly deposited onto substrates by using a sol-gel dip coating technique. The TiO2-PTFE coating exhibited minimal bacterial adhesion against both Gram-negative Escherichia coli WT F1693 and Gram-positive Staphylococcus auerus F1557. Moreover, it was observed that an increasing TiO2 concentration in the bath enhanced antibacterial activity. Benefiting from the synergistic effect between TiO2 and PTFE, the TiO2-PTFE coating showed improved corrosion resistance in artificial body fluids comparing with the sole TiO2 and PTFE coatings. The TiO2-PTFE coating also demonstrated extraordinary biocompatibility with fibroblast cells in culture, making it a prospective useful strategy to overcome current challenges in the use of metallic implants.

Thioridazine (TZ) is an antipsychotic drug that acts against antibiotic resistant bacteria. The main aim of this study was to uncover the mechanism of action of TZ using Salmonella enterica serovar Thypimurium as a model bacterium. The antibacterial activity of TZ was initially determined based on its minimum inhibitory concentration (MIC). Membrane permeability assays were performed and fluorescence measured using the Ethidium Bromide accumulation assay. Salmonella was exposed to TZ and its effects on membrane potential and cell wall assessed by flow cytometry and Transmission Electron Microscopy, respectively. Effects on the bacterial proteome were assessed through 2D gel electrophoresis. Infection assasys were performed in THP-1 ad RAW 264.7 cells treated and non-treated with TZ. The MIC of TZ against Salmonella was 200 mg l−1. Our in vitro data demonstrates that TZ mechanism(s) of action involves primarily Salmonella’s membrane by affecting its permeability and potential after 15 min of exposure to TZ. At half of the MIC, and only after 15 min, TZ disrupts the bacterial membrane leading to leakage of the cellular contents and lysis of Salmonella. Proteomic profiling revealed 75 upregulated and 62 downreuglated proteins. Infected macrophages treated with sub-MIC of TZ, showed a reduction on intracellular c.f.u./mL. This may be indicative of TZ’s ability to enhance the killing activity of infected macrophages. The results obtained suggest that TZ may act in vitro by targeting the bacterial cell-envelope. Due to its effect on infected macrophages, TZ may be considered a useful adjuvant to current therapeutics.