- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Infection Forum
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Augmenting Staphylococcal infection: the importance of timing
More LessStaphylococcus aureus (S. aureus) acts as a commensal in the microbiome of the skin and nasopharynx. However, on gaining access to the bloodstream it can cause an array of pathogenic outcomes. S. aureus can crowdsource the microflora to assist in becoming an opportunistic pathogen as our lab has recently published findings that co-inoculation of S. aureus with commensals, acting as pro-infectious agents, leads to a much more robust, virulent infection. This benefits S. aureus at doses where it would otherwise be cleared by the immune system. Pro-infectious agents do not need to be live commensals as isolated cell wall peptidoglycan also augments infection. This work aimed to assess the effects of inoculation with pro-infectious agents before and after infection with S. aureus. It was found that pro-infectious agents needed to be co-administered in order to fully augment infection. This gives mechanistic insight where S. aureus and the pro-infectious agents need to be in the same local environment or phagocyte to augment infection.
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Clinical evaluation of the novel molecular bacterial load assay for real-time monitoring of tuberculosis treatment response
More LessBackgroundMonitoring the treatment of tuberculosis (TB) relies on less sensitive smear microscopy (SM) and culture methods which are very slow. We evaluated the novel Molecular bacterial load assay (MBLA) for implimentability and real-time monitoring of TB treatment in a clinical setting.
MethodsTherapy naive (Xpert MTB/RIF confirmed) TB positive patients were enrolled in Mbeya, Tanzania. Sputum samples were collected at baseline and thereafter at week 2, month 2, 5 and 6 of treatment. Samples were analysed for M. tuberculosis (M.tb) by MBLA and compared to SM, culture and clinical monitoring.
Results59 TB patients were enrolled for the study. Median age, 37 (18-65) years, 62.7 % (37/59) male, 45.6 % (27/59) HIV positive and 8.47 % (5/59) were re-treatment. Mean BL (± SD) at baseline was 5.48 ± 1.3 declining to 3.42 ± 0.7 at month 2 and 3.51 ± 0.62 log10CFU/ml at month 6 of treatment. This corresponds MBLA positivity of 92.98, 65.5 and 7.84 % at baseline, month 2 and 6 respectively. In contrast, positivity of SM and culture were 78.95, 9.62 and 0 %, and 85.96, 25 and 3.39 % at baseline, month 2 and 6 respectively. Decline in test positivity reflected resolution of clinical signs. While night sweat, and chest pain resolved earlier on in treatment, resolution of cough was slow and consistent with MBLA. Furthermore, the turn-around-time for MBLA results was 24 h compared to median (range) of 14.83 (4.33–42) days for liquid culture.
ConclusionMBLA exhibited higher sensitivity and shorter turn-around-time than standard tests and clinical signs. This demonstrates the potential of MBLA to offer real-time results for clinical decision making.
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Mapping 4D pH evolution in Streptococcus mutans biofilms using fluorescent ratiometric pH-sensitive nanosensors
More LessStreptococcus mutans is a pre-dominant bacterial species found in oral biofilms and participates in the production of dental caries via the generation of organic acids. The production of these acids results from the fermentation of carbohydrates present in a sugar-laden diet. As the acidity of an oral biofilm decreases, the demineralisation of the enamel of a tooth increases; leading to the formation of dental caries. To detect and measure the pH change occurring following a sugar challenge, ratiometric, fluorescent, pH-sensitive nanosensors were incorporated into oral biofilms. Confocal laser scanning microscopy revealed that the addition of glucose (1 % w/v) to an S. mutans biofilm resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was being fermented, resulting in the production and secretion of acids into the extracellular matrix of the biofilm. Additionally, a reduction in pH was detected – using widefield microscopy – in starved, planktonic S. mutans when treated with glucose. Over the course of 30 min, the pH of the medium was reduced from pH 5.3 to pH 3.3 as the glucose was fermented by the bacteria. These findings will help us map pH changes in oral biofilms as we examine potential methods of preventing the acidification of oral biofilms and the eventual demineralisation of the enamel; leading to the reduction in dental caries and an improvement in the standard of living of those effected.
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The O6 antigen of Escherichia coli strain CFT073 is a target for Myoviridae
More LessExtraintestinal pathogenic Escherichia coli strain CFT073 is a prototypic urosepsis isolate. CFT073 is of serotype O6:K2:H1 and thus has an O-antigen. The wzy gene encodes the O-antigen polymerase Wzy, which catalyses the polymerisation of O subunits into a long chain polysaccharide. A CFT073 Δwzy mutant was constructed using the λ Red recombination system. The lack of O-antigen was confirmed by LPS purification and staining with Pro-Q Emerald 300. CFT073 Δwzy and wild-type CFT073 were tested for their bacteriophage sensitivity. ΦEB49, a member of the Myoviridae, can mediate generalised transduction in strain CFT073. CFT073 Δwzy and wild-type CFT073 mixed with the same concentrations of ΦEB49 phage were compared. Confluent growth was evident on all CFT073 Δwzy plates, whilst lysis was apparent on all CFT073 plates. The inability of ΦEB49 to lyse CFT073 Δwzy suggested that this phage binds to O antigen, hence the decreased O antigen of CFT073 Δwzy, compared to wild-type CFT073, prevented phage lysis from occurring. These data indicate that CFT073 Δwzy is not a susceptible host for ΦEB49.
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A micro luminescence-based assay to measure serum susceptibility in Escherichia coli
More LessNeonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.
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Intracellular replication of pneumococcus in ex vivo-perfused human spleens
Recently, we have demonstrated that the pathogenesis of Streptococcus pneumoniae bacteraemia contains a concomitant phase of intracellular replication within splenic macrophages in mice (Ercoli et al. Nat Micro. 2018). In the present study, we aimed to determine if intracellular replication of pneumococci may play a role in the pathogenesis of sepsis in humans, using two innovative approaches. We used a model (Chung et al. ALTREX.2018) involving ex vivo perfusion of human spleens from elective splenectomy patients (REC reference: 18/EM/0057). Organs were infected with 6.5×107 c.f.u. of pneumococci, and serial biopsies and ‘blood’ samples were taken at predetermined times. Samples were analysed by colony counts, confocal microscopy and flow cytometry. Additionally, infected tissue samples were taken for preparation of organotypic slice culture time courses. Bacteria injected into the perfusion circuit were rapidly cleared at early time points post-infection, recapitulating what is observed in experimental murine sepsis. Bacterial counts in the spleen increased, providing initial evidence of intracellular bacterial persistence. Microscopy analysis indicated that bacteria could be localised to splenic macrophages, with the size of infectious foci increased over time. Z-stack microscopy localised bacteria within cell membranes, indicating the infection was predominantly intracellular. In ex vivo slice cultures increasingly large numbers of pneumococci were cultured over time, further indicating intracellular replication. In conclusion, we provide evidence for a role of intracellular replication of pneumococci in human splenic macrophages in the pathogenesis of sepsis.
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Characterization of a bacteriophage from avian Staphylococcus aureus associated with innate immune evasion
More LessStaphylococcus aureus is an important human and livestock pathogen. An S. aureus phage (φAvβ) inserted into the chromosome at the beta toxin gene (beta-converting phage) is present in approx. 90 % of human strains and contributes to human-specific innate immune evasion. Comparative genomic analysis of S. aureus isolates from infected poultry has revealed an avian-specific subfamily of beta-converting phages represented by multiple variants with distinct integrase gene alleles. To investigate the role of the avian beta-converting phages in host-pathogen interactions, a φAvβ-deficient strain with non-functional beta-toxinand a φAvβ-deficient strain with restored beta-toxin were constructed by allele replacement in an avian pathogenic S. aureus strain. Compared to the wild type, both φAvβ-deficient strains have reduced net extracellular growth in vitro in chicken bone-marrow derived macrophages. Further investigation using GFP-tagged bacteria has revealed that both φAvβ-deficient strains show reduced initial phagocytosis and intracellular survival compared to the wild type. Absence of φAvβ is also associated with decreased killing of the chicken bone-marrow derived macrophages. We are currently investigating the mechanism underlying this phenotype using deletion mutants of the candidate phage effector genes. Ongoing work also involves using RNAseq to investigate differential host transcriptional response of the macrophages to S. aureus in presence/absence ofφAvβ. Overall, these data will contribute new information relating to the evolution of avian S. aureus and mechanisms of bacterial host-adaptation.
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Improved molecular typing of toxigenic Clostridium difficile strains affecting animal and human health
More LessClostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.
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Detection and quantification of Staphylococcus aureus heterogeneity to identify antibiotic-induced persistence
More LessPersister cells are characterised as being viable but non-culturable, a state that preserves their metabolic energy to survive the environmental stress, which allows for recurrent infections. Detection of persisters is, therefore, not possible with standard culture-dependent methods. Furthermore, the effect of antibiotics on the induction of persisters has not been assessed. This study aimed to identify antibiotic-induced persistence and determine the percentage of heterogeneity. Vancomycin, daptomycin and dalbavancin were assessed by standard MIC methods against selected Staphylococcus aureus strains. Replicates of MIC assays were stained with propidium iodide to quantify live/dead and a reactive oxygen species (ROS) dye to detect and quantify persisters using culture-independent single-cell sorting, independently. A comparative analysis was then performed. Dalbavancin showed the lowest MIC values against tested S. aureus strains followed by daptomycin and vancomycin. Cell sorting of vancomycin-, daptomycin- and dalbavancin-treated S. aureus strains showed a range of 1.9–10.2 %, 17.7–62.9 % and 7.5–77.6 % live cells based on the strain, respectively, in which daptomycin, in particular, was a strong inducer of a persister population. Persisters represented 3.7–16 % of the bacterial population. The culture-independent identification of antibiotic-induced persistence through studying at the single-cell level showed different efficacy of antibiotics than standard MIC. Vancomycin was the most effective antibiotic against tested strains followed by dalbavancin then daptomycin as assessed by cell sorting. Therefore, re-evaluation of standard MIC methods may be required to assess the efficacy of antibiotics. Additionally, the detection of daptomycin-associated persisters may provide an elucidation to the reported rapid resistance development in vivo.
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In vitro antimicrobial efficacy of Cnidoscolus aconitifolius leaf extract and honey on Staphylococcus epidermidis clinical isolate
More LessThe search for alternative, potent, cost-effective treatment of ailments caused by resistant microorganisms and the role of plants and their products as essential sources of medicinal agents is receiving increasing attention. Several of these natural products are reported to have capacity to produce natural compounds of high structural diversity that serve as defense agents against invading microorganisms. Cnidoscolus aconitifolius also known as tree spinach is an indigenous tropical tree that has gained lot of importance in its nutritive value and traditional use. This study evaluates the possible effect of Cnidoscolus aconitifolius leaf extract alone, extract/honey combination on Staphylococcus epidermidis clinical isolate. Phytochemical analysis and antimicrobial efficacy of the extract were performed using standard methods. The results of phytochemical analysis reveal the presence of carbohydrate, tannins, alkaloids, steroids, flavonoids, anthraquinone, saponins and carotenoids. Antimicrobial activities showed inhibition zone values of 8.0±0.1 mm for aqueous extract alone, 9.0±0.1 mm for aqueous extract/honey combination. The finding suggests that C. aconitifolius might be a good source of compounds that can be used to inhibit the growth of Staphylococcus epidermidis pathogen and further supports its popular and wide traditional applications in the treatment of various illnesses. Hence the need for further research to exploit the full potential of C. aconitifolius tree in order to influence their extensive consumption, storage, improvement and production.
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Novel antimicrobial agents for clinical applications
More LessSince their discovery, antimicrobial compounds have been vital for the treatment and prevention of disease; making many previously fatal diseases treatable or at worst, manageable conditions. The inappropriate use of these compounds has led to the rapid development of resistance mechanisms within bacteria to the majority of compounds currently marketed. A recent UK governmental review predicted that by 2050 global deaths caused by antimicrobial resistant bacteria will outnumber those attributed to cancer [1]. As new resistance mechanisms emerge and resistance within microbial populations increases, so does the need to further understand the molecular basis of resistance, develop new antimicrobial molecules and use better strategies to manage their use [2]. In response to this, we discovered a novel class of antimicrobials and have created 50 structurally related members of this class [3-6]. We sought to understand the structure-activity relationships which will result in the determination of the mode of action of these molecules. Consequently, each variant was screened against Staphylococcus aureus and Escherichia coli and the minimum inhibitory concentration was calculated for effective compounds. This will enable us to identify predictive tools that will aid the synthesis of the next generation of these novel therapeutic molecules. We will present our latest findings in the ongoing analysis of the antimicrobial activity for each variant of this new class of antimicrobial compound. In addition, we will discuss the insights provided by the detailed structure-function analysis. This project is in collaboration with Public Health England and NHS East Kent Trust.
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Prevalence of extended spectrum beta-lactamase producing Enterobacteriaceae in urine samples from Thumbay hospitals, U.A.E
Beta Lactamases is proven to be one of the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. Many up to date researches have shown increase in the incidence and prevalence of ESBL worldwide. This study aimed to determine the prevalence of ESBL strains of Klebsiella spp. and Escherichia coli species in urinary isolates from the patients admitted in Thumbay hospitals around United Arab Emirates. Furthermore, drug resistant genes (SHV and CTX-M) in the ESBL positive samples were detected. 237 urine samples were collected from November 2017 to January 2018. Based on the lactose utilization, colony morphology, and biochemical utilization of the gram negative bacilli were identified as E. coli (53), Klebsiella pneumoniae (10) and Citrobacter species (2). Antibiotic sensitivity test, double disc diffusion test and combination disc tests all confirmed that the 65 (27.4 %) out of 237 isolates were ESBL producing bacteria. There was high prevalence of bacteria in females than male and the number of E. coli strains is higher than Klebsiella spp. DNA isolation was performed on the 65 samples, out of which 50 samples were selected for PCR based on their concentration. The selected DNA samples were used to detect the presence of bla CTX-M and bla SHV genes. Only 24 DNA samples (48 %) contains blaCTX-M genes, bla SHV or both the genes. 14 samples had bla CTX-M gene, 2 bla SHV genes, and 8 with both bla SHV and bla CTX-M. At the rate at which ESBL is spreading, further research, close observation and cautious use of antibiotics is important.
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Harnessing novel bacterial peptides for antimicrobial activity in the gut microbiome
More LessThe Enterococci are a resilient collection of species, found in the human intestine, river sediment and even certain cheeses. Human infection by this genus is dominated by E. faecalis and E. faecium. Vancomycin resistant enterococci (VRE) are associated with higher mortality rates over non-VRE strains. Enterococci can utilise the highly efficient pheromone responsive plasmid (PRP) system to transfer plasmid DNA between cells. Plasmid containing donor cells respond to small peptide pheromones (7–8 amino acids) and transfer plasmid DNA to pheromone-producing plasmid-free recipient cells. PRP can encode antibiotic resistance (including vancomycin) and virulence enhancing factors. Investigation into the PRP system between donor and recipient E. faecalis environmental isolates has indicated a 40 % decrease in PRP transfer in colder environments. Additionally, PRP efficiencies under other conditions, including in presence of synthetic pheromone peptides, have been calculated. Future assays will utilise pheromone imitative fluorescently labelled synthetic peptides to visualise the pheromone binding receptor (PrgZ) on the E. faecalis donor cell membrane. Later experiments will focus on varying the synthetic pheromone amino acid composition so to interfere with the PRP system machinery, with the aim of reducing PRP transfer efficiency or preventing PRP transfer completely.
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Application of furanone compounds for the modulation of biofilm formation in common wound pathogens
More LessChronic 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.
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The effect of local release antibiotic beads on in-vitro bacterial growth from tissue taken from infected diabetic foot ulcers
More LessDiabetic 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.
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Epidemiological characteristics of the Manchurian plague pandemic of 1910-1911
More LessObjectiveTo investigate the epidemiological characteristics of the Manchurian plague pandemic of 1910–1911 with descriptive epidemiological methods.
MethodsThe epidemiological data were distracted from Report of Manchurian Plague, which is a summary of local official reports about plague in 1910–1911. The time distribution by day, and the space distribution by country was recorded, and also the source of infection.
ResultsThe pandemic of plague continued from October 25th, 1910 to April 18th, 1911. There were 46 747 dead cases on record in the three provinces of Manchuria. There were 24 867 dead cases and 7068 dead cases were reported, and the average mortality rates were 41.21 and 10.25 per ten thousands in Jilin and Liaoning province, respectively. In Heilongjiang province, 14 812 dead cases of plague were reported. The huge difference was found in different epidemic regions, the highest mortality rate was 4121 per ten thousands in Binjiang country of Jilin province. Patient zero of pneumonic plague had been infected in Russia and got sick and died in Manzhouli, a northern country in Heilongjiang province. Then the pneumonic plague was mainly spread through railway to other cities.
ConclusionThe epidemiological characteristics of the Manchurian plague pandemic of 1910–1911 were first described with modern epidemiological methods.
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Detection and characterisation of bacteria causing lung infection in people with Cystic Fibrosis (CF) by surface-enhanced Raman spectroscopy (SERS)
More LessRapid and accurate identification of pathogens in CF could ensure prompt treatment with the most appropriate antibiotic; potentially improving outcomes and shortening hospital stays. As traditional culture methods for detecting bacteria are time-consuming there is a growing interest in SERS as a novel culture-free technique that produces a whole-organism spectroscopic fingerprint at high speed. Bacterial isolates including Pseudomonas aeruginosa (n=32), Staphylococcus aureus (n=5), Streptococcus pneumoniae (n=5) were incubated for 6 h at 37 °C/180 r.p.m., with a starting optical density (OD) of 0.15. After adjustment of the OD to 0.3, bacterial cells were harvested by centrifugation at 9000 rcf for 3 min and washed three times with dH2O. Bacterial pellets were mixed with citrate reduced silver colloid (CRSC) and dried. Spectra were recorded (4×10 s at 785 nm) and analysed within GRAMS/Al using Principal Component Analysis (PCA). Spectra of P. aeruginosa isolates (n=32) were separated into two distinct groups; the spectra of one group (n=12) was dominated by the pigment pyocyanin with vibrational brands present at 1350, 1492, 1598 and 1615 cm-1. The other group (n=20) had characteristic vibrational bands at 661, 735 and 800 cm-1 which correspond to guanine, adenine and uracil, respectively. S. aureus has a main characteristic band at 735 cm-1 and S. pneumoniae has a characteristic band present at 480 cm-1. Bacterial species clustered separately when analysed by PCA. Reproducible and distinguishable SERS spectra of bacterial isolates were obtained, and it was possible to differentiate between different bacterial species using PCA. These results suggest SERS has the potential to rapidly detect bacteria.
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Difficulties in diagnosis and treatment of urinary tract infections in an elderly population
More LessBackgroundUrinary tract infection (UTI) contributes significantly to healthcare burden, accounting for 23 % of hospital acquired infections and 2–3 % of general practice consultations. Unfortunately, difficulties exist in obtaining an accurate diagnosis, with studies showing misdiagnosis rates above 40 % in elderly populations. Furthermore, numerous hospitals across the UK still advocate the use of Trimethoprim for UTI, despite high rates of resistance. These factors combined leads to a sub-optimal experience for patients.
AimWe aimed to identify the practices surrounding the diagnosis and treatment of UTIs in elderly patients within Royal Bolton Hospital, a large district general hospital in the North-West of England. We also aimed to identify unique patterns of presentation of UTIs in elderly patients which could lead to diagnostic difficulty. Finally, we assessed local antibiotic resistance rates.
MethodsA retrospective case-note analysis of 100 patients, over the age of 65 years, diagnosed with UTI was carried out in 3 cycles between 2016–2018. The final cycle was conducted following removal of Trimethoprim from antibiotic guidance.
ResultsOf patients diagnosed with UTI and had MSU (mid-stream urine) sample analysed, only 28.8 % displayed microbial growth. 39.1 % of patients with confirmed UTI displayed neither signs nor symptoms of UTI. 20 % diagnosed with UTI did not have a MSU sample requested. Resistance rates of 39.1 % were reported to Trimethoprim, with E. coli accounting for 56.5 % of all UTIs.
ConclusionsDiverse presentation and incomplete diagnostics contributes to misdiagnosis of UTI. Trimethoprim is not an effective treatment option and guidelines should reflect this.
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Shining a light on antibiotic selection: optimised live/dead fluorescence spectrometry for rapid antimicrobial susceptibility testing
More LessAntibiotic 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.
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The present & the past: a review of newly accessioned bacterial strains into the UK’s National Collection of Type Cultures
More LessThe National Collection of Type Cultures (NCTC) is a bacterial culture collection which is taxonomically and biologically diverse. NCTC holds approximately 6000 different strains from over 900 different species; among them strains originally isolated in the 19th century, strains for use as controls as stipulated EUCAST and ISO guidelines, type strains on which the description of bacterial species are based and other strains from a variety of backgrounds. The remit of NCTC is to provide authentic bacterial cultures of medical and veterinary interest to the scientific community, to support and enhance the reproducibility of scientific research and to improve global public health. To fulfil this remit, remain scientifically relevant and to preserve the legacy of contemporary medical bacteriology for future scientists, NCTC accessions strains of clinical significance: such as recently circulating and outbreak strains, diagnostic escape mutants and strains with novel antimicrobial resistance profiles. In 2018, 166 bacterial strains were accessioned into the NCTC and made available to the scientific community. These include NCTC 14052: a reference strain for emergent hyper-virulent K. pneumoniae, 4 type strains of newly described bacterial species, 82 strains accessioned from the Murray Collection of pre-antibiotic era Enterobacteriaceae and 8 strains with antimicrobial resistance mechanisms previously unrepresented in the collection, including NCTC 14208: a N. gonorrhoeae isolated from an instance of combined ceftriaxone and azithromycin treatment failure. Through literature review we have highlighted their value to the scientific community, both in their own right and in the context of bacterial strains already held by the NCTC.
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Efficacy of novel eugenol tosylate congeners as antifungal compounds in combination with fluconazole against Candida albicans
More LessThe incidences of Candida albicans infections and their changing drug resistance patterns have drastically increased in recent years. Therefore, new drugs and alternative treatment strategies are promptly required. Combination therapy and the use of natural products have been extensively studied as alternative treatment. In this study, we synthesized Eugenol Tosylate Congeners (ETCs 1–6) and evaluated their antifungal activity profile alone and in combination with fluconazole (FLC) against four FLC susceptible and three FLC resistant clinical isolates of Candida albicans isolates according to CLSI guidelines. For insight mechanism of antifungal action of ETCs, activity of plasma membrane H+-ATPase pump of these C. albicans isolates was determined by monitoring the pH of the external medium. ETC 1 and ETC 4 were the most active congeners against the resistant isolates with the MIC ranging from 125 to 250 µg ml−1. The MFC of ETCs ranged from 1000 µg ml−1 to 2000 µg ml−1. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms showed 36 % of synergy, 29 % of additive, 33 % of indifferent and 2 % of antagonistic interactions. These compounds also inhibit H+efflux activity of H+-ATPase pump at varying degrees. Our results suggest that these ETCs may be directly binding to this pump and thereby inhibiting H+-efflux in Candida cells. These results advocate the potential of these compounds in developing new antifungal drugs; however, further studies are required to understand the other mechanisms involved and in vivo efficacy and toxicity of these compounds.
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Pseudomonas aeruginosa antibiogram profiles are poor indicators of genetic relatedness
More LessPseudomonas 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.
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The Type III CRISPR-Cas system of Mycobacterium tuberculosis
More LessBackgroundBacteria and archaea have developed a number of strategies to keep the influx of mobile genetic elements (MGEs) such as viruses and plasmids in check. CRISPR-Cas systems provide adaptive immunity; all types of CRISPR-Cas systems have in common the genes for uptake of genetic information from the invading MGE but differ in the composition of the effector complex that directs and enforces the immune response. The genome of Mycobacterium tuberculosis encodes a Class 1, type III-A CRISPR-Cas system that has not been studied in detail.
MethodologyWe used heterologous production of the mycobacterial CRISPR-Cas genes. Purification of the enzymes enabled in vitro biochemical characterisation of the adaptation, maturation and interference proteins. Reconstitution of the system in E. coli was used to demonstrate functionality in vivo.
ResultsMaturation of crRNA through the action of Cas6 proceeded as expected, generating canonical CRISPR RNAs (crRNAs). The type III effector complex, consisting of Csm1-5, was shown to bind crRNA and cleave target RNA with the typical 6 nt spacing, display ssDNase activity and produce the cyclic oligoadenylate signalling molecule. The latter clearly activated the ribonuclease Csm6, an essential element in type III immunity. The M. tuberculosis type III CRISPR-Cas system was also reconstituted in E. coli where it provided plasmid immunity, demonstrating the functionality in vivo.
ConclusionsAll elements of the M. tuberculosis type III CRISPR-Cas system are functional in vitro and in vivo. These studies lay the foundation for further investigations into the mechanism of adaptive immunity and possible applications in biotechnology.
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Pneumococcal invasive disease preceded by intracellular replication within splenic macrophages
During bacteremic pneumonia, the prevailing dogma is that bacteria seed from the lungs into the blood. Recently, we have shown that experimental murine sepsis is preceded by intracellular replication within splenic macrophages (Ercoli Nat Microbiol 2018), which shed into the bloodstream initiating invasive disease. Here we aimed to investigate a role for the spleen in the pathogenesis of bacteraemia following pneumonia. We analysed by confocal microscopy the fate of pneumococci during ex vivo human spleen perfusions (REC reference: 18/EM/0057), in spleens during pneumonia in non-human primates (Reyes PLOS one 2016) and mice. During ex vivohumanspleenperfusion, clusters of pneumococci were observed within macrophages and the size of bacterial clusters increased over time. To associate these infectious foci to invasive pneumococcal disease during pneumonia, we analysed spleens in a baboon pneumonia model, and detected pneumococcal clusters in splenic macrophages. To test the functional relevance of these data, we treated intranasally-challenged mice with a single, non-therapeutic sub-MIC dose of azithromycin, known to concentrate inside macrophages. Data showed that bacterial lung-counts were identical in treated and untreated mice. Untreated mice showed signs of disease, had high blood and spleen-counts, whereas mice treated with the non-therapeutic dose showed no signs of disease, had low spleen-counts and no bacteraemia. Thus, the number of pneumococci in the spleen, not the lung, correlates to blood-counts during bacterial pneumonia. We hypothesise that after initial control of invasive infection by the spleen, bacteraemia associated with pneumonia arises from a sub-set of splenic macrophages that are permissive for bacterial replication.
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Clostridium difficile: cell surface biogenesis
More LessOn the C. difficile cell surface is a proteinaceous paracrystalline array, known as the S-layer. The S-layer of C. difficile is composed of two proteins: the high molecular weight (HMW) and the low molecular weight (LMW) S-layer proteins, derived from the pre-protein SlpA. PS-II, an anionic polymer found in all C. difficile strains examined to date, has been identified as the ligand responsible for the attachment of S-layer and associated cell wall proteins. Early efforts to knock out slpA proved unsuccessful and the genes thought to encode the PS-II synthesis pathway were also thought to be essential. However, by using bacteriocins that specifically target the S-layer, we recently isolated a mutant which had no evident S-layer due to a mutation in the slpA gene. As the S-layer was previously thought to be essential, it now brings into question whether PS-II is also essential. In the strain lacking an S-layer, we have now created a deletion mutant in the putative PS-II polymerase and we are attempting to generate additional mutations in the polysaccharide synthesis pathway. Analysis of these mutants will provide insights into the mechanism of PS-II synthesis and shed light on its function in cell morphogenesis.
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Nature and consequences of Salmonella infections in cattle
More LessSalmonella enterica is a human and veterinary pathogen of global importance. Cattle are a key reservoir of S. enterica serotypes that cause non-typhoidal salmonellosis in humans and infections are often acquired via consumption of contaminated food. Salmonella can survive within the bovine lymphatic system and contaminated peripheral lymph nodes often enter the food chain via ground beef production because they are small and deeply embedded in fat, making it impossible to remove them during food production. S. enterica serotypes can also cause acute enteritis and systemic typhoid-like disease in cattle, thereby exerting a significant burden on bovine welfare and productivity. Existing vaccines confer limited serotype-specific protection and a need exists to better understand the host and bacterial factors involved in pathogenesis and protection to inform the design of new vaccines and other intervention strategies. Most of our knowledge about salmonellosis comes from the mouse typhoid model. Here, we sought to understand the effects of infection by Salmonella Dublin, which causes typhoid-like disease in cattle, in its natural host. By infecting cattle with S. Dublin expressing green fluorescent protein and using flow cytometry we have been able to isolate and characterise the bovine cells infected by S. Dublin, study changes in their cell surface marker expression post-infection and compare tropism in the intestine and draining lymph nodes. We have also studied the survival of S. Dublin in the main infected host cell type in vitro using primary cells to determine the consequences of infection on the pathogen itself.
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A novel deep-sea sponge bacterium producing two promising antimicrobial candidates
More LessBackgroundNatural product screening methods are arguably the most efficient way to identify novel antibiotics. Exploiting obscure, hard to reach environments, implementing the latest high-throughput next generation sequencing techniques, performing in silico analysis and synthesis/recombinant expression of promising candidates may increase the discovery of unique agents.
Materials/methodsDeep-sea sponges were collected (∼1000 m depth) in the North Atlantic Ocean and bacteria were recovered. One strain (EU4) was selected for detailed analysis. Strain EU4 produced an inhibitor into liquid media. This compound was purified using liquid chromatography andmatrix-assisted laser desorption/ionization (MALDI) analysis was performed. In parallel, the draft genome was obtained and analysed using BAGEL-3 and anti SMASH-4.0mining tools. Successful synthetic production was obtained for a candidate identified using antiSMASH.
ResultsAnalysis ofthedraft genome (5.8Mbp) indicates that strain EU4is a novel member of the Bacillaceae. To date, the produced compound showed activity towards Micrococcus luteus only, while the synthetic compound displays a broad spectrum of activity towards Gram positive and negative bacteria. In addition, based on MALDI analysis, the synthetic and the naturally produced compounds possess different molecular weights, being approximately 4 kDa and 1.7 kDa, respectively.
ConclusionsBacteria recovered from deep-sea sponges couldpotentially be a rich source for novel compounds. In silico analysis of producer genomes has provided a means of identifying cryptic compounds, not produced in culture. Further study of both compounds, which showed diverse activity spectra, may lead to promising new candidates for development into clinically relevant therapeutics.
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The role of glutamine synthetase in the pathogenesis of Neisseria meningitidis
More LessNeisseria meningitidisis a cause of meningitis and severe sepsis. A moonlighting protein is a protein with the ability to perform additional task(s) alongside its recognised function. These proteins have been identified in both prokaryotic and eukaryotic cells and they represent a highly conserved subset of proteins that typically are either metabolic pathway-associated enzymes or act as molecular chaperones. To explore the role of GlnA in the pathogenesis of meningococcal disease, the encoding gene designated NMB0359, was amplified from the wild-type meningococcal strain MC58 and cloned into the pQE-30 expression vector. Recombinant glutamine synthetase (rGlnA) was expressed in E. coli and purified by immobilised metal affinity chromatograph. Rabbit antisera was raised against purified rGlnA (RαGlnA) and used to investigate the localisation of GlnA at the cell surface. Attempts were also made to generate glnA knockout and complemented strains of wild-type N. meningitidis. rGlnA was successfully purified from E. coli cell lysates under native conditions. A highly immuno-reactive band of the expected size (52 kDa) was observed when rGlnA immunoblot was probed with RαGlnA. GlnA could be detected on the surface of wild-type encapsulated N. meningitidis MC58 using whole-cell enzyme linked immunosorbent assay (ELISA). Surface localisation of GlnA indicates that it may be a moonlighting protein carrying out function(s) at the cell surface. Future work will investigate possible moonlighting functions which may include adhesion to host cells and proteins, regulation of the host immune response, and contribution to bacterial virulence.
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Investigating the nuclear localisation and proteolytic activity of the meningococcal App and MspA autotransporters
More LessAutotransporter 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.
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Identification of a nitrite reductase in Pseudomonas aeruginosa as a potential antimicrobial target
More LessThe opportunistic pathogen Pseudomonas aeruginosa utilizes a wide range of virulence factors to adapt to the host environment. With the antimicrobial pipeline drying up, understanding and targeting virulence factors for therapeutic development is an exciting alternative for the discovery of novel disease inhibitors. An integrated genome-wide transposon mutagenesis screening approach was performed in P. aeruginosa using multiple in vivo disease models with the aim to identify new virulence factors required for infection. A mutant attenuated in the production of multiple virulence determinants using in vitro assays was identified. This mutant also showed severe attenuation using in vivo models with up to an 80 % increased survival in murine chronic and acute lung infection models. The predicted protein coded by the mutated gene showed homology to nitrite and sulphite reductases. Using a methyl viologen reduction assay, we have shown that this gene encondes a nitrite reductase, operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the requirement of siroheme revealed that product of this gene is an assimilatory nitrite reductase and hence we propose it to be named as NirA. Work is now on-going to understand how NirA contributes to virulence and determine the crystal structure of this protein with a view to screen for novel inhibitors of this enzyme using a drug discovery platform available in our laboratories.
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Investigation of a hospital Enterobacter cloacae NDM-1 outbreak using whole genome sequencing
Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.
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Development of anti-virulence polymers targeting mycobacteria
More LessModern 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.
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The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James’s Hospital, Dublin
More LessTriazole 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.
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A SLIC answer to a continuing problem
More LessBackgroundThe burden of Anti-Microbial Resistance (AMR) is a growing problem globally. Here we present a device that determines susceptibility rapidly from primary human or animal samples and could turn the tide of AMR. SLIC (Scattered Light Integrating Collector) is a sensitive device for the detection of microbes based on the scattering of laser light.
MethodsProof of concept studies were carried out initially to establish the lower limit of detection. This was found to be 10–50 c.f.u. ml−1. This exquisite sensitivity allowed us to commence work establishing rapid MICs. Starting with an inoculum of 105/ml bacteria and using a relevant range of antibiotic concentrations the MIC can be established in less than one microbial doubling period.
ResultsThe rapid and sensitive detection SLIC affords allows for fast growing organisms such as E. coli and S. aureus to have their MICs established in less than 10 min, for any antibiotic. For slow growing organisms such as M. bovis we are able to establish an MIC in H. influenzae and Mycoplasma spp.
ConclusionAs bacterial quantification is continuously monitored we are able to see the action of antibiotics in real time. Using this facility, we can readily distinguish between lytic antibiotics and bactericidal but non-lytic antibiotics. This provides the opportunity to gain new insights into the mechanisms of action and the effect antibiotics have on microbes in a new way in a novel Point-of-Care device.
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Understanding the pathogenic process of uropathogenic Escherichia coli ST127 using proteomics on uroepithelial co-culture samples
More LessBackgroundUropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). Strains of sequence type (ST) 127 exhibit the highest virulence potential of most UPEC strains, but little is known about pathogenicity during infection. We sought to investigate this using a quantitative proteomics approach.
Material/MethodsThree strains of UPEC ST127 (EC18, EC41 and SA189), in addition to non-pathogenic strain E. coli K12, were analysed in co-culture with the uroepithelial cell-line HT1197 for 5 h. We analyzed the bacterial and uroepithelial proteome along with the secreted proteins in the medium (secretome). The digested proteins and peptides from all fractions were separated on a Dionex Ultimate 3000 RSLC nano flow system and analyzed in an Orbitrap Velos Pro FTMS. Data were processed using Persues software.
ResultsLabel free quantitative proteomics revealed different proteomic profiles of the co-cultured strains. Gene Ontology enrichmentanalysis showed upregulation in the pentose phosphate pathway and glycolysis/glycogenesis in EC18 (an O-antigen deficient mutant). These two pathways could be important routes of carbon flux through the central metabolic pathways during growth in urine. Co-culture of SA189 with HT1197 cells leads to apparent cytotoxic effects in HT1197 cells not seen with other UPEC strains. Analysis of the SA189 secretome revealed highly abundant bacterial proteins, some of which (e.g. aromatic-amino-acid aminotransferase) were uniquely found during co-culture conditions.
ConclusionProteomics is crucial towards increasing our understanding of the pathogenic potential of UPEC ST127 strains and may facilitate identification of novel diagnostic or therapeutic targets to reduce UTI.
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Purification and characterisation of antimicrobial agents isolated from a member of the Paenibacillus genus
More LessAntimicrobial 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.
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Induction and characterisation of a 25-hydroxycholesterol associated immune response to Gram positive and negative bacteria in a whole blood model of sepsis
More LessEarly 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.
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Towards a clinically relevant model for investigation of host-microbe interactions in ventilator-associated pneumonia
More LessVentilator-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.
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Development of biofilm-penetrant antimicrobial delivery system to counter Burkholderia infections
More LessMelioidosis 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.
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Acquisition of fluoroquinolone resistance in Campylobacter jejuni leads to an increase in biofilm formation and virulence
More LessCampylobacter 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.
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Investigating the uptake mechanism of S type pyocins
More LessPseudomonas 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.
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Gut microbiota derived mitochondrial inhibitors cross the blood brain barrier and localise white matter
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.
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The UK public health rapid support team: a novel programme integrating outbreak response, operational research, and capacity building
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
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Functionalised liposomal formulations for delivery of antibiotic agents
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.
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Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in the development of resistance
More LessAntibiotic 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.
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Do Streptococcus pneumoniae and respiratory Syncytial virus synergise to promote invasive disease?
More LessStreptococcus 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.
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Effect of metronidazole on microbiomes associated with asymptomatic bacterial vaginosis
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.
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Development of ‘smart’ wound dressings for biofilm sensing and control
More LessChronic 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.
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Advanced titanium dioxide-polytetrafluorethylene (TiO2-PTFE) nanocomposite coatings on stainless steel surfaces exhibit significant antibacterial and anti-corrosion properties
More LessBacterial 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.
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Old drugs learn new tricks – repurposing phenothiazines to uncover effective antimicrobial
More LessThioridazine (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.
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