scholarly journals A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U

2021 ◽  
Vol 478 (3) ◽  
pp. 647-668
Author(s):  
Daniel M. Foulkes ◽  
Keri McLean ◽  
Yalin Zheng ◽  
Joscelyn Sarsby ◽  
Atikah S. Haneef ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
T Cells ◽  
Bacterial Load ◽  
World Health Organisation ◽  
Host Cells ◽  
World Health ◽  
Infection Model ◽  
Compound A ◽  
Type 3

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

scholarly journals Evaluation of ExoU inhibitors in a Pseudomonas aeruginosa scratch infection assay

2020 ◽  
Author(s):  
Daniel M. Foulkes ◽  
Keri McLean ◽  
Joscelyn Harris ◽  
Atikah S. Haneef ◽  
David G. Fernig ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Load ◽  
World Health Organisation ◽  
Host Cells ◽  
World Health ◽  
Infection Model ◽  
Phospholipase Activity ◽  
Compound A ◽  
Corneal Epithelial

AbstractPseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 μM. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa.

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity

2021 ◽  
Author(s):  
Daniel Morgan Foulkes ◽  
Keri McLean ◽  
Marta Sloniecka ◽  
Dominic Byrne ◽  
Atikah S Haneef ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Opportunistic Pathogen ◽  
World Health Organisation ◽  
Host Cells ◽  
World Health ◽  
Corneal Epithelial Cell ◽  
Cell Infection ◽  
Minimal Inhibitory Concentrations ◽  
Line Of Therapy

Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.

scholarly journals The antimicrobial activity of silver acetate against Acinetobacter baumannii in a Galleria mellonella infection model

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11196
Author(s):  
Eden Mannix-Fisher ◽  
Samantha McLean
Keyword(s):  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Galleria Mellonella ◽  
World Health Organisation ◽  
World Health ◽  
Infection Model ◽  
Silver Acetate ◽  
Selective Toxicity ◽  
Carbapenem Resistant

Background The increasing prevalence of bacterial infections that are resistant to antibiotic treatment has caused the scientific and medical communities to look for alternate remedies aimed at prevention and treatment. In addition to researching novel antimicrobials, there has also been much interest in revisiting some of the earliest therapies used by man. One such antimicrobial is silver; its use stretches back to the ancient Greeks but interest in its medicinal properties has increased in recent years due to the rise in antibiotic resistance. Currently antimicrobial silver is found in everything from lunch boxes to medical device implants. Though much is claimed about the antimicrobial efficacy of silver salts the research in this area is mixed. Methods Herein we investigated the efficacy of silver acetate against a carbapenem resistant strain of Acinetobacter baumannii to determine the in vitro activity of this silver salt against a World Health Organisation designated category I critical pathogen. Furthermore, we use the Galleria mellonella larvae model to assess toxicity of the compound and its efficacy in treating infections in a live host. Results We found that silver acetate can be delivered safely to Galleria at medically relevant and antimicrobial levels without detriment to the larvae and that administration of silver acetate to an infection model significantly improved survival. This demonstrates the selective toxicity of silver acetate for bacterial pathogens but also highlights the need for administration of well-defined doses of the antimicrobial to provide an efficacious treatment.

scholarly journals Contribution of swarming motility to dissemination in a Pseudomonas aeruginosa murine skin abscess infection model

2020 ◽  
Author(s):  
Shannon R Coleman ◽  
Daniel Pletzer ◽  
Robert E W Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Bacterial Load ◽  
Infection Model ◽  
Swarming Motility ◽  
Semisolid Medium ◽  
Low Concentrations ◽  
Complex Adaptive

Abstract Swarming motility in Pseudomonas aeruginosa is a multicellular adaptation induced by semisolid medium with amino acids as a nitrogen source. By phenotypic screening, we differentiated swarming from other complex adaptive phenotypes, such as biofilm formation, swimming and twitching, by identifying a swarming-specific mutant in ptsP, a metabolic regulator. This swarming-deficient mutant was tested in an acute murine skin abscess infection model. Bacteria were recovered at significantly lower numbers from organs of mice infected with the ∆ptsP mutant. We also tested the synthetic peptide 1018 for activity against different motilities and efficacy in vivo. Treatment with 1018 mimicked the phenotype of the ∆ptsP mutant in vitro, as swarming was inhibited at low concentrations (<2 μg/mL) but not swimming or twitching, and in vivo, as mice had a reduced bacterial load recovered from organs. Therefore, PtsP functions as a regulator of swarming, which in turn contributes to dissemination and colonization in vivo.

scholarly journals Comprehensive Host Cell-Based Screening Assays for Identification of Anti-Virulence Drugs Targeting Pseudomonas aeruginosa and Salmonella Typhimurium

2020 ◽  
Vol 8 (8) ◽  
pp. 1096
Author(s):  
Julia von Ambüren ◽  
Fynn Schreiber ◽  
Julia Fischer ◽  
Sandra Winter ◽  
Edeltraud van Gumpel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Cell ◽  
Bacterial Infections ◽  
Host Cells ◽  
Infection Model ◽  
Serovar Typhimurium ◽  
New Antibiotics ◽  
Screening Assays ◽  
Screening Platform

The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.

scholarly journals ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Hyung Jun Kim ◽  
Hyunjung Lee ◽  
Yunmi Lee ◽  
Inhee Choi ◽  
Yoonae Ko ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Target ◽  
Bacterial Load ◽  
Thiamine Pyrophosphate ◽  
Infection Model ◽  
Antibiotic Development ◽  
New Drug ◽  
Thiamine Biosynthesis

ABSTRACTThiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. As thiamine biosynthesis occurs in bacteria but not humans, bacterial thiamine biosynthesis is an attractive target for antibiotic development. Among enzymes in the thiamine biosynthetic pathway, thiamine monophosphate kinase (ThiL) catalyzes the final step of the pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. In this work, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen of hospital-acquired infections. We demonstrated that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability, showing growth defects of the ΔthiL mutant even in the presence of thiamine derivatives except TPP. Most importantly, the pathogenesis of the ΔthiL mutant was markedly attenuated compared to wild-type bacteria, with lower inflammatory cytokine induction and 103~104 times decreased bacterial load in an in vivo infection model where the intracellular TPP level is in the submicromolar range. In order to validate P. aeruginosa ThiL (PaThiL) as a new drug target, we further characterized its biochemical properties determining a Vmax of 4.0±0.2 nomol·min−1 and KM values of 111±8 and 8.0±3.5μM for ATP and TMP, respectively. A subsequent in vitro small molecule screening identified PaThiL inhibitors including WAY213613 that is a noncompetitive inhibitor with a Ki value of 13.4±2.3 μM and a potential antibacterial activity against P. aeruginosa. This study proved that PaThiL is a new drug target against P. aeruginosa providing comprehensive biological and biochemical data that could facilitate to develop a new repertoire of antibiotics.

scholarly journals Pharmacodynamics of Polymyxin B against Pseudomonas aeruginosa

2005 ◽  
Vol 49 (9) ◽  
pp. 3624-3630 ◽  
Author(s):  
Vincent H. Tam ◽  
Amy N. Schilling ◽  
Giao Vo ◽  
Samer Kabbara ◽  
Andrea L. Kwa ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bactericidal Activity ◽  
Bacterial Infections ◽  
Bacterial Load ◽  
Polymyxin B ◽  
Dose Fractionation ◽  
Infection Model ◽  
Wild Type ◽  
Clinical Dose

ABSTRACT Despite limited data, polymyxin B (PB) is increasingly used clinically as the last therapeutic option for multidrug-resistant (MDR) gram-negative bacterial infections. We examined the in vitro pharmacodynamics of PB against four strains of Pseudomonas aeruginosa. Clonal relatedness of the strains was assessed by random amplification of polymorphic DNA. Time-kill studies over 24 h were performed with approximately 105 and 107 CFU/ml of bacteria, using PB at 0, 0.25, 0.5, 1, 2, 4, 8, and 16× MIC. Dose fractionation studies were performed using an in vitro hollow-fiber infection model (HFIM) against a wild-type and a MDR strain. Approximately 105 CFU/ml of bacteria were exposed to placebo and three regimens (every 8 h [q8h], q12h, and q24h) simulating the steady-state unbound PB pharmacokinetics resulting from a daily dose of 2.5 mg/kg of body weight and 20 mg/kg (8 times the clinical dose). Samples were obtained over 4 days to quantify PB concentrations, total bacterial population, and subpopulation with reduced PB susceptibility (>3× MIC). The bactericidal activity of PB was concentration dependent, but killing was significantly reduced with a high inoculum. In HFIM studies, a significant reduction in bacterial load was seen at 4 h in all active regimens, but selective amplification of the resistant subpopulation(s) was apparent at 24 h with the clinical dose (both strains). Regrowth was eventually observed in all dosing regimens with the MDR strain, but its occurrence was prevented in the wild-type strain by using 8 times the clinical dose (regardless of dosing intervals). Our results suggested that the bactericidal activity of PB was concentration dependent and appeared to be related to the ratio of the area under the concentration-time curve to the MIC.

Identification of a Potential Inhibitor Targeting MurC Ligase of the Drug Resistant Pseudomonas aeruginosa Strain through Structure-Based Virtual Screening Approach and In Vitro Assay

2019 ◽  
Vol 20 (14) ◽  
pp. 1203-1212
Author(s):  
Abdelmonaem Messaoudi ◽  
Manel Zoghlami ◽  
Zarrin Basharat ◽  
Najla Sadfi-Zouaoui
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virtual Screening ◽  
Homology Model ◽  
Generation Cephalosporin ◽  
World Health ◽  
Resistant Bacteria ◽  
Antimicrobial Drugs ◽  
Vitro Assay ◽  
Priority List

Background & Objective: Pseudomonas aeruginosa shows resistance to a large number of antibiotics, including carbapenems and third generation cephalosporin. According to the World Health Organization global report published in February 2017, Pseudomonas aeruginosa is on the priority list among resistant bacteria, for which new antibiotics are urgently needed. Peptidoglycan serves as a good target for the discovery of novel antimicrobial drugs. Methods: Biosynthesis of peptidoglycan is a multi-step process involving four mur enzymes. Among these enzymes, UDP-N-acetylmuramate-L-alanine ligase (MurC) is considered to be an excellent target for the design of new classes of antimicrobial inhibitors in gram-negative bacteria. Results: In this study, a homology model of Pseudomonas aeruginosa MurC ligase was generated and used for virtual screening of chemical compounds from the ZINC Database. The best screened inhibitor i.e. N, N-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazole-5-sulfonamide was then validated experimentally through inhibition assay. Conclusion: The presented results based on combined computational and in vitro analysis open up new horizons for the development of novel antimicrobials against this pathogen.

scholarly journals Seroprevalence of SARS-CoV-2, Symptom Profiles and Sero-Neutralization in a Suburban Area, France

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1076
Author(s):  
Anne Gégout Petit ◽  
Hélène Jeulin ◽  
Karine Legrand ◽  
Nicolas Jay ◽  
Agathe Bochnakian ◽  
...  
Keyword(s):  
Odds Ratio ◽  
World Health Organisation ◽  
World Health ◽  
Preventive Effect ◽  
Neutralization Activity ◽  
Symptom Profiles ◽  
The World ◽  
Household Members ◽  
Total Immunoglobulin

The World Health Organisation recommends monitoring the circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated anti–SARS-CoV-2 total immunoglobulin (IgT) antibody seroprevalence and in vitro sero-neutralization in Nancy, France, in spring 2020. Individuals were randomly sampled from electoral lists and invited with household members over 5 years old to be tested for anti–SARS-CoV-2 (IgT, i.e., IgA/IgG/IgM) antibodies by ELISA (Bio-rad); the sero-neutralization activity was evaluated on Vero CCL-81 cells. Among 2006 individuals, the raw seroprevalence was 2.1% (95% confidence interval 1.5 to 2.9), was highest for 20- to 34-year-old participants (4.7% (2.3 to 8.4)), within than out of socially deprived area (2.5% vs. 1%, p = 0.02) and with than without intra-family infection (p < 10−6). Moreover, 25% of participants presented at least one COVID-19 symptom associated with SARS-CoV-2 positivity (p < 10−13), with highly discriminant anosmia or ageusia (odds ratio 27.8 [13.9 to 54.5]); 16.3% (6.8 to 30.7) of seropositive individuals were asymptomatic. Positive sero-neutralization was demonstrated in vitro for 31/43 seropositive subjects. Regarding the very low seroprevalence, a preventive effect of the lockdown in March 2020 can be assumed for the summer, but a second COVID-19 wave, as expected, could be subsequently observed in this poorly immunized population.

Pharmacodynamics of once- versus twice-daily dosing of nebulized amikacin in an in vitro Hollow-Fiber Infection Model against 3 clinical isolates of Pseudomonas aeruginosa

2021 ◽  
Vol 100 (2) ◽  
pp. 115329
Author(s):  
Aaron James Heffernan ◽  
Fekade Bruck Sime ◽  
Saiyuri Naicker ◽  
Katherine Andrews ◽  
David Ellwood ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Hollow Fiber ◽  
Clinical Isolates ◽  
Infection Model
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