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Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

ABSTRACT: Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

SUBMITTER: van Tilburg Bernardes E

PROVIDER: S-EPMC5404518 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

van Tilburg Bernardes Erik E Charron-Mazenod Laetitia L Reading David J DJ Reckseidler-Zenteno Shauna L SL Lewenza Shawn S

Antimicrobial agents and chemotherapy 20170424 5

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. <i>Pseudomonas aeruginosa</i> is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of <i>P. aeruginosa</i> biofilms is comprised mainly of exopolysaccharides ...[more]

PMID: 28223377

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Project description:Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens.

Project description:An antivirulence approach targets bacterial virulence rather than cell viability in the antibiotic approach that can readily lead to drug resistance. Opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilm cells of this bacterium are much more resistant to antibiotics than planktonic cells. To identify novel inorganic antivirulence compounds, the dual screenings of thirty-six metal ions were performed to identify that zinc ions and ZnO nanoparticle inhibited the pyocyanin production and biofilm formation in P. aeruginosa without affecting the growth of planktonic cells. Moreover, zinc ion and ZnO nanoparticle markedly reduced the production of 2-heptyl-3-hydroxy-4(1H)-quinolone and siderophore pyochelin, while increased the production of another sideropore pyoverdine and swarming motility. Further, zinc ion and ZnO nanoparticle clearly suppressed hemolytic activity in P. aeruginosa. Transcriptome analyses showed that ZnO nanoparticle induced zinc cation efflux pump czc operon, porin genes (oprD and opdT), and Pseudomonas type III repressor A ptrA, while repressed pyocyanin-related phz operon, which partially explains the phenotypic changes. Overall, ZnO nanoparticle is a potential candidate for use in an antivirulence approach against persistent P. aeruginosa infection. P. aeruginosa Genechip Genome Array (Affymetrix, P/N 900339) was used in order to study the cells after the addition of ZnO nanoparticles. DNA microarray analysis with one biological replicate was performed with an Affymetrix system. P. aeruginosa was inoculated in 25 ml of LB medium in 250 ml shaker flasks with overnight cultures (1 : 100 dilution). Cells were cultured for 5 h with shaking at 250 rpm with and without ZnO nanoparticles (1 mM). Before sample collection, RNase inhibitor (RNAlater, Ambion, TX, USA) was added, and the cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 s before centrifugation at 16,000 g for 2 min. The cell pellets were immediately frozen with dry ice and stored at –80°C. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).

Project description:BackgroundThe biofilm state of pathogens facilitates antimicrobial resistance which makes difficult-to-treat infections. In this regard, it has been found that the compounds screened from plant extracts represent one category of the most promising antibiofilm agents. However, the antibiofilm activities and the active ingredients of plant extracts remain largely unexplored. In this background, the study is (1) to screen out the plant extracts with antibiofilm ability against Pseudomonas aeruginosa, and (2) to identify the active ingredients in the plant extracts and elucidate the underlying mechanism of the antibiofilm activities.MethodsMicro-broth dilution method, in vitro biofilm model, LC-MS/MS analysis and P. aeruginosa-mouse infection model were adopted to assess the antibiofilm activity. GC-MS analysis was performed to detect the active ingredients in plasma. RNA-Seq, GO analysis, KEGG analysis and RT-qPCR were adopted to elucidate the underlying mechanism of antibiofilm activities against P. aeruginosa.ResultsLonicerae Japonicae Flos (LJF) among 13 plants could exert significant inhibitory effects on bacterial biofilm formation, mobility and toxin release in vitro, and it could exert antibiofilm effect in vivo too. Moreover, quinic acid, as one metabolite of chlorogenic acid, was found as an active ingredient in LJF against the biofilm of P. aeruginosa. The active ingredient significantly inhibited EPS secretion in biofilm formation and maturity and could achieve synergistic antibiofilm effect with levofloxacin. It reduced the biofilm formation by regulating core targets in quorum sensing system. In GO process, it was found that the core targets were significantly enriched in multiple biological processes involving locomotion, chemotaxis and motility mediated by flagellum/cilium, which was related to KEGG pathways such as bacterial chemotaxis, oxidative phosphorylation, ribosome, biofilm formation, cyanoamino acid metabolism and quorum sensing. Finally, the binding of quinic acid with core targets rhlA, rhlR and rhlB were validated by molecular docking and RT-qPCR.ConclusionsIn summary, the study verified the in vitro and in vivo antibiofilm effects of LJF against P. aeruginosa and elucidated the active ingredients in LJF and its conceivable pharmacological mechanism, indicating that quinic acid could have the potential of an antibiofilm agent against P. aeruginosa and related infections.

Dataset Information

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

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Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

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