Dataset Information

The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential.

ABSTRACT:

Objectives

To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action.

Methods

Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively.

Results

Here we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms.

Conclusions

Oxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing. As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.

SUBMITTER: Maiden MM

PROVIDER: S-EPMC6735725 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Publications

The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential.

Maiden Michael M MM Zachos Mitchell P MP Waters Christopher M CM

The Journal of antimicrobial chemotherapy 20190401 4

<h4>Objectives</h4>To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action.<h4>Methods</h4>Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using D ...[more]

PMID: 30624737

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Project description:The Pseudomonas aeruginosa LasR-LasI (LasR-I) quorum sensing system regulates secreted proteases that can be exploited by cheaters, such as quorum sensing receptor-defective (lasR) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy. These mutants are exploitative cheaters because they avoid the substantial cost of engaging in quorum sensing. Previous studies showed that quorum sensing increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppressed the emergence of lasR mutants in casein-passaged populations. Several mutations accumulated in those populations, indicating evidence of antibiotic adaptation. We found that mutations in one gene, ptsP, increased antibiotic resistance and also pleiotropically increased production of a quorum sensing-controlled phenazine, pyocyanin. When passaged on casein, ptsP mutants suppressed cheaters in a manner that was tobramycin independent. We found that the mechanism of cheater suppression in ptsP mutants relied on pyocyanin, which acts as a policing toxin by selectively blocking growth of cheaters. Thus, tobramycin suppresses lasR mutants through two mechanisms: first, through direct effects on cheaters and, second, by selecting mutations in ptsP that suppressed cheating in a tobramycin-independent manner. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during interspecies competition or infections. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa is a model for understanding quorum sensing, a type of cell-cell signaling important for cooperation. Quorum sensing controls production of cooperative goods, such as exoenzymes, which are vulnerable to cheating by quorum sensing-defective mutants. Because uncontrolled cheating can ultimately cause a population to collapse, much focus has been on understanding how P. aeruginosa can control cheaters. We show that an antibiotic, tobramycin, can suppress cheaters in cooperating P. aeruginosa populations. Tobramycin suppresses cheaters directly because the cheaters are more susceptible to tobramycin than cooperators. Tobramycin also selects for mutations in a gene, ptsP, that suppresses cheaters independent of tobramycin through pleiotropic regulation of a policing toxin, pyocyanin. This work supports the idea that adaptation to antibiotics can have unexpected effects on the evolution of quorum sensing and has implications for understanding how cooperation evolves in dynamic bacterial communities.

Project description:BACKGROUND:Novel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area. RESULTS:We report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of?~?16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to?~?230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of?~?232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies. CONCLUSIONS:Positive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite-tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

Dataset Information

The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential.

Objectives

Methods

Results

Conclusions

Publications

The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential.

Similar Datasets

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