Eradication of Pseudomonas aeruginosa biofilms and persister cells using an electrochemical scaffold and enhanced antibiotic susceptibility

Abstract

Biofilms in chronic wounds are known to contain a persister subpopulation that exhibits enhanced multidrug tolerance and can quickly rebound after therapeutic treatment. The presence of these "persister cells" is partly responsible for the failure of antibiotic therapies and incomplete elimination of biofilms. Electrochemical methods combined with antibiotics have been suggested as an effective alternative for biofilm and persister cell elimination, yet the mechanism of action for improved antibiotic efficacy remains unclear. In this work, an electrochemical scaffold (e-scaffold) that electrochemically generates a constant concentration of H₂O₂ was investigated as a means of enhancing tobramycin susceptibility in pre-grown Pseudomonas aeruginosa PAO1 biofilms and attacking persister cells. Results showed that the e-scaffold enhanced tobramycin susceptibility in P. aeruginosa PAO1 biofilms, which reached a maximum susceptibility at 40 µg/ml tobramycin, with complete elimination (7.8-log reduction vs control biofilm cells, P ≤ 0.001). Moreover, the e-scaffold eradicated persister cells in biofilms, leaving no viable cells (5-log reduction vs control persister cells, P ≤ 0.001). It was observed that the e-scaffold induced the intracellular formation of hydroxyl free radicals and improved membrane permeability in e-scaffold treated biofilm cells, which possibly enhanced antibiotic susceptibility and eradicated persister cells. These results demonstrate a promising advantage of the e-scaffold in the treatment of persistent biofilm infections.

Fig. 1

E-scaffold enhances tobramycin susceptibility in P. aeruginosa PAO1 biofilms. Bars represent means of logarithms of colony-forming units of viable biofilm cells. Error bars represent the standard error of the means from three biological replicates. The symbols *, **, ***, and **** represent significant differences in tobramycin susceptibility between e-scaffold treated biofilms + tobramycin and untreated biofilms + tobramycin (n = 3, *, P = 0.002; **, ***, **** P ≤ 0.001; paired t-test)

Fig. 2

Exposure to e-scaffold eradicates the persister cells in P. aeruginosa PAO1 biofilms. Bars represent means of logarithms of colony-forming units of viable biofilm cells. Error bars represent the standard errors of the means from three biological replicates. The symbol * denotes a significant difference compared to total biofilm cells (n = 3, P < 0.05, one-way ANOVA), and ** denotes a significant difference compared to control initial persister cells (n = 3, P < 0.001, one-way ANOVA)

Fig. 3

E-scaffold increases OH• formation and membrane permeability. a Increase in fluorescence of HPF-stained e-scaffold treated and exogenous H₂O₂ treated P. aeruginosa PAO1 biofilm cells indicates increased OH• formation compared to untreated biofilms. b Increase in fluorescence of propidium iodide (PI) indicates increased membrane permeability of P. aeruginosa PAO1 cells after exposure to e-scaffold. c Increase in fluorescence of 3, 3′-dipropylthiacarbocyanine iodide, a membrane potential sensitive dye, in e-scaffold treated P. aeruginosa PAO1 cells verifies bacterial membrane depolarization by e-scaffold resulting in increased membrane permeability. Error bars represent standard errors of means for at least three biological replicates. The symbol * indicates a significant difference from the untreated biofilm cells (n = 3, P < 0.001; paired t-test)

Fig. 4

Scanning electron microscopy (SEM) images showing untreated, e-scaffold treated and exogenous H₂O₂ treated P. aeruginosa PAO1 biofilm cells. Three representative images are shown for each treatment. Red arrows indicate cells showing a stressed membrane

Fig. 5

Proposed mechanism for e-scaffold generated H₂O₂ enhancing antibiotic susceptibility. H₂O₂ generated extracellularly by e-scaffold diffuses through the bacterial cell envelope and reacts with intracellular Fe²⁺, forming radicals that oxidize lipids, proteins and DNA, which prompts cell death. Through membrane damage or changing membrane permeability, antibiotic penetration into the bacterial cell is increased, which in turn enhances the antibiotic susceptibility of the cells

Fig. 6

a Schematic of experimental setup for the treatment of biofilm exposed to an e-scaffold with an illustration of electrochemical H₂O₂ production. The electrodes are connected to a potentiostat (not shown in figure). Scientific Reports, 2015. 5, 14908. DOI: 10.1038/srep14908. © Creative Commons Attribution 4.0 International License. b Recovered biofilm cells treated with e-scaffold and tobramycin. c Treatment of biofilms and persister cells with e-scaffold