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.

Hydrogen‐Peroxide‐Generating Electrochemical Scaffold Eradicates Methicillin‐Resistant Staphylococcus aureus Biofilms

“…The electric potential applied to the e‐scaffold is carefully controlled via a potentiostat. Based on our previous studies, the surface of the e‐scaffold was polarized at −0.6 V Ag/AgCl for continuous production of H 2 O 2 . Under these conditions, we observed constant production of H 2 O 2 (≈25 × 10 −6 m concentration) at the e‐scaffold surface, consistent with our previous studies .…”

“…Previously, we developed and studied the anti‐biofilm property of an H 2 O 2 ‐generating electrochemical scaffold (e‐scaffold) with biofilms of Acinetobacter baumannii and Pseudomonas aeruginosa and also determined its biocompatibility with mammalian tissue . In this work, we report application of the H 2 O 2‐ producing e‐scaffold as a novel antibiotic‐free approach to eliminate MRSA biofilms and also explore the potential anti‐biofilm mechanism of the H 2 O 2 ‐generating e‐scaffold.…”

“…Further, any true comparison to conventional antibiotics would have required testing a suite of compounds representing different classes of agents and at varying concentrations. We note that in previous studies, we showed that our e‐scaffold kills persister cells, which are not killed by antibiotics . Another limitation is that we did not include an exogenous H 2 O 2 control; however, we did do this in our prior work …”

“…Based on the results of the live/dead staining assay, the H 2 O 2 ‐producing e‐scaffold appears to damage the membranes of bacteria present in biofilms. H 2 O 2 , along with other members of reactive oxygen species [e.g., hydroxyl free radicals (OH·)] can damage membranes, potentially facilitating entry of propidium iodide into cells . Several recent studies have shown that H 2 O 2 ‐induced intracellular production of hydroxyl free radicals (OH·) can destroy persister cells present in biofilms, resulting in biofilm dispersal and also disrupt global biofilm structures in Gram‐negative bacteria by increasing bacterial membrane permeability .…”

“…Several recent studies have shown that H 2 O 2 ‐induced intracellular production of hydroxyl free radicals (OH·) can destroy persister cells present in biofilms, resulting in biofilm dispersal and also disrupt global biofilm structures in Gram‐negative bacteria by increasing bacterial membrane permeability . In a recent study, e‐scaffold generated H 2 O 2 ‐induced intracellular production of hydroxyl free radicals (OH·) increased membrane permeability of P. aeruginosa biofilms as observed through increased fluorescence of propidium iodide, suggesting that membrane disruption via production of ROS, of which hydroxyl free radicals (OH·) is a member, may be one of the mechanisms through which the e‐scaffold exerts its antibiofilm effect . Since propidium iodide also stains extracellular DNA present in the biofilm matrix, additional quantitative analysis of biofilm biomass matrix through 3D reconstruction of confocal images is warranted for accurate quantification of live and dead cells present in biofilm since the loss of biomass may be due to rinsing away dispersed cells prior to imaging.…”