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

Douglas R Call,Haluk Beyenal,Sujala T Sultana

doi:10.1038/s41522-016-0003-0

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.

Highlights

Chronic and recalcitrant biofilm infections on wounds are often caused by the presence of a bacterial subpopulation of “persister cells” that are tolerant to bacteriostatic antibiotics.[1]
Electrochemical scaffold enhances tobramycin susceptibility in biofilm cells When biofilm treatments were combined with different concentrations of tobramycin, the surviving cells responded differently
The tobramycin susceptibility of P. aeruginosa PAO1 biofilms regrown from fresh culture, untreated biofilm cells, and persister cells isolated from biofilms appeared to follow a dose response at tested concentrations between 0 and 40 μg/ml (Fig. 1)

Summary

Introduction

Chronic and recalcitrant biofilm infections on wounds are often caused by the presence of a bacterial subpopulation of “persister cells” that are tolerant to bacteriostatic antibiotics.[1]. In lieu of classical antibiotics, a number of alternative antimicrobial treatments are being explored either individually (e.g., silver,[6] mannitol7) or in combination with conventional antibiotics.[8,9,10] high concentrations of these antimicrobials/antibiotics have toxic side effects[11,12] while at low concentrations they often decompose before completely eliminating biofilm communities.[13] any persister cells can regrow and form biofilms with potentially enhanced tolerance to antibiotics.[14] Interestingly, the application of an antibiotic in combination with a direct current (DC) (ranging from μA to mA/cm2) can be effective against several Gram-negative bacteria,[15,16] including against putative persister cells.[16] The mechanism underlying this effect was unclear until a recent study demonstrated the presence of electrochemically generated H2O2.17 In that study, an electrochemical scaffold (e-scaffold) made of conductive carbon fabric generated ~25 μM H2O2 near the surface of the e-scaffold and this was sufficient to reduce an A. baumannii biofilm (~3-log) that was established on a dermal explant. The constant but relatively low production of H2O2 did not appear to be cytotoxic to the mammalian tissue.[17]

Methods

Results

Conclusion