Effect of electrical energy on the efficacy of biofilm treatment using the bioelectric effect

Abstract

Background/objectives: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the 'bioelectric effect' (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms.

Methods: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 μg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy.

Results: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P>0.05) for voltages <1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA P<0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment (r² =0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage.

Conclusions: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.

Figure 1

(a) Schematic of experimental setup. Glass chips/coupons placed inside electroporation cuvettes were used as the growth surface for bacterial biofilms. The electrodes of the cuvette were used for easy application of electric fields. (b) Photograph of electroporation cuvette with glass coupon placed inside it.

Figure 2

(a) Table summarizing the voltages and energies used to test the effect of various energies of different signals on BE treatment efficacy to treat mature E. coli biofilms. (b) Plot showing the reduction in viable cells as measured using the colony-forming unit (CFU) assay method. Treatment with SP-BE, that has twice the energy as the AC-BE or DC-BE, results in almost twice the reduction in viable cells (N=4 for each experiment). Furthermore, the reduction in the SP-BE viable cell count is not significantly different from the linear sum of the reduction in the AC-BE and DC-BE viable cell count. The error bars represent the standard deviation across the repeats of the experiments. The error bar for the SP-BE is not large enough to be visible at this scale. AC, alternating current; BE, bioelectric effect; DC, direct current; SP, superpositioned field.

Figure 3

(a) Results of total biomass quantification using the crystal violet staining method. (b) Linear fit of the total biomass for the different energies provided during BE treatment. Plots show the OD at 540 nm after staining the treated biofilms with CV. Results show that the SP-BE shows a 71% reduction in bacterial biomass as compared with the untreated control (analysis of variance P<0.05). The SP-BE, which has higher energy as compared with the AC-BE or DC-BE is also more effective in treating biofilms. The data presented are the average OD₅₄₀ and the error bars represent the standard deviation over repeated experiments (N=6 in each experiment). AC, alternating current; BE, bioelectric effect; DC, direct current; OD, optical density; SP, superpositioned field.

Figure 4

(a) Table summarizing the magnitude of voltages used to test the effect of equivalent energies of different signals on BE treatment efficacy to treat mature E. coli biofilms. (b) Figure plotting OD measured at 540 nm for various biofilms samples treated with BE of equivalent energies. The energy of the electrical signal dictates the efficacy of the BE as observed by the similar reduction in total biomass for the AC-BE, DC-BE and SP-BE treatments. The error bars represent the standard deviation of the experiments performed across eight samples (N=8 for each experiment). AC, alternating current; BE, bioelectric effect; DC, direct current; OD, optical density; SP, superpositioned field.

Figure 5

Fluorescence microscopy images of the biofilm grown on the glass coupon after treatment with DC-BE, AC-BE and SP-BE as compared with untreated biofilms (control). The BE-treated biofilms result in a similar reduction in biomass as observed from the images. AC, alternating current; BE, bioelectric effect; DC, direct current; SP, superpositioned field.

Figure 6

(a) Table summarizing the magnitude of AC voltage used to test the effect of increasing energies of the same type of signal on BE treatment efficacy to treat mature E. coli biofilms. (b) Plot showing the linear relationship between the total biomass of the biofilms as measured using CV staining method and the voltage or energy of the electrical signal applied. The error bars represent the standard deviation across the repeats (N=5 repeats) for each experiment. AC, alternating current; BE, bioelectric effect; CV, crystal violet.