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. 2016 Mar;113(3):643-650.
doi: 10.1002/bit.25818. Epub 2015 Sep 9.

Eradication of multidrug-resistant pseudomonas biofilm with pulsed electric fields

Saiqa I Khan  1 , Gaddi Blumrosen #  2 , Daniela Vecchio #  3   4 , Alexander Golberg  5   6 , Michael C McCormack  1 , Martin L Yarmush  5   7 , Michael R Hamblin  3   4   8 , William G Austen Jr  1
Affiliations

Eradication of multidrug-resistant pseudomonas biofilm with pulsed electric fields

Saiqa I Khan et al. Biotechnol Bioeng. 2016 Mar.

Abstract

Biofilm formation is a significant problem, accounting for over eighty percent of microbial infections in the body. Biofilm eradication is problematic due to increased resistance to antibiotics and antimicrobials as compared to planktonic cells. The purpose of this study was to investigate the effect of Pulsed Electric Fields (PEF) on biofilm-infected mesh. Prolene mesh was infected with bioluminescent Pseudomonas aeruginosa and treated with PEF using a concentric electrode system to derive, in a single experiment, the critical electric field strength needed to kill bacteria. The effect of the electric field strength and the number of pulses (with a fixed pulse length duration and frequency) on bacterial eradication was investigated. For all experiments, biofilm formation and disruption were confirmed with bioluminescent imaging and Scanning Electron Microscopy (SEM). Computation and statistical methods were used to analyze treatment efficiency and to compare it to existing theoretical models. In all experiments 1500 V are applied through a central electrode, with pulse duration of 50 μs, and pulse delivery frequency of 2 Hz. We found that the critical electric field strength (Ecr) needed to eradicate 100-80% of bacteria in the treated area was 121 ± 14 V/mm when 300 pulses were applied, and 235 ± 6.1 V/mm when 150 pulses were applied. The area at which 100-80% of bacteria were eradicated was 50.5 ± 9.9 mm(2) for 300 pulses, and 13.4 ± 0.65 mm(2) for 150 pulses. 80% threshold eradication was not achieved with 100 pulses. The results indicate that increased efficacy of treatment is due to increased number of pulses delivered. In addition, we that showed the bacterial death rate as a function of the electrical field follows the statistical Weibull model for 150 and 300 pulses. We hypothesize that in the clinical setting, combining systemic antibacterial therapy with PEF will yield a synergistic effect leading to improved eradication of mesh infections.

Keywords: biofilm; eradication of multidrug resistant infections; irreversible electroporation; medical device disinfection; pulsed electric fields; treatment of mesh infection.

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Figures

Figure 1
Figure 1
Depiction of experimental setup. (A) Digital image of concentric ring electrodes applied to infected mesh. (B) BTX 830 pulse generator. (C) Theoretical mapping of biophysical effects experienced by cells as a function of electroporation parameters. Our goal was to find the electric field that corresponds to the solid line (red arrow). This represents the minimal electric field needed to kill bacteria and disrupt biofilm. We used the equation displayed to solve for E. (D) Bioluminescent image of infected mesh prior to treatment. (E) Bioluminescent image of infected mesh after treatment with central clearing where biofilm had been eradicated. (F) Mathematical model of the PEF treatment.
Figure 2
Figure 2
Bioluminescent images of mesh before and after treatment. Bioluminescent images of the infected prolene mesh before and after treatment with concentric ring electrodes. It is seen that in all (N = 3 treated mesh) 150 and 300 pulses treatments there is an effect that results in lower bioluminescent intensity in the images after treatment.
Figure 3
Figure 3
Scanning Electron Microscopy confirmed our results. Control untreated infected mesh demonstrated thick biofilm wedged into mesh interstices. Dense bacteria revealed production of exopolysaccharide. After treatment with PEF using the conentric electrodes, the biofilm has been disrupted and debris is left behind. The few remaining scant rods displayed abnormal morphology and exopolysaccharide was not visible. The mesh was not damaged by PEF treatment.
Figure 4
Figure 4
Log (Survival Ratio) of bacteria per treatment group. (A) Bioluminescent values measured from N = 3 mesh pieces per group. The average of all treatments per pulse group compared to the theoretical Weibull distribution as a fucntion of the electric field strength, is demonstrated. Survival rate decreases as the number of pulses and electric field strength increase. (B) An ANOVA test reveals that the P-value between the different conditions (pulse number) is 0.000056, which indicates that treatment efficacy directly correlates to number of pulses delivered. Treatment efficiency is defined by the % of eradicated bacteria, as measured by the % of bioluminescence reduction at the specific location.
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