Date of Award

5-30-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Dacheng Ren

Keywords

Biofilms, Direct Current (DC), Electrodes, Metal cations, Persisters, Rabbit model

Subject Categories

Engineering

Abstract

The emergence of antibiotic-resistant bacteria has presented an increasing challenge to infection control. Conventional methods of antibacterial treatment involving high dose of antibiotics or surgical intervention have proven insufficient for eradicating persistent infections, such as those associated with medical implants. It is well recognized that bacterial populations commonly contain a small percentage of phenotypic variants, known as persister cells, which are metabolically inactive and extremely tolerant to antibiotics. When the antibiotic treatment is stopped, surviving persister cells can regenerate the bacterial population with a similar percentage of persister cells. Thus, persistence presents a great challenge to curing chronic infections.

In this study, we introduced a novel method for controlling bacterial persistence based on a phenomenon we named electrochemical control of persister cells (ECCP). We demonstrate that bacterial persister cells can be effectively eliminated by low-level direct currents (DCs); e.g. treatment with 70 μA/cm2 DC for 1 h using stainless steel (SS) 304 reduced the number of viable planktonic persister cells of Pseudomonas aeruginosa PAO1 by 98% compared to the untreated control. In addition to persister killing by applying DC alone, synergistic effects were observed when treating persister cells with 70 μA/cm2 DC and 1.5 μg/mL tobramycin together using SS 304 electrodes. The same level of DC was also found to be cidal to biofilm associated persister cells of P. aeruginosa PAO1. Based on this discovery, the electrophysiological properties of P. aeruginosa PAO1 cells treated with 70 μA/cm2 DC using carbon and SS 304 electrodes were characterized both at the cellular and genetic levels to understand the mechanism of ECCP. We found that DC treatments affected surface charge and membrane integrity of P. aeruginosa, leading to increase intracellular concentration of metal cations as observed via scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) analysis. Moreover, electrochemical treatments mediated via carbon electrodes provoked the permeabilization of the cells to extracellular materials, and increased their susceptibility to antibiotics, which led to complete eradication of the persisters. These findings are corroborated by DNA microarray analysis, which revealed that DC treatments have profound effects on the physiology of persister cells, altering the regulation of genes involved in antibiotic resistance, pyocin-related functions, and SOS response. Comparative transmission electron microscopy (TEM) studies of the stationary phase P. aeruginosa PAO1 cells confirmed that DC treatments resulted in the compartmentalization of intracellular contents, release of outer membrane vesicles, or cell lysis.

To design novel systems to effectively control infections associated with biofilms and persister cells, the safety and the efficacy of ECCP were evaluated in co-culture models with human epithelial cells and P. aeruginosa PAO1. In addition, a pilot animal study was conducted to investigate the effects of electrochemical currents using a rabbit model of sinus infections. P. aeruginosa PAO1 was used to induce rhinosinusitis in rabbits, which were then treated with antibiotics, or antibiotics with electrical current, and compared with the untreated controls. The results of this study validated the effectiveness of electrochemical treatment in reducing both biofilms and planktonic cells in vivo. Overall, these findings improved the understanding of the electrophysiology of bacterial persister cells, and provided new insight for designing novel systems to effectively control infections associated with biofilms and persister cells.

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