Redox-active conducting polymers modulate Salmonella biofilm formation by controlling availability of electron acceptors

Abstract

Biofouling is a major problem caused by bacteria colonizing abiotic surfaces, such as medical devices. Biofilms are formed as the bacterial metabolism adapts to an attached growth state. We studied whether bacterial metabolism, hence biofilm formation, can be modulated in electrochemically active surfaces using the conducting conjugated polymer poly(3,4-ethylenedioxythiophene) (PEDOT). We fabricated composites of PEDOT doped with either heparin, dodecyl benzene sulfonate or chloride, and identified the fabrication parameters so that the electrochemical redox state is the main distinct factor influencing biofilm growth. PEDOT surfaces fitted into a custom-designed culturing device allowed for redox switching in Salmonella cultures, leading to oxidized or reduced electrodes. Similarly large biofilm growth was found on the oxidized anodes and on conventional polyester. In contrast, biofilm was significantly decreased (52-58%) on the reduced cathodes. Quantification of electrochromism in unswitched conducting polymer surfaces revealed a bacteria-driven electrochemical reduction of PEDOT. As a result, unswitched PEDOT acquired an analogous electrochemical state to the externally reduced cathode, explaining the similarly decreased biofilm growth on reduced cathodes and unswitched surfaces. Collectively, our findings reveal two opposing effects affecting biofilm formation. While the oxidized PEDOT anode constitutes a renewable electron sink that promotes biofilm growth, reduction of PEDOT by a power source or by bacteria largely suppresses biofilm formation. Modulating bacterial metabolism using the redox state of electroactive surfaces constitutes an unexplored method with applications spanning from antifouling coatings and microbial fuel cells to the study of the role of bacterial respiration during infection.

Fig. 1

Chemical structures and electrochemical redox reactions in conducting polymers. a Chemical structure of the conducting polymer polyacetylene (left) and its resonance hybrid (right) depicting the overlapped p-orbitals of the resultant conjugated system. b Flux of ions in a conducting polymer-based electrochemical cell. M⁺ and X⁻ represent positively and negatively charged ions, respectively. The blue color of the anode and purple color of the cathode represent the electrochromism effect occurring in the oxidized and reduced conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT). c Chemical structure of PEDOT. d Chemical structure of heparin. e Chemical structure of sodium dodecylbenzenesulfonate (NaDBS)

Fig. 2

Characterization of electropolymerized composites. a–c Photographs illustrating the opacity of a PEDOT:Cl, b PEDOT:Heparin and c PEDOT:DBS fabricated for indicated time (s) and currents (µA). The resulting polymerization charge (C) is shown in white on each composite. d Electrical sheet resistance (Ω/sq) of PEDOT:Cl, PEDOT:Heparin and PEDOT:DBS composites fabricated at indicated polymerization charges (C). Results expressed as mean ± SEM (n = 3). e–g Cyclic voltammograms of e PEDOT:Cl, f PEDOT:Heparin and g PEDOT:DBS. Each color represents a composite fabricated at specific electropolymerization charges (0.0–1.26 C) as shown in the inset. h Charge storage capacity per electrode surface unit (mF/cm²), calculated from the cyclic voltammograms shown in (e–g), for PEDOT:Cl, PEDOT:Heparin and PEDOT:DBS. Results expressed as mean ± SEM (n = 3). i Surface hydrophobicity, measured as water contact angle (°), of the Orgacon™ starting material (0.0 C) and the PEDOT:Cl, PEDOT:Heparin and PEDOT:DBS composites fabricated at increasing electropolymerization charges. Results expressed as mean ± SEM (n = 3)

Fig. 3

Bacterial cultivation device for integrated redox switching. a Photograph of the bacterial cultivation device, which consists of a modified 12-well plate. The four wells in one row are inoculated with the same bacterial culture, and wells in the other row are inoculated with LB without salt. Electrical connections are made to the two outermost wells, enabling redox switching of the wall-attached electrodes. One well is left without electrical connections to provide the unswitched control. The fourth well contains wall-attached, non-conductive polyester surfaces serving as positive control for biofilm formation. The inoculated, electrically addressed device is covered with its lid and an open transparent box placed upside down as extra protection during incubation. b Close-up on the positioning of two electrodes of the same PEDOT composite inside a well. c Electrical connection of the electrodes made just above the well hinders any contact with the liquid culture. d Characterization of the electrical current circulating in a well modified with two electrodes of PEDOT:Cl (blue), PEDOT:Heparin (red) or PEDOT:DBS (green) upon application of a 0.5 V voltage step (dashed line). LB without salt was used as supporting electrolyte

Fig. 4

Visualization and quantification of Salmonella surface biofilm formed on redox active PEDOT composites. a Photograph of a crystal violet-stained polyester surface showing a narrow, distinct purple band (marked with arrow) representing the biofilm formed after 24 h on the surface at the air-liquid interface. Residues from the pellicle biofilm show as smeared, purple stain in the upper half of the surface. b Schematic, cross-section representation of surface biofilm and pellicle biofilm at the air-liquid interface in a well from the experimental device. c Visual inspection of surface biofilm formed on the three different PEDOT composites in different electrochemical states. Representative photographs of crystal violet-stained PEDOT composites are shown. d Quantification of surface biofilms formed on PEDOT composites under different electrochemical conditions. Absorbance at 595 nm was recorded after extraction of crystal violet bound to each surface biofilm. Results are expressed as mean ± SEM. Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) from ANOVA analysis and Tukey’s HSD. Ox  =  oxidized composite, Red = reduced composite, Uns = unswitched composite. e Photographs of unswitched PEDOT:Cl composites immediately after their removal from a biofilm culture (“Bacteria”) and from LB without salt (“Medium”). f Average color intensity of the red (R), green (G) and blue (B) channels of an area at the air-liquid interface in the PEDOT:Cl composites. Results are expressed as mean ± SEM (n = 3). Statistical significance (*p < 0.05) from MANOVA analysis and Wilks’ lambda. a.u. = arbitrary unit

Fig. 5

Proposed mechanisms for the electrochemical modulation of biofilm formation. a Prior to any bacterial interaction, an external addressing oxidizes the anode (light blue, left electrode), leaving electron holes (dotted-line circle) in the material. Conversely, the cathode (purple, right electrode) becomes reduced, i.e., the material is fully saturated with electrons (dotted-line circle with e⁻). b As the external addressing is continuously applied, the anode acts as a continuously renewable electron sink, always providing available sites for bacterial electron transfer (black arrows). This creates a favorable milieu for bacteria to attach and form biofilms on the anode. Conversely, an electron-saturated interface is presented at the cathode, preventing bacterial electron transfer. This hinders bacterial attachment and biofilm formation. c The biofilm continues to mature in the anode. In the cathode, alternative factors like hydrophobicity, electrostatic interactions and the use of alternative electron acceptors eventually lead to bacterial attachment and biofilm formation, although at a reduced rate. d Pristine, unswitched polymers are in a partially oxidized state (dotted-line circle and dotted-line circle with e⁻) prior to any bacterial interaction. e In the absence of an external addressing, bacterial electron transfer quickly reduces the conducting polymer. As the material now presents an electron-saturated interface that prevents bacterial electron transfer, bacterial attachment and biofilm formation are hindered. f Similarly to the cathode in c, alternative factors like hydrophobicity, electrostatic interactions and the use of alternative electron acceptors eventually lead to bacterial attachment and biofilm formation, although at a reduced rate