Silver-zinc redox-coupled electroceutical wound dressing disrupts bacterial biofilm

Abstract

Pseudomonas aeruginosa biofilm is commonly associated with chronic wound infection. A FDA approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate gets activated to generate electric field (0.3-0.9V), was investigated for its anti-biofilm properties. Growth of pathogenic P. aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the WED. Scanning electron microscopy demonstrated that WED markedly disrupted biofilm integrity in a setting where silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED. Quorum sensing genes lasR and rhlR and activity of electric field sensitive enzyme, glycerol-3-phosphate dehydrogenase was also repressed by WED. This work provides first electron paramagnetic resonance spectroscopy evidence demonstrating that WED serves as a spontaneous source of reactive oxygen species. Redox-sensitive multidrug efflux systems mexAB and mexEF were repressed by WED. Taken together, these observations provide first evidence supporting the anti-biofilm properties of WED.

Conflict of interest statement

Competing Interests: Additional financial competing interest includes ownership of shares and paid consultancy with Vomaris Innovation, Inc. There is no board membership, employment or patent applications involved. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Anti-bacterial properties of WED.

(A) Energy Dispersive X-Ray Spectroscopy (EDS) elemental analysis of Ag/Zn WED. a. Scanning Electron Microscope (SEM) image; b. Light Microscope Image; c. and d. Close-up view of gold and silver dots under light microscope. e-h. EDS element maps of Zn (blue), Silver (red), Oxygen (green) and Carbon (Magenta). (B,C) Absorbance and CFU measurement from planktonic PAO1 culture in presence of placebo or Ag/Zn WED, n = 4 (D) (i) schematic diagram for experimental design showing dressing embedded in the agar plate. (ii, iii) Zone of inhibition above WED is marked with red dotted line, while no such zone was observed over the placebo dressing, n = 4 (E) Biofilm formation measured by absorbance at 540 nm, n = 4 (F) Biofilm co-aggregation observed in the placebo treated overnight PAO1 culture was not observed in WED treated overnight cultures, n = 3.

Fig 2. PAO1 staining demonstrates impaired biofilm formation by WED.

(A) Side-view and thickness of in-vitro static mature biofilm treated with placebo or WED, n = 3 (B) X,Y,Z projection of same (C) Orthogonal view of same at slices 6, 12 and 22 out of total 25 slices acquired.

Fig 3. WED impairs extracellular polymeric substance (EPS) production.

CLSM images of in-vitro static mature PAO1 biofilm grown treated with placebo, WED or Ag control and stained with EPS antibody, n = 3.

Fig 4. WED impairs biofilm structural integrity.

Scanning Electron Microscope images of in-vitro static mature PAO1 biofilm treated with placebo, WED or Ag control shown at 2500X, 5000X and 10000X magnifications, n = 3.

Fig 5. WED impairs cell viability.

CLSM micrographs of mature PAO1 biofilm stained with live-dead stain after treatment with placebo, WED or Ag control. The green fluorescence indicates live bacteria while the red indicates dead bacteria, n = 3.

Fig 6. WED generates superoxide (A) EPR spectra using 20mM DIPPMPO demonstrates spin adduct generation upon exposure to dressings for 40 mins in PBS.

Addition of 500U Catalase does not attenuate EPR signal but is attenuated using both 500U SOD and 500U Catalase, n = 3

Fig 7. WED represses redox-sensitive multidrug efflux genes in P. aeruginosa Real-time PCR was performed to assess mex gene expression post-treatment with placebo, WED or 3mM DTT, n = 3.

Fig 8. WED silences quorum sensing genes and pyocyanin production.

(A) Real-time PCR was performed to assess quorum sensing gene expression with RNA isolated from mature PAO1 biofilm exposed to placebo or WED. rpoD and proC was used as housekeeping control, n = 3. (B) Pyocyanin production (green color) in overnight PAO1 culture followed by exposure to placebo or WED, n = 3.

Fig 9. WED inhibits glycerol-3-phosphate dehydrogenase activity.

Measurement of glycerol-3-Phosphate dehydrogenase enzyme activity from mature PAO1 biofilms treated with placebo or WED A. OD measured in the kinetic mode. B. GPDH activity calculated using the formula: Glycerol-3-Phosphate dehydrogenase activity = B/(ΔT X V) x Dilution Factor = nmol/min/ml, where: B = NADH amount from Standard Curve (nmol). ΔT = reaction time (min). V = sample volume added into the reaction well (ml), n = 3.