Growth medium, chemicals, and antibiotics

For all experiments, 20 g/L (1×) Luria broth (LB) medium (Sigma-Aldrich, catalog #L3522) was used to grow overnight P. aeruginosa PAO1 cultures and 1 g/L (0.05×) LB was used as the growth medium for biofilms. Tobramycin (Sigma Aldrich, catalog #T4014) and ciprofloxacin (Sigma Aldrich, catalog #17850) solutions were diluted in 1 g/L (0.05×) LB for antibiotic susceptibility experiments and persister cell isolation, respectively. Minimum inhibitory concentrations (MICs) were determined for both antibiotics following the Clinical and Laboratory Standards Institute (CLSI) protocol as detailed in Supplementary information.50 Other key compounds included 3′-p-hydroxyphenyl fluorescein dye (Invitrogen, catalog #H36004), propidium iodide (Invitrogen, catalog #L-7012), 3, 3′-dipropylthiacarbocyanine iodide (Sigma Aldrich, catalog #318434), thiourea (AK Scientific, Inc., catalog #S726) and H 2 O 2 (VWR, catalog #RC3819-16).

Culture and biofilm preparation

Frozen stocks of P. aeruginosa PAO1 were cultured overnight in LB at 37 °C on a rotating table (70 rpm). For biofilm experiments, overnight culture was adjusted to OD 600 ≈ 0.5 in LB and used as inoculum.17 Briefly, 2 ml of culture was used to inoculate sterile glass bottom petri dishes (MatTek Corporation, catalog #P35G-1.5-20-C) and allowed to form biofilms for 24 h. These one-day-old biofilms were treated with e-scaffolds for 24 h, as described previously.17 Briefly, untreated and e-scaffold treated biofilm cells were washed twice to remove loosely attached cells and then remaining cells were recovered for antibiotic susceptibility testing. To identify and isolate P. aeruginosa PAO1 persister cells, planktonic culture was grown to the stationary phase and the dose-dependent killing curve for ciprofloxacin (0–200 μg/mL) was investigated (Supplementary information, Fig. S1).34 A plateau of the surviving subpopulation was observed for ciprofloxacin concentrations above 50 μg/mL (200× of MIC), and these were identified as persister cells.34,51 We assessed the antibiotic treatment on biofilms from three different populations: cells recovered after no treatment (“untreated biofilms”), cells recovered after e-scaffold treatment (“e-scaffold treated biofilm”) and “persister cells” that were isolated from biofilms treated with 200 μg/mL ciprofloxacin for 3.5 h according to a published protocol.34 For experiments with persister cells, the ciprofloxacin-treated biofilms were washed and refreshed with 0.9 % NaCl for subsequent experiments. This method for culture, biofilm preparation and treatments is illustrated in Fig. 6.

Fig. 6 a Schematic of experimental setup for the treatment of biofilm exposed to an e-scaffold with an illustration of electrochemical H 2 O 2 production. The electrodes are connected to a potentiostat (not shown in figure). Scientific Reports, 2015. 5, 14908. DOI: 10.1038/srep14908. © Creative Commons Attribution 4.0 International License. b Recovered biofilm cells treated with e-scaffold and tobramycin. c Treatment of biofilms and persister cells with e-scaffold Full size image

Biofilm treatment with e-scaffold

P. aeruginosa PAO1 biofilms were exposed to e-scaffold treatment for 24 h, as described previously.17 Briefly, a custom-built e-scaffold was fabricated using carbon fabric as detailed in Supplementary information.17 Biofilms were carefully washed (2×) with LB (0.05×) and an e-scaffold was overlaid onto the biofilms followed by 4 mL of fresh medium. A standard Ag/AgCl (saturated KCl) reference electrode was introduced to apply a constant potential (-600 mV Ag/AgCl ) to an e-scaffold using a Gamry Series G 300 potentiostat (Gamry Instruments, Warminster, PA, USA) to reduce oxygen, generate a low concentration of H 2 O 2 and deliver it continuously to the biofilms (Fig. 6a). After treatment, the viable cell counts were determined using a modified drop-plate cell counting method.17,52 Loosely attached cells were removed by carefully washing the biofilms with 0.9 % NaCl before they were resuspended in 5 mL of 0.9 % NaCl and vortexed for 30 s. These suspensions were centrifuged (4180 × g for 10 min), and the resulting cell pellet was resuspended in 1 mL of 0.9 % NaCl. Aliquots (250 µL) were then serially diluted, and 10 µL of each dilution was plated onto LB agar. Plates were incubated for 24 h (37 °C), and colony-forming units (CFU) were enumerated.

Tobramycin susceptibility of e-scaffold treated cells

To determine whether e-scaffold treatment altered susceptibility to antibiotics,35 biofilm treatments were combined with tobramycin treatments. For this experiment, the recovered biofilm cells were harvested (Fig. 6b) and adjusted to OD 600 ≈ 0.5 in LB medium (0.05×). A 1-mL cell suspension was used to inoculate a 24-well plate, where biofilms were allowed to form. Biofilms were treated with an e-scaffold for 2 h. Each well was then washed (2×) and challenged with 1 mL of one of the test concentrations (5, 10, 20 and 40 µg/mL) of tobramycin for 6 h. After treatment, the cells were washed (2×), resuspended in 0.9 % NaCl and processed to enumerate CFU.52 Cell counts were compared for e-scaffold and other treated and untreated biofilms. The treatments are summarized in Fig. 6b.

Effect of e-scaffold on persister cells

Biofilms were grown for 24 h in 6-well plates and treated with 200 μg/mL ciprofloxacin for 3.5 h to isolate persister cells from ciprofloxacin-treated biofilms.16 The total number of viable cells was determined for ciprofloxacin-treated and untreated biofilms using the modified drop-plate cell counting method.52 Remaining persister cells were then exposed to either 200 μg/ml ciprofloxacin or e-scaffold treatment for 6 h (in the 0.9 % NaCl solution). Final CFU numbers were then determined following the procedure described above. Figure 6c summarizes the treatment methodology we used.

Hydroxyl free radical detection assay

Intracellular hydroxyl free radical (OH•) formation was detected using 5 µM of a fluorescent reporter dye, 3′-(p-hydroxyphenyl fluorescein) (HPF) (Invitrogen, catalog #H36004) following a published protocol.38 Briefly, e-scaffold treated, exogenous H 2 O 2 -treated and untreated biofilm cells were vortexed in 500 µL of LB in a microcentrifuge tube for 30 s. These samples were centrifuged (10,000 × g for 10 min), and then the medium was replaced with a final concentration of 5 µM HPF prepared in 500 µL of 0.1 M PBS. After staining in the dark at room temperature for 15 min, samples were centrifuged at 10,000 × g for 10 min. Supernatant was removed, and cells were rinsed and resuspended with PBS. An aliquot (100 µL) was added to each well in a 96-well plate, and fluorescence intensity was quantified using a microplate reader (Bio-Tek Cytation 5) with excitation at 490 and emission at 515 nm. For the OH• formation assays, fluorescence was estimated as follows: ((Fluorescence with dye−Fluorescence without dye)/(Fluorescence without dye))×(100).

Membrane permeability

Change in bacterial membrane permeability was evaluated by analyzing the influx of a membrane-impermeable dye, propidium iodide (PI)22,38 according to the manufacturer’s instructions (Invitrogen, catalog #L-7012). E-scaffold treated and untreated biofilm cells were centrifuged (6000 rpm, 10 mins) to cell pellets and supernatant was poured off. The cell pellets were stained with PI for 15 min in the dark, then washed twice with 0.9 % NaCl to remove any unbound dye. Cells were then resuspended in 0.9 % NaCl, and 100 µL of each suspension was transferred in triplicate into wells of a 96-well plate. Fluorescence intensity was quantified (excitation 535 nm, emission 517 nm). Fluorescence was determined as a percentage change compared to untreated sample using the following formula: ((Fluorescence with dye−Fluorescence without dye)/(Fluorescence without dye))×(100).

Additionally, the effect of e-scaffold treatment on bacterial membrane permeability was detected using a membrane potential sensitive dye, 3, 3′-dipropylthiacarbocyanine iodide. The fluorescence intensity of 3, 3′-dipropylthiacarbocyanine iodide changes in response to changes in plasma membrane potential upon structural damage.42,53 Untreated biofilm cells were washed and then resuspended in buffer A (20 mM glucose, 5 mM HEPES, pH = 7.2) with 0.1 M KCl. To achieve a stable signal, 3,3′-dipropylthiacarbocyanine iodide was added to untreated and e-scaffold treated biofilm cell suspensions and incubated for 10 min. Changes in the fluorescence intensity induced by changes in membrane potential after exposure to e-scaffold were measured at an excitation wavelength of 622 nm and an emission wavelength of 670 nm.42 The measurements were performed in triplicate for three biological replicates in wells of a 96-well plate. Fluorescence was determined as a percentage change compared to untreated sample using the following formula: ((Fluorescence with dye−Fluorescence without dye)/(Fluorescence without dye))×(100).

Scanning electron microscopy

For scanning electron microscopy (SEM) imaging, biofilms were grown for 24 h on UV-sterilized, 0.22-µm type GV membrane filters (Millipore, catalog ID #551200401) placed in sterile 6-well plates. Exogenous H 2 O 2 was added continuously at an average 0.008 mmol/h for 24 h to mimic the e-scaffold generated H 2 O 2 treatment described previously.17 After treatment for 24 h, both e-scaffolds and membrane filters with biofilms were aseptically collected from the untreated, e-scaffold treated and exogenous H 2 O 2 treated wells. The membrane filters and e-scaffolds were fixed overnight with 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 M phosphate buffer, followed by rinsing with 0.1 M phosphate buffer (3 × 10 min each). The membranes and e-scaffolds were then dehydrated gradually by being washed sequentially with 10, 30, 50, 70, and 95 % alcohol (10 min each) and 100 % alcohol (3 × 10 min each). Hexamethyldisilizane (HMDS) was used for overnight drying. Samples were then sputter-coated with gold prior to field emission in-lens scanning electron microscopy (FEISEM) (FEI 200F) imaging.

Statistical analysis

All experiments were conducted for at least three biologically independent replicates. Technical replicates were averaged to produce replicate means that were subsequently used for analysis. Mean values were compared within and between groups using one-way ANOVA followed by Bonferroni’s post hoc test for individual comparisons. Differences were considered statistically significant if P < 0.05 (Sigma plot, version 13, Systat Software, Inc., San Jose, CA).