2.3. Biofilm inhibition assay

The long-term macro colony growth assay was performed as recently described by Stepanov et al. (2022) to detect biofilm inhibition by screening for the production of curli fibers and cellulose as these are essential components of E. coli biofilm formation (Stepanov et al., 2022). One milliliter Congo red agar was filled in the wells of 24-well plate containing either test substances (100, 75, 50, 25, 12.5, 10, 7.5, 5, 2.5, 1.25 μg ml⁻¹) or solvent control (SvC DMSO [<2%]). One well without any content was used as a growth control (GC) accounting for 100% of the biofilm production. Five microliter of bacteria suspension with an optical density of 0.5 (approx. 8∙10⁷ CFU ml⁻¹) were dropped into the center of each well. The prepared plates were incubated under aerobic static conditions for 48 h at 28°C. Inhibition of biofilm formation was determined visually based on the colony phenotypes. There was deemed to be no inhibition of biofilm formation if the colonies had the same structure as the growth control (red and rough) after treatment. If the colonies looked smooth and only slightly red, biofilm inhibition was present (see Figure 3A). The experiment was conducted with five independent biological replicates, and the minimal biofilm inhibitory concentration (MBIC) was calculated as the mean of five followed by a statistical one-way ANOVA test with Dunnett’s multiple comparison using GraphPad Prism 10.0.1 software.

(A) Outcome of biofilm inhibition experiments on solid span agar stained with Congo red and Coomassie Brilliant Blue G250 in 24-well plates. The bacteria (E. coli) were grown for 48 h at 28°C. Treatments were filled in wells prior to the addition of liquefied agar and comprised epigallocatechin gallate (EGCG), octyl gallate (Oct), scutellarein (Scu) and wedelolactone (Wed) at decreasing concentrations (100, 50, 25, 12.5, 7.5, and 1.25 μg ml⁻¹). The plates contained one well without anything added (sterility control) and growth controls with 10 μl (final concentration 1%) DMSO. (B) Minimal biofilm inhibitory concentration (MBIC) and minimal inhibitory concentration (MIC) of natural compounds against E. coli PBIO729. MIC was investigated in MH-II-broth and detected visually after growth at 37°C for 24 h, while MBIC was determined in solid agar (see left picture). The dots depict the average of three (MIC) or five (MBIC) independent replicates. * The MIC was not detectable within the chosen concentration.

The same assay conditions were used for the quantification of the biofilm inhibition, but instead of Congo Red and Coomassie Brilliant Blue, Calcofluor White M2R was added to stain the cellulose. Subsequent to incubation, fluorescence intensity was measured using the Omega Fluostar spectrophotometer (BMG LABTECH GmbH, Ortenberg, DE). The excitation wavelength was set at 400 nm and emission was measured at 480 nm with a fixed Gain of 500. A well scanning method with 15 × 15 points was used, and a manually selected matrix of 6 × 6 values formed the basis for an average detection of biofilm formation in the colonies. This is necessary because the colonies do not always have the same position in the well and to ensure, that only the colony is detected without any agar. The test was performed three times independently and the intensity was calculated as mean ± standard deviation. For statistical evaluation a two-way ANOVA test with Dunnett’s multiple comparison was performed with the control DMSO using GraphPad Prism.