Naringin

P. aeruginosa PAO1 was grown overnight in LB medium at 37°C with agitation. After growth, the culture of P. aeruginosa PAO1 was diluted with Biofilm Broth (BB) medium as described by Khalilzadeh et al. [44 (link)] and 25 μl of the diluted culture was added to 470 μl of BB medium (initial A_600nm of culture comprised between 0.14 and 0.16) supplemented with 5 μl of DMSO (1%, v/v) or OALC (200 μM) or naringenin (4mM) or naringin (4mM). Planktonic bacteria were transferred in sterile tube and assessed for cell counting (colony-forming units, C.F.U). Adherent biofilms were washed three times with water (2mL) and fixed with 2 mL of methanol (99%). After 15 min, the methanol was discarded, and the plates were dried at room temperature. Crystal violet (0.1% in water) was then added to each well (2 mL/well), and the plates were incubated for 30 min at room temperature. Crystal violet was then discarded, and stained biofilms were washed three times with 1 mL of water. Acetic acid (33% in water) was added to the stained biofilms (2 mL) in order to solubilize the crystal violet, and the absorbance of the solution was measured at 590 nm with a SpectraMax M2 device (Molecular Devices).
The biofilm formation by PAO1 cells was also examined in glass coverslips cultures by fluorescence microscopy. Two distinct assays were adopted in order to assess the effects of compounds in biofilm development and in one-day-old biofilm. In both cases, the bactericidal activities of tobramycin in one-day-old biofilm-encapsulated PAO1 cells was also assessed. Tobramycin was chosen because it has been shown that QS inhibition greatly enhances the sensitivity of P. aeruginosa to this antibiotic and increases clearance of P. aeruginosa in a foreign-body infection model [28 (link), 45 (link)]. First assay follows the same culture conditions as described above. After 24 h incubation, tobramycin (100 μg mL^-1) was added to 1-day-old treated biofilms. The biofilm development and bacterial viability in biofilms were assessed using the LIVE/DEAD baclight bacterial viability kit (Invitrogen, Molecular probes). The growth medium was removed and replaced by 500 mL of a solution of SYTO 9 and propidium iodide diluted 400 fold in BB medium. Biofilms were incubated for 15 min and PAO1 cells were examined using a Leica DM IRE2 inverted fluorescence microscope coupled to a CCD camera (Leica DC350 FX) and equipped with FITC and Texas red filters. To estimate the % viability of biofilm-encapsulated bacteria for each treatment, the glass coverslip was submerged in 2 mL of PBS solution and sonicated (WVR Ultrasonic cleaner, HF45KHz, 80W) for 1 min in order to unbind the biofilm. The collected biofilm suspension was then assessed for viability using LIVE/DEAD baclight bacterial viability kit (Invitrogen, Molecular probes) following fluorescence microplate reader protocols as described by the manufacturer. The integrated intensities of the green (530 nm) and red (630 nm) emission of suspensions excited at 485 nm were acquired using SpectraMax M2 device, and the green/red fluorescence ratios (Ratio G/R) were calculated and reported to the linear curve obtained from the relationship between % live bacteria and Ratio G/R of biofilm-encapsulated PAO1 cells grown without tobramycin.
For the second assay, PAO1 cells were grown statically in BB medium for 24 hours at 37°C in 24-well polystyrene plates to form biofilm. Tested molecules as described above and/or tobramycin (100 μg mL^-1) were added and incubated for a further 24 hours and the biofilm development and bacterial viability in biofilms were assessed as described for the first assay.

This study was conducted on one candidate biomarker (naringin) identified in Seville oranges (the tolerant cultivar). A 20 000 mg/L stock solution of naringin was prepared and the in vitro exposure to 1 000, 3 000, 4 000, 5 000, 7 500 and 10 000 mg/L concentrations of naringin was used to evaluate the antifungal properties of the candidate biomarker. In addition, three fungicides, Demildex, MHTCO₃ and cuprous oxide, were tested at 2000 mg/L, 10 000 mg/L and 18 000 mg/L respectively and served as positive controls while water was used as negative control. The CSA media and inoculation were performed as mentioned in Sect. 3.9.1. For each compound, 10 replicates of each concentration tested were prepared. After 14 days of incubation at 27 °C ± 2 °C, mycelial growth was measured (in mm) with a digital calliper (Absolute Digimatic-Mitutoyo Corp. Japan). Percentage inhibition of mycelial growth was determined as described by Plaza et al. (2004 (link)).