Egg yolk emulsion

The artificial sputum media was prepared as previously described^{56 (link),57} using DNA from fish sperm (Sigma-Aldrich), mucin from porcine stomach (Sigma-Aldrich), the iron-chelator diethylene triamine pentaacetic acid (Sigma-Aldrich), NaCl (Sigma-Aldrich), KCl (Sigma-Aldrich), egg yolk emulsion (Sigma-Aldrich) and all essential and non-essential amino acids (Fisher Scientific, Loughborough, UK and Sigma-Aldrich). Similarly to the MIC assay in LB, the bacterial isolates LESB65, pmrB WT, pmrB SNP and ∆pmrB were grown in LB agar plates at 37 °C for 18 h. Following incubation, a single colony was inoculated into 5 ml of LB and incubated for 18 h at 37 °C and 180 r.p.m. The overnight culture was diluted into fresh LB broth to an OD₆₀₀ of 0.05. A 1:100 dilution of the bacterial solution was done in sterile ASM and 1.8 ml of the solution was incubated with 200 µl of engineered lysozyme^{25 (link)} or tobramycin (Flynn Pharma LTD, Hertfordshire, UK) at different concentrations for 24 h at 37 °C and 50 r.p.m. Following incubation, the ASM biofilm was dissolved using Sputasol (Oxoid, Basingstoke, UK). The CFU from each biofilm was determined using the Miles and Misra technique^{58 (link)}.

Poly(lactide-co-glycolide) (PLGA) 50:50 (RG 502H) (M_w = 7–17 kDa; lactide/glycolide = 50:50) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (Protasan UP G-113; M_w < 200 kDa) was purchased from Novamatrix, Norway. Docetaxel was kindly donated by ILKO, Turkey. Ethyl acetate, dialysis cellulose tubing membrane (average flat width 25 mm, MWCO 14,000 Da), gelatin type B from bovine skin, mucine from porcine stomach (type II), diethylenetriaminepentaacetic acid (DTPA; min 99%, titration), and egg yolk emulsion were purchased from Sigma-Aldrich, USA. All other chemicals used were of analytical grade and obtained from Sigma-Aldrich.

The poly(lactide-co-glycolide) (PLGA) (Resomer^® RG 502H uncapped PLGA 50:50, inherent viscosity 0.16–0.24 dL/g) was acquired from Boehringer Ingelheim (Ingelheim, Germany). Poly(lactic-co-glycolic acid) copolymer-Rhodamine-B conjugate (Mn 10,000–30,000 Da) (PLGA-Rhod) was purchased from PolyScitech (Akina division, Inc., West Lafayette, IN, USA). Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) with PEG average Mn 2000 Da, PLGA Mn 4500 Da and with PEG average Mn 5000 Da, PLGA Mn 7000 Da, diethylenetriaminepentaacetic acid (DTPA), DNA, egg yolk emulsion, poly(L-lysine), RPMI amino acid solution and type II mucin from porcine stomach were purchased from Merck-Sigma Aldrich (St. Louis, MO, USA). Bacterial alginate from the fermentation of Azotobacter vinelandii or Pseudomonas mendocina was purchased from Carbosynth (UK). Methylene chloride and ethanol were supplied by Carlo Erba (Italy). All salts and reagents were of analytical grade or higher.

The lipase and phospholipase activities were measured by a modification of the method described by Austin et al. (2005) (link). Agar plates for lipase and phospholipase assays were prepared by supplementing Marine Agar with 1% Tween 80 (Sigma-Aldrich, Belgium) and 1% egg yolk emulsion (Sigma-Aldrich, Belgium), each sterilized separately at 121°C for 5 min before mixing. The development of opalescent zones around the colonies was observed and the diameter of the zones was measured after 2–4 days of incubation at 28°C.

All culture media and other chemicals used in this study were of high analytical grade. Sabouraud dextrose broth (Cat No-S3306), Sabouraud dextrose Agar (Cat No-84088), RPMI-1640 Medium (Cat No-R6504), Phosphate buffered saline (Cat No-P4417), Fetal Bovine Serum (Cat No-F3885), Agar (Cat No-05040), Bovine Serum Albumin (Cat No-A9418), Yeast Nitrogen Base without Amino Acids (Cat No-Y0626), Ammonium sulphate (Cat No-A4418), Glucose (Cat No-G8270), Peptone (Cat No-77199), Sodium chloride (Cat No-S1679), Calcium chloride (Cat No-C1016), Egg Yolk Emulsion (Cat No-17148), MOPS (Cat No-M1254), XTT (Cat No-X4626), Menadione (Cat No-M5625), Acetone (Cat No-650501) were purchased from Sigma – Aldrich, St. Louis, MO, USA.

N-acetylcysteine (NAC), bromelain, ascorbic acid, Ribes nigrum, resveratrol, and pelargonium were kindly donated by Anvest Health s.r.l. (Castel San Giorgio, Salerno, Italy). Deoxyribonucleic acid (DNA), mucin from the porcine stomach, diethylenetriaminepentaacetic acid (DTPA), RPMI 1640 amino acids solution, egg yolk emulsion, sodium chloride, and potassium chloride, used for artificial mucus preparation, and barbital buffer were purchased from Sigma-Aldrich (Milan, Italy), while the hydroxyethylcellulose was obtained from A.C.E.F. SpA (Piacenza, Italy). Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were acquired from Biolife (Milan, Italy); trypticase soy broth (TSB) was obtained from Difco Laboratories, and phosphate-buffered saline (PBS) from GIBCO, Thermo Fisher Scientific. All other solvents and chemicals were of analytical grade.