Nisin powder

Nisin was purified as described by Abts et al 2011 [44] (link).
Briefly, commercially available nisin powder (Sigma) was dissolved in 50 mM lactic acid pH 3. The nisin solution was then purified using 5 ml HiTrap SP HP cation exchange column (GE Healthcare) pre-equilibrated with the same buffer. Nisin was eluted with 400 mM NaCl and monitored online at a wavelength of 215 nm, since nisin lacks aromatic amino acids in its sequence. In the last step, nisin was precipitated by TCA and dried out after washing it with cold acetone [44] (link). The concentration of nisin was measured by using RP-HPLC [45] (link).

Nisin–biogel was prepared as described elsewhere [3 (link),19 (link),20 (link),26 (link),51 (link)]. Briefly, a nisin solution of 1000 μg/mL was obtained by dissolving 1 g of nisin powder (2.5% purity, 1000 IU/mg, Sigma-Aldrich, St. Louis, MO, USA) in 25 mL of HCl (0.02 M) (Merck, Alges, Portugal). A 1.5% guar-gum biogel (w/v) solution was obtained by dissolving 0.75 g of guar gum (Sigma-Aldrich, St. Louis, MO, USA) in 50 mL of sterile distilled water, which was then heat sterilized via an autoclave. Afterwards, nisin was incorporated within the biogel in a proportion of 1:1, in order to obtain a gel with a final concentration of 200 µg/mL to be used in the clinical trial [26 (link),31 (link)]. The nisin–biogel concentration was selected based on previous reports regarding its cytotoxicity [20 (link)]. For posology establishment, standard procedures for PD prevention were considered [52 (link),53 (link)].

A stock solution of nisin (1000 μg/mL) was obtained by dissolving 1 g of nisin powder (2.5% purity Sigma-Aldrich, USA) in 25 mL of HCl (0.02 M) (Merck, Germany), filtered using a 0.22 μm cellulose acetate membrane filter and stored at 4°C. The stock solution was then diluted with sterile water to a concentration of 45 μg/mL.
A guar gum gel 1.5% (w/v) was prepared by dissolving 0.6 g of guar gum (Sigma-Aldrich, USA) in 40 mL of sterile distilled water and heat sterilized by autoclave. The solution of nisin was incorporated within the guar gum gel in a proportion of 1:1, obtaining a final 0.75% (w/v) biogel with 22.5 μg/mL of nisin.
Regarding antibiotics solutions, 6.6, 4.76 and 10.62 mg of Clindamycin (Cayman, USA), Gentamicin (PanReac AppliChem, USA) and Vancomycin (PanReac AppliChem, USA), respectively, were dissolved in 10 mL of sterile water and filtered through a 0.22 μm cellulose acetate membrane filter. Stock solutions were kept frozen at -80°C and diluted to the final concentrations of 0.033 μg/mL for Clindamycin, 0.238 μg/mL for Gentamicin and 0.531 μg/mL for Vancomycin, prior to utilization.

A stock solution of nisin (1000 µg/mL) was obtained by dissolving 1 g of nisin powder (2.5% purity, 1000 IU/mg, Sigma-Aldrich, St. Louis, MO, USA) in 25 mL of HCl (0.02 M) (Merck, Darmstadt, Germany). This solution was sterilized by filtration through a 0.22 µm cellulose acetate membrane filter (Millipore, Burlington, MA, USA) and stored at 4 °C [27 (link)].
Brain Heart Infusion (BHI) or Trypticase Soy Broth (TSB) with guar-gum gel at 0.75% (w/v) were prepared by dissolving 3.75 g of guar-gum (Sigma-Aldrich, St. Louis, MO, USA) and 18.5 g of BHI powder (VWR Chemicals, Leuven, Belgium) or 15 g of TSB powder (VWR Chemicals, Leuven, Belgium), respectively, in 500 mL of sterile distilled water, and heat sterilized by autoclave [27 (link)].
Clindamycin is an alternative antibiotic currently used in clinical practice associated with mild DFIs and was used in the present study at 1/2 MIC as a control for comparing the effects of nisin-biogel at sub-MICs on S. aureus DFI isolates virulence factors expression [28 (link)]. A stock solution of clindamycin was obtained by dissolving 6.6 mg of clindamycin powder (Sigma-Aldrich, St. Louis, MO, USA) in 10 mL of sterile water and filtered using a 0.22 μm cellulose acetate membrane filter. This stock solution was kept frozen at −80 °C and diluted with sterile water to the final concentration of 0.0165 µg/mL when required.

Penicillin, ampicillin, gentamicin, kanamycin, roxithromycin, sulfapyridine, streptomycin, vancomycin, chloramphenicol, cefuroxime, cephazolin, ceftriaxone, cefepime, metronidazole, ciprofloxacin, polymyxin, imipenem, and linezolid were prepared at the concentration of 4,096 mg/L (all antibiotics were obtained from the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China). Nisin stock solution was obtained by dissolving 1 g of nisin powder (2.5% purity, 1000 U/mg, Sigma-Aldrich, St. Louis, MO) in 25 ml of dilute HCl (pH 2). All of the antimicrobials were filtered using a Millipore filter with a 0.22 µm pore size.

D-methionine, D-tryptophan, D-leucine, D- or L-cysteine, D- or L-aspartic acid, D- or L-glutamic acid, D-alanine, D-threonine, D-valine, D-isoleucine, D-glutamine, D-phenylalanine, D-histidine, D-proline, D-lysine, D-arginine, and glycine (Sigma-Aldrich, St. Louis, MO, USA) were all prepared at a concentration of 40 mM, and D-tyrosine was prepared at a concentration of 0.8 mM due to its low solubility. Nisin powder (2.5% purity, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a solution at pH 2 to obtain a stock solution with a concentration of 1 g/L.