Tryptic soy broth (tsb)

All seeded samples isolated from fish, water and biofilm were incubated at 22 °C for 48 h. Then, up to five yellow colonies, which were often surrounded by a yellow zone (depigmentation of the GSP medium), were removed per fish organ, and two colonies from biofilm and water samples. Each isolated colony was subcultured in Agar GSP for 48 h at 22 °C in order to obtain the pure colonies [35 ]. After 48 h, the pure colony was inoculated in liquid medium (TSB) (Biokar, Beauvais, France) for 24 h at 22 °C. Aeromonas spp. were identified at the genus level using polymerase chain reaction (PCR) [38 (link)], and cultures were conserved in a cryopreservation tube at −80 °C. By considering the origin and morphology of colonies in order to avoid the cluster-forming units, up to three Aeromonas isolates per sample were selected for antimicrobial susceptibility testing. Then, up to two Aeromonas isolates per sample, depending on the isolate’s antimicrobial susceptibility profiles, were selected for antimicrobial resistance gene studies. These isolates were classified as healthy isolates when no episode of furunculosis or no antibiotic treatment were observed, furunculosis isolates when furunculosis occurred and treated isolates when followed by an antibiotic treatment.

The L. monocytogenes strains (L1 isolated from fermented sausage; L5, L17, L18 and UB2-01-2 (1) isolated from surfaces in the meat industry) used in this research were isolated from the meat industry. These strains were stored as frozen culture at −80 °C in a tryptic soy broth (TSB) (Biokar Diagnostics, Beauvais, France) medium containing 20% glycerol until examination. The strains were cultivated on nutrient agar (Biokar Diagnostics, Beauvais, France) slants for 24 h at 37 °C before the experiments.

Overnight inoculums, incubated at 37 °C (bifidobacteria under an anaerobiotic atmosphere comprised of 10% CO₂, 10% H₂, and 80% N₂ using a Whitley D6250 anaerobic workstation (don Whitley Scientific, West Yorkshire, United Kingdom)) were prepared using tryptic soy broth (TSB, Biokar Diagnostics, Beauvais, France) for E. coli, L. monocytogenes and S. enteritidis, de Mann, Rogosa, and Sharpe broth (MRS broth, Biokar Diagnostics, Beauvais, France) for Lactobacillus, and MRS supplemented with 0.5 g L⁻¹ L-cysteine-HCl (Sigma, St. Louis, MO, USA) for Bifidobacterium. The inocula (10 mL) was centrifuged, washed twice and resuspended in 5 mL of sterile PBS as previously described by Valeriano, et al. [10 (link)].

P. aeruginosa (CIP 103467) and E. faecalis (isolated from French cheese) were used in this study. The strains were maintained in tryptic soy broth (TSB; Biokar Diagnostics, France) supplemented with 40% (v/v) glycerol at -80°C. Prior to use, bacteria were pre-cultivated by inoculating 100 μL of the frozen strain cultures into 5 mL of TSB medium and incubating for 24 h at 37°C. Then 100 μL of the pre-culture were used to inoculate 50 mL of TSB medium in 500 mL sterile flasks, and incubated for 16 h at 37°C under shaking conditions at 160 rpm to prepare the culture. Cells were pelleted by centrifugation for 5 min at 5000 × g. Then, bacteria were washed twice with 20 ml of potassium phosphate buffer (PPB; 100 mM, pH 7) and finally re-suspended in PPB. The cells were dispersed by sonication at 37 kHz (Elmasonic S60H, Elma®) for 5 min at 20°C.

Low-molecular weight (LMW) chitosan (CS) with degree of deacetylation (DD) 75–85%, sodium chloride, hydrochloric acid, hydroxypropylmethyl cellulose (HPMC), 10 mM phosphate-buffered saline (PBS), polyvinyl alcohol (PVA), and mucin from porcine stomach were obtained from Sigma-Aldrich (Irvine, UK). Sodium tripolyphosphate (TPP) was obtained from AppliChem (Darmstadt, Germany). Hyaluronic acid (HA) was a kind gift from Soliance (Paris, France), and ceftazidime (CFT) was offered by Combino Pharm Portugal.
Purified water was of Milli-Rx quality (Merck Millipore, Darmstadt, Germany). All other reagents and solvents were of the purest grade available, and generally were used without further treatment.
Tryptic soy broth and tryptic soy agar were obtained from Biokar (Pantin, France).
Pseudomonas aeruginosa (ATCC 9027), HEK293T (ATCC^® CRL-11268™), and ARPE-19 (ATCC^® CRL-2302™) cell lines were obtained from the American Type Cell Culture collection (Manassas, VA, USA).
Cell culture media and supplements were from Gibco (ThermoFisher Scientific, Paisley, UK).

The antimicrobial activity of the impregnated cotton disks was performed accordingly to the ISO 20743 standard [19 ]. Briefly, inocula were grown overnight, at 37 °C, in Tryptic Soy Broth (Biokar Diagnostics, Beauvais, France) after which the bacterial load was adjusted to a concentration between 1 × 10⁵ to 3 × 10⁵ CFU/mL. Following this, 200 µL of adjusted inoculum was added to 0.40 g (± 0.05 g) of cotton dyed with blue navy everzol NPs (25 mg/mL). Simultaneously, two controls were accessed; one with non-dyed cotton the other with cotton dyed with navy blue everzol at 25 mg/mL. Viable counts were determined at 0 and 24 h and plated by the drop method as previously described by Costa, Silva [20 (link)] in Plate Count Agar (Biokar Diagnostics, Beauvais, France). Plates were then incubated at 37 °C for 24 h and the results were given in log of CFU. All assays were done in sextuplicate.