Minimum essential medium (mem)

BMD was performed at Vanderbilt University Medical Center (VUMC, Nashville, TN) following CLSI methods (36 ). The following antimicrobials were tested: CAZ, LVX, MEM, MIN, and TMP-SMX. Testing for non-CF isolates was performed from October 2021 to March 2022 using custom frozen reference BMD panels prepared by VUMC following CLSI methods (BD Difco). The following antimicrobial dilution ranges were used: 0.06 μg/mL–128 μg/mL, CAZ (Sigma); 0.03 μg/mL–64 μg/mL, LVX (Sigma), MEM (Sigma), and MIN (MedChemExpress); and 0.008/0.14 μg/mL–16/304 µg/mL, TMP-SMX (Sigma). For CF isolates, testing was performed from June 2022 to December 2022 using custom frozen reference BMD panels prepared by ThermoFisher with the same antimicrobial dilution ranges as listed above. Isolates were stored at −70°C. Frozen stocks were sub-cultured onto 5% sheep blood agar (SBAP) and incubated at 35°C in ambient air overnight or until visible colonies were observed. These isolates were sub-cultured onto 5% SBAP and incubated at 35°C in ambient air overnight for 20–24 hours. Isolated colonies were used to make a cell suspension with turbidity equivalent to a 0.5 McFarland standard. BMD panels were inoculated according to CLSI guidelines and incubated at 35°C in ambient air for 20–24 hours. MICs were read at 20–24 hours by two different operators, and disagreement was resolved by a third operator. All non-CF isolates (n = 105) were tested in triplicate on the same day using three separate 0.5 McFarland standards. Due to contamination issues thought to have been encountered during isolate shipment, a subset of CF isolates was tested in triplicate (n = 34 in triplicate, n = 1 in duplicate, n = 65 in single replicate, and two were discarded due to contamination, Table S1). Replicates were performed as described above. QC was performed each day of testing using E. coli ATCC 25922 and P. aeruginosa ATCC 27853.

Beating cell cultures were prepared from rainbow trout hearts (RTC) using a modified protocol for isolating primary cardiomyocytes from zebrafish hearts (Sander et al. 2013 (link)) as published recently by Krebs et al. (2025 (link)). Briefly: A total of 210 hearts from yolk sac stage larvae exhibiting healthy appearance and non‐feeding behaviour were used for each 24‐well plate (RTC‐L). To obtain cell cultures derived from the hearts of one‐year‐old rainbow trout, 15 hearts were utilised for each 24‐well plate (RTC‐A). Prior to extraction of the hearts rainbow trout larvae and one‐year‐old rainbow trout were anaesthetised using an overdose (0.5 g/L) of Tricaine (PHARMAQ). As described in the modified protocol of Krebs et al. (2025 (link)), after several washing steps and a digestion process, cardiomyocytes were plated into all wells of a 24‐well plate containing 500 μL of advanced DMEM/F‐12 plating medium (Thermo Fisher Scientific) and incubated at 15°C and 2% CO₂. On a weekly basis, 300 μL of the medium was removed and replaced with an equal volume of freshly prepared medium. Following a period of two to four weeks, evidence of cell contraction in each well was observed, indicating that the cultures were ready to be used in the planned experiments. In addition, cell cultures of the well‐characterised cell line rainbow trout gonad (RTG‐2) and newly established cell line rainbow trout heart (RTH‐F grown from heart explants of juvenile rainbow trout and passaged over 100 times) from the Fish Disease Research Unit of the University of Veterinary Medicine Hannover, Germany, which is described for the first time in this paper, were used for the planned experiments. RTG‐2 and RTH‐F cells were plated into 24‐well plates, grown for 24 h and utilised upon reaching 95% confluence before infection. The cells were cultured in Minimum Essential Medium Eagle (MEM) (Sigma) medium with 10% FCS, 10 IU/mL penicillin and 100 mg/mL streptomycin. Cultures were incubated at 15°C and 2% CO₂.

Human epithelial Caco-2 cells (KCLB 30037) were maintained at 37°C in a humidified 5% CO₂ atmosphere in minimal essential medium (MEM; Sigma-Aldrich, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Mouse macrophage RAW 264.7 cells (KCLB 40071) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich), supplemented with 10% FBS and 1% L-glutamine, and incubated at 37°C in 5% CO₂. Cytotoxicity was investigated using the Cell Proliferation Assay Kit I, which employs a colorimetric assay based on 2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich) in accordance with the manufacturer’s protocol.