Isoniazid i3377

To test the effectiveness of antimicrobial drugs in vitro, we exposed a bioluminescent M. marinum strain, ATCC BAA535, with the pMV306 plasmid (Addgene plasmid 26161), containing the LuxABDE cassette and kanamycin resistance (Andreu et al., 2010 (link)), to different concentrations of antibiotics (ethambutol E4630, metronidazole M3761, isoniazid I3377 and amikacin A0368000, Sigma-Aldrich) and measured the light emitted by the bacteria with a 2014 EnVision Multilabel Reader (PerkinElmer, Waltham, MA, USA). Antibiotic concentrations were selected based on the literature (de Steenwinkel et al., 2010 (link)). For each tested concentration, six replicate samples were prepared in a white 96-well plate; in each well the desired antibiotic dilution was added to 200 µl of a bacterial dilution (500 cfu/µl). Water was used as a negative control. Mycobacteria were incubated at 29°C for 7 days and the bioluminescence was measured on days 0, 1, 2, 3, 4 and 7. The results were analyzed with EnVision Workstation 1.12 (PerkinElmer).

Immunosuppressive agents and antibiotics were administrated per os in the fish food. The gelatin-coated fish food contained 2.5 mg/g of the immunosuppressant or 12.5 mg/g of the antibiotics. Bovine gelatin (G9391, Sigma-Aldrich; 400 mg) was diluted in 5 ml distilled water by heating (45°C). Then, 25 mg of each immunosuppressant (azathioprine A4638, dexamethasone D4902, prednisolone P6004 and methylprednisolone M1755200, Sigma-Aldrich) or 125 mg of antibiotic (ethambutol E4630, metronidazole M3761, isoniazid I3377 and amikacin A0368000, Sigma-Aldrich) was mixed with 2 ml of 70% EtOH and the suspension was combined with the gelatin solution. The gelatin mixture was spread evenly on 10 g of dry fish food pellets (SDS400, Special Diet Services, Essex, UK) and air dried overnight. The food was weighed and homogenized using a mortar and a pestle. The increase in the weight of the food, from the added gelatin and medicines, was taken in account when the daily doses of prepared food were calculated and weighed, so that each fish received 4 mg of food containing the desired dose of the drug. During the reactivation treatments, the fish were fed once daily. The water circulation was stopped during the feeding to avoid the spreading of chemical-containing food into other tanks of the unit.