Gentamicin

The antibiotic susceptibility profiles for the VT-MRSA isolates were determined using the MIC technique via the microdilution method according to the CLSI 2013 [32 ], as described in the previous section. MIC tests were performed for ampicillin (Sigma-Aldrich), ceftazidime (Pfizer; México DF, México), ceftriaxone (Roche; Mexico DF, México), ciprofloxacin (Bayer; Levenkusen Westfalia, Germany), erythromycin (MP Biomedicals; Solon, OH, USA), kanamycin (Sigma-Aldrich), meropenem (Astra Zeneca; México DF, Mexico), rifampicin (MP Biomedicals), gentamicin (MP Biomedicals), and trimethoprim-sulfamethoxazole (Sigma-Aldrich).

For all experiments, zebrafish embryos were initially derived germ-free using previously described gnotobiotic procedures with slight modification [75 (link)]. Briefly, fertilized eggs from adult mating pairs were harvested and incubated in sterile embryo media (EM) containing ampicillin (100 μg/mL), gentamicin (10 μg/mL), amphotericin B (250 ng/mL; MP Biomedicals, Santa Ana, CA), tetracycline (1 μg/mL; Sigma-Aldrich, St. Louis, MO), and chloramphenicol (1 μg/mL; Amresco, Solon, OH) for approximately 6 hours. Embryos were then washed in EM containing 0.1% polyvinylpyrrolidone-iodine (Syndel, Ferndale, WA) followed by EM containing 0.003% sodium hypochlorite (Fisher Scientific, Hampton, NH). Surface sterilized embryos were distributed into T25 tissue culture flasks (TPP, Trasadingen, Switzerland) containing 15 mL sterile EM at a density of 1 embryo/mL and kept in a temperature-controlled room at 28°C to 30°C with a 14 hours/ 10 hours light/dark cycle. The germ-free status of larval zebrafish was assessed before every experiment by visually inspecting flask water for microbial contaminants using an inverted microscope. Culture-based assessment of germ-free status was done as needed by plating 100 μL flask water on rich media (e.g., tryptic soy agar). Embryos were sustained on yolk-derived nutrients and not fed prior to or during any experiments.

Cytotoxicity of the compounds to HeLa (human cervical cancer) and HEK293A (human embryonic kidney) cell lines was examined using the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria), according to the manufacturer’s protocols. The cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) with 40 μg/mL gentamicin, 50 IU/mL penicillin, 50 μg/mL streptomycin (MP Biomedicals, Waltham, MA, USA), and 10% of fetal bovine serum (Biolot, Saint Petersburg, Russia) in a 5% CO₂ atmosphere. After the formation of a 30–50%monolayer, the tested compounds were added to the medium. TDP1 inhibitors were dissolved in DMSO and topotecan in water. The volume of the added reagents was 1/100 of the total volume of the culture medium, and the amount of DMSO was 1% of the final volume. Control cells were grown in the presence of 1% DMSO. The cell culture was monitored for three days.

PBMC isolated from 7 randomly untreated MS patients were thawed and suspended (1x10⁶ cells mL-1) in pre-warmed RPMI-1640 medium, supplemented with 2 mM l-glutamine (MP Biomedicals, Irvine, CA, USA), 10% Fetal Bovine Serum (FBS) and 0.032 mg mL-1 gentamicin (Normon). Cells were washed by centrifugation and resuspended in RPMI-1640 complete medium with 2% FBS.
To evaluate the ability of recombinant IFN-β to induce the production of sIFNAR2, the cells were stimulated with 20 units/mL of IFN-β (Avonex) during 8 and 24 hours. A non-stimulated control was included each time. After that, the cells were collected for RNA extraction and cDNA conversion, as explained above. sIFNAR2, IFNAR2 and MxA were measured by real time PCR. MxA was considered as positive control since it is well known that its expression is clearly induced after IFN-β stimulation (31 (link)).