Oxytetracycline otc

To culture bacteria from fresh sediment samples, 1 g of the composite sediment was suspended in physiological saline (0.9% NaCl) by vortexing. Serial 10-fold dilutions were cultured on three replicate R2A agar plates to enumerate total bacteria. To enumerate resistant bacteria, serial dilutions were cultured in triplicates on plates supplemented with AZI (15 mg L⁻¹) (Fluka, Germany) for Industry area 1 samples; or sulfamethazine (SMZ; 350 mg L⁻¹) (Sigma, Germany) or oxytetracycline (OTC; 25 mg L⁻¹) (Sigma, Germany) for Industry area 2 samples. Colony forming units (CFU) were counted after a 5 day incubation at 28°C. ARB cultured from sediments from discharge locations were scraped from the plates, pooled and stored in R2A broth containing antibiotic and 15% glycerol at −80°C.

The human lymphoblast derived K562 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured at 37 °C in a humidified atmosphere (5% CO₂) in RPMI (Sigma–Aldrich, St. Louis, Missouri, USA) medium for 48 h. Oxytetracycline (OTC) from Sigma–Aldrich (St. Louis, Missouri, USA) was stocked at 100 mM in DMSO (Sigma–Aldrich, St. Louis, Missouri, USA). Bone powder, achieved from poultry raised with or without the administration of oxytetracycline, was extracted in cell culture medium as previously described [19 (link),20 (link)]. Briefly, the bone powder was dissolved in RPMI at the concentration of 124 mg/mL and kept under continuous stirring at 37 °C for 48 h. After that, the suspension was filtered, and the filtrate neutralized with KOH (Sigma–Aldrich, St. Louis, Missouri, USA) at pH 7.2–7.4. For compound treatment, 0.5–2.0 × 10⁶ cells/mL were supplemented with graded concentration of OTC or graded dilution of bone powder extracts as indicated. The viability of K562 cells was assessed before and after treatments by direct counting Trypan Blue-negative cells with a hemocytometer.