Rifampin

TB47 was synthesized in a batch and supplied by Guangzhou Eggbio Co. Ltd with purity 98.67% by High Performance Liquid Chromatograph (Batch number: TB47160616). It was formulated in 0.05% CMC-Na (sodium salt of carboxy methyl cellulose) for in vivo study. Rifampin and streptomycin were both bought from TCI (Japan) and dissolved in distilled water for in vivo use. streptomycin and isoniazid (Macklin, Shanghai) were dissolved in distilled water, and Rifampin and TB47 were dissolved in dimethyl sulfoxide (DMSO) for in vitro use. All drugs prepared weekly for in vivo studies. Hygromycin was bought from Roche (Swiss) in liquid form and kanamycin was bought from Sigma (USA). They were dissolved in distilled water for in vitro study.

The E. coli strain used in this work is a standard DH5α strain which is commonly used in genetic engineering. The antibiotics tetracycline, streptomycin, roxithromycin, and nalidixic acid were formulated as 2-mg/mL stock solutions in water. Rifampin (Macklin, Beijing) was formulated as a 2-mg/mL stock in dimethyl sulfoxide (DMSO). Azithromycin and chloramphenicol (Macklin, Beijing) were formulated as 2-mg/mL stock solutions in ethanol. Ciprofloxacin (Sigma, Shanghai) was dissolved in 0.15 M sodium hydroxide at 1 mg/mL, and it was neutralized before addition to the medium. Cyanide-m-chlorophenylhydrazone (CCCP; Sigma, Shanghai) was dissolved in DMSO to reach a final concentration of 5 mg/mL, and 20 μg/mL of CCCP was used in the medium to inhibit the activity of efflux pumps (10 (link), 54 (link)). The MFS-specific inhibitor verapamil (Sigma, Shanghai) was formulated at 5 mg/mL in water. Berberine (Sigma, Shanghai) was dissolved in water (1 mg/mL) or DMSO (10 mg/mL) for different applications. LB medium (containing per liter, 10 g NaCl, 10 g peptone, 5 g yeast extract, 15 g agar if necessary; Bioroyee, Beijing) was prepared as broth according to the manufacturer’s instructions. All strains were grown overnight in a 37°C shaking incubator (200 rpm) prior to initiating the experiment.