Streptomycin

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.

Citric acid (CA), uric acid (UA), europium nitrate hexahydrate (Eu(NO₃)₃·6H₂O), nicotinic acid, glucose, D-alanine, L-glutamic acid, L-cysteine and L-leucine were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Antibiotics including oxytetracycline, tetracycline, chlortetracycline, doxycycline, metacycline, minocycline, penicillin, azithromycin, streptomycin, ciprofloxacin and norfloxacin were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). All common metal salts were purchased from Sinopharm Chemical Regents Co., Ltd. (Shanghai, China). HEPES buffer (200 mM) with different pH values was prepared by titrating 200 mM HEPES aqueous solution with 3 M NaOH standard solution. Deionized water (18.2 MΩ cm) prepared from the UPHW-I-90T ultrapure water system was used to prepare standard solutions throughout the experiments. Apparatus and characterization are described in the Supplementary Materials.

Six to eight-week-old male BALB/c mice were purchased from the Experimental Animal Center of Hubei Province, China. All mice were raised under specific-pathogen-free barrier conditions and fed autoclaved food and water according to the institutional guidelines for Animal Care and Use Committee of Tongji Hospital, Huazhong University of Science and Technology, Hubei, China. The body weight of the mice was measured twice a week. All animal protocols were approved by the Ethical Committee on Animal Experiments.
Before intragastric administration of bacteria, streptomycin (2 mg/mL, Shanghai Macklin Biochemical Co., Ltd Shanghai, China) was administered to all mice used in the study via drinking water for three days. To investigate F. nucleatum-associated CRLM, 30 mice were randomly assigned to two groups and 10⁹ colony-forming units of F. nucleatum resuspended in PBS were administered to mice by gavage every day for eight weeks. After the 4-week F. nucleatum or PBS administration, the CT26-Luc cells were injected into the spleens of BALB/c mice to establish a liver metastasis model, which were denoted as F. nucleatum group and PBS group, respectively. Another 30 mice were administered with F. nucleatum or PBS until moribund to investigate F. nucleatum-associated survival.