Vancomycin

Isogenic E. coli K-12 strains (see Table S1 in the supplemental material) were obtained from the Keio collection (52 (link)) or constructed using CRISPR genome editing technology (53 (link)). Plasmids and primers used for this work are listed in Table S4. E. coli strains were cultured by shaking at 200 rpm aerobically at 37°C in Luria-Bertani (LB) broth or by plating on LB agar incubated at 37°C. Yeast extract, tryptone, agar, and 5(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) were obtained from Thermo Fisher Scientific Corp. (Waltham, MA, USA). Ciprofloxacin, ampicillin, and kanamycin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Vancomycin, erythromycin, lysine monohydrochloride, arginine hydrochloride, histidine hydrochloride, malonate, propidium iodide, proteinase K solution, 5× protein loading dye, dimethyl sulfoxide (DMSO), and 2.2′-bipyridyl were purchased from Sangon Biotech Inc. (Shanghai, China). Lipopolysaccharide (LPS) was bought from Beyotime Biotech Inc. (Jiangsu, China). Ceftriaxone (Roche, Shanghai, China), and meropenem (Shenghuaxi Pharmaceutical Co., Chongqing, China) were gifts from the Zhongshan Hospital (Xiamen, China) pharmacy.

The cecal contents of HVEM^−/− mice were harvested into sterile PBS and filtered three times through a 100-μm cell strainer. The filtrate was centrifuged at 200 × g for 5 min at 4°C. The pellet was washed twice with PBS. T. musculis enriched in the pellet was further purified using a 40%/80% Percoll gradient. For T. musculis culture, the isolated T. musculis was suspended with BHI broth (Oxoid, catalog no. CM1135) supplemented with a cocktail of broad-spectrum antibiotics, including 100 mg/mL streptomycin (Sangon Biotech, catalog no. A100382), 100 U/mL penicillin (Sangon Biotech, catalog no. A613460), 50 mg/mL vancomycin (Sangon Biotech, catalog no. A600983), 10 mg/mL ciprofloxacin (Sangon Biotech, catalog no. A600310), 20 mg/mL gentamicin (Sangon Biotech, catalog no. A506614), and 0.5 mg/mL amphotericin B (Sangon Biotech, catalog no. 171375). After suspension, T. musculis was then incubated in an anaerobic workstation (Don Whitley Scientific) at 37°C for 2 days.

For antibiotic treatment, mice were induced CAC with AOM/DSS. At 14 days after AOM administration, mice were placed on antibiotic mixture in autoclaved drinking water (ampicillin, 1 mg/ml; vancomycin, 0.5 mg/ml; neomycin, 1 mg/ml; and metronidazole, 1 mg/ml, all from Sangon Biotech). Fresh antibiotic was supplied every week. This antibiotic mixture was maintained for the duration of the experiment until mice were euthanized for tumor and tissue analysis.
For mice cohousing, age- and sex-matched WT and Tipe^−/− mice were weaned at the age of 4-week old from their parents, and were randomly separated into two cages. In control group, the mice were raised in separate cages to maintain their own microbiota composition, while in cohousing group, WT and Tipe^−/− mice were raised in the same cages at 1:1 ratio (one WT and one Tipe^−/− mouse in each cage). After 6 weeks, the mice were treated with AOM/DSS to induce acute colitis or CAC.

Gut commensal microflora of SPF mice were removed by two methods as previously described31 (link)32 (link). Mice were treated with ampicillin (1 g/L; Sigma-Aldrich), neomycin sulfate (1 g/L; Sigma-Aldrich), vancomycin (500 mg/L; Shanghai Sangon Biotechnology, china) and metronidazole (1 g/L; Sigma-Aldrich) in their drinking water for four weeks31 (link). Another method, mice were orally given neomycin sulfate (100 mg/kg) and streptomycin (100 mg/kg) twice per day for 7 days32 (link).

The predicted antibiotic resistance gene information in the genome was obtained by comparing the amino acid sequences of the strains with the comprehensive antibiotic research database (CARD, http://arpcard.mcmaster.ca, accessed on 24 May 2021) [51 (link)]. The strains were clustered using HemI software [50 (link)].
The microbroth dilution method was used to determine antibiotic resistance of L. sakei according to ISO 10932:2010 [52 ]. The following 11 antibiotics were detected: chloramphenicol, rifampicin, streptomycin, kanamycin, gentamycin, tetracycline, clindamycin, neomycin, erythromycin, ciprofloxacin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). OD₆₂₅ was determined using an enzyme-labeled instrument (Varioskan Lux, Thermo, Waltham, MA, USA) to determine the MIC of strain to antibiotics.

Mice were gavaged with a cocktail of antibiotics (Abx, ampicillin (A610028-0025, 1 g/L), neomycin (A610366-0025, 1 g/L), metronidazole (A600633-0025, 1 g/L), and vancomycin (A600983-0001, 500 mg/L), SangonBiotech, Shanghai, China) in addition with Abx-containing drinking water for 7 days [38 (link)]. Abx-containing water was renewed every 3 to 4 days to maintain efficacy. Mice were treated with 1.5% DSS in drinking water for another 7 days and colon tissues were collected on the seventh day.