Ciprofloxacin

Curcumin (purity ≥ 95.0%) was purchased from Shanghai Yuanye Biotech (Shanghai, China). Carboxymethylcellulose sodium was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Neomycin, streptomycin, penicillin, vancomycin, metronidazole, bacitracin, ciprofloxacin, ceftazidime and gentamycin were purchased from Sangon Biotech (Shanghai, China). TRIzol® Reagent was from Invitrogen (Carlsbad, CA, USA). High-Capacity cDNA Reverse- Transcription Kits were purchased from Applied Biosystems (Foster City, CA, USA). SYBR Green PCR Master Mix was obtained from Promega (Fitchburg, MI, USA). Proteinase inhibitor cocktail and phosphorylase inhibitor were purchased from Roche (Basel, Switzerland). Bicinchoninic acid (BCA) Protein Assay Kits were purchased from Genstar Technologies (Beijing, China). Antibodies against Akt (9272), pAkt (9271) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against FGF15 (ab229630) was obtained from Abcam. The antibody against Tubulin (CW0098) was from Cwbiotech (Beijing, China).

Xanthine (X8030) was purchased from Solarbio Life Science, Beijing, China. Middlebrook 7H9 Broth (271310) was purchased from BD/Difco, Franklin Lakes, NJ, United States. Amikacin (A602232), kanamycin (A100408), gentamicin (A100304), streptomycin (A100382), chloramphenicol (A100230), ciprofloxacin (A600310), rifampicin (A600812), and isoniazid (A600544) were purchased from Sangon Biotech, Shanghai, China. The Anti-His antibody (#9991) was purchased from Cell Signaling Technology, Danvers, MA, United States.

Antibiotic sub-MICs were selected by creating a growth curve. K. pneumoniae SW1780 was grown in the presence of 0 × MIC, 1/2 × MIC, 1/4 × MIC, 1/8 × MIC, 1/16 × MIC, 1/ 32 × MIC, 1/64 × MIC, 1/128 × MIC, 1/256 × MIC, 1/512 × MIC, 1/1024 × MIC, 1/2048 × MIC of meropenem (YuanYeBio-Technology, Shanghai, China), ciprofloxacin (Sango Biotech, Shanghai, China), cefotaxime (Sango Biotech, Shanghai, China) and amikacin (Sango Biotech, Shanghai, China). Samples were collected at one-hour intervals and the optical density was read at 595 nm (OD595) using a microplate reader (Bio-Rad, United States). The logarithmic growth rate of the antibiotic stress group and the non-antibiotic group was calculated by applying the logarithmic growth rate formula: U = [ln(Nt) -ln(N0)]/(t-t0), where Nt is the logarithmic growth late OD₅₉₅ value, N0 is the logarithmic growth early OD₅₉₅ value, and t-t0 is the logarithmic period time. One-way ANOVA was used to determine statistical differences. The highest antibiotic concentration that did not affect the growth of strain SW1780 was used as sub-MIC.

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.

The bacteria isolate was used for antimicrobial susceptibility testing (AST) against 17 antimicrobial agents. Broth microdilution minimum inhibitory concentration (MIC) determination was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and interpretation (CLSI, 2018 ), the European Committee for Antimicrobial Susceptibility Testing (EUCAST, 2018 ), and where no EUCAST or CLSI interpretative criteria were available, breakpoints were harmonized with those of the National Antimicrobial Resistance Monitoring System (NARMS), United States (FDA, 2017 ). The following antimicrobials were tested: fosfomycin, levofloxacin, cefoperazone sulbactam, streptomycin, ampicillin, amoxicillin–clavulanic acid, cefoxitin, imipenem, nalidixic acid, ciprofloxacin, chloramphenicol, tetracycline, kanamycin, gentamicin, trimethoprim–sulfamethoxazole, ceftiofur, and azithromycin (Sangon Biotech, China). Escherichia coli ATCC 25922 were used as a control strain. The AST against all antibiotics were carried out in Muller–Hinton broth (MHB) medium in both aerobic and anaerobic conditions and incubated at 37°C. The AST was also performed in Dulbecco’s modified Eagle’s medium (DMEM) (Dulbecco and Freeman, 1959 (link)) and incubated in a 5% CO₂ incubator.

Metronidazole, ciprofloxacin, clindamycin, neomycin, and ampicillin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Vancomycin was acquired from MSD & Co., Inc. (Hangzhou, China). Luria-Bertani (LB) medium was purchased from Thermo Fisher Scientific (Shanghai, China). Mueller-Hinton (MH) and brain heart infusion (BHI) media were acquired from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). ¹⁸F-FDG and ¹⁸F were obtained from the Department of Nuclear Medicine, First Affiliated Hospital of Xiamen University. Staphylococcus aureus RN450 and Escherichia coli BW25113 were from frozen stocks of the Laboratory of Microbial Pathogens, Xiamen University. Probiotics (30 billion CFU/capsule; Island’s Miracle, USA) were purchased from Amazon. Female C57BL/6 mice and male hamsters were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed at the Laboratory Animal Center of Xiamen University.