Gentamicin

Fresh fecal particles were collected from the DSS group and the DSS-EPP group (feces before the 15th day) as donors, sterile PBS (50 mg/mL) was added, and the mixture was homogenized. After homogenization, the fecal suspension was filtered with a 70 μm screen to remove large particles in the feces and then filtered with a 40 μm screen. The liquid obtained through the sieve was collected and resuspended in a sterile 50 mL centrifuge tube. Then, 10% sterile glycerol was added to the resuspended liquid; the centrifuge tube was sealed with sealing film and stored in a −80 °C freezer [21 (link)]. Seven-week-old BALB/C male mice were treated with a mixture of antibiotics [vancomycin (0.5 mg/mL), streptomycin (1 mg/mL), ampicillin (0.5 mg/mL), and gentamicin (1 mg/mL) acquired from Dalian Meilun Biotechnology Co. (Dalian, China)] in their drinking water for 14 days, after which the water was replaced every two days [22 (link)]. BALB/c male mice were divided into two groups. The F-DSS group received fecal suspensions from the DSS group, and the F-DSS-EPP group received fecal suspensions from the DSS-EPP group. Each animal received a fecal suspension of 200 mg/kg/d once every two days for a total of five treatments in 10 days as shown in Figure 1B. All processes were carried out under aseptic conditions.

The 6-week-old male mice weighing 21.79 ± 0.76 g were given a standard diet and antibiotic treatment (1 g/L streptomycin, 1 g/L gentamicin, 0.5 g/L ampicillin and 0.5 g/L vancomycin, diluted in drinking water, Meilun Bio, Dalian, China) for 1 week to clean their gut microbiota [14 (link)]. Afterwards, the mice were randomized into 3 treatment groups: the antibiotics (anti (CON)), antibiotics + CdCl₂ (anti (Cd)), and antibiotics + CdCl₂ + Mel (anti (Cd + Mel)) group (n = 12/group). Total bacterial 16S rRNA was measured through qPCR, which verified the successful depletion of gut microbiota after antibiotic treatment [16 (link)]. Identical treatments in CON, Cd and Cd + Mel mice were given to anti (CON), anti (Cd) and anti (Cd + Mel) mice. After 10 weeks of Mel, Cd and antibiotic treatments were given once daily, with each mouse subjected to overnight fasting before it was euthanized. Consequently, samples of liver, blood, colonic content and mucosa and ileal mucosa were collected and stored at −80 ℃ until subsequent analyses.

Clinically-used antibacterial agents (Magiorakos et al., 2012 (link)), such as piperacillin, ceftazidime, cefotaxime, tetracycline, minocycline, gentamicin, amikacin, ciprofloxacin, levofloxacin, chloramphenicol, imipenem, meropenem, tigecycline, polymyxin B, polymyxin E, vancomycin, bacitracin, and teicoplanin were purchased from Meilun Biological (Dalian, Liaoning, China). In addition, cation-adjusted MH broth (CAMHB), Muller-Hinton broth (MHB) and tryptic soy broth (TSB) medium were obtained from Haibo Biotechnology (Qingdao, Shandong, China).

The agar dilution method was used to evaluate the antibiotic sensitivity of the isolated strains. The minimum inhibitory concentration (MIC) of each drug was determined in accordance with the method recommended by the Clinical and Laboratory Standards Institute (M100) (Clinical and Laboratory Standards Institute, 2019 ). A total of five isolates and E. coli ATCC25922 as the control strain were diluted to 1 × 10⁷ CFU/mL and inoculated with a sterile inoculation needle on MH agar plates containing meropenem, polymyxin, tetracycline, chloramphenicol, kanamycin, and gentamicin (Dalian Meilun Biotechnology Co., Ltd., Dalian, China). Each plate had a concentration gradient of 1–256 μg/mL. After incubation at 37°C for 48 h, the MIC was determined by observing the bacterial growth.

Mice (28.80 ± 0.30 g) were fed with the Con diet and treated with antibiotics (0.5 g/L vancomycin, 0.5 g/L ampicillin, 1 g/L gentamicin, 1 g/L streptomycin, Meilun Bio, Dalian, China) to clear gut microbiota. The antibiotics were diluted in drinking water and changed daily. After fifteen days of antibiotics treatment, the mice were randomly allocated into three groups: the Con, HFD, and HFD+FML group (n = 7/group). The mice were fed with the respective diets for an additional 2 weeks. Then, blood and tissue samples were collected for analyses.

ZG180, (R)- and (S)-ZG197 were synthesized in lab and fully characterized. ONC212 and ADEP 4 were purchased from Topscience and ChemPartner (Shanghai, China), respectively. Antibiotics of vancomycin, spectinomycin, erythromycin, ciprofloxacin, rifampicin, tetracycline, norfloxacin, clindamycin, and gentamicin, were purchased from Dalian Meilun Biotech. Oxacillin was purchased from Sigma-Aldrich. Anhydrous tetracycline (ATC) was purchased from APExBIO.