Metronidazole

Human faecal samples (preserved in 20% (v/v) glycerol) were transferred to an anaerobic chamber, thawed and sieved through 100 μm meshes, transferred into a GF isolator and introduced into GF mice by oral gavage (200 μl per mouse). For antibiotics treatment, 0.5 g l⁻¹ ampicillin (nacalai tesque), 0.5 g l⁻¹ metronidazole (nacalai tesque) and 1.0 g l⁻¹ tylosin (Sigma-Aldrich) solutions were made using autoclaved tap water. Mice receiving oral gavage of the caecal contents from the donor-C-microbiota-colonized mouse were fed with antibiotic solutions for 12 days. Antibiotic solutions were replaced once per week.

FMT was performed as previously described [20 (link)]. For the bacterial transfer experiments, donor mice received PEG or saline orally for two weeks, and 4–5 fecal pellets (~100 mg) were collected fresh for daily FMT. The collected feces were suspended in 1 mL of phosphate-buffered saline, followed by centrifugation at 2000× g for 10 s. To minimize the changes in microbial composition of the supernatant, the 100 μL aliquots of the supernatant containing fecal bacteria were orally administered to recipient mice immediately after centrifugation. Recipient mice were fed a HFD for four weeks, treated with a mixture of four antibiotics for four days and subsequently rested for three days prior to transfer. For antibiotic treatment, an antibiotics cocktail containing ampicillin, neomycin, metronidazole, and vancomycin (Nacalai Tesque) was diluted in sterilized water to final concentrations of 5, 5, 5, and 2.5 mg/mL, respectively. The antibiotics cocktail (200 mL) was orally administered for four days. Oral antibiotic administration via ad libitum administration via drinking water was avoided to minimize individual differences in body weight change and antibiotic doses [26 (link)]. After three days of rest following antibiotic administration, FMT from donor mice was performed five times weekly for six weeks.

C57BL/6 male mice were randomized into two groups. The AIMD group (n = 13) was housed with access to weakly acidic drinking water containing four types of antibiotic (1 g/L ampicillin sodium, 0.5 g/L vancomycin hydrochloride, 1 g/L neomycin sulfate, and 1 g/L metronidazole; all from Nacalai Tesque Inc., Kyoto, Japan) from 8 weeks of age to deplete their gut microbiota^{6 (link),8 (link),14 (link)}. As several mice initially exhibited low levels of water consumption, metronidazole was excluded from the drinking water following the surgery to implant electrodes until the end of the experiment. The other group of mice was housed as controls (n = 12) with access to weakly acidic drinking water containing no antibiotics.

United States Pharmacopeia (USP) reference standards of omeprazole, sulfamethoxazole, trimethoprim, and clarithromycin-related compound A (6,11-di-o-methyl erythromycin A) were purchased from the Reference Standard Center, Bureau of Drugs and Narcotics, Thailand (Nonthaburi, Thailand). Reference standards of cefixime, clarithromycin, and sildenafil citrate were generously donated by Astellas Pharma Inc. (Tokyo, Japan), Taisho Toyama Pharmaceutical Co, Ltd. (Tokyo, Japan), and Pfizer Japan Inc. (Tokyo, Japan), respectively. Metronidazole and butyl p-hydroxy benzoate were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Primidone and sulfadoxine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Lansoprazole was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals were commercially available and of analytical grade.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Metropolitan University and all experiments were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals (A2-2, A3-22, A4-19).
As shown in Figure 1, mice were randomly divided into three groups, the control group (Con group, n = 5), the antibiotics control group (AB group, n = 5), and the FMT group (n = 5). The FMT group received FMT from S. murinus three times after treatment with antibiotics in the third week. The AB group was treated with a combination of antibiotics without receiving transplantation. The Con group did not receive antibiotic treatment or transplantation. To remove indigenous gut microorganisms, we referred to previous reports on antibiotic administration (Chen et al., 2020 (link); Gudi et al., 2020 (link); Su et al., 2020 (link)). The antibiotics included a combination of ampicillin (1.0 g/L; Nacalai Tesque, Kyoto, Japan), vancomycin (0.5 g/L; Shionogi, Osaka, Japan), neomycin (1.0 g/L; Nacalai Tesque, Kyoto, Japan), and metronidazole (1.0 g/L; Nacalai Tesque, Kyoto, Japan), which was added to drinking water for 10 consecutive days to remove indigenous gut microorganisms. After a 3-day recovery period, FMT was performed on the next 3 consecutive days.