Metronidazole

Antibiotic water was produced by mixing 1 g/L of metronidazole (Fujifilm Wako Pure Chemical Co., Osaka, Japan), 1 g/L ampicillin sodium (Fujifilm Wako Pure Chemical Co.), 1 g/L neomycin sulfate (Fujifilm Wako Pure Chemical Co.), and 0.5 g/L of vancomycin hydrochloride (Fujifilm Wako Pure Chemical Co.) in drinking water. While the antibiotic water treatment, mice had access to water bottle filled with antibiotic water ad libitum, and no access to tap water. To weaken intestinal flora, mice had been treated antibiotic water for at least 2 weeks.

We investigated the role of commensal bacteria in osteoporosis with eight-week-old C57BL/6J female mice that were administered a mixture of antibiotics for four weeks: ampicillin (1 g/L; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), neomycin (1 g/L; FUJIFILM Wako Pure Chemical Corp.), metronidazole (500 mg/L; FUJIFILM Wako Pure Chemical Corp.), and vancomycin (500 mg/L; FUJIFILM Wako Pure Chemical Corp.) mixed in drinking water, to decontaminate the digestive tract (n = 7–10 per group). The control mice received normal drinking water. After the experimental period, blood and the left tibia were collected to assess bone homeostasis.

Two methods were used for the evaluation of antibacterial activity. To measure the MIC value of nisin A (Sigma–Aldrich, St. Louis, USA), the microdilution method was used as described elsewhere [23 (link)]. Bacitracin (Fujifilm Wako chemicals, Osaka, Japan), vancomycin (Sigma–Aldrich), Metronidazole (Fujifilm Wako chemicals), ampicillin (Nacalai Tesque, Kyoto, Japan), chloramphenicol (Wako chemicals), gentamicin (Nacalai Tesque), ofloxacin (Sigma–Aldrich) and imipenem (Fujifilm Wako chemicals) were also used for MIC evaluations.
To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23 (link)]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37°C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (10⁸ cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37°C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.

Antibiotics (ampicillin 1 g/L, neomycin 1 g/L, metronidazole 1 g/L; FUJIFILM Wako Pure Chemical Corporation) were administered in drinking water to depress gut microbiota. Two weeks after initiation of the antibiotics, we administered a single subcutaneous injection of liraglutide or saline to the mice. After the mice were fasted for 16 h, food intake was measured 1, 3, 6, and 24 h later. Mice that drank water without antibiotics were used as controls to perform experiments in the same way.

C57BL6/J mice were housed under a 12-h light–dark cycle and given regular chow (MF, Oriental Yeast Co). All experimental procedures involving mice were performed according to protocols approved by the Committee on the Ethics of Animal Experiments of the Tokyo University of Agriculture and Technology. (Permit Number: 28–87). For HFD studies, 4-week-old male mice were placed on a D12492 diet (60% kcal fat, Research Diets) for 12 weeks. The generation of Gpr43^-/- was described previously[18 (link)]; the mice were maintained on a C57BL6/J genetic background. aP2-Gpr43TG mice were generated as previously[18 (link)]. For antibiotic treatment, 4-6-week-old mice were treated with ampicillin (Nacalai Tesque; 0.4 mg/ml), neomycin (Nacalai Tesque; 0.4 mg/ml), metronidazole (Wako; 0.4 mg/ml), gentamicin (Sigma; 0.4 mg/ml) and vancomycin (Sigma; 0.2 mg/ml) in drinking water for 3 weeks. For collection of blood and tissue samples, mice were sacrificed by anesthesia with somnopentyl. All efforts were made to minimize suffering.

Antibiotic susceptibility was detected using a serial two-fold agar dilution assay to determine the minimum inhibitory concentrations (MICs) of amoxicillin, clarithromycin, metronidazole, levofloxacin, and minocycline (Wako Pure Chemical Industry, Osaka, Japan) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Wayne, PA, USA). Briefly, bacteria were subcultured on Mueller-Hinton II Agar medium (Becton Dickinson, Sparks, MD, USA) supplemented with 5% defibrinated horse blood. The bacterial suspension was adjusted to OD₆₀₀ = 0.1, and a 48-pin inoculator was used to inoculate the culture plate (1 μL per spot, approximately 10⁴ colony forming units [CFU] of bacteria). H. pylori strain 26695 was used as a control strain. MICs were judged according to the presence or absence of growth at the spots at the lowest concentration of antibiotic, followed by checking growth at the spots using 1:1 dilutions after 72-h incubation. Resistance or sensitivity to antibiotics was judged according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST; http://www.eucast.org/). The clinical breakpoints of MICs indicating antibiotic resistance are >0.125 mg/L, amoxicillin; >0.5 mg/L, clarithromycin; >8 mg/L, metronidazole; >1 mg/L, levofloxacin; and >1 mg/L, minocycline. Duplicate agar dilution assays were repeated 2–3 times.