Moxalactam

For strain development and in vitro analyses, C. difficile was routinely maintained on Brain heart infusion medium (Oxoid) supplemented with 5μg/ml yeast extract, 0.1% w/v L-cysteine (BHIS), C. difficile selective supplement comprising 250μg/ml D-cycloserine, and 8 μg/ml cefoxitin (Oxoid) (BHIS CC), and where necessary, an additional supplementation of 15 μg/ml thiamphenicol (BHIS CCTM). C. difficile cultures were grown at 37°C in an anaerobic workstation (Don Whitley) with an anaerobic gas mixture comprising 80% N₂, 10% H₂ and 10% CO₂.
For murine experiments, frozen stocks of wild-type and mutant strains were cultured on CDMN agar plates (C. difficile agar base (Oxoid) supplemented with 7% (v/v) defibrinated horse blood (Lampire Biological Laboratories), 32 mg/L moxalactam (Santa Cruz Biotechnology), 12 mg/L norfloxacin (Sigma-Aldrich) and 500 mg/L cysteine hydrochloride (Fischer) in an anaerobic chamber [27 (link)] at 37°C for 24 hours. Single colonies were picked and grown anaerobically for 16–18 hours at 37°C to saturation in 5 mL of pre-reduced reinforced Clostridial medium (RCM, Oxoid) for inoculation.
For hamster experiments, strains were cultured on blood agar for 5d in order to generate the spore stocks. Terminal colonization was assessed using C. difficile selective agar (Oxoid). In both instances, strains were maintained in an anaerobic workstation, as described above.

For quantification of Cd CFU, 1 μL feces was serially diluted in PBS and plated onto selective media, composed of Cd agar base (OxoiD) with 7% v/v of defibrinated horse blood (Lampire Biological Laboratories), supplemented with 32 mg/L Moxalactam (Santa Cruz Biotechnology) and 12 mg/L Norfloxacin (Sigma-Aldrich) (CDMN). Plates were incubated overnight at 37 °C in an anaerobic chamber (Coy). Identification of colonies as Cd was validated by colony PCR, using the primers Cl1 (5′-TGTTGCAATATTGGATGCTTT) and Cl2 (5′-TGACCTCCAATCCAAACAAA), which target a fragment of tcdB gene.