Clindamycin

The wild-type ETBF strain used in our study was B. fragilis 86-5443-2-2 (bft-2). This wild-type Bacteroides strain was resistant to gentamicin and clindamycin. Colonization of bacteria was monitored by serial dilution and plating of stool on brain heart infusion agar plates containing 50 μg ml^-1 of gentamicin (Corning, USA) and 6μg ml^-1 of clindamycin (Hospira, USA). Characteristic B. fragilis colonies were enumerated after anaerobic culture and shown as colony-forming units (CFU) gram-1 stool. The bacterial strain was a generous gift from Cynthia Sears and Augusto Franco-Mora (Johns Hopkins University, USA).

Antibiotic powders of daptomycin (Merck & Co. Inc., Kenilworth, NJ) and quinupristin/dalfopristin (AG Scientific, San Diego, CA) were dissolved in sterile water for injection and stored as stocks at −20 °C. Sterile liquid formulations of clindamycin (Pfizer, New York, NY), moxifloxacin (Bayer AG, Leverkusen, Germany), gentamicin (GPO, Bangkok, Thailand), and amikacin (Siam Bheasach, Bangkok, Thailand) were stored at 4 °C. Final antibiotic solutions were freshly prepared before use by diluting stock solutions with cell culture medium.

CBM 588 bacterial powder was used for all in vivo studies at a concentration of 2.2*10¹⁰ cfu/g (Lot 61GT: MIYARISAN Pharmaceutical Co. Ltd., Tokyo, Japan). Clindamycin for injection was purchased from Pfizer Japan Inc (Tokyo, Japan). Analytical grade fidaxomicin was purchased from Selleck Chemicals Inc (Houston, TX, USA). and diluted with dimethyl sulfoxide (FUJIFILM Wako Chemical Co. Ltd., Osaka, Japan) with 500 mg/mL used as the stock solution. Immediately before each in vivo experiment, CBM 588 powder was weighed and reconstituted with sterile water. Clindamycin and fidaxomicin were further diluted to achieve the desired concentration with distilled water. fidaxomicin solution was stored 4ºC and discarded 12 h after reconstitution. Neutralizing antibody to IL-17A was generated at BD Biosciences (Franklin Lakes, NJ, USA).

Patients were treated as inpatients at University Hospital Würzburg. They received a single shot of cefazolin 2 g (Hikma, London, UK) and metronidazole 500 mg (B. Braun Supply Solutions, Melsungen, Germany), or ampicillin/sulbactam 3 g (Unacid^®, Pfizer Pharma GmbH, Berlin, Germany), or clindamycin 600 mg (Sobelin^® Solubile, Pfizer Pharma GmbH, Berlin, Germany) intraoperatively, as well as up to 48 h following surgery (cefazolin, ampicillin/sulbactam, and clindamycin administered three times daily, and metronidazole once daily in case of administration in the postoperative course).
The CBCT scan was performed according to a standardized protocol a few days after the operation during the hospital stay depending on the degree of swelling, with the final splint and intermaxillary fixation with elastic bands inserted.
Patients maintained a soft diet for six weeks. The elastic bands remained in place for two to three weeks. We recommended each patient to have the metal plates removed after six to nine months.

Intestinal colonization of select C. difficile strains was assessed using a non-lethal mouse model of infection [30 (link)]. 10-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were pre-treated with cefoperazone (0.5 mg/mL) in the drinking water for 10 days, followed by intraperitoneal clindamycin (10 mg/kg) (Pfizer, New York, NY, USA) on day −1. The animals were administered 10⁶ spores of BI-1, GV106, GV135, or GV148, via oral gavage on day 0 (N = 5/group). Animals were weighed once daily and monitored twice daily for signs of stress. Surviving animals were humanely euthanized at the end of the study using a commercial euthanasia solution described above. Stool pellets or cecal content were collected daily, resuspended, homogenized in PBS, serially diluted, and plated onto TCCFA for isolation and bacterial burden (CFU/g of stool or cecal tissue).

Golden Syrian hamsters were used to assess virulence of select C. difficile strains [27 (link),28 (link)]. Seven to eight-week-old male hamsters (90–100 g; Charles River, Wilmington, MA, USA) were orally administered clindamycin (30 mg/kg) (Pfizer, New York, NY, USA) 72 h prior to infection. For pilot studies, animals (2 hamsters/strain) were orally infected with 100 spores of C. difficile LT-027 strain (GV106, GV135, or GV148) or a high-toxin RT027 strain (BI-1) [29 (link)]. For fully-powered studies, animals were infected with 100 spores of the LT-027 strain GV148 (N = 8), BI-1 (N = 6), or a non-toxigenic strain (T7; N = 2). In all studies, animals were monitored for overt disease every 6–12 h (diarrhoea, inappetence, ruffled fur, lethargy). Surviving animals were humanely euthanized at the end of the study using a commercial euthanasia solution (270 mg/kg sodium pentobarbital: Merck, Kenilworth, NJ, USA). C. difficile burden in stool and cecal contents was determined by plating on TCCFA. Cecal toxin burden was measured by centrifuging the cecal content at 14,000 RPM for 20 min, and using the surface 500 µL for toxin measurements as described above.