Cefoperazone

Six- to seven-week-old male and female mice were housed two per cage and drinking water was supplemented with 0.5 mg/mL cefoperazone (Sigma catalog #62893-20-3) as previously described (Noverr et al., 2005 (link)) on days 0–3 to clear the intestinal bacterial microbiota. Half of the mice further had their water supplemented with a cocktail of SCFAs according to previously established protocols (Smith et al., 2013 (link); Cait et al., 2018 (link)) for the duration of the experiment. The cocktail consisted of sodium acetate (67.5 mM), sodium propionate (25.9 mM), and sodium butyrate (40 mM), and the control animals received water that was pH and sodium matched (Smith et al., 2013 (link)). All water was filter sterilized and had Splenda added (8 g/L) to improve palatability. On day 3, all mice were given an oral gavage with 10⁷ cells of P. kudriavzevii obtained from a 48 hr culture generated from a single colony of yeast and grown at 37°C while shaking. Two days after the gavage, fecal samples were collected for plating. Uncolonized mice were removed from the analysis.

C. jejuni in the colon and cecum were quantified using a standardized semi-quantitative scoring system [30 (link)]. Briefly, colon and cecum tissue segments of the same size were collected at necropsy and were streaked on TSAB containing cefoperazone (2 μg/mL), vancomycin (10 μg/mL), and amphotericin B (2 μg/mL) (all antibiotics were obtained from Sigma-Aldrich, St. Louis MO) agar plates and grown in anaerobic jars equilibrated with CampyGen sachets (Oxoid) at 37 °C for 48–72 h. The resulting growth was assigned a score on a scale of 0–4 based on the density of growth; 0 (no growth), 1 (1–20 CFU), 2 (20–200 CFU), 3 (200–400 CFU), and 4 (confluent growth) as described [30 (link)].

Doxycycline (Dox), amoxicillin (Amo), cefoperazone (CefP), clofazimine (Cfz), miconazole (Mcz), polymyxin B (Pmb), sulfamethoxazole (Smx), daptomycin (Dap), carbomycin (magnamycin A), vancomycin, nisin, carbencillin, ofloxacin, tigecycline, hydroxychloroquine, rifampin, and clarithromycin (Sigma-Aldrich, St. Louis, USA) were dissolved in suitable solvents [38 ] to obtain stock solutions. The antibiotic stocks were filter-sterilized by 0.2 μm filter except clofazimine which was dissolved in DSMO (dimethylsulfoxide) and not filtered. Then the stocks were stored at -20°C.

Apis mellifera venom for in vitro testing and prepared using sterile 1× phosphate buffer saline pH 7.4 (PBS) (Fisher, Waltham, MA, USA). Natural melittin extracted from bee venom was purchased (Sigma, St. Louis, MO, USA) and prepared for testing as directed by the manufacturer. The antibiotics (Doxycycline, Cefoperazone, Daptomycin) were purchased from Sigma and prepared at 10 mg/mL stock per manufacturer’s instructions. In addition, Doxycycline, Cefoperazone and Daptomycin were also combined (D + C + D) to test on B. burgdorferi for the treatment of persister cells. All antimicrobial agents were sterilized using a 0.1 μm filter unit (Millipore, Billercia, MA, USA), aliquoted and stored at −20 °C before further use.

8-week old C57BL/6 mice were given cefoperazone (Sigma-Aldrich) (0.5 mg/ml) in sterile drinking water for five days which was refreshed every other day [31 (link)]. Mice were then switched to regular drinking water, allowed to recover for 2 days prior to C. difficile infection. For ex vivo germination assays, uninfected mice (n = 3) were euthanized, and at the time of necropsy ileal contents were collected, and frozen at -80°C until further analysis. For in vivo infections, groups of mice (n = 8) were inoculated by oral gavage with 50μL of water containing approximately 1500 spores as determined by phase contrast microscopy. Feces were collected daily for 4 days and samples weighed, serially diluted, and plated for total CFU per gram of feces (spores + vegetative cells). Samples from day one were also heat treated and assayed for total heat resistant CFU (spores). Mice were placed on a standard diet of Prolab Isopro RMH 3000 (LabDiet, St. Louis, MO) containing 1.1% calcium. C57BL/6 mice consume an average of ~4 g of chow per day [48 (link)].

Candida albicans ATCC 10231 was purchased from ATCC. Candida albicans SC5314 was kindly provided by Dr. Andrew Koh from University of Texas Southwestern Medical Center^{2 (link)}. Media used in this study included RPMI 1640 (Gibco, MA), MOPS (Sigma, MO), YPD Agar (BD Biosciences, CA), and Fetal Bovine Serum (Atlanta Biologicals, GA). Short-chain fatty acids (acetic acid, butyric acid, and propionic acid) were purchased from Sigma Aldrich (MO). Mice were purchased from The Jackson Laboratory (ME). Cefoperazone was purchased from Sigma Aldrich (MO). Other materials were purchased as indicated: mouse oral gavages (Kent Scientific, MA), vancomycin (Alfa Aesar, MA), gentamicin (Acros Organics, NJ), paraformaldehyde (Alfa Aesar, MA), and glycerol (DOT Scientific, MI).