Brain heart infusion medium

Except for C. difficile strains 11S0047 and 12S0133, which were provided by Christian Seyboldt, Friedrich-Loeffler-Institute Jena, all strains were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH). All cultivation experiments were conducted in an anaerobic workstation (Whitley DG250 anaerobic workstation; 98% N₂, 2% H₂) at 37°C if not stated otherwise. C. difficile spores were inoculated in Brain Heart Infusion medium (Oxoid, Basingstoke, UK) with 0.1% taurocholic acid (Sigma-Aldrich, St. Louis, USA) to allow germination. All other bacteria were inoculated from frozen glycerol stocks.

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.

E. coli TOP10 or DH5α cells were grown in brain heart infusion medium (Oxoid Ltd., Carlsbad, CA) or lysogeny broth at 37°C with shaking. L. plantarum cells were grown in de Man, Rogosa, and Sharpe (MRS) broth (Oxoid) at 37°C without shaking. Solid media were prepared by addition of 1.5% (wt/vol) agar to the broth. L. lactis was used as subcloning host for plasmids containing the SH71_rep replicon and were transformed as previously described (53 (link)). All other plasmids were subcloned in E. coli. The final plasmids were electrotransformed into L. plantarum (54 (link)). When required, antibiotic concentrations were added as follows: for L. plantarum and L. lactis, 10 µg/ml erythromycin and 10 µg/ml chloramphenicol; for E. coli, 200 µg/ml erythromycin and 10 µg/ml chloramphenicol.

Specific pathogen free-grade female NIH HL mice and normal NIH mice (2 or 6 weeks old) were provided by the Changchun Institute of Biological Products Co. Ltd. (SCXK-2016-0008). Two-week-old female NIH HL mice were co-housed with 2-week-old female NIH normal mice for 4 weeks. The bacterial strain used in this study was L. monocytogenes 10403S (serotype 1/2a). L. monocytogenes was cultured in brain heart infusion medium (Oxoid, Hampshire, England) and incubated in a shaker incubator at 37°C and 200 rpm.

All strains used in this study (Table 1) were cultivated on sheep blood agar plates (Oxoid, Basingstoke, United Kingdom) and incubated at 37°C and 5% CO₂. For all non- Escherichia coli bacteria, liquid cultivation was performed in Todd-Hewitt Broth (Oxoid) supplemented with 0.5% yeast extract (THY, Gibco, Waltham, MA) under the same conditions. For E. coli, liquid cultivation was performed in lysogeny broth (LB-Miller) at 37°C while shaking (180 rpm). Listeria monocytogenes harboring pNZ44 or its derivates was cultivated in brain-heart-infusion medium (Oxoid) supplemented with 10 μg/ml chloramphenicol (Sigma-Aldrich Chemie GmbH, Steinheim am Albuch, Germany) at 37°C while shaking (180 rpm).