Histodenz

For C. difficile strain 630 (tcdA⁺, tcdB⁺, tcdC⁺, ctdA⁻, and ctdB⁻)^{22 (link)} two independent biological spore crops were prepared as previously described on BHI agar plates^{14 (link)}. Sporulating cultures were routinely washed with ice cold sterile distilled water, and free spores were separated using 50% HistoDenz and washed five times to eliminate traces of HistoDenz (Sigma). Spore suspensions were further washed 10 times with 150 mM NaCl in milliQ-grade water to remove cytosolic proteins from the exosporium layer. Suspensions were > 99% free of vegetative cells, sporulating cells and cell debris. To remove loosely bound proteins and intracellular contaminants, spores were washed 5 times with 1 M NaCl. Purified spores were quantified with a Neubauer chamber prior to storage at −80°C until use. This storage condition does not affect spore viability and ultrastructural properties (unpublished data).

C. difficile sporulation and preparation was processed as described previously^{76 (link)} with minor modifications. Briefly, single colonies of C. difficile isolates were inoculated in deoxygenated BHIS broth and incubated anaerobically for 40–50 days. C. difficile cells were harvested by centrifugation and five washes with ice-cold water. The cells were then suspended in 20% (w/v) HistoDenz (Sigma, St. Louis, MO) and layered onto a 50% (w/v) HistoDenz solution before centrifugating at 15,000 × g for 15 min to separate spores from vegetative cells. The purified spores pelleted at the bottom were then collected and washed for four times with ice-cold water to remove traces of HistoDenz, and finally resuspended in sterile water. Prepared spores were heated to 60°C for 20 min to kill vegetative cells, diluted and plated on both BHIS agar and BHIS agar containing 0.1% (w/v) taurocholic acid (BHIS-TA) for numeration. Spore stocks for mouse infection were verified to have less than 1 vegetative cell per 200 spores (as the infection dose).

Clostridioides difficile strain UK1 (ribotype 027) was used in all experiments. Vegetative cells were generated by inoculating 10 mL of liquid BHIS (Thermo Fisher Scientific, MA, USA) media, supplemented with the appropriate taurocholate levels (typically 0.1%) or with the metabolic milieu from the stool sample of the antibiotic-treated mouse model. This was followed by growth for 24 hours, in an anaerobic chamber (BACTRON, Sheldon Manufacturing, OR, USA). Following growth, 10 μL of culture was streaked on BHIS agar plates supplemented with 0.1% taurocholate. Bacteria was grown on these plates for 7 days before being washed with ice cold sterile DI water to enumerate spores. In order to generate the spore sample, the spores were concentrated through centrifugation at 15,000 g for 15 minutes, and resuspended in 500 μL of 20% Histodenz (Sigma-Aldrich, USA) with an additional 500 μL of 50% Histodenz added to purify spores through gradient centrifugation at 15,000 g for 15 minutes. Spores were centrifuged and washed 3 times with sterile DI water in order to remove any residual Histodenz. Spores were then heated to 70°C for 20 minutes to kill any remaining vegetative cells and stored for up to 6 months at 4°C. The overall experimental timeline is shown in Fig. 1a.