Spore Isolation and Quantification of Clostridioides difficile

C. difficile isolate DS1813, CD630 and R20291 spores were sourced from the Anaerobic Reference Unit, University Hospital Wales, Cardiff, UK [51 (link)]. All three isolates are clinical isolates, with the DS1813 and R20291 belonging to the hypervirulent 027 ribotype of C. difficile, while the CD630 belong to the 012 ribotype and is a commonly studied and fully gene sequenced [53 (link)]. Spores were grown on BHIS-ST agar (BHI supplemented with 0.5% yeast extract, 1% L-cysteine and 0.1% sodium taurocholate) [54 (link)] at 37 ${}^{\circ }$ C for 4 days under anaerobic conditions (85% N ${}_2$ , 10% CO ${}_2$ , 5% H ${}_2$ ). The colonies were collected, washed with deionised water and left overnight at 4 ${}^{\circ }$ C to release of spores from mother cells. The suspensions were then purified using non-damaging density gradient centrifugation in 50% sucrose as described previously [21 (link), 55 (link), 56 (link)]. Spores were then washed in deionised water and stored at 4 ${}^{\circ }$ C. This method avoids spore purification steps such as lysozyme or proteinase, to ensure that spores and their resilience to chemicals are representative of the spores typically found in hospital environments [57 (link)]. B. thuringiensis (ATCC 35646) spores were sourced from the Swedish Defence Research Agency (FOI), Umeå, Sweden.
We determined the concentration of viable spores in the stock by serially diluting in deionised water down to ${10}^{-7}$ concentration and 10 $\mathrm{\mu }$ l drops plated [58 (link)] onto BHIS-ST agar plates and grown at 37 ${}^{\circ }$ C in anaerobic conditions.