Ribotype

A panel of well characterised C. difficile isolates representing 70 distinct PCR-ribotypes was used in this study [22 (link)]. The panel comprised PCR-ribotypes known to be associated with human CDI in Europe. This collection was assembled using type strains previously shared between two established PCR-ribotyping laboratories (Clostridium difficile Network for England and Northern Ireland (CDRN), and National Reference Laboratory for Clostridium difficile at University Medical Centre, Leiden). All PCR-ribotypes were originally assigned in association with the Anaerobic Reference Laboratory at Cardiff (ARL) using agarose gel-based PCR-ribotyping technique (Table 1). Data on isolates in the panel have been made available on-line in a National Center for Biotechnology Information BioProject database (NCBI) (http://www.ncbi.nlm.nih.gov/bioproject/248340). In addition, a subset (European Centre for Disease Prevention and Control (ECDC)-Brazier collection) is available to all reference laboratories in Europe who participate in the European C. difficile infection study network (ECDIS-NET) [23 ].

A total of 141 well-characterized Austrian clinical isolates (Indra et al., 2008 (link)) collected in the period 2006–2007 at 25 Austrian healthcare facilities were used in this study. Control strains for each of C. difficile ribotypes 001, 014, 017 and 027 were obtained from the Department of Medical Microbiology, University Medical Center, Leiden. The control strain for ribotype 053 was an Austrian isolate ribotyped by J. Brazier, Department of Microbiology, University of Cardiff, Wales, UK.

To improve the efficiency of the allele exchange mutagenesis in C. difficile, we made use of the inducible toxicity of the CD2517.1 type I toxin that we previously reported^{27 (link)}. To construct the pMSR vector, used for allele exchange in C. difficile 630Δerm, the codA gene was removed from the “pseudosuicide” vector pMTL-SC7315^{32 (link)} by inverse PCR, and replaced by a 1169 bp fragment comprising the entire P_tet promoter system and the downstream CD2517.1 toxin gene. This fragment was amplified from pDIA6319 plasmid^{27 (link)} and the purified PCR product was cloned into the linearized plasmid. In parallel, the pMSR0 vector, for allele exchange in C. difficile ribotype 027 strains and other ribotypes, was constructed by removing the codA gene from the vector pMTL-SC7215 by inverse PCR and replacing it with the CD2517.1-RCd8 TA region with CD2517.1 under the control of the P_tet promoter, as described above, and RCd8 under the control of its own promoter. For deletions, allele exchange cassettes were designed to have between 800 and 1050 bp of homology to the chromosomal sequence in both upstream and downstream locations of the sequence to be altered. The homology arms were amplified by PCR from C. difficile strain 630 genomic DNA (Supplementary Data 2) and purified PCR products were directly cloned into the PmeI site of pMSR vector using NEBuilder HiFi DNA Assembly. To insert P_thl-ermB into the phiCD630-1 prophage, within the intergenic region between CD0946.1 and CD0947 genes, homology arms (~900 bp upstream and downstream of the insertion site) were amplified by PCR from strain 630 genomic DNA (Supplementary Data 2). The P_thl-ermB cassette was amplified from the Clostron mutant cwp19^{33 (link),51} . Purified PCR products were all assembled and cloned together into the PmeI site of pMSR vector using NEBuilder HiFi DNA Assembly.
All pMSR-derived plasmids were initially transformed into E. coli strain NEB10β and all inserts were verified by sequencing. Plasmids were then transformed into E. coli HB101 (RP4) and transferred by conjugation into the appropriate C. difficile strains. The adopted protocol for allele exchange was similar to that used for the codA-mediated allele exchange^{32 (link)}, except that counter-selection was based on the inducible expression of the CD2517.1 toxin gene. Transconjugants were selected on BHI supplemented with Cs, Cfx, and Tm, and then restreaked onto fresh BHI plates containing Tm. After 24 h, faster-growing single-crossover integrants formed visibly larger colonies. One such large colony was restreaked once or twice on BHI Tm plate to ensure purity of the single crossover integrant. Purified colonies were then restreaked onto BHI plates containing 100 ng/ml ATc inducer to select for cells in which the plasmid had been excised and lost. In the presence of ATc, cells in which the plasmid is still present produce CD2517.1 at toxic levels and do not form colonies. Growing colonies were then tested by PCR for the presence of the expected deletion.

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.

The study used the C. diff Banana Broth^TM medium (Hardy Diagnostics, Santa Maria, US), which is based on Brucella Broth supplemented with vitamin K and hemin. The L-cystine it contains enables the growth of thiol-dependent microorganisms. The germinating Clostridioides spores cause the fermentation of mannitol, which changes the colour of the medium from red to yellow, a positive result. The swabs were collected with sterile flocked swabs provided by the manufacturer, wetted with sterile 0.85% saline solution, and then placed in vials with the banana broth. The medium was incubated from 3 to 14 days at 37 °C, and its colour and condition were examined every 24h. Positive samples were screened to CLO and CDIFF agar media (BioMérieux, France) and to Columbia Blood Agar medium (Graso Biotech, Poland). The agar media were incubated for 48h at a temperature of 37 °C in anaerobic conditions using anaerobic culture sets—GENbag Atmosphere Generators (BioMérieux, France). Anaerobic conditions were assessed using anaerobic indicator strips (BioMérieux, France). In order to identify the Clostridioides strains, a Vitek 2 Compact device (BioMérieux, France) was used. In the case of detection of Clostridioides strains from the C. difficile species, genetic testing was performed using a GeneXpert device (Cepheid GmbH, Germany). XpertC.difficile BT sets were used for this purpose, which detect a sequence of genes responsible for the production of the B toxin (tcdB), the binary toxin (cdtA) and the tcdC base pair deletion at position 117 related to the hyperepidemic 027 ribotype strain. For toxigenic strains of C. difficile, their susceptibility to antibiotics used in clinical practice, that is, metronidazole and vancomycin, was tested with the use of E-tests (BioMérieux, France). These tests enabled the establishment of the antibiotics’ MIC (Minimum Inhibitory Concentration) value. The susceptibility testing was conducted based on the current version of the EUCAST guidelines (version 12.0, valid as of 01.01.2022).

PCR amplification of toxin genes was performed to detect the toxin genes tcdA, tcdB, cdtA and cdtB (Table 6), using the following primer pairs. The primers NK 2 (5′-CCCAATAGAAGATTCAATATTAAGCTT-3′) and NK 3 (5′-GGAAGAAAAGAACTTCTGGCTCACTCAGGT-3′) served to amplify the non-repetitive region of tcdA, NK 9 (5′-CCACCAGCTGCAGCCATA-3′) and NK 11 (5′-TGATGCTAATAATGAATCTAAAATGGTAAC-3′) the repetitive region of tcdA. For tcdB, the primer pair NK 104 (5′-GTGTAGCAATGAAAGTCCAAGTTTACGC-3′) and NK 105 (5′-CACTTAGCTCTTTGATTGCTGCACCT-3′) was used [52 (link),53 (link)]. For the binary toxin, primers cdtApos (5′-TGAACCTGGAAAAGGTGATG-3′) and cdtArev (5′-AGGATTATTTACTGGACCATTTG-3′) were used to amplify cdtA; cdtBpos (5′-CTTAATGCAAGTAAATACTGAG-3′) and cdtBrev (5′-AACGGATCTCTTGCTTCAGTC-3′) for cdtB [54 (link)].
PCR ribotyping was performed with capillary gel electrophoresis according to a modified version of the protocol of Indra et al. [55 (link)]. Primers 16S 6FAM (5′-6FAM-GTGCGGCTGGATCACCTCCT-3′) and 23S (5′-PET-CCCTGCACCCTTAATAACTTGACC-3′) were used. DNA was diluted to 1 ng/µL and 1 µL was added to 24 µL of a PCR-mix containing 0.2 µL (5 U/µL) DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), 1 µL of each primer (working dilution of 10 pmol/µL), 1 µL of 10 mM dNTP-Mix (Carl Roth, Karlsruhe, Germany), 1 µL MgCl₂ (25 mM) (Qiagen, Hilden, Germany), 2.5 µL DreamTaq buffer (Thermo Fisher Scientific, Waltham, MA USA) and 17.3 µL water.
PCR was performed as follows: 95 °C (2 min) for initial denaturation; 30 cycles of 95 °C (30 s) for denaturation, 52 °C (30 s) for annealing, 72 °C (2 min) for elongation; 72 °C (8 min) for the final extension. PCR products were diluted 1:75 with water and ribotyping was performed by capillary electrophoresis followed by a web-based analysis. The fragment separation was performed with a SeqStudio Genetic Analyzer, SeqStudio™ Cartridge v2 (POP-1 polymer, four capillaries, 28 cm capillary length) and GeneScan™ 600 LIZ™ Size Standard v2.0. The conditions of separation were defined via FragAnalysis Run Module. The fragment length results were converted into a suitable data format and analysed with the publicly accessible WEBRIBO database to assign a ribotype [56 ].