Ridascreen c difficile toxin a b

Free toxin levels produced in vitro were determined at two different time points (24 and 72 h post inoculation), as described previously [19 (link)]. Briefly, pure aliquots from each strain were plated onto Müller Hinton agar (Oxoid, UK) supplemented with 7% horse blood and 0.1% sodium taurocholate (Sigma-Aldrich Co., USA), followed by anaerobic incubation at 37°C for 18–24 h (stationary phase). After incubation, a suspension of C. difficile was prepared in sterile 0.85% saline, using McFarland standard 1; 100 μL of the suspension was transferred to cryotubes containing 500 μL of BHI broth (Oxoid, UK), and the tubes were subjected to anaerobic incubation at 37°C. After incubation for 24 and 72 h, the culture was centrifuged at 16,000 × g for 10 min at 4°C and filtered through a membrane filter with a 0.22 μm pore size. The toxin concentrations in the supernatants were evaluated using a commercial enzyme immunoassay (Ridascreen C. difficile Toxin A/B® - R-Biopharm, Germany) and the absorbance was measured at 450 nm according to the manufacturer´s instructions. Strain CD196 (RT027) was used as the reference strain.

C. difficile was isolated from fecal samples from humans and animals using a direct inoculation in cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) after ethanol shock, as previously described [54 (link)]. Colonies with suggestive morphology (flat, irregular, and with ground-glass appearance) were subjected to a previously described multiplex PCR [29 ] and PCR ribotyping [30 (link)]. RTs not identified in the Brazilian or Leiden (Netherlands) C. difficile library were sent to the National Reference Laboratory for C. difficile (University of Leeds, United Kingdom), and a new type number was assigned when necessary. In addition, toxigenic C. difficile strains, positive for binary toxin-encoding gene (cdtB) and identified as new RTs, were also submitted to MLST, as previously described [55 (link)]. Stool samples positive for toxigenic C. difficile were also subjected to toxin A/B detection using an enzyme-linked immunosorbent assay kit (Ridascreen C. difficile Toxin A/B®, R-Biopharm, Germany).