Enrichment and Identification of Clostridium difficile

Fecal samples were processed within 2 hours after thawing. Enrichment culture was performed as previously described (7 (link),18 (link)). Briefly, ≈1 g of homogenized fecal matter was mixed with 2 mL of 96% ethanol and agitated at room temperature for 50 minutes to select for bacterial spores. The sediment was recovered after centrifugation at 3,800 × g for 10 minutes and resuspended in 5 mL of cycloserine-cefoxitin fructose broth (C. difficile agar and C difficile supplement SR0096; Oxoid, Columbia, MD, USA) that was incubated anaerobically at 37°C for 7 days. This broth was treated with 96% ethanol (1:1 vol/vol), centrifuged at 3,800 × g for 10 minutes, and the sediment was resuspended in 200 μL of sterile deionized water. Thereafter, 200 μL of sediment was streaked onto cycloserine-cefoxitin fructose agar and blood agar that were incubated anaerobically at 37°C. Plates were evaluated in an anaerobic environment daily for <5 days. If present, at least 2 colonies (swarming, flat, rough, nonhemolytic) were subcultured. C. difficile was identified by Gram stain (spore-forming gram-positive rods) and detection of L-proline aminopeptidase activity (Pro Disc, Remel, Lenexa, KS, USA) (19 (link)). Isolates were stored at –70°C until molecular analyses were performed.
Feces were screened for C. difficile toxins A and B by using an ELISA (Tox A/B ELISA, TechLab, Blacksburg, VA, USA) (20 (link)). The test was performed per manufacturer's instructions. Two observers interpreted the reactions in a blinded fashion.