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The alarming emergence of hypervirulent strains ofClostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. TheC. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturingC. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation ofC. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins ofC. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that onlyC. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producingC. difficile strains.

Novel One-Step Method for Detection and Isolation of Active-Toxin-Producing Clostridium difficile Strains Directly from Stool Samples