Effect of metronidazole on growth and toxin production by epidemic Clostridium difficile PCR ribotypes 001 and 027 in a human gut model

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Abstract
We compared the behaviour of Clostridium difficile PCR ribotypes 001 and 027 in a human gut model, and compared the responses to metronidazole exposure. Using a human gut model primed with pooled human faeces, gut flora bacterial counts, C. difficile total viable counts, spore counts and cytotoxin titres were determined, following exposure to clindamycin, in the absence or presence of metronidazole. Duration of cytotoxin production by C. difficile ribotype 027 was markedly longer than that of ribotype 001 (23 versus 13 days, respectively), but peak toxin titres were similar. During toxin production, total C. difficile ribotype 027 populations had higher proportions of vegetative cells than did ribotype 001 (median 56.33 versus 23.54%). Similarly, total C. difficile ribotype 027 populations remained predominantly as vegetative cells for longer than did ribotype 001 (20 versus 9 days). The effects of metronidazole on C. difficile were markedly less than expected. Titres of C. difficile ribotype 001 cytotoxin were reduced but recurred following metronidazole administration. C. difficile ribotype 027 cytotoxin titres in the distal section of the gut model were unaffected by metronidazole. These observations correlated with poor metronidazole concentrations. Duration of cytotoxin production by C. difficile ribotype 027 markedly exceeds that of ribotype 001. Sub-optimal gut concentrations of metronidazole, possibly due to inactivation by components of normal gut flora, are associated with continued toxin production. These findings may help to explain the increased severity of symptoms and higher case-fatality ratio associated with infections due to C. difficile ribotype 027.