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• W2051260712 abstract "Michel Warny and colleagues (Sept 24, p 1079)1Warny M Pepin J Fang A et al.Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.Lancet. 2005; 36: 1079-1084Summary Full Text Full Text PDF Scopus (1183) Google Scholar describe increased toxin production by a strain of Clostridium difficile (NAP1/027) associated with outbreaks of severe disease in North America and Europe. We are concerned with several aspects of the experimental design and interpretation. The in-vitro batch (test tube) culture Warny and colleagues use to investigate C difficile physiology does not reflect the continual replacement of nutrients and metabolites that occurs in vivo. Crucially, substrate limitation affects C difficile physiology.2Dupuy B Sonnenshein AL Regulated transcription of Clostridium difficile toxin genes.Mol Microbiol. 1998; 27: 107-120Crossref PubMed Scopus (207) Google Scholar Several studies that used similar in vitro experimental approaches to measure C difficile toxin production have yielded results that are either contradictory or do not accord with clinical observations.3Barc MC Depitre C Corthier G Collignon A Su WJ Bourlioux P Effects of antibiotics and other drugs on toxin production in Clostridium difficile in vitro and in vivo.Antimicrob Agents Chemother. 1992; 36: 1332-1335Crossref PubMed Scopus (23) Google Scholar Animal (hamster) or human gut chemostat4Freeman J Baines SD Jabes D Wilcox MH Comparison of the efficacy of ramoplanin and vancomycin in both in vitro and in vivo models of clindamycin-induced Clostridium difficile infection.J Antimicrob Chemother. 2005; 56: 717-725Crossref PubMed Scopus (118) Google Scholar models of C difficile infection more closely reflect in-vivo conditions, and are thus more appropriate methods of investigation. Additionally, the use of a proprietary medium, with no description of its constituents nor justification for its use, is a major obstacle to others attempting to reproduce the results reported by Warny and colleagues. Results from batch culture experiments could be improved by use of multiple replicates. Such a practice is widely accepted, since it reduces the risk of anomalous results, which could pass unrecognised in a single experiment. Figure 2 in Warny and colleagues' paper1Warny M Pepin J Fang A et al.Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.Lancet. 2005; 36: 1079-1084Summary Full Text Full Text PDF Scopus (1183) Google Scholar is described as a growth curve, but contains only three observations over 48 h. There is little evidence of corresponding viable counts against which to validate spectrophotometric readings, and Warny and colleagues' determination of the simple presence or absence of spores of each strain is of little value without quantification. Without these crucial data, Warny and colleagues' conclusions about toxin production during logarithmic versus stationary phase are not sustainable. The use of batch culture and an undefined, highly specialised medium, together with absence of essential growth kinetics data cast doubt on the significance of these results in relation to in-vivo bacterial behaviour. Furthermore, some parts of the analysis and interpretation should be regarded with caution. First, Warny and colleagues do not regard a significantly greater 24-h cell density of NAP1/027 than of control strains as a contributory factor for increased toxin production. But greater cell numbers can clearly yield more toxin. Second, the comparison of two groups, one (toxinotype III, n=15) containing only two pulsed-field gel electrophoresis (PFGE) types, the other (toxinotype 0, n=25) containing 12 PFGE types, is inappropriate. Analysis of the results for two groups of strains potentially obscures variation in toxin production across the greater number of PFGE types within the toxinotype 0 group, notably when presenting this as a single median value. The optimum analysis of toxin production is by PFGE-type-specific comparisons with ANOVA, which would not mask interstrain variability. NAP1/027 has been associated with severe symptoms and complication rates that do not match previous clinical experience of C difficile infection.5Pepin J Valiquette L Alary ME et al.Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity.CMAJ. 2004; 171: 466-472Crossref PubMed Scopus (942) Google Scholar We urge caution in the interpretation of preliminary in-vitro data. We encourage study of C difficile virulence determinants in defined models of infection to determine potential intervention and treatment options. We declare that we have no conflict of interest. Measurement of toxin production by Clostridium difficile – Authors' replySubstrate limitation does affect Clostridium difficile physiology, and that, as well as other factors, could limit the ability of any in vitro system to simulate what occurs in-vivo. However, there is no in-vivo model in which toxin production by various strains can be compared. Any human gut chemostat model is restricted by our limited understanding of the human intestinal environment; assumptions made in the design of such chemostats only decrease their potential to represent accurately what occurs in vivo. Full-Text PDF" @default.
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• W2051260712 title "Measurement of toxin production by Clostridium difficile" @default.
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• W2051260712 doi "https://doi.org/10.1016/s0140-6736(06)68417-1" @default.
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