Abstract
BackgroundThe increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. To assess the role of hyper-production of Toxins A and B in clinical isolates of Clostridium difficile, two NAP1-related and five NAP1 non-related strains were compared.MethodsSequencing was performed on tcdC, tcdR, and tcdE to determine if there were differences that might account for hyper-production of Toxin A and Toxin B in NAP1-related strains. Biological activity of Toxin B was evaluated using the HFF cell CPE assay and Toxin A biological activity was assessed using the Caco-2 Trans-membrane resistance assay.ResultsOur results confirm that Toxin A and Toxin B production in NAP1-related strains and ATCC 43255 occurs earlier in the exponential growth phase compared to most NAP1-nonrelated clinical isolates. Despite the hyper-production observed in ATCC 43255 it had no mutations in tcdC, tcdR or tcdE. Analysis of the other clinical isolates indicated that the kinetics and ultimate final concentration of Toxin A and B did not correlate with the presence or lack of alterations in tcdC, tcdR or tcdE.ConclusionOur data do not support a direct role for alterations in the tcdC gene as a predictor of hyperproduction of Toxin A and B in NAP1-related strains.
Highlights
The increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production
The C. difficile strain designated NAP1 was the most prevalent strain associated with the Quebec outbreak of C. difficile-associated diarrhea (CDAD) [4,5,7,8,15] and it has been found in other parts of Canada as well [20]
The objective of this study was to evaluate a variety (Figure 1) of clinical strains of C. difficile (NAP1 related and NAP1 non-related) to determine if there was a correlation between the genetic changes detected in the putative negative regulator gene and the growth kinetics and toxin production
Summary
The increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. The significant increase in incidence and disease severity reported for the Quebec outbreak have prompted investigations to determine if this strain has some unique virulence characteristics. This strain has the gene (cdtB) encoding binary toxin [4,18,21,22] and this has been suggested to contribute to disease severity [15,23,24]. Because there may be differences in the biological activity versus antigen detected, further data evaluating the biological levels of Toxins A and B are needed
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