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Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD)

Department of Biomedical Sciences, Oregon State University, 216 Dryden Hall, Corvallis, OR 97331, USA

Microbiology (2008), 154, part 8, 2241–2250

We were unable to reproduce germination results reported in our previous study (Paredes-Sabja et al., 2008) using Clostridium difficile spores prepared as described by Wilson et al. (1982) and Wilson (1983). Unfortunately, PCR analyses of spores used in our previous study (Paredes-Sabja et al., 2008) revealed that the spore suspensions of C. difficile were cross-contaminated with Clostridium perfringens, and thus the phenotype observed might not be related to C. difficile spores. Consequently, we revisited some of the germination assays with pure C. difficile spores of strains CD630 and 196. Germination was calculated by measuring the percentage of phase dark spores by phase-contrast microscopy. Results showed that fewer than 20 % of spores of both strains became phase dark when germinated with water, 100 mM KCl in 25 mM Tris/HCl (pH 7.4) or 100 mM sodium phosphate (pH 6.0) for 60 min at 40 °C (Fig. 1). However, the majority (75 %) of CD630 and 196 spores became phase dark after 60 min of incubation at 40 °C in sodium taurocholate and glycine (Fig. 1), consistent with results reported elsewhere (Sorg & Sonenshein, 2008). Collectively, these results indicate that C. difficile spores germinate poorly with KCl or sodium phosphate at pH 6.0, and are able to germinate with sodium taurocholate and glycine.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Germination of C. difficile spores. Heat-activated (60 °C, 20 min) spores of strains CD630 (grey bars) and 196 (black bars) were incubated for 60 min at 40 °C with: H2O, sterile distilled water; ST+Gly, 1 % sodium taurocholate and 1.3 mM glycine in 25 mM Tris/HCl (pH 7.0); KCl, 100 mM KCl in 25 mM Tris/HCl (pH 7.4); Pi, 100 mM sodium phosphate (pH 6.0). The percentage of phase dark spores was determined with phase-contrast microscopy by counting at least 200 spores.

REFERENCES

Paredes-Sabja, D., Bond, C., Carman, R. J., Setlow, P. & Sarker, M. R. (2008). Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD). Microbiology 154, 2241–2250.[Abstract/Free Full Text]

Sorg, J. A. & Sonenshein, A. L. (2008). Bile salts and glycine as co-germinants for Clostridium difficile spores. J Bacteriol 190, 2505–2512.[Abstract/Free Full Text]

Wilson, K. H. (1983). Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J Clin Microbiol 18, 1017–1019.[Abstract/Free Full Text]

Wilson, K. H., Kennedy, M. J. & Fekety, F. R. (1982). Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J Clin Microbiol 15, 443–446.[Abstract/Free Full Text]

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