Chenodeoxycholate Is an Inhibitor of Clostridium difficile Spore Germination

Abstract

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.The anaerobic human pathogen Clostridium difficile must be in the spore form to survive for extended periods of time outside the colonic environment (6). Spores are also the form of the organism most likely to be ingested by a host. To cause disease, however, C. difficile spores must germinate in the gastrointestinal tract and reach the anaerobic environment of the colon, where they can grow out as vegetative bacteria (2). The vegetative form produces two toxins that damage the colonic epithelium and lead to C. difficile-associated diseases, such as diarrhea, pseudomembranous colitis, and toxic megacolon (4,15). Extending the work of Wilson and colleagues (17, 18), we have shown that certain bile salts and glycine act as cogerminants for C. difficile spores (13). Primary bile salts produced by the liver are composed mainly of cholate (CA) and chenodeoxycholate (CDCA) derivatives conjugated with either taurine or glycine (11). Since CA derivatives are found in the relatively aerobic proximal ileum (9), we reasoned that C. difficile might benefit if its germination were inhibited until the spores reached the anaerobic environment of the large intestine.Inhibitors of germination are typically structurally similar to the germinant whose activities they inhibit. For example, Lalanine-mediated germination of Bacillus subtilis spores is inhibited by D-alanine (16) and 6-thioguanosine inhibits inosinemediated germination in Bacillus anthracis (1, 16). Since CA and CDCA are structurally similar but CA induces the germination of C. difficile spores (13) and CDCA does not, we tested whether CDCA could act as an inhibitor of germination. C. difficile strain CD196 (10) spores were produced and their concentration determined as described previously (13). After the vegetative bacteria were killed by incubation at 60°C for 20 min, spores were incubated in water containing various concentrations and combinations of bile salts for 10 min. Here we took advantage of the finding by Wilson et al. that C. difficile spores germinate very inefficiently on rich medium plates lacking bile salts (18) unless they are preincubated with bile salts (13, 17). After incubation, spores were serially diluted and plated on brain heart infusion agar supplemented with 5 g yeast extract per liter-0.1% L-cysteine (BHIS) (Difco) in the absence of any bile salt (BHIS contains enough glycine to act as a cogerminant). After overnight growth at 37°C, colonies were enumerated. As a positive or negative control, spores were plated on BHIS containing 0.1% taurocholate (TA) [BHIS(TA)] or on BHIS agar alone, respectively. Preincubation of spores with 0.1% TA in water resulted in the recovery of approximately 0.5% of the tot...

Progesterone Analogs Influence Germination of Clostridium sordellii and Clostridium difficile Spores In Vitro

“…Indeed, recent work has shown that C. difficile spores recognize taurocholate (a bile salt) and glycine (an amino acid) as germinants (53). Furthermore, chenodeoxycholate was shown to be a naturally occurring inhibitor of C. difficile spore germination (54). Work from our laboratory has shown cooperative binding of both taurocholate and glycine, suggesting the presence of unidentified receptors for both germinants (39).…”

Clostridium difficile and the microbiota

“…In vitro studies indicate that germination and outgrowth into the vegetative form depends on the presence of specific primary bile acids, such as taurocholate (20,21). Conversely, other secondary bile acids, such as chenodeoxycholate, inhibit the germination of C. difficile spores (22). Microbes within the gastrointestinal tract play a key role in the metabolism of bile acids (23), and it is hypothesized that the modulation of the microbiota community can impact metabolite availability.…”

Clostridium difficileinfection

“…126 Based on their studies on the differential growth of C. difficile on various bile salts, Sorg & Sonnenshein propose yet another mechanism by which the native flora may impede CDI. 127,128 Their hypothesis is that native flora convert cholate to deoxycholate, a compound that is toxic to vegetative C. difficile and inhibits germination of C. difficile spores.…”