Germinant Synergy Facilitates Clostridium difficile Spore Germination under Physiological Conditions

Abstract

Clostridium difficile is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. C. difficile is the leading cause of nosocomial infectious diarrhea worldwide. C. difficile infection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step in C. difficile pathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest that C. difficile germination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficile is an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of a C. difficile infection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitate C. difficile spore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, but in vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulate C. difficile spore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

Keywords: Clostridium difficile; germination; spore.

FIG 1

C. difficile spores germinate efficiently in the mouse ileum. C. difficile VPI 10463 spores were incubated in various GI contents from antibiotic-treated mice (n = 5) for 1 h at 37°C. Data are presented as percent germination, which was determined by differential plating (CFU on BHIS/total CFU on BHIS-Tc × 100). One hundred percent germination is the amount of CFU when plated on BHIS+Tc. Statistical analyses were done using two-way analysis of variance (ANOVA), and germination in ileal contents was significantly different from all other conditions (P < 0.0001).

FIG 2

pH regulates C. difficile spore germination in vitro. (A) C. difficile VPI 10463 spores were incubated at 37°C in BHIS+Tc at various pH values for 1 h, and germination was monitored by decrease in OD₆₀₀. (C) VPI 10463 spores were incubated at 37°C in BHIS+Tc at various pH values for 15 min, and SleC activation was observed by Western blotting. (B) VPI 10463 spores were incubated at 37°C with 0.2% Tc in BHIS at a pH of 4.5 for 1 h, and optical density was tracked every 30 min. After 1 h, the sample was split into two, and the pH of one sample was adjusted to 7.5, and the other remained at 4.5. Germination was tracked via decreases in the OD at 37°C over the course of 1 h. (D) For observing SleC activation, VPI 10463 spores were incubated as described above, and samples were taken every 30 min and prepared for Western blotting. Germination (loss of OD) assays are the results from three independent spore preparations. Western blots are representative of three independent spore preps. In panels C and D, samples labeled with an asterisk indicate that the sample did not receive taurocholate.

FIG 3

pH regulates C. difficile spore germination in small intestinal contents. Duodenal contents and ileal contents were harvested from mice treated with antibiotics (n = 5), and the contents were then solubilized in PBS. Supernatants were aliquoted into three groups: unadjusted (PBS), adjusted with acid (HCl), or adjusted with base (NaOH) before incubation with VPI 10463 spores for 30 min. Germination was measured by differential plating as described in Materials and Methods. Data are presented as the fold change in percent germination compared to percent germination in unadjusted ileal contents. pH was estimated using phenol red (pH < 6.8 [yellow] and pH > 8.2 [pink]).

FIG 4

C. difficile germination rates are dependent on concentrations of germinant. (A and B) C. difficile VPI 10463 spores were incubated with 0.2% Tc and various concentrations of either glycine (A) or CaCl₂ (B), and rates of release of DPA were measured and presented as percent maximal germination rate. (C) VPI 10463 spores were incubated with 0.2% Tc, 50 μM CaCl₂, and the indicated concentrations of glycine, germination rates were observed by DPA release, and EC50 values were calculated. (D) VPI 10463 spores were also incubated with 0.2% Tc, 500 μM glycine, and the indicated concentrations of calcium, germination rates were observed by DPA release, and EC50 values were calculated. SleC activation was observed by Western blotting, VPI 10463 spores were incubated in Tris-HCl plus 1% Tc, and the indicated concentrations of glycine (D) or CaCl₂ (E). In panels D and E, samples labeled with an asterisk indicate that the sample did not receive taurocholate.

FIG 5

Calcium synergizes with other amino acids to improve C. difficile germination efficiency. (A to D) C. difficile VPI 10463 spores were incubated with 0.2% Tc, 50 μM CaCl₂, and either 1 or 10 mM concentration of glycine (A), serine (B), alanine (C), or histidine (D), and release of DPA was measured over the course of an hour. Data from three independent experiments and two spore preparations were normalized and presented as percent maximal germination.

FIG 6

Germinant synergy overcomes chenodeoxycholate inhibition of germination. (A) C. difficile VPI 10463 spores were incubated with 20 mM glycine in the presence (+) or absence (−) of 250 μM CDCA and the indicated concentrations of taurocholate, and release of DPA was measured after 60 min. (B) VPI 10463 spores were incubated with taurocholate (0.05% [+] or 0.2% [++]) and the indicated concentrations of either glycine, calcium, or CDCA, and release of DPA was measured after 60 min. Data from three independent experiments and two spore preparations were normalized and presented as percent maximal germination. Data were analyzed by one-way ANOVA, and values that are significantly different are indicated by a bar and asterisks as follows: *, P = 0.0269; ****, P < 0.001.