Muricholic Acids Inhibit Clostridium difficile Spore Germination and Growth

Francis, Michael; Allen, Charlotte A.; Sorg, Joseph A.

doi:10.1371/journal.pone.0073653

Cited by 58 publications

(83 citation statements)

References 52 publications

(104 reference statements)

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“…by Francis et al demonstrates that murine bile acids (muricholic acids) inhibit C. difficile spore germination and that there is strain variability in this inhibition (48). In addition, published data show there is significant variability in rates of germination and the compounds that serve as germinants among large sets of C. difficile strains of varying ribotyes, at least in vitro (49).…”

Section: Discussionmentioning

confidence: 99%

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

et al. 2014

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Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.

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Section: Discussionmentioning

confidence: 99%

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

et al. 2014

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“…Wild-type C. difficile UK1 (6,12,15) and C. difficile M68 (15,24,25), C. difficile JSC10 (cspC::ermB) (6), and C. difficile CAA5 (sleC::ermB) were routinely grown in an anaerobic atmosphere (10% H 2 , 5% CO 2 , 85% N 2 ) at 37°C in brain heart infusion agar supplemented with 5 g/liter yeast extract and 0.1% L-cysteine (BHIS). B. subtilis PS533 and B. subtilis FB113 (cwlJ::tet sleB::spc) (26) were a generous gift from Peter Setlow and were routinely grown on Difco sporulation medium (DSM).…”

Section: Bacteria and Strainsmentioning

confidence: 99%

“…C. difficile spores were generated as described previously (6,12,15,29). B. subtilis vegetative cells were spread on DSM agar medium for spore production (30).…”

Section: Bacteria and Strainsmentioning

confidence: 99%

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Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

2015

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Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCEClostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway. Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) has become a significant threat to antibiotic-treated or immunocompromised hosts. Antibiotics are known to disrupt the colonic microbiota, and this perturbation permits C. difficile colonization (1, 2). Due to the strict anaerobic nature of C. difficile cells, spores are generally thought to be the infectious agent (only the spore can survive for extended periods of time in the aerobic environment outside a host) (3, 4). Because the spore form is noninfectious, spores must germinate to actively growing bacteria which initiate infection (5, 6). Thus, germination by C. difficile spores represents one of earliest steps in the pathogenesis of this organism.Endospore germination has been extensively studied in Bacillus sp. and, more recently, in clostridia (7,8). In the spore core,...

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“…C. difficile spore germination is stimulated by cholic acid, while CDCA derivatives inhibit cholic acid-mediated germination (11,19,23,24). Based on previous studies, C. difficile spores correspond to 50% effective concentrations (EC 50 values; the concentrations that achieve a half-maximum germination rate) in the low-millimolar range for TA and the high-micromolar range for CDCA, suggesting that C. difficile spores may have a tighter interaction with inhibitors of germination than with the activators of germination (11,(23)(24)(25).…”

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confidence: 99%

Reexamining the Germination Phenotypes of Several Clostridium difficile Strains Suggests Another Role for the CspC Germinant Receptor

et al. 2016

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Clostridium difficile spore germination is essential for colonization and disease. The signals that initiate C. difficile spore germination are a combination of taurocholic acid (a bile acid) and glycine. Interestingly, the chenodeoxycholic acid class (CDCA) bile acids competitively inhibit taurocholic acid-mediated germination, suggesting that compounds that inhibit spore germination could be developed into drugs that prophylactically prevent C. difficile infection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describing C. difficile strains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms of C. difficile spore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on how C. difficile spores initiate germination. Therefore, we quantified the interaction of these strains with taurocholic acid and CDCA, the rates of spore germination, the release of DPA from the spore core, and the abundance of the germinant receptor complex (CspC, CspB, and SleC). We found that strains previously observed to germinate in the absence of taurocholic acid correspond to more potent 50% effective concentrations (EC 50 values; the concentrations that achieve a halfmaximum germination rate) of the germinant and are still inhibited by CDCA, possibly explaining the previous observations. By comparing the germination kinetics and the abundance of proteins in the germinant receptor complex, we revised our original model for CspC-mediated activation of spore germination and propose that CspC may activate spore germination and then inhibit downstream processes. IMPORTANCEClostridium difficile forms metabolically dormant spores that persist in the health care environment. In susceptible hosts, C. difficile spores germinate in response to certain bile acids and glycine. Blocking germination by C. difficile spores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certain C. difficile strains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors of C. difficile spore germination (calling into question the utility of such strategies). Here, we further investigate these strains and reestablish that bile acid activators and inhibitors of germination affect these strains and use these data to suggest another role for the C. difficile bile acid germinant receptor. S pore formation and germination by Peptoclostridium difficile spores (1) (referred to here as Clostridium difficile for simplicity) are significant hurdles for overcoming the transmission of this pathogen within the hospital environment. Due to the strict anaerobic nature of C. difficile vegetative cells, spores are thought to be the main reservoir for transmission within the health care setting (2, 3). Prior antibiotic treatment ...

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Muricholic Acids Inhibit Clostridium difficile Spore Germination and Growth

Cited by 58 publications

References 52 publications

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

Reexamining the Germination Phenotypes of Several Clostridium difficile Strains Suggests Another Role for the CspC Germinant Receptor

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