Germination efficiency of clinical Clostridium difficile spores and correlation with ribotype, disease severity and therapy failure

Spore germination is an important part of the pathogenesis of Clostridium difficile infection (CDI). Spores are resistant to antibiotics, including those therapeutically administered for CDI and strains with a high germination rate are significantly more likely to be implicated in recurrent CDI. The role of germination efficiency in cases of refractory CDI where first-line therapy fails remains unclear. We investigated spore germination efficiencies of clinical C. difficile isolates by measuring drop in OD600 and colony forming efficiency. Ribotype 027 isolates exhibited significantly higher germination efficiencies in the presence of 0.1 % (w/v) sodium taurocholate (51.66±8.75 %; 95 % confidence interval (CI) 47.37–55.95 %) than ribotype 106 (41.91±8.35 %; 95 % CI 37.82–46 %) (P<0.05) and ribotype 078 (42.07±8.57 %, 95 % CI 37.22–46.92 %) (P<0.05). Spore outgrowth rates were comparable between the ribotype groups but the exponential phase occurred approximately 4 h later in the absence of sodium taurocholate. Spore germination efficiencies for isolates implicated in severe CDI were significantly higher (49.68±10.00 %, 95 % CI 47.06–52.30 %) than non-severe CDI (40.92±9.29 %, 95 % CI 37.48–44.36 %); P<0.01. Germination efficiencies were also significantly higher in recurrent CDI or when metronidazole therapy failed than when therapy was successful [(49.00±10.49 %, 95 % CI 46.25–51.75 %) versus (41.42±9.43 %, 95 % CI 37.93–44.91 %); P<0.01]. This study suggests an important link between C. difficile spore germination, CDI pathogenesis and response to treatment; however, further work is warranted before the complex interplay between germination dynamics and CDI outcome can be fully understood.

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

Journal of Bacteriology ◽

10.1128/jb.00908-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 777-786 ◽

Cited By ~ 28

Author(s):

Disha Bhattacharjee ◽

Michael B. Francis ◽

Xicheng Ding ◽

Kathleen N. McAllister ◽

Ritu Shrestha ◽

...

Keyword(s):

Bile Acid ◽

Clostridium Difficile ◽

Bile Acids ◽

Spore Germination ◽

Germination Rate ◽

Taurocholic Acid ◽

Receptor Complex ◽

Health Care Environment ◽

Content Type ◽

Downstream Processes

ABSTRACTClostridium difficilespore germination is essential for colonization and disease. The signals that initiateC. difficilespore 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 preventC. difficileinfection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describingC. difficilestrains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms ofC. difficilespore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on howC. difficilespores 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 (EC50values; the concentrations that achieve a half-maximum 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 difficileforms metabolically dormant spores that persist in the health care environment. In susceptible hosts,C. difficilespores germinate in response to certain bile acids and glycine. Blocking germination byC. difficilespores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certainC. difficilestrains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors ofC. difficilespore 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 theC. difficilebile acid germinant receptor.

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Inactivation of Clostridium difficile spore outgrowth by synergistic effects of nisin and lysozyme

Canadian Journal of Microbiology ◽

10.1139/cjm-2016-0550 ◽

2017 ◽

Vol 63 (7) ◽

pp. 638-643 ◽

Cited By ~ 4

Author(s):

Changhoon Chai ◽

Kyung-Soo Lee ◽

Goo-Sang Imm ◽

Young Soon Kim ◽

Se-Wook Oh

Keyword(s):

Clostridium Difficile ◽

Synergistic Effects ◽

Sodium Taurocholate ◽

Natural Antimicrobials ◽

Human Contact ◽

Spore Outgrowth

Inactivating Clostridium difficile spores is difficult, as they are resistant to heat, chemicals, and antimicrobials. However, this note describes inactivation of C. difficile spore outgrowth by incubation in a solution containing a germinant (1% (m/v) sodium taurocholate), co-germinants (1% (m/v) tryptose and 1% (m/v) NaCl), and natural antimicrobials (20 nmol·L–1 nisin and 0.2 mmol·L–1 lysozyme). Clostridium difficile spores were resistant to nisin and lysozyme but became susceptible during germination and outgrowth triggered and promoted by sodium taurocholate, tryptose, and NaCl. The degree of inactivation of germinated and outgrowing C. difficile spores by both nisin and lysozyme was greater than the sum of that by nisin and lysozyme individually, suggesting synergistic inactivation of C. difficile spores. The germinant, co-germinants, and natural antimicrobials used in this study are safe for human contact and consumption. Therefore, these findings will facilitate the development of a safe and effective method to inactivate C. difficile spore.

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Seed dormancy in four Tibetan Plateau Vicia species and characterization of physiological changes in response of seeds to environmental factors

Seed Science Research ◽

10.1017/s0960258513000019 ◽

2013 ◽

Vol 23 (2) ◽

pp. 133-140 ◽

Cited By ~ 14

Author(s):

Xiaowen Hu ◽

Tingshan Li ◽

Juan Wang ◽

Yanrong Wang ◽

Carol C. Baskin ◽

...

Keyword(s):

Tibetan Plateau ◽

Seed Dormancy ◽

Germination Rate ◽

Soil Surface ◽

The Tibetan Plateau ◽

Physical Dormancy ◽

High Germination ◽

One Year ◽

Hydrotime Model ◽

Aba Biosynthesis

AbstractAlthough seed dormancy of temperate legumes is well understood, less is known about it in species that grow in subalpine/alpine areas. This study investigated dormancy and germination of four Vicia species from the Tibetan Plateau. Fresh seeds of V. sativa were permeable to water, whereas those of V. angustifolia, V. amoena and V. unijuga had physical dormancy (PY). One year of dry storage increased the proportion of impermeable seeds in V. angustifolia, but showed no effect on seed coat permeability in V. amoena or V. unijuga. Seeds of all four species also had non-deep physiological dormancy (PD), which was especially apparent in the two annuals at a high germination temperature (20°C). After 1 year of storage, PD had been lost. The hydrotime model showed that fresh seeds obtained a significantly higher median water potential [Ψb(50)] than stored seeds, implying that PD prevents germination in winter for seeds dispersed without PY when water availability is limited. After 6 months on the soil surface in the field, a high proportion of permeable seeds remained ungerminated, further suggesting that PD plays a key role in preventing germination after dispersal. Addition of fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, evened-out the differences in germination between fresh and stored seeds, which points to the key role of ABA biosynthesis in maintaining dormancy. Further, fresh seeds were more sensitive to exogenous ABA than stored seeds, indicating that storage decreased embryo sensitivity to ABA. On the other hand, the gibberellic acid GA3 increased germination rate, which implies that embryo sensitivity to GA is also involved in seed dormancy regulation. This study showed that PY, PD or their combination (PY+PD) plays a key role in timing germination after dispersal, and that different intensities of dormancy occur among these four Vicia species from the Tibetan Plateau.

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Cross-Sectional Study Reveals High Prevalence of Clostridium difficile Non-PCR Ribotype 078 Strains in Australian Veal Calves at Slaughter

Applied and Environmental Microbiology ◽

10.1128/aem.03951-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2630-2635 ◽

Cited By ~ 57

Author(s):

Daniel R. Knight ◽

Sara Thean ◽

Papanin Putsathit ◽

Stan Fenwick ◽

Thomas V. Riley

Keyword(s):

Clostridium Difficile ◽

Cross Sectional Study ◽

Sectional Study ◽

Cross Sectional ◽

Adult Cattle ◽

Content Type ◽

Retail Meats ◽

High Prevalence ◽

Pcr Ribotyping ◽

Ribotype 078

ABSTRACTRecent reports in North America and Europe ofClostridium difficilebeing isolated from livestock and retail meats of bovine origin have raised concerns about the risk to public health. To assess the situation in Australia, we investigated the prevalence and genetic diversity ofC. difficilein adult cattle and calves at slaughter. Carcass washings, gastrointestinal contents, and feces were collected from abattoirs across five Australian states. Selective culture, toxin profiling, and PCR ribotyping were performed. The prevalence ofC. difficilewas 56% (203/360 samples) in feces from <7-day-old calves, 3.8% (1/26) in 2- to 6-month-old calves, and 1.8% (5/280) in adult cattle. Three PCR ribotypes (RTs), RT127, RT033, and RT126, predominated in <7-day-old calves and comprised 77.8% (158/203 samples) of isolates. RT056, which has not been reported in cattle before, was found in 16 <7-day-old calves (7.7%). Surprisingly, RT078 strains, which dominate production animal carriage studies in the Northern Hemisphere, were not isolated.

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Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain

Biochimie ◽

10.1016/j.biochi.2015.07.023 ◽

2016 ◽

Vol 122 ◽

pp. 243-254 ◽

Cited By ~ 31

Author(s):

Yuzo Kevorkian ◽

David J. Shirley ◽

Aimee Shen

Keyword(s):

Clostridium Difficile ◽

Spore Germination

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Germinant Synergy FacilitatesClostridium difficileSpore Germination under Physiological Conditions

mSphere ◽

10.1128/msphere.00335-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 13

Author(s):

Travis J. Kochan ◽

Michelle S. Shoshiev ◽

Jessica L. Hastie ◽

Madeline J. Somers ◽

Yael M. Plotnick ◽

...

Keyword(s):

Amino Acids ◽

Clostridium Difficile ◽

Spore Germination ◽

Bile Salts ◽

Gi Tract ◽

Infectious Diarrhea ◽

Content Type ◽

Increased Risk ◽

Calcium Reabsorption

ABSTRACTClostridium difficileis a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host.C. difficileis the leading cause of nosocomial infectious diarrhea worldwide.C. difficileinfection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step inC. difficilepathogenesis 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 thatC. difficilegermination 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 difficileis 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 aC. difficileinfection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitateC. difficilespore 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, butin 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 regulateC. difficilespore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

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A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078

mBio ◽

10.1128/mbio.02790-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 16

Author(s):

Kate E. Dingle ◽

Xavier Didelot ◽

T. Phuong Quan ◽

David W. Eyre ◽

Nicole Stoesser ◽

...

Keyword(s):

Clostridium Difficile ◽

Evolutionary History ◽

Selective Pressure ◽

Tetracycline Resistance ◽

Clonal Expansion ◽

Human Pathogen ◽

Content Type ◽

Resistance Determinants ◽

Ribotype 078 ◽

Human Infections

ABSTRACT The increasing clinical importance of human infections (frequently severe) caused by Clostridium difficile PCR ribotype 078 (RT078) was first reported in 2008. The severity of symptoms (mortality of ≤30%) and the higher proportion of infections among community and younger patients raised concerns. Farm animals, especially pigs, have been identified as RT078 reservoirs. We aimed to understand the recent changes in RT078 epidemiology by investigating a possible role for antimicrobial selection in its recent evolutionary history. Phylogenetic analysis of international RT078 genomes (isolates from 2006 to 2014, n = 400), using time-scaled, recombination-corrected, maximum likelihood phylogenies, revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired a different allele of the tetracycline resistance gene tetM. Consequently, an unusually high proportion (76.5%) of RT078 genomes were tetM positive. Multiple additional tetracycline resistance determinants were also identified (including efflux pump tet40), frequently sharing a high level of nucleotide sequence identity (up to 100%) with sequences found in the pig pathogen Streptococcus suis and in other zoonotic pathogens such as Campylobacter jejuni and Campylobacter coli. Each RT078 tetM clonal expansion lacked geographic structure, indicating rapid, recent international spread. Resistance determinants for C. difficile infection-triggering antimicrobials, including fluoroquinolones and clindamycin, were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid, international spread via the food chain, may explain the increased RT078 prevalence in humans. Our work indicates that the use of antimicrobials outside the health care environment has selected for resistant organisms, and in the case of RT078, has contributed to the emergence of a human pathogen. IMPORTANCE Clostridium difficile PCR ribotype 078 (RT078) has multiple reservoirs; many are agricultural. Since 2005, this genotype has been increasingly associated with human infections in both clinical settings and the community. Investigations of RT078 whole-genome sequences revealed that tetracycline resistance had been acquired on multiple independent occasions. Phylogenetic analysis revealed a rapid, recent increase in numbers of closely related tetracycline-resistant RT078 (clonal expansions), suggesting that tetracycline selection has strongly influenced its recent evolutionary history. We demonstrate recent international spread of emergent, tetracycline-resistant RT078. A similar tetracycline-positive clonal expansion was also identified in unrelated nontoxigenic C. difficile, suggesting that this process may be widespread and may be independent of disease-causing ability. Resistance to typical C. difficile infection-associated antimicrobials (e.g., fluoroquinolones, clindamycin) occurred only sporadically within RT078. Selective pressure from tetracycline appears to be a key factor in the emergence of this human pathogen and the rapid international dissemination that followed, plausibly via the food chain.

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The requirement for co-germinants during Clostridium difficile spore germination is influenced by mutations in yabG and cspA

PLoS Pathogens ◽

10.1371/journal.ppat.1007681 ◽

2019 ◽

Vol 15 (4) ◽

pp. e1007681 ◽

Cited By ~ 11

Author(s):

Ritu Shrestha ◽

Alicia M. Cochran ◽

Joseph A. Sorg

Keyword(s):

Clostridium Difficile ◽

Spore Germination

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Sensitivity of Ulocladium atrum, Coniothyrium minitans, and Sclerotinia sclerotiorum to benomyl and vinclozolin

Canadian Journal of Botany ◽

10.1139/b02-077 ◽

2002 ◽

Vol 80 (8) ◽

pp. 892-898 ◽

Cited By ~ 10

Author(s):

G Q Li ◽

H C Huang ◽

S N Acharya

Keyword(s):

Growth Inhibition ◽

Sclerotinia Sclerotiorum ◽

Spore Germination ◽

Mycelial Growth ◽

Germination Rate ◽

Effective Concentration ◽

Coniothyrium Minitans ◽

Inhibition Concentration ◽

Maximum Inhibition ◽

Germ Tubes

Assays on mycelial growth and spore germination were carried out to determine the sensitivity of the biocontrol agents Ulocladium atrum and Coniothyrium minitans and the plant pathogen Sclerotinia sclerotiorum to benomyl and vinclozolin. Ulocladium atrum was more tolerant to these fungicides than C. minitans and S. sclerotiorum. The 50% effective concentration (EC50) of U. atrum based on the mycelial growth inhibition was 1467.3 µg active ingredient (a.i.)/mL for benomyl and 12.6 µg a.i./mL for vinclozolin, and the maximum inhibition concentration was higher than 4000 µg a.i./mL for both fungicides. For C. minitans and S. sclerotiorum, however, the EC50 based on mycelial growth inhibition was lower than 1 µg a.i./mL. After incubation for 24 h at 20°C, the germination rate of U. atrum conidia was 9099% on potato dextrose agar (PDA) amended with benomyl at 100500 µg a.i./mL or vinclozolin at 10500 µg a.i./mL. At these concentrations, germ tubes of U. atrum developed into long, branched hyphae in benomyl treatments, but they remained short and clustered in vinclozolin treatments. Pycnidiospores of C. minitans and ascospores of S. sclerotiorum germinated on PDA amended with benomyl at 100500 µg a.i./mL, but the germ tubes did not grow further. Spore germination of C. minitans and S. sclerotiorum was less than 3.2% on PDA amended with vinclozolin at 10500 µg a.i./mL after 24 h. This is the first report on the sensitivity of U. atrum and C. minitans to benomyl and vinclozolin. The results suggest that it is possible to control S. sclerotiorum using a combination of U. atrum and benomyl or vinclozolin.Key words: fungicides, mycelial growth, spore germination, integrated pest management.

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