Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Wang, G.; Zhang, P.; Paredes-Sabja, Daniel; Green, C.; Setlow, Peter; Sarker, Mahfuzur R.; Li, Y.-Q.

doi:10.1111/j.1365-2672.2011.05135.x

Cited by 43 publications

(61 citation statements)

References 27 publications

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“…S4c and d). This suggests that sleC spores do not undergo any cortex hydrolysis process, similar to C. perfringens sleC mutant spores (41). It was also notable that the ⌬T release period for CaDPA in the wild-type spores that germinated was ϳ2.5 min, while for the sleC spores that germinated this value was much larger at ϳ40 min.…”

Section: Resultsmentioning

confidence: 76%

“…Previous work has found that bands from CaDPA dominate the Raman spectra of individual spores of Bacillus species and C. perfringens (23,41), and this was also the case for spores of two C. difficile strains (Fig. 1a).…”

Section: Resultsmentioning

confidence: 85%

“…Notably, there also does not appear to be any cortex hydrolysis during C. perfringens spore germination with dodecylamine (41). In Bacillus spore germination with dodecylamine, this agent is thought to bind to and activate the spore's IM channel for CaDPA composed of SpoVA proteins (17,18).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

et al. 2015

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The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ϳ15% concentration of a chelate of Ca 2؉ and dipicolinic acid (CaDPA) at a heterogeneous time T 1 , rapidly released CaDPA at T lag , completed CaDPA release at T release , and finished peptidoglycan cortex hydrolysis at T lysis . T 1 and T lag values for individual spores were heterogeneous, but ⌬T release periods (T release ؊ T lag ) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow T release , in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences. IMPORTANCESpores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination. C lostridium difficile is a Gram-positive, spore-forming, strictly anaerobic bacterium that has become a leading cause of antibiotic-associated diarrhea worldwide (1). Antibiotic treatment disrupts the normal colonic flora that typically suppresses C. difficile growth and allows ingested C. difficile spores to germinate, outgrow...

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 85%

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Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

et al. 2015

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“…This lag period is also seen in germination triggered by HP of ϳ150 megaPascals that activates GRs (43), as well as in germination with CaDPA and dodecylamine that trigger germination in a GR-independent fashion (4). Germination of spores of Clostridium perfringens with various germinants also exhibits this variable lag period (44). The length of the lag period in nutrient germination of spores of bacilli or C. perfringens is determined by multiple factors and is decreased by (i) increasing germinant concentration up to saturation, (ii) heat activation, and (iii) increased GR levels (4).…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

“…There are also some significant differences in the germination of spores of different clostridia, presumably because this is such an extremely diverse group of organisms (66). The similarities between nutrient germination of spores of clostridia and bacilli include the following: (i) spores of clostridia contain multiple SpoVA proteins that are essential at least for CaDPA uptake in sporulation, (ii) spores of clostridia most often contain GRs that are composed of subunits quite similar to those in spores of bacilli (6), and (iii) germination of individual C. perfringens spores in populations with nutrients, CaDPA, and dodecylamine exhibit the same varying lag periods seen during germination of spores of bacilli (44). However, there are also four major differences.…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores' return to life within minutes in the process of germination. This process requires a number of sporespecific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. T he germination of the dormant and highly resistant spores formed by members of the Firmicutes phylum, in particular bacilli and clostridia, has long been of significant research interest for four major reasons, as follows: (i) fascinating regulatory systems allow such spores to remain in their dormant, resistant state for years and yet return to active growth in minutes; (ii) while spores of most Firmicutes do not cause disease, spores of some bacilli and clostridia cause food spoilage and food-borne disease, as well as human diseases like gas gangrene, tetanus, botulism, anthrax, and pseudomembranous colitis; (iii) spores of Bacillus anthracis are a major bioterrorism threat; and (iv) spores of Clostridium difficile are an emerging public health threat (1-3). Invariably, it is the germination of spores of these organisms that leads to disease or food spoilage, and yet, when spores germinate and lose their dormancy, they lose their extreme resistance properties and become relatively easy to kill. Germination is thus both an essential part of disease pathogenesis or food spoilage and a major weak spot in these organisms' life cycle. Consequently, there has long been applied interest in spore germination, with researchers seeking to understand this process better in order to either prevent spore germination or accelerate it and then kill the newly sensitive germinated spores. This review will concentrate on the germination of spores of bacilli, primarily because of the large amount of detailed knowledge of the germination of spores of these species compared to that of spores of clostridia. However, some of the differences and similarities between the germination of spores of these related genera will also be presented. Most discussion will focus on the germination of the model sporeformer, Bacillus subtilis, although the mechanisms of germination of B. subtilis spores appear to be similar for spores of other bacilli. The properties of the various proteins that are specifically involved in the germination process will not be discussed in great detail, since these have recently be...

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Heterogeneity inBacillus subtilisspore germination and outgrowth: an area of key challenges for “omics” in food microbiology

Pandey

Brul

2016

Quantitative Microbiology in Food Processing

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Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Cited by 43 publications

References 27 publications

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Heterogeneity inBacillus subtilisspore germination and outgrowth: an area of key challenges for “omics” in food microbiology

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