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Convenient Protocol for Production and Purification of Clostridioides difficile Spores for Germination Studies.

ABSTRACT: Clostridioides difficile, an obligate anaerobic bacterium, causes infections leading to prolonged diarrhea. The bacterium produces dormant spores that can withstand an aerobic environment, resulting in easy environmental transfer. Here, we present a convenient sporulation and purification protocol that can be practiced in any lab setting using a portable anaerobic glove bag. This protocol also optimizes existing cell growth methods and presents a detailed trouble shooting guide. This protocol is a modification of those previously reported by Edwards and McBride (2016) and Shen et al. (2016).

SUBMITTER: Weldy M

PROVIDER: S-EPMC7580112 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Convenient Protocol for Production and Purification of <i>Clostridioides difficile</i> Spores for Germination Studies.

Weldy Melissa M Evert Clayton C Dosa Peter I PI Khoruts Alexander A Sadowsky Michael J MJ

STAR protocols 20200724 2

<i>Clostridioides difficile</i>, an obligate anaerobic bacterium, causes infections leading to prolonged diarrhea. The bacterium produces dormant spores that can withstand an aerobic environment, resulting in easy environmental transfer. Here, we present a convenient sporulation and purification protocol that can be practiced in any lab setting using a portable anaerobic glove bag. This protocol also optimizes existing cell growth methods and presents a detailed trouble shooting guide. This prot ...[more]

PMID: 33111107

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Project description:Background Clostridioides difficile is a spore forming bacterial species and the major causative agent of nosocomial gastrointestinal infections. C. difficile spores are highly resilient to disinfection methods and to prevent infection, common cleaning protocols use sodium hypochlorite solutions to decontaminate hospital surfaces and equipment. However, there is a balance between minimising the use of harmful chemicals to the environment and patients as well as the need to eliminate spores, which can have varying resistance properties between strains. In this work, we employ TEM imaging and Raman spectroscopy to analyse changes in spore physiology in response to sodium hypochlorite. We characterize different C. difficile clinical isolates and assess the chemical’s impact on spores’ biochemical composition. Changes in the biochemical composition can, in turn, change spores’ vibrational spectroscopic fingerprints, which can impact the possibility of detecting spores in a hospital using Raman based methods. Results We found that the isolates show significantly different susceptibility to hypochlorite, with the R20291 strain, in particular, showing less than 1 log reduction in viability for a 0.5% hypochlorite treatment, far below typically reported values for C. difficile. While TEM and Raman spectra analysis of hypochlorite-treated spores revealed that some hypochlorite-exposed spores remained intact and not distinguishable from controls, most spores showed structural changes. These changes were prominent in B. thuringiensis spores than C. difficile spores. Conclusion This study highlights the ability of certain C. difficile spores to survive practical disinfection exposure and the related changes in spore Raman spectra that can be seen after exposure. These findings are important to consider when designing practical disinfection protocols and vibrational-based detection methods to avoid a false-positive response when screening decontaminated areas. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-02787-z.

Dataset Information

Convenient Protocol for Production and Purification of Clostridioides difficile Spores for Germination Studies.

Publications

Convenient Protocol for Production and Purification of <i>Clostridioides difficile</i> Spores for Germination Studies.

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