Inducing and Quantifying Clostridium difficile Spore Formation

Methods Mol Biol. 2016;1476:129-42. doi: 10.1007/978-1-4939-6361-4_10.

Abstract

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

Keywords: Induction; Phase-contrast microscopy; Spore; Sporulation.

MeSH terms

  • Anaerobiosis
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Load
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Clostridioides difficile / drug effects
  • Clostridioides difficile / growth & development*
  • Clostridioides difficile / ultrastructure
  • Gene Expression
  • Hot Temperature
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Microscopy, Phase-Contrast
  • Mutation
  • Orotate Phosphoribosyltransferase / genetics
  • Orotate Phosphoribosyltransferase / metabolism
  • Spores, Bacterial / drug effects
  • Spores, Bacterial / growth & development
  • Spores, Bacterial / isolation & purification*
  • Spores, Bacterial / ultrastructure
  • Taurocholic Acid / pharmacology*
  • Thiamphenicol / pharmacology
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Transcription Factors
  • Taurocholic Acid
  • Methyltransferases
  • ErmA protein, Bacteria
  • Orotate Phosphoribosyltransferase
  • Thiamphenicol