Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts

PLOS ONE, Dec 2019

Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.

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Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts

Minton NP (2012) Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts. PLoS ONE 7(2): e32381. doi:10.1371/journal.pone.0032381 Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts Daniela Heeg. 0 David A. Burns. 0 Stephen T. Cartman 0 Nigel P. Minton 0 Peter Setlow, University of Connecticut, United States of America 0 Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , United Kingdom Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination. - Funding: The research leading to these results received funding from the UK Medical Research Council (Grant G0601176) and the European Communitys Seventh Framework Programmes CLOSTNET (PEOPLE-ITN-2008-237942) and HYPERDIFF (HEALTH-F3-2008-223585). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journals policy and have the following conflicts: Dr. David Burns is currently employed by Pharmacom Media. All work carried out by Dr. Burns and described in this manuscript has been carried out prior to this employment. Pharmacom Media has had no interest in the work described in this manuscript. . These authors contributed equally to this work. Clostridium difficile is a Gram-positive, anaerobic spore former and the major underlying cause of hospital-acquired diarrhoea. C. difficile infection (CDI) is estimated to affect more than 500,000 people per year in the USA alone and the spread of CDI has led to patient isolation, ward closures and even hospital closures [1]. Infection with C. difficile may manifest as asymptomatic colonisation, but can also lead to severe diarrhoea that may then progress into a potentially fatal pseudo-membranous colitis [2]. Endospores, formed during sporulation, play a pivotal role in the transmission of disease. Spores shed in the faeces are able to withstand a variety of cleaning agents and can reside on hospital surfaces for prolonged periods of time [3]. Therefore, spores are regarded as the infectious stage of C. difficile. However, following ingestion by susceptible individuals, the progression to disease relies first on a return to vegetative cell growth through germination and then on production of the characteristic toxins [4]. Germination is defined as the irreversible loss of spore-specific characteristics and ultimately leads to vegetative cell growth. The mechanisms of germination have been studied extensively in Bacillus spp. in which nutrients and chemicals, termed germinants, can bind to specific receptors at the inner spore membrane [5]. At this point, the spore becomes committed to germination and subsequent events include release of monovalent cations (H+, K+ and Na+) and the spores large depot of calcium dipicolinic acid (CaDPA) [6]. This redistribution of ions and water in the spore core likely precedes the activation of specific lytic enzymes that degrade the spore cortex, a thick layer of peptidoglycan differing subtly from vegetative cell peptidog (...truncated)


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Daniela Heeg, David A. Burns, Stephen T. Cartman, Nigel P. Minton. Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts, PLOS ONE, 2012, Volume 7, Issue 2, DOI: 10.1371/journal.pone.0032381