Ultrastructure Variability of the Exosporium Layer of Clostridium difficile Spores from Sporulating Cultures and Biofilms

Abstract

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202-2209, 2016, http://dx.doi .org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed. IMPORTANCEClostridium difficile spores are recognized as the morphotype of persistence and transmission of C. difficile infections. Spores of C. difficile are intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires a detailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidence that two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, are formed in the same C. difficile sporulating culture or during biofilm development. Infections caused by Clostridium difficile are the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries (1, 2). Mortality rates of C. difficile infections (CDIs) may reach up to 5%; however, the recurrence of the infection, which may reach up to 25, 40, and 65% of cases after a first, second, and third episode of CDI, respectively, has become the current main clinical challenge (3). The main factors in recurrence of CDI include (i) an irreversible loss of function of the gut microbiota due to antibiotic therapy, leading to a loss of the colonization resistance barrier against enteric pathogens (4, 5), and (ii) the formation of metabolically dormant spores during the course of infection (6, 7). These newly formed C. difficile spores have been shown to be essential for the transmission of the disease to a new susceptible host and for the persistence of C. difficile in the host, leading to recurrent infection (6).Biofilms are sessile surface-associated microbial communities that rep...

Clostridioides difficilespore-entry into intestinal epithelial cells contributes to recurrence of the disease

“…14). Electron micrographs demonstrate that, as expected, wild-type R20291 (ΔpyrE/pyrE + ) spores exhibited typical hair-like projections observed in previous reports 1, 11,12 (Fig. 5c).…”

Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells

“…The basic structure of C. difficile spores is similar to the spores of Bacillus subtilis and other related organisms, with the exception of the exosporium, the outermost layer of the C. difficile spore, which is absent in most spore formers (Pizarro-Guajardo et al, 2014, 2016a,b). The spore core, which contains the DNA and other cellular components, is layered by the inner and outer membranes, the cortex and the spore coat (reviewed in Paredes-Sabja et al, 2014).…”

Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease

“…Recent studies demonstrate that C. difficile forms spores with two different morphotypes of the outermost exosporium layer in the same sporulating culture: (i) spores with a thin-exosporium layer; and (ii) spores with a thick exosporium layer (Pizarro-Guajardo et al, 2016a , b ). These studies have demonstrated that ~75% of the spores have a thin exosporium layer, while 25% of the spores have a thick exosporium layer.…”

“…Recently, several studies have highlighted that during sporulation, C. difficile forms spores with two different exosporium morphotypes: (i) spores with an exosporium layer which is thin in the amount of electron-dense material that surrounds the spore coat; and (ii) spores with an exosporium layer thick in electron-dense material surrounding the spore coat (Pizarro-Guajardo et al, 2016a , b ). The relative abundance of each exosporium morphotype is strain-dependent; in the case of strain R20291, spores with a thin- and thick-exosporium morphotype represent ~75 and 25% of the spore population, respectively (Pizarro-Guajardo et al, 2016a , b ). In R20291 spores, both types of spore have hair-like extensions, which is a typical feature of epidemically relevant strains (Pizarro-Guajardo et al, 2016a ).…”