Spore Formation and Toxin Production in Clostridium difficile Biofilms

Abstract

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile

“…In Listeria monocytogenes, an aldehyde alcohol dehydrogenase is a housekeeping enzyme but is also a surface-exposed adhesin commonly referred to as LAP (Listeria adhesion protein) (33). In C. difficile, LdhA participates in a biosynthetic pathway, another member of which, HadA, has been found to be associated with biofilms, a key attribute linked to colonization (34). Both AdhE1 and LdhA may or may not be involved in virulence, and they may or may not be surface exposed, but what is important here is the demonstration that mucosal antibodies can block C. difficile colonization.…”

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores

“…In vitro studies have demonstrated that C. difficile forms spores during biofilm development (20); yet, as demonstrated in that work, the spores formed in these biofilms lack the exosporium layer. Here, we provide evidence that most biofilm-formed spores of strains R20291 and M120 are positive for the exosporium marker CdeC and therefore are likely to have an exosporium layer and that the proportion of the presence/absence of anti-CdeC immunofluorescence signal was dependent on the strain and biofilm culture condition; therefore, it is conceivable that the lack of an exosporium observed by Semenyuk et al in spores formed during biofilm development could be attributable to specific experimental conditions that affected the formation of the exosporium layer of that particular strain (20). Considering these observations, it is likely that the composition of the spore outer layer (i.e., exosporium) might differ markedly, depending on whether spores are formed in the planktonic state or inside a biofilm.…”

“…Several studies have demonstrated that during biofilm development (18)(19)(20), C. difficile spores are also formed, yet whether all spores form an exosporium layer under these conditions remains unclear. In this context, to gain more insight into the variability of the exosporium of C. difficile spores formed during biofilm development, we used CdeC as an exosporium marker (6) to detect the presence/absence of the exosporium layer in C. difficile strain R20291 and M120 spores during in vitro biofilm formation.…”

Analysis of Bacterial Communities during Clostridium difficile Infection in the Mouse

“…Our inability to detect spores with our current methodology is a limitation of the present study and is under investigation in our laboratory. Nonetheless, results of studies of C. difficile communities formed in laboratory cultures (biofilms) suggest that, at least under those conditions, spores are readily formed (45,46). Therefore, it is plausible that spores are present in C. difficilecontaining communities in the mucosa during infection.…”