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Appl Environ Microbiol. 2016 October 1; 82(19): 5892–5898.
Published online 2016 September 16. Prepublished online 2016 July 29. doi:  10.1128/AEM.01463-16
PMCID: PMC5038037

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

D. W. Schaffner, Editor
Rutgers, The State University of New Jersey

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.

IMPORTANCE Clostridium 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.

INTRODUCTION

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 represent the predominant state of bacteria in nature (8). C. difficile biofilms have been shown to be more resistant than planktonic cells to antibiotics (9). During in vivo infection in the mouse model, C. difficile resides in multicellular communities (biofilms) (10) in which spores have been shown to form (11). Although not demonstrated experimentally, it has been suggested that development of spores might be relevant for biofilm formation, since it has been reported that a Spo0A mutant (Spo0A is a transcription factor that controls spore formation) is defective in biofilm formation (12). Recently, the presence of two exosporium proteins (i.e., CdeC and the N-terminal domain of BclA1) was detected by indirect immunofluorescence analysis of spores from C. difficile biofilms (13). However, whether the ultrastructure of biofilm-formed spores is similar to that of sporulating cultures remains unclear.

The imminent relevance of C. difficile spores in the infectious cycle raises numerous questions regarding its assembly, composition, and fate in the host. In this context, recent studies have revealed that the ultrastructure of C. difficile spores is similar to that of other Gram-positive bacteria (13, 14). The outermost layer is an electron-dense layer that, in most epidemic strains, is covered by hair-like projections and has been shown to be ultrastructurally stable (13). Underlying the exosporium layer is the spore coat, which has laminations (i.e., lamellae) similar to those present in other bacterial spores, although it differs in protein composition from the spore coat of other bacterial species (13, 14). The C. difficile spore coat, as similarly reported for Bacillus subtilis (15), exhibits enzymatic digestion resistance to proteases (i.e., proteinase K and trypsin) (16), which might be related to the apparent resistance of C. difficile spores to macrophages (17).

The outermost exosporium layer of C. difficile spores is thought to play important roles in host-spore interaction and to hold spore ligands involved in spore-interaction with host cells (18). However, only recently has the outermost exosporium layer begun to be characterized. The composition of the exosporium layer of C. difficile spores has been recently identified (19), revealing a complex composition of 184 proteins, among which several proteins known to be involved in pathogenesis and evasion of immunity in other pathogens were identified (e.g., elongation factor Tu and alpha-enolase) (19). Notably, our recent results have shown that during spore formation, C. difficile is capable of producing spores with two distinctive morphotypes of the exosporium layer, either thin or thick (13). However, whether these two different morphotypes of the exosporium layer emerge directly from the mother cell and whether these two morphotypes are formed during biofilm development remain unclear. In this work, through transmission electron microscopy, we have evaluated the ultrastructural variability of C. difficile spores formed under sporulation conditions and during biofilm development and demonstrate that two morphotypes of the exosporium layer are formed simultaneously in a sporulating culture or during biofilm development.

MATERIALS AND METHODS

Bacterial strains.

The C. difficile strain used in this study was the epidemic strain R20291 (ribotype 027), which caused an outbreak and has been described elsewhere (20, 21). C. difficile was routinely grown under anaerobic conditions in an anaerobic chamber (Coy, USA) in 3.7% brain heart infusion supplemented with 0.5% yeast extract (BHIS) broth or on BHIS agar plates.

Sporulating culture and spore preparations.

Sporulating cultures were prepared by plating a 1:500 dilution of an overnight culture onto 3% Trypticase soy-0.5% yeast extract (TY) agar plates and incubated for 5 days at 37°C under anaerobic conditions. One-half of the total plates were carefully harvested in sterile distilled water and fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer for further processing for transmission electron microscopy as described below. The other half of the plates were harvested with ice-cold sterile distilled water, washed (18,625 × g for 10 min) five times with sterile distilled water, and purified with 50% Nycodenz as previously described (22). Pure spores were immediately fixed and processed for analysis by transmission electron microscopy as described below.

Biofilm formation.

Biofilm formation was evaluated by previously described protocols (12, 23). Briefly, overnight cultures grown in BHIS (3.7% brain heart infusion broth supplemented with 0.5% yeast extract) were diluted 1:100 in 24-well polystyrene plates (Greiner Bio-One, Stuttgart, Germany) containing fresh BHIS broth and incubated at 37°C under anaerobic conditions for 5 days. Some wells were used to confirm the presence of biofilm biomass, stained with crystal violet, and quantified at an optical density of 570 nm with a spectrophotometer (ELx800; BioTek) as previously described (13). Other wells were used to harvest biofilm material. Briefly, wells were gently rinsed three times with sterile phosphate-buffered saline (PBS) to remove nonbiofilm material, and biofilm mass was removed by mechanical scraping, immediately fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 16 h at 4°C, and processed for transmission electron microscopy as described below.

Transmission electron microscopy.

Purified spores, sporulating culture, and fixed biofilm samples (3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) were stained for 30 min with 1% tannic acid. Samples were further processed and embedded in Spurr resin as previously described (18). Thin sections of 90 nm were obtained with a microtome, placed on glow discharge carbon-coated grids, and double-lead stained with 2% uranyl acetate and lead citrate. Grids were analyzed with a Philips Tecnai 12 Bio Twin electron microscope.

To quantify the percentage of spores with a thick or thin exosporium layer, a total of 100 spores from randomly selected transmission electron micrographs (TEMs) (approximately 10 fields at a magnification of ×10,500) were analyzed. To distinguish spores with a thick and those with a thin exosporium, we established the following criteria: (i) spores with a thick exosporium were defined as having a basal electron-dense exosporium layer at least three-fourths larger than the spore coat while (ii) spores with a thin exosporium layer were defined as those with a basal exosporium layer one-half the size of the spore coat. Two replicates were observed with essentially similar results.

To analyze the length of the spore layers, 60,000× transmission electron micrographs of 19 representative spores with thin and thick exosporia were randomly selected. For each spore, the length of the layers was quantified at 6 different locations.

Statistical analysis.

Data were determined to be nonparametric based on the Q-Q plot and the Shapiro-Wilk test; therefore, to test for statistical significance between two groups, the nonparametric unpaired Mann-Whitney test was used.

RESULTS

Two morphotypes of the exosporium layer are present in sporulating cultures of epidemic C. difficile strain R20291.

Recently, two distinctive thicknesses of the exosporium layer (i.e., a thin and a thick exosporium) were reported to appear in purified spore suspensions of various epidemic C. difficile strains analyzed by transmission electron microscopy (13). In order to rule out the possibility that the spore purification conditions (see Materials and Methods), which include a Nycodenz purification step of C. difficile spores, could be implicated in the removal of exosporium material and lead to the appearance of these two morphotypes, transmission electron micrographs (TEMs) of carefully prepared unwashed sporulating cultures and purified spore suspensions were examined. As expected, TEMs confirmed the presence of both exosporium morphotypes, spores with a thick and those with a thin exosporium-like layer, in the unwashed sporulating culture (Fig. 1A). Depending on how the spores were sliced (i.e., transversal or longitudinal), the distribution of the exosporium layer exhibited notable differences. In transversal spore sections, a regular and homogeneously distributed electron-dense and thick exosporium layer can be observed surrounding the spore (Fig. 1B). In longitudinal spore sections, a thicker electron-dense layer at one of the spore poles becomes evident (Fig. 1C). We also observed that nearly 37% of the spores with a thin exosporium layer were surrounded with cellular debris (Fig. 1D), while 67% were free of cellular debris (Fig. 1E and data not shown). In contrast, none of the analyzed spores with a thick exosporium layer had cellular debris. Spores from both exosporium morphotypes had the distinctive hair-like extensions (Fig. 1B to toE).E). Collectively, these results clearly indicate that both morphotypes of exosporium layers, thick and thin, are formed during sporulation and are not due to an artifact during spore purification.

FIG 1
Presence of two distinctive morphotypes of the exosporium layer in sporulating cultures of C. difficile R20291 spores. (A) Representative electron micrograph of unwashed sporulating cultures of R20291 demonstrates the presence of spores with a thick exosporium ...

C. difficile spores formed during biofilm development exhibit both thick and thin exosporium morphotypes.

To address whether these two exosporium morphotypes were also formed during biofilm development, biofilm biomass was carefully harvested and analyzed for the presence of C. difficile spores by transmission electron microscopy. The presence of C. difficile spores in biofilm biomass was evident, and spores with a thick or a thin exosporium layer were also detectable (Fig. 2A). We also identified spores with a large amount of electron-dense exosporium material, which accounted for 40% of the spores with a thick exosporium morphotype formed during biofilm development (Fig. 2B and andC).C). Similarly, as in unwashed sporulating cultures, in biofilm biomass we observed two types of spores with a thin exosporium layer: (i) those with hair-like extensions and little if any electron-dense exosporium material (Fig. 2D) and (ii) those either surrounded by an aberrantly assembled exosporium material or associated with cellular debris from lysed mother cells (Fig. 2E). In summary, these results clearly indicate that spores formed during biofilm formation (i) have an exosporium layer with hair-like filaments and (ii) may have either a thick or a thin exosporium layer as an outer surface.

FIG 2
Presence of two distinctive morphotypes of the exosporium layer in biofilms of C. difficile R20291 spores. (A) Representative electron micrographs of biofilms of R20291 demonstrate the presence of spores with a thick exosporium layer (black arrows) and ...

Spore purification removes a slight amount of hair-like extensions and electron-dense material.

To more rigorously determine the similarities between spores from unwashed sporulating cultures and purified spores, the lengths of the outermost layers were quantified (Fig. 3A). Notably, the proportion of spores with a thick or a thin exosporium layer in unwashed sporulating cultures (i.e., 29% with a thick layer and 71% with a thin layer) was similar to that of purified spore suspensions (i.e., 23% with a thick layer and 77% with a thin layer) (Fig. 3A). Within the spores with a thick exosporium, those from unwashed sporulating culture had an exosporium layer 60 nm thicker than that of purified spores (Fig. 2B). Differences were observed in both components of the exosporium layer. The hair-like extensions and the electron-dense layer of spores from unwashed sporulating cultures were 27 and 41 nm longer, respectively, than those in spores from purified spores (Fig. 3B). On the other hand, spores with a thin exosporium layer obtained from unwashed sporulating cultures had a slightly thicker exosporium layer (i.e., 12 nm) than those from purified spore suspensions (Fig. 3B), which seems to be due to the slightly shorter hair-like extensions (i.e., 8 nm) observed in purified spores (Fig. 3B). Analysis of the spore coat layer revealed no significant differences between spores with a thick or a thin exosporium layer from unwashed sporulating culture and purified spore suspensions (Fig. 3B). In summary, these observations indicate that spore purification procedures might cause a slight decrease in the length of the hair-like filaments and a reduction in the thickness of the exosporium in both morphotypes.

FIG 3
Analysis of the length of the outermost layers of thick and thin exosporium spores from unwashed sporulating cultures and biofilms. (A) Representative transmission electron micrographs of spores from unwashed sporulating culture, purified spore suspensions, ...

Thinner spore coat layer is evident in biofilm-formed C. difficile spores.

To gain more insight on how the outer spore layers of biofilm-formed spores differ from those formed under traditional sporulation conditions, the lengths of the hair-like extensions, exosporium, and spore coat were analyzed. Electron micrographs of unwashed sporulating cultures prepared at the same time as biofilms were prepared and were the same as those used in the experiment shown in Fig. 3. A significant difference in the thickness of the exosporium layer was evident upon comparison of thick-exosporium spores obtained from biofilms and those from unwashed sporulating cultures, with estimated thicknesses of 150 and 216 nm, respectively (Fig. 3C). Thick-exosporium spores from biofilms had hair-like extensions and an electron-dense layer 34 and 36 nm thinner, respectively, than those from unwashed sporulating cultures (Fig. 3C). In contrast, we found no significant difference in the lengths of the exosporium layers of thin-exosporium spores obtained from biofilms and those from unwashed sporulating cultures (Fig. 3C). Differences were observed between the inner, but not external, coat layer of thick-exosporium spores from biofilms and those from unwashed sporulating cultures, where biofilm spores had a 6-nm-thinner inner coat (i.e., 12 versus 18 nm, respectively) (Fig. 3A and andC).C). Thin-exosporium spores from biofilms had thinner spore coat layers than did spores from unwashed sporulating cultures, with the external and inner coat layers of biofilm spores being 18 nm (i.e., 24 versus 42 nm) and 4 nm (i.e., 9 versus 13 nm) thinner, respectively, than those of spores from unwashed sporulating cultures (Fig. 3C). In summary, these results demonstrate that, during biofilm development, C. difficile also forms spores with a thick or a thin exosporium layer and that, particularly, spores with a thin exosporium layer formed during biofilm development have a thinner spore coat than those formed under standard sporulating conditions.

DISCUSSION

The relevance of C. difficile spores to the pathogenesis of the disease is well known (6, 7, 14), increasing the need to develop spore-based strategies to eradicate C. difficile from the host and clinically relevant surfaces. In this context, we have recently provided evidence of two different morphotypes of the exosporium layer that appear from a monoclonal culture (13). In this work, we have addressed whether these two different exosporium morphotypes are indeed formed during sporulation and whether they also arise in spores formed during biofilm formation.

A first contribution of this work is that the two ultrastructural morphotypes (i.e., thick and thin) of the exosporium layer were detectable in unwashed sporulating cultures and therefore were not an artifact of the extensive washing of spore suspensions. These observations are strongly supported by transmission electron micrographs of unwashed sporulating cultures of the C. difficile R20291 strain showing both morphotypes and reinforce our previous hypothesis that an intrinsic regulatory mechanism(s) might drive the formation of each exosporium morphotype on a cell-to-cell basis (13). Although a slight amount of hair-like extension and exosporium material was removed from spores with a thick exosporium morphotype during the purification process, our electronic micrographs demonstrate that this was not sufficient to contribute to the appearance of the thin exosporium morphotype. Notably, the frequency of appearance of both exosporium morphotypes in our previous work revealed that the frequency was strain dependent and that in R20291 spores nearly one-fourth and three-fourths of the spore population had a thick and a thin exosporium morphotype, respectively (13). Here, we observed a similar proportion of both morphotypes in unwashed sporulating cultures and purified spore suspensions, where one-third and two-thirds of the spores had the thick and thin exosporium morphotypes, respectively. How the assembly of these two morphotypes of the exosporium layer is regulated during sporulation is a question that remains to be answered in order to begin understanding the dynamics of appearance of these morphotypes and their roles during an infection.

Transmission electron micrographs of biofilms of the epidemic R20291 strain demonstrate that (i) C. difficile spores formed during biofilms also have an exosporium layer and hair-like extensions and (ii) spores with both exosporium morphotypes also appear during the development of C. difficile biofilms. These observations are meaningful, since previous evidence (11) suggested that spores formed during biofilm development lacked the outermost exosporium-like layer commonly observed in various epidemic strains (13, 14). However, whether the composition of the outermost surface layers of biofilm-formed spores is similar to that of traditionally prepared spores or to that of in vivo-prepared spores remains unclear. Remarkably, the proportion of thick to thin exosporium spores differed from that observed in sporulating cultures and purified spores, where a 1:1 ratio of spores with a thick and a thin exosporium layer was observed in biofilms. These differences might indicate that slight variations in the regulatory mechanism(s) in exosporium assembly might occur during biofilm development, leading to a variation in the ratio of thick to thin exosporium morphotypes. This hypothesis might be consistent with the thinner spore coat layers observed in biofilm-formed spores than in spores formed under traditional sporulating conditions, particularly between spores with a thin exosporium layer.

In summary, this work complements our previous study (13) by providing convincing evidence that, independently of the conditions under which spores are formed, C. difficile produces spores with two different morphotypes of the exosporium layer. This should be considered in the development of spore removal strategies based on exosporium proteins to ensure efficient development of novel strategies to combat C. difficile infections.

ACKNOWLEDGMENTS

This work was supported by grants from Fondo Nacional de Ciencia y Tecnología de Chile (FONDECYT grant 1151025), from the Research Office of Universidad Andres Bello (DI-641-15/R 2015) (to D.P.-S.), and from the Fondo Nacional de Ciencia y Tecnología de Chile, Doctoral Fellowship CONICYT 21151202 to M.P.-G.

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