Ultrastructural Variability of the Exosporium Layer of Clostridiumdifficile Spores

Marjorie Pizarro-Guajardo, Paulina Calderón-Romero, Pablo Castro-Córdova, Paola Mora-Uribe, Daniel Paredes-Sabja

Gut Microbiota and Clostridia Research Group, Departamento de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developedand developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent mor-photype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact ofC. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on thebiology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863–3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025–1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is alack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electronmicrographs, we analyzed the variability of the spore’s outermost layers in various strains and found distinctive variability in theultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, weobserved that although this layer was stable during spore purification, it was partially lost after 6 months of storage at roomtemperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease inthe levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that thepresence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm cultureconditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of theexosporium surface of C. difficile spores. These findings have direct implications and should be considered in the developmentof novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.

Clostridium difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries (1,

2, 5). Antibiotic treatment results in disturbance of the natural gutmicroflora, allowing germination and outgrowth of C. difficilespores and proliferation and colonization of live cells (3). Duringthe course of the infection, C. difficile initiates a sporulation pro-cess that culminates with the production of metabolically dor-mant spores (3, 4), which are essential for (i) the persistence of C.difficile in the host and (ii) transmission of the disease betweenhosts (4).

C. difficile spore morphology resembles that of other spore-forming bacteria; however, the protein composition of the outerlayers (i.e., the spore coat and exosporium layer) differs greatlyfrom that of other Gram-positive bacteria (13, 14). The composi-tion of the spore coat has been described recently (7); fewer than25% of the spore proteins and several spore coat proteins havebeen functionally characterized (8, 9). Transmission electron mi-croscopy (TEM) reveals the presence of typical laminations (i.e.,lamellae) (6, 10–12) in the spore coat of other spore-forming bac-teria (13). As reported for Bacillus subtilis (14), the spore coat layerof C. difficile spores is resistant to enzymatic digestion with pro-teases (i.e., trypsin and proteinase K) (11). The outermost exospo-rium layer of C. difficile spores is an electron-dense layer withseveral unique features that differentiate this layer, both morpho-logically and functionally, from the spore coat (6, 10, 11). (i) Un-like the outermost layer of members of the B. cereus group, whichexhibits hairlike projections and does not interact directly with thespore coat surface (13), the C. difficile exosporium exhibits hair-like projections in most strains (14) and interacts directly with thespore coat (10, 14). (ii) In contrast to the spore coat, the exospo-rium is easily digested with proteases (i.e., trypsin and proteinaseK) (11). (iii) The exosporium contributes to the spore’s hydro-

phobicity (11). (iv) Its assembly seems to depend, at leastpartly, on the morphogenetic protein CdeC, which is uniquelylocated in the exosporium of C. difficile spores (6, 10) and isessential for the assembly of the exosporium and for correct sporecoat assembly (10).

Recent studies have implicated the exosporium layer in thepathogenesis of C. difficile infections (CDI) and the persistence ofC. difficile spores on stainless steel (15, 16). Recently, Phetchar-aburanin et al. demonstrated that the exosporium collagen-likeBclA1 glycoprotein is required for animal susceptibility to coloni-zation and infection by C. difficile laboratory strain 630. More-over, recent reports have demonstrated that strain 630 spores havea hairless exosporium (6, 11), which also differs in general ultra-structural features from what has been observed in epidemicallyrelevant strains (10, 14, 15). Furthermore, the stability of this layeris also a matter of controversy; several reports suggest that it is afragile and easily lost layer (8, 9), while others provide evidencethat it is robust and remains attached to the spore (6, 10, 11, 14,15). The stability of the exosporium layer seems to be relevant, as

Received 21 October 2015 Accepted 18 January 2016

Accepted manuscript posted online 5 February 2016

Citation Pizarro-Guajardo M, Calderón-Romero P, Castro-Córdova P, Mora-Uribe P,Paredes-Sabja D. 2016. Ultrastructural variability of the exosporium layer ofClostridium difficile spores. Appl Environ Microbiol 82:2202–2209.doi:10.1128/AEM.03410-15.

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

Address correspondence to Daniel Paredes-Sabja, daniel.paredes.sabja@gmail.com.

M.P.-G. and P.C.-R. contributed equally to this work.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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the exosporium layer also contributes to spore adherence to inertsurfaces (i.e., stainless steel) and consequently to the persistence ofspores on hospital surfaces (15). Consequently, the aims of thisstudy were to determine the heterogeneity of the exosporium ul-trastructure of C. difficile spores in various isolates of clinical rel-evance, determine the stability of this layer during long-term stor-age at room temperature, and evaluate whether the exosporiumlayer is present in spores formed during biofilm development.

MATERIALS AND METHODSBacterial strains and plasmids. The C. difficile strains used in this studywere the laboratory strain 630 ribotype 012 (RT012); epidemic strainR20291 (RT027); and strains M120 (RT078-126), TL176 (RT014-020),and TL178 (RT02). C. difficile was routinely grown under anaerobic con-ditions in a Bactron III-2 anaerobic chamber (Shellab) in 3.7% brain heartinfusion broth supplemented with 0.5% yeast extract (BHIS) or on BHISagar plates.

Spore suspensions. Spore suspensions were prepared by plating a1:500 dilution of an overnight culture onto a 3% Trypticase soy (TS)–0.5% yeast extract (TY) agar plate and incubating it for 5 days at 37°Cunder anaerobic conditions. Spores were harvested with ice-cold steriledistilled water, centrifuge washed (5,000 rpm, 5 min) once, and gentlyresuspended in sterile distilled water. Spores were loaded onto a 50%Nycodenz (Sigma-Aldrich) solution, centrifuged (10,000 rpm, 40 min),and centrifuge washed (5,000 rpm, 5 min) twice with ice-cold sterile dis-tilled water to remove Nycodenz remnants.

TEM. Spores (�2 � 108) of C. difficile strains 630, R20291, M120,TL176, and TL178 were fixed with 3% glutaraldehyde in 0.1 M cacodylatebuffer (pH 7.2) overnight at 4°C and stained for 30 min with 1% tannicacid. Samples were further processed and embedded in Spurr resin aspreviously described (12). Thin sections of 90 nm obtained with a micro-tome were placed on glow discharge carbon-coated grids and double leadstained with 2% uranyl acetate and lead citrate. Grids were analyzed witha Philips Tecnai 12 Bio Twin at the electron microscopy facility of thePontificia Universidad Católica de Chile.

Room temperature storage. Suspensions of 5 � 109 spores/mlwere diluted in phosphate-buffered saline (PBS) to a final concentra-tion of 1.7 � 108 spores/ml. Aliquots (50 �l) of spore suspensions insterile distilled water were stored in Eppendorf tubes for up to 6months at room temperature (23°C). Three independent batches ofspores were used, and aliquots were stored in triplicate.

Immunofluorescence microscopy. C. difficile R20291 and M120spores were fixed in 3% paraformaldehyde (pH 7.4) for 20 min on poly-L-lysine-coated glass cover slides. Fixed spores were rinsed three timeswith PBS, blocked with 1% bovine serum albumin (BSA) for 30 min, andfurther incubated overnight at 4°C with 1:50-diluted rabbit antiserumraised against the N-terminal domain of BclA1 (�-NTD BclA1) (17) andrat serum raised against the exosporium marker CdeC (anti-CdeC) (10).Next, coverslips were incubated for 1 h at room temperature with 1:200anti-rabbit or -rat IgG Alexa Fluor 488 conjugate (Invitrogen) in PBS with1% BSA and washed three times with PBS and once with distilled water.Samples were dried at room temperature for 30 min, and then the cover-slips were mounted using Dako Fluorescence Mounting medium (DakoNorth America) and sealed with nail polish. Samples were analyzed withan Olympus BX53 fluorescence microscope. A total of 150 spores wereanalyzed. Control experiments with serum obtained prior to immuniza-tion or in the absence of primary antibodies yielded no fluorescence signalin R20291 and M120 spores (data not shown). Fluorescence intensity wasquantified with ImageJ software (http://rsbweb.nih.gov/ij/).

Biofilm formation. Biofilm was evaluated by using previously de-scribed protocols (18, 19). Briefly, cultures grown overnight in BHIS werediluted 1:100 in fresh BHIS or TS broth and added to 24-well polystyreneplates (Greiner Bio-One, Stuttgart, Germany). Plates were incubated at37°C under anaerobic conditions for 5 days. Some wells were used tomeasure biofilm biomass with crystal violet (CV) as previously described

(19). Briefly, biofilm was stained with 1 ml of filter-sterilized 0.2% CV andincubated for 30 min at 37°C under anaerobic conditions. CV was re-moved from the wells, and then the wells were washed twice with sterilePBS. The dye was extracted by adding 1 ml of methanol to each well, andthen the plates were incubated for 30 min at room temperature. Themethanol-extracted dye was serially diluted, and optical density at 570nm (OD570) was measured with a spectrophotometer (ELx800;BioTek). Other wells were used to remove nonbiofilm material, wellswere rinsed three times with sterile PBS, and the biofilm mass wasremoved by mechanical scraping.

The presence of the exosporium layer on spores formed during biofilmdevelopment was analyzed by immunofluorescence as described above,with some modifications. Briefly, biofilms of strains R20291 and M120were fixed with paraformaldehyde and blocked with 1% BSA for 1 h asdescribed above. Biofilms were subsequently immunostained at roomtemperature for 1 h with 1:100-diluted rat antiserum raised against CdeC,rinsed, incubated for 1 h at room temperature with 1:400-diluted anti-ratAlexa 488 conjugate (LifeScience Technologies) in PBS–1% BSA, washedthree times, and rinsed with sterile distilled water. Samples weremounted with Dako fluorescence mounting medium and analyzedwith an Olympus BX53 fluorescence microscope. Control experimentsincluded the use of rat serum obtained prior to immunization, whichyielded no fluorescence signal in R20291 and M120 spores (data notshown). Control experiments also included purified spores obtainedfrom TY agar plates.

Statistical analysis. Student’s t test was used to detect statistically sig-nificant differences between groups.

RESULTSTwo distinctive exosporium morphotypes within C. difficilespore populations. To gain insight into the variability of the out-ermost exosporium, we evaluated spores of five different strainsby TEM; these included a laboratory strain (630), an epidemicstrain (R20291), and three additional isolates belonging to differ-ent ribotypes (i.e., M120, TL176, and TL178). TEM confirmedpreviously described differences (10, 12, 14) in the exosporiumlayer between C. difficile 630 and R20291 spores. That is, 630spores had a smooth, electron-dense exosporium layer lacking theclassical hairlike extensions observed in exosporium structures ofBacillus anthracis spores (13) (Fig. 1A). In contrast, R20291 sporeshad an exosporium layer with hairlike projections, which werealso observed by TEM of M120, TL176, and TL178 spores (Fig.1A), and depending on the strain, these hairlike projections variedfrom 36 to 65 nm in length (Fig. 1B).

During the ultrastructural analysis, it became evident that all ofthe strains produced spores with two distinctive thicknesses of theexosporium, that is, spores with a thick exosporium and sporeswith a thin exosporium (Fig. 1A). For example, nearly 50% of thestrain 630 and TL176 spores had a thick exosporium, while 75% ofthe M120 and TL176 spores had a thick exosporium. Notably,only 25% of the R20291 spores had a thick exosporium (Fig. 1A).On average, C. difficile spores with a thick exosporium had anexosporium nearly 2-fold thicker than that of spores with a thinexosporium (Fig. 1B). Although significant differences in thelength of the hairlike extensions were observed between thick- andthin-exosporium spores, the magnitude of this change was small(i.e., a 5- to 20-nm difference, depending on the strain) (Fig. 1B).These differences were not observed between M120 spores with athick exosporium and those with a thin exosporium (Fig. 1B).However, significant differences in the thickness of the spore innerand external coat layers were detectable between these two exo-sporium morphotypes, among all of the strains analyzed (Fig. 1B).

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Another noteworthy observation was that only those spores with athick exosporium that had hairlike projections (i.e., strainsR20291, M120, TL176, and TL178) exhibited geometrically dis-tributed bumps in the exosporium layer (Fig. 1A). These bumpswere not observed in spores of strains R20291, M120, TL176, andTL178 with a thin-exosporium morphotype or in either thick- orthin-exosporium strain 630 spores (Fig. 1A). Notably, thesebumps seem to be made of the same electron-dense material as theexosporium of strain 630 spores. Besides these differences, theunderlying layers (i.e., spore outer and inner coats) had similarultrastructural features among the strains analyzed (Fig. 1A); all ofthe strains had spore coats with lamellae, a conserved feature ofBacillus and Clostridium spores (Fig. 1A). Collectively, these re-sults demonstrate the presence of two exosporium phenotypes(i.e., a thick exosporium and a thin exosporium) in the same pop-ulation of C. difficile spores.

Long-term storage decreases the abundance of exosporiumproteins of C. difficile spores. Given the plausible roles of theexosporium in early C. difficile spore-host interactions (16) andspore transmission (15), it is of interest to determine the stabilityof this outer layer. To evaluate the stability of the exosporium,

spore suspensions of strains R20291 and M120 were incubated for6 months at room temperature in PBS and the abundance of twopreviously described markers of the exosporium (i.e., NTD-BclA1and CdeC) (6, 10, 17) were measured by indirect immunofluores-cence assay. Significant fluorescence signals of NTD-BclA1 andCdeC were readily detectable in freshly prepared spores of strainsR20291 and M120 (Fig. 2A to D). However, after 6 months ofstorage at ambient temperature, there was a nearly 2-fold decreasein the fluorescence intensity of both markers in spores of bothstrains (Fig. 2A to D), suggesting that some exosporium proteinmarkers are lost during long-term storage.

Effect of long-term storage on the ultrastructure of C. diffi-cile spores. To gain insight into how long-term storage could af-fect the ultrastructure of the thick- and thin-exosporium morpho-types, new and 6-month-old spores were analyzed by TEM.Representative micrographs of new (i.e., 0-month-old) and6-month-old spores of strains R20291 and M120 are shown in Fig.3. As expected, new spores of R20291 and M120 exhibit a mixedpopulation of thick- and thin-exosporium spores (Fig. 3), which isconsistent with the aforementioned results (Fig. 1A and B). Anal-ysis of 6-month-old spores of strains R20291 and M120 reveals

FIG 1 Ultrastructural variability of the outer layers of C. difficile spores. (A) C. difficile spores of strains 630 (ribotype 012), R20291 (ribotype 027), M120(ribotype 078-126), TL176 (ribotype 014-020), and TL178 (ribotype 002) were analyzed by TEM as described in Materials and Methods. Spore populations (n �100) were analyzed, and two distinct ultrastructural phenotypes became evident in spore samples of all of the strains tested, i.e., spores with a thick exosporiumlayer and spores with a thin exosporium layer. The percentages of spores with a thick exosporium (black bar) and those a thin exosporium (gray bar) are depictedbelow the micrographs. Results are representative of one experiment. Experiments were repeated with two independent spore preparations with essentiallysimilar results. (B) The thickness of the exosporium and the outer and inner coat layers of C. difficile spores with a thick (black bars) or a thin (gray bars)exosporium layer were estimated by TEM of at least 10 individual spores. Scale bars are 100 and 200 nm in micrographs with whole spores and selected sporesections, respectively. Ex, exosporium; EC, external coat; IC, inner coat. Error bars denote standard errors of the means. Asterisks denote statistically significantdifferences (n.s., not significant; *, P � 0.05; ***, P � 0.001). ND, not detected.

FIG 2 Quantitative analysis of loss of exosporium proteins after 6 month of storage. (A, B) Microscopy analysis of purified C. difficile spores of strains R20291and M120 by phase-contrast microscopy and immunofluorescence assay. Spores from fresh crops or from crops stored at room temperature for 6 months weretreated with exosporium protein-specific antibodies. The panels are representative of the fluorescence intensity observed for fresh and 6-month-old spores ofeach strain. (C, D) Quantitative analysis of the fluorescence (Fl.) intensity in fresh and 6-month-old spores of strains R20291 and M120 for each exosporiumprotein-specific antibody was conducted as described in Materials and Methods. The values beside the symbols in the graphs represent the average � the standarddeviation of the fluorescence intensity under each spore condition (n � 150 spores analyzed for each age/strain condition). The data shown are from oneexperiment that is representative of three independent experiments.

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that although thick-exosporium spores were detectable in propor-tions similar to those seen before (Fig. 1A and B), the integrity oftheir exosporium was considerably affected compared to thatof freshly prepared spores (Fig. 3). Six-month-old R20291 sporesof both exosporium morphotypes had hairlike extensions, butthese were less abundant than in newly prepared spores (Fig. 3).Ultrastructural analysis of 6-month-old thick-exosporiumR20291 spores revealed that much of the electron-dense materialhad been peeled off during storage (Fig. 3). Similarly, 6-month-old M120 spores that appeared to be of the thick-exosporiummorphotype had remnants of the electron-dense material of theexosporium layer (Fig. 3). Remarkably, after 6 months of storageat room temperature, thin-exosporium spores of both strainslacked the hairlike projections of freshly prepared spores (Fig. 3).Collectively, these results demonstrate that incubation of C. diffi-cile spores of both ultrastructural morphotypes for 6 months af-fects the exosporium’s integrity.

Presence of exosporium layer in C. difficile spores from bio-films. Several studies have demonstrated that during biofilm de-velopment (18–20), C. difficile spores are also formed, yet whetherall spores form an exosporium layer under these conditions re-mains unclear. In this context, to gain more insight into the vari-

ability of the exosporium of C. difficile spores formed during bio-film development, we used CdeC as an exosporium marker (6) todetect the presence/absence of the exosporium layer in C. difficilestrain R20291 and M120 spores during in vitro biofilm formation.First, as a control, we performed immunofluorescence analysis ofunwashed sporulating cultures and purified spores. R20291 andM120 spores that did not immunoreact with anti-CdeC antibod-ies were classified as CdeCb negative (Fig. 4A and B), suggestingthat they might have either a thin-exosporium phenotype or com-pletely lack the exosporium layer, while those that gave an immu-nofluorescence signal above the background level were classifiedas CdeC positive and presumably have a thick-exosporium phe-notype (Fig. 4A and B). To evaluate if these immunofluorescencephenotypes (i.e., CdeC positive and CdeC negative) were also de-tectable in spores formed during biofilm formation, 5-day-oldbiofilm biomasses with OD570s of 6 to 8 developed in BHIS and TSmedium were analyzed by immunofluorescence assay (Fig. 4Aand B). No biofilm was detectable after 24 h of incubation (datanot shown). Immunofluorescence intensity was quantitative forunwashed, pure spores as well as for spores formed during biofilmdevelopment. Immunoreactive CdeC-positive spores had a fluo-rescence intensity against anti-CdeC antibodies 3- to 5-fold higherthan that of nonimmunoreactive, CdeC-negative spores (Fig. 4Cand D). These observations indicate that, regardless of the methodof preparation (i.e., agar plates or broth in culture wells), C. diffi-cile spores form two spore populations (i.e., CdeC negative andCdeC positive) that can be quantitatively identified.

Next, using the aforementioned approach, we proceeded todetermine the fractions of spores under each culture conditionthat were CdeC positive and CdeC negative. Immunofluorescenceanalysis demonstrated that while similar levels (i.e., 80%) ofCdeC-positive M120 spores were detected in unwashed and puri-fied spores, the levels of CdeC-positive R20291 spores increasedfrom 44 to 87% in washed and purified spores, respectively (Fig.4E and F). Similarly, high levels of CdeC-positive spores wereobserved in M120 spores obtained from BHIS and TS biofilms(i.e., 75 and 86%, respectively) (Fig. 4F). Notably, while a highpercentage (i.e., 87%) of R20291 spores obtained from TS bio-films were CdeC positive, only 59% of R20291 spores obtainedfrom BHIS biofilms were CdeC positive (Fig. 4E), contrasting withthe high levels of M120 CdeC-positive spores obtained under sim-ilar conditions (Fig. 4F). These results suggest that (i) most of theC. difficile spores formed under biofilm conditions are likely tohave an exosporium and (ii) the proportion of exosporium-posi-tive spores depends on the culture medium used to induce biofilmformation, as well as on the strain tested.

DISCUSSION

C. difficile spores have proven to be essential for the initiation,persistence, and dissemination of CDI (3, 4, 14). In this context,the interactions of C. difficile spores with the host and environ-mental surfaces might be essential for spore persistence and trans-mission, respectively. The composition and mechanism of assem-bly of the external surface of C. difficile spores have recently startedto be elucidated (6, 10). In this work, we have addressed severalunexplored features of the outermost exosporium layer of C. dif-ficile spores, specifically, those related to (i) the ultrastructuralvariability of the layer between strains and within spores of thesame strain, (ii) the stability of the exosporium during long-term

FIG 3 TEM of C. difficile spores incubated at room temperature for 6 months.C. difficile spores of strains R20291 and M120 were incubated at room temper-ature (23°C) for 6 months in PBS. Six-month-old spores and newly preparedspore suspensions were prepared and analyzed by TEM as described in Mate-rials and Methods. Representative spores are shown. Scale bar, 200 nm.

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storage, and (iii) the presence of the exosporium layer in C. difficilespores formed during biofilm development.

A first conclusion from this work is that two ultrastructuralmorphotypes of the exosporium layer (i.e., a thick exosporiumand a thin exosporium) were detectable in a spore population ofC. difficile of a single strain. Notably, the remaining spore layersvaried in thickness between spores of these two exosporium mor-photypes, suggesting that slightly different regulatory mecha-nisms might underlie the formation of each morphotype. Theseobservations raise the obvious question of whether these differentmorphotypes appear randomly or are attributable to the differen-tial expression of a protein(s) involved in the assembly of thislayer. It is also unclear whether these two morphotypes might have(i) differential virulence phenotypes that may affect the outcomeof infection or (ii) differential adherence to inert surfaces or hand

skin that might be associated with hospital transmission and/orpersistence.

Another contribution of this work is that C. difficile spores ofmost of the strains analyzed have an exosporium with hairlikeprojections. Most of the previous work on the C. difficile exospo-rium has been conducted with a laboratory strain (i.e., 630) thatlacks hairlike projections (6–9, 12, 16, 17, 21, 22). Indeed, 630spores are substantially different from spores of epidemiologicallyrelevant strains reported here and elsewhere (10, 14). In B. anthra-cis, these hairlike projections are attributed to the presence of thecollagen-like glycoprotein BclA, which is homogeneously distrib-uted on the spore surface (23, 24). Notably, although all three C.difficile BclA homologues (i.e., BclA1, BclA2, and BclA3) are ex-pressed during sporulation and localize to the surface of 630spores (17), TEM of 630 spores indicated the absence of hairlike

FIG 4 Detection of the exosporium marker CdeC in spores from biofilm. (A, B) C. difficile spores of strains R20291 and M120 from 5-day-old BHIS and TSbiofilm cultures were analyzed by immunofluorescence assay with anti-CdeC antibodies as described in Materials and Methods. Unwashed and purified sporecultures from TY agar plates were included as a control. (C, D) Quantitative analysis of the fluorescence (Fl.) intensity in C. difficile spores with and without animmunofluorescence signal against anti-CdeC antibodies. The values beside the symbols in the graphs represent the average � the standard error of thefluorescence intensity for each type of spore. The data shown are from one experiment that is representative of three independent experiments. Asterisks denotestatistically significant differences (**, P � 0.001) between CdeC () and CdeC () spores under each spore condition, as indicated by horizontal brackets. (E,F) The percentages of spores with (gray bars) and without (black bars) a detectable fluorescence signal were calculated for strains R20291 (E) and M120 (F).Experiments were performed two independent times, and more than 300 spores were analyzed under each experimental condition. Values are representative ofthree experimental conditions, and asterisks denote statistically significant differences (*, P � 0.01).

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projections, which are present in epidemiologically relevantstrains (i.e., R20291). These observations suggest that the assem-bly mechanism of the collagen-like BclA glycoproteins in the exo-sporium of epidemiologically relevant spores might be substan-tially different from that in 630 spores. These main ultrastructuraldifferences in the exosporium layer might also have in vivo impli-cations (i.e., persistence in the host or on hospital surfaces). Forexample, a recent study (16) demonstrated that BclA1 affects thesusceptibility and colonization of mice with C. difficile strain 630.However, it is noteworthy that unlike the genome of strain 630,those of most C. difficile isolates that have been sequenced have abclA1 pseudogene because of the appearance of an early stopcodon in the N-terminal domain (17), raising the hypothesis thatBclA2 and BclA3 might have roles in infections with epidemiolog-ically relevant strains.

Previously, we have shown that spores stored for 6 months atroom temperature were viable and exhibited a germination effi-ciency similar to or higher than that of freshly prepared spores(25). Here we demonstrate that 6-month-old spores also retainmost of their exosporium material, yet the apparent loss of thehairlike extensions in the 6-month-old spores analyzed mighthave implications for spore adherence to host mucosal surfaces,inert surfaces, and hand skin that remain to be elucidated.

During in vivo infection, C. difficile initiates a sporulation cyclethat contributes to C. difficile spore persistence and dissemination(4). Recently, Semenyuk et al. demonstrated that C. difficile formsbiofilm communities in vivo in a mouse model of infection (26). Invitro studies have demonstrated that C. difficile forms spores dur-ing 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 ofstrains R20291 and M120 are positive for the exosporium markerCdeC and therefore are likely to have an exosporium layer andthat the proportion of the presence/absence of anti-CdeC immu-nofluorescence signal was dependent on the strain and biofilmculture condition; therefore, it is conceivable that the lack of anexosporium observed by Semenyuk et al. in spores formed duringbiofilm development could be attributable to specific experimen-tal conditions that affected the formation of the exosporium layerof that particular strain (20). Considering these observations, it islikely that the composition of the spore outer layer (i.e., exospo-rium) might differ markedly, depending on whether spores areformed in the planktonic state or inside a biofilm.

In summary, these observations highlight the need to per-form further studies to address whether the variability in theexosporium layer in terms of the presence/absence of hairlikeextensions and thin- and thick-exosporium morphotypes af-fects virulence, persistence in the host, and transmission andpersistence in the hospital environment. Spore removal strate-gies based on exosporium proteins as targets need to considerthe variability in the exosporium layer within and betweenstrains.

ACKNOWLEDGMENTS

This work was supported by grants from the Fondo Nacional de Ciencia yTecnología de Chile (FONDECYT 1151025) and the Research Office ofUniversidad Andres Bello (DI-275-13/R 2013) and by Fondo de Fomentoal Desarrollo Científico y Tecnológico (FONDEF) grant CA13I10077 (toD.P.-S).

FUNDING INFORMATIONFondecyt Regular provided funding to Marjorie Pizarro-Guajardo, Pau-lina Calderón-Romero, Pablo Castro-Córdova, Paola Mora-Uribe, andDaniel Paredes-Sabja under grant number 1151025. Fondef IdeA CienciaAplicada provided funding to Marjorie Pizarro-Guajardo, Pablo Castro-Córdova, and Daniel Paredes-Sabja under grant number CA1310077.

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