For example, depending on the germination ability of Clostridium perfringens spores their fate is significantly different [13]. Isolates with germination proficient spores were efficiently inactivated during macrophage infection, while those that germinated poorly were able to survive for extended periods of time inside macrophages [13]. The main factors involved in resistance of C. perfringens spores are: i) the spore maturation proteins that regulate spore water content [14]; ii) a/b-type small acid soluble proteins (SASPs) that bind and saturate the spores’ DNA [15–17]; and iii) the SpoVA proteins, which are involved in uptake of dipicolinic acid (DPA) and reduction of the spore core water content [18]. However, C. perfringens spores deficient in either of these factors were able to survive similarly as wild-type spores during infection with macrophages, suggesting that other ultrastructural properties of C. perfringens spores are involved in macrophage-resistance [13]. Indeed, studies [19–21] demonstrated that Bacillus anthracis wild- type spores are efficiently phagocytosed and upon germination inside the phagolysosome they were efficiently killed by macro- phages. However, B. anthracis germination deficient (DgerH) spores were able to survive longer periods of time than germination proficient wild-type spores, indicating that spore survival inside macrophages is dependent on the ability of spores to remain dormant [22]. The ability of C. difficile spores to germinate mainly in presence of the co-germinants taurocholate and glycine or other amino acids [8,10], suggests that since at least cholates are not part of the phagolysosomes environment, the fate of C. difficile spores during macrophage infection might be significantly different than that reported for spores of C. perfringens and B. anthracis. Therefore, in this study, we evaluated the interactions of C. difficile spores with Raw 264.7 macrophages. By using electron microscopy, fluores- cence microscopy and cell viability assays we show that C. difficile spores are efficiently recognized and phagocytosed by Raw 264.7 cells. However, C. difficile spores do not germinate inside macrophages and are able to survive, and produce cytotoxic effects to Raw 264.7 cells. These findings indicate that C. difficile spores survive attacks of phagocytic cells. Materials and Methods Bacterial Strains, Cell Lines, and Chemical Reagents C. difficile strains 630 (tcdA+, tcdB+, tcdC+, ctdA2, and ctdB2) [23] and Pitt177 (tcdA+, tcdB+, tcdC+, ctdA+, and ctdB+) are described elsewhere [24,25]. C. difficile strain Pitt177 was isolated from patients presenting clinical symptoms of CDI in a tertiary care hospital in Pittsburg, U.S.A. [25]. Raw 264.7 murine macrophages (ATCC, U.S.A) were routinely grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) (Invitrogen) supple- mented with 10% (V/V) fetal bovine serum (ATCC, Manassas) and incubated at 37uC in 5% CO2 humidified atmosphere. B. subtilis strain PS832 wild-type [26] was kindly provided by Dr. Peter Setlow. Spore Preparation, Purification and Sonication C. difficile spores were prepared as previously described [8]. Briefly, Brain Heart Infusion broth (Difco) supplemented with 0.5% yeast extract (Difco) (BHIS) was inoculated with C. difficile and incubated overnight under anaerobic conditions at 37uC. Overnight cultures were diluted to an OD600 of 0.2, plated onto BHIS agar and incubated under anaerobic conditions at 37uC for 10 days [8]. BHIS agar plates were flooded with 10 ml of ice cold sterile distilled water, washed by repeated centrifugation and resuspension followed by purification through 50% HistoDenz and washed five times to eliminate traces of HistoDenz. Spore C. difficile Spores and Macrophage Interactions suspensions were .99% free of vegetative cells, sporulating cells and cell debris as determined by phase contrast microscopy. Spore suspensions were stored at 280uC until use. B. sutbilis spores were prepared by growing for ,72 h at 37uC on BHI agar plates under aerobic conditions, and spores were purified as described [27]. To remove the C. difficile spore exosporium layer, C. difficile spores were resuspended in 50 mM Tris-HCl 0.5 mM EDTA buffer (pH 7.5) and sonicated with maximum power (20 Watts) for 10 1-min burst separated by 3 min of cooling on ice-cold water as previously described [5]. This sonication protocol has been previously shown by transmission electron microscopy to remove spore surface material affecting the C. difficile spore’s surface hydrophobicity by ,60% and their ability to adhere to epithelial cells [5]. As a marker for the removal of the spores’ exosporium material we measured the change of the spore’s surface hydrophobicity, which was ,60 and 25% in untreated and sonicated spores, respectively. These results were similar to those reported previously [5]. Sonicated C. difficile spores were stored at 220uC until use. Infection of Raw 264.7 Cells with C. difficile Spores For electron microscopy experiments, Raw 264.7 cells (,66105 cells/well) were seeded onto cover slips coated with poly-lysine (BD, USA) on 24-well culture plates for 24-h, rinsed 3 times with Dubelcco’s PBS (DPBS) (Lonza) and infected at a multiplicity of infection (MOI) of 10 for 30 min with 100 ml of DMEM containing C. difficile spores (,66106 spores). Unbound spores were rinsed off with three washes of DPBS, and some samples of infected macrophages further incubated for 24 h at 37uC with 5% CO2. Samples were fixed with freshly prepared 2.5% glutaralde- hyde-1% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) for overnight at 4uC. Secondary fixation was performed with 1% osmium tetroxide-0.1 M cacodylate buffer (pH 7.2), rinsed in cacodelyte buffer and stained for 30 min with 1% tannic acid. Samples were dehydrated with a step-wise acetone gradient of 30% (stained with 2% uranyl acetate at this stage) for 30 min, 50% for 30 min, 70% for overnight, 90% for 30 min, and twice with 100% acetone. Dehydrated samples were embedded in spurs at a ratio of acetone:spurs of 3:1, 1:1 and 1:3 for 40 min each and finally resuspended in spurs for 4 h and baked overnight at 65uC. Thin sections were obtained using a microtome and were placed on a glow discharge carbon-coated grids for negative staining and double lead stained with 2% uranyl acetate and lead citrate. Samples were analyzed at 80 kV with a Philips EM300 trans- mission electron microscopy (TEM) at the Electron Microscopy Facility at Oregon State University and with a Phillips Tecnai 12 Bio Twin at the Electron Microscopy facility at Pontificia Universdad Cato´ lica de Chile. For scanning electron microscopy (SEM), Raw 264.7 cells were infected as described above and fixed with 2.5% glutaraldehyde- 1% paraformaldehyde in 0.1 M cacodylate buffer, and serially dehydrated with acetone: 30% for 20 min, 50% for 20 min, 75% for 20 min, 90% for 20 min and twice with 100% for 20 min. Dehydrated samples were subjected to critical point drying and coated with gold and palladium and analyzed with a FEI Quanta 600 PEG at the electron microscopy facility of Oregon State University. Fluorescent Labeling of C. difficile Spores Purified C. difficile spores were labeled with Alexa Fluor 488 protein labeling kit (Molecular Probes) according to the manu- facture’s specification. Briefly, 1 ml of a spore suspension with a calculated OD600 50 was resuspended in 1 ml of 0.1 M sodium bicarbonate (pH ,8.3), mixed well with 200 ml of 0.1 M sodium PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43635