Figure 1.
Scanning electron microscopic analysis of C. difficile biofilms.
Biofilms were cultured for 3(upper panels) or 6 days (lower panels) from strains BY1 (left panels) and BI17 (right panels). Size bars indicate 1 (BY1-3, 6 days; BI17-6 days) or 10 µm (BI17-3 days).
Figure 2.
Confocal laser-scanning microscopic analysis of C. difficile biofilms.
Biofilms were cultured for 1 (upper panels), 3 (middle panels) or 6 (lower panels) days from strains BI6 (left panels), BI17 (middle panels) and BY1 (right panels). In each panel, X–Y (center), X–Z (top), and Y–Z (right side) sections from a 3D data set are shown. At the upper right corner of each panel in the right column, there is an inset that is an enlargement of the boxed portion of the image. White arrowheads point to cell debris (uppermost panel) or spores (middle and lower panel). Magnifications in each panel have been chosen to assist in interpretation of the data (see the text). Size bars indicate 10 µm.
Figure 3.
Confocal laser-scanning microscopic analysis of C. difficile biofilms stained with nucleic acid stains.
2-old biofilms from strains BI17 (top panels) and BY1 (bottom panels) were stained with propidium iodine (left panels) and Syto-9 (middle panels) and imaged so that the propidium iodine (left panels) or Syto-9 (middle panels) were visualized separately or together (right panels). Size bars indicate 10 µm.
Figure 4.
Transmission electron microscopic analysis of C. difficile spores in biofilms.
A. Spore from a 3-old biofilm. B. Sporulating cell from a 3 day-old biofilm. C. Spore from a 6 day-old biofilm encased in a shroud. D. Matrix from a 6 day-old biofilm. In A, the outer coat (OC), inner coat (IC), cortex (the spore peptidoglycan, Cx) and core (the location of the spore DNA, Cr) are indicated. In B and C, granules (Gr), fiber-like structures (F) and a layer (L) are indicated. E. Spore from a 14 day-old biofilm covered with a thick layer of granules. The granules appear to be in close association with the coat surface. Bars indicate 300 nm (A), 1 µm (B), 1.5 µm (C), 600 nm (D) and 1 µm (E).
Figure 5.
Confocal laser-scanning microscopic analysis of C. difficile biofilms stained with nucleic acid stains and concanavalin A-Texas Red.
3-old biofilms from strains BI6 (top panels), J9 (middle panels) and BY1 (bottom panels) stained with the lectin Concanavalin A and the nucleic acid stain Syto-9, and imaged so that the Concanavalin A (left panels) or Syto-9 (middle panels) were visualized separately or together (right panels). Size bars indicate 10 µm.
Figure 6.
SDS-PAGE analysis of biofilm matrix extracts.
A) Matrix was extracted from 6-day old biofilms of the strains indicated below each lane, electrophoresed on 12% PA gels and stained with Coomassie brilliant blue. Molecular weights are indicated in kD on the left. B) Matrix extracts (ME) or whole cell lysates (WCL) were analyzed from a 5-day old biofilm from strain BI17. C) An extract of surface proteins (SP) or matrix (ME) was analyzed from a 5-day old biofilm from strain J9. Arrows identify bands chosen for analysis by mass spectrometry.
Figure 7.
ELISA, western blot and cytotoxicity analysis of toxin A and B production in biofilms.
A) Toxin A and B levels from the matrix of 3-day old biofilms were measured by the Wampole C. difficile TOX A/B II kit ELISA kit. *indicates that toxin levels differ significantly between BY1 and all other strains. **indicates that strains 630 and K14 differ significantly from all other toxigenic strains. The two sample t test (P<0.05) was used along with the ANOVA test. B) Toxin A and B levels from the matrix of 6-day old biofilms were measured by the Wampole C. difficile TOX A/B II kit ELISA kit. *indicates that toxin levels differ significantly between BY1 and all other strains. **indicates that toxin levels differ significantly between strain K14 and 630, and strains BI6, VPI10463, BI17. C) Cytotoxicity was assayed on 3 days old biofilms, using the Bartels Cytotoxicity Assay for Clostridium difficile Toxin. Cytotoxic titers are shown as the log of the reciprocal of the highest matrix extract dilution resulting in rounding of 100% of cells, divided by the µg of total soluble protein. The assay was performed twice with similar results. A representative experiment is shown. D) Western blot analysis of matrix extracts of 6-day old biofilms, using an anti-toxin A and B antibody. Matrix extracts were prepared from the strains indicated below each lane. A molecular weight standard is indicated on the left.
Figure 8.
Spore production and germination in C. difficile biofilms.
A) For each strain, cells were harvested from 6-day old biofilms and the numbers of spores and vegetative cells counted using phase-contrast light microscopy. Spore % was calculated as the # of spores/# of spores+# of vegetative cells. B) For each strain, spores were harvested from 6-day old cultures on Columbia blood agar plates or biofilms on T soy agar plates, treated with 1% taurocholate, and the numbers of germinated and ungerminated spores counted using phase-contrast light microscopy. Percentage of germinated spores was calculated as # germinated spores/# germinated spores+# ungerminated spores.
Figure 9.
Effects of metronidazole on cell growth in biofilms.
Effects of increasing concentrations of metronidazole on cells of strains J9 (A and C) and BI17 (B and D) grown in liquid culture (A and B) after 6 and 24 hours, or as biofilms (C and D). Biofilms were cultured for 20 hours before transfer to metronidazole. The data are from three experiments and presented as mean cfus.