Spore formation and toxin production in Clostridium difficile biofilms.

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

Confocal laser-scanning microscopic analysis of <i>C. difficile</i> biofilms stained with nucleic acid stains.

<p>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.</p

Transmission electron microscopic analysis of <i>C. difficile</i> spores in biofilms.

<p>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).</p

Scanning electron microscopic analysis of <i>C. difficile</i> biofilms.

<p>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).</p

Spore production and germination in <i>C. difficile</i> biofilms.

<p><b>A)</b> 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>B)</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.</p

Confocal laser-scanning microscopic analysis of <i>C. difficile</i> biofilms stained with nucleic acid stains and concanavalin A-Texas Red.

<p>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.</p

ELISA, western blot and cytotoxicity analysis of toxin A and B production in biofilms.

<p>A) Toxin A and B levels from the matrix of 3-day old biofilms were measured by the Wampole <i>C. difficile</i> 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 <i>C. difficile</i> 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 <i>Clostridium difficile</i> 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.</p

Effects of metronidazole on cell growth in biofilms.

<p>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.</p