Characterization of the virulence of a non-RT027, non-RT078 and binary toxin-positive Clostridium difficile strain associated with severe diarrhea

Figures & data

Fig. 1 Strain LC693 efficiently sporulates.

C. difficile strains were grown in 70:30 liquid sporulation medium and assayed for spore formation after 24 h of incubation. a Sporulation frequencies were plotted from direct enumeration of spores and vegetative cells in micrographs (data not shown). b Sporulation frequency determined by ethanol inactivation of vegetative cells. The means ± standard deviation of the mean from three biological replicates are shown. Student’s t-test was used to assess significance; ns not significant; *p < 0.05

Fig. 2 LC693 spores readily germinate.

Purified C. difficile spores were suspended in buffer supplemented with taurocholic acid and glycine to induce germination. a Germination was monitored by plotting the ratio of the OD₆₀₀ at a given time to the OD₆₀₀ at time zero. **p = 0.0001, LC693 vs CD630 and strain 1379, one-way ANOVA. b CaDPA release from germinating C. difficile spores was monitored using Tb³⁺-fluorescence, and values were normalized to the maximum signal obtained for strain R20291 at 30 min. (****p < 0.0001). Data are reported as the means ± SD from three independent experiments

Fig. 3 LC693 is motile.

A single colony was inoculated in the middle of a swim plate, which was incubated at 37 °C for the indicated time. Swimming motility was quantitatively determined by measuring the radius of the zone of motility derived from a single colony. a–d Bacterial growth resulting from strains LC693 (a), R20291 (b), CD630 (c) and 1379 (d) after 72 h of incubation (e). Radius of the bacterial growth derived from a single colony of each strain. Data are reported as the means ± SD from three independent experiments, and differences were analyzed by Student’s t-test; ns not significant; **p < 0.001

Fig. 4 LC693 forms peritrichous flagella.

Flagella were visualized by transmission electron microscopy. Scale bar represents 1 μM. a LC693; b R20291; c CD630; and d 1379

Fig. 5 LC693 can form a biofilm.

Biofilm formation was measured by crystal violet staining after 5 days of growth in reinforced clostridial medium. Data are from three biological replicates and are reported as the means ± SD. Student’s t-test was used for assess significance; ns not significant; **p < 0.001

Fig. 6 LC693 spores adhere well to human gut epithelial cells.

HCT-8 cells were incubated with spores for 100 min under anaerobic conditions, and the relative amount of adhered spores was subsequently calculated. ns: not significant

Fig. 7 LC693 releases TcdA and TcdB.

Toxin production at four different time-points was measured. a TcdA concentration in culture supernatant as determined by conventional ELISA; b TcdB concentration in culture supernatant as determined by conventional ELISA; c Total toxin released into the supernatant as measured using the C. DIFFICILE TOX A/B II™ assay. Student’s t-test was used for to assess significance; ns not significant; *p < 0.05; **p < 0.001; ***p = 0.0001

Fig. 8 LC693 is virulent in a mouse model of CDI.

Four groups of mice (n = 10) were challenged with 10⁶ spores of UK6, LC693, CD630, or R20291. Mice were monitored for one week for survival (a) and weight changes (b). Animal survival was analyzed by Kaplan–Meier survival analysis with a log-rank test of significance (ns not significant, p > 0.05). The mean relative weight of mice was analyzed for significance using a Student’s t-test. *p < 0.05 between the CD630 and LC693 groups at 1, 2 and 3 days post challenge; p < 0.05, between the CD693 and UK6 groups at 2, 3, 5, 6, and 7 days post challenge; p < 0.05 between the LC693 and R20291 groups at 2 and 3 days post challenge; and p > 0.05 between the UK6 and R20191 groups at all assayed time points