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

Abstract

The expression of the Clostridium difficile binary toxin CDT is generally observed in the RT027 (ST1) and RT078 (ST11) C. difficile isolates, which are associated with severe C. difficile infection (CDI). However, we recently reported that the non-RT027 and non-RT078 C. difficile strain LC693 (TcdA⁺TcdB⁺ CDT⁺, ST201) caused severe diarrhea in a patient in Xiangya Hospital in China. C.difficile LC693 is a member of Clade 3, and in this study, we identified LC693 as RT871 and compared its virulence and pathogenicity to those of C.difficile R20291 (TcdA⁺TcdB⁺CDT⁺, ST1/RT027), UK6 (TcdA⁺TcdB⁺CDT⁺, ST35/RT027), CD630 (TcdA⁺TcdB⁺CDT^-, ST54, RT012), and 1379 (TcdA⁺TcdB⁺CDT^-, ST54/RT012), with strain 1379 being an epidemic C.difficile isolate from the same hospital. LC693 displayed a higher sporulation rate than R20291, CD630 or strain 1379. LC693 was comparable to R20291 with respect to spore germination, motility, and biofilm formation, but showed a faster germination rate, higher motility and a higher biofilm formation capability compared to CD630 and strain 1379. The adherence of spores to human gut epithelial cells was similar for all strains.The total toxin release of LC693 was lower than that of R20291, but higher than that of CD630 and strain 1379. Finally, in a mouse model of CDI, LC693 was capable of causing moderate to severe disease. Our findings demonstrate the pathogenicity of non-RT027 and non-RT078 binary toxin-positive C. difficile strains. Furthermore, our data indicate that LC693 may be more virulent than strain 1379, an epidemic strain from the same hospital, and provide the first phenotypic characterization of a non-RT027 and non-RT078 binary toxin-positive ST201 isolate.

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