Figure 1.
Comparison of CDI sign severity after 48 hours (white bars) and 72 hours (black bars) of animals challenged with C. difficile spores and treated with DMSO, 300 mg/kg taurocholate (TC), 50 mg/kg chenodeoxycholate (CDCA), 50 mg/kg CamSA, or 300 mg/kg ethyl cholate (EC). Non-challenged (NC) animals were used as controls. Clinical endpoint was set as >6 in the CDI sign severity scale (dashed line). None of the animals in the DMSO and EC groups survived to 72 hours post-challenged. Standard deviations represent at least five independent measures.
Figure 2.
Stability of CamSA and taurocholate towards bile salt hydrolases.
CamSA (white bar) and taurocholate (black bars) were incubated with cultures of B. longum or L gasseri. Percent conjugated bile salts were derived by dividing the intensity of TLC spots obtained at different times by the intensity of the TLC spot obtained at the beginning of incubation (time 0). Time 0 was set at 100% and is not shown for clarity. Standard deviations represent at least five independent measures.
Figure 3.
Vero cells (white bars) or Caco-2 cells (black bars) were incubated overnight with 10% DMSO, 10% EtOH, 50 µM CamSA or 200 µM CamSA. Cell viability was determined with the CellTiter Glo viability kit. The luminescence signal from DMSO-treated cells was undistinguishable from untreated cells and was set as 100% cell viability. Percent survival for other conditions was calculated relative to untreated cells. Error bars represent standard deviations from at least five independent measurements.
Figure 4.
Inhibition of C. difficile toxin production by CamSA treatment.
C. difficile spores were incubated overnight in media containing 0 µM CamSA (white bars) or 200 µM CamSA (black bars). The resulting spent media were added to Vero cell cultures and incubated for 24 hours. Cell viability was determined with the CellTiter Glo viability kit. The luminescence signal from untreated cells was set as 100% cell viability. Percent survival for other conditions was calculated relative to untreated cells. Error bars represent standard deviations from at least five independent measurements.
Figure 5.
CDI is established between 6 and 9 hours post-infection.
(A) Survival of infected mice at 48 hours after challenge with C. difficile spores. Mice were treated with 300 mg/kg CamSA at 0, 6, 9, or 12 hours post-challenge. (B) Comparison of CDI severity after 24 hours (white bars) and 48 hours (black bars) for animals challenged with C. difficile spores and treated with 300 mg/kg CamSA at 0, 6, 9, or 12 hours post-challenge. Clinical endpoint was set as >6 in the CDI sign severity scale (dashed line). (C) C. difficile vegetative cell count in feces of untreated, diseased animals. Feces were collected from cages housing five untreated mice challenged with C. difficile spores. Open bars represent C. difficile vegetative cells. The amount of C. difficile spores excreted by untreated animals was negligible (<10% of vegetative cell counts). Standard deviations represent at least five independent measures. Recovered CFU and recovered spores represent mean values from pools of five animals.
Figure 6.
C. difficile spores accumulate in the cecum, colon, and feces of CamSA-treated animals.
(A) Amount of C. difficile spores recovered at different time points following spore challenge from the cecum (white bars) and colon (black bars) of mice treated with 50 mg/kg CamSA. Student’s unpaired t-test was used to determine the significance of difference of means. *indicates recovered spores significantly below 72 hour levels (P = 0.019; Student's t-test). **indicates recovered spores significantly below 72 hour levels (P = 0.049; Student's t-test). (B) Feces were collected from cages housing five mice challenged with C. difficile spores and treated with 50 mg/kg CamSA. Closed bars represent C. difficile spores. The amount of C. difficile vegetative cells in CamSA-treated animals was negligible (<10% compared to spore counts). Standard deviations represent at least five independent measures. Recovered CFU and recovered spores represent mean values from a pool of five animals.
Figure 7.
Time line model for CDI onset in mice.
C. difficile spores (black circles) are ingested by the host. Spores rapidly transit through the upper GI tract and colonize the colon and cecum. Spore shedding begins less than 2 hours post-ingestion. Between 6 and 9 hours after ingestion sufficient numbers of spores germinate to establish infection. The outgrowing C. difficile cells (white circles) proliferate in the lower intestine, are shed, and can re-sporulate. A small amount of ingested spores remain in the lower intestine for more than 96 hours post ingestion.