Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease

Abstract

Recurrent Clostridium difficile infection (CDI) is of particular concern among health care-associated infections. The role of the microbiota in disease recovery is apparent given the success of fecal microbiota transplantation (FMT) for recurrent CDI. Here, we present a murine model of CDI relapse to further define the microbiota recovery following FMT. Cefoperazone-treated mice were infected with C. difficile 630 spores and treated with vancomycin after development of clinical disease. Vancomycin treatment suppressed both C. difficile colonization and cytotoxin titers. However, C. difficile counts increased within 7 days of completing treatment, accompanied by relapse of clinical signs. The administration of FMT immediately after vancomycin cleared C. difficile and decreased cytotoxicity within 1 week. The effects of FMT on the gut microbiota community were detectable in recipients 1-day posttransplant. Conversely, mice not treated with FMT remained persistently colonized with high levels of C. difficile, and the gut microbiota in these mice persisted at low diversity. These results suggest that full recovery of colonization resistance against C. difficile requires the restoration of a specific community structure.

FIG 1

Relapse of C. difficile infection (CDI) in mice treated with vancomycin. (A) Experimental timeline of murine model of CDI relapse. (B) Mean C. difficile colonization levels of untreated mice with primary CDI (no vancomycin; yellow) or vancomycin-treated mice that relapsed (blue). Error bars represent the standard deviations, and a horizontal line indicates the limit of detection. (C) Cytotoxin activity (expressed as the log₁₀ reciprocal cytotoxin dilution per gram of cecal content) following C. difficile infection was determined by using a Vero cell cytotoxicity assay at experimental endpoints on days 4, 9, and 16. Two mice in the vancomycin-treated group fell below weight threshold on day 13. Points represent individual samples, and bars represent median and upper/lower quartiles. (D and E) Summary score of histopathological damage in the cecum (D) and colon (E) after CDI (days 13 to 16), with antibiotic-only treated mice (pink) as a control. Significance was determined by using a Wilcoxon rank sum test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). (F and G) Histopathology of the cecum (F) and colon (G) at days 13 to 16 p.i. for vancomycin-treated mice (top panels) and mice that did not receive vancomycin (bottom panels). All images were taken at ×200 magnification (scale bars, 100 μm).

FIG 2

CDI in mice with or without FMT after vancomycin treatment of primary CDI. (A) Experimental design and timeline of mice with CDI relapse with or without FMT. (B) Mean C. difficile colonization levels (expressed as CFU/g of fecal pellets) of mice with relapse without FMT (blue) or with FMT (green). Error bars represent standard deviations, and the horizontal line indicates the limit of detection. (C) Cytotoxin activity (expressed as the log₁₀ reciprocal cytotoxin dilution per gram of fecal content) was determined using a Vero cell cytotoxicity assay in mice treated with vancomycin only (blue) or vancomycin followed by FMT (green). Points represent samples collected from either mice that received only vancomycin (blue) or mice that also received FMT (green). Bars represent the median per group and lower/upper quartiles. Significance was determined by using a Wilcoxon rank sum test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

FIG 3

Effect of antibiotics and FMT on the microbial community of C. difficile-infected mice. NMDS ordination was calculated from the Yue and Clayton dissimilarity metric (θ_YC) on OTU at a 97% cutoff. Biplot values of the OTU driving the NMDS ordination were derived using a cutoff of P < 0.001 for at least one axis, a total sequence abundance of >100,000 and a length of >0.77.

FIG 4

Changes in community diversity and structure over time during relapse of CDI and FMT. (A) Community diversity of the microbiota in mice with CDI relapse (inverse Simpson index, y axis). Points represent individual samples, and lines represent the mean diversity per group. (B) Mean distance between the community structure on sampling day and conventional/untreated over time in mice without or with FMT, calculated using the Yue and Clayton dissimilarity metric (θ_YC). (C) Mean θ_YC distance between the community structure of mice with or without FMT over time. Error bars represent upper and lower quartiles. A pairwise Wilcoxon rank sum test was used to calculate significance between mice with or without FMT (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

FIG 5

Taxonomic profile of the microbiota in mice treated with FMT after vancomycin treatment of CDI. (A) Median relative abundance (bars) of select dominant phylotypes in microbiota community of untreated mice (left panel, n = 11), during CDI (middle, n = 24, days 1 and 4), and following vancomycin (right, n = 20) before relapse. Error bars represent interquartile range. (B and C) Median relative abundance of dominant phylotypes in mice without FMT (B) and mice with FMT (C) after CDI relapse (left panel, n = 3 without FMT and n = 19 with FMT), 1 day after clindamycin (middle panel, n = 5 and n = 19), and 1 week after clindamycin (right panel, n = 5 and n = 18). A Friedman rank sum test (paired) was used to calculate significance over time in panels B and C (***, P < 0.0005).