Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates

Abstract

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n = 45; 3 to 10 per day, n = 97; >10 per day, n = 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r = 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.

FIG. 1.

Toxin levels in feces versus symptoms in CDAD patients. (A) Toxin levels (U/g feces) in groups of patients having differing diarrhea frequencies. Fecal samples from 20 consecutive patients per group were analyzed. Horizontal bars indicate median values. (B to D) Toxin levels in the same patients as for panel A but divided into groups having abdominal pain (No/Yes, n = 30/18; missing data, n = 12) (B), blood in stool (No/Yes, n = 40/15; missing data, n = 5) (C), or vomiting (No/Yes, n = 35/18; missing data, n = 7) (D). Statistical P values were obtained by one-way ANOVA followed by Bonferroni post hoc test (A) or Student's t test (B to D) of logarithmic toxin level values.

FIG. 2.

Toxin yields in vitro from infecting C. difficile isolates versus fecal toxin levels in corresponding CDAD patients having mild or severe diarrhea (n = 40). For high-level toxin production, the isolates were grown for 24 h in PY medium with 0.4 mM cysteine added (final OD₆₀₀, 1.0 to 1.5).

FIG. 3.

Toxin yields in vitro from C. difficile isolates belonging to major PCR ribotypes. SE21, SE30 (n = 9 each), SE20, and SE22 (n = 10 each) isolates were grown in PY medium without cysteine added for 24 h for maximum toxin production (final OD₆₀₀, 1.0 to 1.5). Each box encloses 50% of the values, and the median is represented by a horizontal line. Error bars include values that extend to the upper quartile value plus 1.5 times the interquartile distance and to the lower quartile value minus 1.5 times the interquartile distance, and open circles represent values outside these ranges (outliers). The P value was derived as described in the legend for Fig. 1 (ANOVA).

FIG. 4.

C. difficile toxin yield in vitro related to count of vegetative bacterial cells (A) and spore count (B). Isolates belonging to PCR ribotypes SE20, SE21 (n = 5 each), SE22 (n = 3), and SE25 (n = 4) were grown for 48 h in PY medium with 0.4 mM cysteine added for high-level toxin production. Counts of vegetative bacteria and cells containing visible spores were obtained by microscopy and were plotted against the toxin yields from the same cultures.