Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition

Abstract

Clostridium difficile is a major enteric pathogen responsible for antibiotic-associated diarrhea. Host susceptibility to C. difficile infections results partly from inability of the intestinal microbiota to resist C. difficile colonization. During early infancy, asymptomatic colonization by C. difficile is common and the intestinal microbiota shows low complexity. Thus, we investigated the potential relationship between the microbiota composition and the implantation of C. difficile in infant gut. Fecal samples from 53 infants, ages 0 to 13 months, 27 negative and 26 positive for C. difficile, were studied. Dominant microbiota profiles were assessed by PCR-temporal temperature gradient gel electrophoresis (TTGE). Bacterial signatures of the intestinal microbiota associated with colonization by C. difficile were deciphered using principal component analysis (PCA). Resulting bands of interest in TTGE profiles were excised, sequenced, and analyzed by nucleotide BLAST (NCBI). While global biodiversity was not affected, interclass PCA on instrumental variables highlighted significant differences in dominant bacterial species between C. difficile-colonized and noncolonized infants (P = 0.017). Four bands were specifically associated with the presence or absence of C. difficile: 16S rRNA gene sequences related to Ruminococcus gnavus and Klebsiella pneumoniae for colonized infants and to Bifidobacterium longum for noncolonized infants. We demonstrated that the presence of C. difficile in the intestinal microbiota of infants was associated with changes in this ecosystem's composition. These results suggest that the composition of the gut microbiota might be crucial in the colonization process, although the chronology of events remains to be determined.

Fig. 1.

TTGE fingerprinting profiles of 16S rRNA gene amplicons (obtained using primers for the V6 to V8 regions) of a C. difficile strain and 18 fecal samples from <1- to 13-month-old infants (lanes 1 to 18). Mq, marker consisting of a PCR amplicon mix of 7 cloned rRNA genes from different bacterial species; Cd+, positive C. difficile culture; Cd−, negative C. difficile culture.

Fig. 2.

Principal component analysis of TTGE fingerprinting profiles of dominant intestinal bacterial species (fecal samples). (A) PCA of TTGE profiles of 53 infants of ages <1 to 13 months. Black circles represent infants colonized by C. difficile, and light-gray circles represent noncolonized infants. (B, C, and D) Interclass PCA of TTGE profiles with instrumental variables. Individuals (represented by black dots or symbols) were clustered (ellipses or bars), and the center of gravity was computed for each class. (B) Interclass PCA of TTGE profiles of 53 infants of ages <1 to 13 months, with C. difficile toxigenic culture status as an instrumental variable. The global Monte Carlo test showed no significant difference between the 3 groups (P = 0.418). Intestinal colonization by a toxigenic strain of C. difficile was not associated with a specific TTGE profile (P = 0.584). Colonization by C. difficile was significantly associated with modifications in the microbiota composition (P = 0.017). (C) Interclass PCA of TTGE profiles of 20 infants of ages 2 to 5 months, with C. difficile culture and age as instrumental variables. Based on a Monte Carlo test with 999 replicates, a significant difference was found between all groups (P = 0.003). Intestinal colonization by C. difficile was significantly associated with a specific TTGE profile (P = 0.02). The age group of the infants was not associated with modifications in the microbiota composition (P = 0.125). (D) Interclass PCA of TTGE profiles of 4 infants of ages 2 to 6 months, with subject and C. difficile culture as instrumental variables. Samples were obtained before (n = 2) and during (n = 2) colonization by C. difficile. One sample collected during colonization was excluded from the analysis for infant S4 as carried out under antibiotic therapy. Intestinal colonization by C. difficile and subject itself were significantly associated with modifications in the microbiota composition as assessed by a Monte Carlo test (respectively, P = 0.012 and P = 0.041). Pos., positive; Neg., negative; AB, toxin A/B. C. difficile −, negative C. difficile culture. C. difficile +, positive C. difficile culture; Comp, component; m, month(s); S, subject.

Fig. 3.

TTGE profiles of 16S rRNA gene amplicons (obtained using primers for V6 to V8 regions) of fecal samples and localization of bands associated with colonization or noncolonization by C. difficile.

Fig. 4.

Frequency and median intensity of TTGE profile bands corresponding to bacterial species associated with presence or absence of C. difficile in infant feces. Median intensity, curves; frequency, bars; Cd−, negative C. difficile culture; Cd+: positive C. difficile culture; SD, standard deviation. *, median intensity is expressed in absolute units. ‡, chi square test on frequency; †, Mann Whitney U test (Wilcoxon) on median intensity.