We read with great interest the recently published study by Rehman and colleagues. 1 Investigating microbial diversity on faecal and ileal samples from 48 mice using Sanger and 454 techniques, Rehman et al detected increased percentages of Bacteroidetes in the faeces of adult Nod2 À/À mice, whereas Firmicutes levels were significantly higher only in the ileum of Nod2 À/À mice. Since we demonstrated that antibiotic treatment of Nod2 À/À mice decreased the number of Peyer’s patches and that suppression of the intestinal microbiota was able to fully reverse Nod2 À/À mice phenotype, 2 we further analysed the faeces of 30 C57BL/6 Nod2 À/À (mean age¼13 weeks; sex ratio¼1) and 30 C57BL/6 wild-type (WT) mice (mean age¼10weeks; sex ratio¼1). Age did not differ significantly between mice groups. The V3eV4 region of the bacterial 16S ribosomal DNA was sequenced on a 454 FLX-Titanium pyrosequencer, and 219 964 sequences (average 3660 sequences/sample) passed the quality trimming (size> 350 bp/base quality> 20). We further used the Ribosomal Database Project Classifier and CD-HIT (Cluster Database at High Identity with Tolerance) Program3 to accurately assign sequences to bacterial genera and down to molecular species (97% similarity clustering). Our results, in agreement with those by Rehman et al, showed a decreased diversity (Simpson index; WT: 0.03; Nod2 À/À: 0.07; p< 0.001) and richness (Shannon index; WT: 3.94; Nod2 À/À: 3.34; p< 0.001) in Nod2 À/À mice microbiota. Analysis of phyla distribution highlighted a depletion in bacteria belonging to Proteobacteria(WT: 2.3861. 82%; Nod2 À/À: 0.3460. 52%; p< 0.001) in Nod2 À/À mice microbiota. Topedown analysis of Proteobacteria shed light on two sequence clusters absent from Nod2 À/À mice microbiota that related to