We compared the microbial composition of stools from 15 preterm neonates with NEC and 15 controls, using 27 different culture conditions (microbial culturomics) and identified the colonies by using mass spectrometry and 16S rRNA sequencing. In parallel, we analyzed the same samples by pyrosequencin

Background. Necrotizing enterocolitis (NEC) is the most common and serious acquired gastrointestinal disorder among preterm neonates. To date, no etiologic organism has been identified although an infectious component is suspected. Methods. We compared the microbial composition of stools from 15 preterm neonates with NEC and 15 controls, using 27 different culture conditions (microbial culturomics) and identified the colonies by using mass spectrometry and 16S rRNA sequencing. In parallel, we analyzed the same samples by pyrosequencing 16S rRNA genes. The whole genome of C. butyricum isolate was sequenced. Then, we investigated the presence of Clostridium butyricum DNA on stool samples from 93 preterm neonates with NEC and 70 controls by specific quantitative real-time PCR (qRT-PCR). We performed a cytotoxic assay with the culture supernatant of C. butyricum and the secretome was analyzed by nano-LC. Results. C. butyricum was the most specific bacteria, identified by pyrosequencing and culturomic, associated with NEC (12/15 vs. 2/15; p<0.001). The C. butyricum q RT-PCR positivity was significantly associated with NEC when compared with controls (p<0.001). A cytotoxic activity of C. butyricum supernatant was observed and found higher than that of Clostridium difficile toxin-producing strain (42% ± 6 vs. 20% ± 5; p<0.001). We identified four genes coding for putative toxins in the genome. Expression of one of these putative toxins, with cytotoxic activity, was confirmed by proteomic analysis. Conclusions. We described, for the first time, a strong association between the presence of a toxin-producing C. butyricum strain and the occurrence of NEC in preterm neonates.