Clostridium and Neonatal Necrotizing Enterocolitis Pathophysiology : Clinical and Molecular Approaches

Necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among preterm neonates. Despite many investigations its pathogenesis remains unclear. The role of intestinal bacteria in NEC development is supported by epidemiologic evidence (outbreaks), the frequent isolation of infectious agents, and a decrease incidence of NEC resulting from preventive antibiotic treatment. Bacterial implication is thought to be due to colonic fermentation of nonhydrolyzed lactose, a consequence of the intestinal immaturity of preterm infants (intestinal lactase deficiency). To date, no specific bacteria or bacterial colonization pattern have been causally associated with the development of NEC. Clinical signs and some epidemiological studies are consistent with clostridia involvement in NEC. Indeed, Clostridium butyricum, Clostridium perfringens, and Clostridium paraputrificum have been isolated from the blood, feces, and peritoneal fluids of preterm neonates suffering from NEC. In addition, a correlation between the presence of C. butyricum and C. paraputrificum and pneumatosis intestinalis in tissue specimens from NEC neonates was reported. Furthermore, the role of these clostridia species in NEC pathogenesis has been demonstrated using NEC animal models (preterm piglet or gnotobiotic quails). Particularly, lactose fermentation end-products (butyrate or iso-butyrate) was linked to cecal NEC-like lesions onset in gnotobiotic quails animal model.

This project is lying within this context, and aims at confirming the clostridial involvement in the pathogenesis of NEC and search for bacterial pathogenicity biomarkers. For this purpose, investigators will use both clinical and experimental approaches.

The clinical study will take the opportunity of previous EPIFLORE project network (ANR 2012) to build during a one-year period a large collection of proved NEC cases. During that period, preterm neonates diagnosed as NEC cases (Bell stage II or III) from 20 French neonatal units will be included and matched to a control group of non-NEC neonates. Anthropometric data, clinical data, drug and feeding intakes will be collected from birth to the neonatal unit discharge. Microbiota analysis of NEC and non-NEC preterm neonates will be performed using culture and 16S rRNA gene pyrosequencing. This case-control study aims at obtaining the largest French collection of clinical and microbiological documented NEC cases that will allow reaching significance in results about the characteristics of the microbiota of NEC neonates. Additionally, it will confirm the involvement of specific bacteria, i.e. clostridia, which will be proposed as a biological marker of high risk to develop NEC.

Experimental approach will consist in the construction of mutants of clinical isolates of C.butyricum, C. perfringens, and C. paraputrificum impaired in butyrate production using the Clostron directed mutagenesis knock out tool recently adapted to clostridia. investigators will inactivate the gene encoding the enzyme responsible for butyrate production. The pathogenicity of wild type and mutants strains will be performed in previous gnotobiotic quail animal model of NEC. This will allow us to verify the hypothesis of clostridia species biological involvement in NEC pathogenesis through fermentation end-product metabolite. This work will be completed by a phenotypic characterization and proteomics analysis of the clinical strains linked to NEC development in order to search for a bacterial pathogenicity biomarker. The absence of a consensus for the prevention of NEC is partly due to the lack of knowledge of its pathogenesis. This project will give important information in terms of clinical and mechanistic data. Looking for biological markers as investigators propose will be helpful for clinical practice and strategies of prevention implementation