Clostridium and neonatal necrotizing enterocolitis pathogenesis: clinical and molecular approaches – ClosNEC

The clinical study is based on a French network of neonatology facilities for the inclusion of preterm neonates with confirmed diagnosed necrotizing enterocolitis and matched (perinatal determinants) with a control group of healthy preterm neonates. Clinical data will be recovered. The microbiological analysis of stool samples of necrotizing enterocolitis cases and control group will be performed by culture and pyrosequencing. For the fundamental study, we will construct by site-directed mutagenesis mutants of different Clostridium species deficient in butyrate production. The pathogenicity of the mutants and their parental strains will be evaluated in our animal model of necrotizing enterocolitis ( gnotobiotic quail ). This work will be complemented by comparison of the characteristics of clinical strains isolated from preterm neonates with or without necrotizing enterocolitis

The clinical study will allow obtaining a collection of clinical and microbiological data to validate the hypothesis of the biological involvement of clostridia in preterm neaonatal necrotizing enterocolitis. The fundamental study will demonstrate the pathogenicity of clostridia in the development of this disease and will search for biomarkers related to this pathogenicity. These results will propose clostridia as a biomarker of high risk of preterm neonatal necrotizing development

The lack of knowledge on the pathophysiology of neonatal necrotizing enterocolitis participates to the absence of consensus for therapeutic and preventive strategies. This project will provide new clinical and mechanistic data in order to improve the management and prevention of this dramatic neonatal digestive disease

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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 non-hydrolyzed lactose, a consequence of the immaturity of the intestinal lactasic equipment in preterm infants. 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.

Our current 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, we will use both clinical and experimental approaches.

The clinical study will take the opportunity of our 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 15 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.

Our 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. We will inactivate the gene encoding the enzyme responsible for butyrate production. The pathogenicity of wild type and mutants strains will be performed in our 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 we propose will be helpful for clinical practice and strategies of prevention implementation.