Characterization of Bifidobacterium spp. strains to be used as probiotics in newborns.

The postnatal period is the critical phase for the formation of the microbiota of the human gut. Bifidobacteria are among the first colonizers in breast-fed infants, whereas the microbiota of bottle-fed infants is different and contains higher counts of Bacteroides, Clostridium and Enterobaceriaceae that are the main responsible for bacterial infant gastrointestinal disorders like diarrhea (Penders et al., 2006). Moreover, recent evidences have indicated gas-forming coliform bacteria and C. difficile as a possible cause of infantile colics, which are widely diffused among newborns (Savino et al., 2009). Several studies have focused on the role of bifidobacteria as antagonist of pathogens in the gut. Therefore, there is a great interest in selecting Bifidobacterium strains to be added to formula-fed infants to improve the intestinal microbial balance. The aim of this work is to use in vitro assays to select bifidobacteria to be used as probiotics in newborns for the treatment of enteric disease. 46 strains of bifidobacteria, isolated from infant faeces, were screened to determine their capability of inhibiting potential pathogenic bacteria in infants by using the spot agar test, which evidenced the inhibition of indicator bacteria by the Bifidobacterium strains. Furthermore, inhibitory potency of bifidobacteria neutralized supernatants was investigated. The 16 strains showing the highest antimicrobial activity were subjected to further investigations, such as the susceptibility to 13 antibiotics to determine MIC breakpoints. Additionally, bacterial adhesion to intestinal epithelium was studied in different experimental in vitro models involving human intestinal epithelial and macrophage cell lines. The strains were also compared for their cytotoxic effects, their ability to stimulate the non specific immune response in the gut as well as the metabolic activity in the human functional cell model. The spot agar test of the selected Bifidobacterium strains against Escherichia coli, C. difficile, Salmonella enteriditis and two gas-forming coliforms isolated from colicky infants, identified as Klebsiella pneumoniae and Enterobacter cloacae , evidenced that a number of the assayed strains were capable of inhibiting a large array of pathogens. Ampicillin, kanamicym and amoxicillin resistances were found to be widely diffused among Bifidobacterium strains, whereas a number of strains possessing sensitivity to the other antibiotics assayed could be evidenced. All bacterial strains examined showed a good ability to adhere to polarized human epithelial cells and macrophages but some of them appeared slightly cytotoxic towards the cell monolayers at the highest bacterial concentration used (107 CFU/mL). In particular the ability to stimulate NO production in eukaryotic cells does not seem to be a common ability of member of the Bifidobacterium genus but rather of few strains, while increased H2O2 production was observed for cells stimulated with all tested strains. The results obtained allowed to identify four strains possessing strong antimicrobial activity against selected pathogens, sensitivity to the majority of the antibiotics tested and a good adhesion on human epithelial cells and no cytotoxic effects on them. These strains, belonging to the species B. breve and B. longum , are therefore potential candidates to be used for in vivo trials in newborns.