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Next-Generation DNA sequencing data from the human intestinal microbiome provides new instruments to explore the correlation between changes in microbiome composition and the host physiologic status. Some studies show that newborns have a highly unstable microbiome composition, but few information are available in literature on the potential effect due to intrapartum antibiotic prophylaxis (IAP) administrated to the mothers positive to the infection by group B Streptococcus, which affects around 10% of pregnant woman. According to this, the aim of the project is to evaluate the main effects of the mentioned IAP on microbiome composition of newborns at seven days after birth, by a massive parallel sequencing of seven different regions of 16S gene. The analysis was performed with Ion PGM platform, taking advantage from “Ion 16s Metagenomics Sequencing Technology Access Program” (LifeTechnologies). Our first results show an evident difference in the microbial composition of treated newborns, resulting in a general decrease of microbial biodiversity and an over representation of Proteobacteria, suggesting that these infants are potentially more exposed to gastrointestinal diseases.

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.

Bifidobacteria are well known for their health promoting properties and are widely used as probiotics for therapeutic purposes (Sanders et al., 2010). A wide literature documentation reports clinical benefits with treatment of infant gastrointestinal disease with probiotics. In addition, the supplementation of infant formula with oligosaccharides stimulates the growth of bifidobacteria in the intestine resembling the effect of breast-feeding (Boehm and Moro, 2008). A recent study in our laboratory has allowed the selection of four Bifidobacterium strains (B. breve B632, B2274 and B7840 and B. longum subsp. longum B1975) as potential probiotics for the treatment of enteric disorders in newborns such as infant colics (Aloisio et al., 2012). This work was therefore aimed at the formulation of a synbiotic product with an appropriate prebiotic fibre capable of supporting the growth of the previously selected Bifidobacterium strains. The bifidogenic potential of eleven commercial fibres was determined. The polysaccharides used included galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulins, glucooligosaccharide, an arabinogalactan and a partially hydrolised guar gum. The growth of the previously selected em>Bifidobacterium strains was evaluated by using the fibres (1% w/v) as the sole carbon source. The assay was performed incubating the bifidobacteria cultures at 37 °C under anaerobic conditions and the bacterial growth was determined by measuring A620 nm at regular intervals. Significant differences were obtained in the growth profiles of the four Bifidobacterium strains on the different fibres, which is not surprising considering that early studies on prebiotics reported that carbohydrate utilization pattern differs greatly among Bifidobacterium species and strains. A common feature was that the 4 strains could grow well on two GOS (Vivinal®GOS and Cup-Oligo®), on the FOS Actilight® and the low molecular weight inulin Orafti®HSI. In addition, B632 could also grow well on the glucooligosaccharide BioEcolians®. The efficacy of a prebiotic fibre has to be evaluated not only by studying its capability of sustaining the growth of a probiotic strain but also taking into consideration the growth stimulation of potentially harmful bacteria (Huebner et al., 2007). A prebiotic activity score was therefore calculated for the four strains on each fibre by comparing the growth of bifidobacteria with that of a mixture of coliform strains isolated from colicky infants and potentially involved in enteric diseases in newborns (E.coli, K.pneumonia, E. cloacae). The results obtained evidenced a significant prebiotic*strain interaction. The highest prebiotic scores were obtained with Orafti®HSI*B632, Orafti®HSI*B2274, Orafti®HSI*B7840 and Actilight®*B7840. These results provide a basis for the formulation of a synbiotic product for the treatment of enteric disorders in newborns. In addition, considering the high prebiotic scores obtained with Vivinal, a mixture of GOS and FOS can be assayed in further studies.

Different factors are known to influence the early gut colonization in newborns, among them the perinatal use of antibiotics. On the other hand, the effect on the baby of the administration of antibiotics to the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has received less attention, although routinely used in group B Streptococcus positive women to prevent the infection in newborns. In this work, the fecal microbiota of neonates born to mothers receiving IAP and of control subjects were compared taking advantage for the first time of high-throughput DNA sequencing technology. Seven different 16S rDNA hypervariable regions (V2, V3, V4, V6 + V7, V8, and V9) were amplified and sequenced using the Ion Torrent Personal Genome Machine. The results obtained showed significant differences in the microbial composition of newborns born to mothers who had received IAP, with a lower abundance of Actinobacteria and Bacteroidetes as well as an overrepresentation of Proteobacteria. Considering that the seven hypervariable regions showed different discriminant ability in the taxonomic identification, further analyses were performed on the V4 region evidencing in IAP infants a reduced microbial richness and biodiversity, as well as a lower number of bacterial families with a predominance of Enterobacteriaceae members. In addition, this analysis pointed out a significant reduction in Bifidobacterium spp. strains. The reduced abundance of these beneficial microorganisms, together with the increased amount of potentially pathogenic bacteria, may suggest that IAP infants are more exposed to gastrointestinal or generally health disorders later in age.