Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study.

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridiumsensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota. Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.