After 1 week, the PRL2010pNZ8048 supplementation was discontinued, and after one additional week, the animals were killed. To follow PRL2010pNZ8048 colonization, fecal samples were collected periodically (on

days 0, 2, 5, 9, 12, and 15), and PRL2010pNZ8048 cell enumeration was performed by plating fecal material on MRS–Cys–Agar supplemented with chloramphenicol. After incubation at 37 °C, the identity of colonies grown on MRS supplemented with chloramphenicol was further evaluated using PCR and employing PRL2010-specific primers that target pili-encoding loci, which have been described previously (Turroni et al., 2010; Foroni et al., 2011). The inoculated bacterial population increased in number (Fig. 2), reaching a maximum of 107 CFU g−1 feces at day 5. Interestingly, following this rapid increase

find more of PRL2010 cell numbers during the period of bacterial supplementation, the level of PRL2010 cells decreased to reach a plateau of approximately 105 CFU that appeared to remain stable during the full length of the post-treatment period (Fig. 2). Notably, the presence of high numbers of PRL2010pNZ8048 cells upon a period of 7 days without any supplementation with bifidobacterial cells reinforces the notion that the plasmid is stable. Altogether these data indicate that PRL2010 is capable of colonizing the intestine of mouse, which will open new avenues in the exploration of host–microbe interactions of this microorganism OSI-744 mw using an in vivo murine model (O’Connell Motherway et al., 2011). This study describes an optimized protocol for the transformation of bifidobacteria MRIP that enables the establishment of plasmid DNA into two very distantly related species, that is, B. bifidum and B. asteroides taxa, where in the

latter case it represents the first report on plasmid-mediated transformability. The transformation rates achieved were sufficiently high for cloning purposes; nonetheless, the experiments so far performed highlighted transformation efficiency of 104 CFU μg−1 which is not yet high enough for site-directed mutagenesis and for an effective selection of transformants in gene knock-out experiments (O’Connell Motherway et al., 2009). The next step will be to improve the transformation efficiency, which could be achieved by overcoming the restriction modification systems of this microorganism (O’Connell Motherway et al., 2009). Genetic tools to manipulate bifidobacteria are still largely undeveloped and represent a bottleneck in the advancing of knowledge on this important group of microorganisms. Thus, the transformation protocol and subsequent colonization model described in this study offer two important adjuncts in exploring genomic functionalities of bifidobacteria under in vitro as well as in vivo conditions. We thank GenProbio srl for the financial support of the Laboratory of Probiogenomics. This work was financially supported by a FEMS Advanced Fellowship 2011 and an IRCSET Embark postdoctoral fellowship to F.T.