35 × 103 CFU per μg DNA when the strain was grown in FOS, and 3.7 × 103 CFU per μg DNA when grown in GOS (Table 2). Plasmid stability was evaluated Apoptosis inhibitor by continuous cultivation for 15 days of five PRL2010 transformants in the

absence of chloramphenicol selection by PCR assays. Notably, all PRL2010 transformants tested did not exhibit any plasmid loss during this period, despite the absence of antibiotic selection. To evaluate the general usefulness of the transformation protocol developed here, we decided to apply it to another Bifidobacterium species, B. asteroides PRL2011, whose genome was recently decoded (F. Bottacini, F. Turroni and M. Ventura, unpublished data). Interestingly, the B. asteroides species represents a distantly related taxon with respect to B. bifidum, while it also occupies a different ecological niche, that is, the hindgut of honeybee (Veerkamp & van Schaik, 1974;

Fischer et al., 1987; Argnani et al., 1996; de Ruyter et al., 1996; Hartke et al., 1996; Rossi et al., 1996; Kullen & Klaenhammer, 2000; Sleator et al., 2001; Schell et al., 2002; Ventura et al., 2006, 2007, 2009; Guglielmetti et al., 2007, 2008; Sela et al., 2008; O’Connell Motherway et al., 2009; Turroni et al., 2010, 2011; Foroni et al., 2011; Serafini et al., 2011). Thus, one may argue that the B. asteroides species possesses a different cell envelope composition (e.g. exopolysaccharides, extracellular proteins) compared to that of B. bifidum. When the transformation protocol optimized on B. bifidum PRL2010 cells was employed for transforming B. asteroides PRL2011 using pNZ8048, a higher transformation efficiency ACP-196 cell line (1.6 × 104 CFU per μg DNA) was obtained as compared to B. bifidum PRL2010. A direct application from the results of the successful transformation protocol described in this study was to monitor the colonization efficiency of B. bifidum PRL2010 in a murine model. In fact, so far, it has been proven impossible to generate stable antibiotic-resistant B. bifidum PRL2010 derivatives

by spontaneous mutation such as those in other bacterial species might be obtained upon repeated cultivation in the presence of antibiotics. Thus, to discriminate the presence of PRL2010 cells from other members of the gut microbiota of mice, we employed a derivative PRL2010 strain Gefitinib in vitro that contained a plasmid carrying an antibiotic resistance gene to act as a selective marker. The normal microbiota of mice encompasses microorganisms that are sensitive to chloramphenicol (Savino et al., 2011), thus indicating that this antibiotic can be used in selective media. Colonization and clearance of PRL2010 were monitored over a 15-day period by determining viable counts recovered from fecal samples. Two groups of six mice were fed orally on a daily basis with either PRL2010 containing pNZ8048 (designated here as PRL2010pNZ8048) or water for 1 week.