garvieae strains was PF-02341066 mouse determined using RAPD and REP-PCR with BOXA1R and (GTG)5 primers. These methods, which use short arbitrary primers or primers targeting short repetitive sequences interspersed throughout the genome are an established approach for delineation

of bacteria at the species and strain-level (Randazzo et al., 2009; Švec et al., 2010). The discriminatory power of these primer sets was similar, with 20 different profiles obtained by BOXA1R and (GTG)5 and 23 different profiles obtained by M13 for a collection of 49 strains. Although isolated at different times, some strains had identical fingerprints with all tested primers; on the contrary, most of the strains grouped at low similarity values. Independently from the primer used, the 49 strains grouped in two distinct selleck chemicals clusters, which we named AT and BT (Fig. 1): one cluster (AT) contained all meat isolates (with the exception of BOXA1R experiment where the meat isolate Sa113 showed a unique fingerprint at a very low similarity value), whereas the other cluster (BT) included all dairy isolates. Unexpectedly, four of 12 strains isolated from fish (V32, V63, Lg23, and V79), always grouped with dairy isolates, whereas the others grouped with meat isolates. Likewise, strains isolated from vegetables allocated between the two main groups. The cluster analysis resulting from the combined profiles of the three primer sets

employed, confirmed the existence of two major divisions, which were separated at a level of similarity of 0.13 (Fig. 1), and did not coincide with the ecological niche of isolation. In particular, the low correlation value between the two clusters suggested the existence of a marked genetic divergence. When we tested several genes belonging to the core genome of L. garvieae, we observed again that all bacterial isolates can be shared out between two clusters, which are correlated to a low similarity level. Specifically, on the basis of conserved regions identified by sequence comparison

of several housekeeping or functional genes in L. garvieae, we selected suitable primers to employ for PCR amplification (Table 2). The expected fragment length of the α-subunit of ATP synthase, elongation factor EF-Tu, D-alanine-D-alanyl carrier Edoxaban protein ligase, α-acetolactate synthase, glyceraldehyde-3-phosphate dehydrogenase, and galactose permease amplicons was observed for all the 49 strains studied. Restriction analysis of each of the loci tested produced one to seven different patterns consisting of one to seven bands, depending on locus, restriction enzyme and strain examined (Table 3). The cluster analysis resulting from the combined restriction profiles of the six amplicons reported in Fig. 2, revealed two distinct L. garvieae clusters at similarity level of approximately 0.12. Notably, the groups obtained were highly similar to PCR-fingerprinting clusters (AT and BT).