Members of the genus Bifidobacterium are Gram-positive, obligate anaerobic, non-motile, non-spore forming bacteria
, and are the most important constituents of human and animal intestinal microbiota
[2, 3]. Recently, news species of bifidobacteria have been described
[4–6] and now more than 30 species have been included in this genus.
Bifidobacterium spp. can be detected in various ecological environments, such as intestines of different vertebrates and invertebrates, dairy products, dental caries and sewage. Considering the increasing application of Bifidobacterium spp. as protective and probiotic cultures
[7–9], and the fast enlargement of the genus, easy identification tools to discriminate new isolates are essential. Moreover, their correct taxonomic identification is of outmost importance for their use as probiotics
. Conventional identification and classification of Bifidobacterium species have been based on phenotypic and biochemical features, such as cell morphology, carbohydrate fermentation profiles, and polyacrylamide gel electrophoresis analysis of soluble cellular proteins
. In the last years several molecular techniques have been proposed in order to identify bifidobacteria. Most available bifidobacterial identification tools are based on 16S rRNA gene sequence analysis, such as ARDRA
[11, 12], DGGE
 and PCR with the use of species-specific primers
[14–16]. However, 16S rDNA of Bifidobacterium spp. has a high similarity, ranging from 87.7 to 99.5% and bifidobacterial closely related species (e.g. B. catenulatum and B. pseudocatenulatum) or subspecies (e.g. B. longum and B. animalis subspecies) even possess identical 16S rRNA gene sequences
[17, 18]. For this reason different molecular approaches have been tested based on repetitive genome sequences amplification, such as ERIC-PCR
[19, 20], BOX-PCR
[21, 22] or RAPD fingerprinting analysis
. These fingerprinting methods have the disadvantage of a low reproducibility, and they need strict standardization of PCR conditions. The use of different polymerases, DNA/primer ratios or different annealing temperatures may lead to a discrepancy in the results obtained in different laboratories
In recent years alternative molecular markers have been proposed for bifidobacteria identification (e.g. hsp60, recA, tuf, atpD, dnaK) and Ventura et al.
 developed a multilocus approach, based on sequencing results, for the analysis of bifidobacteria evolution. The hsp60 gene, coding for a highly conserved 60 kDa heat-shock-protein (a chaperonin), has been evaluated for phylogenetic analysis in bifidobacteria by Jian et al.
. The sequence comparison of this gene has been already used for species identification and phylogenetic analysis of other genera (e.g. Staphylococcus, Lactobacillus) and enteric pathogens
[26–28]. A chaperonin database (cpnDB) is available on line, collecting bacterial and eukaryotic sequences (http://www.cpndb.ca/cpnDB/home.php)
The purpose of this study is the development of a rapid, reproducible and easy-to-handle molecular tool for the identification of Bifidobacterium species isolated from various environments. The protocol is based on the restriction endonuclease analysis of the PCR-amplified hsp60 partial gene sequence (hsp60 PCR-RFLP) with the use of a single restriction enzyme and has been tested on the 30 most widely distributed Bifidobacterium species and subspecies. A diagnostic dichotomous key to speed up profile interpretation has also been proposed.