Identification and tracing of Bifidobacterium species by use of enterobacterial repetitive intergenic consensus sequences

Abstract

Eighty-nine Bifidobacterium strains from 26 species were identified and classified to the species level with an enterobacterial repetitive intergenic consensus (ERIC)-PCR approach. We demonstrated that ERIC-PCR is useful for a phylogenetic and taxonomical analysis but as well as for a species composition analysis of mixed bifidobacterial cultures isolated from dairy products and other environments.

FIG. 1.

ERIC-PCR patterns of reference strains of different Bifidobacterium species. Lane 1, B. lactis DSM 10140; lane 2, B. animalis ATCC 25527; lane 3, B. pseudocatenulatum DSM 20438; lane 4, B. catenulatum ATCC 27539; lane 5, B. subtile DSM 20096; lane 6, B. thermophilum DSM 20212; lane 7, B. minumum DSM 20102; lane 8, B. coryneforme DSM 20216; lane 9, B. angulatum DSM 20098; lane 10, B. choerinum ATCC 27686; lane 11, B. gallinarum DSM 20670; lane 12, B. boum DSM 20432; lane 13, B. mericycum DSM 6492; lane 14, B. suis ATCC 27533; lane 15, B. pullorum DSM 20433; lane 16, B. pseudolongum DSM 20099; lane 17, B. infantis ATCC 15697; lane 18, B. bifidum ATCC 29521; lane 19, B. breve ATCC 15700; lane 20, B. cuniculi ATCC 27916; lane 21, B. dentium ATCC 27534; lane 22, B. asteroides DSM 20089; lane 23, B. ruminantium DSM 6489; lane 24, B. magnum ATCC 27540; lane 25, B. adolescentis ATCC 15703; lane 26, negative control (complete PCR mixture without DNA); lane 27, B. longum ATCC 15707; lane M, 1-kb DNA ladder (Gibco BRL). Strain variation in the ERIC pattern of B. breve species is indicated by the arrows.

FIG. 2.

ERIC-PCR patterns of four Bifidobacterium species and their mixture (mixed strains). Shown are ERIC-PCR patterns of four individual Bifidobacterium strains from the four species (B. bifidum, B. breve, B. longum, and B. lactis) and a combination of these very same applied strains in a mixed approach created by using various template DNA (a) and different quantities of the template DNAs in an ERIC-PCR assay of the two species B. breve and B. longum (b). (a) Lane 1, B. bifidum ATCC 15696; lane 2, B. breve ATCC 15701; lane 3, B. longum ATCC 15708; lane 4, B. lactis DSM 10140; lane 5, B. bifidum and B. breve species; lane 6, B. longum and B. breve species; lane 7, B. longum and B. bifidum species; lane 8, B. bifidum and B. lactis species; lane 9, B. breve and B. lactis species; lane 10, B. longum and B. lactis species; lane M, 1-kb DNA ladder (Gibco BRL). (b) Lane 11, ERIC-PCR with 70 ng of DNA as PCR template for both species; lane 12, 70 ng of B. breve and 7 ng of B. longum as DNA template; lane 13, 70 ng of B. breve and 0.7 ng of B. longum as DNA template; lane 14, 7 ng of B. breve and 70 ng of B. longum as DNA template; lane 15, 0.7 ng of B. breve and 70 ng of B. longum as DNA template; and lane M, 1-kb DNA ladder (Gibco BRL).

FIG. 3.

Quantitative ERIC-PCR results with regard to the detection limit of ERIC-PCR primers that used whole bacterial cells from culture concentrates and subsequent 10-fold dilutions containing 10⁶ to 10 CFU/ml (a). ERIC-PCR patterns obtained from different food samples with declared content of bifidobacteria (b). (a) Lanes 1 to 6, direct detection of B. lactis; lane 7 to lane 12, direct detection of B. bifidum ATCC 35914; lanes 13 to 18, direct detection of B. breve ATCC 15701; and lanes 19 to 24, direct detection of B. longum ATCC 15708. (b) Lane 1, sample 15 containing only B. bifidum; lane 2, sample 16 containing only B. breve; lane 3, sample 13 containing only B. longum; lane 4, sample 1 containing only B. lactis; lane 5, sample 6 containing B. longum and B. bifidum; lane 6, sample 7 containing B. longum and B. bifidum; lane 7, sample 8 containing B. longum and B. bifidum; and lane M, 1-kb DNA ladder (Gibco BRL).