Rapid identification, differentiation, and proposed new taxonomic classification of Bifidobacterium lactis

Abstract

Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140(T), B. animalis ATCC 25527(T), and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.

FIG. 1.

DNA fingerprint analysis of B. lactis and B. animalis strains by ERIC-PCR. Lanes: 1, B. lactis DSM 10140; 2, B. lactis NCC 363; 3, B. lactis NCC 311; 4, B. animalis ATCC 25527; 5, B. animalis ATCC 27672; 6, B. animalis ATCC 27674; 7, B. animalis ATCC 27536; 8, B. animalis ATCC 27673; 9, B. lactis NCC 383; 10, B. lactis NCC 402; 11, B. lactis NCC 239; M, 1-kb DNA ladder (Gibco-BRL).

FIG. 2.

PFGE patterns of genomic DNAs of B. lactis strains after SpeI digestion. Lanes: 1, B. lactis NCC 383; 2, B. lactis NCC 363; 3, B. lactis DSM 10140; 4, B. lactis NCC 402; 5, B. lactis DSM 10140; 6, B. lactis NCC 239; 7, B. lactis NCC 387; 8, B. lactis NCC 311; 9, B. lactis DSM 10140; M, λ DNA ladder (Bio-Rad).

FIG. 3.

(A) Dendrogram based on 16S-23S ITS sequences and showing the relationships of members of the genus Bifidobacterium related to B. animalis and B. lactis strains. The bar at the top indicates 0.02% sequence divergence. The 16S-23S ITS sequences used to draw the phylogenetic tree of the following strains were retrieved from the GenBank database: B. infantis ATCC 15697 (accession no. U09792), B. breve CIP 6470 (accession no. U09521), B. adolescentis CIP 6460 (accession no. U09513), B. adolescentis CIP 6459 (accession no. U09512), B. lactis DSM 10140 (accession no. X89513), B. animalis ATCC 25527 (accession no. U09858), B. choerinum CIP 103378 (accession no. L36968), B. pseudolongum ATCC 25526 (accession no. U09879), and B. globosum ATCC 25865 (accession no. U09524). (B) Overall 16S-23S rRNA spacer region sequence similarities of B. lactis strains.

FIG. 4.

Multiple alignment of the ITS sequences of six B. lactis strains, B. lactis DSM 10140 (accession no. X89513), and B. animalis ATCC 25527 (accession no. U09858). Nucleotides that differ between B. lactis strains and B. animalis ATCC 25527 are shaded in dark grey. Nucleotides that differ among B. lactis strains are shaded in light grey.

FIG. 5.

(A) Primers and amplification systems used to investigate the species B. animalis. (B) PCR products of various Bifidobacterium species obtained by a Multiplex-PCR approach with B. animalis species-specific PCR primers and bifidobacterial genus-specific primers. (A) The position numbering and expected product sizes are given in accordance with the numbering of the B. animalis 16S-23S spacer region (1). (B) Lanes: 1, B. animalis ATCC 25527; 2, B. animalis ATCC 27672; 3, B. lactis DSM 10140; 4, B. animalis ATCC 27673; 5, B. animalis ATCC 27674; 6, B. animalis ATCC 27536; 7, B. lactis NCC 363; 8, B. lactis NCC 387; 9, B. lactis NCC 402; 10, B. lactis NCC 311; 11, B. lactis NCC 383; 12, B. lactis NCC 239; 13, B. bifidum ATCC 29521; 14, B. breve ATCC 15700; 15, B. longum ATCC 15707; 16, B. infantis ATCC 15679; 17, B. adolescentis ATCC 15703; 18, B. catenulatum ATCC 27539. m, 1-kb DNA ladder (Gibco-BRL).