Strain-specific genotyping of Bifidobacterium animalis subsp. lactis by using single-nucleotide polymorphisms, insertions, and deletions

Abstract

Several probiotic strains of Bifidobacterium animalis subsp. lactis are widely supplemented into food products and dietary supplements due to their documented health benefits and ability to survive within the mammalian gastrointestinal tract and acidified dairy products. The strain specificity of these characteristics demands techniques with high discriminatory power to differentiate among strains. However, to date, molecular approaches, such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR, have been ineffective at achieving strain separation due to the monomorphic nature of this subspecies. Previously, sequencing and comparison of two B. animalis subsp. lactis genomes (DSMZ 10140 and Bl-04) confirmed this high level of sequence similarity, identifying only 47 single-nucleotide polymorphisms (SNPs) and four insertions and/or deletions (INDELs) between them. In this study, we hypothesized that a sequence-based typing method targeting these loci would permit greater discrimination between strains than previously attempted methods. Sequencing 50 of these loci in 24 strains of B. animalis subsp. lactis revealed that a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. In addition, the presence of a nonsynonymous SNP within the gene encoding a putative glucose uptake protein was found to correlate with the ability of certain strains to transport glucose and to grow rapidly in a medium containing glucose as the sole carbon source. The method reported here can be used in clinical, regulatory, and commercial applications requiring identification of B. animalis subsp. lactis at the strain level.

FIG. 1.

Representative PFGE patterns of commercial and reference bifidobacterial strains restricted using XbaI (a) and SpeI (b). Lane 1, commercial strain of B. animalis subsp. lactis; lane 2, ATCC 27536; lane 3, DSMZ 10140; lane 4, lambda molecular weight marker.

FIG. 2.

Representative RAPD-PCR pattern with primer 103 and B. animalis subsp. lactis strains. Lanes 1 and 8, 100-bp molecular weight marker; lanes 2 to 7, strains of B. animalis subsp. lactis.

FIG. 3.

Hierarchical clustering analysis of B. animalis subsp. lactis strains across 50 genetic loci. Each row represents a strain and columns represent genetic loci. Numbers on the right indicate strain cluster and numbers on the bottom indicate genetic locus cluster. Colored squares correspond to the sequence type at each loci. Blue represents DSMZ 10140, red represents Bl-04, and gray represents unique sequence.