Species delineation and clonal diversity in four Bifidobacterium species as revealed by multilocus sequencing

The genus Bifidobacterium comprises several species that are important contributors to the gut microbiome, with some strains having beneficial health effects. Understanding the evolutionary emergence of advantageous biological properties requires knowledge of the genetic diversity and clonal structure of species. We sequenced seven housekeeping genes in 119 Bifidobacterium strains of Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum. Phylogenetic analysis of concatenated sequences delineated sequence clusters that correspond to previously named taxa, and suggested that B. longum subsp. infantis is a nascent lineage emerging from within B. longum subsp. longum. Clear traces of recombination among distant bifidobacterial species indicate leaky species borders and warn against the practice of single gene-based identification. Multilocus sequence typing achieved precise strain genotyping, with discrimination indices above 99% in B. bifidum, B. breve and B. longum, providing a powerful tool for strain traceability, colonization dynamics and ecological studies. Frequent homologous recombination accelerates clonal diversification and may facilitate the transfer of biological properties among bifidobacterial strains.