In silico RFLP Analysis of 16S rRNA Genes: A Helpful Application for Distinguishing Bifidobacteria from Human and Animal Source

Abstract

Bifidobacterial species are widespread in gastrointestinal tracts of mammalian and other animals; they can be found in extra body environment only after a fecal contamination or human intentional addition (as the case of probiotics). Interestingly their occurrence is strictly linked to their hosts with a clear demarcation between animal and human species. PCR-restriction fragment length polymorphism (PCR-RFLP) on the 16S rRNA gene, using Alul, and TaqI restriction enzymes, have been utilized to distinguish the animal or human source of 64 strains belonging to 13 Bifidobacterium species (Delcenserie et al. [15]). Our aim was to test this method updating an in silico restriction analysis on the available 16S rRNA gene sequences of all 55 currently described taxa of Bifidobacterium genus. Our results confirmed the reliability of this method, optimized with the use of three restriction enzymes: Alul, TaqI and MaeIII, as a fast and simple strategy to determine the origin (human or animal) of bifidobacteria. Interestingly, the bifidobacterial species recently isolated from non-human primates cluster in the group of animal source except the bifidobacterial species isolated from higher non-human primates closest to humans such as apes (chimpanzee, orangutan and gorilla) that clusters with human group. Moreover, B. minimum, B. subtile and B. mongoliense isolated only from extrabody environment of which the source is unknown clustered with animal species. The in silico RFLP-PCR confirmed its powerful ability to attribute the primary source of occurrence (human or animal) for bifidobacterial species to the human or animal habitat.