Differentiation ofBifidobacterium longumsubspecieslongumandinfantisby quantitative PCR using functional gene targets

Abstract

BackgroundMembers of the genusBifidobacteriumare abundant in the feces of babies during the exclusively-milk-diet period of life.Bifidobacterium longumis reported to be a common member of the infant fecal microbiota. However,B. longumis composed of three subspecies, two of which are represented in the bowel microbiota (B. longumsubsp.longum;B. longumsubsp.infantis).B. longumsubspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of16S rRNAortufgenes that are commonly used in bacterial taxonomy.MethodsWe developed a qPCR method targeting the sialidase gene (subsp.infantis) and sugar kinase gene (subsp.longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested byin silico,pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions.ResultsA pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp.infantiswas detectable (0–32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01–4.6%) compared to subsp.longum(0.1–33.7% abundance; 21.4–100% prevalence).DiscussionOur qPCR method differentiatesB. longumsubspecieslongumandinfantisusing characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.