Abstract
BackgroundMembers of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies 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 of 16S rRNA or tuf genes 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 by in 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. infantis was 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 differentiates B. longum subspecies longum and infantis using 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.
Highlights
Bifidobacteria commonly dominate the fecal microbiota of infants during the exclusively milk-fed period of life (Biavati et al, 1984; Favier et al, 2002; Young et al, 2004; Roger et al, 2010; Grönlund et al, 2011; Tannock et al, 2013; Turroni et al, 2012; Makino et al, 2013; Bäckhed et al, 2015; Milani et al, 2015; Martin et al, 2016)
The primer/probe combinations were tested for reaction efficiency and specificity using genomic DNA purified from bifidobacterial type cultures of species reported to be detected in infant feces: B. adolescentis (DSM 20083T), B. animalis subsp. lactis (DSM 10140T), B. angulatum DSM 20098T, B. bifidum (DSM 20456T), B. breve (ATCC 15700T), B. catenulatum (DSM 20224T), B. dentium (ATCC 27534T), B. longum subsp. infantis (DSM 20088T), B. longum subsp. longum (ATCC 15707T), B. pseudocatenulatum (DSM 20438T), and B. pseudolongum (ATCC 25526T), (Grönlund et al, 2011; Makino et al, 2013; Huda et al, 2014; Bäckhed et al, 2015; Vazquez-Gutierrez et al, 2015; Martin et al, 2016)
Target genes were chosen by submitting gene sequences within the Human Milk Oligosaccharides (HMO) utilization region (Sela et al, 2008) for functional identification of B. longum subspecies infantis and gene sequences within the arabinose utilization region for functional identification of B. longum subsp. longum to BLAST searches against the NCBI nr database
Summary
Bifidobacteria commonly dominate the fecal microbiota of infants during the exclusively milk-fed period of life (Biavati et al, 1984; Favier et al, 2002; Young et al, 2004; Roger et al, 2010; Grönlund et al, 2011; Tannock et al, 2013; Turroni et al, 2012; Makino et al, 2013; Bäckhed et al, 2015; Milani et al, 2015; Martin et al, 2016). Differentiation of B. longum into subspecies has been carried out in relatively few quantitative studies of the infant fecal microbiota, so that subspecies prevalence and relative abundances are not accurately known on a global scale (Grönlund et al, 2011; Huda et al, 2014; Martin et al, 2016) This is probably due to the difficulty of differentiating the B. longum subspecies using 16S rRNA gene sequences (Youn, Seo & Ji, 2008). B. longum subspecies 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 of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy.
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