Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

Abstract

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.

Figures

FIG. 1.

Relative quantification of a mix of 8 different bifidobacterial cultures. “Total” indicates the sum of the different species (98.64% ± 1.67%). Bars represent standard errors.

FIG. 2.

Bifidobacteria as percentage of total bacterial load determined by FISH and real-time PCR (qPCR) in fecal samples of BF infants and infants who received SF or OSF. Bars represent standard errors. @, significant difference (P < 0.05) between the BF and SF groups; *, significant difference (P < 0.05) between the OSF and SF groups; #, significant increase (P < 0.05) during the study period.

FIG. 3.

B. adolescentis and B. catenulatum counts, as determined with a combination of FISH and the duplex 5′ nuclease assays, in feces of BF infants and infants who received OSF or SF. Bars represent standard errors. *, significant difference (P < 0.05) during the study period.