Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR

Abstract

The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.

FIG. 1.

Relative abundances (percentages) of the different bifidobacterial species isolated from infant feces (A) and breast milk samples (B) or detected in the clone library obtained from breast milk samples (C). a, B. adolescentis; b, B. longum; c, B. bifidum; d, B. breve; e, B. pseudocatenulatum; f, B. dentium; g, Bifidobacterium spp.

FIG. 2.

Bifidobacterium species diversity in breast milk samples (lanes 1 to 22) as determined by DGGE. Dominant bands were identified as corresponding to B. adolescentis (a), B. adolescentis (b), B. bifidum (c), B. longum (d), B. adolescentis (e), B. adolescentis (f), Bifidobacterium spp. (97% B. adolescentis) (g), B. longum (h), B. breve (i), B. dentium (j), and B. breve (k). M1 to M4 are markers.