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.

Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota

“…Although earlier studies had concluded that many milk microbes could have origins in maternal skin, the inability of these authors to detect these milk isolates in the mothers' skin prompted them to conclude: "Lactic acid bacteria present in milk may have an endogenous origin and may not be the result of contamination from the surrounding breast skin". This group also cultured bifidobacteria from 8 milk samples and 21 fecal samples of the 23 mother/infant pairs, but not from any breast skin (25 The identification of bifidobacteria in human milk (27) is consistent with the longstanding observation of a much higher content of bifidobacteria in breastfed feces than in those fed artificial formula. The same Spanish laboratory followed up their observations by demonstrating the use of strains isolated from breast milk as potential probiotic bacteria including treatment of mastitis and inhibition of mother-to-infant transfer of HIV (28).…”

Molecular identification of bifidobacteria from infant faeces

“…fed infant faeces in the present study corroborates with Roberts et al (1985), Tamime et al (1995), Silvi et al (1996) and Martin et al (2009) who reported that human milk is favourable for the growth and sustenance of Bifidobacteria in the large intestine of infants. Wasilewska and Bielecka (2003) also isolated and identified fourteen bifidobacterial strains from faeces harbouring the gut of 3-month old breast fed infant.…”

Comparative Analysis of Fecal Microbiota in 5 - 8-Year-old Children with and without Cow Milk Protein Allergy

“…The total DNA was extracted with a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions, and total DNA was amplified with v3 of universal primers, as well as Bifidobacterium, Lactobacillus, and C. coccoides primers (19)(20)(21)(22). The primers and PCR amplifirotocols were performed according to previously reported procedures.…”

“…The stock solutions were a 100 denaturing solution containing 7.0 M of urea and 40 (vol/vol) of formamide, as well as a polyacrylamide solution containing no denaturing components; the desired denaturing gradient was obtained according to percentages. The electrophoresis was performed at 20 v for 10 minutes, 70 v for 18 hours for the universal bacterial primer, 85 v for 16 hours for Bifidobacterium, 70 v for 16 hours for Lactobacillus, and 85 v for 16 hours for C. coccoides at a constant temperature of 60°C (19)(20)(21)(22). After electrophoresis, the DGGE gels were stained for 30 minutes in a GeneFinder (0.5 µg/mL) and photographed under UV transillumination.…”