Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR.
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.
Martín
Rocío
R
Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
Jiménez
Esther
E
Heilig
Hans
H
Fernández
Leonides
L
Marín
María L
ML
Zoetendal
Erwin G
EG
Rodríguez
Juan M
JM
D000446:Aldehyde-Lyases / Q000378:metabolism; D039941:Bifidobacteriales Infections / Q000382:microbiology / Q000635:transmission*; D001644:Bifidobacterium / Q000302:isolation & purification*; D002374:Catalase / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005243:Feces / Q000382:microbiology*; D005260:Female; D020459:Genes, rRNA; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D018445:Infectious Disease Transmission, Vertical; D008895:Milk, Human / Q000382:microbiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D002374:Catalase; D000446:Aldehyde-Lyases; C065556:fructose-6-phosphate phosphoketolase