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Applied and Environmental Microbiology, February 2009, p. 965-969, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02063-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR {triangledown}

Rocío Martín,1 Esther Jiménez,2 Hans Heilig,1 Leonides Fernández,2 María L. Marín,2 Erwin G. Zoetendal,1 and Juan M. Rodríguez2*

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands,1 Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, 28040 Madrid, Spain2

Received 5 September 2008/ Accepted 10 December 2008

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.

* Corresponding author. Mailing address: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Ciudad Universitaria, Avda. Puerta de Hierro, s/n, 28040 Madrid, Spain. Phone: 34-91-3943837. Fax: 34-91-3943743. E-mail: jmrodrig{at}vet.ucm.es

{triangledown} Published ahead of print on 16 December 2008.

Applied and Environmental Microbiology, February 2009, p. 965-969, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02063-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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