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Applied and Environmental Microbiology, May 2009, p. 2668-2676, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.02492-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Responses of Bifidobacteria in Breast- and Formula-Fed Infants
,
Eline S. Klaassens,1,2*
Rolf J. Boesten,1,3
Monique Haarman,4
Jan Knol,4
Frank H. Schuren,3
Elaine E. Vaughan,1,
and
Willem M. de Vos1,5
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands,1 CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Queensland 4067, Australia,2 TNO Nutrition and Food Research, Department of Applied Microbiology and Gene Technology, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands,3 Danone Research, Centre for Specialised Nutrition, P.O. Box 7005, 6700 CA Wageningen, The Netherlands,4 Department of Basic Veterinary Sciences, Helsinki University, FI-00014 Helsinki, Finland5
Received 30 October 2008/ Accepted 6 March 2009
Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.
* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands. Phone: 31-6-28652910. Fax: 31-317483829. E-mail: eline.klaassens{at}live.com
Published ahead of print on 13 March 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Present address: Unilever Food & Health Research Institute (UFHRI), Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.
Applied and Environmental Microbiology, May 2009, p. 2668-2676, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.02492-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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