Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Response of Bifidobacteria in Breast- and Formula-Fed Infants

Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut, and further develop to abundant communities, notably in response to diet. Therefore, the transcriptional response of bifidobacteria was studied in rapidly processed fecal samples of young infants that were either fed with breast-milk or a formula, containing a mixture of galacto- and fructoligosaccharides. The presence and diversity of the bifidobacterial fecal communities was determined using PCR-denaturing gradient gel electrophoresis and quantitative real time PCR for specific species. Changes in total number of bifidobacteria as well as in species diversity were observed indicating their metabolic activity within the infant gut. In addition, total RNA isolated from feces of the infants was labeled and hybridized to a bifidobacterial-specific microarray, comprising approximately 6000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones, that showed the most prominent hybridization with the samples, were sequenced. Less than 10% of the hybridizing clones contained ribosomal RNA 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, most (14 %) of the transcribed genes were predicted to be involved in carbohydrate metabolism while also some were implied in exopolysaccharide production or folate production. A total of 3 of these were selected for qPCR and sequence analysis that confirmed the expression of the corresponding gene and expected nucleotide sequence. In conclusion, this study shows the feasibility of obtaining insight in the transcriptional response of intestinal bifidobacteria by analyzing fecal RNA and highlights the in vivo expression of bifidobacterial genes implied in host-related function