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

Abstract

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.

The human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates

“…Although genes related to formate and acetate production were found to be overrepresented in the metatranscriptome compared with the metagenome, transcripts of other Short Chain Fatty Acids (SCFAs) and lactate fermentation pathways were also frequently detected. Notably, carbohydrate utilization and central metabolism functions were also overrepresented in fecal communities, but appeared to be more dedicated to the degradation of complex carbohydrates rather than simple sugars (Klaassens et al, 2009;Turnbaugh et al, 2010). Moreover, shotgun metaproteomics revealed the enrichment of translation, energy production and carbohydrate metabolism in feces (Verberkmoes et al, 2009).…”

The role of early life nutrition in the establishment of gastrointestinal microbial composition and function

“…Several studies found bifidobacteria to be significantly greater in BF infants 18,23,26,28,33,36,37,39,47,51 ; Fan and colleagues 26 reported roughly double the relative abundance of bifidobacteria in BF than FF infants. Bifidobacteria have been shown to account for 70% of the sequences of exclusively breastfed (EBF) infants 40 and appear earlier in the feces of EBF than in FF infants 38 In addition, infants who were exclusively breastfed in early life maintained greater colonization with bifidobacteria later in infancy.…”

“…From the first wk of life, different colonization patterns are present in BF and FF infants. 26,49 The fecal microbiota of BF infants is more stable over time, 26,39 and characterized by a lower alpha-diversity compared to FF infants. 15,23,31,33 Exclusive breastfeeding is inversely related to alphadiversity in 3 mo-old infants.…”

“…34,50,51 Conversely, Palmer and colleagues 2 reported very low levels of bifidobacteria in all infants with varied dietary patterns; however, these findings are likely due to the 8F universal primer that was used in the study having a three base pair mismatch against B. longum, and that Bifidobacterium genus in general does not have 100% sequence identity to the 8F primer sequence. 58 Infants harboring similar relative abundances of Bifidobacterium still exhibit some variance in diversity, 34,39,42 and stability 51 of populations at the species level. For example, B. longum and B. breve are the dominant species in BF infants, 27,[41][42][43] whereas FF infants also harbor adultassociated bifidobacterial species, such as B. adolescentis.…”

“…For example, B. longum and B. breve are the dominant species in BF infants, 27,[41][42][43] whereas FF infants also harbor adultassociated bifidobacterial species, such as B. adolescentis. 24,39,42 While many studies have focused on bifidobacterial colonization, breastfeeding supports other differences in microbiota composition. Within the phyla Bacteroidetes and Firmicutes, BF infants contain lower levels of Bacteroides, 33,50-53 Atopobium, 51,53 Clostridiales, Lachnospiraceae, and Faecalibacterium than FF.…”