Gut microbiota and host intestinal transcriptome are significantly altered by formula feeding.

Formula fed infants are at increased risk for bacterial mediated diseases such as necrotizing enterocolitis (NEC). However, the impact of diet on infant gut bacterial colonization and host gene expression remains relatively unexplored. Here, we quantitatively compare the colonic microbiota and transcriptional profiles of formula fed mice (FF) and maternal fed mice (MF) to test the hypothesis that diet impacts gut colonization and host gene expression. 7 day old C3HeB/FeJ pups were randomly assigned to FF or MF. After 72h of differential feeding, colonic tissue was collected for nucleic acid extraction. To evaluate the gut microbiota, 16S rRNA was amplified and sequenced on the Roche GS-FLX pyrosequencing platform. Mothur software was utilized to perform operational taxonomical unit (OTU) clustering, Simpson diversity analysis, and principal coordinate analysis (PCA). To evaluate the host transcriptome, cDNA libraries were constructed and sequenced using the Solexa sequencing platform. Reads were annotated by BLAST search against the mouse RNA database (NCBI build 37) and functionally classified using the KOG database (NCBI). The differentially abundant features were identified via Metastats. After only 72h of differential feeding, 16S rRNA pyrosequencing demonstrated that Firmicutes (p<0.001) was the dominant phylum in the MF pups while Proteobacteria (p<0.001) and Bacteroidetes (p<0.001) were dominant in FF mice. At the genus level, the colonic microbiota of FF mice had an increased relative abundance of Serratia (p<0.001) and Lactococcus (p<0.05) while that of MF mice had an increased relative abundance of Lactobacillus (p<0.001). PCA demonstrated distinct clustering by diet. After quality filtering, Solexa sequencing of 8 cDNA libraries resulted in 29,315,823 reads with average read length of 100bp. Blast search against the mouse RNA database yielded 18,014,657 hits to 19,703 mouse mRNAs. Overall, transcriptional patterns between MF and FF mice were relatively similar. However, a statistically significant (p<0.05) difference in mRNA transcript level was detected for 148 genes. Most notable was a 25 fold increase in heme oxygenase 1 (Hmox1), a marker of oxidative stress, in FF mice suggesting that formula feeding alone may induce oxidative stress in the developing gut. Additionally, a decrease in Vinculin (Vcl) (p<0.05), a cytoskeletal protein associated with adherens junctions, and serine peptidase inhibitor, clade H, member 1 (Serpinh1) (p<0.05), a protein that stabilizes collagen cross-linkages, in FF pups suggests that gut structural integrity may be impaired with formula feeding. Proteins involved with immune regulation, cell cycle control/gene expression, cell motility, and vascular function were also impacted by diet. Formula feeding shifted the intestinal microbiota and host genes involved in structural integrity, the oxidative stress response, and immune function presumably making FF mice more vulnerable to disease development. Further in-depth interrogation of microbial and host gene expression patterns in formula fed infants has the potential to shed new light on the pathogenesis of diseases such as NEC and improve current feeding formulations.