Gut microbial gene expression in mother-fed and formula-fed piglets

Abstract

Background: Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants.

Methodology/principal findings: Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sow's milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals.

Conclusions/significance: Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans.

Figure 1. Microbial community structure in cecal contents from mother-fed and formula-fed piglets at 21 days of life.

(A) Mean relative abundances of bacterial taxa within cecal microbiota from 4 MF animals (black bars) and 4 FF animals (white bars) identified by analysis of 16S rDNA amplicon sequences. Sequences were classified to the highest taxonomic level to which they could be confidently assigned using the RDP classification algorithm and taxonomic hierarchy. (B) Relative mean abundances of bacterial taxa within cecal microbiota identified by RDP analysis of unamplified 16S sequences within cDNA libraries. The results demonstrate that all communities are dominated by the phyla Bacteroidetes and Firmicutes; MF data sets are enriched with Prevotella sequences and FF data sets are enriched with Bacteroides sequences. In both panels, differentially abundant phyla (p value<0.05) and genera (p value<0.01) are marked with a * if they were enriched within the MF samples and with a ** if they were enriched within FF samples. Taxa with a mean relative abundance <0.01 in both MF and FF groups are not shown.

Figure 2. A core microbial metatranscriptome in the piglet cecum.

(A) Mean relative abundances of annotated sequences within cDNA libraries from all 8 animals studied. Displayed are the automated SEED Level 1 Subsystem assignments, as determined by MG-RAST . Low standard deviations indicate that variation in the gut metatranscriptome between subjects is low. (B) Projection of the global metabolic profiles onto the KEGG pathways using the iPath tool demonstrates the overall similarity of the MF and FF gut microbial communities. Metabolic pathways common to both diets are shown in blue. Pathways unique to the MF animals are represented in green, and pathways unique to the FF animals are represented in red.

Figure 3. Universal expression of carbohydrate utilization and microbial virulence genes in the piglet cecum.

(A) Relative abundance of cDNA sequences assigned by MG-RAST to the Level 3 SEED Subsystem of carbohydrate utilization. (B) Relative abundance of cDNA sequences assigned by MG-RAST to the Level 3 SEED Subsystem of virulence. Values for mean relative abundances in both (A) and (B) reflect average values across all 8 animals studied.

Figure 4. Relationship between gut microbial community structure and function.

(A) Mean relative abundances of bacterial taxa within cecal microbiota from 4 MF animals (black bars) and 4 FF animals (white bars) identified by analysis of non-ribosomal sequences within cDNA libraries. Taxonomic assignments for sequenced transcripts were made by identifying best BLASTN hits against an in-house database of microbial genomes. Differentially abundant phyla (p value<0.05) and genera (p value<0.01) are marked with a * if they were enriched within the MF samples and with a ** if they were enriched within FF samples. (B) Taxonomic origin of highly expressed genes. Expression levels for the two most abundant general COG hits and two most abundant individual COG hits are represented for each of the four most abundant bacterial phyla.