It is now well established that gut microbes contribute to mammalian physiology 
. Pioneering research has clarified the roles of microbes in the gut in normal development and homeostasis 
, and a new generation of investigations seeks to clarify the role of microbes in disease pathogenesis 
. Although a subset of diseases can be linked to the presence of a single causative pathogen, there is a growing recognition of the need to study complete or near complete microbial communities 
. Prior to the advent of next-generation technologies in nucleotide sequencing and mass spectrometry, molecular studies of entire microbial consortia (rather than selected organisms of interest) were not technically feasible. Although bold advances have been made in understanding the metabolic potential of intestinal microbes based upon a massive amount of DNA sequencing of bacterial isolates and in metagenomic studies 
, few functional studies have been performed characterizing community wide gene and protein expression in the gut.
Here we have presented a metatranscriptomic evaluation of intestinal bacteria in 8 neonatal piglets. In this study, we used an RNA pyrosequencing platform to profile gut microbial gene expression in MF and FF piglets without prior knowledge of which organisms were present. To our knowledge, this study represents the largest number of independent samples (8 subjects) used to date for analysis of community wide gene expression in the gut. Several methodologic considerations of this study warrant mention. First, we performed a single step for mRNA enrichment prior to construction of cDNA libraries, and cDNA sequencing results indicated that the degree of enrichment was modest (19.7% of all RNA sequences were non-ribosomal). In the future, this step in sample processing may not be necessary given the volume and length of sequencing reads available with current sequencing platforms. Second, amplification of cDNA sequences was not necessary in this study, although it has been required in prior metatranscriptomic studies due to low yields of RNA 
. In the piglet, cecal biomass is plentiful and samples can be immediately cryopreserved after collection without exposure to ambient oxygen. As these techniques are inevitably extended to studies of human fecal samples, the need for signal amplification will need to be re-evaluated depending upon RNA yield. Third, we studied the structure of the microbial communities both by sequencing 16S rDNA amplicons and by characterizing unamplified 16S rRNA sequences within the cDNA libraries. In agreement with results from Urich et al. 
, our analyses indicate that taxonomic assignments based upon DNA 16S amplicons correspond well with assignments derived from ribosomal sequences within cDNA libraries. Fourth, gene transcripts were studied without the presence of corresponding metagenomic data sets. The importance of studying paired mRNA and DNA data sets has not yet been clarified and past work in the field has, in fact, demonstrated low levels of homology between metatranscriptomes and metagenomic scaffolds. However, inclusion of whole genome libraries would have likely partially mitigated the large number of unannotated transcripts that we observed in the current study.
A subset of specific transcripts was relatively abundant in all samples studied, indicating that we have begun to define a core neonatal gut microbial metatranscriptome. As expected, a preponderance of mRNA transcripts corresponded to genes related to metabolism of carbohydrates and proteins. These results align well with recent papers that have defined the nature of carbohydrate utilization genes within a core metagenome (9,31,32). Additionally, commonly observed gut microbial transcripts in our study encode for proteins that enable binding to host epithelium, regulate processing of extracellular polysaccharides, and mediate microbial stress response. The validity of our results is supported by a recent proteomic-based study that characterized circulating antibodies against gut bacteria in human subjects 
. The most commonly observed antibodies were active against GroEL, enolase, and elongation factor Tu, which were each observed frequently in our gene expression study. The significance of how and why antibodies are formed against gut bacterial antigens requires further study.
A primary goal of this study was to identify differences in the gut microbial communities of MF and FF neonatal piglets. MF samples were enriched with 16S sequences from the taxa Prevotella, Oscillibacter, and Clostidium, whereas the FF samples were enriched with sequences from Bacteroides, Parabacteroides, and Alistipes. Because the animals in the MF and FF piglets did not originate in the same litter of piglets, it is possible that observed differences between the two groups reflected a litter effect. However, studies from our laboratories have shown that litter effects on the gut flora of piglets are reproducibly overshadowed by diet effects (Wang et al., submitted).
Profiles of gene expression were similar. The abundance of sequences from more than 90% of SEED subsystems and COG clusters did not differ between MF and FF datasets was not statistically different. These results suggest that sow's milk and artificial formula, although chemically distinct, induce relatively subtle changes in the gut microbial gene expression. However, several important differences were noted in the transcriptomes of the MF and FF animals. Marked differences were noted in the expression of genes involved in amino acid metabolism. Interestingly, we observed a clear abundance of enzymes linked to arginine metabolism in the MF group of animals. This finding may have clinical relevance because several reports have indicated that an abnormally low serum concentration of arginine, a precursor for nitric oxide production, confers an increased risk for NEC 
, and it is widely accepted that the incidence of NEC is lower in low birthweight infants receiving breast milk 
. Additionally, the MF data sets were significantly enriched in oxidative stress response genes, which is interesting in light of the oft-stated belief that breast milk provides antioxidant protection for newborns. By contrast, the FF data sets were significantly enriched in sequences encoding the Ton and Tol transport proteins, which have been associated with iron-mediated microbial virulence in animal models of infection 
Recent advances in completing microbial genomes and completing metagenomic surveys have demonstrated the vast metabolic potential of the bacteria and archaea present within the mammalian intestine. As DNA-based studies continue, parallel studies of gut microbial function will be essential. Functional studies of gene expression, protein expression, and metabolite production will make it possible to define what is “normal” in the field of enteric microbiology. Eventually, continued progress in this area will allow us to better understand the contributions of microbes to diseases such as NEC, Crohn's disease, and obesity.