Genome resolved analysis of a premature infant gut microbial community reveals a Varibaculum cambriense genome and a shift towards fermentation-based metabolism during the third week of life

Abstract

Background: The premature infant gut has low individual but high inter-individual microbial diversity compared with adults. Based on prior 16S rRNA gene surveys, many species from this environment are expected to be similar to those previously detected in the human microbiota. However, the level of genomic novelty and metabolic variation of strains found in the infant gut remains relatively unexplored.

Results: To study the stability and function of early microbial colonizers of the premature infant gut, nine stool samples were taken during the third week of life of a premature male infant delivered via Caesarean section. Metagenomic sequences were assembled and binned into near-complete and partial genomes, enabling strain-level genomic analysis of the microbial community.We reconstructed eleven near-complete and six partial bacterial genomes representative of the key members of the microbial community. Twelve of these genomes share >90% putative ortholog amino acid identity with reference genomes. Manual curation of the assembly of one particularly novel genome resulted in the first essentially complete genome sequence (in three pieces, the order of which could not be determined due to a repeat) for Varibaculum cambriense (strain Dora), a medically relevant species that has been implicated in abscess formation.During the period studied, the microbial community undergoes a compositional shift, in which obligate anaerobes (fermenters) overtake Escherichia coli as the most abundant species. Other species remain stable, probably due to their ability to either respire anaerobically or grow by fermentation, and their capacity to tolerate fluctuating levels of oxygen. Metabolic predictions for V. cambriense suggest that, like other members of the microbial community, this organism is able to process various sugar substrates and make use of multiple different electron acceptors during anaerobic respiration. Genome comparisons within the family Actinomycetaceae reveal important differences related to respiratory metabolism and motility.

Conclusions: Genome-based analysis provided direct insight into strain-specific potential for anaerobic respiration and yielded the first genome for the genus Varibaculum. Importantly, comparison of these de novo assembled genomes with closely related isolate genomes supported the accuracy of the metagenomic methodology. Over a one-week period, the early gut microbial community transitioned to a community with a higher representation of obligate anaerobes, emphasizing both taxonomic and metabolic instability during colonization.

Figure 1

Emergent self-organizing map (ESOM) binning of the metagenome assembly. ESOM showing the clustering and binning of de novo assembled metagenomic data. Each point represents a fragment of an assembled scaffold. Clustering of data points is based on the time series abundance pattern of each assembled scaffold. Dark lines between clusters show definitive separation of genome bins. Colors designate the genome bin for each scaffold fragment.

Figure 2

Metabolic analysis of reconstructed community and isolate genomes. Genomes reconstructed from the microbial community were compared with each other and with the genomes of cultured isolates previously sequenced for members of the family Actinomycetaceae. Each genome was annotated with KEGG and the genes that matched specific metabolic features were counted (Additional file 7). The number of genes identified for each group was normalized across genomes to facilitate coloring and clustering. The number of genes identified for each feature in each genome is presented on the heatmap.

Figure 3

Relative abundance of bacterial species over time. Relative abundances were calculated for bacterial species at nine different time points during the third week of life of a premature male infant. (a) Shows dominant taxa and (b) shows low-abundance species across the time series. During this period, the colonization process is defined by two distinct phases based on the dominance of either facultative (phase 1) or obligate (phase 2) anaerobes.

Figure 4

Rank abundance of bacterial species during phases of colonization. Rank abundance was determined from the relative abundance of each species during each phase of microbial colonization. Taxonomic identification and metabolic analysis was completed based on genome reconstructions from the shotgun-sequenced microbial community. The colonization process is broken into two distinct phases defined by the dominance of either (a) facultative anaerobes during phase one or (b) obligate anaerobes during phase two.