16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis

Abstract

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.

Figure 1. Diversity comparison between preterm infants with and without NEC by terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis

(a and b) Representative T-RFLP profiles of intestinal bacteria in infants with and without NEC. N1 (with NEC) and C1 (without NEC) were samples collected from a genetically identical twin pair. CN10 and N10 were samples collected from the same infant before and after the development of NEC. For each profile, the number of peaks (N) and Shannon diversity index (S) are shown. (c) Overall Shannon diversity index between NEC and control groups was calculated from T-RFLP data based on peak number and distribution in T-RFLP profiles from each sample and shows decreased diversity in the NEC patients. (d) Overall diversity by library cloning and sequencing analysis in preterm infants with and without NEC again demonstrates decreased diversity in NEC patients. (e) Comparison of antibiotic exposure time between control and NEC groups. In all panels, * indicates statistical significance at P < 0.05. NEC, necrotizing enterocolitis.

Figure 2. Overview of bacterial composition of all samples by clone library analysis

(a) Relative abundance of Proteobacteria in preterm infants. * Comparison of Proteobacteria between two groups, P < 0.01; ^# Comparison of other phyla (Firmicutes for NEC; Firmicutes, Bacteroidetes and Fusobacteria for control) between the two groups, P < 0.01. (b and c) The genus-level composition of gut microbiota. Genera whose abundance were more than 10% in any sample and its corresponding phyla (P: Proteobacteria; F: Firmicutes; B: Bacteroidetes) are shown as bars (NEC samples top panel, Control samples bottom panel). The dominant genus in each sample is labeled. Only samples from NEC infants had dominance of a single genus of the Proteobacteria phylum (more than 50%). Samples C9 and CN10 are notable for Escherichia dominance, and developed NEC shortly after samples were obtained. NEC, necrotizing enterocolitis.

Figure 3. Phylogenetic relationships among the OTUs detected in fecal samples both from control and NEC infants

The representative 125 clone numbers are listed in the tree. Clones labeled with ECC on blue branches were obtained from control infants. Clones labeled with ECN on red branches were obtained from NEC infants. Sequences with a more than 99% match are given the name of the matched species in GenBank and labeled with the number of identified clones in the libraries. The accession number, the nearest neighbor, the level of similarity and the number of clones in the clone library for each sequence are shown in Supplementary Table 1 and Table 2. Bootstrap values are based on 1000 replications and values above 50% are labeled. OTUs, operational taxonomical units.

Figure 4

Decreased diversity (a) and increased abundance of Proteobacteria (b) are demonstrated between samples collected from infants with and without NEC in samples from 4 sets of twins. (c) Dendrogram demonstrating that even for twin pairs, NEC patients (N1–N4, red) clustered together and distinctly separated from their healthy cohorts (C1–C4, blue) by UniFrac analysis. NEC, necrotizing enterocolitis.

Figure 5. Principal coordinate analysis (PCA) of sequence libraries

Samples collected from infants without NEC (control) are represented by squares (blue); samples from infants with NEC are represented by circles (red). Distribution of samples collected from infants with NEC was distinct from that collected from control infants. Samples C9 and CN10 which were collected from control patients who later developed NEC, notably clustered with the NEC group. NEC, necrotizing enterocolitis.