Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis

Abstract

Background: Necrotizing enterocolitis (NEC) is a catastrophic disease of preterm infants, and microbial dysbiosis has been implicated in its pathogenesis. Studies evaluating the microbiome in NEC and preterm infants lack power and have reported inconsistent results.

Methods and results: Our objectives were to perform a systematic review and meta-analyses of stool microbiome profiles in preterm infants to discern and describe microbial dysbiosis prior to the onset of NEC and to explore heterogeneity among studies. We searched MEDLINE, PubMed, CINAHL, and conference abstracts from the proceedings of Pediatric Academic Societies and reference lists of relevant identified articles in April 2016. Studies comparing the intestinal microbiome in preterm infants who developed NEC to those of controls, using culture-independent molecular techniques and reported α and β-diversity metrics, and microbial profiles were included. In addition, 16S ribosomal ribonucleic acid (rRNA) sequence data with clinical meta-data were requested from the authors of included studies or searched in public data repositories. We reprocessed the 16S rRNA sequence data through a uniform analysis pipeline, which were then synthesized by meta-analysis. We included 14 studies in this review, and data from eight studies were available for quantitative synthesis (106 NEC cases, 278 controls, 2944 samples). The age of NEC onset was at a mean ± SD of 30.1 ± 2.4 weeks post-conception (n = 61). Fecal microbiome from preterm infants with NEC had increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes prior to NEC onset. Alpha- or beta-diversity indices in preterm infants with NEC were not consistently different from controls, but we found differences in taxonomic profiles related to antibiotic exposure, formula feeding, and mode of delivery. Exploring heterogeneity revealed differences in microbial profiles by study and the target region of the 16S rRNA gene (V1-V3 or V3-V5).

Conclusions: Microbial dysbiosis preceding NEC in preterm infants is characterized by increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes. Microbiome optimization may provide a novel strategy for preventing NEC.

Keywords: 16S rRNA sequencing; Intestinal; Microbiome; NEC; Neonate; Preterm.

Fig. 1

PRISMA flow diagram depicts our search results and selection of included studies in this systematic review

Fig. 2

Histogram of necrotizing enterocolitis (NEC) cases by weeks corrected gestational age (CGA). The distribution of 61 cases of NEC plotted against CGA at the time of diagnosis is normal (Shapiro-Wilk test, p = 0.26). The mean ± SD, CGA at the time of NEC diagnosis was 30.1 ± 2.4 weeks.

Fig. 3

Alpha and beta diversity-NEC vs. controls. Alpha-diversity comparison for all corrected gestational ages (CGA) by NEC case vs. control by three metrics. a Observed species, b Shannon diversity, and c Simpson diversity, none of the comparisons are significantly different. Data is represented in box and whisker plots with median and whiskers representing 10–90th centiles. Principal co-ordinate (PCoA) plots of weighted UniFrac distance (d) and unweighted UniFrac distance (e) including all time points from all studies shows a lack of clustering between cases and controls. The figure in parenthesis next the axis labels represents the proportion of variation explained along each axis. Orange circles represent samples from preterm infants with NEC, and green triangles represent samples from control preterm infants

Fig. 4

Comparison of taxonomic profiles between infants with necrotizing enterocolitis (NEC) and controls. a NEC infants had trends of increased relative abundance in Proteobacteria from 24 to 36 weeks corrected gestational age (CGA) accompanied by decreased abundances in Firmicutes and Bacteroidetes, relative to controls. In control infants, the relative abundance of Proteobacteria decreased after 27 weeks and coincided with an increase in Firmicutes and Bacteroidetes. b–d Phylum level differences between NEC cases and controls across CGA (data in means and SD) showed significant differences in Proteobacteria, Firmicutes, and Bacteroidetes (*p < 0.05). e, f Mean relative abundance distributions between NEC cases and controls at the phylum level (e) and genus level (f) when data from all CGAs are included

Fig. 5

Heterogeneity assessment by 16S rRNA target region. Observed species (operational taxonomic unit, OTU) richness (a) and Shannon diversity index (SDI) values (b) in cases and controls are subgrouped by 16S rRNA target region (V1-V3 vs. V3-V5). Data is represented in box and whisker plots with median and whiskers representing 10–90th centiles. Significant differences were observed in SDI between controls of V1-V3 compared to controls of V3-V5 (*p < 0.05) and controls of V1-V3 compared to NEC V3-V5. No other significant differences were observed. c, d Depict weighted and unweighted UniFrac distances in PCoA plots of NEC and controls subgrouped 16S rRNA target region (V1-V3 vs V3-V5), and notable clustering was observed. The proportion of variation explained along each axis is listed in parenthesis with the axis labels. e Represents differences in proportion of sequences based on 16S rRNA target regions; V3-V5 targeting resulted in a significant increase in proportion of sequences of Proteobacteria and significant decrease in proportion of sequences of Firmicutes compared to studies targeting V1-V3.

Fig. 6

Heterogeneity assessment by study. Observed species (operational taxonomic unit, OTU) richness (a) and Shannon diversity index (SDI) values (b) in cases and controls are subgrouped by study. Data is represented in box and whisker plots with median and whiskers representing 10–90th centiles. c, d Depict weighted and unweighted UniFrac distances in PCoA plots of NEC and controls subgrouped by study, and no notable clustering was observed. The proportion of variation explained along each axis is listed in parenthesis with the axis labels. e Represents differences in proportion of sequences based on study. Studies by Normann [34] and Torraza [56] showed significant increase in proportion of sequences of Firmicutes and significant decrease in proportion of sequences of Proteobacteria compared to other studies (*p < 0.05)