Evolution of Gut Microbiome and Metabolome in Suspected Necrotizing Enterocolitis: A Case-Control Study

Abstract

Background: Necrotizing enterocolitis (NEC) is a devastating condition in preterm infants due to multiple factors, including gut microbiota dysbiosis. NEC development is poorly understood, due to the focus on severe NEC (NEC-2/3).

Methods: We studied the gut microbiota, microbiome and metabolome of children with suspected NEC (NEC-1).

Results: NEC-1 gut microbiota had a higher abundance of the Streptococcus (second 10-days of life) and Staphylococcus (third 10-days of life) species. NEC-1 children showed a microbiome evolution in the third 10-days of life being the most divergent, and were associated with a different metabolomic signature than in healthy children. The NEC-1 microbiome had increased glycosaminoglycan degradation and lysosome activity by the first 10-days of life, and was more sensitive to childbirth, low birth weight and gestational age, than healthy microbiome. NEC-1 fecal metabolome was more divergent by the second month of life.

Conclusions: NEC-1 gut microbiota and microbiome modifications appear more distinguishable by the third 10-days of life, compared to healthy children. These data identify a precise window of time (i.e., the third 10-days of life) and provide microbial targets to fight/blunt NEC-1 progression.

Keywords: infant gut; intestinal microbiology; metabolomics; microbiome; necrotizing enterocolitis.

Figure 1

Analysis of gut microbiota, microbiome and metabolome in the first 10-days of life in healthy vs. necrotizing enterocolitis (NEC)-1 children. (A) Gut microbiota analysis via linear discriminant analysis (LDA) score between healthy (H) vs. NEC-1 children, in the first 10-days of life, 1 to 10 days; (B) principal component analysis (PCA) of the gut microbiota; (C) indices of gut microbiota diversity; (D) LDA score for microbial pathways; (E) histogram of the overall fecal metabolites and PCA as inset. **P < 0.01. Two-way ANOVA, followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for multiple comparisons, by controlling the false discovery rate (<0.05); N = 15 for H and N = 4 for NEC-1.

Figure 2

Analysis of gut microbiota, microbiome and metabolome in the second 10-days of life in healthy vs. NEC-1 children. (A) Gut microbiota analysis via LDA score between healthy (H) vs. NEC-1 children, in the second 10-days of life, 11 to 20 days (d) (the LDA score is only shown for NEC-1 children meaning that no bacteria are significantly higher in the H group vs. NEC-1); (B) PCA of the gut microbiota; (C) indices of gut microbiota diversity; (D) null cladogram for microbial pathways; (E) histogram of the overall fecal metabolites and PCA as inset. **P < 0.01. ***P < 0.001. Two-way ANOVA, followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for multiple comparisons by controlling the false discovery rate (<0.05); N = 14 for H and N = 10 for NEC-1.

Figure 3

A specific gut microbiota and microbiome exist in the third 10-days of life in healthy vs. NEC-1 children. (A) Comparative analysis of the gut microbiota by LDA effect size (LEfSe): the cladogram shows bacterial taxa significantly higher in the group of children of the same color, in the fecal microbiota between healthy (H) vs. NEC-1 children, in the third 10-days of life, 21 to 30 days (D) (the cladogram shows the taxonomic levels represented by rings with phyla at the innermost and genera at the outermost ring and each circle is a bacterial member within that level); (B) LDA score used to build the cladogram in (A); (C) PCA of the gut microbiota; (D) indices of gut microbiota diversity; (E) LDA score for microbial pathways. N = 13 for H and N = 7 for NEC-1.

Figure 4

Diseases, host genetic variation and metabolome analysis in the third 10-days of life during NEC-1. (A) Diseases and (B) host genetic variation linked to NEC-1_21–30d associated gut microbiota; (C) histogram of the overall fecal metabolites and PCA, as inset. N = 13 for H and N = 7 for NEC-1.

Figure 5

Analysis of gut microbiota, microbiome and metabolome in the second month of life in healthy vs. NEC-1 children. (A) Gut microbiota analysis via LDA score between healthy (H) vs. NEC-1 children, in the second month of life > 30 days (d); (B) principal component analysis (PCA) of the gut microbiota; (C) indices of gut microbiota diversity; (D) LDA score for predictive microbial pathways (P < 0.01); (E) histogram of the overall fecal metabolites and PCA, as inset. ***P < 0.001. Two-way ANOVA, followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutiel,i to correct for multiple comparisons by controlling the false discovery rate (<0.05); N = 11 for H and N = 6 for NEC-1.