Unraveling the Microbiome of Necrotizing Enterocolitis: Insights in Novel Microbial and Metabolomic Biomarkers

Abstract

Necrotizing enterocolitis (NEC) is among the most relevant gastrointestinal diseases affecting mostly prematurely born infants with low birth weight. While intestinal dysbiosis has been proposed as one of the possible factors involved in NEC pathogenesis, the role of the gut microbiota remains poorly understood. In this study, the gut microbiota of preterm infants was explored to highlight differences in the composition between infants affected by NEC and infants prior to NEC development. A large-scale gut microbiome analysis was performed, including 47 shotgun sequencing data sets generated in the framework of this study, along with 124 retrieved from publicly available repositories. Meta-analysis led to the identification of preterm community state types (PT-CSTs), which recur in healthy controls and NEC infants. Such analyses revealed an overgrowth of a range of opportunistic microbial species accompanying the loss of gut microbial biodiversity in NEC subjects. Moreover, longitudinal insights into preterm infants prior to NEC development indicated Clostridium neonatale and Clostridium perfringens species as potential biomarkers for predictive early diagnosis of this disease. Furthermore, functional investigation of the enzymatic reaction profiles associated with pre-NEC condition suggested DL-lactate as a putative metabolic biomarker for early detection of NEC onset. IMPORTANCE Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease occurring predominantly in premature infants whose etiology is still not fully understood. In this study, the analysis of infant fecal samples through shotgun metagenomics approaches revealed a marked reduction of the intestinal (bio)diversity and an overgrowth of (opportunistic) pathogens associated with the NEC development. In particular, dissection of the infant's gut microbiome before NEC diagnosis highlighted the potential involvement of Clostridium genus members in the progression of NEC. Remarkably, our analyses highlighted a gastrointestinal DL-lactate accumulation among NEC patients that might represent a novel potential functional biomarker for the early diagnosis of NEC.

Keywords: NEC; metagenomics; microbiota; necrotizing enterocolitis; shotgun.

FIG 1

Identification of the five PT-CSTs. Panel a shows a cladogram of the 171 preterm infant fecal samples, obtained through hierarchical clustering (HCL) analysis. The cladogram highlights the five PT-CSTs identified through HCL analysis. Below is reported an overview of the taxonomic composition of the infant population. Panel b displays the average relative abundance of microbial species of the identified PT-CST, with relative abundances on the vertical axis and the sample on the horizontal axis.

FIG 2

Statistically significant differences in the taxonomic composition between NEC and healthy samples of each PT-CST. In detail, for each PT-CST, species with significant P values obtained by comparison between microbial average abundances of healthy and NEC samples have been highlighted in green. Additional taxa above 1% of average abundance have been reported.

FIG 3

Covariance of the most abundant microbial species of the preterm infant gut. The force-driven network was constructed using bacterial taxa as nodes and covariances as edges. Red edges correspond to negative correlations, while green edges represent positive associations. The node size is proportional to the degree of interactions, while node colors represent the 13 obtained clusters of covariating species.

FIG 4

Statistically significant enzymes differentially encoded by gut microbiota of healthy, NEC, and pre-NEC infants. Bar plot depicts the relative average abundance of each considered enzyme in healthy, NEC, and pre-NEC samples on the horizontal axis. The table on the left reports the corresponding EC numbers and enzyme names.

FIG 5

Species-level taxonomic composition of pre-NEC infants. Pre-NEC samples were grouped according to the specific PN-CST to which they belong. Relative abundances are reported on the vertical axis and the samples on the horizontal axis.