Bacterial diversity and Clostridia abundance decrease with increasing severity of necrotizing enterocolitis

Abstract

Background: Necrotizing enterocolitis (NEC) is a devastating neonatal gastrointestinal disease that primarily affects premature infants. It is characterized by bowel inflammation and necrosis. In spite of extensive research, there has been little progress in decreasing the incidence or mortality of NEC over the past three decades. The exact etiology of NEC has not been identified. However, it is believed to result from an inappropriate immune response to gut microbiota. Using 454-pyrosequencing analyses of 16S rRNA genes that were PCR-amplified from stool DNA specimens, we compared the gut microbiota of infants with NEC to matched controls without NEC. The infants with NEC were then categorized into three subgroups based on severity: mild, severe, and lethal. We compared the microbiota among these subgroups and between each severity group and appropriate controls.

Results: Bacterial diversity and the relative abundance of Actinobacteria and Clostridia were significantly lower in NEC specimens compared to controls. The absence of Clostridia was significantly associated with NEC. Microbial diversity and Clostridia abundance and prevalence decreased with increasing severity of NEC.

Conclusions: Low bacterial diversity in stool specimens may be indicative of NEC and the severity of NEC. The low bacterial diversity, and the lack of Clostridia in lethal specimens, could indicate that the presence of a diverse bacterial population in the gut as well as the presence of taxa such as Clostridia may play a role in attenuating inflammation leading to NEC.

Keywords: Clostridia; Microbial diversity; Microbiota; Necrotizing enterocolitis.

Figure 1

Relative abundance of taxonomic differences between NEC stool specimens and controls. Relative abundance of the most abundant taxa at class level (A-C) for NEC specimens (n = 21) compared to controls (n = 74) as well as the abundance at the different severities of NEC (D-F). Clostridia was significantly reduced in NEC patients (C), and as the severity of NEC decreased, the abundance of Clostridia increased. Lethal NEC, n = 6; severe NEC, n = 7; and mild NEC, n = 8. The solid line represents the mean, and the standard deviation is represented by dashed lines when between 0 and 100. (A-C) Significance was determined using a Mann-Whitney U test. For comparison of the NEC levels, Kruskal-Wallis test was used from (D-F). *P value < 0.05, **P value < 0.01. NEC, necrotizing enterocolitis.

Figure 2

Principal coordinate analysis (PCoA) of NEC compared to controls. Unweighted UniFrac uses phylogenetic information to compare specimens that can be visualized with PCoA plots. The gray circles represent the control specimens (n = 74), blue circles represent mild NEC specimens (n = 8), red circles represent severe NEC specimens (n = 7), and green circles represent lethal NEC specimens (n = 6). ANOSIM was used to evaluate the UniFrac distances of all NEC vs controls. Although the P value is statistically significant (P = 0.001), the R value (0.2309) suggests that the communities are essentially not separable. PC, principal coordinate.

Figure 3

Microbial alpha diversity. Chao1 richness estimation (A) and Shannon’s diversity index (B) were used to measure alpha diversity. NEC specimens (n = 21) were significantly lower than controls (n = 74) for both measures of diversity. These measures were also used to compare the different severities of NEC (C, D). Lethal NEC, n = 6; severe NEC, n = 7; and mild NEC, n = 8. Solid lines represent the mean with standard deviation represented by dashed lines. (A, B) were evaluated with a Mann-Whitney U test, and (C, D) were evaluated with a Kruskal-Wallis test. ***P value <0.001, ****P value < 0.0001. NEC, necrotizing enterocolitis.