Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis

Abstract

Necrotizing enterocolitis (NEC) is the most devastating intestinal disease affecting preterm infants. In addition to being associated with short term mortality and morbidity, survivors are left with significant long term sequelae. The cost of caring for these infants is high. Epidemiologic evidence suggests that use of antibiotics and type of feeding may cause an intestinal dysbiosis important in the pathogenesis of NEC, but the contribution of specific infectious agents is poorly understood. Fecal samples from preterm infants ≤ 32 weeks gestation were analyzed using 16S rRNA based methods at 2, 1, and 0 weeks, prior to diagnosis of NEC in 18 NEC cases and 35 controls. Environmental factors such as antibiotic usage, feeding type (human milk versus formula) and location of neonatal intensive care unit (NICU) were also evaluated. Microbiota composition differed between the three neonatal units where we observed differences in antibiotic usage. In NEC cases we observed a higher proportion of Proteobacteria (61%) two weeks and of Actinobacteria (3%) 1 week before diagnosis of NEC compared to controls (19% and 0.4%, respectively) and lower numbers of Bifidobacteria counts and Bacteroidetes proportions in the weeks before NEC diagnosis. In the first fecal samples obtained during week one of life we detected a novel signature sequence, distinct from but matching closest to Klebsiella pneumoniae, that was strongly associated with NEC development later in life. Infants who develop NEC exhibit a different pattern of microbial colonization compared to controls. Antibiotic usage correlated with these differences and combined with type of feeding likely plays a critical role in the development of NEC.

Conflict of interest statement

Competing Interests: The authors would like to acknowledge that one of the coauthors, Dr. Josef Neu is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Dominant OTU's.

Each row represents a separate OTU with the closest match in the database listed on the right. Each column represents an individual sample from both cases and controls at each of the three time points. OTUs are listed in order of dominance with the most dominant on the top. Darker color indicates higher number of sequences in this OTU in an individual sample (see color code in left corner). Darkest shade indicates 50+ sequence reads obtain for that OTU in that sample.

Figure 2. Chao rarefaction diversity.

Chao diversity was calculated from the OTU distribution A) 2 weeks before diagnosis of NEC; B) 1 week before diagnosis of NEC; and C) Week of diagnosis of NEC. As a measure of beta (within sample) diversity it is an estimate of the expected total number of OTUs detected in the sample if sequenced to completion.

Figure 3. Unifrac diversity measures.

Principal component analysis (PCA) of overall diversity based on UniFrac (unweighted) metric A) 2 weeks before; B) 1 week before; and C) week of diagnosis of NEC. Squares represent controls and triangles represent cases. P1 is component 1 and P2 component 2.

Figure 4. Changes in proportions of bacterial phyla.

Proportions of the major bacterial phyla at A) two weeks before, B) one week before, and C) during week of NEC diagnosis for individual samples from controls (c_###) and NEC cases (n_###) and means for samples combined by NEC status (control, NEC).

Figure 5. Differences in the proportions of prevalent bacterial phyla, based on 454 16S rRNA sequencing, in controls at week 0 in A) Jacksonville and B) Gainesville.

Figure 6. Heat map of selected OTU (98% similarity) correlating with NEC status during the week of diagnosis.

Controls are shown on the left (c_###) and NEC cases on the right (n_###). The number of sequences detected per sample for each OTU is indicated by degree of shading, with the darkest shade correlating with the highest number of sequences. OTUs more frequently observed in NEC cases are shown on the top and OTUs less frequently observed are shown on the bottom.

Figure 7. qPCR for fecal counts of Bifidobacteria.

* p-value 2 weeks before NEC