Bacterial community structure and functional contributions to emergence of health or necrotizing enterocolitis in preterm infants

Erika C Claud, Kevin P Keegan, Jennifer M Brulc, Lei Lu, Daniela Bartels, Elizabeth Glass, Eugene B Chang, Folker Meyer, Dionysios A Antonopoulos, Erika C Claud, Kevin P Keegan, Jennifer M Brulc, Lei Lu, Daniela Bartels, Elizabeth Glass, Eugene B Chang, Folker Meyer, Dionysios A Antonopoulos

Abstract

Background: Preterm infants represent a unique patient population that is born functionally immature and must accomplish development under the influence of a hospital environment. Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. The purpose of this study was to evaluate the progression of intestinal microbiota community development between preterm infants who remained healthy compared to preterm infants who developed NEC.

Results: Weekly fecal samples from ten preterm infants, five with NEC and five matched healthy controls were obtained. Bacterial DNA from individual fecal samples was subjected to sequencing of 16S rRNA-based inventories using the 454 GS-FLX platform. Fecal samples from control infants demonstrated a temporal pattern in their microbiota, which converged toward that of a healthy full term breast-fed infant. Microbiota development in NEC patients diverged from controls beginning three weeks prior to diagnosis. Shotgun metagenomic sequencing was performed to identify functional differences in the respective microbiota of fecal samples from a set of twins in which one twin developed NEC and one did not. The majority of the differentially abundant genes in the NEC patient were associated with carbohydrate metabolism and mapped to members of the family Enterobacteriaceae. This may indicate an adaptation of the community to an altered profile of substrate availability for specific members as a first step towards the development of NEC. We propose that the microbial communities as a whole may metabolize milk differently, resulting in differential substrate availability for specific microbial groups. Additional differentially represented gene sets of interest were related to antibiotic resistance and vitamin biosynthesis.

Conclusions: Our results suggest that there is a temporal component to microbiome development in healthy preterm infants. Thus, bacteriotherapy for the treatment or prevention of NEC must consider this temporal component of the microbial community in addition to its taxonomic composition and functional content.

Figures

Figure 1
Figure 1
Timeline of sample collection from NEC and control patients. Included subjects were born between 24–32 weeks gestation and stool samples were collected weekly from birth to 10 weeks of life. Panel A - 16S rRNA-based microbial community analyses were performed on samples from throughout this period and indicated a shift in microbial communities at approximately 2 weeks after birth. Panel B - Shotgun DNA sequencing of metagenomes from samples on the day of NEC diagnosis and one week before showed no significant difference. Panel C - Subsequent shotgun sequencing of metagenomes focused on the interim period.
Figure 2
Figure 2
16S rRNA-based analysis of samples demonstrates a temporal component to healthy development in preterm infants. Samples from a full term breast-fed infant (solid black squares) cluster distinctly from early stages of preterm infant development (up through five weeks of life) along the first principal axis. Additional clusters are detected along the second principal axis (within two weeks of life (orange triangles), between three to five weeks (green circles) along the second axis) denoting a temporal progression (arrows) of the microbial communities towards a stable healthy state represented by the full term breast-fed infant.
Figure 3
Figure 3
16S rRNA-based analysis demonstrates deviation from a healthy state by NEC patients prior to diagnosis. (A) PCoA of control patients after two weeks of life (blue), and NEC patients between three weeks prior to NEC diagnosis up to date of diagnosis demonstrate a separation of the NEC patients. (B) PCoA of control patients after two weeks of life (blue), and NEC patients (red) between three weeks prior to NEC diagnosis and onwards past diagnosis demonstrate increasing clustering and deviation of the NEC patients.
Figure 4
Figure 4
Analysis of microbial communities by shotgun metagenomics between two weeks of life and NEC diagnosis demonstrate functional distinction. Shotgun metagenomes generated from twin patients at times prior to NEC diagnosis (only one of the twins went on to be diagnosed with NEC; labeled ‘pre-NEC’). An expansion of the Proteobacteria is noted in the patient that went on to develop NEC.
Figure 5
Figure 5
Differences in gene abundances between pre-NEC sample versus other samples. Heat map (A) and pie charts (B) displaying significant differences in gene abundances between later pre-NEC time point of NEC diagnosed patient versus other samples.

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