Longitudinal analysis of the premature infant intestinal microbiome prior to necrotizing enterocolitis: a case-control study

Abstract

Necrotizing enterocolitis (NEC) is an inflammatory disease of the newborn bowel, primarily affecting premature infants. Early intestinal colonization has been implicated in the pathogenesis of NEC. The objective of this prospective case-control study was to evaluate differences in the intestinal microbiota between infants who developed NEC and unaffected controls prior to disease onset. We conducted longitudinal analysis of the 16S rRNA genes of 312 samples obtained from 12 NEC cases and 26 age-matched controls with a median frequency of 7 samples per subject and median sampling interval of 3 days. We found that the microbiome undergoes dynamic development during the first two months of life with day of life being the major factor contributing to the colonization process. Depending on when the infant was diagnosed with NEC (i.e. early vs. late onset), the pattern of microbial progression was different for cases and controls. The difference in the microbiota was most overt in early onset NEC cases and controls. In proximity to NEC onset, the abundances of Clostridium sensu stricto from Clostridia class were significantly higher in early onset NEC subjects comparing to controls. In late onset NEC, Escherichia/Shigella among Gammaproteobacteria, showed an increasing pattern prior to disease onset, and was significantly higher in cases than controls six days before NEC onset. Cronobacter from Gammaproteobacteria was also significantly higher in late onset NEC cases than controls 1-3 days prior to NEC onset. Thus, the specific infectious agent associated with NEC may vary by the age of infant at disease onset. We found that intravenously administered antibiotics may have an impact on the microbial diversity present in fecal material. Longitudinal analysis at multiple time points was an important strategy utilized in this study, allowing us to appreciate the dynamics of the premature infant intestinal microbiome while approaching NEC at various points.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Premature gut microbiota variation in the first two month of life.

The gut microbiota variation at class level over 0–60 days of life from all the samples is visualized by NMDS plot. The color gradient from red to blue represents the day of life of the babies. The microbiota from early day of life is distinguished from the elder days.

Fig 2. NMDS of early and late onset NEC and controls at the genus level.

The difference between NEC and controls is displayed for early onset NEC (Fig. 2A) and late onset NEC (Fig. 2B) with their controls by NMDS plot. Each dot represents one sample. Green dots represent the controls and red dots represent the NEC samples. Early NEC subjects and control subjects have a clear separation at second week of the life. The distinction between late onset NEC and controls is less obvious except at the third week of life.

Fig 3. Microbiota progression before early onset NEC and late onset NEC at class and genus level.

The relative abundances of four most dominant classes (Fig. 3A-early onset at class level, 3B-late onset at class level) and the genera (Fig. 3C-early onset at genus level, 3D-late onset at genus level) that are significantly different between NEC and controls are plotted at 7–9 days, 4–6 days and 1–3 days prior to NEC onset. In early onset NEC category, 3,6, 8 NEC samples were included at 7–9 days, 4–6 days and 1–3 days; 8, 5, 6 control samples were included in the above time points. In late onset NEC category, 7, 7,10 NEC samples and 6, 8, 4 control samples were included in the above time points. Red and green of the boxplots indicate NEC and control samples, respectively. The asterisks indicate the significant difference between NEC and control.