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

Yanjiao Zhou,George Weinstock,Gururaj Shan,Katherine E Gregory,Erica Sodergren,W Allan Walker,David A Lightfoot

doi:10.1371/journal.pone.0118632

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.

Highlights

Premature infants are disproportionally at risk for morbidity and mortality as a result of organs that are immature and ill equipped for extrauterine life
The sample distribution in this dataset as a function of day of life was summarized in S1 Fig. The median day of life of Necrotizing enterocolitis (NEC) onset for the 12 NEC subjects was 25.5 (IQR: 16.8–37.0)
In analyzing the data in this manner, we found that prior to early onset NEC, four classes of bacteria showed interesting patterns in the microbiome 9 days prior to disease (Fig. 3A): (1) The amount of Gammaproteobacteria in controls maintained the same predominance for 9 days before NEC onset, yet it is significantly higher in controls than in NEC samples at 7–9 and 4–6 days prior to NEC onset (p = 0.01 and 0.008, respectively), and not significantly different just before disease onset as a result of Gammaproteobacteria from NEC cases showing an increasing trend while approaching disease

Summary

Introduction

Premature infants are disproportionally at risk for morbidity and mortality as a result of organs that are immature and ill equipped for extrauterine life. These children are especially prone to inflammatory disease as a result of a poorly-regulated immune system and an inappropriate inflammatory response[1,2,3,4,5]. Better understanding the pattern of intestinal colonization and community structure of the microbiome associated with NEC is an important area of study in the etiology of the disease. Some studies have been unable to demonstrate clear differences among cases and controls[10,11], while others have shown that the community structure of the microbiome of the premature infant gut prior to and at the time of disease onset is unique[12,13,14,15,16]

Methods

Results

Conclusion