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.

Necrotizing Enterocolitis, Gut Microbiota, and Brain Development: Role of the Brain-Gut Axis

“…However, some studies demonstrated that the specific microbiota profile was dependent on the age of disease onset. Infants developing early NEC (within 10 days of life) demonstrated a dominance of Firmicutes, largely attributable to increased abundances of bacilli class members [33-35]. Different bacteria of the Clostridia class, also within the phylum of Firmicutes, were also associated with early-onset NEC.…”

“…Concurrent with this increase in Firmicutes, a decrease in Gammaproteobacteria was observed in these early-onset cases. Late-onset NEC (after 10 days of life) was associated with an increase in Gammaproteobacteria with an accompanying decrease in Firmicutes (especially Negativicutes) [35, 36]. …”

The Microbiome and Biomarkers for Necrotizing Enterocolitis: Are We Any Closer to Prediction?

“…Despite this limited diversity, longitudinal studies in the preterm population have demonstrated a dynamic but choreographed pattern of early intestinal colonization. Initial colonization begins with Gram-positive cocci (within the Bacilli class), soon overtaken by Gram-negative facultative anaerobic organisms (within the Gammaproteobacteria class), counterbalanced by a gradually increasing abundance of anaerobes (within the Clostridia and Negativicutes class) (5–10). These limited taxa account for >90% of the taxa present (6).…”

“…Longitudinal studies that utilized 16S or WGS sequencing on stool demonstrate a relative increase in Gram-negative bacteria (class Gammaproteobacteria) before the onset of NEC (4, 8–10, 12, 15, 16) (Table III; available at www.jpeds.com) and an associated decrease in anaerobes (classes Clostridia and Negativicutes) (10, 12, 14, 16). These findings were confirmed by a recent meta-analysis that showed that prior to the onset of clinical NEC there was a predominance of the Gram-negative phylum Proteobacteria (including the class Gammaproteobacteria) that was offset by a decrease in the relative abundance of the anaerobe containing phyla Firmicutes (including the class Clostridia and Negativicutes) and Bacteroidetes (17).…”

Necrotizing enterocolitis and preterm infant gut bacteria

“…Even though only one of the papers in Table 2 exclusively focused on defining the pattern of progression in children without NEC [20], data supplied in the others [16,18,21] were sufficient to confirm the findings of La Rosa et al [20]. In that comprehensive study of preterm infants, 16S rRNA gene sequencing demonstrated a remarkably choreographed pattern: namely, the early-in-life gut bacterial content is predominated by Bacilli (despite their name, Bacilli are Gram-positive cocci such as staphylococci, streptococci, and enterococci).…”