Zhuoteng Yu

Early microbial and metabolomic signatures predict later onset of necrotizing enterocolitis in preterm infants

BackgroundNecrotizing enterocolitis (NEC) is a devastating intestinal disease that afflicts 10% of extremely preterm infants. The contribution of early intestinal colonization to NEC onset is not understood, and predictive biomarkers to guide prevention are lacking. We analyzed banked stool and urine samples collected prior to disease onset from infants <29 weeks gestational age, including 11 infants who developed NEC and 21 matched controls who survived free of NEC. Stool bacterial communities were profiled by 16S rRNA gene sequencing. Urinary metabolomic profiles were assessed by NMR.ResultsDuring postnatal days 4 to 9, samples from infants who later developed NEC tended towards lower alpha diversity (Chao1 index, P = 0.086) and lacked Propionibacterium (P = 0.009) compared to controls. Furthermore, NEC was preceded by distinct forms of dysbiosis. During days 4 to 9, samples from four NEC cases were dominated by members of the Firmicutes (median relative abundance >99% versus <17% in the remaining NEC and controls, P < 0.001). During postnatal days 10 to 16, samples from the remaining NEC cases were dominated by Proteobacteria, specifically Enterobacteriaceae (median relative abundance >99% versus 38% in the other NEC cases and 84% in controls, P = 0.01). NEC preceded by Firmicutes dysbiosis occurred earlier (onset, days 7 to 21) than NEC preceded by Proteobacteria dysbiosis (onset, days 19 to 39). All NEC cases lacked Propionibacterium and were preceded by either Firmicutes (≥98% relative abundance, days 4 to 9) or Proteobacteria (≥90% relative abundance, days 10 to 16) dysbiosis, while only 25% of controls had this phenotype (predictive value 88%, P = 0.001). Analysis of days 4 to 9 urine samples found no metabolites associated with all NEC cases, but alanine was positively associated with NEC cases that were preceded by Firmicutes dysbiosis (P < 0.001) and histidine was inversely associated with NEC cases preceded by Proteobacteria dysbiosis (P = 0.013). A high urinary alanine:histidine ratio was associated with microbial characteristics (P < 0.001) and provided good prediction of overall NEC (predictive value 78%, P = 0.007).ConclusionsEarly dysbiosis is strongly involved in the pathobiology of NEC. These striking findings require validation in larger studies but indicate that early microbial and metabolomic signatures may provide highly predictive biomarkers of NEC.

Intestinal microbiota of preterm infants differ over time and between hospitals

BackgroundIntestinal microbiota are implicated in risk of necrotizing enterocolitis (NEC) and sepsis, major diseases of preterm infants in neonatal intensive care units (NICUs). Rates of these diseases vary over time and between NICUs, but time and NICU comparisons of the intestinal microbiota of preterm infants are lacking.MethodsWe included 66 singleton infants <29 weeks gestational age with stool samples collected between postnatal days 3 to 21 of life who survived free of NEC and sepsis. Infants were enrolled during 2010 and 2011. Twenty-six infants were enrolled at hospital 1 in Cincinnati, OH, and 40 infants were enrolled at hospital 2 in Birmingham, AL. Samples collected from days 3–9 (“week 1”) and days 10–16 (“week 2”) were compared between years and hospitals. Microbial succession was compared between hospitals in 28 infants with samples from the first 3 weeks of life. DNA extracted from stool was used to sequence the 16S rRNA gene by Illumina MiSeq using universal primers. Resulting operational taxonomic unit tables were analyzed for differences between years and hospitals using linear discriminant analysis effect size algorithm (LEfSe; significance, p < 0.05).ResultsSignificant variation was observed in infant microbiota by year and hospital. Among hospital 1 infants, week 1 samples had more phylum Firmicutes (class Bacilli, families Clostridiaceae and Enterococcaceae) in 2010 and more phylum Proteobacteria (family Enterobacteriaceae) in 2011; week 2 samples did not significantly vary over time. However, among hospital 2 infants, the week 1 shift was nearly opposite, with more Proteobacteria (Enterobacteriaceae) in 2010 and more Firmicutes (Bacilli) in 2011; week 2 samples exhibited the same pattern. Regression analysis of clinical covariates found that antibiotic use had an important influence but did not explain these observed shifts in microbiota over time and between hospitals. Microbial succession also differed by hospital, with greater change in microbiota in hospital 1 than hospital 2 infants (p < 0.01, Jaccard distance).ConclusionColonizing microbiota differ over time and between NICUs in ways that could be relevant to disease. Multi-site, longitudinal studies are needed to reliably define the impact of intestinal microbiota on adverse outcomes of preterm infants.

Center Variation in Intestinal Microbiota Prior to Late-Onset Sepsis in Preterm Infants

Objective Late onset sepsis (LOS) contributes to mortality and morbidity in preterm infants. We tested the hypotheses that microbes causing LOS originate from the gut, and that distortions in the gut microbial community increases subsequent risk of LOS. Study Design We examined the gut microbial community in prospectively collected stool samples from preterm infants with LOS and an equal number of age-matched controls at two sites (Cincinnati, OH and Birmingham, AL), by sequencing the bacterial 16S rDNA. We confirmed our findings in a subset of infants by whole genome shotgun sequencing, and analyzed the data using R and LEfSe. Results Infants with LOS in Cincinnati, as compared to controls, had less abundant Actinobacteria in the first samples after birth (median 18 days before sepsis onset), and less abundant Pseudomonadales in the last samples collected prior to LOS (median 8 days before sepsis onset). Infants with LOS in Birmingham, as compared to controls, had no differences identified in the first sample microbial communities, but Lactobacillales was less abundant in the last samples prior to LOS (median 4 days before sepsis onset). Sequencing identified detectable levels of the sepsis-causative organism in stool samples prior to disease onset for 82% of LOS cases. Conclusions Translocation of gut microbes may account for the majority of LOS cases. Distortions in the fecal microbiota occur prior to LOS, but the form of distortion depends on timing and site. The microbial composition of fecal samples does not predict LOS onset in a generalizable fashion.

Relative fermentation of oligosaccharides from human milk and plants by gut microbes

Gut microbiota is important to human health. Specific dietary glycans promote favorable microbiota growth and inhibit pathobionts. Dietary glycans most relevant to adults and weaned infants are derived from plants or lactose; human milk oligosaccharides (HMOS) are most relevant to breastfed infants. Their efficacy in supporting bacterial growth is compared to determine their potential roles in the initiation and maintenance of colonization. Bioactivities of gluco-manno-oligosaccharides (GMOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), cellobiose (CBS), HMOS, and the most prominent individual HMOS, 2′-fucosyllactose (2′-FL) were contrasted. Two representative gut microbiota mutualists, Bifidobacteria longum ATCC15697 and Lactobacillus acidophilus NRRL B-4495, and two non-mutualists, Campylobacter jejuni S107 and Escherichia coli K12, were used to assess the in vitro prebiotic potential of these oligosaccharides. All oligosaccharides afforded growth of B. longum and L. acidophilus, with HMOS supporting the most robust growth, while none of these oligosaccharides afforded meaningful growth of non-mutualists. B. longum efficiently converted HMOS, GMOS, GOS, and XOS into organic acid fermentation products, and, to a lesser degree, L. acidophilus metabolized HMOS, GMOS, and GOS. Fermentation of these glycans by C. jejuni and E. coli was sparse. B. longum fermentation products inhibited C. jejuni and E. coli. Thus, HMOS most strongly promoted growth of the two mutualists, and both HMOS and GMOS were efficiently fermented by these mutualists into organic acids. This is consistent with a primary role of HMOS in guiding early colonization of the infant microbiota by mutualist symbionts, and of plant oligosaccharides, especially GMOS, in maintaining a favorable microbiota through adulthood.