Dysbiosis anticipating necrotizing enterocolitis in very premature infants

Abstract

Background: Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants.

Methods: Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization-time of flight. Clostridial isolates were typed and toxin genes detected.

Results: A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk.

Conclusions: Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC. Clinical Trials Registration. NCT01102738.

Keywords: NEC; fecal microbiota; necrotizing enterocolitis; premature infant.

Figure 1.

Cumulative cases of necrotizing enterocolitis (NEC) over 24 months. Hatching indicates hospital site and Bell staging. Bar length indicates duration of infant admission on the neonatal unit and circles indicate date of NEC diagnosis. Asterisked cases were excluded from the analyses due to lack of samples (n = 2), sequencing data (n = 4), or controls (n = 2).

Figure 2.

Bacterial community structure in control infants by postnatal week and mode of delivery. Data were generated using 1 sample per week from control infants using mean number of reads. When the same descriptive label (genus, family) is attached to >1 operational taxonomic unit (OTU; defined by 97% sequence similarity), these are numbered sequentially; no OTUs are combined.

Figure 3.

Bacterial communities in necrotizing enterocolitis (NEC) infant samples collected closest to the day of diagnosis of NEC (D₀), and their matched controls. Samples are categorized along the x-axis. Color intensity indicates the number of rarefied reads from each operational taxonomic unit (OTU) that is found in a sample, as shown by the colored bar. Mean bacterial community diversity was not found to differ significantly between NEC and control infant samples.

Figure 4.

Relationship between Klebsiella operational taxonomic unit (OTU) reads and days of continuous positive airway pressure (CPAP) oxygen requirement by quartiles. Black circles indicate “Clostridium-associated” necrotizing enterocolitis (NEC); black X's indicate “Klebsiella OTU-associated” NEC. Control (white circles) and “Clostridium-associated” NEC samples are evenly spread over the quartiles. In contrast, “Klebsiella OTU-associated” NEC samples cluster above the third quartile.

Figure 5.

Evolution of the fecal microbiota in infants with necrotizing enterocolitis (NEC) Bell stage 2/3. The day of NEC diagnosis is shown at the bottom right of each bar chart. Cases defined as “Clostridium-associated” NEC are N7, N15, N21, and N26. Cases defined as “Klebsiella OTU-associated” NEC are N2, N6, N11, N14, N18, N23, and N24. N19 does not fit into either group. Abbreviation: OTU, operational taxonomic unit.