Defining the gastrointestinal microbiota in premature neonates: Its development and relation to necrotizing enterocolitis

Abstract

Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel disease affecting premature infants. The disease can progress rapidly and is associated with high mortality. Early diagnosis can be difficult as the early manifestations of NEC can be very similar to sepsis. There is currently no pre-symptomatic screening method available. The aim of the work reported in this thesis was to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. Methods: Longitudinal (≥ daily) faecal samples and daily clinical data from throughout their neonatal intensive care unit stay were collected from 369 premature infants born at <32 weeks gestation over a two-year period. The faecal microbiota of 12 infants with definite/severe NEC, 8 with suspected NEC, and 44 control infants was characterised using next-generation sequencing of 16S ribosomal RNA gene regions. Clinical characteristics and operational taxonomical units (OTUs) that discriminated cases from controls were identified using logistic regression. Bacteria isolated from stored faecal samples underwent selective culture, and the resulting isolates were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight. Isolates identified as Clostridium perfringens were additionally typed and screened for the presence of toxin genes. Results: Faecal samples collected from four NEC infants just prior to diagnosis were found to contain a higher abundance of a clostridial OTU compared to matched control samples. Culture investigation identified this OTU as Clostridium perfringens. No two Clostridium perfringens isolates were found to be identical, as determined by fluorescent amplified fragment length polymorphism. Infants with NEC who did not have an over-abundance of the clostridial OTU (n=7) prior to diagnosis had a faecal microbiota dominated by a Klebsiella OTU. Only one infant did not have an abundance of either the clostridial OTU or the Klebsiella OTU. The use of prolonged continuous positive airway pressure therapy with supplemental oxygen (CPAP oxygen) was also found to be associated with NEC risk. Conclusions: Two OTUs (Clostridium and Klebsiella) were identified at high levels in faecal samples collected from infants prior to NEC diagnosis. A screening tool incorporating these biomarkers with the duration of CPAP oxygen use may aid the clinician in making an early diagnosis of NEC.