Fecal microbiota and metabolomics as preclinical diagnostic biomarkers in neonatal microbiome-driven diseases

This thesis focusses on the potential of gut microbiota and fecal volatile organic compounds (VOC) as novel preclinical biomarkers for microbiome-driven neonatal diseases. Fecal volatile organic compounds are hypothesized to reflect the function of the gut microbiota, host and interaction between both. Previous studies have demonstrated that preterm infants who developed late onset sepsis (LOS), can be differentiated from non-LOS cases based on their fecal VOC profile. In this thesis, it has been demonstrated that LOS cases can be differentiated from controls based on their gut microbiota composition in a preclinical phase. Necrotizing enterocolitis (NEC), a devastating gastro-intestinal disease, occurring in preterm infants, could also be differentiated from controls based on their fecal VOC profiles and microbiota composition. This difference was mostly profound in severe NEC. Before clinical implementation of fecal VOC as preclinical biomarker, a uniform analytical protocol should be developed. Therefore, potential influencing factors on fecal VOC outcome were assessed. This thesis proposes an analytical protocol for fecal VOC analysis in preterm born infants

Fecal amine metabolite analysis before onset of severe necrotizing enterocolitis in preterm infants: a prospective case-control study

Infants developing necrotizing enterocolitis (NEC) have a different metabolomic profile compared to controls. The potential of specific metabolomics, i.e. amino acids and amino alcohols (AAA), as early diagnostic biomarkers for NEC is largely unexplored. In this multicenter prospective case-control study, longitudinally collected fecal samples from preterm infants (born <30 weeks of gestation) from 1-3 days before diagnosis of severe NEC (Bell's stage IIIA/IIIB), were analyzed by targeted high-performance liquid chromatography (HPLC). Control samples were collected from gestational and postnatal age-matched infants. Thirty-one NEC cases (15 NEC IIIA;16 NEC IIIB) with 1:1 matched controls were included. Preclinical samples of infants with NEC were characterized by five increased essential amino acids-isoleucine, leucine, methionine, phenylalanine and valine. Lysine and ethanolamine ratios were lower prior to NEC, compared to control samples. A multivariate model was rendered based on isoleucine, lysine, ethanolamine, tryptophan and ornithine, modestly discriminating cases from controls (AUC 0.67; p < 0.001). Targeted HPLC pointed to several specific AAA alterations in samples collected 1-3 days before NEC onset, compared to controls. Whether this reflects metabolic alterations and has a role in early biomarker development for NEC, has yet to be elucidated

Fecal Volatile Metabolomics Predict Gram-Negative Late-Onset Sepsis in Preterm Infants: A Nationwide Case-Control Study

Early detection of late-onset sepsis (LOS) in preterm infants is crucial since timely treatment initiation is a key prognostic factor. We hypothesized that fecal volatile organic compounds (VOCs), reflecting microbiota composition and function, could serve as a non-invasive biomarker for preclinical pathogen-specific LOS detection. Fecal samples and clinical data of all preterm infants (≤30 weeks' gestation) admitted at nine neonatal intensive care units in the Netherlands and Belgium were collected daily. Samples from one to three days before LOS onset were analyzed by gas chromatography-ion mobility spectrometry (GC-IMS), a technique based on pattern recognition, and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), to identify unique metabolites. Fecal VOC profiles and metabolites from infants with LOS were compared with matched controls. Samples from 121 LOS infants and 121 matched controls were analyzed using GC-IMS, and from 34 LOS infants and 34 matched controls using GC-TOF-MS. Differences in fecal VOCs were most profound one and two days preceding Escherichia coli LOS (Area Under Curve; p-value: 0.73; p = 0.02, 0.83; p < 0.002, respectively) and two and three days before gram-negative LOS (0.81; p < 0.001, 0.85; p < 0.001, respectively). GC-TOF-MS identified pathogen-specific discriminative metabolites for LOS. This study underlines the potential for VOCs as a non-invasive preclinical diagnostic LOS biomarker