The Impact of Storage Conditions on Stool Smellprints as Assessed by an Electronic Nose

Necrotizing enterocolitis (NEC) is a devastating disease of the neonatal gastrointestinal tract. Volatile organic compounds (VOCs), odoriferous compounds released as a byproduct of bacterial metabolism, can be used as a proxy for gut health. We hypothesized that patients with NEC would have different microbial profiles and elicit different VOC signatures as assessed by gas chromatography/mass spectrometry (GC/MS) or an electronic nose compared to controls. Furthermore, we hypothesized that the temperature of sample storage and the length of time in storage would impact the VOC signatures. Forty-five human stool samples were obtained from Neonatal Intensive Care Units. They were stored at -80 °C as part of the Necrotizing Enterocolitis Biorepository. The microbiome composition was determined by 16S-rRNA gene sequencing and VOC profiles were obtained with GC-MS and by analysis with the Cyranose 320 electronic nose. In separate experiments, fresh stool samples were collected from three different strains of mice. Samples were stored for different times and different temperatures, and VOC signals were compared. A p-value less than 0.05 was considered significant. 16S-rRNA sequencing found a difference in the microbiome composition (p = 0.025) between human NEC and control samples. There was also a difference observed between NEC and control samples identified by GC-MS (p = 0.001). However, there were no differences in VOC smellprints between NEC and controls when analyzed with an electronic nose. When mouse specimens were analyzed, principal component values changed significantly over time and with different storage temperatures. NEC is associated with a different gut microbiome and the VOC profile compared to age-matched controls. However, this difference was not appreciated when biobanked stool samples were compared via an electronic nose. Older samples may experience VOC decay, or the electronic nose may not be sensitive enough to detect NEC in stool samples. Further studies on fresh human stool samples are needed, but the findings herein may limit the use of electronic noses as diagnostic tools for NEC.