Using formalin fixed paraffin embedded tissue to characterize the preterm gut microbiota in necrotising enterocolitis and spontaneous isolated perforation using marginal and diseased tissue

Abstract

Background: Necrotising enterocolitis (NEC) is a common cause of death in preterm infants and is closely linked to the gut microbiota. Spontaneous intestinal perforation (SIP) also occurs in preterm neonates, but results in lower mortality and less adverse neonatal outcomes than NEC. Existing studies are largely limited to non-invasive stool samples, which may not be reflective of the anatomical site of disease. Therefore, we analysed historical formalin-fixed paraffin-embedded (FFPE) tissue from NEC and SIP preterm infants. A total of 13 NEC and 16 SIP infants were included. Extracted DNA from FFPE tissue blocks underwent 16S rRNA gene sequencing. For a subset of infants, diseased tissue and marginal healthy tissue from the same infant were compared. Results: Xylene provided a cost and time effective means of deparaffinization. Tissue from the site of disease was highly comparable to adjacent healthier tissue. Comparing only diseased tissue from all infants showed significantly lower Shannon diversity in NEC (P = 0.026). The overall bacterial communities were also significantly different in NEC samples compared to SIP (P = 0.038), and large variability within NEC infants was observed. While no single OTU or genus was significantly associated with NEC or SIP, at the phylum level Proteobacteria (P = 0.045) and Bacteroidetes (P = 0.024) were significantly higher in NEC and SIP infants, respectively. Conclusions: Existing banks of intestinal FFPE blocks provide a robust and specific sample for profiling the microbiota at the site of disease. We showed preterm infants with NEC have lower diversity and different bacterial communities when compared to SIP controls.