Distinct microbiota profiles in non-survivors in preterm infants with surgical necrotizing enterocolitis: Insights from FFPE intestinal tissue analysis

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Background The variant microbiome is associated with necrotizing enterocolitis (NEC). We aimed to analyze remnant formalin fixed paraffin-embedded (FFPE) intestine tissue for microbiome profiling in preterm infants with surgical NEC. Methods We analyzed FFPE small intestine tissues from 16 infants with NEC (8 survivors and 8 non-survivors). Extracted DNA from FFPE tissue blocks underwent 16S rRNA sequencing. We compared the microbiota profiles in survivors and non-survivors. Alpha- and beta diversity metrics were calculated using QIIME2. To assess differences in overall microbial community structure, we performed a Permutational Multivariate Analysis of Variance (PERMANOVA). The analysis was performed in MaAsLin2 R package to determine the specific microbial taxa whose relative abundances were significantly associated with survival status in a multivariable linear model. Results Sequencing of FFPE extracted DNA resulted in high-quality sequence reads in 16 cases. Analysis Analysis of microbial communities from 16 cases revealed a significant association between microbiome structure and survival status. Beta diversity analysis demonstrated distinct clustering of microbiome profiles between survivor and non-survivor groups. Alpha diversity metrics further characterized these differences: the non-survivor group exhibited a more complex and even microbiota (Shannon entropy, p = 0.02; Pielou’s evenness, p = 0.017), whereas the survivor group’s microbiome was significantly richer in observed features ( p = 0.004). Notably, this association was specific to survival outcome, as overall community structure did not significantly differ when grouped by histological features of disease severity such as necrosis, inflammation, or hemorrhage. Linear mixed effect models and direct comparisons further identified numerous taxa potentially associated with survival status. Conclusion FFPE intestinal tissue enabled retrospective and spatially relevant microbiota assessment at the disease site. The non-survivors had complex microbiota and had distinct bacterial communities compared to survivors. Our findings suggest that the gut microbiome is a key factor related to prognosis, independent of other measures of NEC severity.

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