Zhang Pengbo, Garg Padma P, Liu Liang, Zhang Wei, Kavanagh Kylie, Shetty Avinash, Howard Tim, Varshney Neha, Sawaya David, Hawkins Greg, Schwartz Drew J, Garg Parvesh Mohan
Center for Precision Medicine, Wake Forest University School of Medicine, Winston Salem, NC, USA.
Department of Pediatrics/Neonatology, University of Mississippi Medical Center, Jackson, MS, USA.
J Neonatal Perinatal Med. 2025 Jun 24:19345798251353777. doi: 10.1177/19345798251353777.
BackgroundThe 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.MethodsWe 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.ResultsSequencing of FFPE extracted DNA resulted in high-quality sequence reads in 16 cases.AnalysisAnalysis 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, = 0.02; Pielou's evenness, = 0.017), whereas the survivor group's microbiome was significantly richer in observed features ( = 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.ConclusionFFPE 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.
背景
微生物群变异与坏死性小肠结肠炎（NEC）相关。我们旨在分析用于手术治疗的NEC早产儿的残留福尔马林固定石蜡包埋（FFPE）肠组织的微生物群谱。
方法
我们分析了16例NEC婴儿（8例存活者和8例非存活者）的FFPE小肠组织。从FFPE组织块中提取的DNA进行16S rRNA测序。我们比较了存活者和非存活者的微生物群谱。使用QIIME2计算α和β多样性指标。为了评估整体微生物群落结构的差异，我们进行了置换多元方差分析（PERMANOVA）。在MaAsLin2 R包中进行分析，以确定在多变量线性模型中其相对丰度与生存状态显著相关的特定微生物分类群。
结果
对FFPE提取的DNA进行测序，16例均获得了高质量的序列读数。
分析
对16例病例的微生物群落分析显示，微生物群结构与生存状态之间存在显著关联。β多样性分析表明，存活者组和非存活者组的微生物群谱有明显聚类。α多样性指标进一步表征了这些差异：非存活者组的微生物群更复杂且更均匀（香农熵， = 0.02；皮洛均匀度， = 0.017），而存活者组的微生物群在观察到的特征方面明显更丰富（ = 0.004）。值得注意的是，这种关联特定于生存结果，因为按疾病严重程度的组织学特征（如坏死、炎症或出血）分组时，整体群落结构没有显著差异。线性混合效应模型和直接比较进一步确定了许多可能与生存状态相关的分类群。
结论
FFPE肠组织能够在疾病部位进行回顾性和空间相关的微生物群评估。与存活者相比，非存活者具有复杂的微生物群和独特的细菌群落。我们的研究结果表明，肠道微生物群是与预后相关的关键因素，独立于NEC严重程度的其他指标。
