Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
Institute of Cellular Medicine, Newcastle University, Medical School, Leech Building, M3.121, Newcastle upon Tyne, NE2 4HH, UK.
BMC Microbiol. 2019 Mar 4;19(1):52. doi: 10.1186/s12866-019-1426-6.
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.
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.
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.
坏死性小肠结肠炎（NEC）是早产儿死亡的常见原因，与肠道微生物群密切相关。自发性肠穿孔（SIP）也发生在早产儿中，但死亡率和不良新生儿结局低于 NEC。现有研究主要局限于非侵入性粪便样本，这些样本可能无法反映疾病的解剖部位。因此，我们分析了 NEC 和 SIP 早产儿的历史福尔马林固定石蜡包埋（FFPE）组织。共纳入 13 例 NEC 和 16 例 SIP 早产儿。从 FFPE 组织块中提取的 DNA 进行 16S rRNA 基因测序。对于一部分婴儿，比较了同一婴儿患病组织和边缘健康组织。
二甲苯提供了一种经济有效的脱蜡方法。疾病部位的组织与相邻的健康组织高度相似。仅比较所有婴儿的患病组织，NEC 组的 Shannon 多样性显著降低（P=0.026）。与 SIP 相比，NEC 样本的整体细菌群落也存在显著差异（P=0.038），并且在 NEC 婴儿中观察到很大的变异性。虽然没有单个 OTU 或属与 NEC 或 SIP 显著相关，但在门水平上，NEC 婴儿的变形菌门（P=0.045）和拟杆菌门（P=0.024）显著升高，SIP 婴儿的厚壁菌门（P=0.002）显著升高。
现有的肠道 FFPE 块库为在疾病部位对微生物群进行分析提供了一个强大而特异的样本。我们表明，与 SIP 对照组相比，NEC 婴儿的多样性较低，细菌群落也不同。
