ABSTRACT

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxin-encoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca, a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

摘要

坏死性小肠结肠炎（NEC）是与肠道细菌性营养不良相关的早产儿的毁灭性肠炎性疾病。使用基于16S rRNA的方法，我们的实验室在NEC粪便样品中鉴定了一个高丰度的未分类肠杆菌科序列（NEC_unk_OTU）。我们旨在鉴定这种细菌并确定其在疾病中的潜在作用。通过NCBI数据库搜索16S序列，选择培养系统，生物分型和聚合酶链反应来完善NEC_unk_OTU的分类并从NEC病例中鉴定出毒素编码基因。质谱和凋亡分析证实了细菌细胞毒素的产生。使用类似方法分析了来自9个NEC和5个非NEC对照的其他粪便样本，并进行了多位点序列分型（MLST），以研究克隆关系并确定分离株的序列类型。NEC_unk_OTU被标识为产酸克雷伯菌（Klebsiella oxytoca），一种病原菌，已知会引起抗生素相关的出血性结肠炎，但以前并未与NEC相关。包括索引病例在内，从10例NEC受试者中分离出6例产产产氧合杆菌的菌株。在这些中，催产克雷伯氏菌16S序列占据了粪便微生物群的大部分。的细胞毒素产生菌催娩克氏也从5所控制的4隔离; 但是，在这些序列中，相应的16S序列的丰度非常低。毒素阳性分离物的MLST分析表明，病例与对照之间无克隆关系和相似的基因簇。这些结果表明细胞毒素的生产菌株催娩克氏定居在大部分早产儿中。这种机会病原体的生长可能加剧了某些（也许很多）NEC病例。