丁酸梭菌致婴儿肉毒中毒及肠炎发生的生态学及分子基础研究

Infant botulism (IB) and necrotising enterocolitis (NEC) are potentially lethal and serious gastrointestinal disorder among infants and preterm neonates, respectively. Previous data indicated that Clostridium butyricum is highly correlated to both diseases. This project will be conducted as follows with the aim of decreasing the incidence of NEC and IB: (1) We will explore the source of contamination, changes in distribution in nature and geographical diversity of C. butyricum based on the case-control study in infants and preterm neonates. Meanwhile, diversity of intestinal microflora of infants with either IB or NEC will also be carried out. (2) The same C. butyricum strain will be identified at the species level by an original culture-based method in combination with molecular biological techniques, followed by analyzed by both pulsed field gel electrophoresis and multilocus sequence typing in order to elucidate the homeology and phylogenetic distance among the strains obtained. The strains that harbor the bont/e gene will be screened as well. (3) A Caco-2 cell monolayer model of intestinal barrier will be employed in the study of the effect of butyrate produced by C. butolinum on intestinal barrier. Additionally, apoptosis in Caco-2 cell lines caused by the metabolite of C. butolinum will be studied by flow cytometry in order to clarify the mechanism of NEC. (4) Genes coding botulinum type E neurotoxin, new gens and functional single nucleotide polymorphism will be explored by whole genome sequencing. This will give an answer of how IB and NEC occur. On the other hand, the possibility of whether the same strain of C. butolinum could result in IB and NEC simultaneously will be probed. All data obtained in this study will be helpful for developing the advanced control measures against C. butolinum contamination. It is also to provide a scientific base for developing the effective diagnosis and treatment measures of IB and NEC.

婴儿肉毒中毒（IB）和新生儿坏死性小肠结肠炎（NEC）严重危害婴儿健康。研究显示，丁酸梭菌与两病发生高度相关。本研究以降低IB和NEC发生率为目标，①通过病例对照研究，探索丁酸梭菌污染、分布、地域多态性变化轨迹；开展NEC患儿肠道菌群多样性差异研究；②传统法结合分子生物学方法，对丁酸梭菌和肉毒梭菌进行种水平鉴定、PFGE和MLST分型，揭示菌株间同源性和进化关系，筛选bont/e基因携带株；③利用肠粘膜屏障模型和流式细胞技术，阐明丁酸梭菌致NEC的发生机制；④运用全基因组测序技术，探索丁酸梭菌中相关毒力基因、新基因和功能性SNP，解析丁酸梭菌致IB和NEC的遗传基础，探究丁酸梭菌同时致IB和NEC两病发生的可能性。本项目的实施，可为发展先进的丁酸梭菌污染控制措施、制定有效的IB和NEC临床诊治方案提供依据。

婴儿肉毒中毒（IB）和新生儿坏死性小肠结肠炎（NEC）严重危害婴儿健康，丁酸梭菌感染可能与两病发生密切相关。本项目通过病例对照研究在我国十省范围内对婴儿人群进行流行病学调查，采集人群粪便、食品、环境样品并同时采集市售婴儿配方粉样品进行梭状芽胞杆菌分离鉴定、毒素分型、溯源分析、致病机制和遗传基础等研究。本项目研究结果如下：（1）建立了不同样品中梭状芽胞杆菌的分离体系、属和部分种鉴定用荧光PCR方法、四型肉毒毒素毒力基因检测荧光PCR方法。（2）本项目共调查婴儿人群213例，采集样品942份，分离出梭状芽胞杆菌173株，其中包括丁酸梭菌87株，肉毒梭菌4株。丁酸梭菌分离株在NEC病例粪便样品中检出率最高（33.3%），接下来依次为对照组粪便样品（11.7%）、NEC病例食品样品（8%）、NEC病例环境涂抹样品（4.8%），市售婴儿配方粉中检出率最低（1.4%）。经统计分析发现，丁酸梭菌在NEC病例粪便样品中的检出率与对照组相比存在显著性差异（P＜0.05），因此可认为NEC病例组粪便中丁酸梭菌的污染水平明显高于对照组。（3）两株丁酸梭菌全基因组测序结果显示，分离自NEC患儿粪便和暖箱内壁涂抹样品的两株丁酸梭菌基因组序列上均携带溶血素、内毒素和唾液酸酶的编码毒力基因，尤其是唾液酸酶编码基因nanH为两菌特有基因，且该基因位于可移动基因原件上，推测其可能是从其它致病菌转移而来。（4）Caco-2体外肠道模型实验结果发现，高浓度的丁酸可致Caco-2细胞凋亡率增加，且增加水平与丁酸浓度正相关。本项目的实施，可保障婴儿人群健康，为两病的诊断和治疗，污染的预防和控制等提供技术支持。