一种新K抗原血清型副溶血弧菌的生物学特征和致病机制研究

Vibrio parahaemolyticus (VP) is the main pathogen of infectious diarrhea. Our recent research found a novel K antigen VP. The isolation rate of this novel VP increased significantly in acute diarrhea patients recently, and became second only to O3:K6 serotype. The genome sequencing analysis showed that the novel strain had 10 specific genes in capsule gene clusters and an insertion at recA gene. To clear biological characteristics and pathogenicity of the novel strain, we intend to use electron microscopy and gas chromatography-mass spectrometry technology to analyze the structure and composition of capsule of the novel VP; carry out bacteria and cell experiment to study the characteristics of growth, resistance to the environment, as well as adhesion and damage to epithelial cells; establish an animal model to observe the symptoms of infectious diarrhea, the intestinal tissue lesions and the expression of inflammatory factors. Furthermore, we will construct specific gene deletion and recovery strains by gene knockout and complementation, respectively, and observe the changes of serum agglutination, biological characteristics and pathogenicity of the strains to analyze the function of these specific genes. Finally, we plan to prepare the new K antigen serum by immunizing rabbits to detect the novel VP and submit the specific serum and bacterial to VP serotype typing committee to name the serotype. This study will provide the technical methods and the scientific basis for the diagnosis, prevention and control of VP infection.

副溶血弧菌是导致感染性腹泻的主要病原菌。课题组近期发现一种具有新型K抗原的副溶血弧菌，近两年该菌在急性腹泻患者中分离率呈明显上升趋势，已成为仅次于O3:K6的血清型。全基因组测序显示该菌K抗原决定簇中存在10个特异性基因，并在recA处存在插入序列。本项目拟采用电镜和质谱技术分析该新型菌株的微荚膜结构和成分；采用培养技术研究该菌的生长特点、环境抵抗力及对上皮细胞的黏附和损伤能力；建立动物模型，观察该菌致泻的严重程度、肠组织病变及炎症因子表达情况；并与主要流行株比较，明确该新型菌株的生物学特征和致病性。采用基因敲除和回补技术，构建新型K抗原决定簇特异性基因和recA处插入基因的缺失和回补株，观察菌株血清凝集、生物学特征和致病性改变，阐明这些基因的功能。采用灭活新型菌株免疫兔子制备新型K抗原特异性血清，并申请命名。本研究旨在为该新型副溶血弧菌相关疾病的诊断和防控提供技术手段和理论依据。

副溶血弧菌是感染性腹泻的主要病原，也是食源性疾病暴发中最重要的生物因素。在病原变异变迁研究中发现一种具有新型K抗原的副溶血弧菌（O4:Knew-recAin），近年来该菌在急性腹泻患者中分离率呈明显上升趋势，成为仅次于O3:K6血清型的新流行株。全基因组序列分析显示该菌K抗原决定簇中存在10个特异性基因，并在管家基因recA处存在插入序列。本项目研究O4:Knew-recAin副溶血弧菌的生物学特征和致病性、新型K抗原决定簇特异性基因和插入基因的功能，及建立新型细菌的检测方法。结果显示，O4:Knew-recAin菌株在20℃～37℃，pH 7.5～10.5范围内生长良好，在pH= 4.9时能够生长而大部分O3:K6菌株不能生长；电镜下清晰看到鞭毛和微荚膜；除氨苄西林外，对临床其他常见抗菌药物的敏感性高。构建进化树分析，O4:Knew-recAin与O4:K8亲缘性最近。在动物感染性腹泻模型中，口服O4:Knew-recAin菌株6～16小时后，乳兔出现体重减轻、水样便或粘液便，甚至生死亡。解剖后发现肠道肿胀变红，病理改变以回肠末端最为明显，表现为肠腔内绒毛破损、断裂，在固有层有中性粒细胞浸润，破损处有大量副溶血弧菌，炎症因子表达水平升高。通过构建基因缺失株和回补株，未发现新型K抗原10个特异性基因和recA处插入基因对细菌生物学特点和致病性有明显影响。针对新型K抗原决定簇和recA处插入基因的特异性序列分别设计引物，建立O4:KUT-recAin细菌核酸检测方法，回顾性分析新型菌株分布；用灭活的O4:Knew-recAin副溶血弧菌免疫兔子，8周后取全血分离血清，并用其它血清型副溶血弧菌制备抗原对抗血清进行吸附，获得特异性K血清。本研究为该新型副溶血弧菌相关疾病的诊断和防控提供技术手段和理论依据。