鳖源柠檬酸杆菌属新种的分离鉴定与多位点序列

为确定2020年10月湖北一中华鳖发病的病原体及病原特征，实验从该养殖场患病中华鳖个体肝脏中分离获得1株优势菌A3，经理化性状及16S rRNA序列鉴定，表明菌株A3属于柠檬酸杆菌。进一步通过recN序列鉴定，发现A3与布氏柠檬酸杆菌recN碱基一致性为95.58%～96.01%，与柠檬酸杆菌属其他种的recN碱基一致性均低于93.72%，表明菌株A3为柠檬酸杆菌属潜在新种。人工回归感染实验证实菌株A3是引起本次中华鳖患病的病原菌。与嗜水气单胞菌相比，A3感染病程偏慢但致死率较高。药敏实验结果显示，菌株A3对美罗培南等10种抗生素敏感，对氟苯尼考等9种抗生素耐药。多位点序列分型(MLST)鉴定显示，菌株A3属于新序列型(ST)。对柠檬酸杆菌多位点序列分型的7个管家基因序列进行级联并构建系统发育树，结果显示，柠檬酸杆菌的进化与菌株的分离源及分离地点没有明显关系；菌株A3与分离自欧洲人源的ST120型菌株Citfre2580亲缘关系最近。本研究对中华鳖的病原进行了分离鉴定，并阐述了一种更可靠的柠檬酸杆菌鉴定方法，旨在引起人们对柠檬酸杆菌致病性的重视，为水产品健康养殖提供一定的参考依据。

Identification and multilocus sequence analysis of a novel species of Citrobacter isolated from diseased Pelodiscus sinensis

In October 2020, some Pelodiscus sinensis died in a farm in Hubei Province, China. To determine the pathogen and pathogenic characteristics of the disease, a dominant strain A3 was isolated from the liver of the diseased P. sinensis. Based on physiological and biochemical characteristics and 16S rRNA gene sequence identification, it was identified as Citrobacter sp.. Further identification of the recN gene showed that the recN gene sequence identities between A3 and C. braakii were 95.58%-96.01%, while the identities between A3 and other species of Citrobacter were less than 93.72%, which indicated that A3 belonged to a novel species of Citrobacter. Artificial infection tests confirmed its pathogenicity to P. sinensis. Compared with Aeromonas hydrophila, the course of A3 infection was slower, but the mortality was as high. Drug sensitivity tests showed that strain A3 was sensitive to 10 kinds of antibiotics such as meropenem, and resistant to 9 kinds of antibiotics such as florfenicol. Multilocus sequence typing (MLST) showed that strain A3 belonged to a new sequence type (ST). A phylogenetic tree based on the 7 housekeeping gene sequences of Citrobacter from MLST database was constructed. The results showed that the evolution of Citrobacter spp. had no significant relationship with the source and location of isolates. Moreover, strain A3 has the closest genetic relationship with ST120 strain Citfre2580 isolated from human in Europe. In this study, the pathogen of P. sinensis was isolated and identified, and a more reliable method for the identification of Citrobacter was described, in order to arouse people''s attention to the pathogenicity of Citrobacter, and provide some reference for the healthy aquaculture of aquatic products.