一株自中国大鲵的副炭疽芽孢杆菌分离和鉴定  

Isolation and identification of a Bacillus paranthracis strain from Chinese giant salamander

作　　者：袁璐 张晓妍 吕宇飞 郭艳 陈杰 汪立平[1] 王恒樑[1,2] 王东澍[2] 刘先凯 YUAN Lu;ZHANG Xiaoyan;LYU Yufei;GUO Yan;CHEN Jie;WANG Liping;WANG Hengliang;WANG Dongshu;LIU Xiankai(College of Food Sciences and Technology,Shanghai Ocean University,Shanghai 201306,China;State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]军事科学院军事医学研究院生物工程研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《微生物学报》2022年第9期3613-3630,共18页Acta Microbiologica Sinica

基　　金：国家自然科学基金(81871619,82102412)。

摘　　要：【目的】对陕西某大鲵养殖场患病的中国大鲵腹水中分离培养得到的一株蜡样芽孢杆菌群细菌疑似菌株进行鉴定,明确该菌生长特性和种类。【方法】无菌解剖患病大鲵,取肠道、腹水、皮肤等各部位的样品均质稀释并分离纯化,从腹水中获得疑似蜡样芽孢杆菌群细菌的纯菌株,命名为SHOU-BC01。对该菌株进行形态与染色特性、培养与生化特性、生物膜形成能力、芽孢形成、药敏检测、全基因组测序等试验鉴定,并根据测序结果进行平均核苷酸相似度(average nucleotide identity,ANI)、数字DNA-DNA杂交(digital DNA-DNA hybridization,dDDH)、多位点序列分型(multilocus sequence typing,MLST)、全基因组SNP聚类和毒力因子分析。【结果】菌株SHOU-BC01为革兰氏阳性杆菌,表面粗糙;具有蛋白酶、卵磷脂酶和溶血酶活性;能够发酵L-阿拉伯糖、D-核糖、D-木糖等多种糖类,能利用色氨酸、丙酮酸盐等;有较强生物膜形成能力;120h的芽孢形成率达到70.60%;该菌株对青霉素G、头孢噻吩、万古霉素等15种抗生素耐药,对哌拉西林、头孢唑啉、庆大霉素等25种抗生素敏感;根据生物学特性结合ANI、dDDH及全基因组SNP聚类分析,鉴定菌株SHOU-BC01为副炭疽芽孢杆菌(Bacillus paranthracis),经MLST分型,该菌株属于ST205序列型;该菌株含有鞘磷脂酶、CytK和NheC毒素、多糖荚膜、PlcR-PapR群感效应系统及Ⅶ型分泌系统等毒力因子。【结论】成功从中国大鲵腹水中分离出副炭疽芽孢杆菌,丰富了大鲵副炭疽芽孢杆菌数据。[Objective] A bacterial strain likely affiliated with the Bacillus cereus group was isolated from the ascites of Chinese giant salamander from a giant salamander farm in Shaanxi Province and was investigated for the biological characteristics. [Methods] The diseased giant salamander was aseptically dissected, and the samples taken from intestine, ascites, skin and other parts were homogeneously diluted,separated and purified. A pure strain of suspected B. cereus group bacteria was obtained from the ascites,which was named SHOU-BC01. The strain was identified for morphological and staining characteristics,culture and biochemical characteristics, biofilm formation ability, spore formation, drug susceptibility, and whole genome sequencing. Based on the sequencing results, average nucleotide identity(ANI), digital DNA-DNA hybridization(dDDH), multilocus sequence typing(MLST), whole-genome SNP based clustering and virulence factor analysis were performed. [Results] Strain SHOU-BC01 is a Gram-positive bacillus with rough surface, with protease, lecithinase and fibrinolysin activities. It can ferment L-arabinose,D-ribose, D-xylose and other carbohydrates, and can utilize tryptophan and pyruvate, with strong biofilm formation ability. Its spore formation rate reached 70.60% in 120 h. The strain was resistant to 15 antibiotics including penicillin G, cephalothin and vancomycin, and sensitive to 25 antibiotics including piperacillin,cefazolin and gentamicin. According to biological characteristics combined with ANI, dDDH and whole-genome SNP based clustering analysis, strain SHOU-BC01 was identified as B. paranthracis, and the MLST typing showed that the strain belonged to ST205 sequence type. It contains virulence factors such as sphingomyelinase, CytK and NheC toxin genes, polysaccharide capsular, PlcR-PapR quorum-sensing system and type Ⅶ secretion system. [Conclusion] B. paranthracis was successfully isolated from the ascites of Chinese giant salamander, which enriched the data of B. paranthracis.

关 键 词：中国大鲵 副炭疽芽孢杆菌 分离 鉴定 

分 类 号：S943[农业科学—水产养殖]