一株弗兰西斯菌的鉴定及其微生物学特性研究

Identification and characterization of the Francisella sp. strain 08HL01032 isolated in air condition systems

【目的】对广州市某空调冷却水中分离的一株L-半氨酸刺激生长性细菌08HL01032进行分类学鉴定及微生物学特性研究。【方法】通过培养基生长状况、菌株形态、生理生化特性、PCR鉴定、16SRNA基因序列分析、RNA聚合酶β亚单位(rpoB)基因序列分析、动物实验、药敏实验等多种表型与基因相结合的方法,探讨该菌株的分类进化地位及一些基本的生物学特性。【结果】该菌株一些生理生化特征与军团菌十分相似,为革兰氏阴性杆菌,氧化酶阴性,不还原硝酸盐,不分解尿素,血平板缓慢生长、易忽略,酵母活性炭缓冲(BCYE)琼脂48h内生长良好,最初错误鉴定为军团菌。但16SrRNA基因(GenBank登陆号:FJ591095)、rpoB基因(GenBank登陆号:FJ939309)系统发育进化显示,该菌株为弗兰西斯(Francisella)属细菌,与蜃楼弗兰西斯菌(F.philomiragia)最相近,相似度分别为95.3%,87.3%,菌落形态、表型特征亦与弗兰西斯菌属相符。其最适生长温度在25℃～28℃之间,42℃不生长;能耐受pH2.2酸液10min;万古霉素、青霉素、多粘霉素B等耐药,军团菌甘氨酸-万古霉素-多粘霉素B-放线菌酮(GVPC)选择性添加剂无生长抑制作用;菌体无芽孢、无鞭毛动力,透射电镜下观察到荚膜样结构,但动物实验初步显示对小鼠无致病性。【结论】菌株08HL01032为弗兰西斯菌属一潜在的新种,其典型特征为L-半胱氨酸刺激生长性,不同于军团菌的L-半胱氨酸依赖性生长特性。

[ Objective] To identify and characterize the strain 08H101032 was isolated from air condition systems in the routine investigations of Legionella in Guangzhou, China, in 2008. [ Methods ] We adopted several phenotypic and genotypieal methods, such as the growth status on various media, morphological, physical and biochemical characteristics, animal test, antibiotic susceptivities, PCR identification, sequence analysis of 16S RNA and RNA polymerase β-subunit （ropB） gene etc, to determinate the phylogenetic position and outline the basic biological characteristics. [ Results] Strain 08H101032 was Gramnegative with polymorphic short rods or eoccobacillus; with no flagella; devoid of spores; well growth on buffered charcoal yeast extraction （BCYE） agar and BCYE supplemented with glycine （3 g/L）, polymyxin B sulfate （80000 iu/L）, vancomycin （1 mg/L） and cycloheximide （80 mg/L） （GVPC medium） within 2 days, but delayed growth on ordinary sheep blood agar untill 5 - 7 days; catalase positive; oxidase negative; no reduction of nitrate; no hydrolysis of urea; delayed fermention of glucose to produce acid; which was primarily considered as Legeionella. It was lastly identified to the genus FransiceUa, charaeterized by a variety of biochemical and molecular phylogenetic tests, which shared the highest similarities to F. Philomiragia with 95.3% to 16S rRNA gene of 1377 oligo nucleotides and 87.3 % to ropB gene of 367 oligo nucleotides （GenBank accession number： FJ591095, FJ939309）. Growth were observed after a treatment for 10 minutes with the KCI-HCI buffer of pH 2.2, 20℃, and at 25℃, 37℃ （optimum 25℃ - 28℃）, but not at 42℃. The cells had capsule-like construction by transmission electron microscopy, however no virulence found to mice. [ Conculsions] Strain 08H101032 was a potential new species of the genus Frartsicella with a typical characteristic of L-cysteine growth stimulating activity, distinguishingly to LegioneUa with L-cysteine growth dependent activity.