中国家猫克氏巴尔通体分离株生物学及分子特征分析

Biological and molecular characteristics of a cat-borne Bartonella clarridgeiae

【目的】分析中国家猫中分离的巴尔通体菌株M9HN-SHQ生物学性状和分子特征。【方法】应用含5%羊血的胰酶大豆琼脂培养基在5%CO2培养箱中37℃培养6～7d,革兰氏和吉姆尼茨染色镜下观察形态;应用VITEK ANI厌氧菌鉴定卡进行生化反应鉴定;气相色谱分析方法获取菌体脂肪酸成份组成(CFA);Etest药敏试条测定对10种抗生素的敏感性;分别应用随机扩增多态性DNA(RAPD)和脉冲场凝胶电泳(PFGE)技术对目标菌株和其他国际标准菌株等绘制DNA指纹图谱;对16S rRNA,gltA,groEL,ftsZ,rpoB,ribC基因和16S～23S rRNAITS测序并根据序列进行系统发育分析。【结果】菌株M9HN-SHQ的菌落呈圆形、灰白色,较小;镜下观察为革兰染色阴性、细小、微弯曲的杆状菌体。生化反应不活跃,氧化酶等阴性;CFA主成份组成与克氏巴尔通体一致;体外培养对头孢噻肟钠、利福平等多种抗生素敏感;RAPD和PFGE指纹图能够很好的与汉赛巴尔通体等4种国际标准菌株区别,具有独特带型;16SrRNA等多个基因序列与克氏巴尔通体同源性为100%。【结论】综合生物学性状和分子特征分析,菌株M9HN-SHQ是克氏巴尔通体,这是我国首次从宿主动物中分离到该病原菌,证明在中国家猫中存在这种能够引起人猫抓病的病原菌。

[ Objective] To characterize a Bartonella strain M9HN-SHQ from a blood culture of cat from Henan Province, China. [Methods] The organisms were subcuhured in 5% CO2 at 37℃ on trypticase soy agar containing 5% sheep blood for 6 to 7 days. We analyzed the isolate using whole-cell fatty acid analysis, Etest for susceptibility testing, random amplified polymorphic DNA（RAPD） ,pulsed-field gel electrophoresis（PFGE） and sequence analysis of 16S rRNA, gltA, groEL,fisZ, rpoB, ribC and 16S - 23S rRNA intergenic spacer region. [ Results] Isolate M9HN-SHQ stained faintly as a gram-negative rod but was easier to visualize when stained by the Gimenez technique. Most of the biochemical and cellular fatty acid properties of strain M9HN-SHQ were typical for bacteria of the Bartonella genus. The strain was susceptible to Cefotaxime sodium, Rifampin, Ciprofloxacin and other four antibiotics. Genotypic characterization of strain M9HN-SHQ, including RAPD, PFGE was distinguishable from the reference strains of B . henselae , B. elizabethae , B . vinsonii subsp, berkhoffii and B . grahamii . Sequence analysis of the genes from the seven chromosomal regions identified the strain M9HN-SHQ as B. clarridgeiae. [ Conclusion ] To our knowledge, this is the first report that documents Bartonella clarridgeiae infections of domestic cats in China.