集中空调系统中弗朗西斯菌的分离与鉴定  

Isolation and identification of Francisella-like strains from central air conditioning system in Guang-zhou

作　　者：李营[1] 屈平华[2] 张颖[3] 李工厂 叶大柠 陈守义[2] 陈茶[1] 吴尚为[6] 

机构地区：[1]天津医科大学检验学院,300070 [2] 广东省中医院检验科,广州510006 [3]广州市疾病预防控制中心微生物检验科,510006 [4] 郑州大学第二附属医院检验科 [5]广州中医药大学第二临床医学院,510006 [6]广州金域医学检验中心有限公司,510006

出　　处：《中华微生物学和免疫学杂志》2015年第10期729-734,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（3130004）;广东省教育厅育苗工程基金（2013LYM_0015）

摘　　要：目的 对集中空调系统中的弗朗西斯菌进行分离鉴定. 方法 收集2008—2014年广州市集中空调系统的324份冷却水和36份冷凝水水样,经负压膜过滤集菌、酸处理后接种军团菌选择性甘氨酸-万古霉素-多黏霉素B-放线菌酮（ BCYEα-GVPC）培养基进行细菌分离培养,通过培养特征、生化反应特性等表型分析及弗朗西斯菌属特异性引物PCR鉴定、16S rRNA 基因序列分析、基因组DNA-DNA杂交等方法进行菌种鉴定. 结果 从14份集中空调冷却水中分离到15株菌,经表型分析和弗朗西斯菌属特异性引物PCR扩增确定为弗朗西斯菌,水样的阳性率分别为：冷却水4.3%,冷凝水0%. 15个分离菌株的16S rRNA 基因序列分析及系统发育分析显示,其可分为4个分支,包括为广州弗朗西斯菌9株,蜃楼弗朗西斯菌1株,另2个分支的5株菌与广州弗朗西斯菌亲缘关系最接近,但基因组DNA-DNA杂交证明与广州弗朗西斯菌同源性〈70%,初步确定为弗朗西斯菌属内的候选新种. 结论 广州市集中空调系统水样弗朗西斯菌阳性率低,且未发现强致病性的土拉热弗朗西斯菌.Objective To isolate the Francisella-like strains from central air conditioning system in Guangzhou and to identify their species .Methods Three hundred and twenty-four samples of cooling wa-ter and 36 samples of condensed water were collected from the air conditioning system in Guangzhou from year 2008 to 2014.The samples were cultured in BCYE supplemented with glycine （3 g/L）, polymyxin B sulfate （80 000 U/L）, vancomycin （1 mg/L） and cycloheximide （80 mg/L） （BCYEα-GVPC） medium af-ter concentrated by membrane filtration and pretreated by KCl-HCl buffer of pH2.2 for 10 minutes.The Francisella-like isolates were identified according to their cultural characteristics , biochemical test , PCR identification, 16S rRNA gene sequencing analysis and genomic DNA-DNA hybridization.Results Fifteen stains were isolated from 14 samples of cooling water and were identified as Francisella by PCR identification and phenotype analysis .The Francisella strains were detected in 4.3%of cooling water samples , but not in the condensed water samples .The phylogenetic tree constructed based on the sequence of 16S rRNA showed that the 15 strains formed four clusters including one cluster of nine Francisella guangzhouensis isolates, one cluster of one Francisella philomiragia isolate and two clusters of five isolates closest to the Francisella guan-gzhouensis strain.The five isolates represented two novel genospecies sharing a genomic homology of less than 70%with the Francisella guangzhouensis strain as indicated by the DNA-DNA hybridization analysis . Conclusion Water samples collected from the air conditioning system in Guangzhou city showed a lower positive rate for Francisella strain and no Francisella tularensis strain with strong pathogenicity was found in those samples .

关 键 词：集中空调系统 弗朗西斯菌 基因序列分析 

分 类 号：R440[医药卫生—诊断学]