一例败血症患者解甘露醇罗尔斯顿菌的分离与鉴定及其耐药相关基因分析  

Isolation and identification of Ralstonia mannitofilyfica strains from a patient with septicaemia andanalysis of their drug resistance genes

作　　者：寿晓岚 胡玮琳[1] 邵泓瑜 吕火烊[3] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058 [2]浙江省德清县人民医院检验科,湖州313200 [3]浙江省人民医院检验科,杭州310014

出　　处：《中华微生物学和免疫学杂志》2016年第1期57-63,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的对病因不明败血症患者外周血中病原菌进行分离与鉴定，了解分离菌株耐药性并分析耐药相关基因。 方法采用双份外周血标本血平板分离划线培养、菌落革兰染色镜检及VITEK 2-compact全自动细菌检测分析系统和16S rRNA基因测序鉴定病原菌。采用微量稀释法对分离菌株进行药敏试验。采用β-内酰胺酶、超广谱β-内酰胺酶确证试验以及AmpC酶和碳青酶烯酶表型试验，了解分离菌株产酶情况及类型。分别采用肠杆菌科细菌19种常见β-内酰胺酶、AmpC酶和碳青酶烯酶基因以及解甘露醇罗尔斯顿菌21个注释为β-内酰胺酶基因引物，用PCR检测分离菌株基因组中各靶基因。PCR产物T-A克隆后测序。 结果上述血标本中均分离出解甘露醇罗尔斯顿菌。该菌株对头孢曲松、头孢吡肟、环丙沙星、左旋氧氟沙星、替加环素、复方新诺明敏感，但对其他11种抗生素耐药。所有肠杆菌科细菌常见β-内酰胺酶基因PCR扩增结果阴性，但扩增出TK49_09850、TK49_12955、TK49_14470、TK49_14495、TK49_18990这5个解甘露醇罗尔斯顿菌β-内酰胺酶基因条带，其序列与肠杆菌科细菌常见β-内酰胺酶基因相似性较低。 结论该患者感染的病原体为解甘露醇罗尔斯顿菌。该解甘露醇罗尔斯顿菌株有较高耐药性且对不同β-内酰胺类抗生素耐药性有较大差异，其β-内酰胺酶基因与肠杆菌科细菌常见β-内酰胺酶基因完全不同。To isolate and identify the pathogenic bacteria from peripheral blood of a patient with septicemia of unknown etiology and to analyze their drug resistance genes. MethodsTwo peripheral blood samples were collected from the patient after having fever. Several assays including culturing bacteria on blood agar plates by using streaking technique, Gram-staining of bacterial colonies and microscopic observation, VITEK 2-compact automatic bacterium detection and analysis system as well as a sequencing analysis of the 16s rRNA gene were performed to identify the bacterial pathogens in blood samples. Microdilution test was performed to detect the drug susceptibilities of isolated bacteria to antibiotics. Confirmatory tests were performed to detect the production of β-lactamase and extended spectrum β-lactamase by the isolated strains and the phenotypes of AmpC enzyme and carbapenemase. PCR was used to identify the β-lactamase-encoding genes in the isolated strains by using the primers of 19 common β-lactamase-, AmpC enzyme- and carbapenemase-encoding genes in Enterobacteriaceae strains and the primers of 21 annotated gene sequences encoding the β-lactamase of a Ralstonia mannitolilytica strain. The PCR products were sequenced and analyzed after T-A cloning. ResultsRalstonia mannitolilytica strains were isolated from the two peripheral blood samples. The isolated strains were sensitive to ceftriaxone, cefepime, ciprofloxacin, ofloxacin, tigecycline and compound sulfamethoxazole （SMZ-TMP）, but resistant to the other 11 tested antibiotics. Results of PCR amplification by using the primers of common genes encoding β-lactamase of Enterobacteriaceae strains were all negative. Fragments of genes encoding the β-lactamase of the isolated Ralstonia mannitolilytica strain were successfully amplified, which were TK49_09850, TK49_12955, TK49_14470, TK49_14495 and TK49_18990. The sequences of the amplified gene fragments were not similar to those of the common β-lactamase-encoding genes in Enterobacteriaceae strains.

关 键 词：解甘露醇罗尔斯顿菌 败血症 分离鉴定 耐药性 Β-内酰胺酶基因 

分 类 号：R440[医药卫生—诊断学]