烧伤患者中产VIM-2型金属β内酰胺酶鲍氏不动杆菌耐药机制及同源性分析
Analysis of the mechanism of drug resistance of VIM-2-type metallo-β-lactamase-producing Acinetobacter baumannii isolated from burn patients and its homology
目的 探讨我院烧伤患者中产VIM-2型金属β内酰胺酶(MBL)的鲍氏不动杆菌(AB) l对碳青霉烯类抗生素的耐药机制及同源性. 方法 2011年9月-2014年3月,收集从笔者单位收治的烧伤住院患者痰液、尿液、血液、脓液、引流液中分离的400株AB(经鉴定).采用全自动微生物鉴定及药敏分析系统测定菌株对复方磺胺甲(口恶)唑、氨曲南等15种抗生素的耐药性.针对抗碳青霉烯类抗生素菌株,采用改良Hodge试验筛选产碳青霉烯酶菌株.PCR法及测序检测产碳青霉烯酶AB的碳青霉烯酶基因、携带blaVIM-2基因的产碳青霉烯酶AB的可移动基因元件1类整合子(Intl1)及其保守片段(CS).针对携带blaVIM-2基因产碳青霉烯酶AB行亚胺培南-乙二胺四乙酸(EDTA)协同试验以及亚胺培南-EDTA与头孢他啶-EDTA增效试验验证其产MBL情况,并分析产VIM-2型MBL的AB的耐药情况.对产VIM-2型MBL的AB行质粒接合试验检测质粒转移情况,PCR法检测外膜蛋白CarO基因.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测携带CarO基因的产VIM-2型MBL的AB菌株的CarO蛋白表达量.肠杆菌基因间重复一致序列(ERIC)-PCR法对产VIM-2型MBL的AB菌株进行基因分型,分析同源性. 结果 (1)400株AB对左氧氟沙星和复方磺胺甲(口恶)唑的耐药率低.共筛选出381株抗碳青霉烯类抗生素AB,其中240株产碳青霉烯酶.(2)240株产碳青霉烯酶AB中检出18株携带VIM-2酶基因blaVIM-2,占7.5%;133株携带bla TEM-1基因,占55.42%;195株携带blaOXA-23基因,占81.25%;188株携带bla armA基因,占78.33%.(3)18株携带blaVIM-2基因的产碳青霉烯酶AB均携带Intl1基因,呈现Intl1-VIM连锁携带型,Intl1可变区CS呈现多样性.(4)经验证,18株携带blaVIM-2基因的产碳青霉烯酶AB均为产VIM-2型MBL的AB.该18株AB菌株对复方磺胺甲(口恶)唑的耐药率最低,其次为左氧氟沙星和头孢哌酮/舒巴坦,对其余抗生素的耐药率均大于60.00%.(5)18株产VIM-2型MBL的AB经多次质粒接合试验均未见耐药基因阳性转移菌株.(6)18株产VIM-2型MBL的AB中9株携带CarO基因,携带CarO基因的产VIM-2型MBL的AB菌株外膜蛋白CarO缺失或表达量减少.(7)18株产VIM-2型MBL的AB ERIC-PCR指纹图谱共分6个谱型,A、B、C、F型菌株分别为6、4、3、l株,D、E型菌株各为2株. 结论 bla TEM-1、bla OXA-23和bla armA基因仍是引起AB耐药的主要原因之一;与此同时,产VIM-2型MBL联合外膜蛋白CarO缺失或改变也是导致烧伤患者AB对碳青霉烯类抗生素耐药的重要机制之一,其中Intl1基因也可能参与了bla VIM-2基因的传播.
更多Objective To study the drug resistance of Acinetobacter baumannii (AB) producing VIM-2-type metallo-β-lactamase (MBL) isolated from burn patients of our ward against carbapenem antibiotics and its homology.Methods A total of 400 strains of AB (identified) were isolated from sputum,urine,blood,pus,and wound drainage of burn patients hospitalized in our ward from September 2011 to March 2014.Drug resistance of the 400 strains of AB to 15 antibiotics,including compound sulfamothoxazole,aztreonam,etc.,was tested using the automatic microorganism identifying and drug sensitivity analyzer.Among the carbapenems-resistant AB isolates,modified Hodge test was applied to screen carbapenemase-producing strains.The carbapenemase genes of the carbapenemase-producing strains,and the mobile genetic elements class 1 integron (Intl1) gene and conserved sequence (CS) of carbapenemase-producing strains carrying bla VIM-2 gene were determined with PCR and DNA sequencing.For carbapenemase-producing strains carrying bla VIM-2 gene,synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to verify the MBL-producing status.Drug resistance of the VIM-2-type MBL-producing AB strains was analyzed.For VIM-2-type MBL-producing AB strains,plasmid conjugation experiment was used to explore the transfer of plasmid;outer membrane protein (OMP) CarO gene was detected by PCR.For VIM-2-type MBL-producing AB strains carrying CarO gene,the protein content of CarO was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis.The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing of VIM-2-type MBL-producing AB strains to analyze their homology.Results (1) The resistant rates of the 400 strains of AB against levofloxacin and compound sulfamethoxazole were low.A total of 381 carbapenems-resistant AB strains were screened,including 240 carbepenemase-producing strains.(2) Out of the 240 carbepenemase-producing strains,18 strains were found to harbor the bla VIM-2 gene,accounting for 7.5%;133 strains carried the bla TEM 1 gene,accounting for 55.42%;195 strains carried the bla OxA-23 gene,accounting for 81.25%;188 strains carried the bla armA gene,accounting for 78.33 %.(3)Eighteen carbepenemase-producing strains which carried the bla VIM-2 gene were found to carry the Intl1 gene,showing the Intl1-VIM linkage.Simultaneously,Intl1 variable area CS showed diversity.(4) Eighteen carbepenemase-producing strains which carried the bla VIM-2 gene were verified to produce MBL.The resistant rates of the 18 strains of AB against compound sulfamethoxazole were the lowest,followed by levofloxacin and cefoperazone/sulbactam,and those against the other antibiotics were above 60.00%.(5) Through multiple joint tests,plasmid conjugation experiment positive transfer strain was not found in 18 VIM-2-type MBL-producing AB strains.(6) Nine out of the 18 VIM-2-type MBL-producing AB strains were found to carry CarO gene.The OMP CarO of VIM-2-type MBL-producing AB strains carrying CarO gene was lost or lowered in the protein content.(7) The 18 VIM-2-type MBL-producing AB strains were classified into 6 genotypes by the ERIC-PCR.There were respectively 6,4,3,and 1 stain (s) in genotypes A,B,C,and F,and there were 2 strains in genotypes D and E respectively.Conclusions The resistance mechanism of AB against carbapenems is mainly mediated by bla TEM-1,bla OXA-23,and bla;meanwhile,VIM-2-type MBL-producing and lack or change in OMP CarO are attributable to carbapenems resistance of clinically isolated AB from burn wards,and the Intl1 gene may take a part in bla VIM-2 gene transmission.
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