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
소상환자중산VIM-2형금속β내선알매포씨불동간균내약궤제급동원성분석
Analysis of the mechanism of drug resistance of VIM-2-type metallo-β-lactamase-producing Acinetobacter baumannii isolated from burn patients and its homology
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2015年 3期 205-210 ,共6页

    杨喜丽%李悦%詹剑华%郭菲%闵定宏%王年云%李国辉%郭光华

    양희려%리열%첨검화%곽비%민정굉%왕년운%리국휘%곽광화

    烧伤%感染%鲍氏不动杆菌%抗药性%β内酰胺酶类%细菌外膜蛋白质类%碳青霉烯酶 燒傷%感染%鮑氏不動桿菌%抗藥性%β內酰胺酶類%細菌外膜蛋白質類%碳青黴烯酶
    소상%감염%포씨불동간균%항약성%β내선알매류%세균외막단백질류%탄청매희매
    Burns%Infection%Acinetobacter baumannii%Drug resistance%Beta-lactamases%Bacterial outer membrane proteins%Carbapenems
    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.
    목적 탐토아원소상환자중산VIM-2형금속β내선알매(MBL)적포씨불동간균(AB) l대탄청매희류항생소적내약궤제급동원성. 방법 2011년9월-2014년3월,수집종필자단위수치적소상주원환자담액、뇨액、혈액、농액、인류액중분리적400주AB(경감정).채용전자동미생물감정급약민분석계통측정균주대복방광알갑(구악)서、안곡남등15충항생소적내약성.침대항탄청매희류항생소균주,채용개량Hodge시험사선산탄청매희매균주.PCR법급측서검측산탄청매희매AB적탄청매희매기인、휴대blaVIM-2기인적산탄청매희매AB적가이동기인원건1류정합자(Intl1)급기보수편단(CS).침대휴대blaVIM-2기인산탄청매희매AB행아알배남-을이알사을산(EDTA)협동시험이급아알배남-EDTA여두포타정-EDTA증효시험험증기산MBL정황,병분석산VIM-2형MBL적AB적내약정황.대산VIM-2형MBL적AB행질립접합시험검측질립전이정황,PCR법검측외막단백CarO기인.십이완기류산납-취병희선알응효전영검측휴대CarO기인적산VIM-2형MBL적AB균주적CarO단백표체량.장간균기인간중복일치서렬(ERIC)-PCR법대산VIM-2형MBL적AB균주진행기인분형,분석동원성. 결과 (1)400주AB대좌양불사성화복방광알갑(구악)서적내약솔저.공사선출381주항탄청매희류항생소AB,기중240주산탄청매희매.(2)240주산탄청매희매AB중검출18주휴대VIM-2매기인blaVIM-2,점7.5％;133주휴대bla TEM-1기인,점55.42％;195주휴대blaOXA-23기인,점81.25％;188주휴대bla armA기인,점78.33％.(3)18주휴대blaVIM-2기인적산탄청매희매AB균휴대Intl1기인,정현Intl1-VIM련쇄휴대형,Intl1가변구CS정현다양성.(4)경험증,18주휴대blaVIM-2기인적산탄청매희매AB균위산VIM-2형MBL적AB.해18주AB균주대복방광알갑(구악)서적내약솔최저,기차위좌양불사성화두포고동/서파탄,대기여항생소적내약솔균대우60.00％.(5)18주산VIM-2형MBL적AB경다차질립접합시험균미견내약기인양성전이균주.(6)18주산VIM-2형MBL적AB중9주휴대CarO기인,휴대CarO기인적산VIM-2형MBL적AB균주외막단백CarO결실혹표체량감소.(7)18주산VIM-2형MBL적AB ERIC-PCR지문도보공분6개보형,A、B、C、F형균주분별위6、4、3、l주,D、E형균주각위2주. 결론 bla TEM-1、bla OXA-23화bla armA기인잉시인기AB내약적주요원인지일;여차동시,산VIM-2형MBL연합외막단백CarO결실혹개변야시도치소상환자AB대탄청매희류항생소내약적중요궤제지일,기중Intl1기인야가능삼여료bla VIM-2기인적전파.
    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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