铜绿假单胞菌耐亚胺培南现状与分子水平研究
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摘要
目的：1) 探讨耐亚胺培南铜绿假单胞菌在医院内的感染现状及耐药谱特性，以利于临床合理选用抗生素：2) 选择基于肠杆菌科基因间重复一致序列(enterobacterial repetitive intergenic consensus，ERIC)的聚合酶链反应(polymerasechain reaction，PCR)分型方法，对耐亚胺培南铜绿假单胞菌临床株进行分子生物学分型，以监测耐药菌株的流行状况；3) 探讨并建立可在临床微生物室常规应用的一种检测产金属β-内酰胺酶菌株的纸片协同试验方法；4) 探讨铜绿假单胞菌临床分离株中金属β-内酰胺酶流行的主要基因型，并分析产酶株耐药特性。
方法：1) 用VITEK 32型全自动细菌分析系统筛选武汉大学人民医院2001年1月～2002年12月期间耐亚胺培南铜绿假单胞菌，用琼脂稀释法检测耐药菌株的最低抑菌浓度(minimal inhibitory concentrations，MIC)；2) 采用ERIC-PCR分型方法对临床分离耐亚胺培南铜绿假单胞菌的DNA进行PCR扩增，用phylip 3.5c软件分析扩增产物电泳图谱，得到菌株间相似性聚类图；3) 以头孢他啶、头孢噻肟和亚胺培南为金属β-内酰胺酶底物，分别以乙二胺四乙酸二钠(EDTA·Na_2)和2-巯基丙酸为金属β-内酰胺酶抑制剂，在MH平板上用纸片扩散法对耐亚胺培南铜绿假单胞菌进行协同试验；4) 采用金属β-内酰胺酶流行基因型别的引物对耐亚胺培南铜绿假单胞菌进行PCR扩增并对PCR产物进行DNA测序，确定本地区金属β-内酰胺酶流行的主要基因型，并对其耐药性进行分析。
结果：1) 2001年、2002年分别从本院临床各类标本中分离到铜绿假单胞菌172株、226株，其中耐亚胺培南菌株数分别为21株(12.2％)、35株(15.5％)。总分离率为14.1％。其来源按标本类型来分以痰液最多，占58.9％(33／56)，按病区来分以重症监护病房(ICU)最高，占35.7％(20／56)。体外药物敏感试验所测10种抗菌药物中，耐亚胺培南铜绿假单胞菌对氯霉素耐药率最高，达85.7％；对头孢吡肟、氨曲南和阿米卡星的耐药率较低，分别为33.9％、32.1％和35.7％：对其余抗生素耐药率均在50％-70％之间；2) ERIC-PCR方法能很好的对铜绿假单胞菌进行分子生物学分型，56株耐药菌分为33型。3) 头孢他啶、头孢噻肟和亚胺培南三者
中任一药敏纸片与含酶抑制剂纸片间出现抑菌环扩大现象为协同试验阳性，提
示受试菌产金属p一内酞胺酶。在56株受试菌中，分别以EDTA.NaZ和2一琉基丙酸
为酶抑制剂进行协同试验，结果一致，阳性均为5株，但用EDTA.NaZ做酶抑制剂
产生的协同效应更清晰明显:4)5株协同试验阳性菌株blav，M_2基因扩增阳性，
其他菌株扩增均阴性。将PCR扩增阳性产物纯化后进行DNA测序，经序列同源
性比较，证实5株菌所产金属p一内酞胺酶基因型均为VIM一2型。产酶株对临床常
用的卜内酞胺类抗生素均不敏感，对非p一内酞胺类抗生素如环丙沙星、庆大霉
素和氯霉素也高度耐药，对单环p一内酞胺类抗生素(氨曲南)和阿米卡星相对较
敏感。
结论:1)耐亚胺培南铜绿假单胞菌在武汉大学人民医院内的分离率有随年
度增加趋势，其主要来源为ICU病房和老年病房，主要感染部位为呼吸道和伤口
分泌物;2)耐亚胺培南铜绿假单胞菌对常用抗生素耐药现象十分严重，治疗此
类细菌感染宜参考微生物室的细菌药敏结果，选用敏感的抗生素，并对其加强
临床检测和监控;3)E租C一PCR分型技术简便易行，适合于医院感染菌株的分
子流行病学研究，武汉大学人民医院2001一2002年分离的56株耐亚胺培南铜绿
假单胞菌被分为33型，其中NZ、N3;N4、N49;N 12、N34;N 15、N 19;N 18、
N54:N25、N56;N26、N47;N39、N48;N40、N55;NS、N43、N44:N17、
N23、N31;N24、N32、N41:N3O、N35、N36;N38、N42、N52;N6、N7、
Ng、N10、Nn分别是同一克隆的耐药菌株。4)以EDTA.NaZ为酶抑制剂的纸片
协同法是一种简便、敏感的筛选产金属p一内酞胺酶细菌的检测方法，可作为临
床微生物室常规初筛试验;5)武汉大学人民医院临床分离铜绿假单胞菌所产金
属卜内酞胺酶为VIM一2型。产酶株呈高度多重耐药，应注意对其进行临床检测
和监控。
Objective 1) To investigate the drug resistance of Imipenem-resistant Pseudomonas aeruginosa to instruct clinical application of antibiotics. 2) To establish a convenient polymerase chain reaction(PCR)method for bacterial DNA fingerprints analysis in epidemiology research of Imipenem-resistant Pseudomonas aeruginosa. 3) To explore and establish a simple and convenient method for screening metallo- β-lactamase-producing Pseudomonas aeruginosa in the clinical microbe laboratory. 4) To investigate the genotype characteristics and antibiotics sensitivity of clinical Pseudomonas asaeruginosa isolates producing metallo-B -lactamase.
Methods 1) Imipenem-resistant Pseudomonas aeruginosa isolated from 2001 to 2002 were identified by VITEK 32 system. The minimal inhibitory concentrations (MlC)of them to 10 antibiotics, including Imipenem, Piperacillin, Cefotaxime, Ceftazidime, Cefepime, Aztreonam, Ciprofloxacin, Gentamycin, Amikacin and Chloromycetine were detected by agar dilution method. 2) Strains were typed by rep-PCR with the primer enterobacter repetitive intergenic consensus(ERIC) following electrophoresis in agarose gel. 3) Using substrate Ceftazidime, Cefotaxime and Imipenem, enzyme inhibitor EDTA Na2 and 2-mercaptopropionic acid, the microbe sensitivity synergic tests were processed by KB method in MH agar. 4) Metallo-β-lactamase gene was detected by PCR method using primers specific for blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2, respectively, and the PCR products were purified and sequenced.
Results 1) Among 398 strains of Pseudomonas aeruginosa, 56 of them were resistant to Imipenem, mainly isolated from ICU ward. The resistance rate to Chloromycetine was the highest(85.7%). The resistance rates to Cefepine, Aztreonam and Amikacin were 33.9%, 32.1% and 35.7%, respectively. The resistance rates to
other 6 antimicrobial agents were from 50.0% to 70.0%. 2) 56 Imipenem-resitant Pseudomonas aeruginosa strains were typed to 33 genotype by ERIC-PCR. 3) 5 metallo-β-lactamase-producing Pseudomonas aeruginosa were detected among 56 strains. 4) Amplification of blaVIM-2 gene was positive in those 5 strains, and negtive in other strains. All 5 strains were resistant to most β-lactams antibiotics. Only was it rather sensitive to Aztreonam and Amikacin.
Conclusion 1) Pseudomonas aeruginosa strains resisted to Imipenem remains a problem. And they show increasing tendency year by year. 2) The method of ERIC-PCR is practical and economical for epidemiology research in nosocomial infection. 3) The synergic test was simple, convenient and sensitive for screening metallo- β-lactamase-producing bacteria. It was suitable for screening metallo-β-lactamase- producing Pseudomonas aeruginosa routinely in the clinical microbe laboratory. 4) VIM-2 was the genotype of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa in this research. Multiple drugs resistant occurred seriously in these strains.
方法：1) 用VITEK 32型全自动细菌分析系统筛选武汉大学人民医院2001年1月～2002年12月期间耐亚胺培南铜绿假单胞菌，用琼脂稀释法检测耐药菌株的最低抑菌浓度(minimal inhibitory concentrations，MIC)；2) 采用ERIC-PCR分型方法对临床分离耐亚胺培南铜绿假单胞菌的DNA进行PCR扩增，用phylip 3.5c软件分析扩增产物电泳图谱，得到菌株间相似性聚类图；3) 以头孢他啶、头孢噻肟和亚胺培南为金属β-内酰胺酶底物，分别以乙二胺四乙酸二钠(EDTA·Na_2)和2-巯基丙酸为金属β-内酰胺酶抑制剂，在MH平板上用纸片扩散法对耐亚胺培南铜绿假单胞菌进行协同试验；4) 采用金属β-内酰胺酶流行基因型别的引物对耐亚胺培南铜绿假单胞菌进行PCR扩增并对PCR产物进行DNA测序，确定本地区金属β-内酰胺酶流行的主要基因型，并对其耐药性进行分析。
结果：1) 2001年、2002年分别从本院临床各类标本中分离到铜绿假单胞菌172株、226株，其中耐亚胺培南菌株数分别为21株(12.2％)、35株(15.5％)。总分离率为14.1％。其来源按标本类型来分以痰液最多，占58.9％(33／56)，按病区来分以重症监护病房(ICU)最高，占35.7％(20／56)。体外药物敏感试验所测10种抗菌药物中，耐亚胺培南铜绿假单胞菌对氯霉素耐药率最高，达85.7％；对头孢吡肟、氨曲南和阿米卡星的耐药率较低，分别为33.9％、32.1％和35.7％：对其余抗生素耐药率均在50％-70％之间；2) ERIC-PCR方法能很好的对铜绿假单胞菌进行分子生物学分型，56株耐药菌分为33型。3) 头孢他啶、头孢噻肟和亚胺培南三者
中任一药敏纸片与含酶抑制剂纸片间出现抑菌环扩大现象为协同试验阳性，提
示受试菌产金属p一内酞胺酶。在56株受试菌中，分别以EDTA.NaZ和2一琉基丙酸
为酶抑制剂进行协同试验，结果一致，阳性均为5株，但用EDTA.NaZ做酶抑制剂
产生的协同效应更清晰明显:4)5株协同试验阳性菌株blav，M_2基因扩增阳性，
其他菌株扩增均阴性。将PCR扩增阳性产物纯化后进行DNA测序，经序列同源
性比较，证实5株菌所产金属p一内酞胺酶基因型均为VIM一2型。产酶株对临床常
用的卜内酞胺类抗生素均不敏感，对非p一内酞胺类抗生素如环丙沙星、庆大霉
素和氯霉素也高度耐药，对单环p一内酞胺类抗生素(氨曲南)和阿米卡星相对较
敏感。
结论:1)耐亚胺培南铜绿假单胞菌在武汉大学人民医院内的分离率有随年
度增加趋势，其主要来源为ICU病房和老年病房，主要感染部位为呼吸道和伤口
分泌物;2)耐亚胺培南铜绿假单胞菌对常用抗生素耐药现象十分严重，治疗此
类细菌感染宜参考微生物室的细菌药敏结果，选用敏感的抗生素，并对其加强
临床检测和监控;3)E租C一PCR分型技术简便易行，适合于医院感染菌株的分
子流行病学研究，武汉大学人民医院2001一2002年分离的56株耐亚胺培南铜绿
假单胞菌被分为33型，其中NZ、N3;N4、N49;N 12、N34;N 15、N 19;N 18、
N54:N25、N56;N26、N47;N39、N48;N40、N55;NS、N43、N44:N17、
N23、N31;N24、N32、N41:N3O、N35、N36;N38、N42、N52;N6、N7、
Ng、N10、Nn分别是同一克隆的耐药菌株。4)以EDTA.NaZ为酶抑制剂的纸片
协同法是一种简便、敏感的筛选产金属p一内酞胺酶细菌的检测方法，可作为临
床微生物室常规初筛试验;5)武汉大学人民医院临床分离铜绿假单胞菌所产金
属卜内酞胺酶为VIM一2型。产酶株呈高度多重耐药，应注意对其进行临床检测
和监控。
Objective 1) To investigate the drug resistance of Imipenem-resistant Pseudomonas aeruginosa to instruct clinical application of antibiotics. 2) To establish a convenient polymerase chain reaction(PCR)method for bacterial DNA fingerprints analysis in epidemiology research of Imipenem-resistant Pseudomonas aeruginosa. 3) To explore and establish a simple and convenient method for screening metallo- β-lactamase-producing Pseudomonas aeruginosa in the clinical microbe laboratory. 4) To investigate the genotype characteristics and antibiotics sensitivity of clinical Pseudomonas asaeruginosa isolates producing metallo-B -lactamase.
Methods 1) Imipenem-resistant Pseudomonas aeruginosa isolated from 2001 to 2002 were identified by VITEK 32 system. The minimal inhibitory concentrations (MlC)of them to 10 antibiotics, including Imipenem, Piperacillin, Cefotaxime, Ceftazidime, Cefepime, Aztreonam, Ciprofloxacin, Gentamycin, Amikacin and Chloromycetine were detected by agar dilution method. 2) Strains were typed by rep-PCR with the primer enterobacter repetitive intergenic consensus(ERIC) following electrophoresis in agarose gel. 3) Using substrate Ceftazidime, Cefotaxime and Imipenem, enzyme inhibitor EDTA Na2 and 2-mercaptopropionic acid, the microbe sensitivity synergic tests were processed by KB method in MH agar. 4) Metallo-β-lactamase gene was detected by PCR method using primers specific for blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2, respectively, and the PCR products were purified and sequenced.
Results 1) Among 398 strains of Pseudomonas aeruginosa, 56 of them were resistant to Imipenem, mainly isolated from ICU ward. The resistance rate to Chloromycetine was the highest(85.7%). The resistance rates to Cefepine, Aztreonam and Amikacin were 33.9%, 32.1% and 35.7%, respectively. The resistance rates to
other 6 antimicrobial agents were from 50.0% to 70.0%. 2) 56 Imipenem-resitant Pseudomonas aeruginosa strains were typed to 33 genotype by ERIC-PCR. 3) 5 metallo-β-lactamase-producing Pseudomonas aeruginosa were detected among 56 strains. 4) Amplification of blaVIM-2 gene was positive in those 5 strains, and negtive in other strains. All 5 strains were resistant to most β-lactams antibiotics. Only was it rather sensitive to Aztreonam and Amikacin.
Conclusion 1) Pseudomonas aeruginosa strains resisted to Imipenem remains a problem. And they show increasing tendency year by year. 2) The method of ERIC-PCR is practical and economical for epidemiology research in nosocomial infection. 3) The synergic test was simple, convenient and sensitive for screening metallo- β-lactamase-producing bacteria. It was suitable for screening metallo-β-lactamase- producing Pseudomonas aeruginosa routinely in the clinical microbe laboratory. 4) VIM-2 was the genotype of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa in this research. Multiple drugs resistant occurred seriously in these strains.
引文
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16．韩立中，蒋燕群，蒋晓飞等．介导VIM-2型金属β内酰胺酶的整合子的研究．中国抗感染化疗杂志，2003，3：347-350．
17．张怡滨，宋诗铎，祁伟等．临床分离耐亚胺培南铜绿假单胞菌金属酶基因的PCR检测．天津医药，2002，30：131-133．
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13. Spyros P, Athanassios T, Maria M, et al. Novel variant (blaVIM-4) of the Metallo-β-lactamasc Gene blaVIM-1 in a clinical strain of Pseudomonas aeruginosa. Antimicrob Agents Chemother, 2002, 46:4025-4028.
14. Kyungwon L, Jong BL, Jong HY, et al. blaVIM-2 cassette-containing novel integron in mctallo-β-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in Korean hospital. Antimicrob Agents Chemother, 2002, 45: 1053-1058.
15. Jong HY, Keonsoo Y, Kyungwon L, et al. Molecular Characterization of metallo-β-lactamasc-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korean: identification of two new integrons carrying the blaVIM-2 gene cassettes. Journal of Antimierobial Chemotherapy, 2002, 49: 837-840.
16．韩立中，蒋燕群，蒋晓飞等．介导VIM-2型金属β内酰胺酶的整合子的研究．中国抗感染化疗杂志，2003，3：347-350．
17．张怡滨，宋诗铎，祁伟等．临床分离耐亚胺培南铜绿假单胞菌金属酶基因的PCR检测．天津医药，2002，30：131-133．