鲍曼不动杆菌ATCC19606多重耐性分析及耐药基因的克隆

          作者：苏显中  张兴  陈彦土 屋友房

【摘要】  鲍曼不动杆菌已成为重要的院内感染病菌。我们测定了鲍曼不动杆菌ATCC19606的药物最小抑制浓度(MIC)，结果显示该菌株对多种抗菌药物都有很高的耐性，如链霉素、诺氟沙星、氯霉素、红霉素、四环素、氨苄西林及一些其他的抗菌染剂。利用大肠埃希菌超敏菌株KAM32作为宿主，从鲍曼不动杆菌ATCC19606染色体DNA中克隆耐药基因，共获得9个使宿主细胞产生耐药性的杂合质粒，其中1个为单一耐药，其余全部为多重耐药。根据药物特异性分析可知，具有不同耐药图谱的杂合质粒携带不同类型的耐药基因。由此揭示鲍曼不动杆菌ATCC19606的多重耐药有多种机制参与。

【关键词】  多重耐药； 基因克隆； 鲍曼不动杆菌

Multiple resistance in Acinetobacter baumannii ATCC 19606　and cloning of genes responsible for the resistance

　　ABSTRACT  Drug resistance of Acinetobacter baumannii ATCC 19606 was tested in this study. The result showed fairly high resistance to many antimicrobial agents tested including streptomycin, norfloxacin, chloramphenicol, erythromycin, tetracycline, ampicillin, and antimicrobial dyes. Using the drug?hypersensitive strain of Escherichia coli KAM32 as the host, we cloned the genes responsible for multiple resistance from chromosomal DNA of A.baumannii ATCC 19606. We obtained 9 hybrid plasmids that made host cells resistant to several antimicrobial agents. Many of the transformants harboring each of the plasmids showed multiple resistance, and one showed resistance to specific drug. The hybrid plasmids were classified into several groups based on their drug specificity. It appears that each class of plasmid carries different types of drug resistance genes. Analysis of such genes will reveal the various mechanisms involved in multiple resistance in A.baumannii ATCC 19606.
   
　　KEY WORDS  Multiple resistance;  Gene cloning;  Acinetobacter baumannii
　　The appearance and spread of drug resistance in pathogenic organisms have made many available antibiotics ineffective［1, 2］. Drug resistance, especially multiple resistance, in bacterial cells has currently become an increasing serious clinical problem. An extensive knowledge of molecular mechanisms underlying bacterial drug resistance is required to control multiple resistant bacteria and to treat patients infected with multiple?resistant bacteria successfully.
   
　　Certain strains of A.baumannii are now resistant to all commonly prescribed antibiotics (including imipenem and the new fluoroquinolones), and reports of a high prevalence of fluoroquinolone resistance among Acinetobacter isolates have appeared［3,4］.
   
　　It is important to investigate the mechanisms by which A.baumannii acquires drug resistance. In this study, we report patterns of drug resistance in strain A.baumannii ATCC 19606 and functional gene cloning of the drug?resistance systems.

　　1  Materials and methods

　　1.1  Bacteria and growth
   
　　A.baumannii ATCC 19606 (generously provided by Professor Shigeo Yamamoto, Faculty of Pharmaceutical Sciences, Okayama University, Japan) and Escherichia.coli KAM32 were used in this study. E.coli TG1 and Pseudomonas aeruginosa PAO1 were also used. E.coli KAM32, a derivative of E.coli TG1, lacks the major multiple efflux pumps AcrB［5］ and YdhE［6］, and shows hypersensitivity to many antimicrobial agents and is useful as a host for gene cloning of drug?resistance systems. Bacterial cells were grown in LB medium (Difco) with aeration at 37℃ overnight. The growth of cells was monitored by measuring optical density at 650nm.

　　1.2  Drug susceptibility sest
   
　　The minimal inhibitory concentrations (MICs) of various drugs were determined by serial two?fold dilution of antimicrobials in the Mueller?Hinton broth (Difco) containing different drugs at various concentrations. MIC values were determined as a concentration of an antimicrobial that completely prevented cell growth during an 24h incubation at 37℃ with aeration.

　　1.3  Gene cloning
   
　　Chromosomal DNA was prepared from cells of A.baumannii ATCC 19606 by the method of Chen and Kuo［7］. The DNA was partially digested with Sau3AI, and fragments of 4 to 10 kbp were separated by sucrose density gradient centrifugation. The DNA fragments were ligated into plasmid pBR322 (which was digested with BamHI and dephosphorylated with shrimp alkaline phosphatase) using a ligation kit, Ver. 2 (TaKaRa Co.). The ligated hybrid DNA was transformed into competent cells of E.coli KAM32, then the cells were spread onto agar (1.5%) plates containing LB broth, ampicillin100 μg/ml, and one of the following drugs: norfloxacin (0.05 μg/ml), ethidium bromide (10 μg/ml) or 4′, 6?diamidino?2?phenylindole (DAPI) (0.5 μg/ml). The plates were incubated at 37℃ for 24 h. We obtained 9 candidate colonies on the plates. Each candidate colony was purified by single colony isolation on the plate containing each drug. Plasmids were isolated from each of the candidates. Plasmid DNAs were retransformed in the KAM32 cells, spread on the same type of plate, and their resistance against various antimicrobial agents tested. We confirmed that one type of plasmid was harbored in each candidate clone by the retransformation and by checking the restriction fragment of each plasmid. All hybrid plasmids carried DNA inserts of 3.0 to 11 kbp.

　　2  Results and discussion

　　2.1  Multiple resistance in A.baumannii
   
　　To investigate drug resistance patterns, we determined the MICs of various antimicrobial agents in A.baumannii ATCC 19606. We compared the MICs with those in E.coli TG1 and P.aeruginosa PAO1 that showed intrinsic multiple resistance. The results showed this strain was resistant to a variety of antimicrobial agents including aminoglycosides, quinolones, β?lactams, detergents, dyes and antiseptics (Tab.1). In addition, the
       
　　Tab.1        Susceptibility of several bacteria to diverse antimicrobial agents　略

　　strain exhibited a lower level of resistance to impenem and carbenicillin, but not ampicillin in β?lactams, as well as to nalidixic acid and not norfloxacin in quinolones. Thus it is clear that the clinically isolated strain of A.baumannii are resistant to many antimicrobial agents, and the level of resistance is higher than that in E.coli TG1, but similar substrates specificity with P.aeruginosa PAO1, a well?known multiple resistant bacterium.

　　2.2  Functional cloning of genes for drug resistance and classification
   
　　We tried to clone A.baumannii ATCC 19606 genes responsible for drug resistance using E.coli KAM32, a drug hyper?sensitive strain, as the host. We obtained 9 candidate clones harboring recombinant plasmids using some antimicrobial agents for the selection of drug?resistant clones. Three clones were obtained from the norfloxacin plate, four clones from the ethidium bromide plate, two clones from the DAPI plate (Tab.2). They showed different patterns of drug resistance. One clone obtained from the norfloxacin plate showed resistance only to norfloxacin among the antimicrobial agents tested, and others showed resistance not only to norfloxacin, but also to kanamycin, DAPI, chloramphenicol, and ethidium bromide. Drug resistance patterns among clones obtained from the ethidium bromide plate were versatile and classified into three types. The first type (one clone) showed resistance to kanamycin, norfloxacin, DAPI, and ethidium bromide. The second type (one clone) was resistant to kanamycin, norfloxacin, erythromycin, DAPI and ethidium bromide. The third type (two clones) was resistant to all the antimicrobial agents tested. The two clones obtained from the DAPI plate showed resistance not only to DAPI but also to kanamycin, norfloxacin, and ethidium bromide. We checked the restriction fragments of each plasmid and confirmed that plasmids that conferred the same drug specificity possessed a common DNA fragment. Thus, it appears that we cloned five different types of drug resistance gene (s). Among them, one type was specific to norfloxacin, and others were resistant to multiple
   
　　Tab.2        Patterns of drug resistance in each candidate　Drug used for    略

　　structurally and functionally unrelated antimicrobial agents. Those four types may represent multiple efflux pumps, because only the multiple efflux pump is involved in all multiple resistance among known drug?resistance mechanisms. In E.coli or P.aeruginosa, in which the existence of more than 30 multiple efflux pumps is suggested from their genome sequences, it is clear that multiple efflux pumps are mainly involved in intrinsic and acquired multiple resistance. Although we do not know at present how many systems of drug resistance are present in A.baumannii, it appears that cloned genes of A.baumannii contribute to the observed multiple resistance of this microorganism. Genetic analysis of the cloned genes and biochemical analysis of the gene products are now in progress.

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