志贺菌耐多药相关基因水平转移的验证及其耐药性研究

目的对志贺菌耐多药相关基因(T1C4)水平转移进行验证,并对其与细菌耐药性的关系进行初步判定。方法培养已筛选出的包含T1C4基因片段的阳性克隆,提取其质粒,PCR扩增T1C4基因片段、纯化、制备探针,与志贺菌敏感株SS23、志贺菌耐多药转移株(ST11)、大肠埃希菌耐多药株(E667)的基因组DNA和质粒DNA进行斑点杂交。采用药敏纸片法研究含T1C4基因片段大肠埃希菌的耐药性。结果应用PCR方法可以从ST11和E667菌株基因组DNA中扩增出T1C4基因片段,而SS23菌株不含该基因片段;应用T1C4基因探针进行斑点杂交显示,ST11和E667菌株的全基因组和质粒DNA均可显示杂交信号,而SS23菌株的基因组和质粒DNA均不产生杂交信号;14种药物的药敏试验表明,含有T1C4基因的大肠埃希菌(DH5α)对四环素、先锋V、头孢噻吩、氟哌酸、复方新诺明等五种药物的抑菌环直径明显小于(相差3mm以上)不含T1C4基因的DH5α菌株。结论 T1C4基因片段为ST11菌株从E667菌株获得,且存在于两菌株的质粒上;T1C4基因可能介导细菌对多种药物的抗性。

Identification on horizontal transfer of multi-drug resistance related gene fragment of Shigella and its drug-resistance

The objective of the present study was to identify horizontal transfer of gene fragment（T1C4） from E.coli to Shigella and its relationship with multi-drug resistance.Screened positive clone which contains T1C4 gene fragment was cultured and the plasmid was extracted.The fragment of T1C4 gene was then amplified by PCR and purified by gel purification kit.The probe was prepared and the dot hybridization was conducted for genomic DNA and plasmid DNA of Shigella sensitive strain（SS23）,transferred multi-drug resistant Shigella strain（ST11） and Escherichia colimulti-drug resistant clinical strain（E667）.Finally,antimicrobial sensitivity of DH5α,DH5α（pUC18）,DH5α（pUC18-T1C4） and ATCC25922 were detected by disk diffusion testing.The procedure was carried out in accordance with the guidelines in standard of antibiotics susceptibility test（Kirby-Bauer method） document.Results displayed that T1C4 gene fragment was amplified from genomic DNA of ST11 and E667 by PCR.The genomic DNAs and plasmid DNAs of transferred strain（ST11） and clinical strain（E667） all gave positive signals in dot hybridization,whereas both of genomic DNA and plasmid DNA of SS23 gave negative hybridization signal.The bacteriostatic diameters of Tetracycline,Cefazolin,Cefalotin,Norfloxacin and SMZ-TMP to DH5α（pUC18-T1C4） were significantly shorter than those to DH5α（pUC18）.It＇s concluded that gene fragment（T1C4） was transferred from E667 to ST11 with plasmid.Moreover,T1C4 gene fragment might be closely related to multi-drug resistance of bacteria.