多重耐药大肠杆菌耐药基因与整合子遗传标记基因间的相关性

摘要

[Objective]To investigate co-existence of resistance genes ( β-lactamases, BLs, and aminoglycoside-modifying enzymes, AMEs ) and their association with the genetic marker genes of Class Ⅰ ,Ⅱ , Ⅲ integrons carried by multiresistant Escherichia coli isolates.[Methods]We used VITEK-GNS to determine the susceptibility of 136 isolates to 14 antibiotics, disc agar diffusion test to confirm ESBL-producing isolates, PCR to analyze BLs, AMEs and integrons genes, conjugation and plasmids extraction to locate the methylase genes.[Results]We found that 70.59％ of the isolates produced ESBLs.They showed stronger resistance against 9 antibiotics than isolates without ESBLs in 14 antibiotics.PCR amplification showed that the positive rate of BLs, AMEs and qacEΔ1-sul1 was 96.32％ , 100％ and 94.12％ , respectively, but Class Ⅱ ,Ⅲ integrons genes were negative.Only one strain was oprD2 gene negative.90.44％ of the isolates were both positive for BLs and qacEΔ1-sul1 genes, and 94.12％ for AMEs and qacEΔ1-sul1 genes, but there was no statistical significance.90.44％ of the isolates were all positive for the 3 genes.12 strains carried 16S rRNA methylase genes including armA (2.21％ ) , rmtB (7.35％ ) while rmtA, rmtC, rmtD were negative.The conjugation assay and plasmids mapping results showed that the methylase genes were located on the 23 kb plasmid, and the efficiency of transformation was 83.3％.[Conclusions]The results suggested that there was a tight correlation between the 3 genes (BLs, AMEs and qacEΔ1-sul1)and the incidences of multi-resistance of Escherichia coli, but there was no correlation of the incidence of multi-resistance with Class Ⅱ ,Ⅲ integrons.16S rRNA methylase genes harboured plasmids of ～ 23 kb which transformed other isolates within the same strains efficiently.%[目的]对多重耐药大肠埃希菌(Escherichia coli)临床株的β-内酰胺酶类(β-lactamases,BLs)和氨基糖苷钝化酶类(aminoglycoside modifying enzymes,AMEs)耐药基因与Ⅰ-Ⅲ类整合子遗传标记基因之间的相关性进行研究.[方法]采用VITEK-GNS药敏卡测定136株E.coli对14种抗菌素的敏感性;纸片扩散法确认超广谱β-内酰胺酶(extended-spectrum beta-lactamases,ESBLs)产酶株;PCR法检测BLs、AMEs相关耐药基因及Ⅰ-Ⅲ类整合子遗传标记基因;接合传递试验和质粒提取对16S rRNA甲基化酶基因进行初步定位.[结果]136株多重耐药的E.coli ESBLs产生率高达70.59%(96/136),产酶株对氨苄西林、庆大霉素、妥布霉素、亚胺培南和哌拉西林/他唑巴坦以外的其余9种抗菌素的耐药率显著高于非产酶株(P<0.05).BLs耐药基因总检出率为96.32%(131/136),1株外膜通道蛋白oprD:基因缺失,AMEs耐药基因总检出率为100%(136/136),Ⅰ类整合子遗传标记基因qacE△1-sull的检出率为94.12%(128/136),未检出Ⅱ类与Ⅲ类整合子基因.BLs基因和AMEs基因与qacE△l-sull遗传标记基因的同时携带率分别为90.44%(123/136)和94.12%(128/136),两类同时携带率之间的差异不具统计学意义(P>0.05),上述两类耐药基因与qacEAl-sull遗传标记基因的三者同时携带率为90.44%(123/136).此外,还检出16S rRNA甲基化酶基因12株(8.82%),其中,armA与rmtB的检出率分别为2.21%和7.35%,未检出rmtA、rmtC和rmtD.接合试验与质粒图谱结果初步表明:armA和rmtB编码基因位于约23 kb的质粒上,其耐药质粒在同种菌间的传递率高达83.3%(10/12).[结论]多重耐药E.coli临床株的BLs基因、AMEs基因与qacE△1-sull遗传标记基因三者同时携带率高达90.44%,表明多重耐药的形成在三者间具有密切的相关性;实验结果还显示:E.coli临床株的多重耐药性形成和传播与Ⅱ和Ⅲ类整合子基因无关.另外,16S rRNA甲基化酶基因携带率为8.82%(armA 2.21%和rmtB 7.35%),并初步证明armA和rmtB编码基因位于约23 kb的质粒上,其耐药质粒在同种菌间的传递率高达83.3%(10/12).