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

Co-existence of resistance genes and their association with the genetic marker of integrons among multi-resistant Escherichia coli isolates

【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oprD2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qacEΔ1-sul1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qacEΔ1-sul1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qacEΔ1-sul1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qacEΔ1-sul1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)。

[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 ClassⅡ,Ⅲ integrons.16S rRNA methylase genes harboured plasmids of ～ 23 kb which transformed other isolates within the same strains efficiently.