东北地区狐源大肠杆菌耐药性、整合酶及整合子基因的检测与相关分析

Detection and correlation analysis of drug resistance,integrase and integron genes of Escherichia coli from foxes in Northeast China

为了解东北地区狐源大肠杆菌的耐药性及其整合子—基因盒系统的流行特征,本研究采用K-B纸片法检测东北地区分离的330株狐源大肠杆菌对6类15种抗菌药物的敏感性;通过PCR检测这些分离菌株中I类整合酶基因(intI1)、II整合酶基因(intI2)和I类、II类整合子基因并测序,采用DNAStar软件分析I类、II类整合子基因携带基因盒的组成方式,根据PCR结果分析整合酶及整合子基因与大肠杆菌耐药表型的相关性。药敏试验结果显示:330株狐源大肠杆菌对β-内酰胺类药物和四环素类药物耐药性较高,耐药菌株均超过80%;其次是对氨基糖苷类药物和磺胺类药物,耐药菌株超过60%;对β-内酰胺类药物亚胺培南、氨基糖苷类药物阿米卡星相对敏感,敏感菌株分别占86.97%(287/330)和83.94%(277/330),且87.88%(290/330)的狐源大肠杆菌表现为多重耐药性。整合酶和整合子基因的检测结果显示,intI1和intI2基因的检出率分别为63.64%(210/330)和15.45%(51/330),且有36株(10.9%)狐源大肠杆菌同时携带intI1和intI2基因。I类整合子基因的检出率为62.86%(207/330),其中33.81%(70/207)I类整合子基因中携带基因盒,其余66.19%(137/207)I类整合子基因中未携带基因盒,为空整合子,且未检出II类整合子基因;70个I类整合子基因的测序结果显示,共检测出10种基因盒,且存在6种不同的基因盒组成方式,其中aadA22基因盒数量最多,达48.57%(34/70),其余基因盒组成分别为dfrA17-aadA5、dfrA27-aadA2、dfrA5、dfrA1-aadA1、dfrA12-orfF-aadA2。且上述整合酶基因(与氨基糖苷类和磺胺类耐药基因相关)及整合子基因(与甲氧苄啶类和氨基糖苷类药物的耐药性相关,但未做甲氧苄啶类的耐药性试验)与狐源大肠杆菌对氨基糖苷类和磺胺类药物的耐药表型基本一致。以70株携带基因盒的I类整合子的狐源大肠杆菌为供体菌,E.coli NK5449株作为受体菌进行接合转移试验,提取接合子质粒并经PCR测序后统计接合率;通过PCR分别检测接合子携带的整合酶和整合子基因,分析接合子中耐药基因的水平转移情况。结果显示,共获得23株接合子,接合率为32.86%(23/70),23株接合子中I类整合酶基因的检出率为34.78%(8/23),I类整合子基因检出率为43.48%(10/23)。上述结果表明,耐药基因具有水平转移的能力,但并不能将全部的耐药基因转移到受体菌中。本研究为东北地区狐源大肠杆菌的风险评估和抗菌药的合理应用提供参考依据。

To understand the resistance of E.coli from foxes and the prevalence characteristics of its integron-cassette system in Northeast China,the sensitivity of 330 fox-derived E.coli isolates to 6 classes and 15 kinds of antibiotics was detected by K-B disk method.The class I integrase gene(intI1),class II integrase gene(intI2)and class I and II integron genes were detected by PCR and sequenced.The composition of class I and II integron gene carrying cassette was analyzed by DNAStar software.The correlation between integrase and integron genes with drug resistance phenotype of E.coli was analyzed.The drug sensitivity test showed that more than 80%of the 330 strains were resistant toβ-lactam and tetracycline,and more than 60%strains were resistant to paraminoglycosides and sulfonamides,and there were 86.97%(287/330)and 83.94%(277/330)susceptible strains sensitive toβ-lactam imipenem and aminoglycoside amikacin,respectively,while 87.88%(290/330)strains showed multiple drug resistance.The detection rate of integrase genes intI1 and intI2 was 63.64%(210/330)and 15.45%(51/330),respectively,and 36 strains(10.9%)carried both intI1 and intI2 genes.The detection rate of class I integron genes was 62.86%(207/330),among which 33.81%(70/207)of class I integron genes carried gene cassette,and the remaining 66.19%(137/207)of class I integron genes did not carry gene cassette,were empty integrons,and no class II integron genes were detected.Sequencing of 70 strains carrying class I integron genes showed that a total of 10 loci were detected,and there were 6 different loci composition modes,among which aadA22 loci accounted for 48.57%(34/70).The other loci were dfrA17-aadA5,dfrA27-aadA2,dfrA5,dfrA1-aadA1 and dfrA12-orfF-aadA2.The above integrase genes(associated with aminoglycoside and sulfonamide resistance)and integron genes(related to the resistance to trimethoprim and aminoglycosides,but the resistance to trimethoprim was not tested)were related to the resistance to aminoglycosides and sulfonamides in the E.coli strains from foxes.The 70 strains carrying class I integrons containing gene cassette were used as donor and E.coli NK5449 strain was used as recipient in the conjugation transfer experiment,the zygote plasmid DNA was extracted and the conjugation rate was calculated after PCR sequencing.The conjugation integrase gene and the integron gene were detected by PCR,and the horizontal transfer of resistance genes in the conjugants was analyzed.The results showed that a total of 23 strains of zygotes were obtained,the conjugation rate was 32.86%(23/70),and the detection rate of class I integrase gene and class I integron gene was 34.78%(8/23)and 43.48%(10/23),respectively.The above results showed that the drug resistance genes had the ability of horizontal transfer,but not all of them can be transferred to recipient bacteria.The results of this study provide scientific basis for the risk assessment of E.coli from foxes in Northeast China and rational application of antibacterial drugs.