猪源肠外致病性大肠杆菌耐药性分析及耐药基因和I类整合子检测

Analysis of drug resistance and detection of drug resistance gene and class I integron in porcine ExPEC

摘要:

[背景] 由于抗生素的长期大量且不合理使用，猪源肠外致病性大肠杆菌（extraintestinal pathogenic Escherichia coli，ExPEC）多重耐药性日趋严重。[目的] 探究猪源ExPEC的耐药性及其与耐药基因和I类整合子的相关性。[方法] 采用微量肉汤稀释法测定54株猪源ExPEC对22种抗生素的最低抑菌浓度（minimal inhibitory concentration，MIC）和最低杀菌浓度（minimum bactericidal concentration，MBC）；依据药敏试验结果确定相关耐药基因，采用PCR方法检测染色体DNA和质粒DNA上的耐药基因及I类整合子分布情况。[结果] 54株猪源ExPEC对青霉素、氟苯尼考、氨苄西林、阿莫西林高度耐药，其中52株对甲氧苄氨嘧啶、复方新诺明高度耐药，它们的MIC值均大于256 μg/mL，无MBC值；对头孢唑林、四环素、头孢氨苄、大观霉素、链霉素的MIC值在1-256 μg/mL之间，MBC值分别为8、16、32、64、128、256 μg/mL，均可耐受11种以上抗生素，其中以耐受17种为主，占比为18.52%，耐药谱有47种。确定了7类相关耐药基因20种，染色体DNA除aph（3'）-IIa基因外的19种耐药基因均可检出，以aadA1和tetA检出率最高，所有菌株均携带5种以上耐药基因；质粒DNA除tetB、tetC、SHV、qnrA基因外的16种耐药基因均可检出，以floR和parC检出率最高，所有菌株均携带5种以上耐药基因。I类整合子在染色体DNA和质粒DNA中阳性检出率分别为96.3%和98.15%，阳性菌株均为多重耐药。[结论] 猪源ExPEC多重耐药性严重，耐药谱复杂多样，耐药基因与I类整合子在染色体DNA和质粒DNA中分布率极高，耐药性的产生与耐药基因的存在具有一定相关性，而I类整合子的存在又增加了耐药基因水平转移的风险。

Abstract:

[Background] The long-term massive and irrational use of antibiotics has led to increasing multi-drug resistance of extraintestinal pathogenic Escherichia coli (ExPEC) isolated from pigs. [Objective] To investigate the drug resistance of ExPEC isolated from pigs and its correlation with drug resistance genes and class I integrons. [Methods] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 54 strains of ExPEC isolated from pigs against 22 antimicrobial drugs were determined by micro-broth dilution method. The drug resistance genes were identified based on the results of drug sensitivity tests, and the distribution of drug resistance genes and class I integrons on chromosomal DNA and plasmid DNA were detected by PCR. [Results] The 54 strains of porcine ExPEC were highly resistant to penicillin, florfenicol, ampicillin and amoxicillin, of which 52 strains of porcine ExPEC were highly resistant to trimethoprim and cotrimoxazole, and their MIC values were greater than 256 μg/mL, no MBC value; and MICs to cefazolin, tetracycline, cefadroxil, daikonin, and streptomycin were 1-256 μg/mL with MBC values of 8, 16, 32, 64, 128, and 256 μg/mL, all tolerating more than 11 antimicrobial drugs, of which 17 were predominantly tolerated, accounting for 18.52%, with 47 drug resistance spectrums. We identified 20 drug resistance genes in 7 major categories, 19 drug resistance genes in chromosomal DNA except aph(3')-IIa gene were detected, with the highest detection rate of aadA1 and tetA, and all strains carried more than 5 drug resistance genes; 16 drug resistance genes in plasmid DNA except tetB, tetC, SHV and qnrA genes were detected, with the highest detection rate of floR and parC, and all strains carried more than 5 drug resistance genes. The positive detection rates of class I integrons in chromosomal DNA and plasmid DNA were 96.3% and 98.15%, respectively, and all positive strains were multi-drug resistant strains. [Conclusion] ExPEC isolated from pigs has serious multi-drug resistance, the drug resistance profiles are complex and diverse, a very high distribution of resistance genes and class I integrons in chromosomal DNA and plasmid DNA, and a correlation between the development of resistance and the presence of resistance genes, which in turn increases the risk of horizontal transfer of resistance genes.