食品动物源大肠杆菌耐药性分析及16S rRNA甲基化酶基因的检测

Analysis on resistance of Escherichia coli from food of animal origin and detection of its 16S rRNA methylation enzymes

【目的】对四川地区临床分离的437株健康动物源及82株患病动物源大肠杆菌(E.coli)的耐药性及其机制进行研究,为临床合理用药提供理论依据。【方法】用3种氨基糖苷类药物和11种非氨基糖苷类药物对分离的大肠杆菌进行药物敏感性试验(K-B纸片法)。选取至少耐1种氨基糖苷类药的大肠杆菌作为供试菌,通过PCR检测其16SrRNA甲基化酶基因armA、rmtA、rmtB、rmtC、rmtD、rmtE和npmA;对阳性菌株进行质粒接合试验;并用K-B纸片法分析临床菌和接合子对抗生素的敏感性。【结果】437株健康动物源和82株患病动物源大肠杆菌对14种抗菌药物表现出不同程度的耐药性。健康动物源大肠杆菌对氨苄西林、氯霉素、磺胺甲恶唑的耐药率较高,分别为69.79%,50.8%和68.72%,对庆大霉素、卡那霉素、阿米卡星比较敏感,敏感率分别为75.97%,67.73%和97.02%;其中有134株分离菌至少对1种氨基糖苷类药物表现出耐药性。患病动物源大肠杆菌除对美罗培兰耐药率较低(为3.66%)外,对其余药物的耐药率在32.93%~100%,且都对至少1种氨基糖苷类药物表现出耐药性。多重耐药分析表明,健康动物源大肠杆菌主要分布于0~4耐,而患病动物源大肠杆菌至少耐7种抗菌药物,主要分布于10~14耐。在134株耐氨基糖苷类抗生素大肠杆菌中,检测到5株含rmtB基因,检测率为3.73%,未检测到armA、rmtA、rmtC、rmtD、rmtE和npmA基因。在82株患病动物源大肠杆菌中,检测到1株含armA基因,10株含rmtB基因,检出率分别为1.22%和12.2%,未检测到rmtA、rmtC、rmtD、rmtE及npmA基因;接合试验的耐药质粒传递率为100%,受体菌接合频率在(9.2×10-9)^(4.8×10-6)。【结论】四川地区健康动物源大肠杆菌对抗生素呈中低水平耐药,多重耐药以4耐及以下为主,占63.84%,主要由rmtB基因引起;而患病动物源大肠杆菌对抗生素则呈高水平耐药,89.02%为10~14耐,主要由rmtB和armA基因引起。

【Objective】The resistance of 437 E.coli strains isolated from healthy animals and 82 E.coli strains separated from sick animals in Sichuan and the mechanism were investigated to provide basis for its clinical rational use.【Method】Sensitivity test was conducted to examine the separated E.coli strains with three Aminoglycosides and 11non-Aminoglycosides.The strains with resistant to at least one Aminoglyco-side drug were selected and their 16 SrRNA methylase genes（armA,rmtA,rmtB,rmtC,rmtD,rmtEand npmA）were detected by PCR.Plasmid conjugation experiment was also carried out to the positive strains and the drug susceptibility of clinical isolated bacteria and zygote to antimicrobial agents was executed by the Kirby-Bauer method.【Result】A total of 437 strains of healthy animal origin and 82 strains of sicken animal origin showed resistance to 14 antibacterial drugs.The resistant rates of E.coli strains of healthy animal origin against ampicillin,chloramphenicol and sulfamethoxazole were higher than other tested drugs with the rates of 69.79%,50.8%and 68.72%,respectively.They were more sensitive to gentamycin,kanamycin and amikacin with sensitive rates of 75.97%,67.73% and 97.02%,respectively.There were 134 strains with resistance to at least one aminoglycoside drug.The resistant rates of sick animal derived E.coli to 14 antibacterial drugs were 32.93%to 100%except for meropenem（3.66%）.They were all resistant to at least one aminoglycoside drug.Strains separated from health animal were resistant to 0to 4drugs at the same time but those from sick animal were resistant to 10 to 14drugs.A total of 5 E.coli strains containing rmtBgene were detected among the 134 strains with detection rate of 3.73%,while armA,rmtA,rmtC,rmtD,rmtE,and npmA gene were not detected.One strain was detected to contain armA gene and 10 strains were detected to contain rmtBgene among the 82 strains from diseased animal,with detection rates of 1.22%and 12.2%,respectively.But rmtA,rmtC,rmtD,rmtE,and npmAgene were not detected.Transconjugation test showed that resistant plasmids pass rate was 100%,and conjugation frequency of each receptor cell was（9.2×10-9）-（4.8×10-6）.【Conclusion】In Sichuan,E.coli strains from healthy animals had low-level resistance and 63.84% were resistant to 4or less antibiotics,which was mainly caused by rmtB.The E.coli separated from sick animals showed high-level resistance to antibiotics with 89.02%were resistant to 10-14 drugs,which was caused by rmtBand armAgenes.