毛皮动物源大肠杆菌毒力和耐药基因检测及药物敏感性分析
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摘要
为了解秦皇岛昌黎地区毛皮动物大肠杆菌毒力基因和耐药基因的分布及药物敏感性情况,以60只临床患病毛皮动物为试验菌株的分离宿主,采用常规分离纯化、生化鉴定以及分子生物学的方法对病原菌进行鉴定,动物致病性试验检测其致病性,PCR方法检测毒力基因和耐药基因,K-B法对常用西药进行药物敏感性检测,琼脂平板打孔法对中草药药物敏感性进行检测,试管二倍稀释法检测中草药的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果显示:此次分离到的28株大肠杆菌均使小鼠发病死亡;其中,24株大肠杆菌检测到irp2、fyuA毒力基因,检出率为85.7%;5株大肠杆菌临床分离菌株检出Ler、eaeA毒力基因,检出率为17.9%;耐药基因blaTEM、aadA1、blaCTX-M、blaTEM1、blaSHV-1、strA-strB、blaPSE1和blaOXA1的检出率分别为85.7%,89.3%,75.0%,71.4%,25.0%,24.1%,14.3%,14.3%;西药药敏试验结果显示,28株菌对链霉素、头孢拉定、多西环素、复方新诺明、林可霉素、替米考星、青霉素同时耐药;中草药药敏试验结果显示,诃子、乌梅、五味子对这28株大肠杆菌均具有较好的体外抑菌效果,抑菌圈直径为18～31 mm,MIC和MBC为15.63～500.00 g/L。
In order to understand the distribution of virulence genes and drug resistance genes and drug sensitivity in fur animal E.coli strains from Changli,Qinhuangdao,60 diseased fur-bearing animals were used as the isolation hosts for the test strains,and methods of routine isolation,purification,biochemical identification,and molecular biological analysis were used to identify pathogenic bacteria,animal pathogenicity test was used to detect E.coli pathogenicity,PCR methods were used to detect virulence genes and drug resistance genes,K-B methods were used to detect drug susceptibility of commonly used antibiotic,agar plate drilling method was used to detect the sensitivity of Chinese herbal medicines,and test tube double dilution method was used to detect the minimum inhibitory concentration(MIC) and the minimum bactericidal concentration(MBC) of Chinese herbs.The test results showed that 28 strains of E.coli were isolated and detected,which further resulted in the occurrence of serious illness and death in experimental mice after intraperitoneal injection;the detection rate of virulence genes of Ler and eaeA virulence genes were 14.3% and 14.3%,respectively; the detection rates of blaTEM,aadA1,blaCTX-M,blaTEM1,blaSHV-1,strA-strB,blaPSE1 and blaOXA1 were 85.7%,89.3%,75%,71.4%,25%,24.1%,14.3% and 14.3%,respectively;commonly used antibiotic susceptibility test showed that 28 strains were resistant to streptomycin,cephradine,doxycycline,cotrimoxazole,lincomycin,tilmicosin and penicillin;Chinese herbal medicine susceptibility test showed that Terminalia chebula,Prunus mume,and Schisandra chinensis displayed good antibacterial effect on E.coli strains all in vitro,the inhibition zone diameter were from 18 to 31 mm,and MIC and MBC were from 15.63 to500.00 g/L.
In order to understand the distribution of virulence genes and drug resistance genes and drug sensitivity in fur animal E.coli strains from Changli,Qinhuangdao,60 diseased fur-bearing animals were used as the isolation hosts for the test strains,and methods of routine isolation,purification,biochemical identification,and molecular biological analysis were used to identify pathogenic bacteria,animal pathogenicity test was used to detect E.coli pathogenicity,PCR methods were used to detect virulence genes and drug resistance genes,K-B methods were used to detect drug susceptibility of commonly used antibiotic,agar plate drilling method was used to detect the sensitivity of Chinese herbal medicines,and test tube double dilution method was used to detect the minimum inhibitory concentration(MIC) and the minimum bactericidal concentration(MBC) of Chinese herbs.The test results showed that 28 strains of E.coli were isolated and detected,which further resulted in the occurrence of serious illness and death in experimental mice after intraperitoneal injection;the detection rate of virulence genes of Ler and eaeA virulence genes were 14.3% and 14.3%,respectively; the detection rates of blaTEM,aadA1,blaCTX-M,blaTEM1,blaSHV-1,strA-strB,blaPSE1 and blaOXA1 were 85.7%,89.3%,75%,71.4%,25%,24.1%,14.3% and 14.3%,respectively;commonly used antibiotic susceptibility test showed that 28 strains were resistant to streptomycin,cephradine,doxycycline,cotrimoxazole,lincomycin,tilmicosin and penicillin;Chinese herbal medicine susceptibility test showed that Terminalia chebula,Prunus mume,and Schisandra chinensis displayed good antibacterial effect on E.coli strains all in vitro,the inhibition zone diameter were from 18 to 31 mm,and MIC and MBC were from 15.63 to500.00 g/L.
引文
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[15] 史秋梅,张艳英,房海,等.腹泻水貂检出携带耶尔森菌HPI毒力岛的大肠杆菌[J].中国兽医学报,2012,32(6):55-59.
[16] 赖婧,刘洋,汪宇,等.800株不同动物源大肠杆菌的耐药性监测[J].中国兽医杂志,2011,47(4):12-14.
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[18] 王艳萍,董林,郭时金,等.禽源性大肠杆菌HPI毒力岛irp2基因PCR检测方法的建立与应用[J].中国兽医杂志,2017(1):86-89.
[19] 徐丰都,彭丽园,蔡婷,等.中草药对水产动物致病菌抑杀作用的研究进展[J].水产养殖,2016,37(10):48-52.
[20] 向双云,周珍辉,李玉冰,等.8种中草药对大肠杆菌的体外抑菌试验[J].黑龙江畜牧兽医,2016(5):167-169.
[21] 李树明.50味中药对8种畜禽病原菌抑菌对比研究[J].中兽医学杂志,2000(4):5-8.
[2] 于杰,曲海娜,张灿,等.毛皮动物大肠杆菌对氟喹诺酮类药物及中药的耐药性研究[J].中国畜牧兽医,2015,42(6):1597-1601.
[3] 朱僧,邢艳萍,胡腾,等.15株临床分离耐药大肠杆菌耐氟喹诺酮类抗生素基因的检测与序列分析[J].中国兽医学报,2011,31(6):875-879.
[4] 冯敏燕,傅剑,杨世发,等.山东省鸡源性大肠杆菌耐药变化趋势监测[J].中国兽医学报,2017,37(2):250-253.
[5] 李晓娜,杨慧君,余婷,等.牛源大肠杆菌耐药表型与Ⅰ型整合子-基因盒的关系[J].中国兽医学报,2017,37(3):348-442.
[6] 索朗斯珠,王刚,罗润波,等.西藏牦牛源大肠杆菌耐药表型及耐药基因检测[J].中国兽医学报,2017,37(8):1501-1506.
[7] 教郁,高维凡,胡彩光.大肠杆菌耐药性研究进展[J].现代畜牧兽医,2013(5):53-58.
[8] 杨小平,徐建国.携带小肠结肠炎耶尔森菌HPI毒力岛大肠杆菌的毒力基因分析[J].中国热带医学,2007,7(9):1508-1510.
[9] 洪婷,缪萍,林云,等.尿感方对大肠杆菌生物膜形成及与毒力基因fim、usp、hlyA表达的影响[J].辽宁中医杂志,2012(11):2319-2323.
[10] 庄林林.三重LAMP检测禽源沙门菌、大肠杆菌磺胺耐药基因(sul1、sul2、sul3)研究[D].江苏扬州:扬州大学,2016.
[11] 孟春萍,陈燕杰,吴华,等.鸭源大肠杆菌对氟喹诺酮类耐药机制的研究[J].江西农业学报,2012,24(1):128-130.
[12] 何先元,李坤,喻录容,等.53味清热类中药对绿脓杆菌抑菌作用的聚类分析[J].中国实验方剂学杂志,2014,20(20):222-225.
[13] 代如意,李莉,殷中琼,等.5味中药对鸡白痢沙门菌的体外联合抑茵研究[J].西北农林科技大学学报(自然科学版),2015,43(2):34-37.
[14] 王凯,刘琪琦.致泻性大肠杆菌毒力基因及检测方法研究进展[J].军事医学,2013,37(3):234-237.
[15] 史秋梅,张艳英,房海,等.腹泻水貂检出携带耶尔森菌HPI毒力岛的大肠杆菌[J].中国兽医学报,2012,32(6):55-59.
[16] 赖婧,刘洋,汪宇,等.800株不同动物源大肠杆菌的耐药性监测[J].中国兽医杂志,2011,47(4):12-14.
[17] 展天松,王彦红,许梦怡,等.肠致病性大肠杆菌的分离鉴定和耐药性分析[J].黑龙江畜牧兽医,2015(2):67-69.
[18] 王艳萍,董林,郭时金,等.禽源性大肠杆菌HPI毒力岛irp2基因PCR检测方法的建立与应用[J].中国兽医杂志,2017(1):86-89.
[19] 徐丰都,彭丽园,蔡婷,等.中草药对水产动物致病菌抑杀作用的研究进展[J].水产养殖,2016,37(10):48-52.
[20] 向双云,周珍辉,李玉冰,等.8种中草药对大肠杆菌的体外抑菌试验[J].黑龙江畜牧兽医,2016(5):167-169.
[21] 李树明.50味中药对8种畜禽病原菌抑菌对比研究[J].中兽医学杂志,2000(4):5-8.