致病性大肠埃希菌ESBLs的基因检测与耐药性控制
摘要
动物专用药物头孢噻呋,具有抗菌活性强、药代动力学特征优良、毒副作用小、残留低等优点。但近年来,随着第三代头孢菌素的广泛应用和滥用,细菌尤其革兰氏阴性菌迫于生存压力对其已经产生耐药性,且耐药菌株逐年增多。研究表明:革兰阴性菌对第三代头孢和新的β‐内酰胺环类抗生素耐药的主要机制,是产生了超广谱β‐内酰胺酶(extended spectrumβ‐ lactamase, ESBLs)和Ampc酶(Ampcβ‐l actamase);产生ESBLs细菌携带ESBLs质粒的同时,可能带有对喹诺酮类、氨基糖苷类和磺胺类等药物的多种耐药基因,使其具有多重耐药表型,给临床治疗带来严重的威胁,成为临床关注的焦点。为指导兽医临床科学、合理地使用兽药,降低产生ESBLs耐药菌带来的危害,进行了本论文试验研究。
1.致病性大肠埃希菌ESBLs的检测及质粒传播研究
(1)用试管二倍稀释法测定了177株猪、鸡大肠埃希菌(E.coli)临床分离株对头孢噻呋、阿莫西林、头孢曲松、头孢噻肟的敏感性。结果表明,它们对临床分离株的(minimal inhibitory concentration, MIC)值高于对大肠埃希菌标准菌株的MIC值。
(2)用双纸片协同法进行了ESBLs的检测,ESBLs的阳性率是20.34 %;用PCR方法进行了ESBLs的DNA扩增,19.77 %菌株扩增出ESBLs的目的条带。以上两种检测方法检测结果差异不显著。PCR检测出2株菌含有TEM型ESBLs和SHV型ESBLs,1株含有TEM型ESBLs和CTX型ESBLs。
(3)不同地区的TEM型ESBLs基因分析结果发现,大肠埃希菌(E.coli)新郑猪、鸡分离株产生的ESBLs和鸡大肠埃希菌(E.coli)中牟分离株产生的是同一种ESBLs;大肠埃希菌(E.coli)荥阳猪、鸡分离株产生的ESBLs关系很近。
(4)以氨苄西林作为选择抗生素,以大肠埃希菌DH5α为受体菌,进行的ESBLs耐药质粒接合、传递试验结果表明,猪源、鸡源大肠埃希菌(E.coli)均能传递ESBLs耐药质粒至受体菌中。
2.他唑巴坦的抑酶保护抗生素作用研究
(1)用试管二倍稀释法测定了头孢噻呋(CEF)、头孢噻呋‐他唑巴坦(CEF-T)复方及头孢噻呋-他唑巴坦-恩诺沙星(CEF-T-EN)复方制剂对标准大肠埃希菌(C83845和C83907)及产生ESBLs大肠埃希菌荥阳分离株(E.coli荥阳-ESBLs)的最小抑菌浓度(MIC)值;结果表明:CEF-T复方对E.coli荥阳-ESBLs的MIC值是1.2μg/mL,较头孢噻呋单药降低了12.5倍,CEF-T-EN复方对该菌株的MIC值是1.2μg/mL,抗菌活性更高。
(2)绘制出的CEF-T复方对E.coli荥阳-ESBLs的杀菌动力学曲线与头孢噻呋对C83845的杀菌动力学曲线相似,杀菌效果略差于CEF-T-EN复方制剂对E.coli荥阳-ESBLs的杀菌效果。
(3)头孢噻呋和CEF-T复方对标准菌株C83845的抗菌后效应作用非常相似。但CEF-T-EN复方在相同MIC值的PAE明显强于头孢噻呋。对产酶耐药菌株E.coli荥阳-ESBLs的抗菌后效应图三种药之间表现出了相似性;他唑巴坦对E.coli荥阳-ESBLs的β-内酰胺酶抑制剂后效应(postβ-lactamase inhibitor effect, PLIE)曲线图显示:他唑巴坦有轻微的β-内酰胺酶抑制剂后效应(PLIE)。
(4)用E.coli荥阳-ESBLs菌液(0.4mL∕只)对7日龄雏鸡腹腔注射(intraperitoneal injection, IP),制备耐药大肠埃希菌病模型。以感染不给药组为对照,用头孢噻呋单药粉针剂、混悬剂及CEF-T和CEF-T-EN复方混悬剂分别进行治疗。药物治疗结果,经SPSS11.5统计软件进行分析、X2检验表明:与感染对照组鸡只死亡率90%相比较,各治疗组雏鸡死亡率下降均显著(P<0.05)。头孢噻呋钠粉针治疗组的治愈效果优于头孢噻呋混悬液;CEF-T的治疗效果优于头孢噻呋;CEF-T-EN的治疗效果显著。
3. ESBLs耐药质粒的逆转研究
(1)头孢噻呋做底物,用亚抑菌浓度法对标准大肠埃希菌C83907和C83845进行诱导至产生ESBLs后,用传统质粒消除剂SDS做对照,进行了氯丙嗪、异丙嗪、地西泮、恩诺沙星、环丙沙星等质粒消除剂对ESBLs耐药质粒的体外消除研究。四环素(Tetracycline , TC)、红霉素(Erythromycin, EM)、利福平(Rifampin, RIF)对小白鼠去污染后,以SDS作对照,进行了氯丙嗪等质粒消除剂对ESBLs质粒的体内消除效果研究。结果表明,各种质粒消除的体内消除率与体外消除率基本相同,消除剂之间的消除效果相比较,三环类中枢神经系统抑制药物的消除效果好,适合用来阻止细菌耐药性的传播和发展.
(2)收集体内和体外消除试验中得到的消除子,测定并比较了它们和质粒消除前原耐药菌对头孢噻呋等药物的MIC值,并以氨苄西林(ampicillin, AP)做诱导剂,进行了消除子耐药性的稳定性试验。结果表明:质粒消除子恢复了对头孢噻呋等的敏感性,但诱导50代后,对氨苄西林和阿莫西林耐药,诱导100代后,仍然对头孢噻呋和头孢曲松敏感。
Ceftiofur is a high activity, good pharmacokinetic, little side effects, and low residual, animal-specific medicine, which is used to treat bacterial diseases in swine, chickens and ruminants. But in recent years, bacteria, especially Gram - negative bacteria has generated resistance for its pressure of survival, with the third - generation cephalosporin widely used and abused, and the resistant strains increased year by year. Results showed that: the main resistant mechanism of gram-negative bacteria to the third generation cephalosporins and the new class ofβ-lactam antibiotics is producing extended spectrumβ‐lactamase (ESBLs) and Ampc enzyme, bacteria producing ESBLs may carry other resistant plasmid to quinolones, aminoglycosides, and sulfa drugs so on, showed cross-resistance and multiple-resistance, lead to serious threat to clinical treatment, and it attracted the focus of clinical attention. In order to guide farmers to use drugs scientific and legitimately in clinics, to reduce the harm leaded from bacteria producing ESBLs, some experiment had been done in this paper.
1. Detection of ESBLs in pathogenic E. coli and research on plasmid dissemination
(1)To the 177 clinical E.coli strains, the minimal inhibitory concentrations (MICs) of ceftiofur, ceftriaxone, amoxicillin and cefotaxime were determined with the two-fold dilution method. Results showed that MICs of ceftiofur, amoxicillin, ceftriaxone, and cefotaxime to the clinical E. coli in chicken and swine were higher than their MICs to the standard strains.
(2)Detection of ESBLs were done by double disc synergy test(DDST), three primers were designed: SHV, TEM and CTX, ESBLs gene type was amplified,two strains from dofferent citys were selected, their ESBLs were sequenced and analyzed by PCR. For all E. coli strains, ESBL-positive rate with the double disk synergy test was 20.34%, ESBL-positive rate with PCR was 19.77%. There was not significant difference between the two results. And two strains were detected to have produced TEM type ESBLs and SHV type ESBLs; one strain was detected to have produced TEM type ESBLs and CTX type ESBLs.
(3)Results of analysis about TEM type ESBLs gene showed that: E. coli in swine and in chicken from Xinzheng and E. coli in swine from Zhongmou produced the same ESBLs; there were close relationship between ESBLs produced by E. coli in swine and in chicken from Xingyang.
(4)Conjugation and transformation about the plasmid of ESBLs were tested, as ampicillin used as an alternative antibiotic, E. coli DH5αwas used as the recipient bacteria in the test. Results showed that E. coli in swine and chicken can transfer the resistant ESBLs plasmid to the recipient strain.
2. Study on the inhibited and protective effect of tazobactam
(1)MICs of ceftiofur, CEF-T and CEF-T-EN to C83907, C83845 and E.coli Xingyang-ESBLs were determined. Results showed that compound of ceftiofur with tazobactam showed higher activity to the E.coli Xingyang-ESBLs, the MIC reduced for 12.5 times than ceftiofur, compound of ceftiofur - tazobactam -enrofloxacin showed the highest activity, the MIC was 1.2μg/mL.
(2)The killing curve map of CEF-T to E.colixingyang-ESBLs was similar with CEF to C83845, but it was worse than CEF-T-EN slightly.
(3)PAE of ceftiofur to the standard E.coli were similar to CEF-T, but compand of CEF-T-EN was longer than them at same MICs. PAE of ceftiofur, CEF-T and CEF-T-EN to E.colixingyang-ESBLs were similar. The PLIE map of Tazobactam showed that it have轻微的PLIE.
(4)The 7 day old chicks were infected to make into artifical E.coli infection model successfully by E.coli Xingyang-ESBLs (0.4mL, IP). The chickens infected but not treated were used as control, other infected chickens were treated by ceftiofur powder, ceftiofur suspension, CEF-T and CEF-T-EN. The results of drug treatment were analyzed by SPSS11.5 software and X2 test. The mortality rates of every treatment group were lower than the control group significantly. The cure effect of ceftiofur sodium powder was higher than ceftiofur suspension, the cure effect of CEF-T was higher than CEF, and the cure effect of CEF-T-EN was the highest of all those drugs.
3. Research on reversal of ESBLs- resistant plasmid
(1)After the standard E.coli were induced to produce ESBLs by ceftiofur with sub-MIC method, with SDS as control, the elimination rate of chlorpromazine, promethazine, diazepam, enrofloxacin ciprofloxacin to the ESBLs plasmid in vitro were determined. After the mice were got rid of pollution by tetracycline (TC), erythromycin (EM), rifampicin (RIF), elimination rate of chlorpromazine, promethazine, diazepam, norfloxacin, ciprofloxacin to the induced strains in vivo were studied. Results showed that the elimination rate of every plasmid-cure agent in vitro were almost the same as it’s in vivo. And the the tricyclic psychotropic drugs showed better elimination effect to ESBLs, they were suitable for to prevent bacterial resistance from spreading and developing.
(2)Bacter eliminated in vivo and in vitro were collected, the MICs of them and original bacter to ceftiofur, ceftriaxone, amoxicillin, ampicillin were determined. Results of MICs showed bacteria eliminated have restored to be sensitive to ceftiofur etc. Ampicillin was used as inducer; resistance stability of bacteria eliminated was study. They were resistant to amoxicillin and ampicillin after they were induced for 50 generations, but they were sensitive to ceftiofur and ceftriaxone after they were induced for 100 generations.
1.致病性大肠埃希菌ESBLs的检测及质粒传播研究
(1)用试管二倍稀释法测定了177株猪、鸡大肠埃希菌(E.coli)临床分离株对头孢噻呋、阿莫西林、头孢曲松、头孢噻肟的敏感性。结果表明,它们对临床分离株的(minimal inhibitory concentration, MIC)值高于对大肠埃希菌标准菌株的MIC值。
(2)用双纸片协同法进行了ESBLs的检测,ESBLs的阳性率是20.34 %;用PCR方法进行了ESBLs的DNA扩增,19.77 %菌株扩增出ESBLs的目的条带。以上两种检测方法检测结果差异不显著。PCR检测出2株菌含有TEM型ESBLs和SHV型ESBLs,1株含有TEM型ESBLs和CTX型ESBLs。
(3)不同地区的TEM型ESBLs基因分析结果发现,大肠埃希菌(E.coli)新郑猪、鸡分离株产生的ESBLs和鸡大肠埃希菌(E.coli)中牟分离株产生的是同一种ESBLs;大肠埃希菌(E.coli)荥阳猪、鸡分离株产生的ESBLs关系很近。
(4)以氨苄西林作为选择抗生素,以大肠埃希菌DH5α为受体菌,进行的ESBLs耐药质粒接合、传递试验结果表明,猪源、鸡源大肠埃希菌(E.coli)均能传递ESBLs耐药质粒至受体菌中。
2.他唑巴坦的抑酶保护抗生素作用研究
(1)用试管二倍稀释法测定了头孢噻呋(CEF)、头孢噻呋‐他唑巴坦(CEF-T)复方及头孢噻呋-他唑巴坦-恩诺沙星(CEF-T-EN)复方制剂对标准大肠埃希菌(C83845和C83907)及产生ESBLs大肠埃希菌荥阳分离株(E.coli荥阳-ESBLs)的最小抑菌浓度(MIC)值;结果表明:CEF-T复方对E.coli荥阳-ESBLs的MIC值是1.2μg/mL,较头孢噻呋单药降低了12.5倍,CEF-T-EN复方对该菌株的MIC值是1.2μg/mL,抗菌活性更高。
(2)绘制出的CEF-T复方对E.coli荥阳-ESBLs的杀菌动力学曲线与头孢噻呋对C83845的杀菌动力学曲线相似,杀菌效果略差于CEF-T-EN复方制剂对E.coli荥阳-ESBLs的杀菌效果。
(3)头孢噻呋和CEF-T复方对标准菌株C83845的抗菌后效应作用非常相似。但CEF-T-EN复方在相同MIC值的PAE明显强于头孢噻呋。对产酶耐药菌株E.coli荥阳-ESBLs的抗菌后效应图三种药之间表现出了相似性;他唑巴坦对E.coli荥阳-ESBLs的β-内酰胺酶抑制剂后效应(postβ-lactamase inhibitor effect, PLIE)曲线图显示:他唑巴坦有轻微的β-内酰胺酶抑制剂后效应(PLIE)。
(4)用E.coli荥阳-ESBLs菌液(0.4mL∕只)对7日龄雏鸡腹腔注射(intraperitoneal injection, IP),制备耐药大肠埃希菌病模型。以感染不给药组为对照,用头孢噻呋单药粉针剂、混悬剂及CEF-T和CEF-T-EN复方混悬剂分别进行治疗。药物治疗结果,经SPSS11.5统计软件进行分析、X2检验表明:与感染对照组鸡只死亡率90%相比较,各治疗组雏鸡死亡率下降均显著(P<0.05)。头孢噻呋钠粉针治疗组的治愈效果优于头孢噻呋混悬液;CEF-T的治疗效果优于头孢噻呋;CEF-T-EN的治疗效果显著。
3. ESBLs耐药质粒的逆转研究
(1)头孢噻呋做底物,用亚抑菌浓度法对标准大肠埃希菌C83907和C83845进行诱导至产生ESBLs后,用传统质粒消除剂SDS做对照,进行了氯丙嗪、异丙嗪、地西泮、恩诺沙星、环丙沙星等质粒消除剂对ESBLs耐药质粒的体外消除研究。四环素(Tetracycline , TC)、红霉素(Erythromycin, EM)、利福平(Rifampin, RIF)对小白鼠去污染后,以SDS作对照,进行了氯丙嗪等质粒消除剂对ESBLs质粒的体内消除效果研究。结果表明,各种质粒消除的体内消除率与体外消除率基本相同,消除剂之间的消除效果相比较,三环类中枢神经系统抑制药物的消除效果好,适合用来阻止细菌耐药性的传播和发展.
(2)收集体内和体外消除试验中得到的消除子,测定并比较了它们和质粒消除前原耐药菌对头孢噻呋等药物的MIC值,并以氨苄西林(ampicillin, AP)做诱导剂,进行了消除子耐药性的稳定性试验。结果表明:质粒消除子恢复了对头孢噻呋等的敏感性,但诱导50代后,对氨苄西林和阿莫西林耐药,诱导100代后,仍然对头孢噻呋和头孢曲松敏感。
Ceftiofur is a high activity, good pharmacokinetic, little side effects, and low residual, animal-specific medicine, which is used to treat bacterial diseases in swine, chickens and ruminants. But in recent years, bacteria, especially Gram - negative bacteria has generated resistance for its pressure of survival, with the third - generation cephalosporin widely used and abused, and the resistant strains increased year by year. Results showed that: the main resistant mechanism of gram-negative bacteria to the third generation cephalosporins and the new class ofβ-lactam antibiotics is producing extended spectrumβ‐lactamase (ESBLs) and Ampc enzyme, bacteria producing ESBLs may carry other resistant plasmid to quinolones, aminoglycosides, and sulfa drugs so on, showed cross-resistance and multiple-resistance, lead to serious threat to clinical treatment, and it attracted the focus of clinical attention. In order to guide farmers to use drugs scientific and legitimately in clinics, to reduce the harm leaded from bacteria producing ESBLs, some experiment had been done in this paper.
1. Detection of ESBLs in pathogenic E. coli and research on plasmid dissemination
(1)To the 177 clinical E.coli strains, the minimal inhibitory concentrations (MICs) of ceftiofur, ceftriaxone, amoxicillin and cefotaxime were determined with the two-fold dilution method. Results showed that MICs of ceftiofur, amoxicillin, ceftriaxone, and cefotaxime to the clinical E. coli in chicken and swine were higher than their MICs to the standard strains.
(2)Detection of ESBLs were done by double disc synergy test(DDST), three primers were designed: SHV, TEM and CTX, ESBLs gene type was amplified,two strains from dofferent citys were selected, their ESBLs were sequenced and analyzed by PCR. For all E. coli strains, ESBL-positive rate with the double disk synergy test was 20.34%, ESBL-positive rate with PCR was 19.77%. There was not significant difference between the two results. And two strains were detected to have produced TEM type ESBLs and SHV type ESBLs; one strain was detected to have produced TEM type ESBLs and CTX type ESBLs.
(3)Results of analysis about TEM type ESBLs gene showed that: E. coli in swine and in chicken from Xinzheng and E. coli in swine from Zhongmou produced the same ESBLs; there were close relationship between ESBLs produced by E. coli in swine and in chicken from Xingyang.
(4)Conjugation and transformation about the plasmid of ESBLs were tested, as ampicillin used as an alternative antibiotic, E. coli DH5αwas used as the recipient bacteria in the test. Results showed that E. coli in swine and chicken can transfer the resistant ESBLs plasmid to the recipient strain.
2. Study on the inhibited and protective effect of tazobactam
(1)MICs of ceftiofur, CEF-T and CEF-T-EN to C83907, C83845 and E.coli Xingyang-ESBLs were determined. Results showed that compound of ceftiofur with tazobactam showed higher activity to the E.coli Xingyang-ESBLs, the MIC reduced for 12.5 times than ceftiofur, compound of ceftiofur - tazobactam -enrofloxacin showed the highest activity, the MIC was 1.2μg/mL.
(2)The killing curve map of CEF-T to E.colixingyang-ESBLs was similar with CEF to C83845, but it was worse than CEF-T-EN slightly.
(3)PAE of ceftiofur to the standard E.coli were similar to CEF-T, but compand of CEF-T-EN was longer than them at same MICs. PAE of ceftiofur, CEF-T and CEF-T-EN to E.colixingyang-ESBLs were similar. The PLIE map of Tazobactam showed that it have轻微的PLIE.
(4)The 7 day old chicks were infected to make into artifical E.coli infection model successfully by E.coli Xingyang-ESBLs (0.4mL, IP). The chickens infected but not treated were used as control, other infected chickens were treated by ceftiofur powder, ceftiofur suspension, CEF-T and CEF-T-EN. The results of drug treatment were analyzed by SPSS11.5 software and X2 test. The mortality rates of every treatment group were lower than the control group significantly. The cure effect of ceftiofur sodium powder was higher than ceftiofur suspension, the cure effect of CEF-T was higher than CEF, and the cure effect of CEF-T-EN was the highest of all those drugs.
3. Research on reversal of ESBLs- resistant plasmid
(1)After the standard E.coli were induced to produce ESBLs by ceftiofur with sub-MIC method, with SDS as control, the elimination rate of chlorpromazine, promethazine, diazepam, enrofloxacin ciprofloxacin to the ESBLs plasmid in vitro were determined. After the mice were got rid of pollution by tetracycline (TC), erythromycin (EM), rifampicin (RIF), elimination rate of chlorpromazine, promethazine, diazepam, norfloxacin, ciprofloxacin to the induced strains in vivo were studied. Results showed that the elimination rate of every plasmid-cure agent in vitro were almost the same as it’s in vivo. And the the tricyclic psychotropic drugs showed better elimination effect to ESBLs, they were suitable for to prevent bacterial resistance from spreading and developing.
(2)Bacter eliminated in vivo and in vitro were collected, the MICs of them and original bacter to ceftiofur, ceftriaxone, amoxicillin, ampicillin were determined. Results of MICs showed bacteria eliminated have restored to be sensitive to ceftiofur etc. Ampicillin was used as inducer; resistance stability of bacteria eliminated was study. They were resistant to amoxicillin and ampicillin after they were induced for 50 generations, but they were sensitive to ceftiofur and ceftriaxone after they were induced for 100 generations.
引文
边保华,林平,汪旭明,包兆胜,项昌富,朱敏. 2006.克痢痧对致病性大肠埃希菌R质粒消除的作用.中国老年学杂志, 26(1): 46-47
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