【作者】 仲崇岳；

【导师】 汪铭书； 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 鸭疫里默氏杆菌(R.anatipestifer)是引起水禽细菌性疾病的主要病原之一。目前公认的血清型众多,各血清型之间很少有交叉保护作用,虽然疫苗的效果很显著,但受血清型的局限,R.anatipestifer感染还是很难得到控制,所以实际生产中使用大量的化学药物对鸭疫里默氏杆菌病进行治疗。但是到目前为止,关于R.anatipestifer耐药机理的文献有限,因此本论文根据目前我国用药的现状,对分离自我国不同地区和时间的R.anatipestifer的耐药性、基因型和耐药机理进行了系统的研究,报告如下:1.为了研究分离自我国RA临床株的耐药性和耐药变迁规律有,采用K-B纸片法对1998-2005年分离的224株RA对9大类36种常用药物的耐药性进行了检测,使用WHONET 5.3软件对结果进行分析。结果表明RA临床株对氨曲南、头孢吡肟、青霉素G、苯唑西林、头孢他啶和复方新诺明耐药率最高,分别高达87.8%、64.3%、86.9%、88.6%、75.9%和79.2%,而对阿米卡星、头孢哌酮、亚胺培南和新霉素的敏感率最高,依次为85.1%、70.6%、95%和61.8%,耐药率低于10%。比较耐药谱发现R.anatipestifer耐药谱以10～29种药物为主,即呈现泛耐药现象。比较耐药率变化规律发现除氨曲南、大观霉素、头孢哌酮和哌拉西林外,RA临床株对其它药物耐药率并无规律可寻,这可能是由于各地区血清型和用药习惯不同造成分离的RA耐药性不同有关,因此在用药前应先进行药物筛选。2.抽取72株耐β-内酰胺类药物的菌株,建立快速脉冲电泳分型方法,经限制性内切酶SmaⅠ酶切,将R.anatipestifer基因组切成6-19条带。以80%相似性为界将多重耐药菌株分为60个脉冲电泳型,与耐药谱型相比两者没有相关性。本实验建立的快速样品处理方法,可以将菌体蛋白消化时间缩短为1.5h,从菌体沉淀到获得脉冲电泳图片整个过程可以在30h之内完成,经重复性实验,证明该方法快速稳定,重复性好,解决了使用脉冲电泳方法在R.anatipestifer流行病学和遗传学的研究中出现的耗时长的确定。3.通过对R.anatipestifer携带1、2和3型整合子检测和sul1、sul2和sul3基因检测,结果表明,共检测出4种结构的1型整合子:aadAl1-qacE△1-sul1(17株),dhffⅠ-aadA2-qacE△1-sul1(1株),sul(29株)和非典型整合子(2株),1型整合子携带率为61.11%,有5株同时携带2种1型整合子,其中4株同时携带aadAl1-qacE△1-sul1和sul1,1株同时携带dhfrⅠ-aadA2-qacE△1-sul1和sul1,所有的菌株均未检测到2型和3型整合子。通过sul基因检测发现,有53株RA携带sul基因,其中50株只携带sul1基因,2株同时携带sul1和sul2基因,1株只携带sul2基因,所有的菌株均未检测到sul3基因,sul基因携带率为73.61%,这与RA临床株对磺胺类药物耐药率为88.89%相接近,因此可以推断携带sul基因是RA临床株耐磺胺类药物药主要原因之一。通过整合子检测得出25%的RA临床株携带aadA基因,耐药泵表型检测得出65.28%的菌株携带有耐药泵,比较CCCP作用前后RA对链霉素MIC值的变化得出RA对氨基糖苷类药物的耐药性主要由整合子携带aadA基因和耐药泵共同作用引起。4.经PCR检测,四环素耐药基因tetC携带率为16.67%,其中1株同时也携带tetA基因;tetM基因携带率为11.11%,其中1株同时也携带tetC基因。将K-B纸片法和MIC实验结果对比发现在耐药率上相差很大,可能是由于所采用的判断标准不适于R.anatipestifer,通过建立荧光染料定量RT-PCR法对R.anatipestifer四环素耐药基因mRNA水平的研究和MIC实验结果,认为R.anatipestifer对四环素类药物的抗药性与携带tet基因有关,但是也存在其它的耐药机制。5.选取5株氧氟沙星耐药株,通过CCCP耐药泵表型检测,左氧氟沙星和新喹诺酮药物CO-06NF诱导,抽提外膜蛋白和全菌蛋白电泳,SSCP对GyrA基因诱导前后突变检测,结果显示5株菌有4株携带耐药泵,对诱导成功的1株菌株外膜蛋白和全菌蛋白进行SDS-PAGE电泳后发现,左氧氟沙星诱导耐药株外膜蛋白内有变化,CO-06NF诱导耐药株在接近21KD处的一条蛋白质条带表达量明显增加,全菌蛋白电泳发现,左氧氟沙星诱导耐药株和CO-06NF诱导耐药株在45KD处有一条共同条带表达量高于诱导前。而SSCP电泳检测GyrA基因发现诱导前后没有变化,这株细菌耐药泵检测为阳性,因此由结果推测R.anatipestifer对喹诺同类药物的耐药主要由耐药泵引起,而且最先发生突变耐药基因可能不是GyrA基因。6.72株临床株对苯唑西林、青霉素G和头孢吡肟耐药率分别为100%、97.22%和95.83%,对头孢噻肟的耐药率也达到70.83%,MIC检测结果与纸片法检测结果相一致。加入CCCP后,RA对苯唑西林、青霉素G、头孢噻肟和头孢吡肟的耐药率依次下降为91.67%、55.56%%、21.97%和44.44%,MIC值最低可降至处理前的1/2048。而β-内酰胺酶表型和基因型检测均为阴性,说明不存在β-内酰胺酶耐药机制,而耐药泵介导耐药是R.anatipestifer主要原因之一,通过头孢吡肟诱导试验,和外膜蛋白电泳发现在14.4～20KD和45KD处各有两条条带发生缺失。诱导株加入CCCP后,MIC值只下降为原来一半,说明可能这株诱导株对头孢吡肟的耐药性是由膜通透性降低引起,因此R.anatipestifer耐药株存在耐药泵出机制,推测也存在膜通透性降低引起的耐药机制。更多还原

【Abstract】 R.anatipestifer infection(RAI) is probably the most economically important disease of farm ducks.So far twenty one serotypes of R.anatipestifer have been reported.Many reports support that the vaccines could provide significant protection against RAI.But unfortunately,no cross-protection has been observed with inactivated bacterins made from different serotypes of.R.anatipestifer.Because of the limitation of vaccines protection,drugs were used in treatment of RAI frequently. But so many antibiotics were used in breeding industry that the the study on the usage of antibiotics,multi-drug resistance(MDR) mechanism and genotyping of MDR R.anatipestifer were carried out,and contents are summarized as following:1.To get the exact datas of drug resistance status and the regulation of drug resistance of R.anatipestifer isolated from five regions of China during 1998-2005. The resistance rates of 36 antibiotics were detected,and the data were nanlysisted by EWHONET5.3 software.The results supported that most isolates(50%) examined were resistant to ampicillin,ceftazidime,aztreonam,cefazolin,cefepime,cefuroxime, oxacillin,penicillin G,rifampin and trimethoprim/sulfamethoxazole.Resistance rates to aztreonam,cefepime,oxacillin,penicillin G,ceftazidime,and trimethoprim/ sulfamethoxazole were 87.8,64.3,88.6,86.9,75.9 and 79.2%,respectively. Antibiotics to which resistance was low included amikacin(9.5%),cefoperazone (7.2%),imipenem(3.2%),and neomycin(9.5%).Most of resistant to 10-29 antimicrobials.The resistance rates were changed year by year.2.A rapid method for genotyping R.anatipestifer isolates by PFGE were established and 72 isolates were random selected for evaluating the protocol. Among the isolates sixty different pulsetypes(1-60) predominantly using the limit of 80%similar and 6-19 bands resulted from SmaI digestion..With this method,only 2h cost in lysis time.The whole protocol time that may be completed,from a turbid tube of culture media to a picture of a gel,between 30h and the time in preparion of genomic DNA and restriction endnuclease digestion of DNA was less than 8h.the protocol time ensured that less time we spent,but a batch of high reproducible resultsstudies on this species.3.In this part,class 1,2 and 3 integron and sul1,sul2,sul3gene were detected by PCR.The result showed that there were four structure of Class 1 integrons were found: aadA11-qacE△1-sul1(17 isolates),dhfrI-aadA2-qacE△1-sul1(1 isolate),sul(29 isolates) and untypical integron(2 isolates),the percentage of islolates carried Class 1 integron is 61.11%.Five isolates carried two kinds of Class 1 integron,among the five isolates,four isolates carried aadA11-qacE△1-sul1 and sul at the same time,and 1 isolate carried dhfrI-aadA2-qacE△1-sul1 and sul1.There were no class 2 and 3 integron were detected.There are 53 isolates carried sul gene,among them,50 isolates carried sul1 gene,3 isolates carried sul2 gene.In the 3 isolates,2 carried sul1 and sul2 gene at same time.There was no sul3 were detected.The percentage of islolates carried sul gene is 73.61%,that was near to the resistance rate of RA to sulfonamides(88.89%),so we can conclude that the sul gene was one of main factors in sulfonamides resistance.According to the result that 25%islolates carried aadA gene and 65.28%RA carried efflux pump,we can conclude that aadA gene and efflux pump were the main factors in aminoglycosides resistance.4.In this part,tet gene were detected by PCR.tetC were detected from 16.67%And among them,a isolate RA45 carried both tetC and tetA at the same time.And 11.11%isolates carried tetM gene,and one of isolates(RA62) carried tetM and tetC gene at same time.the resistance rates of K-B diffusion and MIC methods were compared,but there were much more difference between the two results.And MIC method were more close to the fact.A real time RT-PCR for detecting the expression level of tet gene.And through the detecting the induced expression levels of tet gene by concentrationgradient of doxycycline between 0.03μg/ml～32μg/ml.The optimal inducted concentration of doxycycline was decided(0.0625μg/ml).Through the concentration gradient induction of doxycycline,the cDNA of tetC could be detected at allconcentrations but there were no cDNA of tetM could be detected.In contrast, the cDNA of tetA and tetC of RA45 both could detected at all concentrations.And at same time,we detected the expression level of all isolates carried tet gene,the exression level at mRNA of each isolate were proportional to their MIC values.So we can conclude that the expression level of tet gene was closely related to their own drug resistance level.And the tet gene introduced resistance was one of a mainly resistance mechanisms to tetracyclines.5.In this part,five ofloxacin resistant isolates were selected for studying the resistance mechanism of quinolinones.Through detection of the affection of CCCP to MIC values,i inducted mution experiments by levofloxacin and new drug-CO-06NF,the electrophoresis of OMP and whole proteins by SDS-PAGE, and SSCP of gyrA.Compared the change of MIC values of the 5 isolates,we can conclude that the that the resistance introduced by efflux pump was the mainly mechanism.And through the inducted mutation by levofloxacin and new drug-CO-06N,we drawed the OMP and whole protein of the mutation isolates and electrophoresis by SDS-PAGE.We got at leatest two protain(21KD and 445KD) were related with drug resistance.But there were no gene mutions of gyrA were observed by SSCP.So we could conclude that there are some other gens were mutated and led to drug resistance,but not the first combining site gene of quinolinones-gyrA.6.The resistance rates of 72 isolates to oxacillin,penicillin and cefepime were 100%,97.22%and 95.83%,and the resistance rate to cefotaxime were arrived to 70.93%too,this was similar to the result of MIC methods.And after added CCCP,the resistance rate to oxacillin,penicillin,cefotaxime and cefepime were decreased to 91.67%、55.56%%、21.97%和44.44%.and the Some MIC values were reduced to 1/2048,and there were noβ- Lactamase were detected on both phenotype and gene type.Then we can concluded that the resistance introduced by efflux pump was the mainly mechanism.Through the induction of cefepime,and SDS-PAGE electrophoresis of OMP,there are two protain strap were absent(14.4～20KD and 45KD).We detected the change of MIC values by CCCP,the MIC value were reduced to half of before,so we coald conclude that except the efflux pump mechanism,the lwer permeability of OMP were another resistance mechanism.But if there was the ther reisitance mechanism,that’ need a deeply and perseverativly study.更多还原