Study On Multi-Drug Resistance Mechanism And Genotyping Of R. Anatipestifer

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.