Differential Expression Of AphA And HSP70 Of E.coli Isolates From Ducks And The Elimination Of Drug Resistance Of E.coli By Traditional Chinese Drug

Colibacillosis is an infectious disease caused by the bacterium Escherichia coli,or E.coli as it is commonly known,and is seen in ducks flocks worldwide.E.coli can cause an infection under the skin,known as cellulitis,and is commonly associated with respiratory disease in ducks,which in severe cases leads to septicaemia and death.Colibacillosis of ducks primarily affects ducks between the ages of 4 and 6 weeks and is responsible for a significant proportion of the mortality found in ducks flocks,and result in significant costs to the ducks industry.Antimicrobial plays very important role incontrolling diseases caused by E.coli,along with the widely use of antibiotics.Antibitic resistance to bacteria is getting worse.There are a significantly increasing tendency of multi-resistance.The resistance of E.coli has raised several concerns relatied to human health and the development of poultry industry.Therefore,it was very important to investigate the resistance of E.coli,to analyze the molecular mechanism of their drug resistance and study the elimination agent for the resistance of bacteria,which will be of great significance for monitoring and controlling of bacterial resistance.1 Identification and drug resistance analysis of E.coli isolates from ducks52 strains of E.coli were isolated from the infected duck in large-scale ducks farms in Chongqing area in an epidemic investigation from November 2007 to August 2008,and they were isolated and identified by the culture,microscope and biochemistry test in the lab.Twenty-one antibiotic scrips popular in clinic were selected to the antibiotics sensitivity test.By the K-B round scrips method,52 bacteria strains were detected the antibiotic sensitive.And the data were analysed according to the standard recommended by NCCLS(National committee of clinical and laboratory standard).The results were compared with the result of ATCC25922 which was set as control strain. The antibiotics sensitivity test indicated that antibiotic-resistance of the strains is very popular.All 52 strains E.coli were multi-drug-resistance and 39 strains were even resistant to more than 10 antibiotics.2 Differential expression of aphA and HSP70 of E.coli isolates from du(?)ksThis experiment is to construct recombinant plasmid as the standards for aphA and HSP70 gene detection by real time fluorescence quantitative PCR.The DNA fragment of aphA and HSP70 was amplified by PCR,cloned into pMD19-T vector,transformed into bacterium DH5αand restriction enzyme digestion.The concentration of purified plasmid DNA solution was determined and ten-fold serially diluted into 10~8 to 10~4 copy/μL.Standard curves were constructed by real-time PCR detection with the series of plasmid standards.The results indicated that the standard curves were shown to be of high linearity.The method is to compare the levels of aphA mRNA and HSP70 mRNA of multidrug strain and ATCC25922 with quantitative RT-PCR.The results were as follows: the aphA mRNA level of multidrug strain is 1202 times as high as that of ATCC25922,the HSP70 mRNA level of multidrug strain is 34.21 times as high as that of ATCC25922.The results showed that there was a correlation between the aphA mRNA and HSP70 mRNA levels and resistance level of the multidrug strain.Detect the expression of the HSP70 mRNA of isolates by RT-PCR,the result shows that HSPT0 can be used to biomarker for drug resistance.3 The elimination of drug resistance of E.coli from ducks by traditional Chinese drugTo observe the cancellation effect on drug resistance E.coli,this test has selected three kinds traditional Chinese medicine monomer of bacteriostasis and three kinds of traditional Chinese medicine monomer without antibacterial,and two chemical agents as a control.The minimal inhibitory concentration(MIC) test indicated that the MIC were 200μg/mL in SDS,100μg/mL in Glibenclamide,500μg/mL in Matrine,250μg/mL in Berberine hydrochloride,500μg/mL in Artemisinin,respectively.The antibiotics sensitivity tests showed that,compared with the E.coli strains,the inhibition zone of the strains with drugs were increased to Aztreonam,Fortum,Amikacin, Tobramycin,Erythromycin,Ciprofloxacin,Ofloxacin,Rifampicin. The result of PCR detection demonstrated that the drug Resistance Gene(rpsL and intⅠ) not be eliminated by the traditional Chinese drug monomer,but the Matrine and Evodiamine could eliminated the blaTEM of the multi-drug strain.The results suggested that some resistance gene could be eliminated by the traditional medicine monomer of no antifungal effect.The method is to compare the levels of aphA mRNA of multidrug strain and drugs strains with quantitative RT-PCR. The result of the aphA mRNA indicated that gene expression were 0.873%in Matrine,4.78%in Berberine hydrochloride,28.0%in Artemisinin,0.952%in Sinomenine,6.92%in Evodiamine, 0.923%in TanshinoneⅡA,respectively.