Molecular Mechanism Of Aminoglycoside Resistance In Escherichia Coli Isolated From Animals

Aminoglycoside antibiotics are a kind of high performance and broad-spectrum drugs. Because of antibiotics are in use widely, the phenomenon of drug resistance in bacteria become more serious than before, which lead to the use of those antibiotics in therapy were suffered limited. However, the research on structure, activity and drug kinetics of those antibiotics is maturation. The structure of semisynthetic Aminoglycoside antibiotics cannot be changed so easily; therefore, the mechanisms of this drug resistance must be reconstructed for research.2 strains of higher-level drug resistance, 4 strains of midrange level drug resistance, and 1 strains of lower level drug resistance were selected from 15 strains Escherichia coli isolated. To monitor and control the drug resistance of aminoglycoside antibiotics, we investigate the mechanisms of drug resistance to those strains from several aspects.Firstly, we select acc(6' )-Ib, acc(3)-TV, aadA, aphA, aph(2 ") which are the gene of usual inactive enzymes to amplify, but only the gene of aphA is amplified successfully, so we think the drug resistance mechanisms of those strains are caused by phosphotransferase which is coded by aphA.Secondly, we detect the mRNA expression of aphA gene by real time RT-PCR to those strains, the approach is to construct standard plasmid using reference primers, extract RNA from those strains, reverse transcription and run real time RT-PCR using detected primers. The results proved that there are differences in the mRNA expression of aphA gene within those strains; they are correlated to each other. And through the differences between drug resistance and expression, we detect the major mechanisms of those strains were caused by phosphotransferase which could make antibiotics inactivation.Meanwhile, the proteinic expression of aphA gene was qualitated by western blotting, the approach is to clone and express aphA gene, to immune animals using the expressed protein, to get antibody blood serum, and at last, to detect the proteinic expression by western blotting. The results of aphA proteinic expression is positive correlated to drug resistance intensity, thus, the conclusion is resembled to the results of real time RT-PCR.To analyze the results further, 4 strains of midrange level drug resistance have the equivalent drug resistance intensity, and the level of expression are corresponded with each other, so we think the major...