The Mechanism Of The Free Radicals Formation In E. Coli Induced By QdNOs

Quinoxaline 1,4-dioxide (QdNOs) is a kind of chemosynthesis antibacterial angents. Due to its excellent antibacterial activity, growth improvement and low rate of resistance, QdNOs is widely used in livestock and aquafarm all over the world. There’s a confusion "how the QdNOs kill the bacteria" and even now the drug’s antibacterial mechanism is not been elaborated clearly. Our research aimed at free radical which is the connection between bacteria and drug, and the key to the mechanism of QdNOs. HF Zhang (2012) detected large amounts of ROS which may induced DNA damage in E. coli (Escherichia. coli) after incubation with cyadox. The researchers hypothesise that the QdNOs could be reduced to drug radical which may cause DNA damage. What we need to do is to identify if it happens in bacteria and to analysis the mechanism of its formation. In this research we used E. coli CVCC2943 as the experimental strain due to its high susceptible to cyadox. Cyadox and mequindox had been chosen as the experimental drug for their high susceptibility to bacteria and the widely use in production. Olaquindoxin and carbadox are only tested in some parts of the research. There’s two parts in our research:first part is to detect the free radical pattern and structure in E. coli CVCC2943 after the other QdNOs’ affection; the second part was to determine the formation mechanism of the free radical.1. Free radical detection in bacteria affected by QdNOsIn this section we tested the MIC and MBC under anaerobic and aerobic condition respectively for a reference of concentration. Then we determined ROS and superoxide in bacteria which was incubated with MEQ and OLA under anaerobic and aerobic condition respectively. And we also identify the structure of drug radical by electron paramagnetic resonance.The results of the QdNOs sensitive test reveal that the QdNOs except QCT are susceptible to E. coli CVCC2943 under anaerobic condition. Contrasts to the anaerobic test, most of the QdNOs aren’t sensitive to E. coli under the aerobic condition. There’s a situation that the ROS production of the CYA affected bacteria could reach the peak at about 1h and then descend slowly under the anaerobic condition in Zhang’s paper. A similar pattern was found in the test of OLA and MEQ that the ROS reach the peak at 30 min. We also identified the drug radical intermediate through the incubation of drug with bacteria protein extraction not in the bacteria. The spectra showed us a triplet and it maybe the complex of drug radical anion and its proton product.2. The mechanism of the formation of free radical induced by QdNOsSome papers suggest that the QdNOs could be activated by the metabolism enzymes to drug radical intermediates. We have found that drug radical but the enzymes which activate the drug to radical formation still a puzzle to us. In this section, we used many kinds of metabolism enzyme inhibitors to inhibit the enzymes’ activity and through bacterial counting and free radical/metabolites detection to confirm which enzymes participate in the activation of QdNOs.We chose the xanthine oxidase/alcohol dehydrogenase/aldehyde dehydrogenase/lipoamide dehydrogenase/uricase/thioredoxin as the candidates after papers reading. At last we found that XOR and proteins contain ferrous could influence the drug’s activity to E. coli, in which maybe cytochrome oxidase or peroxidase involved. The XOR then was determined by a series of test:superoxide measurement, drug radical intermediates detection, metabolites detection, DNA damage of XOR and QdNOs to confirm its key role to QdNOs.There’s a phenomenon that the electron could be leaked by the disorder of electron transfer chain (respiratory chain) which could be caused by antibiotics. In this section, we will study if this situation appears in our research. The answer is positive, but the influence by QdNOs is much less than ENR.For a conclusion of the above, we determined the pattern of QdNOs caused free radical in E. coli and the structure of drug radical intermediates in the incubation with bacteria protein extraction. The XOR is confirmed as one of the key enzymes of QdNOs and the XOR was able to metabolise the drug to superoxide and drug radical at the condition of anaerobic and aerobic respectively. Also the XOR could activate the drug and induce damage of DNA. At last, we find the free radical caused by ETC disorder which influenced by the QdNOs, but the ETC radical only possesses small portion in total ROS.