Mechanism Of Antibacterial Action Of Quinoxaline1,4-dio-Xides Against Clostridium Perfringens And Brachyspira Hyodysenteriae

Quinoxaline 1,4-dioxide derivatives(QdNOs)with a wide range of biological activities are used in animal husbandry worldwide,including carbadox,olaquindox(OLA),mequindox,quinocetone and cyadox(CYA).QdNOs are active against Gram-positive and Gram-negative bacteria,and are highly active under anaerobic conditions.QdNOs with good antimicrobial activity,the mechanism of which is ot clear yet.Early study showed that,the mechanism of antibacterial action is damaging DNA of bacteria,but it is still not clear how does it damage DNA and the study bacteria is Escherichia coli only.In order to study the mechanism of action of antibacterial QdNOs clearly,two anaerobic bacterial strains which were both highly sensitive to QdNOs were selected for this experiment;one strain was Clostridium perjfringens CVCC1125,gram-positive while the other strain was Brachyspira hyodysenteriae B204.Cyadox,the new member of QdNOs,and olaquindox,the old member of QdNOs,were selected as the representatives of QdNOs.In this study,morphological changes such as cell wall and cell membrane integrity along with DNA damage were detected under anaerobic conditions.Moreover,high performance liquid chromatography(HPLC)was used to detect the metabolites of QdNOs and fluorescence probe was used to identify ROS and hydroxyl free radical.We hope that this study would state the mechanism of antibacterial action of QdNOs under anaerobic conditions and speed up the developmental pace of new member of QdNOs as well as provide more reliable information for new drugs in future clinical use.1.Metabolites in bacterial and antibacterial activity of QdNOs and their metabolitesUnder anaerobic conditions,antibiotic susceptibility test showed that the MICs of cyadox against C.perfringens and Brachyspira hyodysenteriae B204 was 1 ?g/mL and 0.031 ?g/mL respectively.The MICs of olaquindox against Clostridium perfringens and Brachyspira hyodysenteriae was 1?g/mL and 0.0625 ?g/mL respectively,which indicated that both two bacterial strains were highly sensitive to cyadox and olaquindox.We detected two kinds deoxidation metabolites of cyadox(Cyl,bidesoxycyadox;Cy2,N4-desoxycyadox)in broth supernatant by high performance liquid chromatography(HPLC)when Clostridium perfringens and Brachyspira hyodysenteriae were incubated with cyadox for some time respectively,but did not found Nl-desoxycyadox(Cy10).We also detected two kinds of deoxidation metabolites of olaquindox(O1,N1-desoxyolaquindox;O2,bidesoxy desoxyolaquindox)with the same method.Antibiotic susceptibility test showed that all the deoxidation metabolites of cyadox and olaquindox had no antibacterial activity which means that QdNOs killed bacteria during deoxidatio process.2.Effects of QdNOs on morphological change of C.perfringens and B.hyodysenteriaeIn this experiment,C.perfringens and B.hyodysenteriae treated by subinhibitory concentration of QdNOs became longer and appeared in a form of filament by optical microscope,the phenomenon of which is the response of DNA damage.Well,SEM photographs showed that C.perfringens treated by QdNOs by SEM appeared longer,rough surface,and even fractured.B.hyodysenteriae treated by QdNOs appeared aggregation,curly and malformation.TEM photographs showed that C.perfringens treated by QdNOs appeared unequal cell division and cytoplasm leakage.TEM photographs showed that B.hyodysenteriae treated by QdNOs appeared serious cytoplasm leakage.3.Effects of QdNOs on integrity of cell wall and cell membrane of the two strainsIn our study,the alkaline phosphatase(ALP)levels of C.perfringens and B.hyodysenteriae were increased upon treatment with QdNOs,and C.perfringens produced higher amounts of ALP than B.hyodysenteriae;this may be due to overcome the oxidative stress,C.perfringens generated spore during which bacteria produce higher amounts of ALP.The leakage of intracellular material after exposure to the different concentrations of QdNOs was assessed by measuring the optical density at 260 nm(OD260).An increase in OD260 is indicative of leakage of intracellular nucleic acids and consequently,reflects a loss in membrane integrity.The data showed that the increase of OD260 was dependent on the drug concentration and incubation time.4.Intracellular free radicals of C.perfringens and B.hyodysenteriaeUnder anaerobic conditions,MIC and 4-fold MIC of cyadox and olaquindox caused a release of ROS in C.perfringens and B.hyodysenteriae.And the production of ROS in C.perfringens treated by QdNOs increased when the drug concentration increased.While the ROS level in C.perfringens treated by different concentration of QdNOs decreased between 90 to 120 min.On the contrary,the ROS level in B.hyodysenteriae had been accumulated from 30 min to 120 min,this may be caused by higher scavenging activity of superoxide dismutase(SOD)and catalase.According to the previous reports,QdNOs are enzymatically reduced in vivo to yield a radical intermediate that causes DNA damage.Under anaerobic conditions,this radical intermediate may produce hydroxyl radical via chemical reaction.In this experiment,MIC and 4-fold MIC of cyadox and olaquindox caused a release of hydroxyl radical in C.perfringens and B.hyodysenteriae under anaerobic conditions,which led to damage in cell membrane,DNA and intracellular respiratory system,ultimately affected the cell wall integrality.5.QdNO-induced chromosome DNA damage of the two strainsThe 8-Hydroxy-desoxyguanosine(8-OHdG)level of DNA in bacteria represents degree of the oxidative damage of DNA.In the experiment,8-OHdG level in bacteria treated by QdNOs,significantly increased compared with the level observed in the non-treated group.The formation of 8-OHdG can cause DNA breakage,which would be observed in agarose gel electrophoresis.The results showed that chromosome DNA of both of the two strains in agarose gel electrophoresis appeared trailing and smear phenomenon which indicated the DNA degradation and breakage.In addition,it was found that there was a fragment of 6?8kb in chromosomal DNA,the virus particle of which induced by DNA damaging agent was identified as prophageVSH-1.In conclusion,QdNOs are a class of hypoxia-selective and redox-activated DNA damaging agents which not only lead to oxidative damage to DNA but also damage to cell wall and cell membrane of bacteria via ROS and hydroxyl radical generation during metabolism process in bacteria under anaerobic conditions,resulting in bacterial cells death in the end.This study confirms that oxygen-free environment contributes to effective generation of free radicals during QdNOs metabolisms and the antimicrobial mechanisms of QdNOs against Gram-positive bacteria and Gram-negative bacteria are nearly the same.