Study On The Combined Toxic And Resistance Of Polypeptides And Sulfonamides On Bacteria

Antibiotics are widely used in medical and health,livestock and aquaculture fields because of their good bacteriostatic and bactericidal properties.However,the abuse of antibiotics has led to the production of a large number of drug-resistant bacteria.One of the important conditions for bacterial drug resistance is to obtain antibiotic resistance genes?AntibioticResistanceGenes,ARGs?.The more common ways of acquisition are plasmid conjugation transfer and gene mutation.In the face of the problem of bacterial drug resistance,it is urgent to explore and develop substitutes for antibiotics.Polypeptide antibiotics?Antimicrobialpeptides,AMPs?is an important defense molecule against the invasion of pathogenic microorganisms,which widely exists in the biological world,and is an important defense substance of biological innate immunity.It not only has broad-spectrum antibacterial activity and anti-viral,anti-fungal,anti-parasite and anti-tumor biological activities,but also has structural diversity with species adaptation to the environment and unique bactericidal mechanism different from traditional antibiotics,and is not easy to produce drug resistance.It is a kind of peptide antibiotics with great potential.Polypeptide antibiotics are also recommended to be combined with low concentrations of antibiotics to reduce the use of antibiotics.However,the mechanism of combined toxicity of antimicrobials is more complex,and the results of the combined effects of different types of antimicrobials are difficult to predict.However,the effects of combined exposure of peptides and antibiotics on bacterial growth and drug resistance,and the mechanism of their combined toxicity are unknown.In this study,polypeptide antibiotics?Antimicrobialpeptides,AMPs?,sulfonamides?Sulfonamides,SAs?and sulfa synergist?Sulfonamidepotentiators,SAPs?were used as test compounds,and Bacillus subtilis?Bacillus subtilis,B.subtilis?and Escherichia coli?Escherichiacoli,E.coli?,which are representative of gram-positive bacteria?Gram-positive bacteria?and gram-negative bacteria?Gram-negative bacteria?,were used as model organisms.The effects of polypeptides and sulfonamide/sulfonamide synergist binary mixtures on the combined toxicity of B.subtilis and E.coli,the conjugation transfer effect of E.coliRP4 plasmid and the mutation effect of E.coliMG1655 were studied.The main results and conclusions are as follows:?1?The toxic effect of the binary combination of antibiotic AMPs-SAs/SAPs on the growth of two kinds of bacteria was studied.the results showed that the dose-effect curve of the binary mixture of AMPsSAs/SAPs on the growth of B.subtilis showed an obvious"S"shape,that is,it had only inhibitory effect on the growth of B.subtilis.When the binary mixture of AMPs-SAs/SAPs acts on E.coil,it can produce an obvious Hormesis effect of"low concentration promotion,high concentration inhibition",of which the maximum promoting effect is 23.57%.In addition,most of the joint toxicity tables of AMPs-SAs/SAPs binary mixtures to bacteria showed synergistic and antagonistic effects.We speculate that the synergistic effect of mixed drugs on bacteria may be due to their ability to induce the formation of ROS.The antagonistic effect of some mixtures on bacteria may be due to the different binding numbers of polypeptide antibiotics to B.subtilis and E.coli and the structural particularity of some peptide antibiotics.Meanwhile,the combined toxicity of the mixture on the two bacteria is also different.The experimental data show that the toxicity of E.coli is greater than that of B.subtilis.The reason for this phenomenon may be that the cell wall of B.subtilis is thicker than that of E.coli,and the drug is less likely to pass through the cell wall of B.subtilis.?2?The effects of 24 groups of AMPs-SAs/SAPs mixed compounds on the conjugation transfer effect of RP4 plasmid were studied.The results showed that all the 24 groups showed an obvious inverted"U"dose-response curve,that is,the effect of mixed antibiotics on the conjugation transfer effect of RP4plasmid showed a Hormesis phenomenon.The results showed that the RU₂ of AMPs-SAs was mostly synergistic,and the RU₁ of AMPs-SAPs was antagonistic,which was consistent with the combined toxicity of II and AMPs-SAs/SAPs,which might be related to the number of binding sites between single component and target protein in the mixed system,so we speculated that the conjugation transfer effect might be related to the combined toxicity.In addition,most of the combined effects of AMPs-SAs/SAPs mixed compounds on the conjugation transfer effect of RP4 plasmid were synergistic and antagonistic.Previous studies have shown that polypeptide antibiotics enter the cell by producing small pores in the bacterial cell wall,while sulfonamides may hinder nucleic acid synthesis by competitive binding to DHPs/DHFR,triggering a SOS?stress response?response.In that case,the synergistic effect between the two may be due to the fact that polypeptide antibiotics can produce holes in the cell wall,making more sulfonamides enter the cells,and then induce the SOS reaction to occur faster,thus promoting conjugation transfer.On the other hand,the different concentrations of some peptides and their special structures may eliminate the synthesis of nucleic acids by sulfonamides and show antagonism.?3?The effect of the combined action of 14 groups of AMPs-SAs/SAPs mixed compounds on the mutation effect of MG1655E.coli was studied.The results showed that the mixture could promote the mutation of MG1655E.coli resistant to rifampicin.With the increase of the concentration of the mixture,the mutation promotion effect increased at first and then decreased,and the mutation effect curve showed an inverted"U"shape.In this study,the joint conjugation transfer effect was expressed by MU.The MU at the mixed system RC_0-1?the minimum concentration for promoting conjugation transfer?was set to MU₁,and the MU at the mixed system RC₀₂?the maximum concentration for promoting conjugation transfer?was set to MU₂.Among them,the joint action mode of AMPs-SAs/SAPs on Escherichia coli mutation is mainly synergistic and antagonistic,which is consistent with the combined toxicity of AMPs-SAs/SAPs.Among them,the discriminant results of MU₁ and MU₂ of 7 groups of mixtures were the same,that is,the combination of AMPs and SAs/SAPs could promote the mutation of Escherichia coli,and the mode of combined action was the same in the increasing stage and decreasing stage.Based on the analysis in?1?,we speculate that sulfonamides may promote mutation by promoting the expression of SOS gene and synergistically with polypeptides,while the inhibitory effect of xanthamine antibiotics on nucleic acid synthesis will hinder the promotion of mutation by polypeptides.