| Polymyxin has been re-used in clinical practice due to the treatment of infections caused by multi-drug-resistant gram-negative pathogens.In 2018,the World Health Organization classified antibiotics such as polymyxin as "vital antimicrobial drugs" for use in humans and animals.And 30% to 90% of the drugs taken by organisms enter the environment through excretion.Therefore,it is expected that the consumption of polymyxin will increase in the near future,and the environmental pollution caused by these compounds will also increase.These compounds have significant persistence and the ability to accumulate in the soil.Clay minerals are the most common substances in the surface environment of the earth and coexist with antibiotics and bacteria for a long time.However,there are a series of complex interface reactions among clay minerals,antibiotics and bacteria.Clay minerals affect the stress behavior and resistance mechanism of bacteria under antibiotic stress.To this end,this paper studied the mechanism of action of different concentrations of polymyxin B on E.coli and the regulation effect of clay minerals on bacteria under the stress of gradient concentration of polymyxin B.The research in this paper mainly achieved the following results:(1)Using the polymyxin B medium additive as an inducer,E.coli was continuously induced in the medium containing 0,1.0,5.0,10.0 mg/L of polymyxin B.The results of confocal laser microscopy showed that as the concentration of polymyxin B increased,the degree of damage to the bacterial cell membrane became more serious.The proteomic analysis results showed that the protein expression of bacteria was significantly different under the induction of 1.0 and 10.0 mg/L polymyxin B.Among them,the bacteria continuously induced in 10.0 mg/L polymyxin B had higher levels of multidrug resistance efflux transporter and efflux pump membrane transporter expression,and high concentration of polymyxin B further increased multidrug resistance of bacteria.With the increase of the concentration of polymyxin B,bacteria will up-regulate the expression of Fad D protein.The protein participates in the aerobic β-oxidative degradation of fatty acids,which in turn leads to the overproduction of lipopolysaccharides,damages and even kills bacteria.(2)Using polymyxin B sulfate as an inducer,the E.coli cultured with or without clay minerals was continuously induced with gradient concentration of polymyxin B.The potential mechanism of kaolinite and halloysite on E.coli under polymyxin B stress was analyzed by means of proteomics,antibiotic resistance detection,confocal laser scanning electron microscopy,scanning electron microscope and Fourier transform infrared.The research results showed that kaolinite had a significant promoting effect on the growth of bacteria.Compared with halloysite,kaolinite could stimulate the overexpression of polymyxin B resistance-related proteins Arn A,Arn B and Ept A in E.coli under polymyxin B stress,and could promote the synthesis of peptidoglycan and activate the glycolytic pathway to produce energy.Halloysite could regulate the production of low-molecular-weight thiols by E.coli to prevent the bacteria from producing excessive reactive oxygen species,activate the oxidative phosphorylation pathway to provide energy for bacterial life activities,and reduce the multidrug resistance of E.coli through a variety of ways. |
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