Study On The Mechanism Of Luteolin Reducing The Drug Resistance Of Trueperella Pyogenes To Tetracycline Antibiotics

Trueperella pyogenes(T.pyogenes)is a gram-positive pathogen that leads to various pyogenic infections in economic animals.This disease has become a difficult problem in clinical treatment which caused by antibiotic-resistant T.pyogenes.In previous study,we found that efflux genes tetK and tetL could mediate the development of antibiotic resistance of T.pyogenes to tetracycline antibiotics.Similarly,efflux pump inhibitors can inhibit the efflux effect of drug-resistant strains on antibiotics,and restore the sensitivity of drug-resistant strains to antibiotics to a certain extent.The screening of high-efficiency and low-toxicity efflux pump inhibitors has become a hotspot of research currently.Luteolin,as a typical flavonoid,not only has anti-bacterial,anti-allergic,anti-inflammatory,anti-tumour and other pharmacological activities,can it also reduce the resistances of several bacteria to macrolides and aminoglycoside antibiotics.In this research,we will determine the drug resistance of T.pyogenes to tetracycline antibiotics as well as the expression of tetracycline efflux genes and proteins of T.pyogenes before and after the treatment of Luteolin.It will be analysed the mechanism of luteolin reducing the antibiotic resistance of T.pyogenes to tetracycline antibiotics,and that will lay a theoretical foundation for the development and application of luteolin as a new drug against drug-resistant bacteria.The Minimal Inhibitory Concentration(MIC)values of five tetracycline antibiotics(tetracycline,aureomycin,doxycycline,metacycline and oxytetracycline)against T.pyogenes before and after the treatment of luteolin by using the broth microdilution method.After comparing the alteration of MIC values,the optimal conditions for luteolin to reduce the drug resistance of T.pyogenes were found to be 1/2 MIC for 36 h.It showed that the MICs of five tetracycline antibiotics against drug resistance T.pyogenes carrying tetK and tetL efflux genes were decreased to different levels.Nevertheless,the MIC values of five tetracycline antibiotics did not change which the strains carry the tetK efflux genes but were sensitive to tetracycline antibiotics.It suggested that luteolin could cut the resistance of T.pyogenes to tetracycline antibiotics by inhibiting the tetK and tetL efflux genes.The functions of luteolin on mRNA expression of tetK and tetL efflux genes was detected by fluorescence quantitative PCR.The results showed that luteolin can remarkably reduce the mRNA expression level of tetK and tetL efflux gene of drug resistance T.pyogenes.However,luteolin showed no effect on the mRNA expression level of tetK efflux gene of a subtype of T.pyogenes,which the strains carry the tetK efflux genes but were sensitive to tetracycline antibiotics.The mRNA expression level of tetK and tetL gene of most strains has the same trend as the MICs value of antibiotic.Reserpine is a control drug,and the results were the same as luteolin.In this study,the prokaryotic expression vector of tetK gene was constructed to prepare TetK protein.Rabbit polyclonal antibodies against T.pyogenes TetK protein were prepared by immunizing rabbits.TetK expression in T.pyogenes was detected before and after luteolin action by Western Blot.The results showed that luteolin can remarkably reduce the TetK expression level of drug resistance T.pyogenes,However,luteolin had no effect on the TetK expression level of T.pyogenes,which the strains were sensitive to tetracycline antibiotics.The TetK expression level of most strains has the same trend as the MICs value of antibiotic.Reserpine is a control drug,and the results were the same as luteolin.It is revealed that luteolin may reduce the resistance of T.pyogenes to tetracycline antibiotics by inhibiting the expression of TetK.This study indicated that luteolin may reduce tetracycline antibiotic resistance of T.pyogenes by inhibiting the expression of efflux genes tetK and tetL mRNA and efflux protein TetK,which will lay a solid theoretical foundation for the in-depth study of the resistance mechanism of luteolin inhibiting bacteria and the development of many new antimicrobial drugs.