Study On Antimicrobial Active Ingredients Of Euphorbia Heyneana Spreng. And Its Mechanism Against Multidrug-resistant Escherichia Coli

Escherichia coli is the most common pathogen that causes animal infectious and finally evolved into multidrug-resistant or extensively drug-resistant E.coli under the pressure of antibiotics,leading to the treatment failure of bacterial infection in veterinary medicine.In order to combat the escalating antibiotic resistance,it is vital to focus on finding or developing new antibacterial.Traditional Chinese Medicine,as a natural reservoir of small molecule com-pounds,exert a rich variety of activities,lower environmental pollution with less drug residues,which expands the source of new antibacterial screening and provides a new strategy for the treatment of MDR bacterial infection.Euphorbia heyneana previously has been shown to pos-sess good antidiarrheal and antimicrobial activity,but its antimicrobial active ingredients and mechanism of action remain unclear.Therefore,MDR-E.coli carrying the mcr-1 gene was isolated and identified,and resistance phenotype,sequence type and genomic characteristics were analyzed in detail.Meanwhile,MDR-E.coli carrying the mcr-1 gene was used as the tested strains to track the strongest active extract,and the antibacterial active ingredients of that was isolated,purified and identified.The most active compound screened was used to study the molecular mechanism of its antimicrobial effect.The main results are as follows:(1)Isolation,resistance detection and sequence type analysis of MDR E.coli:The resistance spectrum and the diversity of ST type of MDR E.coli carrying-mcr-1 gene isolated from animal lesions organs were determined using PCR amplification,broth microdilution method and MLST.The results showed that a total of 24 E.coli strains carrying-mcr-1 gene were isolated,all of which were resistant to 3 or more antibiotics,and 9 of which showed resistance to all tested antibiotics.The resistance rate of 24 strains for CAZ,CIP,ENR,COL,SMZ was 100%,95.8%for AMP,TET and FFC,91.7%for CTX,ATM and DOX,79.2%for ENR and FOS,37.5%for MEM.24 strains belonged to nine STs and classified into eight clonal complexes,of which MDR E.coli carrying-mcr-1 gene ST10596 was newly discovered.24 E.coli strains carrying the mcr-1 gene exhibited MDR and XDR.The clonal complexes were mainly concentrated in ST156 CC,ST448 CC,and ST10 CC,ST10596 was the newly discovered ST type in this study.(2)Genomic features of MDR E.coli S-4:Broth mating assay,S1-PFGE,Southern hy-bridization and WGS were used to investigate conjugation and transfer of mcr-1 gene,the location of mcr-1 gene and the genomic features of S-4 strain.The results showed that mcr-1gene in E.coli S-4 located on approximately 33.3 kb plasmid and could be successfully trans-ferred to the recipient EC600 conferring at least 8-fold colistin resistance.WGS analysis of S-4 strain showed that this strain contains one chromosome and two plasmids p S4-mcr-1.1(Inc X4)and p S4-aph(Inc FII-FIB).The chromosome contained two acquired resistance genes[mdf(A)and tet(A)],two points mutations(83 and 87 of gyr A gene,57 and 80 of par C gene)and 54 genes associated with antimicrobial resistance annotated in CARD database.The re-sistance genes bla TEM-1B,aph(4)-Ia,aac(3)-IV,sul3,tet(M)and mef(B)were located on Inc FII-FIB-type plasmid and one composite transposon ISEc59-aph(4)-Ia-aac(3)-IV-IS15was found on this plasmid;mcr-1 gene and two insert sequences IS26,IS2 all were located on the Inc X4-type plasmid,on which IS2 inserted and truncated the vir B6 gene in T4SS.These results indicated that the acquired resistance gene,the point mutation on the chromosome and the acquired resistance genes on the plasmid collectively conferred the strain S-4 with MDR.The insertion sequence and transposon could mediate the translocation of resistance gene and enriched the types and characteristics of mobile element.(3)Isolation and identification of antimicrobial active components from E.heyneana against MDR E.coli isolates:Tracking and isolating the antimicrobial active extract of E.heyneana were subjected to the systematic solvent extraction method,macro-porous adsorbent resin and column chromatography,and characterizing of the compounds isolated were per-formed by ¹H-NMR,¹³C-NMR and ESI-MS.The results showed that the ethyl acetate extract from E.heyneana exhibited the strongest antibacterial activity against five MDR E.coli mcr-1-carrying strains,with the MIC of 12.8 mg/m L,and further enriched in 30%ethanol eluent,showing the strongest antibacterial activity with an MIC of 5.12 mg/m L.Five compounds were isolated and identified from the 30%ethanol eluate,namely quercetin,kaempferol,lute-olin,apigenin-7-O-?-D-glucopyranoside and gallic acid,and the MICs were 0.256-0.512,1.024,2.048,2.048,5.12 mg/m L,respectively.The ethyl acetate extract of E.heyneana pos-sessed the strongest antimicrobial activity against MDR E.coli carrying the mcr-1 gene,quer-cetin among the five compounds isolated from the ethyl acetate extract showed the strongest activity against MDR-E.coli.(4)The effect of quercetin from E.heyneana on MDR E.coli S-4 isolate and the expression level of plasmid-mediated drug-resistant genes:The bacteriostatic and bacteri-cidal activity,antibacterial synergism,and the effect on expression level of plasmid-mediated drug resistance genes in E.coli S-4 were performed by time-kill kinetic assay,checkerboard broth microdilution method,and q RT-PCR.The results showed that quercetin exhibited bac-tericidal effect at 2×MIC concentration,bacteriostatic effect at 1/2×MIC and 1×MIC concen-tration.Quercetin in combination with CAZ,CTX,ATM,TET,DOX,ENR,AZM,FOS,FFC,AMP,CIP,COL,MEM exhibited synergistic effects,combined with SMZ showing additive effect,combined with GEN showing irrelevant effect.mcr-1,bla TEM-1B,aph(4)-Ia,tet(M),and sul3 genes on the plasmid were significantly down-regulated by quercetin at 1×MIC(P<0.01).Quercetin exhibited the bactericidal activity at a high concentration and the bacteri-ostatic activity at a low concentration.Quercetin in combination with?-lactams,tetracyclines,fluoroquinolones,fosfomycins,chloramphenicol,and polymyxin antibiotics showed synergis-tic effects,combined with sulfonamides showing additive effect.Meanwhile,quercetin could down-regulate the m RNA expression level of resistance genes on the plasmid.(5)Study on antibacterial mechanism of quercetin from E.heyneana against MDR E.coli:The antimicrobial mechanism of quercetin was investigated by LIVE/DEAD assay,SEM,TEM,the extracellular AKP activity,SDS-PAGE,flow cytometry and RNA-seq.The results showed that the green fluorescence intensity of S-4 cells in the control group and the treatment group with 1/2×MIC was significantly stronger than the red fluorescence(P<0.01),and the difference between the green fluorescence intensity and red fluorescence intensity of S-4 cells treated with 1×MIC quercetin was not significant(P>0.05),in contrast,the red fluo-rescence intensity of treatment group with 2×MIC quercetin was significantly stronger than the green fluorescence intensity(P<0.01).SEM and TEM of the treatment group with 1×MIC quercetin showed swelling,recession and shrinkage and leakage of contents,and the ultra-microstructure of the bacteria showed the phenomenon of plasmolysis,the contents reduction,the inner and outer membranes of cells blurred,thinned or even disappeared,and vacuolar structure.The extracellular AKP activity of the bacteria treated with quercetin at 1/4×MIC,1/2×MIC and 1×MIC was significantly higher than that of the control group(P<0.01).The gray values of protein bands(about 40 k Da size)in the treatment group of quercetin at1/2×MIC and 1×MIC showed that significant differences(P<0.01).The apoptotic rates of the1/4×MIC,1/2×MIC,and 1×MIC treatment groups were significantly different from the control group(P<0.01).A total of 549 DEGs were identified by transcriptome sequencing.Among them,308 genes were significantly down-regulated and 241 genes were significantly up-reg-ulated.Down-regulated DEGs significantly enriched the GO terms,such as biological pro-cesses,outer membrane,plasma membrane,cellular components,and precursor metabolites and energy,and the KEGG pathway was significantly enriched in the TCSs(Pho P),NM(nar-GHJI operon),and CAMP resistance(arn BCA operon,pag P and ept A)(P<0.05).The up-regulated DEGs significantly enriched GO terms,such as cellular response to stimulus,trans-porter activity and carboxylic acid metabolic process;KEGG pathway significantly enriched glyoxylate and dicarboxylate metabolism and ABC transporter(P<0.05).The antibacterial mechanism of quercetin against MDR-E.coli carrying mcr-1 gene was to down-regulate the expression of related genes in TCSs,nitrogen metabolism and CAMP pathway,which caused the bacterial energy generation disorders and finally led to the integrity and permeability of cell wall/membrane destroyed and the inhibition of related protein synthesis.In addition,the expression of mcr-1 gene and genes related to CAMP resistance were down-regulated by quer-cetin to reduce polymyxin resistance.