| Salmonella is aimportant zoonotic pathogen,it can cause disease in animals,and can also cause disease in humans by contaminating animal products such as meat,eggs,and milk.In recent years,with the rapid development of animal husbandry,a large number of antibacterial drugs have been used to prevent and treat Salmonella infections.However,due to their widespread and unreasonable use in animal clinics,not only a large number of antibacterial drugs remain in animal foods,but also It has also led to the emergence of multi-drug resistant strains of Salmonella,posing a serious threat to people’s health.Therefore,in-depth understanding of the multidrug resistance mechanism of Salmonella and protecting the safety of animal food has become a top priority.It had been confirmed that two-component signal transduction system and multiple drug-resistant efflux pump play an indispensable role in the mechanism of Salmonella resistance.The Bae SR two-component system is a membrane stress response system that can control the adaptive response of bacteria to environmental changes.It plays an important role in regulating the resistance of Salmonella.In addition,the presence of acr B may mask the function of other efflux pumps,making Bae SR unable to fully function.Therefore,in the present study,a bae R complementary strain(CRcbae R△bae SR△acr B)and a bae R overexpression strain(CRpbae R△bae SR△acr B)were construced based on a bae SR and acr B double-deletion strains of Salmonella typhimurium(CR△bae SR△acr B),and antimicrobial susceptibility and biological characteristics were tested.Furthermore,RNA-seq,the Lac Z fusion assay experimental,and EMSA were performed to investigate the molecular mechanism of Bae SR regulating drug resistance in Salmonella typhimurium.The main research contents and results are as follows:1.Minimum Inhibitory Concentration and biological characteristics of CR△bae SR△acr B,CRcbae R△bae SR△acr B and CRpbae R△bae SR△acr B strains were detected.The MIC,growth characteristics,biofilm formation ability,and motility of the tested strains were tested respectively.The MIC of amoxicillin,ampicillin,ceftazidime,ceftiofur,florfenicol,mequindox,ofloxacin,and enrofloxacin was 256-,256-,16-,16-,4-,4-,2-,and 2-fold higher,respectively,in CRpbae R△bae SR△acr B,than in CR△bae SR△acr B.The MIC of spectinomycin and apramycin decreased 2-fold,whereas the MIC of other drugs remained unchanged.The MIC of ceftazidime,ceftiofur,florfenicol,and enrofloxacin was two-fold higher in CRcbae R△bae SR△acr B,than that in CR△bae SR△acr B,but the MIC of other drugs remained unchanged.The growth curves of CR△bae SR△acr B,CRpbae R△bae SR△acr B,and CRcbae R△bae SR△acr B showed no significant difference.However,the biofilm-forming ability and motility of CRpbae R△bae SR△acr B and CRcbae R△bae SR△acr B were significantly higher than those of CR△bae SR△acr B(P< 0.01).Indicating that bae R affects the drug resistance of Salmonella typhimurium by affecting its biofilm-forming ability and motility.2.The transcriptomics datas of CR△bae SR△acr B,CRcbae R△bae SR△acr B and CRpbae R△bae SR△acr B were analyzed.The differentially expressed genes were identified by RNA-seq of CR△bae SR△acr B,CRcbae R△bae SR△acr B and CRpbae R△bae SR△acr B on the basis of the criteria | log2(Fold Change)| > 1 and padj < 0.05 simultaneously.A total of 743 differentially expressed genes were identified between CRpbae R△bae SR△acr B and CR△bae SR△acr B,of which 19 genes were downregulated and 724 genes were upregulated.Bioinformatics analysis of the differential genes showed that the differential genes were mainly enriched in pathways such as Bacterial secretion system,β-lactam resistance,quorum sensing,two-component system,TCA cycle and lipopolysaccharide biosynthesis.A total of 3073 differentially expressed genes were identified between CRcbae R△bae SR△acr B and CR△bae SR△acr B,of which 1606 genes were downregulated and 1467 genes were upregulated.Bioinformatics analysis of the differential genes showed that the differential genes were mainly enriched in pathways such as carbon metabolism,biosynthesis of amino acids,Biosynthesis of antibiotics,ABC transporters and Biosynthesis of secondary metabolites.According to gene annotation and literature reports,a total of 15 drug resistance-related differentially expressed genes were screened,involving efflux pump,biofilm,two-component system and so on.Random screening of 5 differentially expressed genes was basically consistent with the q RT-PCR detection results,which confirmed the reliability of RNA-Seq analysis results.3.The Lac Z fusion assay experimental proved the regulation relationship of Bae R on target genes.Select omp W,sod B,tol C,mdt D,mar R and spy as the potential target genes of Bae R,design specific primers according to their promoter regions,and connect the promoters of each potential target gene to the p ACYC184-lac Z plasmid without a promoter region.Recombinant plasmids were then transferred into CRpbae R△bae SR△acr B and CR△bae SR△acr B,and the difference in β-galactosidase activity was measured to verify the regulation of Bae R on target genes.The results show that Bae R can positively regulate the expression of omp W,sod B,tol C,mdt D,mar R and spy,thereby regulating the resistance of Salmonella to antibiotics.4.The electrophoretic mobility shift assay to explore the target genes regulated by Bae R.omp W,sod B,tol C,mdt D,mar R and spy were selected as target genes of Bae R.The electrophoretic mobility shift assay was carried out according to the promoter region of the DNA fragment containing biotin labelled.The results showed that the expression of mar R genes was regulated by the Bae R protein.In this study,we constructed bae R gene overexpression strains under the condition of acr B and bae SR deletion.Through MIC,biological function validation,transcriptome,the Lac Z fusion assay experimental,EMSA,and other experimental means,we explored the changes of drug resistance before and after the bae R gene overexpression,explored the mechanism of drug resistance,and provided scientific basis for further study on the mechanism of Bae SR resistance to Salmonella typhimurium. |
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