Molecular Mechanism Of Reversing Synergistically Resistance Of Salmonella To Colistin By The Combination Of Baicalin And EDTA

Colistin is a cationic peptide antibacterial drug and currently used as a last-line drug for treating MDR bacterial infections.However,its resistance has been increasing worldwide in recent years and has become the focus of global attention.Much progress has been made in the research on the mechanism of colistin resistance.The reported mechanisms include: chromosomal-mediated two-component signal transduction system(TCSs)PhoPQ and PmAB,plasmid-mediated colistin resistance gene mcr9 and the unelucidated molecular mechanism of active efflux system,etc.TCS CpxAR is one of the common two-component systems in Gram-negative bacteria,and it has an important regulatory effect on bacterial resistance and pathogenicity.AcrABTolC is the most important active efflux system of Salmonella,which can excrete exogenous substances(such as antibiotics)and metabolites,and plays an important regulatory role in MDR.Preliminary studies in our laboratory found that CpxR has a regulatory effect on the MDR of Salmonella typhimurium(JS).When acrB and cpxR are both missing and then replenished cpxR {cpxR activation),strain JSAacrBAcpxR:.Mon/pcpxR can significantly increase the sensitivity of Salmonella typhimurium to colistin(16 times),and the resistance can be reversed.Based on this important finding,the CpxAR system activator EDTA and the efflux pump inhibitor CCCP and the traditional Chinese medicine monomer baicalin with the function of inhibiting the efflux pump were further selected,to confirm its synergistic effect on reversing bacterial colistin resistance in clinical isolation of Salmonella^ and to study its molecular mechanism in order to explore the strategy of combination medication to control the infection of colistin-resistant bacteria.From 2019 to 2020,a total of 153 samples(liver and spleen)of sick chicken suspected of infecting Salmonella from chickens were collected.After preliminary screening of SS medium and PCR amplification and sequencing of Salmonella-specific invasion genes invA and 16S rRNA,some strains were identified by MALDI-TOF-MS,and finally 32 clinical isolates of Salmonella from chicken were obtained,with an isolation rate of 20.9%;Five of the 32 Salmonella strains were resistant to colistin,the detection rate of colistin resistance gene mcr-1 was 0.The detection rate of mcr-1 gene in 107 strains of colistin-resistant Salmonella from pigs preserved in the laboratory was30.8%.The susceptibility tests of EDTA、CCCP and baicalin single and their combination with colistin against 5 strains of Salmonella from chicken and 27 strains of Salmonella from pigs were carried out respectively.The test results showed that the combination of EDTA and CCCP^ EDTA and baicalin significantly reduced the MIC of colistin to Salmonella^ and the fold change is greater than that of EDTA> CCCP、and baicalin alone combined with colistin.The combination of EDTA and CCCP,EDTA and baicalin reversed the resistance of Salmonella to colistin.The time sterilization curve results are consistent with the susceptibility results;In vivo test results show that baicalin and EDTA combined with colistin significantly reduced bacterial counts in the liver and spleen of clinical5a/mone//a-infected mice.RT-qPCR results showed that baicalin and EDTA inhibited the expression of acnD、tolC、acrAs acrB、mar A > ramA ^ soxS and rob A genes in the efflux pump system AcrAB-TolC;increased the expression of the colistin resistance-related gene mgrB gene,and significantly reduced pmrA、phoP、phoQ、pmrH、pmrC、pmrD、cptA and mcr-1 gene expression;significantly increased the expression of cpxR and cpxA genes of the CpxAR system,and significantly reduced the mRNA expression of H-NS nucleoprotein gene h-ns.Transcriptomics studies showed that a total of 391 differential genes were detected in the co-induced group with EDTA and baicalin,including 182 up-regulated genes and209 down-regulated genes.EDTA and baicalin down-regulated the transcription of ABC protein transport system,Salmonella type III secretion system and invasion system,antioxidant function,and LPS modification related genes in Salmonella;Up-regulated the transcription of TCA cycle and the two-component signal transduction system(CpxAR)related genes in Salmonella.EDTA and baicalin enhance the antibacterial activity of colistin by promoting oxidative damage,activating TCS CpxAR and inhibiting the efflux pump function in Salmonella,and affect the invasion function of Salmonella.The PUC18-mcr-1 recombinant strain was successfully constructed,baicalin and EDTA can synergistically reversed the colistin resistance of the mcr-1 recombinant strain;RTqPCR results showed that baicalin and EDTA can extremely significantly reduced the mRNA expression of the mcr-1 gene of the mcr-l recombinant strain.In conclusion,baicalin and EDTAsynergistically reversed the resistance of clinical isolates of Salmonella to colistin,and significantly reduced the number of bacteria in the liver and spleen of clinical Salmonella infected mice.Its molecular mechanism is to enhance the antibacterial activity of colistin by promoting oxidative damage,activating TCS CpxAR and inhibiting the efflux pump function.The results of this study can provide a basis for formulating a combined treatment plan for colistinresistant Salmonella infections.