Molecular Mechanism Of Synergistically Reversing Resistance Of Bacteria To Colistin By The Combination Of Berberine And EDTA

With the emergence and prevalence of multi-drug resistant(MDR)bacteria,the world is facing a growing threat.The combination of antimicrobial adjuvants to restore the activity of the original antimicrobial drugs has become a current research hotspot.Considering the role of MDR efflux pumps and cell membrane permeability in innate and evolved resistance,berberine,a plant-derived efflux pump inhibitor with small toxic and side effects and membrane permeability accelerator EDTA were chosed to investigate the reversal effect and mechanism of colistin resistance of E.coli and Salmonella by combination of two drugs.Nine strains of Salmonella and nineteen strains of E.coli from chicken were isolated and identified by PCR and mass spectrometry.The effects of BBR and EDTA on colistin sensitivity of Salmonella and E.coli with different resistance mechanisms were observed by drug susceptibility test and time-bactericidal test in vitro.The resistance mechanism of mcr-1 negative colistin resistant strains was analyzed by whole genome sequencing and quantitative fluorescence PCR.qRT-PCR and transcriptome sequencing were used to study the molecular mechanism of BBR and EDTA in reversing colistin resistanc of Salmonella..Nine strains of Salmonella and nineteen strains of E.coli were isolated and identified from 103 dead chickens from small and medium-sized farmers in Henan province,with isolation rates of 8.74%and 18.47%,respectively.The broth microdilution method was used to determine the sensitivity of isolates to colistin.Then the colistin resistance gene mcr-l～mcr-8 were amplified.The results showed that the resistant rate of Salmonella was 55.56%(5/9)and all of them were mcr negative strains.The resistant rate of E.coli was 63.16%(12/19),with a detection rate of 47.37%(9/19)of mcr-1.All isolates were not detected mcr-2～mcr-8.Sequence alignment of mcr-1 negative and colistin resistant Salmonella showed that some amino acids of pmrB,pmrC and pmrFJHIKM which were two-component signal transduction system related genes were mutated.The qRT-PCR results showed that the expression of efflux pump genes such as acrA,acrB,tolC,acrD and marA were up-regulated.These results suggest that in mcr-1 negative and colistin resistant Salmonella,the mutation of the genes associated with lipid A modification and the up-regulation of the expression level of the efflux pump may mediate the significant increase of the resistance to colistin.In vitro susceptibility test and time-killing test showed that resistance of E.coli and Salmonella to colistin could be reversed synergistically by BBR and EDTA in a concentration-dependent manner.The synergistic effect of BBR and EDTA on colistin was significantly better than that of single drug on colistin,and significantly better than on other antimicrobial agents,suggesting that BBR and EDTA could synergistically enhance the antimicrobial activity of colistin and reverse colistin resistance of the strain.qRT-PCR and transcriptome data showed that the transcription of genes that modifies outer membrane lipid A,such as mcr-1,pmrAB,pmrC,pmrHFIJK,1pxM were down-regulated and the transcription of efflux pump gene tolC was down-regulated.The transcription of genes associated with TCA cycle and oxidative phosphorylation were up-regulated.The above results indicate that BBR and EDTA could reverse resistance of Salmonella to colistin mainly by affecting the genes transcription related to bacterial metabolism,LPS modification,and efflux pump system.This study identifies and provides a potential combination regimen using existing antimicrobials in combination with antimicrobial adjuvants such as plant-derived efflux pump inhibitors and membrane permeabilizers to response to infections caused by colistin resistant MDR Gram-negative bacteria.