CRISPR-associated Protein Csy1 Of Acinetobacter Baumannii Inhibits Drug Resistance And Study On The Antibacterial Effect And Mechanism Of Capsaicin Combined With Colistin

In this work,the CRISPR-Cas system deletion strain was constructed to study the regulation effect on the resistance of A.baumannii.The cooperative antibacterial experiment was conducted to investigate the effect of capsaicin combined with colistin on the CRISPR-Cas negative strains of A.baumannii.By measuring biofilm formation,membrane permeability and ATP content to study the mechanism of capsaicin enhancing the antibacterial activity of colistin.1.The CRISPR-Csyl negative strains of A.baumannii are highly resistantIn order to study the regulation of the CRISPR-Cas system on the drug resistance and virulence of A.baumannii,and explore the unknown biological function of the Csyl protein in the CRISPR-Cas system,the csy1 gene of clinical isolate AB43 carrying the I-Fb subtype CRISPR Cas system was knocked out by RecAB homologous recombination system in this study,and phenotypic changes such as drug resistance,biofilm formation,and survival rate of infected mice of the wild strain(AB43)and gene-deleted strain(AB43Δcsy1)were observed,and the differentially expressed genes between the two were analyzed by transcriptome sequencing.The results showed that the deletion of csy1 significantly increased the resistance level of the mutant strain(still sensitive to colistin,but the tolerance has increased slightly),biofilm formation ability and the mortality rate of infected mice compared with the parent strain AB43.More importantly,the survival rate of mice in the AB43Δcsy1 infection group was higher than that of the wild strain AB43 infection group.In addition,through RNA-seq analysis,we found that the csy1 gene affects the expression of a series of genes related to antibiotic resistance,virulence and metabolic processes(marR,acrB,pgaC,plcN,astE,fur,etc.),resulting in the phenotypic changes of AB43Δcsy1.This study suggests that Csyl inhibits the drug resistance,biofilm formation and pathogenicity of A.baumannii by regulating the expression of related genes.In clinical practice,an environment conducive to maintaining the CRISPR-Cas system should be created and maintained to ensure CRISPR-Cas positive strains are in an evolutionary superiority position,which enables A.baumannii to maintain a low virulence and antibiotic sensitivity.2.Antibacterial effect of capsaicin combined with colistin on A.baumanniiA.baumannii has a high resistance rate to carbapenem antibiotics which are first-line antibacterial drugs.Although it is highly sensitive to colistin,there have been reports about its resistance in recent years.In order to develop a new drug combination therapy to deal with the failure of colistin monotherapy and delay the development of colistin resistance,three highly colistin-resistant strains were induced in vitro by the concentration gradient method in this study,and the mechanism of colistin resistance was preliminarily evaluated.In addition,the antibacterial activity of capsaicin combined with colistin against colistin-resistant A.baumannii was also tested in vitro and in vivo.The results showed that the expression of colistin resistance genes lpxB,lpxA and pmrAB were up-regulated compared with the wild-type strains,and the expression of efflux pump genes adeB and adeJ was down-regulated,while the expression of these genes showed a completely opposite trend in the AB13-R.The checkerboard test and time killing test showed that capsaicin combined with colistin have a synergistic effect on colistin-resistant strains of A.baumannii.The murine model of bacteremia proved that the combination of capsaicin and colistin can significantly reduce the number of A.baumannii in different organs of the host.In short,this strategy may help reduce the clinical dosage of colistin.Therefore,the combination of capsaicin and colistin may be an alternative for the treatment of A.baumannii infection.3.Study on the mechanism of capsaicin enhancing the antibacterial activity of colistinIn order to clarify the potential molecular mechanism of capsaicin enhancing the antibacterial activity of colistin,this study tested the effects of capsaicin on biofilm formation,membrane permeability,intracellular ATP content,reactive oxygen and cell surface morphology.The gene expression levels of bacteria after colistin treatment alone and colistin-capsaicin combined treatment were analyzed by transcriptome sequencing.The results showed that capsaicin inhibited the biofilm formation of A.baumannii,destroyed the bacterial outer membrane,reduced the level of intracellular ATP and oxidative stress.Capsaicin also destroyed the cell surface structure of ATCC19606-R.In addition,the results of RNA-Seq analysis showed that capsaicin combined with colistin significantly up-regulated the ribosome synthesis genes of ATCC19606-R,and down-regulated the expression of multi-drug efflux genes and outer membrane protein-related genes compared with the group of colistin treatment alone.This study proved that capsaicin enhances the antibacterial effect of colistin by inhibiting biofilm and efflux pump,as well as destroying cell membrane structure.Therefore,the combination of capsaicin and colistin has clinical application potential for the treatment of severe A.baumannii infection.