Study On Drug Resistance Mechanism Of Shigella Sonnei Regulated By Cfa Gene

Shigella is an important pathogen causing bacillary dysentery.In recent years,with the development of the economy,Shigella sonnei has gradually surpassed Shigella flexneri to become the main epidemic flora.At present,Shigella sonnei is not only resistant to traditional antibiotics,such as ampicillin,penicillin,chloramphenicol,streptomycin,etc.,but also resistant to commonly used antibiotics such as cephalosporin and ciprofloxacin.The rate is also getting higher and higher,and solving the problem of Shigella Sonnei resistance has become an important public health problem in the world.In the early stage of our research,Shigella Sonnei was induced by doubling the concentration of ampicillin to make it progress from sensitive to resistant.Whole-genome sequencing and resequencing showed that,compared with the original strain,the cfa gene of the resistant strain had a non-synonymous mutation,and its expression was significantly higher than that of the original,suggesting that the cfa gene may be related to the resistance of Shigella sonnei.In this study,we mainly knocked out the cfa gene of drug-resistant bacteria and studied the regulation mechanism of its resistance to Shigella,and provided new ideas for solving the problem of Shigella resistance.ObjectiveThe regulation effect of cfa gene on drug resistance of Shigella sonnei was analyzed,and the differentially expressed genes after cfa gene knockout were differentially expressed by RNA-seq technology,so as to provide new ideas and drug targets for preventing Shigella infectious diseases.Methods1.The cfa gene knockout strain(Res/△cfa)was constructed by λ-red homologous recombination technique.2.The micro-broth dilution method was used to detect the minimal inhibitory concentrations(mic)of Shigella sonnei drug-resistant strains and the cfa knockout strains.The antibiotics included ampicillin,penicillin,amoxicillin,aztreonam,ceftriaxone,cefotaxime,tetracycline,meropenem,imipenem,kanamycin,and norfloxacin,chloramphenicol,polymyxin B,etc.3.The absorbance values of drug-resistant strain Res,cfa gene knockout strain Res/△ cfa,and the original strain without ampicillin induction under different antibiotic concentrations were measured by spectrophotometer.The growth curve of the strains was plotted to reveal the growth status of the three strains under different antibiotic concentrations.4.Transcriptome sequencing was performed on drug-resistant strains of Shigella sonnei and cfa gene knockout strains,and GO annotation and KEGG pathway analysis were performed on differentially expressed m RNAs to screen m RNAs related to drug resistance.5.Total RNA of drug-resistant strains of Shigella sonnei was extracted and q RT-PCR was used to verify the expression level of differential genes.Results1.The cfa gene knockout strain of Shigella sonnei was successfully constructed and named Res/△cfa.2.The resistance of knockout strains to ampicillin decreased significantly,and the minimum inhibitory concentrations against antibiotics such as penicillin,amoxicillin,aztreonam,ceftriaxone,cefotaxime,and tetracycline were lower than those of resistant strains.The minimum inhibitory concentration of meropenem,imipenem,kanamycin,norfloxacin,chloramphenicol,polymyxin B,etc.did not change.3.Transcriptome sequencing showed that compared with drug-resistant strains,the expression levels of 166 genes were significantly up-regulated and 136 genes were significantly down-regulated.The deletion of cfa resulted in significant changes in the expression levels of β-lactamase amp C,membrane porin omp D,ABC transporter mal E,rbs A,rbs B,rbs C,and ribosomal function related genes rpl V,rps J,and rps B,thereby regulating the drug resistance of Shigella sonnei.Conclusions1.cfa gene regulates the resistance of Shigella sonnei: knockout of cfa gene causes the resistance of drug-resistant Shigella to decline in ampicillin and other penicillin antibiotics,and the sensitivity to antibiotics such as cephalosporins and tetracyclines decline.The sensitivity to carbapenems,aminoglycosides,quinolones,polymyxin B and other antibiotics has not changed.2.Transcriptome sequencing results showed that cfa gene knockout increased the expression levels of ABC transport system genes rbs B,rbs C,mgl B,and porin omp C,and decreased β-lactamase amp C,ribosomal function-related genes rps B,rpl V,etc.,thereby regulating the process of Shigella resistance.