Drug-Resistance Inducing Model In Vivo Of Salmonella Enterica- Caenorhabditis Elegans And Transcriptome Sequencing Analysis Of The Inducing Drug-Resistance Salmonella Enterica In Vitro

Salmonella is a common type in the production process and the clinical treatment of zoonotic pathogens,the vast majority of it are pathogenic to man and animal.Typhoid,paratyphoid fever and sepsis were caused by Salmonella in animal,miscarriage would happen especially when sepsis in pregnant female animal.In man,the most common disease were bromatoxism and local infection.With the widespread use of quinolones drugs,the occurrence rate of quinolone resistance was rising,drug resistance problem was becoming more and more serious.The mechanism of antimicrobial resistance was usually studied by using the fluoroquinolone to inducing sensitive Salmonella strain,but induction model in vitro could not reflect the antimicrobial resistance generation mechanism.Because there was a complex biological environment inside of the body,the metabolism and immunoreaction could affect the mechanism of drug resistance.The Caenorhabditis elegans(C.elegans)was a classic model organism that in pathogen infection process,activation mechanism,immune response and regulation mechanism of life activities and other aspects were highly conserved in mammals.In this study,the C.elegans wild-type N2 as the host,the Salmonella enterica Typhimurium(S.Typhimurium)ATCC13311 was applied to colonize in worms,and then gradient increasing the concentration of ciprofloxacin in the supernatant to induce parasitizing S.Typhimurium.An in vitro induction experiment by increasing concentrations of ciprofloxacin to induce a drug-resistance strain processed at the same time.Quinolone-resistant determining region(QRDR)of gyrA,gyr B,parC and parE and the efflux pump inactivation genes(acrR,marR,ramR and soxR)of the drug-resistance inducing S.Typhimurium in vivo and in vitro were analyzed by BLAST,after PCR amplification.To gain an insight of the difference of transcriptomes of the S.Typhimurium ATCC13311 and its in vitro drug-resistance strain,two transcriptome sequencing libraries were constructed and sequenced by Illumina RNA sequencing technique,and the global transcriptome information was analyzed subsequently.By using Real Time PCR to detecte the result of RNA-seq.The research confirmed: S.Typhimurium ATCC13311 could colonized the alimentary canal of C.elegans N2,the bacteria in the digestive tract of the worm were concentrated in the pharynx and gut,and furthermore the drug-resistance mutant could be selected by gradient increasing the concentration of ciprofloxacin.The results indicated that the bacteria could product resistance in vivo and in vitro induced,the MIC(minimum inhibitory concentration)of the drug-resistant inducing S.Typhimurium in vivo TN4 and the S.Typhimurium in vitro TW4 both were 4 ?g/mL.In TN4,the mutation of Asp87 Asn of the gyrA QRDRs was observed.In TW4,there were two mutations of QRDRs in gyrA,Ser83 Phe and Asp87 Val.The TW4 also contained a 20 bp deficiency of the ramR.RNA-seq sequencing was performed on ATCC13311 and TW4,4.46 G available transcriptome data were acquired,311 unigene were obtained by de novo assembly method and the average length was 15587 bp.The length of the whole genome sequence is 4847532 bp,annotation analysis indicated 4902 genes of 4998 genes had homolog in public protein database,all genes with annotation were classified as 25 Clusters of Orthologous Groups of protein.Compared with ATCC13311,upregulation of 3434 genes,downregulation of 32 genes were observed and 7 metabolic pathways markedly changed in TW4.The result of RT-PCR showed the expression levels of acrB,acrD and soxS were consistent with RNA-seq result.This research established a drug-resistant inducing model of S.Typhimurium in vivo,analyzed the differences of mutations of TN4 and TW4,which laid the foundation of further research of in vivo bacterial drug-resistance mechanism.By using the RNA-seq of ATCC13311 and TW4,this study gave a global insight into the characteristics of inducing drug-resistance strain transcriptome and pivotal genes expression that leading the drug-resistance character,provided an important basis for the way in-depth study of bacterial antimicrobial resistance mechanism and connection of bacterial metabolism.