| Salmonella species widely spread and distribution in the world that can cause typhoid fever,paratyphoid and sepsis and other diseases in human and animals,and are also the main pathogen of bacterial food poisoning in food hygiene.With the extensive and unreasonable use of fluoroquinolones(FQs)in clinical treatment,Salmonella resistance to FQs is becoming increasingly serious.At present,the resistance mechanism of FQs was studied by using single or multiple drugs selected susceptible bacteria to produce stable drug resistance in vitro,but this approach does not accurately reflect the Salmonella resistance mechanism in the complex biological environment.Hence,our laboratory in the previous time constructed the drug resistance selected model in vivo of "Caenorhabditis elegansSalmonella typhimurium",and ciprofloxacin-resistant strain selected in vivo was obtained.In this study,Salmonella typhimurium parental strain and ciprofloxacin-resistant strain selected in vivo were used to analysis the differences of selected in vivo on growth performance,invasiveness and intracellular viability,differential expression genes(DEGs)and enrichment analysis,resistant gene and drug resistance regulation and other aspects,and had a preliminary understanding of the metabolic,drug resistance and drug resistance regulation and virulence on drug-resistant strain.It provides effective experimental methods and means for the study of drug resistance mechanism in vivo,and to lay the foundation for further study of the mechanism of Salmonella resistance in vivo.The main research contents and results shown as follows:Firstly,detection of biological characteristics of resistant strain.The drug sensitivity,growth ability,invasiveness and intracellular viability of the two strains were measured.The results showed that the resistant strain showed intermediary or resistance to FQs(ciprofloxacin,ofloxacin,and enrofloxacin)and ceftriaxone,and reduced susceptibility to other drugs,but did not become resistant.Compared with parental strain,the resistant strain in growth performance,the invasion of epithelial cells,and the viability of epithelial cells and macrophages were all decreased.It is suggested that Salmonella can survive by various physiological reactions against the changes of external environmental factors.Secondly,RNA-seq analyzed on two strains and RT-qPCR verification of DEGs.Total RNA from two strains was extracted and sequenced,the results showed that the differential expression of 2,277 of the 3,844 genes predicted in the Salmonella typhimurium genome,of these,1,833 genes were up-regulated and 444 genes were down-regulated.GO function analysis of DEGs showed that 1946 GO terms were classified into 48 functional categories,the most important of which were "cell physiological processes","cells" and "binding".Pathway enrichment analysis showed that 1,601 DEGs mapped to 126 KEGG pathways,including 757 genes that were distributed in seven significantly affected metabolic pathways.Classification of DEGs according to their gene annotation showed that 15 DEGs were associated with drug resistance and were directly related to drug delivery systems,which the expression of 11 drug transporter genes were up-regulated,two drug target protein genes were up-regulated and two efflux pump inhibitor regulation genes were down-regulated.To verify the RNA-seq results,six of the DEGs were analyzed with RT-qPCR.Similar patterns of DEGs were observed in RT-qPCR and RNA-seq analyses,which confirmed the reliability of transcriptome sequencing and data analysis.It is indicating that the changes in drug resistance can lead to changes in metabolic pathways,which can also affect the expression of genes;overexpression of efflux pumps play a major role in the resistance of FQs to drug resistant strain in vivo.Thirdly,resistance regulation analysis of resistant strain.Classification statistical analysis of DEGs found that 19 genes related to the two-component system,including 14 up-regulated genes and five down-regulated genes.Prediction of sRNA,757 and 394 were found in parental and resistant strains,respectively,among which 338 sRNA expression levels were significantly different.The candidate sRNA was aligned and screened knew database,and the annotation information of 101 candidate sRNAs was obtained.The candidate sRNA matched to the Rfam database to show that 795 candidate sRNAs were aligned to 10100 families in the Rfam database.Analysis of candidate sRNA target genes showed that each candidate sRNA corresponded to multiple target genes.It is suggested that drug resistance can cause the expression changes of genes related two-component system and sRNA,and these changes may have a certain regulatory role in drug resistance. |
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