Study On The Resistance Mechanism Of Salmonella In Vitro Induced Strains To Polymyxin

Salmonella multi-drug resistance is becoming increasingly serious and has become a global health concern.Polymyxin,a class of peptide antibiotics,has been revalued and used in recent years,and its increased use in the clinic has led to a gradual increase in the resistance of Gram-negative bacteria such as Salmonella to polymyxin,but its resistance mechanism is not fully understood,so it is important to study its resistance mechanism.In this study,Salmonella typhimurium ATCC13311(AT)was induced by the concentration increment method,and the highly resistant strain of polymyxin AT-P128 was obtained.First,the antimicrobial drug susceptibility and other biological properties of the two strains were compared.Then,the genomes of the two strains were analyzed by comparative genomics,and the differences in gene expression between the two strains were compared by transcriptomics and fluorescence quantitative PCR.Finally,the pho P gene of AT-P128 was knocked out by CRISPR/Cas9 gene editing technology,and the drug susceptibility of the strains to polymyxin before and after pho P gene deletion was comparatively analyzed to investigate the resistance mechanism of Salmonella to polymyxin.The main study contents and results are as follows:1.Induction and biological function studies of in vitro drug-resistant strains of Salmonella.A highly resistant strain of polymyxin,AT-P128,was obtained by using AT as the parental strain and using the concentration-increasing method to induce AT.The ultrastructure of the bacterium was observed under transmission electron microscopy,and the drug susceptibility,growth characteristics,motility and biofilm formation ability of the two strains were examined.The results showed that the resistant bacteria AT-P128 showed not only high levels of resistance to polymyxin B and colistin but also changes in susceptibility to several other antimicrobial drugs compared to AT.Among them,the MIC of amoxicillin increased more than 64-fold,that of chloramphenicol increased 16-fold,that of ceftiofur increased 8-fold,that of florfenicol and ampicillin increased 4-fold and more,and that of ampicillin and tetracycline increased only 2-fold;while the susceptibility of sulfamethoxazole decreased 16-fold.Transmission electron microscopy revealed a decrease in the bacterial hairs of AT-P128,indicating that in vitro drug induction reduced the growth ability of the hairs of drug-resistant bacteria.There was no significant difference in the growth rate of the strains before and after induction,indicating that in vitro drug induction had almost no effect on the growth rate of the strains.Measurement of the bacteriophage diameter and biofilm assay of both strains revealed that the motility(P<0.01)and biofilm formation(P<0.05)of AT-P128 were significantly decreased,indicating that the strains acquired polymyxin resistance through in vitro drug induction,which caused some degree of impairment to their motility and biofilm formation.2.Whole-genome sequencing and comparative genomics analysis of AT and AT-P128.The complete genome sequence obtained by triple genome sequencing of AT and second generation genome sequencing of drug-resistant bacterium AT-P128.After assembling the sequencing data,the genome size of AT was 4 679 519 bp with 52.05 % GC content,and that of AT-P128 was 4 643 663 bp with 52 % GC content.The coding genes,repetitive sequences,non-coding RNAs,gene islands,prephages,and CRISPR were predicted for both genomes,and the characteristics of the two genomes were found to be largely consistent.Both AT and AT-P128 were annotated to 50 resistance genes,which were mainly exocytosis pump-encoding genes,genes regulating antibiotic exocytosis,aminoglycoside resistance genes,peptide resistance genes,fosfomycin resistance genes,fluoroquinolone resistance genes,aminocoumarin resistance genes,genes encoding antibiotic target protection proteins and antibiotic inactivating enzymes,and some variants or mutants of antibiotic resistance genes.Single nucleotide polymorphism analysis revealed a total of eight genomic missense mutations in AT-P128(ccm B,ccm H,ccm G,ccm C,pho P,ybj Z,lpt D,bas S(pmr B)),with lpt D,pho P and bas S associated with polymyxin resistance.3.Transcriptomic analysis of AT and AT-P128.Differentially expressed genes were screened with |Fold-change| ? 2 and Q-value<0.005 as the criteria.A total of 1 380 differentially expressed genes were screened by AT-P128/AT,among which 319 genes were up-regulated,1 061 genes were down-regulated,and most of the differentially expressed genes were down-regulated.Screening of some drug resistance and pathogenicity-related differentially expressed genes from the above results revealed that these genes were mainly enriched in pathways such as two-component system,active efflux system,bacterial chemotaxis,streptomycin biosynthesis,lipopolysaccharide biosynthesis,outer membrane,flagellar assembly,bacterial invasion of bacterial hairs,epithelial cells,bacterial secretion system,and Salmonella infection.Eleven differentially expressed genes were selected for fluorescent quantitative PCR analysis,and the results were consistent with the transcriptome sequencing results,confirming the reliability of the sequencing results.Both RNA-seq and q RT-PCR results showed elevated expression of pho P and pho Q genes of Pho PQ TCS,indicating that Pho PQ may be involved in regulating the polymyxin resistance mechanism of induced bacteria in vitro.4.Construction of pho P gene deletion strain and drug sensitivity analysis.The pho P gene deletion mutant of drug-resistant bacterium AT-P128 was constructed by CRISPR/Cas9 gene editing technology,and the pho P gene deletion strain was successfully constructed by PCR identification and sequencing verification.The polymyxin susceptibility assay revealed that the resistance of AT-P128?pho P to both polymyxin B and mucilage was reduced 32-fold compared with that of AT-P128,and the results indicated that the pho P gene deletion could substantially reduce the polymyxin resistance of AT-P128.In this study,a polymyxin-resistant strain of Salmonella was constructed in vitro.After determining its genetic stability,we investigated the changes in biological properties,genomic components,functions and gene expression levels of Salmonella after it acquired polymyxin resistance through biological characterization,whole genome sequencing,transcriptomics and gene deletion assays.To reveal the mechanism of in vitro induction of polymyxin resistance in bacteria,and to provide scientific basis for finding new targets of anti-polymyxin resistance and control of polymyxin-resistant bacterial infections.