| Salmonella,as an important zoonotic pathogen,can cause a variety of intestinal diseases,typhoid fever and food poisoning in humans.In particular,the prevalence of multi-drug resistant strains poses a great threat to public health.Plasmid-mediated drug resistance is more likely to spread rapidly among bacteria than chromosomal drug resistance genes.Large plasmids with multiple replicons have more drug-resistant genes and mobile elements,and their horizontal transfer poses a more serious threat.Therefore,it is important to fully understand the genomic characteristics of multi-drug resistant strains,plasmid-mediated resistance,and the structure and evolution of multi-replicon resistant plasmids to grasp the epidemiological characteristics and evolution of multi-drug resistant Salmonella.ObjectivesTo understand the drug resistance genes,virulence genes and mobile element carriage of multi-drug resistant Salmonella in Jiangsu Province by whole genome sequencing;and to explore the correlation between drug resistance genes and insertion sequences.To analyze the genomic features of Salmonella Indiana and the role of plasmids on its multi-drug resistance formation.To analyze the structural features and evolutionary traceability of a multi-replicon HI2 type plasmid in conjunction with public databases.Methods1.With screening multi-drug resistant strains by drug sensitivity assay,25 multi-drug resistant Salmonella strains were subjected to second generation whole genome sequencing,and their resistance genes,virulence genes on virulence islands SPI1-SPI5 and transposons were analyzed in combination with CARD resistance gene database,VFDB virulence gene database and,IS finder mobile element database and INTEGRALL database We also identified core genes and constructed correlation co-occurrence networks of resistance genes and mobile elements.2.Three generations of whole genome sequencing were performed on seven strains of multi-drug resistant Salmonella Indiana and one strain of antibiotic-sensitive Salmonella typhimurium to compare their chromosomes and plasmid structures.Plasmid elimination assays were performed on six multi-drug resistant Salmonella strains(three Indiana,one Enteritidis,one Salmonella typhimurium and one Salmonella delbe),and the resistance genes,resistance phenotypes and plasmid sequence changes after plasmid elimination were analyzed by polymerase chain reaction(PCR),drug sensitivity assays and whole genome sequencing.3.Compared similar plasmids in the NCBI public database by sequencing data of plasmid S15p1,and performed structural analysis and evolutionary tracing of a multi-replicon HI2-type plasmid by sequence comparison,SNP data to construct a phylogenetic evolutionary tree,and principal component analysis(PCA).Plasmid transfer experiments were performed using Salmonella Indiana S15 carrying this plasmid as the donor bacterium and three Enterobacteriaceae(Escherichia coli,Salmonella,and Klebsiella pneumoniae)as the recipient.Results1.25(16 Indiana,2 Enterobacteriaceae,4 S.typhimurium,2 Thompson and 1 Risen)multi-drug resistant strains were compared to a total of 73 resistance genes in 13 categories.9 regulators and efflux pump resistance genes were present in all strains,and the major resistance genes were aminoglycosides,P-lactams and fluoroquinolones.The virulence genes on SPI1 and SPI2 were more complete in all strains,only two fragments of genes mediating metal toxicity were missing,the main deletions were concentrated in SPI3-SPI5,and only genes related to type Ⅲ secretion system and flagella were present.four insertion sequences,IS6,IS66,IS5 and IS214,were present in all strains,and all transposons belonged to the Tn3 transposon family,and 20 sequenced strains carried class Ⅰ integrons.The co-occurrence network results of drug-resistant genes and mobile elements mainly formed three large clusters,two of which were composed of drug-resistant genes and one of which was mainly composed of mobile elements,and some insertion sequences were closely related to drug-resistant genes.2.The results of three generations of whole genome sequencing showed that the chromosomal structures of seven multi-drug resistant strains and one antibiotic-sensitive strain were highly similar,all carrying the same 14 resistance genes on the chromosomes,and all seven multi-drug resistant Salmonella strains carried 1-5 plasmids,with one 179kb-223kbp multi-replicon HI2 type being found in all multi-drug resistant strains.After plasmid elimination,S15,S38 and S222 of Salmonella Indiana all had 4-5 resistance phenotypes and 7-8 resistance genes altered,while only S163 of Delbe regained susceptibility to imethiomycin in the other serotypes,all of which showed 1-2 loss of resistance genes.Sequencing results after elimination showed that S15 carried a 153510 bp IncHI2/IncHI2A type plasmid,with 61 kb of sequence missing and a 45 kb section of sequence inverted compared to the original 214 kb large plasmid.3.The multi-replicon HI2-type plasmids are mainly found in E.coli and Salmonella and are composed of a highly stable IncHI2-type plasmid backbone and a multivariate region.The highly stable region contains two replicons,IncHI2/IncHI2A,and is highly similar to an independent E.coli plasmid MK574666.1(Query Cover>99%,Per.Ident>99%).The multivariate region contained multiple drug resistance genes such as β-lactams,sulfonamides and aminoglycosides and mobile elements such as transposons,class Ⅰ integrons,insertion sequences and plasmid replicons such as IncN and Incl1.The results of plasmid splice transfer experiments showed that this plasmid could be transferred to Salmonella,Klebsiella pneumoniae and Escherichia coli at a frequency of 10-7 to 10-6.Conclusion1.The multi-drug resistant strains carried mainly aminoglycosides,β-lactams and fluoroquinolones,and the remaining types of resistance genes were also detected,which were basically consistent with their resistance phenotypes.The virulence genes associated with the type Ⅲ secretion system were very complete in the structures of SPI1-SPI5 of Salmonella,indicating that these strains possessed multiple drug resistance along with many virulence genes.The large number of mobile elements explains the origin of the multiple resistance genes and predicts that these resistance genes possess the ability to continue to transfer horizontally between strains.The presence of multiple resistance genes and insertion sequences are closely related,suggesting that these genes do not exist independently and that a fixed cluster of resistance genes may exist.2.The multi-drug resistant Salmonella Indiana in this study all carried plasmids ranging from 1-5 in number,and multi-replicon complex HI2 plasmids were found in all strains.Resistance genes on the primary chromosome were consistent with antibiotic-sensitive strains,with differences concentrated in plasmid-carried resistance genes.The alteration of resistance genes,resistance phenotypes and genomic sequences after plasmid elimination experiments further suggested that the plasmids mediated resistance in these Salmonella Indiana.3.HI2-type plasmids with multiple replicons are present in Salmonella,E.coli and Klebsiella pneumoniae.Sequence structure comparisons suggest that this plasmid was initially composed of a single IncHI2-type independent plasmid that evolved into a complex multi-drug resistant plasmid with multiple replicons by capturing resistance genes in the environment or fusing with plasmids present in other strains and was able to continue to transfer to other Enterobacteriaceae. |
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