Characterization Of Novel Genetic Mobile Elements Carrying Bla_VIM-4/-24 And Mcr-9.1/-9.2 And The Resistance Accessary Modules

Antibiotic resistance,a global public health problem,is posing new challenges to human health: continuous dissemination of carbapenemase-producing clinical common strains;newly discovered antibiotic resistance genes such as mcr,which mediate polymyxin resistance,appear continuously and spread rapidly,which even spread to rare clinical strains and carbapenemase-producing bacteria.Antibiotic resistance genes are one of the main causes of bacterial drug resistance.Plasmids,transposons,integrons and other mobile elements play a crucial role in facilitating horizontal genetic exchange,serving as the carriers which promote the acquisition and spread of resistance genes.In this study,clinical isolates collected from many large hospitals across the country were collected,and the strains were screened through phenotypic experiments such as antimicrobial drug susceptibility tests,phenotypic assays,drug resistance genes screening,etc.Furthermore,novel mobile elements carrying bla_VIM and mcr were studied through genome determination,phylogenetic analysis,bioinformatics analysis,etc.The purpose was to analyze their detailed genetic characterization,investigate their origin and evolution,and explore their role in mediating the dissemination of antibiotic resistance genes.In the first chapter,two clinical isolates 1160 and 6762 of pseudomonas aeruginosa producing bla_VIM were screened out.The complete nucleotide sequence of Inc P-7 plasmid p1160-VIM from the 1160 isolate and that of the 6762 chromosome carrying integrative and conjugative element Tn6413 were determined,then comparison of p1160-VIM or Tn6413 with related sequences was performed.A series of novel mobile elements were discovered and designated: 1)p1160-VIM carrying drug resistance gene was discovered among Inc P-7 plasmids for the first time;2)the ICE Tn6413 carrying bla_VIM-4 was found for the first time;3)Tn3 family unit transposon Tn6392(carrying bla_VIM-24 and other antibiotic resistance genes),Tn6393（aminoglycoside and other antibiotc resistance genes）and Tn6403(carrying bla_VIM-4 and other antibiotc resistance genes);4)integrons In1394(carrying bla_VIM-24 and other gene cassettes),In1395（aminoglycoside and other drug resistance genes）and In1443(carrying bla_VIM-4 and other gene cassettes);5)insert sequences ISpa75,ISpa79 and ISpa82.This was the first report of a bla_VIM-24-carrying P.aeruginosa isolate and the first fully sequenced Inc P-7 resistance plasmid in clinical isolates.Based on nucleotide sequences of rep A,Inc P-7 plasmids are further divided into two subtypes,Inc P-7 α and Inc P-7 β.p1160-VIM belonged to Inc P-7 β plasmid and carried Tn6392 and Tn6393.Tn6413 was a large chromosome-borne ICE carrying Tn6403.Tn6392,Tn6393 and Tn6403 were generated from integration of bla_VIM-24-carrying In1394,In1395 and bla_VIM-4-barboring In1443 into prototype Tn3-family unit transposons Tn5563,Tn1403 and Tn6346,respectively.In the second chapter,11 clinical isolates of Leclercia species were studied.In the first section,an evolutionary tree was built on the genome of the 11 strains and the mobile elements carrying polymyxin resistance genes on chromosomes were analyzed in detail.5 strains of 11 were mcr positive,of which 2 strains cocarried mcr-9.1 and bla NDM,in different plasmids of one isolate or in the same plasmid.All the 11 strains carried a total of 32 mobile elements,including 28 plasmids（type of Inc HI2/Inc FII,etc.）and four transposons integrated into the chromosome.A new gene allele and a series of novel mobile elements were discovered and designated: 1)mcr-9.2 gene,a variant of mcr-9.1,was found and named for the first time;2)Four novel transposons of three different types were discovered and named: Tn6573 family ICE Tn6573 and Tn6572（carrying mcr-9.2）,Tn7 family transposon Tn6574（carrying mcr-9.1）,and IS26 mediated composite transposon Tn6579（carrying amr A）;mcr-9.1 was found on the chromosome for the first time.Tn6573 and Tn6572 of Tn6573 family were analyzed in detail.Comparison of Tn6574 with related sequences of the same family was performed,and it was found that both ends of this kind of transposons were the core transposon structures of the Tn7 family Tns ABCD and mcr-related region,respectively,while the intermediate regions constructed by recombination were different.The genetic environment of mcr-9.1 was orf168–rcn R–rcn A–copE1–?copS–IS903B–mcr-9.1–wbu C（–qse B–qse C）.In the second section,the detailed comparative genomic analysis was carried out on three Inc HI2 plasmids,the reference plasmid R478,and mcr-9.1-carrying plasmids p1106151-mcr and p707804-mcr.It was found that the backbone region of Inc HI2 type plasmids was very conservative,while the accessory regions were different from each other mainly due to the polymyxin-related regions and the derived regions of Tn1696.The polymyxin-related region is closely related to the Tn6574 transposon structure,and the core genetic structure of mcr-9.1 was also orf168–rcn R–rcn A–copE1–?copS–IS903B–mcr-9.1–wbu C/（–qse B–qse C）.Studies have shown that both bla_VIM and mcr-9 can exist in plasmids and chromosomes.Both bla_VIM-24 and bla_VIM-4 were integrated into type 1 integrons,and then carried by conjugative plasmids and ICE integrated into chromosomes as part of unit transposons,which could further cause dissemination of bla_VIM.Host of Inc P-7 plasmids have spread from the previous environmental strains to the clinical isolates,and Inc P-7 plasmids have developed from traditional degradable plasmids to the fields involving carbapenem resistance,which clinical attention and monitoring are desperately needed.The mcr and carbapenemase genes could coexist in different plasmids of the same isolate or even in the same plasmid,and that is a major concern in the field of bacterial resistance.Inc HI2 plasmids and Tn6574 transposon of Tn7 family are both important vectors for mcr-9.1 transmission,and the genetic environment in which mcr-9.1 is located mostly exists in the core structure orf168–rcn R–rcn A–copE1–?copS–IS903B–mcr-9.1–wbuC/（–qse B–qseC）.Tn7 family transposon Tn6574 and Inc HI2 plasmids have undergone large-scale complex recombination during the evolution of host bacteria,which greatly promotes the transmission and dissemination of mcr-9.1 gene.