Characterization Of Novel Bla_NDM-carrying Mobile Genetic Elements In Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae（CRE）are distributed worldwide.This represents an urgent problem for clinical and public health departments because few drugs are available to treat infections caused by these pathogens.Producing hydrolase is one of the core resistance mechanisms of CRE,with NDM one of the important metalloenzymes allowing this.Mobile genetic elements are important vectors that mediate the dissemination of the bla _NDMgene.Of the mobile genetic elements, plasmids play an extremely important role.IncFⅡ multi-replicon plasmids occur in Enterobacteriaceae widely and typically carry the bla _NDMgenes in CRE.In addition,bla _NDM-carrying mobile genetic elements exist on the chromosomes of CRE that have complex structures and diverse types.However,research on chromosome-borne mobile genetic elements is lacking.In this study,CRE isolates were collected throughout China,and screened using strain identification and drug susceptibility tests,carbapenemases activity assays, PCR screening for drug resistance genes,etc.Then,whole-genome sequencing was used to analyze the genetics and genomics of the bla _NDM-carrying mobile genetic elements.Mobile genetic elements were defined by type,and the complex structures and evolutionary relationship analyzed to explore their role in mediating the spread of bla _NDMgenes in Enterobacteriaceae. In the first chapter,four strains of Enterobacteriaceae with bla _NDMlocated on their chromosomes were screened for further study.Six chromosome-borne accessory resistance regions were identified for use,along with six related resistant mobile genetic elements retrieved from Gen Bank.These 12 genetic elements were divided into five groups:1)a novel integrative mobilizable element（IME）Tn6588;2)two related IMEs Tn6523（SGI1）and Tn6589;3)four related integrative conjugative elements（ICEs）Tn6512（R391）,Tn6575（ICEPvu Chn BC22）,Tn6576 and Tn6577;4)Tn7 and its two derivatives Tn6726 and Tn7-related element₅₁₀₀₃;and 5)two related IMEs Tn6591（GIsul2）and Tn6590.An extensive analysis of the 12 chromosomal genetic elements,representing at least 51 resistance genes,uncovered resistance to 18different categories of antibiotics and heavy metals.Six chromosomal bla _NDM-carrying genetic elements,Tn6575,Tn6726,Tn6588,Tn6589,Tn6576,and Tn7-related element₅₁₀₀₃,had large multidrug resistance（MDR）regions,each of which had a complex mosaic structure composed of intact or residual mobile genetic elements,including ISs,unit or composite transposons,integrons and putative resistance units,and likely assembled from complex transposition and homologous recombination.Core bla _NDMgenetic environments manifested as four different Tn125derivatives that integrated into the mobile genetic elements of chromosomes in two ways.1)Through transposition,whereby Tn125 was integrated into the mobile genetic element of the chromosome,followed by further modular modifications such as insertion and truncation.2)The Tn125 derivative was captured by ISCR1,then integrated into a complex integron within the mobile genetic element of the chromosome.Notably,two or more copies of relevant Tn125 derivatives were found in each of Tn6576,Tn6588,Tn6589,and Tn7-related element₅₁₀₀₃.In this chapter,the novel bla _NDM-carrying IME Tn6588,Tn6523-related IME Tn6589 and Tn6512-related ICE Tn6576 were first found in Providence,Proteus mirabilis,and Klebsiella pneumonia,respectively.Eight novel mobile genetic elements were identified for the first time in this study:three ICEs,Tn6576,Tn6577 and Tn6582;two IMEs,Tn6588and Tn6589;two composite transposons,Tn6580a and Tn6580b;and one integron,In1718.An additional nine mobile genetic elements(IME:Tn6590;composite transposons:Tn6578,Tn6581,and Tn6728;unit transposons:Tn6727,Tn6909,and Tn6911;IS:ISPvu1;and Tn7-related element₅₁₀₀₃)were newly designated（first designated in this study,but with previously determined sequences）.In the second chapter,Escherichia coli 28078 and Klebsiella pneumonia A1706were screened for further study.Two bla _NDM-carrying IncFⅡ_R100multi-replicon plasmids,p28078-NDM and p A1706-NDM,were obtained.Another twoIncFⅡ_R100-type multi-replicon plasmids,p5571-MDR and p61806-dfr A,were previously screened in our laboratory and the reference plasmid R100 was included.Compared with the reference plasmid R100（screened previously in our laboratory）,p5571-MDR carried IncFⅡ_R100-,IncFIA-and IncFIB-type backbones,p61806-dfr（A）and p28078-NDM carried IncFⅡ_R100-and IncFIA-type backbones,and p A1706-NDM carried IncFⅡ_R100-and Inc R-type backbones.Interestingly,p A1706-NDM has an incomplete Inc R backbone,located in the composite transposon Tn7061,which is integrated into p A1706-NDM through homologous recombination mediated by IS26.All of the IncFⅡ_R100-type multi-replicon plasmids contain multiple insertion sites,and the core drug-resistant region contains multiple copies of IS26,forming a complex MDR region or IS26-mediated composite transposon.In addition to complex drug-resistant regions,plasmids p61806-DFRA,p28078-NDM and p A1706-NDM also carried virulence gene clusters.bla _NDMin P28078-NDM and p A1706-NDM were found in two different truncated Tn125 variants.Both of them were captured by insertion sequence ISCR1 and then integrated into complex integrons.In this chapter,two new mobile genetic elements Tn7065 and Tn7061 carrying bla _NDMwer discovered and named.In summary,multiple classes of novel bla _NDM-carrying mobile genetic elements in the chromosomes of Enterobacteriaceae bacteria were identified in this study.The structures of these mobile genetic elements are extremely complex;in addition to carrying bla _NDMgenes,these mobile genetic elements carry a large number of other resistance genes.The backbone of IncFⅡ_R100multi-replicon plasmids is a complex mosaic structure with multiple exogenous insertion sites,mostly due to the presence of multiple backbones.IS26 plays an important role in mediating the formation of the MDR region of IncFⅡ_R100-type multi-replicon plasmids through complex homologous recombination and transposition.In addition to carrying drug resistance genes, IncFⅡ_R100multi-replicon plasmids often carry virulence genes,making them important vectors for the transmission of drug resistance and virulence genes.The core bla _NDM-carrying element Tn125 has different truncated variants integrated into drug-resistant elements by direct transposition or captured by ISCR1.As these drug-resistant elements spread among various bacterial species,they will inevitably produce multi-drug-resistant or even pan-drug-resistant bacteria,causing great harm to public health safety.Monitoring of these elements should thus be prioritized.This study provides an important theoretical basis for follow-up research on drug resistance mechanisms,epidemiology,prevention and treatment of CRE.