Genetic Characterization Of IncHI5?Inc_pRBL16?Inc_pCT14 And Inc_pND6-2 Resistant Plasmids

Carbapenems have broad antibacterial spectrum and strong antibacterial activity.In recent years,the number of clinical strains resistant to carbapenem antibiotics has gradually increased,which brings great difficulties and challenges to the treatments of infectious diseases.Production of carbapenenase is one of the important mechanisms of carbapenem resistance.Plasmids carrying genes encoding carbapenemases are important carriers for the rapid spread of drug-resistance genes among strains.The main purpose of this study is to explore the genetic characteristics of known IncHI5 plasmids,and three novel types(Inc_{p RBL16},Inc_pCT14 and Inc_{p ND6-2})plasmids from Pseudomonas.Bacterial species are preliminarily identified by 16S rDNA.Activity of carbapenemases are determined through a modified Carbapenemase Nordmann Poirel test.Bacterial antimicrobial susceptibility is tested by VITEK 2.Based on the background information of strains,genome sequencing is performed with a paired-end library with an average insert size of 350bp for the strains producing carbapenemase.After the whole genome sequence is obtained,bacterial species is validated by average nucleotide identity value,resistance genes are screened via Res Finder,and replication initiation genes are screened by Plasmid Finder.For strains carrying targeted or novel replication initiation genes,sequencing is performed to obtain complete sequences of plasmids.Detailed annotation and comparative genomics analysis of plasmids are carried out.Conjugal transfer or electroporation experiments are performed of selected strains to obtain corresponding transconjugants or electroporants.Chapter one:four isolates from Klebsiella pneumoniae producing carbapenemase B and containing IncHI5 plasmid were further studied.A324-IMP-EC600 and A324-IMP-TOP10 were obtained via conjugal transfer and electroporation experiments,respectively.pA324-IMP,p11219-IMP,p12208-IMP and p13190-VIM were all IncHI5plasmids.Further comparative genomics analysis was applied to six IncHI5 plasmids,including the above four plasmids sequenced this study and two plasmids?p Kp NDM1and p KP04VIM?derived from Gen Bank.Conserved IncHI5 backbone sequences including rep HI5B and rep FIB-like,par ABC,tra1 and tra2 were observed among all these six plasmids.pA324-IMP was the IncHI5 reference plasmid due to its most complete IncHI5 backbone sequence.IncHI5 plasmids could integrate multiple types of accessory modules through horizontal transfer.The truncations or deletions of surrounding backbone sequences caused by exogenous insertions were the main reasons of the diversification of backbones.Various types of accessory modules were observed among IncHI5 plasmids.The resistance accessory modules were composed of Tn10 and two antibiotic resistance islands designated ARI-A and ARI-B.ARI-A and ARI-B were inserted downstream of orf342 and within xer C2,respectively.ARI-A and ARI-B were derived from Tn1696 and Tn6535,respectively,and were discriminated as intact transposons or transposon-like structures.ARI-A and ARI-B from different IncHI5plasmids contained different profiles of resistance genes and associated mobile elements,promoting the rapid spread of resistance genes.Complex transposition and homologous recombination were the main driving forces for the assembly of these regions.Chapter two:eight isolates from Pseudomonas producing carbapenemase B and containing Inc_{p RBL16} plasmid were further studied.A total of 17 plasmids were included in this study,including the above eight plasmids sequenced in this study,and the other nine plasmids derived from Gen Bank?including p12969-DIM and p SY153-MDR from our previous studies?.All the 17 plasmids were assigned into the novel Inc_{p RBL16} group.Each of the 17 plasmids contained the conserved Inc_{p RBL16}backbone including rep A_{Incp RBL16} together with its iterons,parB2–parAB,che,pil and ter.pRBL16 had the most complete backbone with no exogenous insertions,and was defined as the reference plasmid.At least 18 regions or sites across their genomes exhibited the modular differences among these 17 plasmids,involving insertion,truncation or deletion resulted from insertions,and substitution of multiple-gene backbone regions.These 17 plasmids contained at least 71 non-redundant accessory modules,such as:Tn1403-related regions,Tn7-family transposons,Tn6571-family transposons,integrative and conjugative elements and integrative and mobilizable element.40 known resistance genes,which were involved in resistance to 15 categories of antibiotics and heavy metals,were present in these 17 plasmids.Different Inc_{p RBL16} plasmids contained different accessory modules,and thus different profiles of resistance genes and related mobile elements.Inc_{p RBL16} plasmids were important carriers for the spread of resistance genes among Pseudomonas.In addition,p12939-PER was the first reported bla _OXA-246-carrying Inc_{p RBL16} plasmid,and p12939-PER and p A681-IMP were the first reported bla _PER-carrying Inc_{p RBL16} plasmids.This study included a total of 14 firstly discovered and 31 newly named mobile elements.Chapter three:Pseudomonas sp.918607 producing carbapenemase B and containing Inc_pCT14 plasmid was further studied.Detailed comparative genomic analysis was applied to p918607-IMP and pCT14,both of which were novel Inc_pCT14 plasmids and contained conserved Inc_pCT14 backbones including rep A_IncpCT14and its iterons,krf A–vag CD–stb AB and cpl–rlx.pCT14 was the first sequenced Inc_pCT14plasmid,and thus defined as the reference plasmid.In994,and ISPa17 and cbz region were identified in p918607-IMP and pCT14,respectively.Bla _IMP-1from p918607-IMP was identified within In994.The cbz region carried several chlorocatechol-degradation genes.Tn6729was the newly designated transposon in this study.Inc_pCT14backbone could integrate different types accessory modules,contributing to the dissemination of resistance genes among Pseudomonas.Chapter four:P.aeruginosa PA15W and 201330 producing carbapenemase B and containing Inc_{p ND6-2} plasmid were further studied.Two Inc_{p ND6-2}plasmids,namely p PA15W-NR and p201330-IMP were obtained.Detailed comparative genomic analysis was applied to the three plasmids,including p PA15W-NR and p201330-IMP,and p ND6-2 derived from Gen Bank.All the three plasmids were assigned into novel Inc_{p ND6-2}groups.pND6-2 was the reference plasmid because it was the first sequenced Inc_{p ND6-2}plasmid.Conserved Inc_{p ND6-2}backbone including rep _{Incp ND6-2} and its iterons,par BA,mva T,top A,ssb and maz EF,dot DCBA,pilus and icm BJCGEKL.Only p201330-IMP contained two accessory modules,namely Tn6411 and Tn5563-related region.Bla _IMP-1was identified within In992,and In992 was embedded in Tn6411.Inc_{p ND6-2}backbone could integrate resistance accessory modules,leading to the dissemination of resistance genes among Pseudomonas.In summary,through sequencing,detailed annotation and comparative genomics analysis of these four types plasmids,we have better dissected the backbone and accessory modules of each type plasmid.Data presented here provided a more comprehensive understanding of the conservatism and diversification,and a deeper understanding of the evolutionary history and genetic characteristics of each type plasmid,which will offer public health departments and clinicians theoretical guidances to delay the development trend of drug resistance.