Sequencing And Comparative Analysis Of IncN2 Plasmids Carrying Bla_IMP Genes And Class 1 Integrons

Carbapenem antibiotics show great efficacy against Gram-negative bacteria,especially the enterobacterial species.Many carbapenem-resistant bacteria have been reported in recent years,which challenges the use of this class of lifesaving drugs.Carbapenemases can hydrolyze carbapenems.Carbapenemases can be mainly divided into three classes,namely A,B and D.Classes A and D are serine-?-lactamases,whereas class B is metallo-?-lactamases.The production of carbapenemases is one of the most important mechanisms of enterobacterial carbapenem resistances.In this study,we intend to identify the detailed mechanisms of the productions of plasmid-encoding carbapenemases IMP in a Klebsiella pneumonia strain 0801,a Klebsiella oxytoca strain 7121,and a Citrobacter freundii strain 17285.K.pneumoniae 0801 and K.oxytoca 7121 which were from the 307 th Hospital of the People's Liberation Army were isolated from the blood specimens and the sputum specimens,respectively.C.freundii 17285 which was from the First Affiliated Hospital of Anhui Medicial University was isolated from the voided midstream urine specimens.The species of the strains were distinguished by 16 S r RNA gene sequencing.The major plasmid-borne carbapenemase genes were screened for by PCR,followed by amplicon sequencing on ABI 3730 Sequencer.Activities of class A/B/D carbapenemases in bacterial cell extracts were determined via modified Carba NP tests.Bacterial antimicrobial susceptibilities were tested by Bio Mérieux VITEK 2 and interpreted as per Clinical and Laboratory Standards Institute(CLSI)guidelines[1].Plasmid conjugal transfer experiments were carried out with the rifampin-resistant Escherichia coli EC600.Plasmid DNA was isolated from E.colitransconjugant using a Qiagen Large Construct Kit,and then sequenced with a mate-pair library with average insert size of 5,000 bp through an Illumina Mi Seq sequencer(Illumina).K.pneumoniae 0801,K.oxytoca 7121 and C.freundii 17285 produced IMP,which were encoded by three different plasmids p0801-IMP(accession number KT345947),p7121-IMP(accession number KX784502),and p17285-IMP(accession number KX784503).All of these plasmids could be transferred into strain EC600 through conjugation experiments,and generated E.coli transconjugants 0801-IMP-EC600,7121-IMP-EC600 and 17285-IMP-EC600,respectively,indicating that all these three plasmids were conjugative.Both 0801 and 7121 harbored bla IMP,bla TEM,and bla CTX-M-1 group,while 17285 containted bla IMP and bla TEM.Only bla IMP was detected in 0801-IMP-EC600,7121-IMP-EC600 and 17285-IMP-EC600.All the above wild-type and transconjugant strains had the class B carbapenemase activity,and were resistant to ceftriaxone,ceftazidime,imipenem and meropenem.Strains 0801,7121 and 17285 were resistant to gentamicin but the other strains remained susceptible to this drug,and all of them were susceptible to amikacin.p0801-IMP,p7121-IMP and p17285-IMP are the first fully sequenced bla IMP-carrying Inc N2 plasmids.There are 6 plasmids in total included in our study,namely the Inc N2 reference plasmid p YNKP001-NDM and the five integron-carrying Inc N2 plasmids(p0801-IMP,p7121-IMP,p17285-IMP,p JIE137,and p34983-59.134kb).Further comparative genomics analysis of all the six Inc N2 plasmids shows that the modular structure of each plasmid is divided into the Inc N2 backbone as well as one or more accessory modules inserted at different sites of the backbone.Four class 1 integrons(In1223 in p0801-IMP/p7121-IMP,In655 in p17285-IMP,In27 in p JIE137,and In1130 in p34983-59.134kb),two insertion sequence-based transposition units(ISEcp1-orf RA1-14 in p17285-IMP,and ISEcp1-bla CTX-M-62-?orf477-orf RA1-14 in p JIE137),and a novel Tn1696-related transposon Tn6325 in p34983-59.134 kb areindentified in the integron-carrying plasmid accessory regions.In1223 and In655 represent ancestral Tn402-associated integrons,while In27 and In1130 belong to complex class 1 integrons.The relatively small Inc N2 backbones are able to integrate different mobile elements which carry various resistance markers,promoting the accumulation and spread of antimicrobial resistance genes among enterobacterial species.