Molecular Characteristics Of NDM-5 Carbapenemase-Producing Enterobacteriaceae

ObjectiveTo analyze the antibiotic resistant mechanism of carbapenem resistant enterobacteriaceae(CRE)isolated from clinical environment and elaborate the molecular mechanism of blaNDM-5 dissemination by analyzing molecular genetic level of strains,plasmids and gene transfer elements systematically,thus providing theoretical basis for clinical antibiotic therapy.Methods35 unduplicated strains of carbapenem resistant enterobacteriaceae(CRE)were isolated from First people’s hospital of Foshan from 2013 to 2016.The minimal inhibitory concentration(MIC)was used to test the susceptibility of common used antibiotics in clinics.Three-dimensi onal test and polymerase chain reaction(PCR)was used to identify the different kinds of carbapenemase and corresponding genotypes.For 12 NDM-5-positive strains,the cloning correlation of these strains was detected by MLST method.The transferability of plasmids carrying blaNDM-5 was confirmed by conjugation tests.The identification of plasmid incompatibility groups was performed by PCR to tetect the type of plasmids replicon.Whole-genome sequencing of NDM-5-producing S81741 was performed by using a single-molecule real-time(SMRT)technique based on a PacBio RS Ⅱ platform(Pacific Biosciences,California,USA).Assembled sequence was analyzed by bioinformaitic tools,such as ResFinder、PlasmidFinder,ISFinder,MLST Cgview 和NCBI Blast.For analysis the genetic environment of blaNDM-5,PCR was used to amplify the blaNDM-5 flanking sequence of other 11 NDM-5-positive strains.Results35 strains of CRE presented multidrug resistance to the common antibiotics used in clinics.The results of three-dimensional test and PCR of carbapenem genes detection showed that the CRE producing metalloenzyme are 85.71%(30/35)and carbapenemase genes are mainly blaNDM-1 and blaNDM-5.Among them,36.36%are blaNDM-s-positive strains,include 8 strains of E.coli,2 strains of K.pneumoniae,1 E.cloacae and 1 S.typhi.Among 8 escherichia coli strains,besides two strains of ST5706 and three strains of ST224,the other three E.coli isolates were not clonally related.12 plasmids carrying NDM-5 could be conjugected to the recipient E.coil C600 successfully.After identification of plasmid incompatibility groups of NDM-5-positive plasmids,10 of them are IncX3 plasmid,the other 2 strains:1 K.pneumoniae and 1 S.typhi are IncFⅡ plasmid.According to the plasmid MLST,these 2 IncFII plasmid are not clonally related,which are K9:A-:B-and S2:A-:B-.Whole-genome sequencing revealed that a 84,565-bp NDM-5-positive IncFII plasmid pST41-NDM is carried by S81741,which possesses a similar structure identity to the IncFIl-type plasmid backbone by comparing with two blaNDM-5-positive plasmids and one blacTx-M-27-positive plasmid.But,lots of transpose elements IS26 were concentrated in variable region,and several antibiotic resistant genes were integrated into this area.Interestingly,analysis of the variable region on pST41-NDM revealed that the blaNDM-5 gene was likely transferred by two flanking IS26 elements,which shared the same flanking sequence(IS3000-△ISAba125-IS5-blaNDM-5-ble-trpF-dsbC-IS26)with the blaNDM-5 gene-bearing IncX3 plasmids reported previously,and the rest 11 strains of CRE mentioned above also have the identical structure.ConclusionDissemination of the blaNDM-5 in Enterobacteriaceae collected in this hospital was mainly due to IncX3 plasmid diffusion or IS26-mediatied horizontal gene transfer(HGT)between different plasmid types.The blaNDM-5-positive Salmonella was first reported in guangdong.The plasmid carrying blaNDM-s in S81741 was found to be IncFII-type,which could be a potential factor in the transmission of blaNDM-5 in Enterobacteriaceae.