Coexistence of blaNDM-1 and blaIMP-4 in One Novel Hybrid Plasmid Confers Transferable Carbapenem Resistance in an ST20-K28 Klebsiella pneumoniae

ObjectivesWe identified a novel hybrid plasmid simultaneously carrying bla(NDM-1) and bla(IMP-4) in an ST20-K28 carbapenem-resistant Klebsiella pneumoniae (CRKP) strain AZS099 and reported its detailed genetic and phenotypic characterization. MethodsAntimicrobial susceptibility was characterized using broth microdilution method. Complete genome characteristics and plasmid detailed analysis were carried out by PacBio Sequel and Illumina sequencing and further bioinformatics analysis. Conjugation assay, S1-PFGE, Southern blot, plasmid stability, and fitness cost were conducted to the phenotypic characterization of this novel hybrid plasmid. ResultsAZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. Susceptibility testing showed that AZS099 was resistant to almost all beta-lactams examined, including cephalosporins, combinations of beta-lactams and beta-lactamase inhibitors, carbapenems, and aztreonam. PacBio and Illumina sequencing together with S1-PFGE and Southern blot showed that bla(NDM-1) and bla(IMP-4) were simultaneously located on a 296 kb IncFIB(K)/IncHI1B/IncX3 plasmid (pAZS099-NDM-IMP), which consists of four main parts that came from four different types of plasmids. The region harboring bla(IMP-4) is located in a class 1 integron designated as In0, which is located in an IS6100-IS26 transposon-like structure with a total length of ~5 kb. The region harboring bla(NDM-1) is located in the Tn125 transposon remnant. Conjugation and transformation assay confirmed that the plasmid pAZS099-NDM-IMP has the potential for horizontal transfer and displayed high stability (retention rate > 95%). Furthermore, growth curve assessment confirmed that the presence of pAZS099-NDM-IMP exhibits no growth pressure on bacteria. ConclusionOur research reported a hybrid plasmid coharboring bla(NDM-1) and bla(IMP-4) in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid could threaten the control of antimicrobial resistance and should be closely supervised.