Evolution of ceftazidime–avibactam resistance driven by mutations in double-copy bla KPC-2 to bla KPC-189 during treatment of ST11 carbapenem-resistant Klebsiella pneumoniae

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC) variants can contribute to resistance to ceftazidime–avibactam (CZA) in Klebsiella pneumoniae (KP). However, two-copy KPC variant-mediated resistance to CZA has rarely been reported to date. Here, we aimed to clarify the evolutionary trajectory of CZA resistance driven by mutations in double-copy bla KPC-2 to bla KPC-189 carried by the tandem core structure (IS Kpn6-bla KPC -IS Kpn27-tnpR -IS 26 ) during treatment of ST11 carbapenem-resistant K. pneumoniae (CRKP). The CZA-resistant KP strain carried double-copy bla KPC-189 , a variant with alanine–threonine and aspartate–tyrosine substitutions at Ambler amino acid positions 172 (A172T) and 179 (D179Y) of bla KPC-2 . Clone experiments confirmed that, compared with that of the wild-type bla KPC-2 clone strain, the minimum inhibitory concentration of CZA increased 16-fold in the bla KPC-189 -mutant strain. Furthermore, protein structure analysis revealed the A172T and D179Y mutations of bla KPC-189 can have a direct effect on the binding affinity of CAZ and AVI for KPC. Sequence comparison revealed that bla KPC-189 was mutated in a double-copy format upon CZA exposure, which was carried by the IS 26 -mediated tandem core structure IS Kpn27-bla KPC -IS Kpn6 . This tandem core structure apparently evolves in vivo during infection, although not by self-transferring, and multiple IS Kpn27-bla KPC -IS Kpn6 copy numbers could mediate transferable CZA resistance upon mobilization. In addition, compared with the wild-type bla KPC-2 gene, the bla KPC-189 gene had no fitness cost. In summary, our study highlighted the emergence of CZA-resistant bla KPC-189 variants in the ST11 clone and the presence of a double-copy bla KPC-189 in the IncFII-type plasmid, which is carried by a tandem core structure (IS 26- IS Kpn6-bla KPC-189 -IS Kpn27-tnpR -IS 26 ). IMPORTANCE To date, ceftazidime–avibactam (CZA) resistance caused by double-copy Klebsiella pneumoniae carbapenemase (KPC) variants has not been elucidated. The multicopy forms of carbapenem resistance genes carried by the same plasmid are relatively rare in most carbapenem-resistant Enterobacteriaceae . In this study, we elucidate the evolutionary trajectory of CZA resistance in ST11 carbapenem-resistant K. pneumoniae harboring a double-copy blaKPC and provide new insights into the mechanisms of acquired resistance to CZA.