Evolution of ceftazidime–avibactam resistance driven by mutations in double-copy <i>bla</i> <sub>KPC-2</sub> to <i>bla</i> <sub>KPC-189</sub> during treatment of ST11 carbapenem-resistant <i>Klebsiella pneumoniae</i>
Xiaofan Zhang, Yinrong Xie, Ying Zhang, Tailong Lei, Longjie Zhou, Jiayao Yao, Lin Liu, Haiyang Liu, Jintao He, Yunsong Yu, Yuexing Tu, Xi Li
Abstract
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.