Dynamic evolution of ceftazidime-avibactam resistance from a single patient through the IncX3_NDM-5 plasmid transfer and blaKPC mutation.

• We identified interspecies IncX3_NDM-5 plasmid transfer and bla KPC mutation under ceftazidime-avibactam pressure in vivo. • The IncX3_NDM-5 plasmid showed strong transmissibility and stability. • We simulated in vitro what happens in vivo using conjugation, single-step selection, and competition experiments. • Further plasmid analysis revealed the diverse origin and genetic structure of plasmid carring bla NDM-5. The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and bla KPC mutation from bla KPC-2 to bla KPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10−3–10−5, while the mutation frequency of K. pneumoniae 5298 was 10−6–10−8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of bla NDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: IS Kox3 -IS 26 - bla NDM-5 -IS 5 -IS Aba125 -Tn 3000 -Tn 3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including bla NDM-5 tranfer and bla KPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment. [Display omitted] [ABSTRACT FROM AUTHOR]