From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA) resistant
Klebsiella pneumoniae
strains were isolated in a university hospital in Rome, Italy. These strains belonged to the ST512, ST101 and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among them, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69 and KPC-70 showed amino acid substitutions, insertions and deletions in the Ω loop of the protein. KPC-29 has the duplication, while the novel KPC-67 has the triplication of the KDD triplet in the 270-loop of the protein. Genomics performed on contemporary resistant and susceptible clones underlined that those novel mutations emerged in
bla
KPC-3
genes located on conserved plasmids: in ST512, all
bla
KPC-3
mutant genes were located in pKpQIL plasmids, while the three novel
bla
KPC-3
mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids.
When expressed in
Escherichia coli
recipient cells cloned in the high-copy number pTOPO vector, the Ω loop mutated variants showed CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems.
The investigation of CZA-resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of
K. pneumoniae
high-risk clones.
Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period
