Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates

Abstract

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.

Place in Therapy of the Newly Available Armamentarium for Multi-Drug-Resistant Gram-Negative Pathogens: Proposal of a Prescription Algorithm

“…Some isolates retained carbapenem-susceptibility with meropenem MIC <8 mg/L. Worryingly, the vertical evolutionary trajectory of some clones as well as the transmission of CZA-resistant strains to untreated patients was observed [ 37 ].…”

Isolation of a Ceftazidime-Avibactam-Resistant bla KPC-71 -Positive Klebsiella pneumoniae Clinical Isolate

“…However, following the wide clinical application of ceftazidime-avibactam, K. pneumoniae acquired resistance rapidly. Currently, 98 bla KPC subtypes have been reported in the world ( 5 ); all of the novel bla KPC variants reported in China were mutated from bla KPC-2 , while those in the United States were mainly from bla KPC-3 ( 6 – 9 ).…”

“…Problems have arisen in clinical microbiology because of these novel KPC variants, owing to their inconspicuous characteristic of carbapenem resistance and lack of an effective detection method. Routine detection methods for carbapenemase, including the Carba NP test, the modified carbapenem inactivation method (mCIM)/EDTA-modified CIM (eCIM) recommended by the CLSI, the 3-aminophenylboronic acid (APB)/EDTA synergy test, and the NG-Test Carba 5, usually show false-negative results for these isolates ( 6 ). These novel KPC variants may fail to grow on selective chromogenic medium ( 6 ).…”

“…Routine detection methods for carbapenemase, including the Carba NP test, the modified carbapenem inactivation method (mCIM)/EDTA-modified CIM (eCIM) recommended by the CLSI, the 3-aminophenylboronic acid (APB)/EDTA synergy test, and the NG-Test Carba 5, usually show false-negative results for these isolates ( 6 ). These novel KPC variants may fail to grow on selective chromogenic medium ( 6 ). Interestingly, bla KPC-71 variants conferred resistance to ceftazidime-avibactam but restored susceptibility to imipenem.…”

First Description of Ceftazidime/Avibactam Resistance in a ST13 KPC-70-Producing Klebsiella pneumoniae Strain from Portugal

“…A high rate of clinical success and survival with ceftazidime/avibactam treatment regimen has been demonstrated in patients with infections caused by K. pneumoniae carbapenemase-producing Enterobacterales [ 8 , 9 , 10 ], and it has become an important first-line option [ 11 ]. However, despite the limited use of CZA worldwide, emerging ceftazidime/avibactam-resistant strains that produce variants of KPC genes have already been reported [ 12 , 13 , 14 , 15 , 16 , 17 ] and may represent a serious cause of concern [ 18 ], despite not being reported to date in Portugal. Thus, this article aims to describe the genomic molecular characterization of a CZA-resistant K. pneumoniae strain that was identified at a Tertiary University Hospital Center in Lisboa, leading, to the best of our knowledge, to the first description of a CZA-resistant strain in Portugal.…”