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 sequence type 512 (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 the nine, 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 a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these 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 the 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.

Keywords: Klebsiella pneumoniae; antimicrobial resistance; carbapenemase.

FIG 1

KPC-3 variants identified in the ceftazidime-resistant Klebsiella pneumoniae strains. Protein sequence alignment of amino acid residue positions 156 to 298 of KPC-3 variants is shown with respect to the KPC-3 reference protein sequence (NCBI reference sequence WP_004152396.1 [59]). Mutation mapping was performed on the MMDB 6QWD crystal structure of KPC-3 downloaded at the NCBI Cn3D macromolecular structure database visualized by the Cn3D 4.3.1 viewer tool (60) at the Cn3D home page (https://www.ncbi.nlm.nih.gov). Amino acid residues involved in the two loops highlighted in yellow in the KPC-3 three-dimensional model are indicated in blue letters. Amino acid substitutions, insertions, and deletions in ceftazidime-avibactam-resistant KPC variants are indicated in red letters.

FIG 2

Epidemiological analysis of isolates along the time of surveillance and distribution of the ceftazidime-avibactam-resistant KPC variants per Klebsiella pneumoniae sequence type. The blue curves represent the number of isolates per month of KPC producers and CZA-resistant KPC producers (blue line with blue dots), respectively, during the January 2019 to April 2020 period. Orange dots represent the CZA-susceptible strains collected during the surveillance and used as controls for genomic comparison. Bars represent the distribution of the variants per ST in the three surveillance periods: January to June 2019, July to December 2019, and January 2020 to April 2020.

FIG 3

Phylogenetic analysis of ST512 (A) and ST101 (B) K. pneumoniae lineages. The left side shows the unrooted maximum likelihood phylogenetic trees based on a concatenated core gene alignment. The middle represents metadata, each block indicating the presence or absence of a specific resistance gene and of the plasmid replicon (some of which are not shown for the sake of clarity). A legend shows the colors assigned to each KPC variant in the respective column. The right side shows a gene possession matrix, with each row representing the respective strain gene content. Each of the blue blocks on the right represents the presence of a gene. (A) Phylogenetic comparison of ST512 genomes performed using 10 CZA-resistant and 3 CZA-susceptible strains, isolated in the same surveillance period in the hospital and on 7 genomes of epidemiologically unrelated ST512 strains. Among them, four genomes were downloaded from the collection of ST512 strains at the Klebsiella Pasteur MLST isolate database (https://bigsdb.web.pasteur.fr/klebsiella), and three were KPC-3 ST512 producers isolated at the University Hospital Policlinico Umberto I of Rome in 2012 (KP-1, KP-2, KP-3). The blue line localizes the branches of the tree generated by genomes of the CZA-resistant strains under study (except strain 9 producing KPC-31, which was located in a different branch). (B) Phylogenesis of ST101 strains performed on three CZA-resistant strains (strains 16, 10, and 31) and one CZA-susceptible strain (strain 33) isolated during the surveillance period and for comparison on 11 ST101 genomes downloaded from the NCBI GenBank, including two ST101 strains isolated from patients hospitalized in the ICU ward dedicated to COVID-19 patients and identified in our hospital in April 2020 (strain Co_1, producing OXA-48, and strain Co_2, producing KPC-3).

FIG 4

Visualization by BRIG (BLAST Ring Image Generator) of the major plasmid scaffolds identified in this study. A comparison of plasmid sequences using BRIG v.0.95 is shown. The colored rings represent similarities to the reference sequence. (A) Reference DNA sequence and annotation plasmid pKpQIL carrying bla_KPC-29 from strain 6, compared with pKpQIL plasmids carrying bla_KPC-49 (strain 4), bla_KPC-66.(strain 24), bla_KPC-67 (strain 24-2), bla_KPC-69 (strain 34), bla_KPC-70 (strain 32), and the reference pIT01C22 plasmid (HG969997.1). (B) Reference DNA sequence and annotation plasmid FIA(HI1)-FIIk-R carrying bla_KPC-68 from ST101 (strain 31) compared with plasmids carrying bla_KPC-39 (strain 16) and bla_KPC-66.(strain 19). (C) Reference DNA sequence and annotation of plasmid ColRNAI carrying bla_KPC-3 from strain 7 compared with ColRNAI carrying bla_KPC-31 (strain 21). (D) Reference DNA sequence and annotation of the plasmid fusion pKpQIL-pKPN carrying bla_KPC-31 from ST307 strain 23, compared with plasmid pKpQIL from strain 6 and plasmid pKPN from strain 24-2. (E) Reference DNA sequence and annotation of the plasmid fusion pKpQIL-pKPN carrying bla_KPC-67 from ST512 strain 20-2, compared with plasmid pKpQIL (strains 20 and 24-2) and plasmid pKPN (strains 20 and 24-2), respectively. Coding DNA sequences (CDS) are represented by arrows in the inner ring.

FIG 5

bla_KPC gene variant phenotypes expressed in Escherichia coli. MICs were measured by Etest and MicroScan for carbapenems (imipenem [IMI] and meropenem [MEM]; blue bars) and ceftazidime (CAZ; yellow bars), ceftazidime-avibactam (CZA; red bars), and cefoxitin (FOX; green bars) antibiotics. Strains tested were TOP10 Escherichia coli transformed with bla_KPC variants cloned in pCR-Blunt II TOPO vector (ThermoFisher). TOP10 cells carrying the empty pTOPO vector were also tested for comparison. Phenotypic tests were also performed on pKpQIL transformants obtained by transformation of E. coli DH5α chemically competent recipient cells, using purified plasmid DNAs from strains 32, 24-2, 6, and 15 and from the multicopy ColRNAI plasmid carrying bla_KPC-3 obtained by transformation from strain 7 in DH5α recipient cells.