KPC-Mediated Resistance to Ceftazidime-Avibactam and Collateral Effects in Klebsiella pneumoniae (2025)

KEYWORDS: Klebsiella pneumoniae, ceftazidime, avibactam, KPC, antibiotic resistance, carbapenemase

Phenotypes and molecular profiling of CZA-resistant K. pneumoniae mutants.

Considering that mutations in bla_KPC-2 and bla_KPC-3 genes have previously been shown to be involved in resistance to CZA in K. pneumoniae (9–15), sequencing of the bla_KPC genes was performed for all the CZA-resistant colonies obtained by single-step mutation selection. A total of 16 bla_KPC-2 mutant alleles (M1a to M16a) and 10 bla_KPC-3 mutant alleles (M1b to M10b) were identified. Twenty-one contained amino acid substitutions (12 KPC-2 variants and 9 KPC-3 variants), 3 contained insertions (2 KPC-2 variants and 1 KPC-3 variant), and the remaining 2 were deletions in the bla_KPC-2 gene (Table 1). Twelve of those identified mutations had been previously identified in other studies, four corresponding to clinical isolates (16–18) and eight to in vitro-obtained mutants (19–21). A total of 14 newly identified KPC variants were thus identified in this study. All strains producing KPC variants recovered in our assays exhibited a concomitant increase in resistance to both CZA and CAZ, with MICs ranging from 64 μg/ml to ≥256 μg/ml for CAZ and 8 μg/ml to 64 μg/ml for CZA (Table 1). Conversely, all mutants also exhibited a significant reduction in the MICs of carbapenems, including imipenem, meropenem, and ertapenem, and to the combinations of carbapenems and inhibitors. A significant increase (≥2 dilutions) in susceptibility to piperacillin-tazobactam and aztreonam was also observed for 20 (12 KPC-2 and 8 KPC-3) and 16 variants (12 KPC-2 and 4 KPC-3), respectively.

The majority of substitutions (16/26) were found in the omega loop, which forms the basis of the KPC active site (Table 1) (22). Four of the omega loop mutants actually corresponded to previously reported inhibitor-resistant variants, namely, KPC-31, KPC-33, KPC-57, and KPC-61 (16–18), another eight had been identified previously in in vitro studies, and one was a novel variant (KPC-2; R164H) (19–21). Positions D179 and R164 in the KPC enzyme are known to form the omega loop salt bridge, and substitutions in either of these residues can result in disruption of the salt bridge and enhanced activity toward broad-spectrum cephalosporins while reducing carbapenemase activity (22). We also found mutations at position 243, located between the β7 and β8 strands, T243M and T243P from both KPC-2 and KPC-3 parent enzymes, which had previously been observed in KPC-2-derived variants (20). Additionally, 2 of the novel variants exhibited 4-amino-acid deletions in the KPC-2 omega loop, and 3 isolates had insertions of 2, 8, and 20 amino acids (2 in KPC-2 and 1 in KPC-3, respectively). Previous studies have identified indels in KPC enzymes identified in CZA-resistant isolates, most often in the immediate vicinity of the omega loop or active site, being, however, relatively small, ranging from 1 to 5 amino acids (11, 12, 21, 23). One notable exception is in KPC-44, a variant obtained from a patient following CZA treatment, with a 15-amino-acid deletion (from 278 to 292) and exhibiting CZA resistance while also a reduction in carbapenem MICs (24). The insertions observed in this study were diverse in both size and location (2, 8, and 20 amino acids) and, despite being located outside the omega loop or active site vicinity, still resulted in increased CZA resistance while negatively impacting carbapenem resistance. It could be hypothesized that despite their physical location, such deletions must have a downstream effect on the structure of the KPC binding site. Examples of indels, rather than point mutations, causing an extended-spectrum β-lactamase (ESBL) phenotype in carbapenemases are relatively rare; however, there have been reported instances such as the aforementioned KPC-44, and also in OXA-163, an OXA-48-like enzyme with a 5-amino-acid deletion resulting in an ESBL phenotype and carbapenem-reduced susceptibility (25).

Phenotypes of the recombinant E. coli and K. pneumoniae strains with cloned bla_KPC-2 and bla_KPC-3 mutant alleles.

In order to further confirm that the identified bla_KPC-2 and bla_KPC-3 mutant alleles were indeed the main sources of resistance to CZA and to rule out possible associated resistance mechanisms (such as decreased outer membrane permeability), the 26 bla_KPC-2 and bla_KPC-3 mutant alleles, as well as the parental bla_KPC-2 and bla_KPC-3 genes, were cloned into reference E. coli TOP10. Parent alleles and four selected mutant alleles, M1a (T243M), M6a (A35G), M1b (8-amino-acid [aa] insertion), and M2b (S171P; KPC-61) were also cloned into K. pneumoniae CIP53 (Table S1 in the supplemental material). E. coli TOP10 (Table 2) and K. pneumoniae CIP53 (Table S1) recombinant strains producing all the selected KPC mutant alleles showed MICs of CZA ranging from 2 μg/ml to 128 μg/ml and significantly decreased carbapenem MICs. These data, corresponding to both E. coli and K. pneumoniae backgrounds, confirm that the identified bla_KPC-2 and bla_KPC-3 mutant alleles were the dominant mechanism responsible for the CZA resistance phenotype observed in the mutant isolates. It is also important to note that even the novel mutants with single substitutions at distinct locations from those described previously in the literature (M6a, A35G; M3a, S109P; M4b, P67Q) still exhibited the same elevated CZA MICs and decreased carbapenem MICs, indicating that these sites possibly play an important role in the formation and/or structure of the KPC active site.

Enzyme kinetic measurements of the mutant KPC variants.

The enzyme kinetics of the identified mutant KPC carbapenemases were compared to the wild-type KPC-2 and KPC-3 enzymes. Kinetic data showed that all selected mutant KPC enzymes conferring resistance to CZA had significantly lower hydrolysis activities against carbapenems, namely, either imipenem, meropenem, and ertapenem, than KPC-2 and KPC-3. Similarly, lower hydrolysis rates were noticed for the different penicillin substrates tested, namely, benzylpenicillin, piperacillin, and ticarcillin, than those obtained with KPC-2 and KPC-3 (Table 3). It should also be noted that similar decreased hydrolytic properties toward carbapenems have been previously reported for other KPC-2 and KPC-3 variants conferring resistance to CZA, such as KPC-41 and KPC-50.

Hydrolysis experiments were performed in order to evaluate the ability of the KPC mutants obtained to compromise the efficacy of CAZ. Noticeably, although CAZ was significantly hydrolyzed by both KPC-2 and KPC-3, almost no hydrolysis could be detected for all the KPC mutants obtained in our study conferring resistance to CZA under normal conditions (measurement made over 5 min). In fact, it was shown that those given KPC mutants indeed conferred resistance to CAZ when produced by recombinant E. coli strains. By measuring the affinity of those KPC variants toward CAZ using various concentrations of this β-lactam, and by comparing with values obtained with KPC-2 and KPC-3, with all experiments being performed with benzylpenicillin as a reporter substrate, we could explain these paradoxical observations. Hence, a much stronger affinity of the KPC variants toward CAZ was evidenced than KPC-2 and KPC-3, explaining the inefficacity of CAZ to retain its bactericidal activity. Overall, our analysis of 26 KPC mutants showed that the decreased hydrolytic properties toward β-lactams and increased affinity toward CAZ was a systematic trait among KPC variants conferring resistance to CZA.

Inhibitory activities measured for clavulanic acid, tazobactam, vaborbactam, and AVI toward the KPC mutants were compared with the wild-type KPC-2 and KPC-3 enzymes (Table 4). While the inhibitory activity of clavulanic acid, tazobactam, and vaborbactam toward those KPC mutants was comparable or slightly higher than KPC-2 and KPC-3, that of AVI toward most of the identified KPC mutants was slightly lower. Therefore, the resistance conferred to CZA was not related to changes in the inhibitory effect of AVI but, rather, to the significant increased affinity toward CAZ. Similar results have been observed in previous studies of both in vivo- and in vitro-obtained CZA-resistant KPC mutants (12, 26).

Conclusions.

A series of 16 bla_KPC-2-derived and 10 bla_KPC-3-derived mutant alleles conferring resistance to CZA in K. pneumoniae was identified and further characterized in this study, 14 of which corresponded to novel mutants. The mutations identified within the bla_KPC-2 and bla_KPC-3 gene sequences led to some collateral effects with respect to resistance to some other β-lactam molecules, with significant reductions of the MIC values of carbapenems and piperacillin-tazobactam. It is clear that the KPC omega loop is a hot spot for mutations capable of conferring CZA resistance, although it is also important to note that mutations found elsewhere in the protein that were not described previously could also confer resistance. Such modification of the β-lactamase hydrolytic properties was systematically observed for all KPC variants conferring resistance to CZA.

There has been a reported case where a patient was treated for a KPC-Kp infection with CZA, the corresponding strain developing CZA resistance due to KPC mutations, the patient being subsequently treated and cured with meropenem (27). This highlights that while the CZA-resistant phenotype developed through mutations in the KPC encoding gene is not desirable, it can still allow carbapenem therapy to be resumed. However, in vitro mutation studies have shown that some CZA-resistant KPC mutants were capable of reverting to wild type upon exposure to carbapenems, highlighting the flexible nature of such mutations (20, 28). In a Galleria mellonella infection model, Gottig et al. showed that when used together, CZA and imipenem resulted in the prevention of the development of resistance (20), possibly illustrating the limitations of resistance development. Our present data support the use of carbapenems and carbapenem-containing antibiotic combinations for treating infections due to KPC mutants as has previously reported (27), albeit with caution and awareness of the potential for KPC reversion.

Bacterial isolates.

Two clinical K. pneumoniae isolates that had previously been sequenced were used as the parental strains in this study, P-KPC-2, a sequence type 34 (ST34) strain harboring the wild-type bla_KPC-2, and P-KPC-3, an ST405 strain harboring the wild-type bla_KPC-3 allele. The additional β-lactamase content of both isolates was as follows: P-KPC-2 harbored bla_TEM-1 and bla_SHV-26, while P-KPC-3 harbored bla_CTX-M-15, bla_TEM-1, bla_OXA-1, and bla_SHV-76. Both isolates had been recovered from patients hospitalized in Switzerland and from rectal swabs.

Antimicrobial susceptibility testing.

MICs of antimicrobial agents were determined by broth microdilution according to CLSI methodology M07 (29). Antimicrobial agents were obtained from Sigma-Aldrich (Buchs, Switzerland) and Roche (Basel, Switzerland). The preparation of the different β-lactam/β-lactamase inhibitor combinations, namely, piperacillin-tazobactam, CZA, aztreonam-AVI, imipenem-relebactam, and meropenem-vaborbactam, was performed according to the CLSI guidelines (29), with a fixed concentration of the inhibitor at 4 mg/liter for tazobactam, AVI, and relebactam and 8 mg/liter for vaborbactam. Data were interpreted according to EUCAST guidelines (30).

CZA mutant selection.

K. pneumoniae mutant strains were selected by single-step mutation selection. Briefly, both K. pneumoniae strains harboring the wild-type bla_KPC-2 and bla_KPC-3 gene alleles were separately grown overnight in a liquid culture (Mueller-Hinton [MH]) on a rotatory shaker (150 rpm) at 37°C. One hundred microliters of overnight culture were spread on Luria-Bertani agar plates containing CZA at the breakpoint concentration (8 mg/liter for CAZ and 4 mg/liter for AVI). CZA-resistant mutants were selected for further analysis following overnight incubation at 37°C.

Sequence analyses.

The bla_KPC-2 and bla_KPC-3 wild type and mutant alleles were amplified using primers KPC-Fw (5′-TATATGAATTCAAGGGCGGCTGAAGGAATAC-3′) and KPC-Rev (5′-ATATAGAATTCCGCCATCGTCAGTGCTCTAC-3′). Sequencing of the amplicons and recombinant plasmids was performed by Microsynth AG (Balgach, Switzerland). Sequences were analyzed with Expasy tool of the Bioinformatics Resource Portal (https://web.expasy.org/translate/) and Clustal Omega tool of the European Molecular Biology Laboratory of the European Bioinformatics Institute (EMBL-EBI) (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Cloning experiments.

The bla_KPC alleles were amplified using FIREPol DNA polymerase (Solis BioDyne) and primers KPC-Fw and KPC-Rev. Amplicons were purified with the ExoSAP-IT PCR product cleanup reagent (Thermo Fisher) before being cloned into pCR-BluntII-Topo (Invitrogen, Thermo Fisher) and transformation into Escherichia coli TOP10. Transformants were selected on plates supplemented with ampicillin (100 mg/liter) and kanamycin (30 μg/ml) and were confirmed by sequencing of the bla_KPC allele. Recombinant plasmids were then transformed into K. pneumoniae CIP53 and selected as described above.

Enzyme kinetic measurements.

β-Lactamases from crude extracts were used for the measurement of the specific activity at 30°C in 100 mM sodium phosphate (pH 7.0). A Genesys 10S UV-visible (UV-Vis) spectrophotometer (Thermo Scientific) was used to determine the initial rates of hydrolysis. The following wavelengths and absorption coefficients were used: for benzylpenicillin, 232 nm and −1,100 M⁻¹ cm⁻¹; for cephalothin, 62 nm and −7,960 M⁻¹ cm⁻¹; for ceftazidime, 260 nm and −8,660 M⁻¹ cm⁻¹; for ertapenem, 295 nm and −10,940 M⁻¹ cm⁻¹; for imipenem, 297 nm and −9,210 M⁻¹ cm⁻¹; for meropenem, 298 nm and −10,940 M⁻¹ cm⁻¹; for ceftazidime, 260 nm and −8,660 M⁻¹ cm⁻¹; for piperacillin, 235 nm and −1,070 M⁻¹ cm⁻¹; for ticarcillin, 235 nm and −660 M⁻¹ cm⁻¹. To compare the affinities of the purified β-lactamase extract toward CAZ, we performed competitive inhibition of 50 μM benzylpenicillin using 0.1-μM KPC enzymes and various concentrations of CAZ, as described previously (11). The 50% inhibitory concentrations (IC₅₀) were determined for the different β-lactamase inhibitors, namely, clavulanic acid, tazobactam, vaborbactam, and AVI, as described previously (11). All measurements were performed in triplicate.

Data availability.

The sequence data for the isolates P-KPC-2 and P-KPC-3 were deposited in the Sequence Read Archive under BioProject accession number PRJNA738811.

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Supplementary Materials

Data Availability Statement

The sequence data for the isolates P-KPC-2 and P-KPC-3 were deposited in the Sequence Read Archive under BioProject accession number PRJNA738811.