KPC-50 Confers Resistance to Ceftazidime-Avibactam Associated with Reduced Carbapenemase Activity

INTRODUCTION

The occurrence of multidrug-resistant Enterobacterales, especially carbapenemase-producing isolates, is increasingly reported, leaving very few therapeutic options for treating related infections (1). Interestingly, the recently marketed ceftazidime-avibactam (CZA) drug combination offers novel perspectives (2). This β-lactam/β-lactamase inhibitor combination provides a therapeutic alternative for treating infections caused by KPC-like and OXA-48-like producers, whereas producers of carbapenemases of the metallo-β-lactamase type remain resistant to that combination (1, 2). Despite CZA being rarely prescribed worldwide, KPC-like-producing isolates resistant to this drug combination have already been reported (3–7). Several reports identified KPC variants exhibiting single-amino-acid substitutions in their omega-loop (amino acid positions 164 to 179), particularly the Asp179Tyr substitution, leading to enhanced affinity toward ceftazidime with a concomitant reduced binding to avibactam (AVI) (8–12). In addition, we recently identified KPC-41, possessing a three-amino-acid insertion in the KPC-3 protein sequence and being distantly located from the omega loop (namely, between positions 269 and 270), that conferred high levels of resistance to CZA in a clinical Klebsiella pneumoniae isolate recovered in Switzerland (13).

K. pneumoniae isolate N869 was recovered from a patient repatriated from Greece to Switzerland after a traffic accident. In the Greek hospital, the patient developed ventilator-associated pneumonia, for which he received a treatment made of clindamycin, linezolid, and meropenem for 2 days, to which colistin was added on day 5. A few days later, he was transferred to Switzerland, where all antibiotics but meropenem (as monotherapy) were discontinued. Rectal swabs performed at admission grew K. pneumoniae isolate N859 using the Chrom ID Carba Smart selective plate (bioMérieux, La Balme-les-Grottes, France). According to the EUCAST 2020 breakpoints (14), K. pneumoniae isolate N859 was resistant to all β-lactams, including imipenem and ertapenem, but remained susceptible to meropenem (Table 1). The carbapenemase activity was evaluated by using the Rapid Carba NP test, which gave a positive result (15).

K. pneumoniae N859 was also resistant to aminoglycosides (kanamycin, tobramycin, and netilmicin), to fluoroquinolones, and to colistin (MIC at 128 μg/ml). It remained susceptible to tetracycline, tigecycline, chloramphenicol, trimethoprim-sulfamethoxazole, and fosfomycin and was of intermediate susceptibility to amikacin and gentamicin. It also showed resistance to CZA (MIC of >256 μg/ml) and to a ceftolozane-tazobactam combination (>256 μg/ml), using inhibitor concentrations of 4 μg/ml.

PCR identified a bla_KPC-like gene, and sequencing of the corresponding amplicon identified a gene encoding a KPC variant possessing a three-amino-acid insertion (Glu-Ala-Val) between amino acids 276 and 277 (Ambler numbering), leading to a novel variant named KPC-50 (see Fig. S1 in the supplemental material). A search for additional β-lactamase resistance genes, as reported previously (16), identified a bla_SHV-like gene (intrinsic to K. pneumoniae) but no additional extended-spectrum β-lactamase gene. Mating-out assays performed using K. pneumoniae N859 as the donor and azide-resistant Escherichia coli J53 strain as the recipient (13) were successful and confirmed the plasmid location of the bla_KPC-50 gene, being ca. 60 kb in size (data not shown). No other antibiotic marker was cotransferred along with the bla_KPC-50 gene. PCR-based replicon typing showed that this plasmid belonged to the IncFIB incompatibility group (17). Multilocus sequence typing, performed as described previously (18), showed that isolate N859 belonged to sequence type ST258, which corresponds to the worldwide spread of the KPC-producing K. pneumoniae background (19, 20).

To confirm whether the amino acid substitutions identified within the KPC sequence was responsible for the CZA resistance phenotype observed in K. pneumoniae N859, the bla_KPC-50 gene was cloned and expressed in E. coli TOP10. MIC values then were compared with those of the previously obtained KPC-3-producing E. coli TOP10 (13). In such an E. coli background, KPC-3 conferred resistance to all β-lactams, including ceftazidime, but remained susceptible to CZA, as previously shown (13). Conversely, although KPC-50 also conferred high-level resistance to ceftazidime, it additionally conferred high-level resistance to CZA (Table 1). It is worth noting that KPC-50 conferred much lower levels of resistance to cefoxitin, cefotaxime, and cefepime than KPC-3. One of the most marked features of KPC-50 compared to KPC-3 was that its production did not lead to resistance to carbapenems (Table 1). Indeed, E. coli expressing the bla_KPC-50 gene remained susceptible to ertapenem and meropenem (MICs of 0.25 μg/ml and 0.5 μg/ml, respectively), while the MIC of imipenem observed for the KPC-50-producing E. coli recombinant strain was 4 μg/ml (breakpoint value). Low MICs were observed when testing the new carbapenem–β-lactamase inhibitor combinations, such as imipenem-relebactam, and, more specifically, meropenem-vaborbactam showed an excellent capacity to inhibit the growth of KPC-50 producers (Table 1).

The enzymatic properties of KPC-50 were determined using purified extracts and compared to those of KPC-3 previously obtained under the same conditions (13). Kinetic data showed that KPC-50 has a lower hydrolysis activity of cefalotin, cefotaxime, aztreonam, and imipenem than those of KPC-3 (Table 2). Similar decreased hydrolytic properties toward β-lactams have been reported previously for those KPC variants conferring resistance to the CZA combination, such as KPC-41 (13) or the Asp179Tyr KPC-2 mutants (21–23). Furthermore, the activity of KPC-50 toward aztreonam was not detectable, in contrast to KPC-3 (Table 2) and also contrasting with data obtained for KPC-41 (13).

Kinetic activities toward ceftazidime were measured and compared for KPC-50 and KPC-3 enzymes. As expected, a significant hydrolysis rate was detected with KPC-3, but no hydrolysis could be detected with KPC-50 under normal conditions (measurement made during 5 min). Another assay was performed for 1 h, showing that ceftazidime was indeed hydrolyzed by KPC-50, but the hydrolysis rate was much lower than that of KPC-3 (Fig. 1). Therefore, we observed a paradoxical situation here, with KPC-50 conferring high-level resistance to ceftazidime once produced by a recombinant E. coli clone but a weak hydrolysis rate as measured by UV spectrophotometry. Thus, affinities of KPC-50 and KPC-3 compared to those of the ceftazidime substrate were measured using various concentrations of ceftazidime to inhibit the hydrolysis of a reporter substrate (nitrocefin), as published previously (10, 13). At the same ceftazidime concentrations, a higher inhibition level of nitrocefin was observed with KPC-50 than with KPC-3 (Fig. 1), showing that KPC-50 exhibited a higher affinity toward ceftazidime than KPC-3.

• Open in new tab
• Download powerpoint

FIG 1

Analysis of ceftazidime hydrolysis. (A) KPC-50 and KPC-3 (1 μM enzyme) hydrolysis of 25 μM ceftazidime (CAZ) at room temperature. (B and C) Competitive inhibition curves determined with 50 μM nitrocefin and increasing concentrations of CAZ with 0.1 μM KPC-50 (B) and 0.1 μM KPC-3 (C) at room temperature. Nitrocefin absorbance was measured.

Comparative inhibitory activities of clavulanic acid, tazobactam, and AVI were determined for KPC-50 and KPC-3, showing a 4-fold lower inhibitory activity of AVI toward KPC-50 than KPC-3; conversely, those of tazobactam and clavulanic acid were higher toward KPC-50 than KPC-3 (Table 2).

Overall, these results indicated that the 276-Glu-Ala-Val-277 insertion observed in the KPC-50 sequence was responsible for the reduced hydrolysis of cefalotin, cefotaxime, and carbapenems, associated with a higher affinity toward ceftazidime and a reduced sensitivity to AVI.