Introduction

Carbapenem-resistant K. pneumoniae (CRKP) strains are among the most serious antimicrobial resistant strains worldwide, leading to high morbidity and mortality.1 The main carbapenemase in CRKP is Klebsiella pneumoniae carbapenemase (KPC), which hydrolyzes almost all cephalosporins and carbapenems, resulting in a lack of effective treatment options.2 As a result, tigecycline and polymyxin B have become the final resorts for defense against multidrug-resistant gram-negative bacteria.3 However, side effects, such as toxicity and induced resistance, limit their clinical application.4

Ceftazidime–avibactam (CZA) is a novel combination of β-lactam and β-lactamase inhibitors, and offers a valuable alternative strategy against KPC-producing K. pneumoniae infections.5 Avibactam protects ceftazidime from hydrolysis by KPCs, OXA-48, AmpC, and extended-spectrum β-lactamases (ESBLs) but not metallo-β-lactamases.6 Reports of resistance to CZA have increased in recent years since clinical approval.7,8 The main resistance mechanism of CZA is mutation, which is derived from the blaKPC-2 or blaKPC-3 genes, causing abnormal amino acids in the protein sequences, and loss of outer membrane proteins, mutations in other β-lactamases genes and overexpression of KPC enzyme and efflux pumps could also lead to CZA resistance.7–9 Moreover, the number of newly identified KPC variants increases within the last 2 years, more than 190 blaKPC subtypes have been reported in the world according to the NCBI database until Jan. 2024.

Here, we describe the characterization of blaKPC-179, a novel blaKPC variant that confers resistance to CZA while restoring susceptibility to certain carbapenems.

Patients and MethodsPatient

Clinical data were extracted from the hospital information system (HIS) were extracted, including age, sex, specimen origin, antibiotic exposure, duration of hospitalization, in-hospital stay, and disease prognosis.

Phenotypic Detection and Antimicrobial Susceptibility Testing

Two CRKP strains, HX1032 and HX1192, were isolated from the sputum specimens of a patient hospitalized at West China Hospital in Sichuan, China. The isolates were confirmed as K. pneumoniae using a Bruker Biotyper MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). Minimum inhibitory concentration (MIC) results were determined by VITEK-2 automatic microbial analyzer (BioMerieux, Marcy-l’Étoile, France) and broth microdilution, K. pneumoniae ATCC 700603, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC27853 were used as quality control strains, and the results were interpreted by CLSI 2022 for all agents, except for tigecycline and eravacycline, which were interpreted according to the guidelines of the Food and Drug Administration, and aztreonam-avibactam was interpreted by European Committee on Antimicrobial Susceptibility Testing. Carbapenemase production was phenotypically detected using APB/EDTA10 (carbapenemase inhibitor APB [3-aminophenylboronic acid] and EDTA enhancement method) and NG-Test CARBA 5.11

Conjugation Experiments

A conjugation experiment12 was conducted with rifampicin-resistant E. coli EC600 as a recipient strain to explore the transferability of CZA resistance. The transconjugants were screened on Luria–Bertani agar plates containing rifampicin (600 mg/L) and CZA (8 mg/L). The plates were incubated at 37°C for 16–18 h. PCR was performed to confirm the presence of blaKPC.

Whole-Genome Sequencing (WGS) and Analysis

Genomic DNAs of the CRKP strains were extracted using a QIAamp1 DNA Mini Kit (QIAGEN, Hilden, Germany) and subsequently sequenced using the Illumina HiSeq X10 platform (Illumina Inc., San Diego, CA). The sickle tool (GitHub) and SPAdes 3.8 were utilized for sequence trimming and de novo genome assembly. Sequence typing (ST) was performed using Institut Pasteur (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Antimicrobial resistance genes were identified using the ABRicate program (https://github.com/tseemann/abricate) to query the ResFinder database (http://genomicepidemiology.org/).13

Results

The patient was a 40-year-old male with bacterial pneumonia (Carbapenem-resistant Klebsiella pneumoniae), IVB stage NK/T-cell lymphoma, septic shock, sepsis, and hemophagocytic syndrome. He was in poor condition and experienced recurrent fever. Piperacillin-tazobactam (4.5 g every 8 h) was administered on Jun. 14, Meropenem (1000 mg every 8 h) and sulfamethoxazole/trimethoprim (0.48 g once a day) were substituted with piperacillin-tazobactam because of fever and immunosuppression after 8 days. On day 16, the antibiotic regimen was changed to imipenem-cilastatin (500 mg every 6 h). On Sep. 17, the first CRKP isolate (HX1032, blaKPC-2 positive) was recovered from the sputum sample. Due to the patient’s symptoms and drug susceptibility results, imipenem was replaced with tigecycline (100 mg every 12 h), and CZA (2.5 g every 8 h) was added due to rising body temperature after six days. The second CRKP strain (HX1192, blaKPC-179 positive) was isolated from the sputum on Nov. 15. Antibiotics were adjusted multiple times, considering side effects, recurrent clinical symptoms, and the patient’s economic condition. However, the patient eventually died. Microbiological details, timelines, and antibiotic therapies used are summarized in Figure 1.

Figure 1 Antimicrobial treatments of the patient and isolation of K. pneumoniae strains.

Abbreviations: TZP, piperacillin-tazobactam; SXT, sulfamethoxazole/trimethoprim; IMP, imipenem-cilastatin; MEM, meropenem; ATM, aztreonam; TGC, tigecycline; VAN, vancomycin; LZD, linezolid; AK, amikacin; CZA, ceftazidime-avibactam; LVF, levofloxacin; FOX, cefoxitin.

HX1032 and HX1192 were blaKPC-positive isolates highly resistant to β-lactams, aminoglycosides, quinolones, and eravacycline. However, HX1032 was sensitive to colistin, tigecycline, CZA, and aztreonam-avibactam, and phenotypic detection revealed that HX1032 produces serine-carbapenemase (KPC). In contrast, while HX1192 was resistant to CZA, the hydrolytic ability of imipenem was restored and the MIC of meropenem decreased. APB/EDTA and NG-Test CARBA 5 were unable to distinguish the carbapenemase of HX1192 carbapenemases. The plasmid harboring blaKPC-179 from strain HX1192 was successfully transferred into the E. coli EC600 recipient strain, rendering the transconjugants resistant to CZA, quinolones, aminoglycosides, and cephalosporins (Table 1). Despite HX1192 CRKP was resistant to ertapenem, we retested HX1192 using APB/EDTA with ertapenem. However, the enzyme type remained undetectable (Figure 2).

Table 1 Minimal Inhibitory Concentrations (MICs) of K. Pneumoniae Strains HX1032, HX1192, and the blaKPC-179-Positive E. Coli Transconjugant of HX1192

Figure 2 Carbapenemase enzyme detection with APB/EDTA (A) and NG-Test CARBA 5 (B).

Notes: (A); i, carbapenem; ii, carbapenem + APB; iii, carbapenem + EDTA; iv, carbapenem + APB+ EDTA. HX1032 strain produced serine-carbapenemase (KPC) and HX1192 strain could not be distinguished by APB/EDTA and NG-Test CARBA 5.

Based on the results of WGS analysis, K. pneumoniae HX1032 belonged to clonal lineage ST11 and carried the blaKPC-2 gene. Isolate HX1192 also belonged to ST11 and harbored blaKPC-179 (a variant of blaKPC-2). Other resistance genes explaining the resistance phenotype of the two CRKP isolates are shown in Table 2.

Table 2 Molecular Characteristics of CRKP Isolates from the Present Study

Discussion

As one of the few effective treatment alternatives against KPC-producing K. pneumoniae strains, CZA has been widely used clinically since its approval.14 However, acquired resistance has been increasingly reported in multiple independent occurrences. The primary resistance mechanisms include (1) the co-production of serine-carbapenemase and metallo-β-lactamases,15 (2) overexpression of blaKPC, researches showed increased expression of blaKPC-2 and blaKPC-3 could contribute to resistance to CZA,16–18 (3) membrane impermeability of porin mutations, including mutation of the ompK35/36 gene resulting in a significant increase (4→32 mg/L) in the MIC value of CZA in K. pneumoniae,18 and OprD loss with elevated AmpC could cause resistance to CZA in P. aeruginosa,19 (4) overexpression of efflux pumps, increased activity of the MexAB–OprM efflux system, resulting from overexpression of mexA and overproduction of AmpC may be the mechanism underlying the resistance of CZA,8 (5) mutations in other β-lactamases genes, such as blaCTX‐M, blaSHV, blaOXA, AmpC enzyme, and the Leu169Pro amino acid substitution in PBP3 mutations were related to CZA resistance,8 and (6) mutations derived from the blaKPC-2 or blaKPC-3 gene, resulting in amino acid substitutions at positions 164, 167, 169, and 179 within the Ω-loop of class A β-lactamases, which are the most frequent mutations, followed by amino acid insertions and deletions.12,20,21

In the present study, we identified a CRKP (HX1192) isolate harboring blaKPC-179 (NCBI number, OR115556.1), a novel KPC-2 variant of clinical origin associated with reduced susceptibility to CZA (MIC ≥ 256 mg/L) during the treatment of CRKP infections, and its MICs of imipenem and meropenem decreased to 0.25 mg/L and 2 mg/L respectively, which was consistent with previous reports that strains restored susceptibility to imipenem/meropenem or low-level resistance to meropenem,12,13 the reason was the mutations within the blaKPC Ω-loop (positions 165–179) enhance ceftazidime affinity and restrict avibactam binding.12 However, HX1192 carried an A133T substitution and an insertion of S at Ambler position 183 in the protein sequence. Whether these mutations could cause a structural change in the KPC protein, similar to previously observed mutations, resulting in CZA resistance and the recovery of carbapenem susceptibility warrants further investigation.

In our case, blaKPC-179-positive CRKP was isolated after using CZA for more than two weeks, and we previously identified a CRKP carrying blaKPC-78 after 14 days of CZA treatment in a 58-year-old male,13 Shi Q12 reported that after 16 days of CZA use, a blaKPC-33-harboring Klebsiella pneumoniae was detected in sputum. Three patients carrying blaKPC-7, blaKPC-179 and blaKPC-33 CRKP isolates were treated with CZA for more than seven days. We speculate that the duration of CZA usage may be associated with the selective pressure of bacteria, thus causing CZA resistance, and more evidences were needed. Therefore, clinicians should be vigilant for CZA resistance when CZA is used to treat infections caused by blaKPC-2-positive isolates.

The Infectious Diseases Society of America (IDSA) recommended CZA, eravacycline, colistin, tigecycline, CZA, and aztreonam (exert the action of aztreonam-avibactam) for the treatment of carbapenem-resistant Enterobacterales -associated infections.22 However, colistin and tigecycline have limited clinical use because of their side effects. Newer drugs, such as aztreonam-avibactam, have emerged as alternatives, specifically targeting CZA-resistant strains. HX1032 and HX1192 are resistant to β-lactams, aminoglycosides, and quinolones. In this case, colistin, tigecycline, CZA, aztreonam-avibactam, and eravacycline may have been therapeutic agents. Aztreonam-avibactam is considered useful for the treatment of infections caused by gram-negative organisms, particularly those that produce MBLs and serine carbapenemases.23 In this study, aztreonam-avibactam demonstrated potent antibacterial activity against KPC-mutant CRKP strains in vitro.

KPC serine carbapenemases, MBLs, and the coproduction of MBLs and KPC enzymes can be distinguished using APB/EDTA in combination with imipenem or meropenem.10 HX1032 carries serine carbapenemase via APB/EDTA with imipenem, meropenem, and ertapenem. However, the presence of serine carbapenemase in HX1192 was not confirmed by APB/EDTA with imipenem, meropenem, and ertapenem, which could be attributed to blaKPC-2 mutations resulting in the loss of carbapenemase activity. Our results suggested that strains harboring KPC variants may escape through surveillance procedures, thus facilitating their spread in hospital settings, making it particularly important to select suitable negativity of phenotypical carbapenemase detection methods. Immunochromatographic assay NG-Test CARBA 5 detected common carbapenemase genes (KPC, NDM, VIM, IMP, and OXA-48) and some KPC mutations, such as blaKPC-79 (262V_268N dup) and blaKPC-35 (L169P), but not blaKPC-33 (D179Y), blaKPC-71(181S_182P insertion), blaKPC-76 (D179Y+262V_268N dup), or blaKPC-179 (A133T+183S insertion).24 These findings suggested that position 179 or the surrounding amino acids in KPC are crucial for accurate enzyme immunodetection. However, other resistance mechanisms such as porin loss or mutations in other β-lactamases genes cannot be detected by immunochromatographic assays, and alternative methods such as Chromatic Super CAZ/AVI® medium25 could be considered, using molecular biological approaches if necessary.

This study has some limitations. First, other resistance genes in HX1032 and HX1192 K. pneumoniae strains were not analyzed. Second, our study did not use structural and biochemical assays to characterize the functional consequences of blaKPC-179.

Conclusions

In summary, we identified a novel KPC-2 variant, KPC-179, that emerged during CZA treatment. Clinicians must tailor the treatment plans for critically ill patients, closely monitor the susceptibility of KPC-producing strains to CZA, and promptly detect KPC variants at an early stage of therapy. This approach will help prevent treatment failure and the rapid dissemination of CZA-resistant strains.

Data Sharing Statement

The datasets presented in this study are available in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1086695.

Ethical Statement and Informed Consent

All experimental protocols were approved by the Ethics Committee of the West China Hospital of Sichuan University, and the number was 2021623. Institutional approval wasn’t required to publish the case details. The patient provided written informed consent for publication of this case report.

Funding

This study was sponsored by grants from the Special Foundation for the National Science and Technology Basic Research Program of China (2019FY101200).

Disclosure

The authors declare that they have no conflict of interest.

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