In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae

Abstract

Objectives To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. Methods The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. Results We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Conclusions Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

Therapeutic Options for Metallo-β-Lactamase-Producing Enterobacterales

“… 68 , 69 MBL-producing Enterobacterales that co-harbor an AmpC, such as bla CMY , may be particularly prone to developing ATM-AVI resistance as mutations in the gene encoding for this enzyme have also been shown to cause ATM-AVI resistance. 70 , 71 The insertion is not associated with MBL β-lactamases and appears limited to E. coli isolates.…”

CRISPR-Cas9-Mediated Carbapenemase Gene and Plasmid Curing in Carbapenem-Resistant Enterobacteriaceae

“…The plasmid pSGKp-km (9), containing a single artificial chimeric guide RNA (sgRNA) under the control of the synthetic constitutive J23119 promoter, a sucrose-sensitive sacB gene, and a kanamycin resistance marker, was used as the backbone to construct pCasCure. Initially, the kanamycin resistance gene was replaced by rifampin resistance gene arr-3, amplified from a clinical isolate, Kp202 (15), to generate plasmid pSGKp-rif. The gene encoding the Cas9 nuclease was amplified from plasmid pCasKp (9) and cloned into NotI and XbaI sites in pSGKp-rif, resulting in the plasmid pSGKp-Cas-rif.…”

Genetic Features Leading to Reduced Susceptibility to Aztreonam-Avibactam among Metallo-β-Lactamase-Producing Escherichia coli Isolates

“…On the other hand, AVI is an excellent inhibitor of many non-MBL ␤-lactamases, including most ESBL and AmpC enzymes. Therefore, the recently developed ATM-AVI combination has been proposed for the treatment of MBL-producing Enterobacterales and is under commercial development (5)(6)(7)(8)(9). Considering that most MBL-producing Enterobacterales (including E. coli), and particularly those producing the NDM-type enzymes, are highly resistant to other non-␤-lactam antibiotics (aminoglycosides, trimethoprim-sulfamethoxazole, tetracycline, and fluoroquinolones), this ATM-AVI combination therapy is among the last-resort options against MBL-producing E. coli.…”