Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne bla _KPC-3 Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections

Abstract

Ceftazidime-avibactam is a novel ␤-lactam/␤-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidimeavibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Wholegenome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne bla KPC-3 , which were not present in baseline isolates. bla KPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and bla KPC cloning into competent Escherichia coli. In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution Ͼ D179Y Ͼ V240G. Remarkably, mutations reduced meropenem MICs Ն4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced Ն4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of bla KPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to bla KPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring bla KPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

In Vitro Selection of Meropenem Resistance among Ceftazidime-Avibactam-Resistant, Meropenem-Susceptible Klebsiella pneumoniae Isolates with Variant KPC-3 Carbapenemases

“…Likewise, our bla KPC data are in keeping with results from numerous studies demonstrating that KPC mutations alter enzyme activity (12,13). The D179Y KPC-3 substitution alone or in combination with other substitutions, such as T243M, is the most common mutation among clinical ceftazidime-avibactamresistant K. pneumoniae isolates at our center (1,2) and in resistant Enterobacteriaceae isolates selected by in vitro drug exposure (14). Substitutions in the KPC ⍀-loop (amino acid positions 165-179) enhance affinity for ceftazidime, which is postulated to prevent subsequent binding of avibactam (15).…”

“…Among isolates with D179Y substitutions in KPC-3, meropenem selection resulted in a reversion to wild-type KPC-3 or, in some cases, replacement of baseline mutations with an A177E or T243A substitution. We previously demonstrated that substitutions at T243A had less impact on carbapenem or ceftazidime-avibactam MICs than did D179Y (2). As such, the high-level ceftazidime-avibactam MICs exhibited here by isolates expressing KPC-3 or an A177E or T243A variant were likely caused by as yet unidentified resistance mechanisms.…”

“…Genome libraries were prepared using a Nextera DNA library preparation kit and sequenced using the Illumina NextSeq platform (with paired-end reads of 150 bp). Analysis was performed as previously reported (2). In total, 7 isolates were tested (2 isolates with wild-type bla KPC-3 and 5 isolates with different bla KPC-3 mutations) ( Table 1).…”

“…All isolates harbored genes encoding SHV-11, TEM-1, and OXA-9 ␤-lactamases, a mutated ompK35 gene encoding a truncated porin (premature stop codon at amino acid position 89), and a wild-type ompK36 porin gene. Phylogenetic analysis of whole-genome sequences (WGS) demonstrated that isolates clustered tightly within a distinct sequence type 258 (ST258) sublineage, which was previously shown to include longitudinal K. pneumoniae isolates from patients in whom ceftazidime-avibactam resistance had emerged (2).…”

Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

“…There is continued emergence and reemergence of antibiotic-resistant bacteria, including carbapenem-resistant K. pneumoniae. For example, ceftazidime-avibactam was shown recently to be effective for treatment of carbapenem-resistant K. pneumoniae (32,33), but, as with many antibiotics, resistance can develop during treatment (34). Therefore, new preventive or therapeutic approaches are needed.…”