Successive Emergence of Ceftazidime-Avibactam Resistance through Distinct Genomic Adaptations in bla _KPC-2 -Harboring Klebsiella pneumoniae Sequence Type 307 Isolates

Abstract

Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistant (CRE). Despite improved treatment outcomes compared to those achieved with aminoglycoside- and colistin-based regimens, the rapid evolution of CAZ-AVI resistance during treatment has previously been reported in sequence type 258 (ST258) -harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistant isolates from a patient with pancreatitis. All susceptible ( = 3) and resistant ( = 5) isolates were of the ST307 clonal background, a rapidly emerging clone. Taking advantage of short-read Illumina and long-read Oxford Nanopore sequencing and full-length assembly of the core chromosome and plasmids, we demonstrate that CAZ-AVI resistance first occurred through a 532G → T point mutation in (D179Y protein substitution) following only 12 days of CAZ-AVI exposure. While subsequent isolates exhibited substantially decreased meropenem (MEM) MICs (≤2 μg/ml), later cultures demonstrated a second CAZ-AVI resistance phenotype with a lower CAZ-AVI MIC (12 μg/ml) but also MEM resistance (MIC > 128 μg/ml). These CAZ-AVI- and MEM-resistant isolates showed evidence of multiple genomic adaptations, mainly through insertions and deletions. This included amplification and transposition of wild-type into a novel plasmid, an IS insertion upstream of , and disruption of the gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non- ST258 clonal backgrounds and alternative variants. These results raise concerns about the strong selective pressures incurred by novel carbapenemase inhibitors, such as avibactam, on isolates previously considered invulnerable to CAZ-AVI resistance. There is an urgent need to further characterize non-KPC-mediated modes of carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance during treatment.

Pharmacodynamics of Ceftazidime plus Avibactam against KPC-2-Bearing Isolates of Klebsiella pneumoniae in a Hollow Fiber Infection Model

“…Resistance emergence during CAZ/AVI has been described during therapy (9)(10)(11) in Klebsiella pneumoniae due to alterations to the ␤-lactamase (KPC-2 and KPC-3), leading to high-level resistance (11). Mutations in ompK36 have also been described to shift CAZ/AVI MIC's in Klebsiella.…”

Beware of broad-spectrum generalizations: ceftazidime-avibactam compared to meropenem for the treatment of gram-negative pneumonia

“…Because CA use will largely be used in infections from CRE as opposed to MERO-susceptible isolates, this paper provides important and relevant data when considering its use in a real life manner. Lastly, de novo resistance (11) and evolution of resistance during CA treatment (including for pneumonia) have also been reported from other centers (3,12). This raises the possibility of a relatively low barrier to resistance for CA and has important implications for antibiotic stewardship programs.…”

“…The exclusions were unfortunate because these populations represent the highest at risk individuals for MDR-GNR infections in general and CRE infections in particular (2). Notably, the study also excluded patients with a creatinine clearance (CrCl) less than 16 mL/min and those receiving hemodialysis (HD) or other renal support, which are also populations of key interest since CA resistance has been documented to occur in patients receiving renal replacement (3,4). Additionally, patients previously treated with CA were excluded, but there was no such exclusion for MERO.…”

Critical analysis of antibacterial agents in clinical development

“…New β lactamase variants with a range of amino acid substitutions, increased hydrolysis of the β lactam antibiotic, or amplification of the bla gene are commonly described. In addition, a diversity of KPC2 and KPC3 variants conferring resistance to ceftazidime-avibactam are circulating in the United States and Europe [22][23][24] . The production of β lactamases may be accompanied by other resistance mechanisms -such as porin deficien cies with a similar, highly diverse epidemiology, upregu lated efflux pumps in E. coli and, rarely, insertions into E. coli penicillin-binding protein 3 (PBP3) -that affect the microbiological activity of cephalosporins and monobac tams 25,26 .…”