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

Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistantEnterobacteriaceae (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 inKlebsiella pneumoniae sequence type 258 (ST258)bla KPC-3 -harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistantKlebsiella pneumoniae isolates from a patient with pancreatitis. All susceptible (n = 3) and resistant (n = 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 → Tbla KPC-2 point mutation inbla KPC-2 (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-typebla KPC-2 into a novel plasmid, an IS1 insertion upstream ofompK36 , and disruption of therfb gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non-K. pneumoniae ST258 clonal backgrounds and alternativebla KPC 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.