Successive emergence of ceftazidime-avibactam resistance through distinct genomic adaptations in blaKPC-2-harboring Klebsiella pneumoniae ST307 [PublishAheadOfPrint]

Ceftazidime-avibactam (CAZ/AVI) is a promising novel treatment for carbapenem-resistant Enterobacteriaceae (CRE). Despite improved treatment outcomes compared to aminoglycoside- and colistin-based regimens, rapid evolution of CAZ/AVI resistance during treatment has previously been reported in Klebsiella pneumoniae ST258 bla_KPC-3 harboring isolates. Here, we report the step-wise evolution and isolation of two phenotypically distinct CAZ/AVI resistant Klebsiella pneumoniae phenotypes 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->T bla_KPC-2 point mutation in bla_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-type bla_KPC-2 into a novel plasmid, IS1 insertion upstream of ompK36 and disruption of the rfb gene locus in these isolates. Our findings illustrate the potential of CAZ/AVI resistance to emerge in non-K. pneumoniae ST258 clonal backgrounds and alternative bla_KPC variants. These results raise concerns about 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 rapid emergence of resistance during treatment.

Importance Few treatment options are available for carbapenem-resistant Enterobacteriaceae infections, leading to high morbidity and mortality. Novel beta-lactam/beta-lactamase inhibitor combinations such as ceftazidime/avibactam (CAZ/AVI) promise better clinical outcomes. In K. pneumoniae ST258 CAZ/AVI resistance due to bla_KPC-3 mutations following in vivo exposure has been reported. Our report demonstrates that diverse mechanisms of CAZ/AVI resistance can develop in K. pneumoniae ST307, an emerging multi-drug resistant clone. We studied sequential K. pneumoniae ST307 isolates that rapidly developed CAZ/AVI resistance during treatment. We initially noted a 532G->T point mutation in bla_KPC-2 leading to a D179Y protein substitution, associated with decreased carbapenem MIC. After cessation of CAZ/AVI a novel phenotype emerged, characterized by both CAZ/AVI and high-level meropenem resistance. Using short- and long-read sequencing, we detected a transposed copy of bla_KPC-2 and additional transposon insertions into the bacterial chromosome. Our findings highlight the vulnerability of novel carbapenemase inhibitors to resistance and demonstrate that numerous resistance mechanisms may evolve during antibiotic exposure.

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