Increased Expression and Amplification of bla(KPC-2) Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain

Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the bla(KPC-2) expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the bla(KPC-2) expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated bla(KPC-2) gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate bla(KPC-2) replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones. IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by bla(KPC-2) overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying bla(KPC-2) could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of bla(KPC-2) during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.