Decoding Ceftazidime-Avibactam Resistance in Newly Identified <i>Klebsiella pneumoniae</i> Carbapenemase (KPC) 107 Variant by Modulating the Active Site Environment

Antibiotic resistance is a serious health concern worldwide, predominantly driven by β-lactamase enzymes that can inactivate most prescribed β-lactam drugs. Carbapenemases are specialized β-lactamases that hydrolyze carbapenems, considered last-resort antibiotics. Insertion variants of <i><i>Klebsiella pneumoniae</i></i> carbapenemase (KPC) play a major role in this threat, showing alarming resistance to ceftazidime-avibactam (CZA), a key combination therapy to treat this menace. KPC-107 is a variant with an exceptional 28-residue insertion raised by duplication mutation (Ser-Ser-Pro-Arg-Ala-Val-Thr-Glu-Ser-Leu-Gln-Lys-Leu-Thr-Leu-Gly-Ser-Ala-Leu-Ala-Ala-Pro-Gln-Arg-Gln-Gln-Phe-Val) between Ambler positions 180 and 181, conferring resistance to CZA. Our study employs comprehensive biophysical, biochemical, and biological analyses accompanied by molecular docking and MD simulations, reporting that KPC-107 exhibits 10 times enhanced catalytic efficiency and 4-fold increased MIC value for ceftazidime compared to KPC-2, while its activity against other tested β-lactams is diminished. The reduced <i>K</i>_i value (0.23 ± 0.05 μM) of avibactam against KPC-107, in contrast with KPC-2 (<i>K</i>_i = 0.83 ± 0.1 μM), is further explained by carbamylation and decarbamylation rates. Insertion-induced active site modification might lead to a better binding with ceftazidime, which is reduced in the case of avibactam, which could possibly explain the contrasting behavior of KPC-107 against these ligands, as confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Molecular docking and MD simulations, including free energy landscape (FEL), principal component analysis (PCA), and radial distribution function (RDF), may further support the binding affinity and the mechanisms underlying ceftazidime resistance. Our findings suggest that KPC-107 adapts distinct mechanistic strategies to evade CZA armor by enhanced ceftazidime hydrolysis while compromising increased inhibition by avibactam, strengthening its role in antibiotic resistance evolution.