Precise Detection of Carbapenem-Resistant and Hypervirulent <i>Klebsiella pneumoniae</i> Using MOF-Derived Bimetallic Nanocube Hybrid Nanosheet

Carbapenem resistance and hypervirulence represent two distinct evolutionary pathways in <i>Klebsiella pneumoniae</i>, posing significant challenges in clinical settings. Of particular concern are convergent strains that combine both traits, complicating timely diagnosis and treatment. Herein, we present a novel MOF-derived bimetallic nanocube hybrid nanosheet (denoted Pt-G@Cu₅Zn₈C@Au) designed to enhance laser desorption/ionization mass spectrometry (LDI-MS) in distinguishing convergent strains from other variants. The novel material, synthesized through the pyrolysis of pristine MOFs, features uniformly distributed Cu and Zn synergistic metal sites within the carbon matrix, addressing critical limitations of current nanomatrices for in situ extraction of metabolic fingerprints from microbial cells, such as limited sensitivity (e.g., amorphous silicon, TiO₂, and metal nanoparticles) or relatively weak conductivity and stability (MOF-based materials). Utilizing this advanced matrix, the metabolic fingerprints of 248 <i>K. pneumoniae</i> isolates were rapidly extracted, identifying 23 top VIP-score peaks as potential biomarkers for differentiating convergent strains from their variants. Combined with machine learning, the prediction model achieved 100% accuracy in distinguishing convergent strains from carbapenem-sensitive isolates (CS_cKP) or hypervirulent isolates (hvKP) using the SVM model, while achieving 78.26% accuracy in differentiating them from carbapenem-resistant isolates (CR_cKP) with the KNN/NB models. These findings highlight the high accuracy and efficacy of our assay in distinguishing convergent strains from their variants.