β-lactams represent cornerstone antibiotics, but resistance towards them is widespread [1]. Of particular concern are β-lactam-resistant Gram-negative bacteria, for which alternative therapeutic options are extremely limited. To address this unmet medical need, we developed a natural product screening platform for searching antibiotic adjuvants to be used in combination with β-lactams against multidrug-resistant Gram-negative (MDR-GN) bacteria. To this end, we first selected five clinically-relevant models of MDR-GN characterized by β-lactam resistance (with MICs of ceftriaxone, meropenem, and/or ceftazidime–avibactam in the range 8-1024 µg/ml) and we identified the β-lactamases acting as resistance determinants (namely, CTX-M, KPC-3, KPC-31, VIM-1, and VIM-2). Then, we performed the high-throughput biological activity-guided screening of a microbial library comprising 39,000 crude extracts mainly obtained from actinomycetes’ and filamentous fungi’s culture broths. The screening assay was developed to select putative HITs with no or negligible direct antimicrobial activity, but capable of counteracting resistance mechanisms by inhibiting β-lactamase activity. Notably, >200 HITs capable of restoring the activity of β-lactams against the selected resistant isolates, even when the antibiotics were used at 1/4 or 1/8 the MIC value, were identified. The candidates were further subjected to activity-guided purification, resulting in the isolation of several compounds with the desired adjuvant activity, which underwent chemical and biological characterization for dereplication and novelty assessment. Preliminary findings suggested that the observed bioactivities were mostly associated to metal chelating agents. As further perspectives, an in-depth characterization of these compounds is being conducted, to better elucidate their mode of action through in vitro and in silico studies, to test their efficacy in conjunction with β-lactams against a broader panel of MDR-GN clinical isolates, and to assess their cytotoxicity on various eukaryotic cell models, ultimately paving the way for their possible preclinical development and prospective therapeutic application.

[1] GBD 2021 Antimicrobial Resistance Collaborators. (2024). The Lancet. 404(10459):1199-1226.