Identificazione di nuovi target nel genoma batterico di batteri Gram-negativi

Acinetobacter baumannii is one of the most clinically relevant multidrug-resistant (MDR) pathogens responsible for severe hospital-acquired infections, particularly in critically ill patients. Carbapenem resistance in this organism is commonly associated with carbapenemhydrolysing β-lactamases; however, additional mechanisms, including alterations of antibiotic targets and complex genomic resistance backgrounds, significantly contribute to therapeutic failure. In this context, Cefiderocol (FDC) has emerged as a novel therapeutic option, although heterogeneous microbiological responses have been increasingly reported. The aim of this work is to investigate the determinants of β-lactam activity and FDC response in clinical A. baumannii isolates through an integrated genomic, phenotypic, and biochemical approach, with particular focus on penicillin-binding protein 3 (PBP3), a key antibiotic target whose structural alterations may critically influence β-lactam efficacy and contribute to resistance development. Five clinical strains obtained from critically ill patients treated with FDC plus ampicillin/sulbactam (AMP/SUL) therapy were analyzed. Whole-genome sequencing (WGS) was performed to characterize resistome composition, virulence determinants, and mutations affecting functionally relevant genomic regions. Antimicrobial susceptibility testing and time-kill assays were used to evaluate antibiotic activity and combination effects under dynamic conditions. Phenotypic analyses revealed isolatedependent susceptibility profiles, with partial restoration of FDC activity following sulbactam (SUL) addition in selected strains during early incubation phases, while sustained bactericidal activity was not consistently observed after prolonged exposure. To investigate the contribution of target modification, penicillin-binding protein 3 (PBP3) variants identified in clinical isolates were cloned, expressed, and purified using a heterologous overexpression system followed by affinity chromatography. Antibiotic–protein interactions were characterized through fluorescence-based bocillin competition assays. Biochemical experiments demonstrated measurable binding of carbapenems to wild-type PBP3, enabling determination of IC₅₀ values, whereas mutant variants carrying K235N and K235N/H370Y substitutions showed markedly reduced or abolished carbapenem interaction. In contrast, limited apparent binding was observed for ampicillin (AMP) and FDC under the experimental conditions employed, suggesting that target engagement may depend on structural or dynamic features not fully reproduced in the in vitro assay system. Overall, this study demonstrates that antibiotic response in MDR A. baumannii results from a multilayer resistance architecture integrating genomic background, phenotypic behavior, and target-site alterations. These findings emphasize the importance of combining WGS, pharmacodynamic analyses, and biochemical characterization of antibiotic targets to improve interpretation of antimicrobial susceptibility and to support more rational antimicrobial stewardship strategies against difficult-to-treat A. baumannii infections.