Overcoming the challenge of antimicrobial resistance: in vitro evaluation of zosurabalpin, a novel LPS transporter inhibitor, against carbapenem-resistant Acinetobacter baumannii

Acinetobacter baumannii is a clinically significant opportunistic pathogen and is classified by the World Health Organization (WHO) as a microorganism of critical priority due to its remarkable ability to develop resistance to multiple classes of antibiotics, particularly carbapenems. Infections caused by A. baumannii, such as ventilator-associated pneumonia (VAP) and bloodstream infections, are especially common in patients admitted to intensive care units (ICUs) and represent a major public health concern, as therapeutic options remain extremely limited. The aim of this study was to evaluate the in vitro activity of a novel, not yet commercially available molecule, zosurabalpin (ZAB), as a potential therapeutic strategy against carbapenem-resistant A. baumannii (CRAB). ZAB is a tethered macrocyclic peptide that exerts antibacterial activity through a novel mechanism of action by inhibiting the lipopolysaccharide (LPS) transport system via direct targeting of the LptB₂FGC complex, an essential transporter required for outer membrane biogenesis in Gram-negative bacteria. Particular attention was given to determining whether ZAB could overcome resistance mechanisms associated with cefiderocol (FDC), a siderophore cephalosporin with a distinct mechanism of action that exploits bacterial iron transport systems to gain intracellular access and subsequently inhibits penicillin-binding proteins. FDC is currently used in clinical practice for the treatment of severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, including CRAB. In the first part of the research, the in vitro efficacy of FDC was assessed on a collection of 100 clinical CRAB strains, isolated from respiratory specimens and blood cultures obtained from patients admitted to ICU at Sapienza University Hospital Policlinico Umberto I in Rome. Minimum inhibitory concentrations (MICs) were determined, with values ranging from 0.06 mg/L to >32 mg/L. Among the 100 CRAB strains, 89 isolates exhibited FDC MICs values of ≤2 mg/L, whereas 11 isolates showed MICs of 4 mg/L or ≥32 mg/L. According to the clinical breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), 89 strains were classified as susceptible to FDC, while the remaining 11 isolates were classified as resistant. These findings demonstrate that, although FDC exhibited strong activity against the majority of isolates, 11 strains presented markedly elevated MICs values, highlighting the emergence of FDC-resistant CRAB strains. Two representative FDC-resistant strains, one with an MIC of 4 mg/L and the other with an MIC >32 mg/L, and one FDC-susceptible control isolate (MIC 1 mg/L) were subjected to whole-genome sequencing (WGS) to identify potential genetic determinants of FDC resistance, including β-lactamase production (particularly OXA-type enzymes), target-site modifications, reduced membrane permeability, efflux pump overexpression, and biofilm formation. All three isolates belonged to the same sequence type, harbored nearly identical antimicrobial resistance genes across multiple antibiotic classes, and shared a comparable set of mobile genetic elements, including numerous insertion sequences. They also carried a capsule and a similar complement of virulence-associated genes, indicating a largely conserved genomic and pathogenic profile. Given their high genomic similarity, the marked differences observed in FDC MICs could not be explained by the features analyzed. Analysis of penicillin-binding protein 3 (PBP3), a known FDC target, revealed no single nucleotide polymorphisms (SNPs) in the resistant isolates. Clinical profiles of the corresponding patients were also comparable, supporting the hypothesis that FDC resistance in these strains likely results from a multifactorial process rather than a single identifiable determinant. Other mechanisms, such as alterations in transport systems, regulatory pathways, or mutations outside known virulence, may contribute to resistance and remain to be further investigated. In the second part of the study, the in vitro activity of ZAB was evaluated to determine its potential as a novel therapeutic option for A. baumannii infections. ZAB showed potent and selective activity against all 100 CRAB isolates, with MICs values ranging from ≤0.015 mg/L to 0.5 mg/L. Importantly, when MICs values were compared with those of FDC, ZAB demonstrated potent activity against all 11 FDC-resistant strains, which exhibited MICs of 4 mg/L or >32 mg/L. Remarkably, these strains showed low MICs values for ZAB, ranging from 0.06 mg/L to 0.5 mg/L. Overall, this study provides real-life evidence based on clinical isolates from a large Italian hospital and highlights the strong potential of ZAB as a future therapeutic option for severe A. baumannii infections. These findings support further preclinical development and contribute to the advancement of innovative antimicrobial therapies urgently needed to address the global threat posed by CRAB strains.