Multidrug-resistant (MDR) bacterial infections pose a significant global health threat, exacerbated by the limited development of new antibiotics and the overuse of existing ones. Gram-negative bacteria, particularly *Pseudomonas aeruginosa*, present a major challenge due to their complex cell structures and efficient efflux pump systems that hinder antibiotic penetration. *P. aeruginosa*, a prominent ESKAPE pathogen, is highly resistant to many antibiotics due to its outer membrane permeability and efflux pump expression. Therefore, developing new antibiotics or enhancing the effectiveness of existing ones is crucial. One promising approach is the Trojan Horse strategy, which uses siderophores (iron-chelating molecules produced by microorganisms) to deliver drugs into bacterial cells. Siderophore-drug conjugates, known as sideromycins, utilize active transport pathways for bacterial uptake. Cefiderocol (CEF), a clinically used sideromycin, is effective against severe drug-resistant Gram-negative infections; however, cefiderocol-resistant strains have emerged. To overcome this, the study explores the potential of combining sideromycins with metal compounds, which have known antimicrobial properties and are being explored as resistance breakers. This combination therapy, termed metallo-sideromycins, aims to leverage a dual Trojan Horse strategy, synergistically enhancing bacterial killing and delaying resistance development.

The literature extensively documents the challenges posed by antimicrobial resistance, particularly in Gram-negative bacteria. The lack of new antibiotics and the widespread misuse of existing ones have fueled the rise of drug-resistant strains. *P. aeruginosa*’s intrinsic resistance mechanisms, including its outer membrane and efflux pumps, are well-established. The Trojan Horse strategy, employing siderophores to deliver antibiotics, has shown promise. Sideromycins like albomycins, naturally occurring antibiotics, have demonstrated high efficacy due to their active transport mechanisms. Cefiderocol, the first clinically approved sideromycin, represents a significant advancement, but resistance development is an ongoing concern. Several studies have documented the emergence of cefiderocol resistance through various mechanisms, including mutations in siderophore receptors and increased efflux pump expression. Concurrently, metal compounds have shown potential as antimicrobial agents and resistance breakers due to their multi-targeted modes of action, particularly bismuth(III) and gallium(III) compounds. These metals can compete with Fe²⁺ for cellular uptake and disrupt Fe³⁺ functions. The potential of a dual Trojan Horse strategy combining sideromycins and metals to synergistically enhance antibacterial activity and slow down resistance development forms the basis of this study.

This study employed a range of methodologies to investigate the synergistic effects of metallo-sideromycins. Initial screening involved examining the minimum inhibitory concentrations (MICs) of cefiderocol (CEF) against various Gram-negative bacteria in the presence of different metal compounds (colloidal bismuth citrate (CBS), Ga(NO₃)₃, FeCl₃, Ti(IV)-citrate, Mn(OAc)₂, Co(OAc)₃, and Cr₂(SO₄)₃). Checkboard microdilution assays determined the fractional inhibitory concentration indices (FICIs) to assess synergistic interactions between CEF and the most promising metal compounds (CBS and GaNit). Time-kill assays were conducted to evaluate the bactericidal activity of CEF and CBS individually and in combination. Biofilm formation was assessed using crystal violet staining and plate counting methods, along with confocal laser scanning microscopy (CLSM) to visualize biofilm architecture. To examine resistance development, the mutant prevention concentration (MPC) of CEF was determined, with and without CBS. Serial passaging experiments were performed to monitor the development of CEF resistance in the presence and absence of CBS. The efficacy of CEF and CBS against clinically isolated, CEF-resistant strains was determined using checkboard microdilution assays. UV-vis spectroscopy, NMR spectroscopy, and mass spectrometry (MS) were used to investigate the interaction between Bi³⁺ (from CBS) and CEF. The intracellular iron and bismuth concentrations in *P. aeruginosa* were measured using calcein-AM fluorescence and inductively coupled plasma mass spectrometry (ICP-MS), respectively. A *P. aeruginosa* mutant lacking the *piu*A gene (a siderophore transporter) was generated to assess the role of this transporter in the synergistic effect. Finally, a murine acute pneumonia model was used to evaluate the *in vivo* efficacy of the CEF-CBS combination therapy, assessing survival rates and bacterial loads in the lungs.

The study revealed strong synergistic effects between bismuth/gallium compounds and cefiderocol against *P. aeruginosa* and *B. cepacia*. CBS and GaNit demonstrated the most significant enhancement of CEF's antimicrobial activity, reducing MICs by 32–64 folds. Checkboard microdilution assays confirmed strong synergy (FICI < 0.25) between CBS and CEF, with similar results observed for GaNit. Time-kill assays showed a significant reduction in *P. aeruginosa* population with the CEF-CBS combination compared to monotherapy. CBS also substantially inhibited *P. aeruginosa* biofilm formation in combination with CEF, demonstrating over 80% reduction in biofilm viability. Furthermore, CBS significantly suppressed the development of CEF resistance in *P. aeruginosa*, substantially lowering the mutant prevention concentration (MPC) of CEF. Interestingly, the CEF-CBS combination resensitized clinically isolated, CEF-resistant *P. aeruginosa* strains to CEF. Spectroscopic and MS analyses revealed the formation of a 1:1 Bi³⁺-CEF complex, supporting the hypothesis that Bi³⁺ competes with Fe³⁺ for binding to CEF and transport into bacterial cells. This competition led to reduced intracellular Fe³⁺ levels and enhanced Bi³⁺ uptake. The study also demonstrated that the *piu*A gene, encoding a siderophore transporter, plays a role in the transport of both Bi-CEF and Fe-CEF complexes. Finally, *in vivo* studies in a murine pneumonia model showed a significant increase in survival rate and decrease in bacterial load in the lungs for mice treated with the CEF-CBS combination compared to monotherapies, demonstrating the efficacy of this combination therapy.

The findings demonstrate the potential of metallo-sideromycins as a novel strategy to combat antimicrobial resistance. The strong synergistic effect observed between CEF and CBS, along with the suppression of resistance development and resensitization of resistant strains, highlights the therapeutic potential of this combination therapy. The mechanism underlying this synergy involves Bi³⁺ competing with Fe³⁺ for binding to CEF and transport into bacterial cells, leading to reduced intracellular iron levels and enhanced bismuth uptake. The disruption of bacterial iron homeostasis and possible additional effects of bismuth on bacterial enzymes likely contribute to the observed synergistic antibacterial activity. The successful translation of *in vitro* findings to a murine infection model underscores the potential clinical applicability of this approach. Future studies should explore the use of other sideromycins and metal compounds to determine the generality of this approach. More research is necessary to fully elucidate the mechanisms of action and to optimize the combination therapy for clinical use. Additional studies focusing on pharmacokinetics, pharmacodynamics, and potential toxicity in humans are crucial before this metallo-sideromycin strategy can be widely applied.

This study demonstrates the significant potential of metallo-sideromycins, specifically the combination of cefiderocol and colloidal bismuth citrate (CBS), to combat antimicrobial resistance. The strong synergy observed *in vitro* and *in vivo*, along with the suppression of resistance development and resensitization of resistant strains, offers a promising new therapeutic strategy. Future research should explore the application of this approach to other sideromycins and metal compounds to broaden its applicability. Optimization of the combination therapy and further preclinical and clinical evaluation are needed to establish its effectiveness and safety for widespread use.

While this study provides compelling evidence for the synergistic effects of cefiderocol and CBS, further research is required to fully elucidate the underlying mechanisms and to address potential limitations. The murine pneumonia model, while valuable, may not fully replicate the complexity of human infections. The study focused primarily on *P. aeruginosa*, and further investigations are necessary to determine the effectiveness of this combination therapy against other Gram-negative pathogens. Long-term effects of the combination therapy, including potential toxicity and resistance development over extended periods, require further investigation.