Metallo-sideromycin as a dual functional complex for combating antimicrobial resistance

Multidrug resistant bacterial infections, particularly those caused by Gram-negative pathogens like Pseudomonas aeruginosa, are difficult to treat due to outer membrane permeability barriers and active efflux systems that prevent antibiotic accumulation. The limited pipeline of new antibiotics and misuse of existing drugs have accelerated resistance. Trojan Horse strategies exploit bacterial nutrient uptake systems, especially siderophore-mediated ferric iron transport, to deliver antibiotics as sideromycins into cells. Cefiderocol, a chlorocatechol-ceftazidime sideromycin, was approved clinically but resistance has emerged via mechanisms such as mutations in siderophore receptors and increased efflux. Metal compounds (e.g., Bi(III), Ga(III)) have multi-target antimicrobial activity and can interfere with iron metabolism; Bi3+ can compete with Fe2+ for uptake and Ga3+ can mimic Fe3+. The authors hypothesize that simultaneous delivery of sideromycins and metals (Bi3+/Ga3+) via a dual Trojan Horse metallo-sideromycin strategy will enhance bacterial killing synergistically and slow the development of resistance. They use cefiderocol as a showcase to evaluate synergy with metal compounds against P. aeruginosa and Burkholderia spp., assess resistance suppression, and investigate mechanisms and in vivo efficacy.

Natural sideromycins such as albomycins, composed of ferrichrome-like siderophores linked to antibacterial warheads, historically demonstrated superior efficacy through active uptake and intracellular release, achieving much lower MICs and in vivo activity. Synthetic sideromycin libraries have been developed to broaden or narrow spectrum activity in Gram-negative bacteria. Cefiderocol utilizes ferric iron transporters for entry and has shown potent activity against P. aeruginosa and other Gram-negative pathogens, yet resistance—linked to siderophore receptor mutations (e.g., cirA) and efflux—has arisen clinically. Metals have a long history as antimicrobials through multi-target actions; bismuth and gallium can disrupt iron-dependent processes, serving as antimicrobial agents or resistance breakers. Prior studies indicate Bi3+ competes with Fe2+ for uptake and Ga3+ mimics Fe3+, motivating the concept of combining metals with sideromycins to leverage siderophore pathways for co-delivery.

• Primary screening: Assessed MICs of cefiderocol (CEF) against multiple Gram-negative strains (Pseudomonas aeruginosa, Burkholderia cepacia, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Enterobacter aerogenes, Aeromonas hydrophila, Vibrio cholerae, Proteus mirabilis) in the presence of 50 μM metal compounds (colloidal bismuth citrate, Ga(NO3)3, FeCl3, Ti(IV)-citrate, Mn(OAc)2, Co(OAc)2/3, Cr2(SO4)3) using CLSI broth microdilution.
• Drug interaction: Checkerboard microdilution to determine fractional inhibitory concentration index (FICI) for CEF with CBS or gallium nitrate under iron-sufficient (CA-MHB) and iron-poor (M9) conditions. Time-kill assays with PAO1 at sub-MIC concentrations (e.g., CBS 16 μM, CEF 0.5 μM) over 24 h.
• Biofilm assays: Crystal violet staining and CFU enumeration after 48 h to quantify biofilm formation of PAO1 with CEF and CBS alone or in combination; confocal laser scanning microscopy using PAO1-GFP to visualize biofilm integrity and thickness.
• Resistance studies: Mutant prevention concentration (MPC) determination for CEF with varying CBS; serial passaging of PAO1 for 16 days under CEF alone vs CEF+CBS to monitor MIC evolution. Generation of laboratory CEF-resistant strains (PAO1 and clinical isolate PA1882) by 12 serial passages with sub-inhibitory CEF; evaluated resensitization by CBS via checkerboard.
• Mechanistic studies: UV–vis titration (HEPES pH 7.4) of CEF with CBS to derive binding stoichiometry and affinity (Ryan–Weber analysis); 1H NMR titration to monitor aromatic proton shifts upon Bi3+ binding to the chlorocatechol; high-resolution MS to detect Bi–CEF complexes. Iron uptake monitored with calcein-AM fluorescence quenching upon addition of CEF with/without Bi3+ and Fe3+. Intracellular bismuth quantified by ICP–MS after exposure to CBS ± CEF; simultaneous assessment of cellular Fe (calcein-AM) and Bi (ICP–MS) across CBS titration. Constructed PAO1ΔpiuA (iron transporter for Fe–CEF) to test requirement for transporter in synergy; assessed drug interaction by checkerboard. SEM imaging to evaluate cell morphology and membrane integrity after treatments.
• Cytotoxicity: MTT assays in human A549 and HEK293T cells exposed to combinations of CEF (up to 100 μM) and CBS (100 μM).
• In vivo efficacy: Murine acute pneumonia model (female BALB/c). Intranasal infection with P. aeruginosa PAO1 (lethal or sublethal doses). Treatments delivered intranasally 30 min post-infection: PBS, CEF (0.25 mg/kg), CBS (5 mg/kg), or CEF+CBS. Endpoints: 7-day survival, lung bacterial loads (CFU), and histopathology (H&E).

• CBS and Ga(NO3)3 synergized with cefiderocol against P. aeruginosa and B. cepacia, reducing CEF MICs by 32–64-fold; no substantial MIC change with other metals tested across strains.
• Against P. aeruginosa PAO1: CBS alone showed minimal inhibition up to 64 μM; CEF+CBS lowered CEF MIC to 0.06 μM in iron-sufficient medium and 0.125 μM in iron-poor medium. FICI values indicated strong synergy: 0.24 (iron-sufficient) and 0.125 (iron-poor). Ga nitrate also synergized (FICI 0.312).
• Time-kill: Combination (CEF 0.5 μM + CBS 16 μM) reduced PAO1 counts by >4 logs within 24 h relative to monotherapies.
• Biofilm: At sub-inhibitory doses (CEF 0.25 μM; CBS 16 μM), combination reduced relative biofilm viability by >80% and decreased biofilm bacterial loads to ~10^3 CFU/cm^2; CLSM showed reduced biofilm integrity/thickness.
• Resistance suppression: CEF alone drove MIC increase to 64× after 16 passages; CEF+CBS limited MIC rise to 2×. CBS reduced CEF MPC from 32× MIC to 2× MIC at 128 μg/ml.
• Resensitization: In CEF-resistant strains (lab-evolved and clinical), CBS restored CEF susceptibility with FICI < 0.5. Clinical isolates PA247 and PA245: CEF MIC decreased from 16 to 1 μM with 32 μM CBS (FICI 0.125 and 0.188).
• Mechanism: Bi3+ binds CEF to form a 1:1 complex. UV–vis showed new absorbance (314 nm) with a binding constant Ka ≈ 10.35 μM and Bi:CEF molar ratio ~0.7±0.2; 1H NMR aromatic shifts; HRMS detected Bi–CEF species (m/z 958.1157 [Bi–CEF+H]+, 479.5638 [Bi–CEF+2H]2+). Calcein-AM assays showed CEF enhanced Fe uptake; addition of Bi3+ reversed Fe uptake (higher fluorescence), indicating competition. ICP–MS showed increased intracellular Bi with increasing CEF; increasing CBS decreased cellular Fe and increased Bi. Deletion of piuA elevated CEF MIC and abolished synergy with CBS (FICI ~0.5), implicating siderophore transporter involvement. SEM suggested cell elongation and membrane disruption under combination treatment.
• In vivo: In a murine acute pneumonia model, a single intranasal dose of CEF (0.25 mg/kg) + CBS (5 mg/kg) increased survival to 75% versus monotherapies and significantly reduced lung bacterial loads; lung histology showed recovery with combination.
• Cytotoxicity: Combination showed low toxicity in A549 and HEK293T cells with viability >78% up to 128 μM.

The study addresses the need to preserve and enhance the efficacy of sideromycin antibiotics by demonstrating that metal drugs, particularly bismuth (CBS) and gallium, can synergize with cefiderocol to combat Gram-negative pathogens. The results support the dual Trojan Horse hypothesis: Bi3+ competes with Fe3+ for binding to the chlorocatechol moiety of CEF, forming a Bi–CEF complex that is taken up via siderophore transporters (e.g., PiuA), thereby increasing intracellular bismuth while decreasing iron availability. This dual effect enhances antibacterial activity, inhibits biofilm formation, and suppresses the evolution of resistance. The ability to resensitize CEF-resistant clinical isolates underscores clinical relevance. The low mammalian cytotoxicity and significant in vivo efficacy in a pneumonia model indicate translational potential. The findings suggest that leveraging metal–sideromycin interactions can both potentiate antibiotic activity and mitigate resistance development by limiting iron-dependent pathways and delivering multi-target metal ions into bacterial cells.

Bismuth drugs, exemplified by colloidal bismuth citrate, markedly potentiate cefiderocol against P. aeruginosa and B. cepacia, reduce biofilm formation, suppress the emergence of high-level CEF resistance, and restore susceptibility in resistant clinical isolates. Mechanistically, Bi3+ binds the siderophore moiety of CEF, facilitating bismuth uptake via siderophore transporters while reducing iron influx, contributing to bacterial stress and enhanced antibiotic efficacy. The combination shows low cytotoxicity and strong efficacy in a murine lung infection model, supporting its potential clinical utility. Future work should explore other sideromycin–metal combinations, delineate transporter contributions across species, and further validate mechanisms and dosing strategies to extend the life-span of sideromycins.

Synergy and efficacy were most pronounced in P. aeruginosa and B. cepacia, with limited effects observed in some E. coli strains, indicating pathogen-dependent variability. Mechanistic attribution is partial: while Bi–CEF binding and transporter involvement (piuA) are supported, additional pathways may contribute and require further clarification. In vivo assessment was limited to a single murine pneumonia model and dosing regimen. Cytotoxicity was evaluated in two cell lines and may not capture all safety aspects.