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Animal histological images for in vivo maximum tolerated dose and toxicity

Yang, Yanxi1; Chen, Irene1

Published Sep 02, 2025 on Dryad. https://doi.org/10.5061/dryad.47d7wm3mw

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Abstract

The rising rate of antibiotic resistance is a widely acknowledged challenge, especially for gram-negative bacterial infections. Some drugs, such as the lipopeptide polymyxin B (PMB), have high antimicrobial activity against gram-negative organisms but also high toxicity owing to low specificity, resulting in a low therapeutic index and poor clinical utility. Due to the high rate of nephrotoxicity, systemic use of PMB is presently restricted to last-line therapy, with adverse clinical tradeoffs. To salvage such antibiotics, we propose specific drug delivery to bacterial cells using engineered phage as a carrier. We replaced the receptor-binding protein of phage M13 with an antibody fragment (scFab) recognizing the core antigen of lipopolysaccharide (LPS), creating a phage that bound a wide range of clinically important gram-negative pathogen species. We then cross-linked thousands of PMB molecules per virion, making ‘PMB-Phage’. PMB-Phage reduced the minimum inhibitory concentration (MIC) by up to 2 orders of magnitude for a variety of species, including the ESKAPEE pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. To test efficacy, immunocompetent mice were infected with multidrug-resistant P. aeruginosa in a pneumonia model. PMB-Phage treatment was effective, causing bacteriostasis and up to 500-fold reduction in bacterial counts compared to a negative control, with a ~90-fold increase in potency compared to PMB. Similarly, P. aeruginosa infection of the cornea (keratitis) was treated effectively by PMB-Phage, with ~10,000-fold reduction in bacterial counts and a >10-fold increase in potency compared to PMB. PMB-Phage was well-tolerated, with no toxic effects observed in vitro or in vivo, including by serum biomarkers and histology. This work demonstrates a proof-of-concept of phage-antibiotic conjugates as a strategy to improve cell targeting for bacteria over mammalian cells, combining engineerable phage-based targeting with large payload capacity. Phage-drug conjugates may represent a next-generation strategy to greatly improve potency and therapeutic index for otherwise toxic molecules.