{ "abstract": "Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are the etiological agents of the fatal diseases melioidosis and glanders, respectively. No licensed vaccine is available to protect against these facultative intracellular bacteria and treatment of infection is difficult, requiring intensive and prolonged antimicrobial therapy with low success rates due to intrinsic resistance of Bp and Bm to most antibiotics. This, combined with concerns regarding their adversarial use as biological warfare agents, emphasize an immediate need to identify novel and effective countermeasures for the organisms. Herein, we developed an in vitro high-content imaging, high-throughput screening platform and tested a Library of Pharmacologically Active Compounds (LOPAC) and the Repurposing, Focused Rescue, and Accelerated MEdchem (ReFRAME) library of small molecules for bioactive compounds protecting against bacterial intracellular replication, which is a key pathogenicity trait of Bp and Bm. We first analyzed confocal microscopy images of Burkholderia-infected monolayers of Vero E6 cells and established a reproducible endpoint readout that quantifies cytopathic effects and the formation of plaques during infection. Following this, we screened ~14,800 small molecules and identified 170 unique structures providing ≥ 75% inhibition of intracellular replication of Bp and Bm with EC50 values ranging from 1 pM to 9.99 µM. Of these hits, 23 were found to disrupt the intracellular replication of both organisms including 8 compounds targeting dihydrofolate reductase, 3 tetracycline-class antibiotics, an inhibitor of bacterial leucyl-tRNA synthetase (epetraborole), and 2 host-directed compounds targeting receptors on the surface of mammalian cells. Selected compounds were evaluated in vivo using a BALB/c mouse model of aerosol infection and we discovered that daily treatment with epetraborole (20 mg/kg) and the fluoroquinolone levofloxacin (5 mg/kg) completely protected mice against exposure to lethal doses of Bm and Bp, reducing bacterial burdens in the lungs and spleen to undetectable levels in all animals by day 14 post-challenge. Author summary: Pathogens that can survive inside host cells such as Burkholderia pseudomallei and Burkholderia mallei are difficult to treat because they damage host cells from within and are resistant to many conventional antimicrobial therapies. We developed an in vitro high-throughput, high‑content imaging platform that allows us to monitor this process and measure how well different compounds protect infected cells. Using this system, we screened nearly 14,800 pharmacologically active molecules. Many caused a measurable reduction in cell damage, revealing both known antibiotic classes and host‑directed compounds with activity against intracellular Burkholderia. We selected a small subset of candidates for in vivo testing and found that epetraborole and levofloxacin provided full protection against both Burkholderia species in a mouse aerosol infection model, eliminating detectable bacteria from target organs. Our study shows how image‑based phenotypic screening can rapidly identify new therapeutic opportunities, including drugs already available for other uses. This approach may help accelerate treatment discovery for pathogens that depend on intracellular survival and cause severe disease.", "abstract_shown": true, "affiliations": [ "Department of Biomedical Sciences, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America", "Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America", "Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America", "Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America", "Division of Scripps Research, Calibr-Skaggs Institute for Innovative Medicines, La Jolla, California, United States of America" ], "article_type": "Research Article", "arxiv": null, "author_total": 7, "authors": [ { "affs": [ 0 ], "name": "Nichole Orr-Burks" }, { "affs": [ 1 ], "name": "Sarah R. Hosking" }, { "affs": [ 2 ], "name": "Steven P. Maher" }, { "affs": [ 3 ], "name": "Dennis E. Kyle" }, { "affs": [ 4 ], "name": "Malina A. Bakowski" }, { "name": "Robert J. Hogan" }, { "name": "Eric R. Lafontaine" } ], "bibtex": "https://research.draconitic.com/paper/e3j2rz5jj2x8a.bib", "cited_by": 0, "doi": "10.1371/journal.ppat.1014596", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014596", "id": "e3j2rz5jj2x8a", "issns": [ "1553-7374" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014596", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2713|Burkholderia infections and melioidosis" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Bacterial pathogens/Burkholderia" ], [ "plos", "/Biology and life sciences/Microbiology/Microbial control/Antimicrobials/Antibiotics" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral transmission and infection/Host cells" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Molecular biology assays and analysis techniques/Library screening" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Burkholderia" ], [ "plos", "/Medicine and health sciences/Medical conditions/Infectious diseases/Bacterial diseases/Burkholderia infection" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Intracellular pathogens" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Bacterial pathogens/Burkholderia" ], [ "plos", "/Medicine and health sciences/Pharmacology/Drug screening" ], [ "plos", "/Medicine and health sciences/Pharmacology/Drugs/Antimicrobials/Antibiotics" ], [ "plos", "/Research and analysis methods/Animal studies/Experimental organism systems/Animal models/Mouse models" ], [ "plos", "/Research and analysis methods/Animal studies/Experimental organism systems/Model organisms/Mouse models" ], [ "plos", "/Research and analysis methods/Model organisms/Mouse models" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Molecular biology assays and analysis techniques/Library screening" ] ], "openalex": "W7213424972", "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014596&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-16", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/e3j2rz5jj2x8a.ris", "sources": [ { "first_seen": "2026-10-30T12:17:49Z", "native_id": "10.1371/journal.ppat.1014596", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014596" } ], "subjects": [ { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" }, { "field": "medicine", "origin": "plos:pharmacology", "subfield": "pharmacology-toxicology" } ], "title": "Discovery of repurposed drugs that disrupt intracellular replication of Burkholderia pseudomallei and Burkholderia mallei and protect against lethal aerosol infection", "title_text": "Discovery of repurposed drugs that disrupt intracellular replication of Burkholderia pseudomallei and Burkholderia mallei and protect against lethal aerosol infection", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:49Z", "url": "https://research.draconitic.com/paper/e3j2rz5jj2x8a", "venue": "PLOS Pathogens", "versions": [] }