Hydrophobic Agent Permeability Assay (HAPA) for Rapid Antibiotic Susceptibility Testing

Hydrophobic Agent Permeability Assay (HAPA) for Rapid Antibiotic Susceptibility Testing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hydrophobic Agent Permeability Assay (HAPA) for Rapid Antibiotic Susceptibility Testing Dayong Jin, Yunpeng Guan, Le Zhang, Jian Li, Amy Cain, Dan Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7373783/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Timely and accurate antibiotic susceptibility testing (AST) is essential for effective infection management and combating antimicrobial resistance. Standard clinical methods, such as broth microdilution (BMD) and disk diffusion, depend on monitoring optical density or inhibition zone diameters and typically require 24 hours or longer to complete. Recent approaches that track single-cell division and morphological changes within the first four hours have shown promise for rapid AST but suffer from limited throughput. Here, we introduce a subcellular phenotyping strategy, termed the Hydrophobic Agent Permeability Assay (HAPA), that monitors bacterial outer membrane permeability dynamics, as a direct functional readout of antibiotic efficacy. In details, we find that susceptible Gram-negative bacteria will rapidly increase membrane permeability upon antibiotic exposure, while resistant cells are impermeable, which difference can be visualized by hydrophobic dyes. Remarkably, such a straightforward readout, as a unique indicator, at the cell envelope coincides with MIC-level inhibition, enabling discrimination of breakpoint responses, which only takes from minutes, to two hours, and four hours, for carbapenem, β-lactam, and aminoglycoside and quinolone drugs, respectively. From the tested 75 clinical isolates of the most representative Gram-negative members of the ESKAPE pathogens, including Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa , HAPA achieved 97.1% categorical agreement with BMD, with low minor error (1.33%), major error (2.50%), and very major error (1.23%) rates, exceeding established regulatory performance benchmarks. By quantitatively correlating bacterial sensitivity to antibiotics to the dynamic membrane permeability responses, HAPA provides a high-throughput, accurate, and clinically translatable solution for rapid AST. Biological sciences/Microbiology/Clinical microbiology Biological sciences/Microbiology/Infectious-disease diagnostics Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7373783","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":556358827,"identity":"7ef2080b-838f-400a-af53-7a2e3fffa4d9","order_by":0,"name":"Dayong 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Standard clinical methods, such as broth microdilution (BMD) and disk diffusion, depend on monitoring optical density or inhibition zone diameters and typically require 24 hours or longer to complete. Recent approaches that track single-cell division and morphological changes within the first four hours have shown promise for rapid AST but suffer from limited throughput. Here, we introduce a subcellular phenotyping strategy, termed the Hydrophobic Agent Permeability Assay (HAPA), that monitors bacterial outer membrane permeability dynamics, as a direct functional readout of antibiotic efficacy. In details, we find that susceptible Gram-negative bacteria will rapidly increase membrane permeability upon antibiotic exposure, while resistant cells are impermeable, which difference can be visualized by hydrophobic dyes. 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