Development of a Rapid Antibiotic Susceptibility Test Capable of Detecting Heteroresistance

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The World Health Organization (WHO) has highlighted antibiotic resistance as one of the greatest medical challenges of the 21st century. Without new antibiotics, it is predicted that antibiotic resistant infections will cause 10 million annual deaths worldwide by the year 2050 (surpassing cancer). These infections are poised to negate many advances of modern medicine that rely on antibiotics. Thus, choosing effective antibiotics based on bacterial susceptibilities would provide the greatest therapeutic benefit. One form of resistance that is overwhelmingly undetected is heteroresistance, in which a minor subpopulation of bacterial cells is phenotypically resistant. These resistant cells rapidly expand in the presence of an antibiotic and thus can cause treatment failure. As heteroresistance is common (>25% of antibiotic-bacteria combinations), detecting heteroresistance is critical to address the growing crisis of antibiotic resistance. Indeed, it was recently demonstrated that knowledge of heteroresistance can be used to guide successful combination therapy, and can thus even treat pan-resistant bacteria. Unfortunately, clinical susceptibility tests lack the resolution to identify heteroresistance, and the laboratory test for heteroresistance is time consuming and slow. Further, the next-generation susceptibility tests currently under development are largely focused on rapid diagnostics performed on a small number of cells, making detection of rare cells impossible. Developing a new susceptibility test is further complicated by the requirements of the clinical microbiology laboratory; to be adopted, a susceptibility test must require minimal manual labor, work robustly, and be inexpensive – properties that are often at odds with high sensitivity. Based on these issues, there is broad agreement that a new technological approach is required. Here the development of a new, more sensitive susceptibility test using interferometry to measure bacterial population topography is proposed. The use of interferometry to measure topography has no precedence in antibiotic susceptibility testing, and little precedence in biomedical research. However, its commonly used in physical sciences as it is rapid, inexpensive, robust, doesn’t require dyes or stains, and provides super-resolution measurements of topography. Preliminary results demonstrate that interferometry has the resolving power to rapidly detect heteroresistance and distinguish it from susceptibility. Our interferometry-based approach is a substantial improvement over clinical susceptibility tests that cannot detect heteroresistance, research laboratory tests that can detect heteroresistance but are too slow for clinical application, and traditional topographic imaging approaches, e.g. confocal microscopy, that are expensive and lack the resolving power to distinguish heteroresistance from susceptibility. Building off our promising preliminary results, this proposal will determine the underlying biophysics relating bacterial topography to death and reproduction. This work is pivotal to reliably convert topography into susceptibility profiles across a broad range of clinically relevant bacteria and antibiotics.