Rapid, High-Throughput, and Real-time Assessment of Antibiotic Effectiveness against Pathogenic Biofilms

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Bacterial infections from biofilms are a major threat to human health because biofilm bacteria become very resistant to antibiotics and human immune responses. Effective and rapid antibiotic-susceptibility testing (AST) for biofilms is urgently required to guide effective antibiotic use and to survey the spread and emergence of antimicrobial resistance. Conventional AST techniques are not generally suitable for biofilms, thus the overall objective of this project is to provide an innovative, practical, and reliable AST for disease-causing biofilms. This AST will enable rapid, high-throughput, and real-time monitoring along with controllable manipulation of bacterial microenvironments and rapid biofilm formation from a low volume sample. This is accomplished by continuously monitoring bacterial extracellular electron transfers (EEFs) through their metabolic activities, which are impaired by effective antibiotics. Furthermore, a novel strategy will be created to rapidly construct a 3-D polymicrobial biofilm and to establish various biofilm models that mimic natural polymicrobial communities. The project will address grand challenges in microbial infections critical to U.S. healthcare and the economy. Findings will first be disseminated within the discipline through local and international conferences and journal publications; then they will be distributed through educational venues maximizing the project’s reach and impact. This project aims to provide a new strategy for rapid and high-throughput assessment of antibiotic effectiveness against pathogenic biofilms by monitoring the energy output of bacteria in a 3-D multi-laminate structure of papers as a scaffold to support bacterial biofilms. Studies are designed to test a two-fold central hypothesis that: (1) the electrons collectively harvested from a group of cells in a biofilm can be strong enough as a transducing signal to sensitively and continuously monitor both bacterial growth and antibiotic susceptibility, and (2) a 3-D multi-laminate paper stack can provide a new strategy for rapid layer-by-layer biofilm formation in a high-throughput format. The research plan is organized under three aims: (1) create a real-time, sensitive biosensing platform to electrically evaluate antibiotic effectiveness of bacteria in a high-throughput (96 wells) and rapid (5 hours) manner using microbial fuel cell (MFC) based biosensors previously developed by the investigator; (2) develop a multi-layer hydrophilic paper-based culturing platform for rapid biofilm formation with control of biofilm thickness and microbial concentration; and (3) demonstrate integration as a system and practical use with model biofilms of Pseudomonas aeruginosa and Enterococcus faecalis. In summary, the AST array developed will contribute to an in-depth understanding of the underlying dynamics of antibiotic resistance evolution in biofilms and test the effectiveness of an antibiotic regime for treating biofilm-associated infections.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.