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

生物膜细菌感染是对人类健康的主要威胁，因为生物膜细菌对抗生素和人体免疫反应具有很强的耐药性。迫切需要有效和快速的生物膜药敏试验（AST）来指导有效的抗生素使用，并调查抗菌素耐药性的传播和出现。传统的AST技术一般不适用于生物膜，因此本项目的总体目标是为致病生物膜提供一种创新、实用、可靠的AST。该AST将实现快速、高通量和实时监测，同时可控制细菌微环境的操作，并从小体积样品中快速形成生物膜。这是通过持续监测细菌细胞外电子转移（EEFs）的代谢活动来实现的，这些代谢活动被有效的抗生素破坏。此外，将创建一种新的策略来快速构建三维多微生物生物膜，并建立模拟天然多微生物群落的各种生物膜模型。该项目将解决对美国医疗保健和经济至关重要的微生物感染方面的重大挑战。研究结果将首先通过当地和国际会议和期刊出版物在学科内传播；然后，它们将通过教育场所分发，最大限度地扩大项目的覆盖范围和影响。该项目旨在通过监测细菌在三维多层纸结构中的能量输出，作为支撑细菌生物膜的支架，为快速和高通量评估抗生素对致病性生物膜的有效性提供一种新的策略。研究旨在测试一个双重中心假设：(1)从生物膜中的一组细胞中收集的电子可以作为一个足够强的转导信号，以敏感和连续地监测细菌生长和抗生素敏感性；(2)3-D多层纸堆栈可以为高通量格式的快速层层生物膜形成提供一种新策略。该研究计划有三个目标：(1)创建一个实时、灵敏的生物传感平台，利用研究者先前开发的基于微生物燃料电池（MFC）的生物传感器，以高通量（96孔）和快速（5小时）的方式对细菌的抗生素有效性进行电评估；(2)开发多层亲水性纸基生物膜快速培养平台，控制生物膜厚度和微生物浓度；(3)展示铜绿假单胞菌和粪肠球菌模型生物膜的系统整合和实际应用。总之，AST阵列的开发将有助于深入了解生物膜中抗生素耐药性演变的潜在动力学，并测试抗生素治疗生物膜相关感染的有效性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.