High-Throughput, Massively Parallel Antimicrobial Resistance Surveillance Using Drop-Based Microfluidics

对细菌引起的传染病进行有效治疗的一个根本挑战是快速确定抗菌素耐药性和单一或联合药物治疗的有效性，包括防止细菌生长所需的最低浓度或联合抗生素。我们建议通过设计一个易于使用的平台来克服这一点，该平台基于使用微流控液滴的微生物培养的微型化，该微流控液滴可以在可扩展的大规模平行设计中进行标记、跟踪和评估。标准的抗生素敏感性检测(AST)平台通常需要18-72小时才能产生药敏结果。这项建议的主要研究目标是应用基于液滴的微流控技术在四小时内对铜绿假单胞菌临床分离株进行快速AST筛选。基于液滴的微流体是一种创建皮升大小的体积并以每秒数千次的速度进行分析的技术。这些液滴充当单独的微反应器，可以包含单个细胞。在微升大小的微反应器体积内分离单个细胞和离散的抗生素组合的能力将允许以高分辨率和保真度快速和早期地检测抗生素敏感性。这种方法将能够检测和量化通常低于标准药物检测极限的单细胞亚群。为了实现这一目标，我们有两个具体目标：(1)通过与条形码液滴文库进行多路分析，进行高通量最低抑菌浓度筛选；(2)将设计一种集成光纤的微流控设备，用于对条形码和细胞信号的灵敏检测，这将使该平台易于携带，易于适应临床设置。这项用于细菌高通量抗生素敏感性测试的新颖、可扩展的技术可以极大地缩短细菌感染(如败血症或尿路感染)的诊断时间。

A fundamental challenge in administering effective treatments for infectious diseases caused by bacteria is the rapid identification of antimicrobial resistance and the efficacy of single or combinatorial drug treatments, including the lowest concentrations or combinations of antibiotics required to prevent bacterial growth. We propose to overcome this by engineering an easy-to-use platform based on miniaturization of microbial cultivation using microfluidic drops that can be tagged, tracked, and evaluated in scalable and massively parallel designs. Standard antimicrobial susceptibility testing (AST) platforms typically require 18-72 hours to generate susceptibility results. The primary research objective of this proposal is to apply droplet-based microfluidics to perform rapid AST screening of P. aeruginosa clinical isolates in under four hours. Droplet-based microfluidics is a technology in which picoliter-sized volumes are created and assayed at rates of up to thousands per second. These drops serve as individual microreactors that can contain single cells. The ability to isolate single cells and discrete combinations of antibiotics within picoliter-sized microreactor volumes will allow for rapid and early detection of antibiotic susceptibility with high resolution and fidelity. This method will enable the ability to detect and quantify subpopulations of single cells that are normally below the limit of detection of standard drug assays. To achieve this goal, we have two specific aims: (1) High-throughput minimum inhibitory concentration screening will be performed by multiplexed assaying with a barcoded droplet library, and (2) A microfluidic device with integrated optical fibers will be engineered for sensitive detection of barcode and cell signals, which will enable the platform to be portable and readily adaptable to clinical settings. This novel, scalable technology for high-throughput antimicrobial susceptibility testing in bacteria can greatly decrease the diagnosis time of bacterial infections such as sepsis or urinary-tract infections.