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迅速查明抗菌素耐药性和单一或组合药物治疗的疗效，包括防止细菌生长所需的最低浓度或组合抗生素。我们建议通过设计一个易于使用的平台来克服这一问题，该平台基于微流控滴的微生物培养小型化，可以在可扩展和大规模并行设计中进行标记，跟踪和评估。标准的抗菌素药敏试验（AST）平台通常需要18-72小时才能产生药敏结果。本课题的主要研究目标是应用基于微流控的微流体技术在4小时内对铜绿假单胞菌临床分离株进行快速AST筛选。基于液滴的微流体技术是一种以每秒数千升的速度创建和分析皮升大小的体积的技术。这些液滴作为单个的微反应器，可以容纳单个细胞。在皮升大小的微反应器体积内分离单细胞和抗生素的离散组合的能力将允许以高分辨率和保真度快速和早期检测抗生素敏感性。该方法将能够检测和量化通常低于标准药物分析检测极限的单细胞亚群。为了实现这一目标，我们有两个具体的目标：(1)高通量最小抑制浓度筛选将通过条形码液滴库的多路分析进行；(2)集成光纤的微流控装置将被设计用于条形码和细胞信号的敏感检测，这将使该平台易于携带并易于适应临床环境。这种新颖的、可扩展的细菌高通量抗菌药物敏感性检测技术可以大大缩短脓毒症或尿路感染等细菌感染的诊断时间。

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.