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

对由细菌引起的感染性疾病进行有效治疗的根本挑战是快速鉴定抗微生物剂耐药性和单一或组合药物治疗的功效，包括防止细菌生长所需的最低浓度或组合的抗生素。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体液滴的微生物培养的小型化，可以在可扩展和大规模并行设计中标记，跟踪和评估。标准的抗菌药物敏感性测试（AST）平台通常需要18-72小时来生成敏感性结果。该提案的主要研究目标是应用基于液滴的微流体技术在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技术，其中皮升大小的体积以每秒数千的速率产生和分析。这些液滴作为单独的微反应器，可以包含单个细胞。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定量通常低于标准药物测定检测限的单细胞亚群。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与条形码液滴库的多重测定来进行，以及（2）具有集成光纤的微流体装置将被设计用于条形码和细胞信号的灵敏检测，这将使平台能够便携并易于适应临床环境。这种用于细菌高通量抗菌药物敏感性测试的新型可扩展技术可以大大缩短细菌感染（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.