CAREER: A Multi-phase Biosensing Approach towards Point-of-Care Evaluation of Pseudomonas aeruginosa Virulence in Infected Chronic Wounds

对抗生素有耐药性的细菌感染是医疗费用（每年46亿美元）和导致死亡的治疗并发症的主要来源。美国每年发生超过280万例耐药感染，尽管抗生素广泛使用，但仍有超过3.5万人死亡，主要是因为抗生素对耐药细菌无效。对于免疫系统较弱的患者，并发症或死亡的可能性增加。细菌形成一种叫做生物膜的复杂结构，保护自己免受抗生素治疗。这些生物膜是通过一种叫做群体感应的细菌细胞通信策略形成的。该项目旨在研究群体感应如何通过一种廉价、快速、灵活的传感器来跟踪，这种传感器可以提供细菌生长速度和生物膜形成速度的信息。在这项研究中开发和使用的传感器是独一无二的，因为它的灵活性和测量与细菌生长有关的电和化学活动的能力。该项目的长期研究目标是实现快速（不到5分钟）确定感染，以便在最合适的时间提供正确的抗生素和剂量。重要的是，这种传感方法也可以用于其他应用，如水过滤和农业。该项目的教育目标是支持代表性不足的学生，特别是退伍军人和非传统学生的研究和研究生教育。这些学生在参与传统的本科研究经历时往往面临独特的障碍，例如工作、家庭或军事承诺。该项目将通过课程作业、研究和研讨会经验的结合来应对这些挑战，旨在让学生接触和参与新的想法和工作机会。该项目的主要动机是开发一种快速测量慢性非愈合伤口细菌感染的策略，以抑制抗生素耐药性/耐受性，这既是一个重要的社会问题，也是一个基本的科学兴趣。美国每年发生280多万例抗微生物药物耐药性感染，尽管抗生素广泛可用，但仍有超过3.5万人死亡，这在很大程度上是因为它们对耐药菌株和生物膜无效，尤其是（但不仅限于）免疫功能低下的患者。该项目的研究目标是利用浓度依赖的群体感应（QS）分子来量化与细菌病原体毒力进展相关的生物膜形成中的关键转变。本项目重点研究铜绿假单胞菌的生物膜形成和毒力，作为慢性伤口中常见的其他细菌病原体的模型系统。本项目采用无纺布纳米纤维复合电极设计，在电化学阻抗谱和伏安实验中，通过pyocyanin和3OC12HSL（用于介导毒力的QS分子）的检测和定量来量化毒力的进展。本工作将产生以下科学贡献：1)细菌浓度（P. aeruginosa）、QS分子浓度（Pyocyanin和AHLs - 3OC12HSL）与生物膜发育阶段之间的直接定量关系；2)实现一种灵活的、可调的伏安传感器，该传感器提供对氧化还原物质的高度敏感和特定的电化学检测，同时由于其生物纺织品设计，它很容易融入可穿戴织物或伤口敷料中；3)纳米纤维复合适体传感器的功能化，能够产生可量化的电化学信号，大大降低了检测限（LOD），提高了特异性。这项工作解决了量化与细菌负荷（生物负担）进展相关的物种特异性信号分子的关键挑战。这项工作的长期重要性在于增加了对更有效治疗时机的了解，同时降低了产生耐药性的风险。对于任何可能被这些细菌感染的生物，包括植物、动物和人类，了解细菌病原体可能在毒力上进步的精确时刻是很重要的。该项目由生物传感计划和促进竞争研究的既定计划（EPSCoR）共同资助。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。

Infections from bacteria that are resistant to antibiotics are a major source of healthcare costs ($4.6B annually) and treatment complications that lead to death. More than 2.8 million drug-resistant infections occur each year in the US, with more than 35,000 deaths despite widespread availability of antibiotics, mostly because they are not effective against drug-resistant bacteria. The likelihood of complication or death is increased for patients with weakened immune systems. The bacteria form complex structures called biofilms that protect themselves from antibiotic treatment. These biofilms are developed through a bacteria cell communication strategy called quorum sensing. This project seeks to study how quorum sensing can be tracked by an inexpensive, rapid, and flexible sensor that provides information on how quickly bacteria are growing and how fast biofilms are being developed. The sensor developed and used in this study is unique because of its flexibility and ability to measure electrical and chemical activity related to bacteria growth. The long-term research goal of this project is to make possible the fast (less than 5 minutes) determination of infection so that the correct antibiotics and dosage can be delivered at the most opportune time. Importantly, this sensing approach can also be used in other applications, like water filtering and agriculture. The educational goal of this project is to support research and graduate education for underrepresented students, especially veterans and non-traditional students. These students often face unique obstacles to participating in traditional undergraduate research experiences, such as work, family, or military commitments. This project will address these challenges through a combination of coursework, research, and workshop experiences designed to expose and engage students in new ideas and job opportunities.The primary motivation for this project is the development of a strategy to quickly measure bacterial infections in chronic, non-healing wounds for the inhibition of antibiotic resistance/tolerance, which is both an important societal problem and of fundamental scientific interest. More than 2.8 million antimicrobial-resistant infections occur each year in the US, with more than 35,000 deaths despite widespread availability of antibiotics in large part because they are ineffective against resistant strains and biofilms, especially but not only in immune compromised patients. The research objective of this project is to leverage concentration-dependent quorum sensing (QS) molecules to quantify key transitions in biofilm formation that relate to the progression of virulence in bacterial pathogens. This project focuses on Pseudomonas aeruginosa biofilm formation and virulence, as a model system for other bacterial pathogens commonly found in chronic wounds. This project uses a nonwoven nanofiber composite electrode design in electrochemical impedance spectroscopy and voltammetric experiments to quantify virulence progression via pyocyanin and 3OC12HSL (QS molecules used to mediate virulence) detection and quantification. The following scientific contributions will result from this work: 1) A directly quantifiable relationship between bacterial concentration (P. aeruginosa), QS molecule concentration (Pyocyanin and AHLs - 3OC12HSL), and stage of biofilm development; 2) Enablement of a flexible, tunable voltametric sensor that offers highly sensitive and specific electrochemical detection of redox species while being easily incorporated into wearable fabrics or wound dressings given its bio-textile design; and 3) The functionalization of nanofiber composite aptasensors, enabling generation of quantifiable electrochemical signals to greatly reduce the Limit of Detection (LOD) and improve specificity. This work addresses the critical challenge of quantifying species-specific signaling molecules associated with progression of bacterial load (bioburden). The long-term importance of this work is increased understanding of more effective treatment timing, while reducing the risk for development of drug resistance. Understanding the precise moments in which a bacterial pathogen may be advancing in virulence is important for any organism that may be infected by these bacteria, including plants, animals, and humans.This project is jointly funded by the Biosensing Program and the Established Program to Stimulate Competitive Research (EPSCoR).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.