Study of bacterial clustering and chemotaxis using novel optical tweezers

使用新型光镊研究细菌聚类和趋化性

• 批准号: 8773455
• 负责人: Zhigang Chen
• 金额: $ 35.11万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8773455
• 关键词: Academic Research Enhancement Awards; Accounting; Address; Adhesives; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Drug Resistance; Biological Assay; Cell Aggregation; Cell Communication; Cells; Characteristics; Chemotaxis; Clinical; Colistin; Culture Media; Data; Databases; Dependence; Development; Dimensions; Disease; Dose; Drug Exposure; Drug Interactions; Drug resistance; Environment; Exhibits; Exposure to; Frequencies; Goals; Health; Human; Image; Infection; Knowledge; Label; Lasers; Life; Measurement; Measures; Mediating; Melilotus; Methods; Microbe; Microbial Biofilms; Microscopy; Mission; Monitor; Motion; Nature; Nosocomial Infections; Optics; Outcome; Pharmaceutical Preparations; Polysaccharides; Population; Pseudomonas aeruginosa; Public Health; Research; Research Training; Resistance development; Response to stimulus physiology; Role; Science; Shapes; Silicon Dioxide; Sinorhizobium meliloti; Soil; Stimulus; Stretching; System; Techniques; Technology; Testing; Translating; United States; War; Water; bacterial resistance; cell motility; design; drug development; extracellular; graduate student; improved; laser tweezer; microorganism; novel; optical traps; pathogen; response; retinal rods; technique development

DESCRIPTION: Antibacterial resistance is a serious and growing public health threat. In the United States, each year at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23,000 people die as a direct result of these infections. Pseudomonas aeruginosa, an antibiotic resistant bacterium commonly found in soil and water, has been recognized as one of the most commonly-isolated nosocomial pathogen accounting for over 10 percent of all hospital-acquired infections. Understanding bacterial extracellular interaction of P. aeruginosa and its response to drug exposure is thus highly desired and important. The long-term goals of our research are (1) to develop an effective optical tweezers technique that can be used to quantify the bacterial- bacterial cell interaction and the stimulus-response relation; and (2) to build up a bacterial chemotaxis database that will be useful for understanding antibacterial resistance and for drug development. The objective of the current application is to design and set up an optical tweezers for manipulating especially rod-shaped bacteria such as a P. aeruginosa cell, and for investigating its aggregation with neighboring cells and interaction with different stimuli (drugs). We hypothesize that a rod-shaped bacterium can be stably trapped in three dimensions yet examined in an observing plane by a "tug-of-war" optical tweezers (which can apply much stronger pulling forces than that in conventional tweezers), and its clustering with neighboring cells and response to a stimulus can be quantitatively measured. Guided by strong preliminary data, these hypotheses will be tested through the following Specific Aims: (1) Design and set up an optical "tug-of-war" tweezers system that allows for stable in- plane trapping and stretching of rod-shaped bacteria. Such a system with dark-field microscopy enables high- contrast imaging and direct monitoring the bacterial motility without the need of fluorescent labeling. (2) Employ the "tug-of-war" tweezers to study bacterial extracellular interaction with two specific examples. The first is adhesive Sinorhizobium meliloti bacterial cells grown under different growth media (say PYE vs. TY), and the second is P. aeruginosa bacterial clusters prepared with or without Pel polysaccharide. This novel tweezers with tunable pulling forces will be used to characterize stable and unstable cell aggregations in these two cases, thus identifying the role of a particular medium in bacterial-bacterial cell interaction and cellular aggregation. (3) Employ the "tug-of-war" tweezers to study bacteria-drug interaction. By varying the strength (doses) and/or the frequency of exposure to a drug (e.g., Colistin), the motion of a P. aeruginosa cell in the optical trap due to bacteria-drug interaction can be quantified through direct chemotactic force measurement. Successful completion of these studies will help us understand bacterial extracellular interaction in biofilm formation and bacterial resistance to drug exposure. Thus, it will have significant clinical impact

描述：抗菌抗性是一种严重且日益严重的公共卫生威胁。在美国，每年至少有200万人感染了对抗生素耐药性的细菌，至少有23,000人死亡，这是这些感染的直接结果。铜绿假单胞菌是一种在土壤和水中常见的抗生素耐药细菌，已被认为是最常见的医生病原体之一，占所有医院获得的感染的10％以上。因此，了解铜绿假单胞菌的细菌细胞外相互作用及其对药物暴露的反应是高度期望和重要的。我们研究的长期目标是（1）开发有效的光学镊子技术，该技术可用于量化细菌细菌细胞相互作用和刺激 - 反应关系； （2）建立一个细菌趋化数据库，该数据库将有助于理解抗菌耐药性和药物开发。当前应用的目的是设计和建立一个光学镊子，以操纵，尤其 并与不同的刺激（药物）相互作用。我们假设可以稳定地将棒状细菌稳定地捕获在三个维度中，但通过“拖船”光学镊子在观察到的平面中检查（在常规镊子中，可以施加强大的拉力），并且它与相邻细胞的聚类和对刺激的聚类和响应的响应能够定量测量。在强大的初步数据的指导下，这些假设将通过以下特定目的进行检验：（1）设计并建立了光学的“拖船”镊子系统，该系统允许稳定的杆形细菌捕获和拉伸杆状细菌。具有深色场显微镜的系统可实现高对比度成像并直接监测细菌运动性，而无需荧光标记。 （2）用“拖船”镊子使用两个特定示例研究细菌细胞外相互作用。第一个是在不同生长培养基下生长的粘合性sinorhizobium meliloti细菌细胞（例如Pye vs. Ty），第二个是铜绿假单胞菌细菌簇，以有或没有PEL多糖制备。在这两种情况下，这种具有可调拉力的新型镊子将用于表征稳定且不稳定的细胞聚集，从而确定特定培养基在细菌 - 细菌细胞相互作用和细胞聚集中的作用。 （3）使用“拖船”镊子研究细菌 - 药物相互作用。通过改变暴露于药物的强度（剂量）和/或频率（例如colistin），可以通过直接的趋化力测量来量化由于细菌 - 药物 - 药物相互作用而导致的铜绿假单胞菌细胞在光学陷阱中的运动。这些研究的成功完成将有助于我们了解生物膜形成中细菌细胞外相互作用和对药物暴露的细菌耐药性。因此，它将具有重大的临床影响