Microfluidic platform for stress-induced rapid antibiotic susceptibility testing

描述(申请人提供)：目前检测抗生素敏感性的标准方法是基于细菌在有抗生素的情况下增殖的能力，因此这些技术耗时、昂贵且不敏感，特别是在评估生长缓慢的生物体时。为了开发一种真正快速的药敏试验，人们必须绕过增长的需要。我们正在开发一种微流体测试，它可以询问细胞在机械和/或可溶应激源存在的情况下对抗生素的反应，从而将结果的时间降至最低。该假说的核心是，通过对细胞进行筛选，我们可以 诱导细胞修复过程和相关的生化途径。这些途径通常是抗生素的靶标(例如，细胞壁生物合成、蛋白质合成、DNA转录)。如果抗生素阻碍了这些修复过程，细胞将在继续使用抗生素的情况下死亡 压力。我们假设，在抗生素存在的情况下监测应激状态下的细胞死亡可以在超快的时间范围内提供表型信息，从而使医生能够更快地做出适当的抗生素治疗选择。我们预计，这种方法将补充现有的基于分子诊断的快速检测(例如，聚合酶链式反应)，因为它将提供一种传递表型信息的低成本快速方法。虽然基因检测为流行病学研究提供了准确的信息，但高昂的试剂成本、相对较高的操作技能要求以及有限的临床实用性继续限制了广泛的常规使用。此外，分子诊断在非无菌样本(例如，多菌样本)中存在大量的假阳性和不可接受的性能。由于我们的方法可以自动化，并基于表型变化，我们认为它作为一种常规临床诊断方法是优越的，它为医生提供治疗患者所需的信息，即使用什么抗生素来杀死感染的病原体。该方法可以是多重的(多种抗生素和多种微生物)，并可以与目前的细菌鉴定方法相结合，因此它有可能成为一种新的诊断系统的基础，该系统可以在比目前可能的时间短得多的时间内为临床医生提供鉴定和抗生素敏感性曲线。该方法还为研究压力如何增强抗生素的效果开辟了新的途径。一旦开发出来，这项技术还可以作为一种新的抗生素候选药物的快速筛选技术。

DESCRIPTION (provided by applicant): Current standard methods for detecting antibiotic susceptibility are based on the ability of the bacteria to proliferate in the presence of antibiotis, and thus these techniques are time-consuming, costly, and insensitive, particularly for evaluation of slow-growing organisms. To develop a truly rapid susceptibility test, one must circumvent the need for growth. We are developing a microfluidic test that interrogates the response of cells to antibiotics in the presence of mechanical and/or soluble stressors and thereby minimizes the time to results. The core of the hypothesis is that by straining the cell, we induce the cellular repair processes and associated biochemical pathways. These pathways are often targets of antibiotics (e.g., cell wall biosynthesis, protein synthesis, DNA transcription). f the antibiotic hinders those repair processes, the cell will die under the continued application of stress. We posit that monitoring cell death under stress in the presence of antibiotics can provide phenotypic information in an ultra-rapid time frame allowing physicians to make appropriate antibiotic treatment choices sooner. We envisage that this methodology would complement the existing rapid tests based on molecular diagnostics (e.g., PCR) because it would provide a low-cost rapid method that delivers phenotypic information. While genetic tests provide precise information for epidemiological studies, the high reagent costs, relatively high operator skills required, and limited clinical utility continue to limit widespread routine use. Furthermore, the molecular diagnostics suffer from a high number of false positives and unacceptable performance in non-sterile specimens (e.g., polymicrobial samples). As our method could be automated and is based on phenotypic changes, we believe it is superior as a routine clinical diagnostic providing physicians with the information they need to treat their patients, namely what antibiotic to use to kill the infecting pathogen. The method can be multiplexed (multiple antibiotics and multiple organisms) and can be integrated with current bacterial identification methodologies, thus it has the potential to be the basis of a new diagnostics system that rapidly provides clinicians with both identification and antibiotic susceptibility profiles in a timeframe that is much shorter than is currently possible. The method also opens new avenues of research into how stress can potentiate the effects of antibiotics. Once developed, the technique could also be used as a rapid screening technology for new antibiotic drug candidates.