Department of Food Science and Technology, University of California, Davis, Davis, California, United States of America.
Department of Biological and Agricultural Engineering, University of California, Davis, Davis, California, United States of America.
PLoS One. 2020 Jun 5;15(6):e0233853. doi: 10.1371/journal.pone.0233853. eCollection 2020.
Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.
快速检测细菌病原体是食品和环境样本（如灌溉水）的一项关键未满足需求。作为《食品安全现代化法案》（FSMA）的一部分，《农产品安全法》规定了对水中普通大肠杆菌存在情况进行测试的若干要求，但目前可用于测试的方法（EPA M1603）需要指定的多个菌落验证和经过高度培训的人员来进行这些测试。本研究旨在评估噬菌体诱导的细菌裂解，使用定量图像分析在 8 小时内实现低浓度大肠杆菌的快速检测。本研究旨在开发一种简单但高度敏感和特异的方法，以检测复杂基质中的目标细菌。在研究中，首先在胰蛋白酶大豆肉汤（TSB）中富集大肠杆菌细胞，然后用 T7 噬菌体诱导裂解、浓缩、染色和荧光成像。进行图像分析，包括图像预处理、图像分割和细胞形态特征（面积、偏心率和半峰全宽）的定量分析。使用现实基质（包括模拟新鲜农产品洗涤水、椰子水和菠菜洗涤水）进行的挑战实验表明，该方法可用于食品加工设施中发生的情况。结果表明，被 T7 噬菌体裂解的大肠杆菌细胞表现出明显（P < 0.05）更高的胞外 DNA 释放、改变的细胞形状（从杆状变为圆形）和扩散荧光信号强度。使用这种生物传感策略，在 8 小时内可以在实验室培养基和复杂基质中检测到 10 CFU/ml 的大肠杆菌，灵敏度达到 10 CFU/ml。该基于噬菌体的生物传感策略可实现细菌的快速检测，适用于食品系统的分析。此外，该测定中涉及的步骤可以自动化，以便在无需大量资源的情况下在食品设施中检测目标细菌。
