Microfluidic droplet application for bacterial surveillance in fresh-cut produce wash waters

Abstract

Foodborne contamination and associated illness in the United States is responsible for an estimated 48 million cases per year. Increased food demand, global commerce of perishable foods, and the growing threat of antibiotic resistance are driving factors elevating concern for food safety. Foodborne illness is often associated with fresh-cut, ready-to-eat produce commodities due to the perishable nature of the product and relatively minimal processing from farm to the consumer. The research presented here optimizes and evaluates the utility of microfluidic droplets, also termed ultra-miniaturized bioreactors, for rapid detection of viable Salmonella enterica ser. Typhimurium in a shredded lettuce wash water acquired from a major Mid-Atlantic produce processing facility (denoted as Producer) in the U.S. Using a fluorescently-labeled anti-S. Typhimurium antibody and relative fluorescence intensities, paired with in-droplet incubation, S. Typhimurium was detected and identified with 100% specificity in less than 5 h. In initial optimization experiments using S. Typhimurium-spiked sterile water, the relative fluorescence intensity of S. Typhimurium was approximately two times that of the observed relative intensities of five non-S. Typhimurium negative controls at 4-h incubation in droplets containing Rappaport-Vasiliadis (RV) broth at 37˚C: relative fluorescence intensity for S.

Emerging Bioanalytical Devices and Platforms for Rapid Detection of Pathogens in Environmental Samples

“…Salmonella at the single-cell level could be specifically detected by employing a novel microdroplet approach [ 176 ]. In addition to these, microfluidic droplet technology has the potential to do surveillance for infectious pathogens such as bacterial in fresh-cut wash water [ 177 ], a virus such as SARS-CoV-2 [ 178 ], and automated detection and monitoring of chemical and biological warfare agents [ 179 ].…”

Microfluidic devices for studying bacterial taxis, drug testing and biofilm formation

“…Biomolecular techniques have been applied within this system, mainly in order to detect the presence of bacteria in samples. For example, using fluorescent antibodies against Salmonella enterica serovar typhimurium in freshcut produce water allowed the detection of contamination and avoided foodborne contamination (Harmon et al ., 2020 ). Fluorescence resonance energy transfer (FRET) based on RNA has also been used to analyse samples.…”

Investigation of Different Free Image Analysis Software for High-Throughput Droplet Detection

“…The analysis has been implemented in different types of image data, from single static image up to real-time data, either by bright-field or fluorescence microscopy . This approach has been used for a wide range of experiments, such as bacterial surveillance of foodborne contamination, screening of specific substrates, single-cell analysis, and detecting viable bacteria or viruses (e.g., SARS-CoV-2). , Image-based droplet analysis (IDA) often requires specific skills in programming that are not widely available in non-specialist laboratories. Most of the published articles in droplet detection use scripted programs, such as Circular Hough Transform in Python programming language, Mathematica, , Scikit-image in Python, Image Processing Toolbox from MATLAB, OpenCV and Keras in Python, and OpenCV in C++ .…”

“…Most of the published articles in droplet detection use scripted programs, such as Circular Hough Transform in Python programming language, Mathematica, , Scikit-image in Python, Image Processing Toolbox from MATLAB, OpenCV and Keras in Python, and OpenCV in C++ . There are some user-friendly software that may be used for droplet microfluidic image analysis, such as the Zen imaging program and NIS-Elements from NIKON . However, these kinds of programs are only commercially available.…”