A portable, easy-to-use paper-based biosensor for rapid in-field detection of fecal contamination on fresh produce farms.

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations are towards clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

Bacteroidales as a fecal contamination indicator in fresh produce industry: A baseline measurement.

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in California's Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The quantification of Bacteroidales concentration was performed using qPCR, revealing a notably low concentration (0-2.00 copies/cm2) in the commercial fields. To further enhance the applicability of our findings, we developed a user-friendly method for real-world fecal contamination risk assessment that seamlessly integrates with industry practices. Through the generation of heatmaps that visually illustrate varying risk levels across fields, this approach can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their land. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.

A loop-mediated isothermal amplification assay to detect Bacteroidales and assess risk of fecal contamination.

Fecal contamination of fresh produce from human and animal sources is a public health concern due to the risk of foodborne illnesses. The current standard laboratory procedures for microbiological analyses usually require an enrichment step that involves several hours. Molecular techniques such as polymerase chain reaction (PCR) have been used to directly detect pathogens from the samples, however, due to the low quantity of pathogen present and small volumes used for PCR, enrichment is usually required. Additionally, the need for specialized equipment and experienced workers hinders the use of these molecular techniques for field testing. Here, we developed a rapid risk-assessment assay for fecal contamination by targeting Bacteroidales using loop-mediated isothermal amplification (LAMP). The assay allows for naked-eye observation of reactions with as few as ∼8 copies of Bacteroidales per cm2 of the surface in the field. We evaluated this assay with complex field samples as well as on-site field studies. Our on-field studies demonstrated that the Bacteroidales LAMP assay enables us to easily and quickly (<50 min) assess the risk of fecal contamination from animal operations, with a concordance of 85.3% when compared to lab-based qPCR. These results were obtained without expensive equipment (when compared to standard laboratory procedures). These assays could be used to determine site-specific risk and help the decision-making process of fresh produce growers.

Paper-Based Biosensors for the Detection of Nucleic Acids from Pathogens.

Paper-based biosensors are microfluidic analytical devices used for the detection of biochemical substances. The unique properties of paper-based biosensors, including low cost, portability, disposability, and ease of use, make them an excellent tool for point-of-care testing. Among all analyte detection methods, nucleic acid-based pathogen detection offers versatility due to the ease of nucleic acid synthesis. In a point-of-care testing context, the combination of nucleic acid detection and a paper-based platform allows for accurate detection. This review offers an overview of contemporary paper-based biosensors for detecting nucleic acids from pathogens. The methods and limitations of implementing an integrated portable paper-based platform are discussed. The review concludes with potential directions for future research in the development of paper-based biosensors.

Engineering modeling frameworks for microbial food safety at various scales.

The landscape of mathematical model-based understanding of microbial food safety is wide and deep, covering interdisciplinary fields of food science, microbiology, physics, and engineering. With rapidly growing interest in such model-based approaches that increasingly include more fundamental mechanisms of microbial processes, there is a need to build a general framework that steers this evolutionary process by synthesizing literature spread over many disciplines. The framework proposed here shows four interconnected, complementary levels of microbial food processes covering sub-cellular scale, microbial population scale, food scale, and human population scale (risk). A continuum of completely mechanistic to completely empirical models, widely-used and emerging, are integrated into the framework; well-known predictive microbiology modeling being a part of this spectrum. The framework emphasizes fundamentals-based approaches that should get enriched over time, such as the basic building blocks of microbial population scale processes (attachment, migration, growth, death/inactivation and communication) and of food processes (e.g., heat and moisture transfer). A spectrum of models are included, for example, microbial population modeling covers traditional predictive microbiology models to individual-based models and cellular automata. The models are shown in sufficient quantitative detail to make obvious their coupling, or their integration over various levels. Guidelines to combine sub-processes over various spatial and time scales into a complete interdisciplinary and multiphysics model (i.e., a system) are provided, covering microbial growth/inactivation/transport and physical processes such as fluid flow and heat transfer. As food safety becomes increasingly predictive at various scales, this synthesis should provide its roadmap. This big picture and framework should be futuristic in driving novel research and educational approaches.

A Mechanistic Model for Bacterial Retention and Infiltration on a Leaf Surface during a Sessile Droplet Evaporation.

Evaporation of sessile droplets on the surface of plant leaves is a process that frequently occurs during plant growth as well as postharvest processes. Evaporation-driven internal flows within sessile droplets can transport microorganisms near the leaf surface, facilitating their adhesion to surface microstructures such as trichomes, and infiltration into available openings such as stomata and grooves. A mechanistic model for this retention and infiltration pathway was developed. Solution domain is a sessile droplet located on a leaf surface, as well as its surrounding gas. The model includes fluid flow within the droplet and gas phases, gas-water interface tracking, heat transfer, transport of vapor in gas, and transport of sugar and bacteria within water. The model results are validated based on available literature data and experimental images. The results showed that a hydrophilic surface would promote bacterial retention and infiltration. Evaporation-driven flows increase concentration of bacteria around or inside microstructures at the leaf surface, facilitating their adhesion and infiltration. Larger microstructures having wider spacing between them increased the retention. A higher evaporation rate led to higher infiltration. Chemotaxis toward nutrients at the leaf surface and random motility were shown to decrease the retention and infiltration during evaporation.

Improving vascular imaging with co-planar mutually guided photoacoustic and diffuse optical tomography: a simulation study.

Diffuse optical tomography (DOT) and photoacoustic tomography (PAT) are functional imaging modalities that provide absorption coefficient maps of the tissue. Spatial resolution of DOT is relatively low due to light scattering characteristics of the tissue. On the other hand, although PAT can resolve regions of different absorptions with a high spatial resolution, measuring the absolute value of optical absorptions using PAT is challenging due to unknown light fluence distribution in the tissue. Development of image guidance techniques using a priori information of imaging target structure has been shown to increase the accuracy of DOT. PAT is one such method that can be used as a complementary modality to serve as a guide for DOT image reconstruction. On the other hand, estimated fluence map provided by DOT can be used to quantitatively correct PAT images. In this study we introduce a mutually-guided imaging system for fast and simultaneous optical and photoacoustic measurements of tissue absorption map, where DOT is guided by the PAT image and vice versa. Using the obtained absorption map of the tissue, we then estimate the tissue scattering map. We conducted this study using a series of simulations on digital phantoms and demonstrated the effectiveness of the proposed method.

Mechanistic modeling of light-induced chemotactic infiltration of bacteria into leaf stomata.

Light is one of the factors that can play a role in bacterial infiltration into leafy greens by keeping stomata open and providing photosynthetic products for microorganisms. We model chemotactic transport of bacteria within a leaf tissue in response to photosynthesis occurring within plant mesophyll. The model includes transport of carbon dioxide, oxygen, bicarbonate, sucrose/glucose, bacteria, and autoinducer-2 within the leaf tissue. Biological processes of carbon fixation in chloroplasts, and respiration in mitochondria of the plant cells, as well as motility, chemotaxis, nutrient consumption and communication in the bacterial community are considered. We show that presence of light is enough to boost bacterial chemotaxis through the stomatal opening and toward photosynthetic products within the leaf tissue. Bacterial chemotactic ability is a major player in infiltration, and plant stomatal defense in closing the stomata as a perception of microbe-associated molecular patterns is an effective way to inhibit the infiltration.

A Study on actinobacterial diversity of Hampoeil cave and screening of their biological activities.

Caves are oligotrophic, dark and less-explored environments and are considered as sources of promising microbial strains in biotechnology. Hampoeil Cave is located in massive dolomite with thin bedded limestone in northwestern of Iran. In an isolation and screening program, various samples from soil, water, floor, wall and ceiling of Hampoeil cave and its invertebrates were collected. Four various treatments and 10 different isolation media were used for the isolation of the actinobacteria. Screening of the isolates for antimicrobial activity against 10 bacteria and fungi, 5 hydrolytic enzymes production and resistance to 5 heavy metals have been performed. Among 33 various samples, 76 actinobacteria from various genera, including Streptomyces, Micromonospora, Micrococcus, Kocuria and Corynebacterium were isolated. Eighty percent of the strains had one of the studied hydrolytic enzyme activity. At least one type of antimicrobial activity was seen in 25.3% of the isolates. Resistance to one metal or more was seen in 26.32% of the isolates. The ratio of rare-actinobacteria in the oligotrophic samples to enriched samples is 20% more than Streptomyces. Percentage of strains with the highest activity in esterase, amylase, DNase, protease or lipase activity that were isolated from organic-rich environmental samples were 100, 100, 100, 82 and 82%, respectively. Also, 26.32% of the actinobacterial isolates resisted to heavy metals. It was concluded that Hampoeil cave is a good source in finding cave-living actinobacteria potent in producing hydrolytic enzymes and bioremediation.