Abstract
Rapid detection of bacteria in water and food samples is a critical need. The current molecular methods like real-time PCR can provide rapid detection after initial enrichment. However, these methods require significant preparation steps, specialized facilities to reduce contamination, and relatively expensive reagents. This study evaluates a novel approach for detecting bacteria based on imaging of bacteriophage amplification upon infection of the target host bacteria to mitigate some of these constraints and improve the specificity of discriminating live vs. dead bacteria. Thus, this research leverages the natural ability of lytic bacteriophages to rapidly amplify their genetic material and generate progeny phages upon infecting the host bacterium. This study uses a nucleic acid staining dye, a conventional fluorescence microscope, and quantitative image analysis for imaging the amplification of bacteriophages. The sensitivity and assay time for imaging-based quantification of phage amplification for detecting Escherichia coli were compared with RT-PCR and the standard plaque-forming assay for detection phage amplification in model systems, including coconut water and spinach wash water. The results demonstrate that the imaging approach matches both the sensitivity and speed for detecting E. coli using the RT-PCR method without requiring isolation of nucleic acids, expensive reagents, and specialized facilities. The quantitative imaging results demonstrate the detection of 10 CFU/ml of E. coli in coconut water and simulated spinach wash water with a chemical oxygen demand (COD) of 3,000 ppm within 8 h, including initial enrichment of the bacteria. In summary, the results of this study illustrate a novel phage amplification-based approach for detecting target bacteria in complex food and water samples using simple sample preparation methods and low-cost reagents.
Highlights
Detection of pathogens and indicator bacteria in food and water systems is one of the essential tools to manage the risk of foodborne outbreaks
No significant increase in the phage particles per image was observed with an extended incubation time greater than 2 h for the spinach wash water samples. For both coconut water and spinach wash water samples without E. coli inoculation, there was no significant increase in the number of phage particles after 4 h of infection. These results demonstrate that the imaging method can detect amplification of phages in coconut water and spinach wash water, and 10 CFU/ml of E. coli in coconut water and spinach wash water can be detected with an infection period of 2 and 1 h, respectively, using the imaging approach
The number of the phage particles in the fluorescence images can be enumerated using image processing software, which allows the rapid enumeration of phage amplification upon infection of the target bacteria
Summary
Detection of pathogens and indicator bacteria in food and water systems is one of the essential tools to manage the risk of foodborne outbreaks. Common pathogenic bacteria, including Escherichia coli, Listeria monocytogenes, and Salmonella spp., have been reported as the major causes of bacterial foodborne illness outbreaks (Bintsis, 2017). Among diverse bacteria associated with foodborne illnesses, Escherichia coli are a Gram-negative bacterium typically found in the enteric tract of humans and warm-blooded animals (Kaper et al, 2004). Even though most E. coli strains are not pathogenic, E. coli presence is an indicator for fecal contamination and, their presence indicates the potential contamination of water and food supplies by other pathogenic bacteria (Krumperman, 1983; Molina et al, 2015; Yang et al, 2017). Improving detection of E. coli can enable monitoring of general sanitation and hygiene in food environments and enable detection of specific pathogens in food and water systems
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