Salmonella, Escherichia coli O157, and Shigella flexneri are typical foodborne pathogens in ground beef, which can cause severe infection even when present as a single cell. Flow cytometry (FCM) methods are widely applied in the rapid detection of pathogens in food products. In this study, we report an FCM-based method for detecting single cells of Salmonella, E. coli O157, and S. flexneri in 25 g ground beef samples. We fluorescently labeled specific antibodies that could effectively identify bacterial cells, prepared single-cell samples by serial dilution, and optimized the pre-enrichment time. The results showed that 7 h of pre-enrichment is appropriate for sensitive single-cell detection by FCM. Finally, we evaluated this method in artificially contaminated and retail beef samples. This study outlines a novel highly sensitive FCM-based method to detect Salmonella, E. coli O157, and S. flexneri in beef samples within 8 h that can be applied to the rapid and multiplexed detection of foodborne pathogens.
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