Abstract
The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared
Highlights
Listeria monocytogenes (Lm) is a foodborne pathogen found in a variety of foods; outbreaks of listeriosis have been linked to the consumption of contaminated raw milk, ready-to-eat deli meats, cantaloupes, hot dogs, smoked fish, mushrooms, eggs, soft cheeses, frozen vegetables, packaged salads, ice cream, caramel apples, and bean sprouts [1]
By using BacLightTM staining in conjunction with flow cytometry and comparing it with direct viable counts, the membrane integrity of viable but non-culturable (VBNC) Lm populations can be demonstrated, even as plate-count cultivability was reduced to nearly 0% [20]
Esterase activity produces a fluorescent product a Ethidium Monoazide- and Propidium Monoazide-stained real-time Polymerase Chain Reaction. b Ethidium monoazide (EMA) and propidium monoazide (PMA) are both DNA-binding agents used with quantitative PCR for Direct Viable Count. c 5-cyano-2,3-ditolyl tetrazolium chloride—40,6-diamidino-2-phenylindole double staining. d carboxy-fluorescein diacetate staining
Summary
Listeria monocytogenes (Lm) is a foodborne pathogen found in a variety of foods; outbreaks of listeriosis have been linked to the consumption of contaminated raw milk, ready-to-eat deli meats, cantaloupes, hot dogs, smoked fish, mushrooms, eggs, soft cheeses, frozen vegetables, packaged salads, ice cream, caramel apples, and bean sprouts [1]. Any Lm contaminated RTE products are considered adulterated in inter-state commerce if they contain Lm or come into direct contact with a food contact surface that is contaminated with Lm. If RTE food products are exposed to the environment after a lethal processing step, they are required to adhere to one of three Listeria control alternatives to comply with USDA-FSIS guidelines. If RTE food products are exposed to the environment after a lethal processing step, they are required to adhere to one of three Listeria control alternatives to comply with USDA-FSIS guidelines These alternatives include using a post-lethality treatment, antimicrobial agents or processes, or strict sanitation standards. The inability of VBNC Lm cells to grow on traditional plating media can lead to false negatives during routine testing of products or food contact surfaces.
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