Survival of Listeria monocytogenes and Salmonella on surfaces found in the dry packinghouse environment and effectiveness of dry-cleaning processes on pathogen reduction

Salmonella and Listeria monocytogenes are important foodborne pathogen involved in foodborne outbreaks linked to the consumption of produce and fresh fruits. Contamination of fresh produce is problematic since these products are usually consumed without heating. To avoid contamination events, the packing industry must rely on rigorous sanitation practices including in the dry areas of the packinghouse. This study proposes to develop informational tools regarding the die off rates of the pathogens exposed to matric stress. Experiments will reassemble packinghouse conditions. Dried planktonic cells and dried biofilms formed by the packing house microbiota and L. monocytogenes or Salmonella will simulate packing industry surfaces and environmental conditions. Experiments will investigate the conditions that favor transition of the planktonic cells present on surfaces to form attached embedded communities or biofilms, and formation of VBNC cells. Inactivation studies will provide data for best practices regarding dry cleaning/sanitation methodology in the packing house and elimination of the foodborne pathogens. These findings will be validated for practical use in the packing house, in a large pilot plant study to reduce the load of microorganisms on equipment and produce. Results from this study will provide improved pathogen control in addition to basic good agricultural practices.

The percentage of produce commodities contaminated with foodborne pathogens has increased in the past decade. These products are often consumed raw or with minimal processing or preparation, which contributes to the risk of food-borne disease. Salmonella and Listeria monocytogenes are two bacterial foodborne pathogens of concern for the produce industry. A packing company issued in 2016 the first recall of certain stone fruits because of concern about contamination with Listeria monocytogenes. Another 2020 interstate recall of peaches involved contamination with Salmonella. Additionally, Salmonella and L. monocytogenes are significant pathogens for other commodities such as leafy greens, tomatoes, cantaloups and mango. In the absence of any practices that prevent pathogen survival on the product, the exposure of the fresh produce to contaminated postharvest contact surfaces will increase the likelihood of contamination and foodborne outbreaks. In certain areas of the packinghouse, FSMA grants the use of dry cleaning techniques such as vacuuming or scrapping followed by the use of dry sanitizers for food contact surfaces, or Zone 1 areas. The use or presence of water could be a significant risk for foodborne pathogen growth, cross-contamination, and dissemination in the facility. However, microorganisms have multiple mechanisms for microbial adaptation and survival in dry conditions. Microbial survival in low moisture and desiccation conditions present in the packinghouse can lead to cross-contamination. In addition, adverse dry conditions could force bacteria to enter an inactive physiological state, such as viable but nonculturable (VBNC). The presence of VBNC cells has food safety implications since these microorganisms may be undetected during routine sampling for environmental monitoring. In the proposed research we aim to determine survival of dry surface-associated planktonic and biofilms of L. monocytogenes and Salmonella grown in combination with background microflora on surfaces typically found in the packinghouse. In Objective 1, pathogen die off rates will be determined for mixed planktonic and biofilms dried on packinghouse surfaces. Multiple analytical methods of analysis (plate count, microscopy, real-time PCFR) for the dry films will avoid false negative results (e.g., low number in the mixed film and possibly injured cells that are not recovered for the quantification assay) and determine the effects of matric stress on the formation of VBNC microorganisms. Experiments will investigate the conditions that favor transition of the planktonic cells present on surfaces to form attached embedded communities or biofilms (the main question is when and how a dried cell becomes embedded biofilm). Inactivation studies in Objective 2 will provide data for best practices regarding dry cleaning/sanitation methodology in the packing house and elimination of the foodborne pathogens. Objective 3, a pilot plant study, will validate findings from Objectives 1 and 2 for practical use in the packing house, to reduce the load of microorganisms on equipment and produce. Together the laboratory data and pilot plant trials can identify management practices associated with reduced pathogen presence in the dry environment. This project is intended for CPS 2021 Research Priorities-Part 3a. Packing, Cooling and Storage.

1. Determine die-off curves of dry surface-formed biofilms and dried planktonic cells of Listeria monocytogenes and Salmonella (in single and cultures with environmental isolates) on surfaces commonly found in the packinghouse. 

2. Test the efficacy of commonly used dry cleaning and sanitation methods on dry surface biofilms and desiccated planktonic cells of L. monocytogenes and Salmonella on surfaces found in the packinghouse. 

3. Validate, in pilot plant trials*, the laboratory die-off rates of the surface-associated microflora isolated from the dry areas of the packinghouse, and the inactivation through dry cleaning/sanitation methodology. *Objective 3 was modified to remove pilot plant trials; instead, to determine the specific variables that are associated with the microflora on dry surfaces, two large packinghouse facilities in the Central Valley of California were sampled over the summer 2022, before and after sanitation.

Listeria monocytogenes is a pathogen of concern in dry environments since its survival rates were comparable with Salmonella viability on dry surfaces. L. monocytogenes and Salmonella can survive for extended times (days and weeks) in relative humidity as low as 25%. Microbial viability is influenced by the physiological state: e.g., if cells dry on surfaces as free-floating planktonic cells or in the form of biofilms, as polymer-embedded groups of cells. Most notable is the long persistence of more than a month of L. monocytogenes biofilms at 30 32°C and 70-72% RH, a combination of environmental conditions frequently found in the summer in the packinghouse. The dry environment by itself was deleterious only for microorganisms present as planktonic cells without the protection of compatible solutes. The tested dry sanitation compounds were effective against planktonic cells; in most cases cells were below the detection limit of the assay (1.3 log CFU/test surface) after 1 minute of exposure. However, the sanitation agents failed to achieve complete elimination of the pathogens except in the case of Salmonella biofilms. The most efficient sanitizer was hypochlorous acid followed by quaternary ammonium. The sanitizers’ efficacy increased on samples incubated at 65% RH. Our findings suggest that application of the sanitizing agent should be accompanied by some physical cleaning (e.g., scraping) to remove microbial biomass. Finally, sampling of the major facilities in the Central Valley of California indicated that microorganisms survive in the dry area of the plant. These strains withstand the sanitation treatment and the in vitro biofilm experiments showed that they are more resistant to the sanitizers than the laboratory strains. Some of these microorganisms persist on surfaces as biofilms, which can protect the incidental L. monocytogenes or Salmonella.