Listeria monocytogenes growth potential, kinetics, and factors affecting its persistence on a broad range of fresh produce

Listeria monocytogenes (Lm) is one of the most dangerous foodborne pathogens that can cause diseases from diarrhea to death. It is widespread in the natural and food production environments including soil and water bodies. It also has a unique ability to grow at temperatures of refrigeration. In recent decades, Lm has been associated with several produceassociated foodborne outbreaks and become a major concern for the fresh produce industry. In developing guidelines to effectively manage risks of Lm, both FDA and the fresh produce industry urgently need information on the potentials and the underlying factors of Lm growth on a large variety of fresh produce. In this proposal, we outline research aimed at providing such information. We will examine Lm growth potentials and kinetics on a broad range of whole and fresh-cut vegetables and fruits, under typical pre-market storage and/or retail display conditions, as well as under elevated abusive temperatures. We will investigate the effects of produce nutritional and physiochemical characters, and produce microbial community, on Lm growth.

Listeria monocytogenes (Lm) is a major foodborne pathogen that has been characterized as ubiquitously present in the natural and food production environments, and has been implicated in multiple fresh produce related listeriosis outbreaks. Although a mesophilic bacterium, its ability of growth under refrigeration makes Lm an especially dangerous contaminant for vulnerable ready to eat (RTE) foods, including fresh-cut fruits and vegetables. Lm is also known for deficiencies in the synthesis of multiple vitamins and amino acids and sensitivity to high acidity. Therefore, Lm growth potential on given fresh produce is dependent of produce intrinsic characteristics and environmental factors. Research outlined in this proposal is aimed at addressing CPS 2018 RFP Research Priorities 1.1.5: Expanding knowledge on Listeria growth potential and kinetics; and 2.3.3: Factors affecting human pathogen persistence. The objectives of this research are: 1) to assess Lm growth potentials on a broad range of fresh fruits and vegetable under simulated normal storage and retail display conditions; 2) to examine temperature abuse on Lm growth potentials on selected whole and fresh-cut produce and develop a Lm growth potential-based means for quantifying temperature abuse; 3) to investigate produce intrinsic characteristics, including nutrient (specifically, vitamins and amino acids required for Lm growth), acidity, and potential anti-listerial substances, on Lm growth potentials, and 4) to explore the potential role of produce microbiota on Lm growth and persistence on fresh produce. A large variety of whole and fresh-cut fruits and vegetables representing multiple produce categories will be selected for testing in consideration of multiple factors including means of consumption, market volume, and typical storage/display conditions. Lm growth potential and kinetics will be determined using conventional microbiological analyses by inoculation multiple Lm strains on whole and fresh cut fruits and vegetables, and enumerating the surviving or grown Lm cells at time intervals appropriate for the storage conditions. Uninoculated controls will be used for assessing the behaviors of produce microbiota under the same conditions. Produce quality assessment will be conducted in parallel to microbiological analyses to make sure that the data is relevant to industry practices. Lm growth potentials and kinetics and the effects of produce intrinsic characteristics will also be determined using diluted sterile extracts from relevant fresh-cut produce. Produce microbiome shifts during storage/display will be determined using 16S rDNA survey-based metagenomic analyses. Effectively managing Lm food safety risks is a high priority for both fresh produce industry and regulatory agencies. Recent FDA draft guidelines for Lm risk management recommended different industrial actions based on commodity-specific assessment of Lm growth potentials. Information from this proposed research can be of great importance to both fresh produce industry and regulatory agencies for developing commodity specific guidelines and practices to minimize the food safety risks resulted from Lm contamination and growth on fresh produce.

1. Determine Listeria monocytogenes (Lm) growth potential and kinetics on major classes of whole and fresh-cut produce under normal storage/retail display conditions. 

2. Determine Lm growth potential and kinetics under temperature abuse conditions, and develop an indexing system for quantifying temperature abuse. 

3. Determine produce nutritional and physiochemical characteristics on Lm growth potential and kinetics. 

4. Evaluate the effects of the indigenous microbial community from produce on Lm growth.

L. monocytogenes growth potential and kinetics on a broad range of fresh produce at normal and abusive temperatures. Rapid declines in L. monocytogenes counts were observed on all whole fruits tested, including green pepper and tomato, likely caused by a lack of accessible nutrients on the fruit surface. L. monocytogenes growth and survival varied for the fresh-cut products. Fresh-cut cantaloupe and most fresh-cut vegetables but none of the fresh-cut acidic fruits supported significant L. monocytogenes growth. Most of these observations were also corroborated when corresponding sterile juices were used for assessing the effect of produce intrinsic characteristics on L. monocytogenes growth and survival. Therefore, all fresh-cut produce with higher pH, such as cantaloupe and vegetables, should be considered highly conducive to L. monocytogenes growth, especially at abusive temperatures. Low pH is a primary deterrent for L. monocytogenes growth. Acidic fruits did not support L. monocytogenes either on commodity surfaces or in sterile juices. Exposing inoculated produce to abusive temperatures did not change the trend of L. monocytogenes growth on whole fruits. Effective strategies for mitigating L. monocytogenes risks on such commodities should focus on preventing the initial contamination instead of temperature control. L. monocytogenes cultivability. A large proportion of L. monocytogenes cells rapidly lose cultivability after inoculation onto the surface of whole fruits, including non-acidic fruits such as avocado. The same was observed when L. monocytogenes was exposed to the surface of carrot and carrot-soaking water. It is not clear whether this large portion (99%) of unrecoverable L. monocytogenes cells die or survive in a VBNC state. Transmission electron microscopy showed that carrot-exposed L. monocytogenes cells form progressive membrane invagination. Although these observation were beyond the scope of the current project, more research should be conducted to assess the food safety implications of foodborne pathogens with reduced cultivability. Intrinsic characteristics of fresh produce and effects on L. monocytogenes growth. In addition to pH as a primary determinant for L. monocytogenes growth, other fresh produce intrinsic characteristics potentially affecting L. monocytogenes growth include the presence of potential anti-listerial substances or the absence of essential micronutrients. We observed preliminary evidence of such substances playing a role in determining L. monocytogenes growth in neutralized peach juice (likely undissociated organic acids), in cauliflower juice (likely glucosinolates or degradation products), and fresh-cut carrots. We did not observe evidence of L. monocytogenes failing to grow due to a lack of micronutrients. These naturally occurring antilisterial substances can be further explored for mitigating L. monocytogenes risks. Microbiome of fresh produce and L. monocytogenes growth. In addition to the intrinsic factors of fresh produce, L. monocytogenes growth on a given product can be strongly affected by its microbiome. This was consistent with our observations of the overwhelming growth of L. monocytogenes on fresh-cut cantaloupe and the strong growth inhibition in raw romaine lettuce juice. We also isolated a Lactococcus lactis strain that strongly inhibited L. monocytogenes growth when co-cultured in a liquid medium at a low initial inoculation ratio. Further research needs to be conducted to explore the use of such antagonist isolates for manipulating fresh produce microbiomes. Microbiome research. Fresh produce microbiome research can contribute to a better understanding of the interactions between foodborne pathogens and indigenous microbiota on produce. Such efforts are often discouraged due to limited access to fresh produce growth, processing, and distribution systems. Closer industry collaboration and expanded access will greatly help such research for finding more useful tools against foodborne pathogens.