Effect of dehydration on the inactivation of Listeria monocytogenes and Salmonella enterica on enoki and wood ear mushrooms.

Foodborne illness outbreaks in the U.S. associated with consumption of both fresh and dried specialty mushrooms have recently occurred. Dried wood ear mushrooms were implicated in a salmonellosis outbreak in 2020, while fresh enoki mushrooms were associated with two listeriosis outbreaks in 2020 and 2023. These specialty mushrooms are commercially available in both their fresh and dried states. Due to the short shelf life of mushrooms, dehydration is a common method used in both industry and by consumers to extend the shelf life and preserve quality. Therefore, the aim of this study was to evaluate the use of dehydration on the inactivation kinetics of both Listeria monocytogenes and Salmonella enterica on enoki and wood ear mushrooms. Fresh mushrooms were inoculated with four strain cocktails of either L. monocytogenes or S. enterica and dried at ambient conditions for 10 min. Following drying of the inoculum, mushrooms were placed into food dehydrators preheated to 70, 80, or 90°C and treated for up to 24 h. At treatment intervals, mushrooms were removed from the dehydrators for pathogen enumeration. Inactivation kinetics for both pathogens were modeled using the Weibull, log-linear with tail, and log-linear with shoulder models. Pathogen reductions of >4 log CFU/g were achieved on both enoki and wood ear mushrooms during dehydration at 90°C after only 2-4 h. At 70 and 80°C, log reductions of >4 log CFU/g were observed on wood ear mushrooms after 4-8 h. On enoki mushrooms, a tailing effect was observed with residual populations (>2 log CFU/g) of L. monocytogenes and S. enterica remaining even after 24 h of treatment at both 70 and 80°C. This study emphasizes the need for an individualized dehydration strategy for each mushroom type to ensure the effectiveness of dehydration as a process to reduce pathogen populations. Results of this study will aid in informing proper time and temperature combinations for dehydration of specialty mushrooms to ensure product safety.

Survival kinetics of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during long-term storage.

Two specialty mushrooms have recently become novel vectors for foodborne outbreaks in the U.S.: fresh enoki and dried wood ear mushrooms were linked to a listeriosis and salmonellosis outbreak, respectively. The aim of this study was to evaluate the survival kinetics of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during long-term storage. Following heat dehydration, mushrooms were inoculated with either L. monocytogenes or S. enterica, allowed to dry for 1 h, and then stored for up to 180 d at 25 °C and 33% relative humidity. Both pathogens were enumerated from the mushrooms at intervals during the storage period. Survival kinetics of both pathogens were modeled using both the Weibull and log-linear with tail models. After inoculation and 1 h drying, both pathogen populations decreased 2.26-2.49 log CFU/g on wood ear mushrooms; no decrease was observed on enoki. Both pathogens survived during storage on both mushroom types. On wood ear mushrooms, a 2-log decrease of both pathogens occurred during storage. On enoki mushrooms, 4-log decreases of both pathogens were modeled to occur after 127.50-156.60 d. The results of this study suggest that L. monocytogenes and S. enterica can persist on dehydrated specialty mushrooms during long-term storage.

Modeling the Fate of Listeria monocytogenes and Salmonella enterica on Fresh Whole and Chopped Wood Ear and Enoki Mushrooms.

Two recent foodborne illness outbreaks linked to specialty mushrooms have occurred in the United States, both representing novel pathogen-commodity pairings. Listeria monocytogenes and Salmonella enterica were linked to enoki and wood ear mushrooms, respectively. The aim of this study was therefore to examine the survival of both L. monocytogenes and S. enterica on raw whole and chopped enoki and wood ear mushrooms during storage at different temperatures. Fresh mushrooms were either left whole or chopped and subsequently inoculated with a cocktail of either S. enterica or rifampicin-resistant L. monocytogenes, resulting in an initial inoculation level of 3 log CFU/g. Mushroom samples were stored at 5, 10, or 25°C for up to 7 d. During storage, the population levels of S. enterica or L. monocytogenes on the mushrooms were enumerated. The primary Baranyi model was used to estimate the growth rates of both pathogens and the secondary Ratkowsky square root model was used to model the relationship between growth rates and temperature. Both L. monocytogenes and S. enterica survived on both mushroom types and preparations at all temperatures. No proliferation of either pathogen was observed on mushrooms stored at 5°C. At 10°C, moderate growth was observed for both pathogens on enoki mushrooms and for L. monocytogenes on wood ear mushrooms; no growth was observed for S. enterica on wood ear mushrooms. At 25°C, both pathogens proliferated on both mushroom types with growth rates ranging from 0.43 to 3.27 log CFU/g/d, resulting in 1 log CFU/g increases in only 0.31 d (7.44 h) to 2.32 d. Secondary models were generated for L. monocytogenes on whole wood ear mushrooms and S. enterica on whole enoki mushrooms with goodness-of-fit parameters of r2 = 0.9855/RMSE = 0.0479 and r2 = 0.9882/RMSE = 0.1417, respectively. Results from this study can aid in understanding the dynamics of L. monocytogenes and S. enterica on two types of specialty mushrooms.

Strain, Soil-Type, Irrigation Regimen, and Poultry Litter Influence Salmonella Survival and Die-off in Agricultural Soils.

The use of untreated biological soil amendments of animal origin (BSAAO) have been identified as one potential mechanism for the dissemination and persistence of Salmonella in the produce growing environment. Data on factors influencing Salmonella concentration in amended soils are therefore needed. The objectives here were to (i) compare die-off between 12 Salmonella strains following inoculation in amended soil and (ii) characterize any significant effects associated with soil-type, irrigation regimen, and amendment on Salmonella survival and die-off. Three greenhouse trials were performed using a randomized complete block design. Each strain (~4 log CFU/g) was homogenized with amended or non-amended sandy-loam or clay-loam soil. Salmonella levels were enumerated in 25 g samples 0, 0.167 (4 h), 1, 2, 4, 7, 10, 14, 21, 28, 56, 84, 112, 168, 210, 252, and 336 days post-inoculation (dpi), or until two consecutive samples were enrichment negative. Regression analysis was performed between strain, soil-type, irrigation, and (i) time to last detect (survival) and (ii) concentration at each time-point (die-off rate). Similar effects of strain, irrigation, soil-type, and amendment were identified using the survival and die-off models. Strain explained up to 18% of the variance in survival, and up to 19% of variance in die-off rate. On average Salmonella survived for 129 days in amended soils, however, Salmonella survived, on average, 30 days longer in clay-loam soils than sandy-loam soils [95% Confidence interval (CI) = 45, 15], with survival time ranging from 84 to 210 days for the individual strains during daily irrigation. When strain-specific associations were investigated using regression trees, S. Javiana and S. Saintpaul were found to survive longer in sandy-loam soil, whereas most of the other strains survived longer in clay-loam soil. Salmonella also survived, on average, 128 days longer when irrigated weekly, compared to daily (CI = 101, 154), and 89 days longer in amended soils, than non-amended soils (CI = 61, 116). Overall, this study provides insight into Salmonella survival following contamination of field soils by BSAAO. Specifically, Salmonella survival may be strain-specific as affected by both soil characteristics and management practices. These data can assist in risk assessment and strain selection for use in challenge and validation studies.

A Predictive Model for Survival of Escherichia coli O157:H7 and Generic E. coli in Soil Amended with Untreated Animal Manure.

This study aimed at developing a predictive model that captures the influences of a variety of agricultural and environmental variables and is able to predict the concentrations of enteric bacteria in soil amended with untreated Biological Soil Amendments of Animal Origin (BSAAO) under dynamic conditions. We developed and validated a Random Forest model using data from a longitudinal field study conducted in mid-Atlantic United States investigating the survival of Escherichia coli O157:H7 and generic E. coli in soils amended with untreated dairy manure, horse manure, or poultry litter. Amendment type, days of rain since the previous sampling day, and soil moisture content were identified as the most influential agricultural and environmental variables impacting concentrations of viable E. coli O157:H7 and generic E. coli recovered from amended soils. Our model results also indicated that E. coli O157:H7 and generic E. coli declined at similar rates in amended soils under dynamic field conditions.The Random Forest model accurately predicted changes in viable E. coli concentrations over time under different agricultural and environmental conditions. Our model also accurately characterized the variability of E. coli concentration in amended soil over time by providing upper and lower prediction bound estimates. Cross-validation results indicated that our model can be potentially generalized to other geographic regions and incorporated into a risk assessment for evaluating the risks associated with application of untreated BSAAO. Our model can be validated for other regions and predictive performance also can be enhanced when data sets from additional geographic regions become available.

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production.

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech's Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields.

Comparison of U.S. Environmental Protection Agency and U.S. Composting Council microbial detection methods in finished compost and regrowth potential of Salmonella spp. and Escherichia coli O157:H7 in finished compost.

Bacterial pathogens may survive and regrow in finished compost due to incomplete thermal inactivation during or recontamination after composting. Twenty-nine finished composts were obtained from 19 U.S. states and were separated into three broad feedstock categories: biosolids (n=10), manure (n=4), and yard waste (n=15). Three replicates of each compost were inoculated with ≈ 1-2 log CFU/g of nonpathogenic Escherichia coli, Salmonella spp., and E. coli O157:H7. The U.S. Environmental Protection Agency's (EPA) protocols and U.S. Composting Council's (USCC) Test Methods for the Examination of Composting and Compost (TMECC) were compared to determine which method recovered higher percentages of inoculated E. coli (representing fecal coliforms) and Salmonella spp. from 400-g samples of finished composts. Populations of Salmonella spp. and E. coli O157:H7 were determined over 3 days while stored at 25°C and compared to physicochemical parameters to predict their respective regrowth potentials. EPA Method 1680 recovered significantly (p=0.0003) more inoculated E. coli (68.7%) than TMECC 07.01 (48.1%) due to the EPA method using more compost in the initial homogenate, larger transfer dilutions, and a larger most probable number scheme compared to TMECC 07.01. The recoveries of inoculated Salmonella spp. by Environmental Protection Agency Method 1682 (89.1%) and TMECC 07.02 (72.4%) were not statistically significant (p=0.44). The statistically similar recovery percentages may be explained by the use of a nonselective pre-enrichment step used in both methods. No physicochemical parameter (C:N, moisture content, total organic carbon) was able to serve as a sole predictor of regrowth of Salmonella spp. or E. coli O157:H7 in finished compost. However, statistical analysis revealed that the C:N ratio, total organic carbon, and moisture content all contributed to pathogen regrowth potential in finished composts. It is recommended that the USCC modify TMECC protocols to test larger amounts of compost in the initial homogenate to facilitate greater recovery of target organisms.

Effect of repeated irrigation with water containing varying levels of total organic carbon on the persistence of Escherichia coli O157:H7 on baby spinach.

The California lettuce and leafy greens industry has adopted the Leafy Greens Marketing Agreement (LGMA), which allows for 126 most-probable-number (MPN) Escherichia coli per 100 ml in irrigation water. Repeat irrigation of baby spinach plants with water containing E. coli O157:H7 and different levels of total organic carbon (TOC) was used to determine the epiphytic survival of E. coli O157:H7. Three irrigation treatments (0 ppm of TOC, 12 or 15 ppm of TOC, and 120 or 150 ppm of TOC) were prepared with bovine manure containing E. coli O157:H7 at either low (0 to 1 log CFU/100 ml) or high (5 to 6 log CFU/100 ml) populations, and sprayed onto baby spinach plants in growth chambers by using a fine-mist airbrush. MPN and direct plating techniques were used to determine the E. coli O157:H7 populations on the aerial plant tissue. Plants irrigated with high E. coli O157:H7 populations, regardless of TOC levels, showed a 3-log reduction within the first 24 h. Low levels of E. coli O157:H7 were observed for up to 16 days on all TOC treatments, ranging from 76.4 MPN per plant (day 1) to 0.40 MPN per plant (day 16). No viable cells were detected on spinach tissue 24 h after irrigation with water containing fewer than 126 CFU/100 ml E. coli O157:H7. Under growth chamber conditions in this study, E. coli O157:H7 populations in irrigation water that complies with the LGMA standards will not persist for more than 24 h when applied onto foliar surfaces of spinach plants.

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce.

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.

A novel approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and hydroponic medium.

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. A Tn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein-labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 10(3) or 10(7) CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 10(7) CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.