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Controlling Listeria in produce packinghouses can be challenging due to the large number of potential contamination routes. For example, repeated isolation of the same Listeria subtype in a packinghouse could indicate persistence in the packinghouse or reintroduction of the same Listeria from an upstream source. To improve understanding of Listeria transmission patterns in packinghouses, we performed a longitudinal study in four apple packinghouses, including testing of 1,339 environmental sponges and whole genome sequencing (WGS)-based characterization of 280 isolates. Root cause analysis and subsequent intervention implementation were also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5–31% for individual packinghouses). Sites that showed high Listeria prevalence included drains, forklift tires and forks, forklift stops, and waxing area equipment frames. A total of 240/280 WGS-characterized isolates were represented in 41 clusters, each containing two or more isolates that differed by ≤50 high-quality single nucleotide polymorphisms (hqSNPs); 21 clusters were isolated from one packinghouse over ≥2 samplings (suggesting persistence or possibly reintroduction), while 11 clusters included isolates from >2 packinghouses, suggesting common upstream sources. Some interventions successfully (i) reduced Listeria detection on forklift tires and forks (across packinghouses) and (ii) mitigated packinghouse-specific Listeria issues (e.g., in catch pans). However, interventions that lacked enhanced equipment disassembly when persistence was suspected typically appeared to be unsuccessful. Overall, while our data suggest a combination of intensive environmental sampling with subtyping and root cause analysis can help identify effective interventions, implementation of effective interventions continues to be a challenge in packinghouses.

Abstract Enteric pathogens can enter a persister state in which they survive exposure to antibiotics and physicochemical stresses. Subpopulations of such phenotypic dormant variants have been detected in vivo and in planta in the laboratory, but their formation in the natural environment remains largely unexplored. We applied a mathematical model predicting the switch rate to persister cell in the phyllosphere to identify weather-related stressors associated with E. coli and S. enterica persister formation on plants based on their population dynamics in published field studies from the USA and Spain. Model outputs accurately depicted the bi-phasic decay of bacterial population sizes measured in the lettuce and spinach phyllosphere in these studies. Predicted E. coli persister switch rate on leaves was positively and negatively correlated with solar radiation intensity and wind velocity, respectively. Likewise, predicted S. enterica persister switch rate correlated positively with solar radiation intensity; however, a negative correlation was observed with air temperature, relative humidity, and dew point, factors involved in water deposition onto the phylloplane. These findings suggest that specific environmental factors may enrich for dormant bacterial cells on plants. Our model quantifiably links persister cell subpopulations in the plant habitat with broader physical conditions, spanning processes at different granular scales.

Pathogen contamination of agricultural water has been identified as a probable cause of recalls and outbreaks. However, variability in pathogen presence and concentration complicates the reliable identification of agricultural water at elevated risk of pathogen presence. In this study, we collected data on the presence of Salmonella and genetic markers for enterohemorrhagic E. coli (EHEC; PCR-based detection of stx and eaeA) in southwestern US canal water, which is used as agricultural water for produce. We developed and assessed the accuracy of models to predict the likelihood of pathogen contamination of southwestern US canal water. Based on 169 samples from 60 surface water canals (each sampled 1–3 times), 36% (60/169) and 21% (36/169) of samples were positive for Salmonella presence and EHEC markers, respectively. Water quality parameters (e.g., generic E. coli level, turbidity), surrounding land-use (e.g., natural cover, cropland cover), weather conditions (e.g., temperature), and sampling site characteristics (e.g., canal type) data were collected as predictor variables. Separate conditional forest models were trained for Salmonella isolation and EHEC marker detection, and cross-validated to assess predictive performance. For Salmonella, turbidity, day of year, generic E. coli level, and % natural cover in a 500–1,000 ft (~150–300 m) buffer around the sampling site were the top 4 predictors identified by the conditional forest model. For EHEC markers, generic E. coli level, day of year, % natural cover in a 250–500 ft (~75–150 m) buffer, and % natural cover in a 500–1,000 ft (~150–300 m) buffer were the top 4 predictors. Predictive performance measures (e.g., area under the curve [AUC]) indicated predictive modeling shows potential as an alternative method for assessing the likelihood of pathogen presence in agricultural water. Secondary conditional forest models with generic E. coli level excluded as a predictor showed < 0.01 difference in AUC as compared to the AUC values for the original models (i.e., with generic E. coli level included as a predictor) for both Salmonella (AUC = 0.84) and EHEC markers (AUC = 0.92). Our data suggests models that do not require the inclusion of microbiological data (e.g., indicator organism) show promise for real-time prediction of pathogen contamination of agricultural water (e.g., in surface water canals).

While the Food Safety Modernization Act established standards for the use of surface water for produce production, water quality is known to vary over space and time. Targeted approaches for identifying hazards in water that account for this variation may improve growers' ability to address pre-harvest food safety risks. Models that utilize publicly-available data (e.g., land-use, real-time weather) may be useful for developing these approaches. The objective of this study was to use pre-existing datasets collected in 2017 (N = 181 samples) and 2018 (N = 191 samples) to train and test models that predict the likelihood of detecting Salmonella and pathogenic E. coli markers (eaeA, stx) in agricultural water. Four types of features were used to train the models: microbial, physicochemical, spatial and weather. “Full models” were built using all four features types, while “nested models” were built using between one and three types. Twenty learners were used to develop separate full models for each pathogen. Separately, to assess information gain associated with using different feature types, six learners were randomly selected and used to develop nine, nested models each. Performance measures for each model were then calculated and compared against baseline models where E. coli concentration was the sole covariate. In the methods, we outline the advantages and disadvantages of each learner. Overall, full models built using ensemble (e.g., Node Harvest) and “black-box” (e.g., SVMs) learners out-performed full models built using more interpretable learners (e.g., tree- and rule-based learners) for both outcomes. However, nested eaeA-stx models built using interpretable learners and microbial data performed almost as well as these full models. While none of the nested Salmonella models performed as well as the full models, nested models built using spatial data consistently out-performed models that excluded spatial data. These findings demonstrate that machine learning approaches can be used to predict when and where pathogens are likely to be present in agricultural water. This study serves as a proof-of-concept that can be built upon once larger datasets become available and provides guidance on the learner-data combinations that should be the foci of future efforts (e.g., tree-based microbial models for pathogenic E. coli).

There is a need for science-based tools to (i) help manage microbial produce safety hazards associated with preharvest surface water use, and (ii) facilitate comanagement of agroecosystems for competing stakeholder aims. To develop these tools an improved understanding of foodborne pathogen ecology in freshwater systems is needed. The purpose of this study was to identify (i) sources of potential food safety hazards, and (ii) combinations of factors associated with an increased likelihood of pathogen contamination of agricultural water. Sixty-eight streams were sampled between April and October 2018 (196 samples). At each sampling event separate 10-L grab samples (GS) were collected and tested for Listeria, Salmonella, and the stx and eaeA genes. A 1-L GS was also collected and used for Escherichia coli enumeration and detection of four host-associated fecal source-tracking markers (FST). Regression analysis was used to identify individual factors that were significantly associated with pathogen detection. We found that eaeA-stx codetection [Odds Ratio (OR) = 4.2; 95% Confidence Interval (CI) = 1.3, 13.4] and Salmonella isolation (OR = 1.8; CI = 0.9, 3.5) were strongly associated with detection of ruminant and human FST markers, respectively, while Listeria spp. (excluding Listeria monocytogenes) was negatively associated with log10E. coli levels (OR = 0.50; CI = 0.26, 0.96). L. monocytogenes isolation was not associated with the detection of any fecal indicators. This observation supports the current understanding that, unlike enteric pathogens, Listeria is not fecally-associated and instead originates from other environmental sources. Separately, conditional inference trees were used to identify scenarios associated with an elevated or reduced risk of pathogen contamination. Interestingly, while the likelihood of isolating L. monocytogenes appears to be driven by complex interactions between environmental factors, the likelihood of Salmonella isolation and eaeA-stx codetection were driven by physicochemical water quality (e.g., dissolved oxygen) and temperature, respectively. Overall, these models identify environmental conditions associated with an enhanced risk of pathogen presence in agricultural water (e.g., rain events were associated with L. monocytogenes isolation from samples collected downstream of dairy farms; P = 0.002). The information presented here will enable growers to comanage their operations to mitigate the produce safety risks associated with preharvest surface water use.