A longitudinal study on the bacterial quality of baby spinach cultivated in Arizona and California.

In the U.S., baby spinach is mostly produced in Arizona (AZ) and California (CA). Characterizing the impact of growing region on the bacterial quality of baby spinach can inform quality management practices in industry. Between December 2021 and December 2022, baby spinach was sampled after harvest and packaging for microbiological testing, including shelf-life testing of packaged samples that were stored at 4°C. Samples were tested to (i) determine bacterial concentration, and (ii) obtain and identify bacterial isolates. Packaged samples from the Salinas, CA, area (n = 13), compared to those from the Yuma, AZ, area (n = 9), had a significantly higher bacterial concentration, on average, by 0.78 log10 CFU/g (P < 0.01, based on aerobic, mesophilic plate count data) or 0.67 log10 CFU/g (P < 0.01, based on psychrotolerant plate count data); the bacterial concentrations of harvest samples from the Yuma and Salinas areas were not significantly different. Our data also support that an increase in preharvest temperature is significantly associated with an increase in the bacterial concentration on harvested and packaged spinach. A Fisher's exact test and linear discriminant analysis (effect size), respectively, demonstrated that (i) the genera of 2,186 bacterial isolates were associated (P < 0.01) with growing region and (ii) Pseudomonas spp. and Exiguobacterium spp. were enriched in spinach from the Yuma and Salinas areas, respectively. Our findings provide preliminary evidence that growing region and preharvest temperature may impact the bacterial quality of spinach and thus could inform more targeted strategies to manage produce quality. IMPORTANCE: In the U.S., most spinach is produced in Arizona (AZ) and California (CA) seasonally; typically, spinach is cultivated in the Yuma, AZ, area during the winter and in the Salinas, CA, area during the summer. As the bacterial quality of baby spinach can influence consumer acceptance of the product, it is important to assess whether the bacterial quality of baby spinach can vary between spinach-growing regions. The findings of this study provide insights that could be used to support region-specific quality management strategies for baby spinach. Our results also highlight the value of further evaluating the impact of growing region and preharvest temperature on the bacterial quality of different produce commodities.

Phenotypic and genomic characterizations of Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata strains reveal no clear association between genetic content and ropy phenotype.

Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize 3 K. pneumoniae ssp. pneumoniae strains and 2 R. inusitata for (1) their ability to grow and cause ropy defect in milk at 6°C and 21°C and to (2) probe the genetic basis for observed ropy phenotype. Although all K. pneumoniae ssp. pneumoniae and R. inusitata strains showed net growth of >4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-d incubation period at 6°C (>6 log10). Two out of 3 K. pneumoniae ssp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these 2 strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6°C and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae ssp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae ssp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the 2 strains that produced ropy defect, and for R. inusitata a set of 2 glycosyltransferase- and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. Although these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.

Assessment of the exposure to cytotoxic Bacillus cereus group genotypes through HTST milk consumption.

This study addresses the limited tools available for assessing food safety risks from cytotoxic Bacillus cereus group strains in contaminated food. We quantified the growth, in skim milk broth, of 17 cytotoxic B. cereus strains across 6 phylogenetic groups with various virulence gene profiles. The strains did not grow in HTST milk at 4 or 6°C. At 10°C, 15 strains exhibited growth; at 8°C, one strain grew; and all strains grew at temperatures ≥ 14°C. Using growth data from 16 strains, we developed linear secondary growth models and an exposure assessment model. This model, simulating a 5-stage HTST milk supply chain and up to 35 d of consumer storage with an initial contamination of 100 cfu/mL, estimated that 2.81 ± 0.66% and 4.13 ± 2.53% of milk containers would surpass 105 cfu/mL of B. cereus by d 21 and 35, respectively. A sensitivity analysis identified the initial physiological state of cells (Q0) as the most influential variable affecting predictions for specific isolates. What-if scenarios indicated that increases in mean and variability of consumer storage temperatures significantly affected the predicted B. cereus concentrations in milk. This model serves as an initial tool for risk-based food safety decision making regarding low-level B. cereus contamination.

Utilizing whole genome sequencing to characterize Listeria spp. persistence and transmission patterns in a farmstead dairy processing facility and its associated farm environment.

Farmstead dairy processing facilities may be particularly susceptible to Listeria spp. contamination due to the close physical proximity of their processing environments (PE) to associated dairy farm environments (FE). In this case study, we supported the implementation of interventions focused on improving (i) cleaning and sanitation efficacy, (ii) hygienic zoning, and (iii) sanitary equipment/facility design and maintenance in a farmstead dairy processing facility, and evaluated their impact on Listeria spp. detection in the farmstead's PE over 1 year. Detection of Listeria spp. in the farmstead's PE was numerically reduced from 50% to 7.5% after 1 year of intervention implementation, suggesting that these interventions were effective at improving Listeria spp. control. In addition, environmental samples were also collected from the farmstead's FE to evaluate the risk of the FE as a potential source of Listeria spp. in the PE. Overall, detection of Listeria spp. was higher in samples collected from the FE (75%, 27/36) compared with samples collected from the PE (24%, 29/120). Whole genome sequencing (WGS) performed on select isolates collected from the PE and FE supported the identification of 6 clusters (range of 3 to 15 isolates per cluster) that showed ≤ 50 high quality single nucleotide polymorphism (hqSNP) differences. Of these 6 clusters, 3 (i.e., clusters 2, 4, and 5) contained isolates that were collected from both the PE and FE, suggesting that transmission between these 2 environments was likely. Moreover, all cluster 2 isolates represented a clonal complex (CC) of L. monocytogenes commonly associated with dairy farm environmental reservoirs (i.e., CC666), which may support that the farmstead's FE represented an upstream source of the cluster 2 isolates that were found in the PE. Overall, our data underscore that, while the FE can represent a potential upstream source of Listeria spp. contamination in a farmstead dairy processing facility, implementation of targeted interventions can help effectively minimize Listeria spp. contamination in the PE.

Troubleshooting high laboratory pasteurization counts in organic raw milk requires characterization of dominant thermoduric bacteria, which includes nonsporeformers as well as sporeformers.

Laboratory pasteurization count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. No regulatory limit has presently been set for LPC, but LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Although it is occasionally used as a proxy for levels of bacterial spores in raw milk, limited knowledge is available on the types of organisms that are enumerated by LPC in contemporary raw milk supplies. Although historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as gram-positive sporeformers, gram-positive nonsporeformers, and gram-negatives, respectively; 0.5% could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as gram-positive sporeformers were predominantly Bacillus (168/200), and gram-positive nonsporeformers were predominantly Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk, and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using MALDI-TOF mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. Although our data indicated agreement between 16S rDNA sequencing and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF MS, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.

Selection of mutant Listeria phages under food-relevant conditions can enhance application potential.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

Low-cost, on-farm intervention to reduce spores in bulk tank raw milk benefits producers, processors, and consumers.

Bacterial spores, which are found in raw milk, can survive harsh processing conditions encountered in dairy manufacturing, including pasteurization and drying. Low-spore raw milk is desirable for dairy industry stakeholders, especially those who want to extend the shelf life of their product, expand their distribution channels, or reduce product spoilage. A recent previous study showed that an on-farm intervention that included washing towels with chlorine bleach and drying them completely, as well as training milking parlor employees to focus on teat end cleaning, significantly reduced spore levels in bulk tank raw milk. As a follow up to that previous study, here we calculate the costs associated with that previously described intervention as ranging from $9.49 to $13.35 per cow per year, depending on farm size. A Monte Carlo model was used to predict the shelf life of high temperature, short time fluid milk processed from raw milk before and after this low-cost intervention was applied, based on experimental data collected in a previous study. The model predicted that 18.24% of half-gallon containers of fluid milk processed from raw milk receiving no spore intervention would exceed the pasteurized milk ordinance limit of 20,000 cfu/mL by 17 d after pasteurization, while only 16.99% of containers processed from raw milk receiving the spore intervention would reach this level 17 d after pasteurization (a reduction of 1.25 percentage points and a 6.85% reduction). Finally, a survey of consumer milk use was conducted to determine how many consumers regularly consume fluid milk near or past the date printed on the package (i.e., code date), which revealed that over 50% of fluid milk consumers surveyed continue to consume fluid milk after this date, indicating that a considerable proportion of consumers are exposed to fluid milk that is likely to have high levels spore-forming bacterial growth and possibly associated quality defects (e.g., flavor or odor defects). This further highlights the importance of reducing spore levels in raw milk to extend pasteurized fluid milk shelf life and thereby reducing the risk of adverse consumer experiences. Processors who are interested in extending fluid milk shelf life by controlling the levels of spores in the raw milk supply should consider incentivizing low-spore raw milk through premium payments to producers.

Population dynamics of Listeria spp., Salmonella spp., and Escherichia coli on fresh produce: A scoping review.

Collation of the current scope of literature related to population dynamics (i.e., growth, die-off, survival) of foodborne pathogens on fresh produce can aid in informing future research directions and help stakeholders identify relevant research literature. A scoping review was conducted to gather and synthesize literature that investigates population dynamics of pathogenic and non-pathogenic Listeria spp., Salmonella spp., and Escherichia coli on whole unprocessed fresh produce (defined as produce not having undergone chopping, cutting, homogenization, irradiation, or pasteurization). Literature sources were identified using an exhaustive search of research and industry reports published prior to September 23, 2021, followed by screening for relevance based on strict, a priori eligibility criteria. A total of 277 studies that met all eligibility criteria were subjected to an in-depth qualitative review of various factors (e.g., produce commodities, study settings, inoculation methodologies) that affect population dynamics. Included studies represent investigations of population dynamics on produce before (i.e., pre-harvest; n = 143) and after (i.e., post-harvest; n = 144) harvest. Several knowledge gaps were identified, including the limited representation of (i) pre-harvest studies that investigated population dynamics of Listeria spp. on produce (n = 13, 9% of pre-harvest studies), (ii) pre-harvest studies that were carried out on non-sprouts produce types grown using hydroponic cultivation practices (n = 7, 5% of pre-harvest studies), and (iii) post-harvest studies that reported the relative humidity conditions under which experiments were carried out (n = 56, 39% of post-harvest studies). These and other knowledge gaps summarized in this scoping review represent areas of research that can be investigated in future studies.

Laboratory Misidentifications Resulting from Taxonomic Changes to Bacillus cereus Group Species, 2018-2022.

Whole-genome sequencing (WGS) is being applied increasingly to Bacillus cereus group species; however, misinterpretation of WGS results may have severe consequences. We report 3 cases, 1 of which was an outbreak, in which misinterpretation of B. cereus group WGS results hindered communication within public health and industrial laboratories.

Nonsynonymous Mutations in fepR Are Associated with Adaptation of Listeria monocytogenes and Other Listeria spp. to Low Concentrations of Benzalkonium Chloride but Do Not Increase Survival of L. monocytogenes and Other Listeria spp. after Exposure to Benzalkonium Chloride Concentrations Recommended for Use in Food Processing Environments.

Selection for Listeria monocytogenes strains that are tolerant to quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a concern across the food industry, including in fresh produce processing environments. This study evaluated the ability of 67 strains of produce-associated L. monocytogenes and other Listeria spp. ("parent strains") to show enhanced BC tolerance after serial passaging in increasing BC concentrations and to maintain this tolerance after substreaking in the absence of BC. After serial passaging in BC, 62/67 "BC passaged cultures" showed higher MICs (4 to 20 mg/L) than parent strains (2 to 6 mg/L). After the substreaking of two isolates from BC passaged cultures for each parent strain, 105/134 "adapted isolates" maintained MICs (4 to 6 mg/L) higher than parent strain MICs. These results suggested that adapted isolates acquired heritable adaptations that confer BC tolerance. Whole-genome sequencing and Sanger sequencing of fepR, a local repressor of the MATE family efflux pump FepA, identified nonsynonymous fepR mutations in 48/67 adapted isolates. The mean inactivation of adapted isolates after exposure to use-level concentrations of BC (300 mg/L) was 4.48 log, which was not significantly different from inactivation observed in parent strains. Serial passaging of cocultures of L. monocytogenes strains containing bcrABC or qacH did not yield adapted isolates that showed enhanced BC tolerance in comparison to that of monocultures. These results suggest that horizontal gene transfer either did not occur or did not yield isolates with enhanced BC tolerance. Overall, this study provides new insights into selection of BC tolerance among L. monocytogenes and other Listeria spp. IMPORTANCE Listeria monocytogenes tolerance to quaternary ammonium compounds has been raised as a concern with regard to L. monocytogenes persistence in food processing environments, including in fresh produce packing and processing environments. Persistence of L. monocytogenes can increase the risk of product contamination, food recalls, and foodborne illness outbreaks. Our study shows that strains of L. monocytogenes and other Listeria spp. can acquire heritable adaptations that confer enhanced tolerance to low concentrations of benzalkonium chloride, but these adaptations do not increase survival of L. monocytogenes and other Listeria spp. when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Overall, these findings suggest that the emergence of benzalkonium chloride-tolerant Listeria strains in food processing environments is of limited concern, as even strains adapted to gain higher MICs in vitro maintain full sensitivity to the concentrations of benzalkonium chloride used for food contact surface sanitation.

Whole-Genome Sequencing-Based Characterization of Listeria Isolates from Produce Packinghouses and Fresh-Cut Facilities Suggests Both Persistence and Reintroduction of Fully Virulent L. monocytogenes.

The contamination of ready-to-eat produce with Listeria monocytogenes (LM) can often be traced back to environmental sources in processing facilities and packinghouses. To provide an improved understanding of Listeria sources and transmission in produce operations, we performed whole-genome sequencing (WGS) of LM (n = 169) and other Listeria spp. (n = 107) obtained from 13 produce packinghouses and three fresh-cut produce facilities. Overall, a low proportion of LM isolates (9/169) had inlA premature stop codons, and a large proportion (83/169) had either or both of the LIPI-3 or LIPI-4 operons, which have been associated with hypervirulence. The further analysis of the WGS data by operation showed a reisolation (at least 2 months apart) of highly related isolates (<10 hqSNP differences) in 7/16 operations. Two operations had highly related strains reisolated from samples that were collected at least 1 year apart. The identification of isolates collected during preproduction (i.e., following sanitation but before the start of production) that were highly related to isolates collected during production (i.e., after people or products have entered and begun moving through the operation) provided evidence that some strains were able to survive standard sanitation practices. The identification of closely related isolates (<20 hqSNPs differences) in different operations suggests that cross-contamination between facilities or introductions from common suppliers may also contribute to Listeria transmission. Overall, our data suggest that the majority of LM isolates collected from produce operations are fully virulent and that both persistence and reintroduction may lead to the repeat isolation of closely related Listeria in produce operations. IMPORTANCE Listeria monocytogenes is of particular concern to the produce industry due to its frequent presence in natural environments as well as its ability to survive in packinghouses and fresh-cut processing facilities over time. The use of whole-genome sequencing, which provides high discriminatory power for the characterization of Listeria isolates, along with detailed source data (isolation date and sample location) shows that the presence of Listeria in produce operations appears to be due to random and continued reintroduction as well as to the persistence of highly related strains in both packinghouses and fresh-cut facilities. These findings indicate the importance of using high-resolution characterization approaches for root cause analyses of Listeria contamination issues. In cases of repeat isolation of closely related Listeria in a given facility, both persistence and reintroduction need to be considered as possible root causes.

A Validated Preharvest Sampling Simulation Shows that Sampling Plans with a Larger Number of Randomly Located Samples Perform Better than Typical Sampling Plans in Detecting Representative Point-Source and Widespread Hazards in Leafy Green Fields.

Commercial leafy greens customers often require a negative preharvest pathogen test, typically by compositing 60 produce sample grabs of 150 to 375 g total mass from lots of various acreages. This study developed a preharvest sampling Monte Carlo simulation, validated it against literature and experimental trials, and used it to suggest improvements to sampling plans. The simulation was validated by outputting six simulated ranges of positive samples that contained the experimental number of positive samples (range, 2 to 139 positives) recovered from six field trials with point source, systematic, and sporadic contamination. We then evaluated the relative performance between simple random, stratified random, or systematic sampling in a 1-acre field to detect point sources of contamination present at 0.3% to 1.7% prevalence. Randomized sampling was optimal because of lower variability in probability of acceptance. Optimized sampling was applied to detect an industry-relevant point source [3 log(CFU/g) over 0.3% of the field] and widespread contamination [-1 to -4 log(CFU/g) over the whole field] by taking 60 to 1,200 sample grabs of 3 g. More samples increased the power of detecting point source contamination, as the median probability of acceptance decreased from 85% with 60 samples to 5% with 1,200 samples. Sampling plans with larger total composite sample mass increased power to detect low-level, widespread contamination, as the median probability of acceptance with -3 log(CFU/g) contamination decreased from 85% with a 150-g total mass to 30% with a 1,200-g total mass. Therefore, preharvest sampling power increases by taking more, smaller samples with randomization, up to the constraints of total grabs and mass feasible or required for a food safety objective. IMPORTANCE This study addresses a need for improved preharvest sampling plans for pathogen detection in leafy green fields by developing and validating a preharvest sampling simulation model, avoiding the expensive task of physical sampling in many fields. Validated preharvest sampling simulations were used to develop guidance for preharvest sampling protocols. Sampling simulations predicted that sampling plans with randomization are less variable in their power to detect low-prevalence point source contamination in a 1-acre field. Collecting larger total sample masses improved the power of sampling plans in detecting widespread contamination in 1-acre fields. Hence, the power of typical sampling plans that collect 150 to 375 g per composite sample can be improved by taking more, randomized smaller samples for larger total sample mass. The improved sampling plans are subject to feasibility constraints or to meet a particular food safety objective.

Structural Equation Models Suggest That On-Farm Noncrop Vegetation Removal Is Not Associated with Improved Food Safety Outcomes but Is Linked to Impaired Water Quality.

While growers have reported pressures to minimize wildlife intrusion into produce fields through noncrop vegetation (NCV) removal, NCV provides key ecosystem services. To model food safety and environmental tradeoffs associated with NCV removal, published and publicly available food safety and water quality data from the Northeastern United States were obtained. Because data on NCV removal are not widely available, forest-wetland cover was used as a proxy, consistent with previous studies. Structural equation models (SEMs) were used to quantify the effect of forest-wetland cover on (i) food safety outcomes (e.g., detecting pathogens in soil) and (ii) water quality (e.g., nutrient levels). Based on the SEMs, NCV was not associated with or had a protective effect on food safety outcomes (more NCV was associated with a reduced likelihood of pathogen detection). The probabilities of detecting Listeria spp. in soil (effect estimate [EE] = -0.17; P = 0.005) and enterohemorrhagic Escherichia coli in stream samples (EE = -0.27; P < 0.001) were negatively associated with the amount of NCV surrounding the sampling site. Larger amounts of NCV were also associated with lower nutrient, salinity, and sediment levels, and higher dissolved oxygen levels. Total phosphorous levels were negatively associated with the amount of NCV in the upstream watershed (EE = -0.27; P < 0.001). Similar negative associations (P < 0.05) were observed for other physicochemical parameters, such as nitrate (EE = -0.38). Our findings suggest that NCV should not be considered an inherent produce safety risk or result in farm audit demerits. This study also provides a framework for evaluating environmental tradeoffs associated with using specific preharvest food safety strategies. IMPORTANCE Currently, on-farm food safety decisions are typically made independently of conservation considerations, often with detrimental impacts on agroecosystems. Comanaging agricultural environments to simultaneously meet conservation and food safety aims is complicated because farms are closely linked to surrounding environments, and management decisions can have unexpected environmental, economic, and food safety consequences. Thus, there is a need for research on the conservation and food safety tradeoffs associated with implementing specific preharvest food safety practices. Understanding these tradeoffs is critical for developing adaptive comanagement strategies and ensuring the short- and long-term safety, sustainability, and profitability of agricultural systems. This study quantifies tradeoffs and synergies between food safety and environmental aims, and outlines a framework for modeling tradeoffs and synergies between management aims that can be used to support future comanagement research.

Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond.

The Bacillus cereus group, also known as B. cereus sensu lato (s.l.), is a species complex that contains numerous closely related lineages, which vary in their ability to cause illness in humans and animals. The classification of B. cereus s.l. isolates into species-level taxonomic units is thus essential for informing public health and food safety efforts. However, taxonomic classification of these organisms is challenging. Numerous-often conflicting-taxonomic changes to the group have been proposed over the past two decades, making it difficult to remain up to date. In this review, we discuss the major nomenclatural changes that have accumulated in the B. cereus s.l. taxonomic space prior to 2020, particularly in the genomic sequencing era, and outline the resulting problems. We discuss several contemporary taxonomic frameworks as applied to B. cereus s.l., including (i) phenotypic, (ii) genomic, and (iii) hybrid nomenclatural frameworks, and we discuss the advantages and disadvantages of each. We offer suggestions as to how readers can avoid B. cereus s.l. taxonomic ambiguities, regardless of the nomenclatural framework(s) they choose to employ. Finally, we discuss future directions and open problems in the B. cereus s.l. taxonomic realm, including those that cannot be solved by genomic approaches alone.

A perspective on data sharing in digital food safety systems.

In this age of data, digital tools are widely promoted as having tremendous potential for enhancing food safety. However, the potential of these digital tools depends on the availability and quality of data, and a number of obstacles need to be overcome to achieve the goal of digitally enabled "smarter food safety" approaches. One key obstacle is that participants in the food system and in food safety often lack the willingness to share data, due to fears of data abuse, bad publicity, liability, and the need to keep certain data (e.g., human illness data) confidential. As these multifaceted concerns lead to tension between data utility and privacy, the solutions to these challenges need to be multifaceted. This review outlines the data needs in digital food safety systems, exemplified in different data categories and model types, and key concerns associated with sharing of food safety data, including confidentiality and privacy of shared data. To address the data privacy issue a combination of innovative strategies to protect privacy as well as legal protection against data abuse need to be pursued. Existing solutions for maximizing data utility, while not compromising data privacy, are discussed, most notably differential privacy and federated learning.

Associations between Listeria monocytogenes genomic characteristics and adhesion to polystyrene at 8 °C.

Listeria monocytogenes remains a threat to the food system and has led to numerous foodborne outbreaks worldwide. L. monocytogenes can establish itself in food production facilities by adhering to surfaces, resulting in increased resistance to environmental stressors. The aim of this study was to evaluate the adhesion ability of L. monocytogenes at 8 °C and to analyse associations between the observed phenotypes and genetic factors such as internalin A (inlA) genotypes, stress survival islet 1 (SSI-1) genotype, and clonal complex (CC). L. monocytogenes isolates (n = 184) were grown at 8 °C and 100% relative humidity for 15 days. The growth was measured by optical density at 600 nm every 24 h. Adherent cells were stained using crystal violet and quantified spectrophotometrically. Genotyping of inlA and SSI-1, multi-locus sequence typing, and a genome-wide association study (GWAS) were performed to elucidate the phenotype-genotype relationships in L. monocytogenes cold adhesion. Among all inlA genotypes, truncated inlA isolates had the highest mean adhered cells, ABS595nm = 0.30 ± 0.15 (Tukey HSD; P < 0.05), while three-codon deletion inlA isolates had the least mean adhered cells (Tukey HSD; P < 0.05). When SSI-1 was present, more cells adhered; less cells adhered when SSI-1 was absent (Welch's t-test; P < 0.05). Adhesion was associated with clonal complexes which have low clinical frequency, while reduced adhesion was associated with clonal complexes which have high frequency. The results of this study support that premature stop codons in the virulence gene inlA are associated with increased cold adhesion and that an invasion enhancing deletion in inlA is associated with decreased cold adhesion. This study also provides evidence to suggest that there is an evolutionary trade off between virulence and adhesion in L. monocytogenes. These results provide a greater understanding of L. monocytogenes adhesion which will aid in the development of strategies to reduce L. monocytogenes in the food system.

Adjacent Terrestrial Landscapes Impact the Biogeographical Pattern of Soil Escherichia coli Strains in Produce Fields by Modifying the Importance of Environmental Selection and Dispersal.

High-quality habitats for wildlife (e.g., forest) provide essential ecosystem services while increasing species diversity and habitat connectivity. Unfortunately, the presence of such habitats adjacent to produce fields may increase risk for contamination of fruits and vegetables by enteric bacteria, including Escherichia coliE. coli survives in extrahost environments (e.g., soil) and could be dispersed across landscapes by wildlife. Understanding how terrestrial landscapes impact the distribution of soil E. coli strains is of importance in assessing the contamination risk of agricultural products. Here, using multilocus sequence typing, we characterized 938 E. coli soil isolates collected from two watersheds with different landscape patterns in New York State, USA, and compared the distribution of E. coli and the influence that environmental selection and dispersal have on the distribution between these two watersheds. Results showed that for the watershed with widespread produce fields, sparse forests, and limited interaction between the two land use types, E. coli composition was significantly different between produce field sites and forest sites; this distribution appears to be shaped by relatively strong environmental selection, likely from soil phosphorus, and slight dispersal limitation. For the watershed with more forested areas and stronger interaction between produce field sites and forest sites, E. coli composition between these two land use types was relatively homogeneous; this distribution appeared to be a consequence of wildlife-driven dispersal, inferred by competing models. Collectively, our results suggest that terrestrial landscape attributes could impact the biogeographic pattern of enteric bacteria by adjusting the importance of environmental selection and dispersal.IMPORTANCE Understanding the ecology of enteric bacteria in extrahost environments is important for the development and implementation of strategies to minimize preharvest contamination of produce with enteric pathogens. Our findings suggest that watershed landscape is an important factor influencing the importance of ecological drivers and dispersal patterns of E. coli Agricultural areas in such watersheds may have a higher risk of produce contamination due to fewer environmental constraints and higher potential of dispersal of enteric bacteria between locations. Thus, there is a perceived trade-off between priorities of environmental conservation and public health in on-farm food safety, with limited ecological data supporting or refuting the role of wildlife in dispersing pathogens under normal operating conditions. By combining field sampling and spatial modeling, we explored ecological principles underlying the biogeographic pattern of enteric bacteria at the regional level, which can benefit agricultural, environmental, and public health scientists who aim to reduce the risk of food contamination by enteric bacteria while minimizing negative impacts on wildlife habitats.

In Silico Models for Design and Optimization of Science-Based Listeria Environmental Monitoring Programs in Fresh-Cut Produce Facilities.

Food facilities need time- and cost-saving methods during the development and optimization of environmental monitoring for pathogens and their surrogates. Rapid virtual experimentation through in silico modeling can alleviate the need for extensive real-world, trial-and-error style program design. Two agent-based models of fresh-cut produce facilities were developed as a way to simulate the dynamics of Listeria in the built environment by modeling the different surfaces of equipment and employees in a facility as agents. Five sampling schemes at three time points were evaluated in silico on their ability to locate the presence of Listeria contamination in a facility with sample sites for each scheme (i.e., scenario, as modeled using scenario analysis) based on the following: the facilities' current environmental monitoring program (scenario 1), Food and Drug Administration recommendations (scenario 2), random selection (scenario 3), sites exclusively from zone 3 (i.e., sites in the production room but not directly adjacent to food contact surfaces) (scenario 4), or model prediction of elevated risk of contamination (scenario 5). Variation was observed between the scenarios on how well the Listeria prevalence of the virtually collected samples reflected the true prevalence of contaminated agents in the modeled operation. The zone 3 only (scenario 4) and model-based (scenario 5) sampling scenarios consistently overestimated true prevalence across time, suggesting that those scenarios could provide a more sensitive approach for determining if Listeria is present in the operation. The random sampling scenario (scenario 3) may be more useful for operations looking for a scheme that is most likely to reflect the true prevalence. Overall, the developed models allow for rapid virtual experimentation and evaluation of sampling schemes specific to unique fresh-cut produce facilities. IMPORTANCE Programs such as environmental monitoring are used to determine the state of a given food facility with regard to the presence of environmental pathogens, such as Listeria monocytogenes, that could potentially cross-contaminate food product. However, the design of environmental monitoring programs is complex, and there are infinite ways to conduct the sampling that is required for these programs. Experimentally evaluating sampling schemes in a food facility is time-consuming, costly, and nearly impossible. Therefore, the food industry needs science-based tools to aid in developing and refining sampling plans that reduce the risk of harboring contamination. Two agent-based models of two fresh-cut produce facilities reported here demonstrate a novel way to evaluate how different sampling schemes can be rapidly evaluated across multiple time points as a way to understand how sampling can be optimized in an effort to locate the presence of Listeria in a food facility.

Nature versus Nurture: Assessing the Impact of Strain Diversity and Pregrowth Conditions on Salmonella enterica, Escherichia coli, and Listeria Species Growth and Survival on Selected Produce Items.

Inoculation studies are important when assessing microbial survival and growth in food products. These studies typically involve the pregrowth of multiple strains of a target pathogen under a single condition; this emphasizes strain diversity. To gain a better understanding of the impacts of strain diversity ("nature") and pregrowth conditions ("nurture") on subsequent bacterial growth in foods, we assessed the growth and survival of Salmonella enterica (n = 5), Escherichia coli (n = 6), and Listeria (n = 5) inoculated onto tomatoes, precut lettuce, and cantaloupe rind, respectively. Pregrowth conditions included (i) 37°C to stationary phase (baseline), (ii) low pH, (iii) high salt, (iv) reduced water activity, (v) log phase, (vi) minimal medium, and (vii) 21°C. Inoculated tomatoes were incubated at 21°C; lettuce and cantaloupe were incubated at 7°C. Bacterial counts were assessed over three phases, including initial reduction (phase 1), change in bacterial numbers over the first 24 h of incubation (phase 2), and change over the 7-day incubation (phase 3). E. coli showed overall decline in counts (<1 log) over the 7-day period, except for a <1-log increase after pregrowth in high salt and to mid-log phase. In contrast, S. enterica and Listeria showed regrowth after an initial reduction. Pregrowth conditions had a substantial and significant effect on all three phases of S. enterica and E. coli population dynamics on inoculated produce, whereas strain did not show a significant effect. For Listeria, both pregrowth conditions and strain affected changes in phase 2 but not phases 1 and 3.IMPORTANCE Our findings suggest that inclusion of multiple pregrowth conditions in inoculation studies can best capture the range of growth and survival patterns expected for Salmonella enterica and Escherichia coli present on produce. This is particularly important for fresh and fresh-cut produce, where stress conditions encountered by pathogens prior to contamination can vary widely, making selection of a typical pregrowth condition virtually impossible. Pathogen growth and survival data generated using multiple pregrowth conditions will allow for more robust microbial risk assessments that account more accurately for uncertainty.

Recent Evolution and Genomic Profile of Salmonella enterica Serovar Heidelberg Isolates from Poultry Flocks in Brazil.

Salmonella enterica serovar Heidelberg is isolated from poultry-producing regions around the world. In Brazil, S. Heidelberg has been frequently detected in poultry flocks, slaughterhouses, and chicken meat. The goal of the present study was to assess the population structure, recent temporal evolution, and some important genetic characteristics of S. Heidelberg isolated from Brazilian poultry farms. Phylogenetic analysis of 68 S. Heidelberg genomes sequenced here and additional whole-genome data from NCBI demonstrated that all isolates from the Brazilian poultry production chain clustered into a monophyletic group, here called S. Heidelberg Brazilian poultry lineage (SH-BPL). Bayesian analysis defined the time of the most recent common ancestor (tMRCA) as 2004, and the overall population size (Ne) was constant until 2008, when an ∼10-fold Ne increase was observed until circa 2013. SH-BPL presented at least two plasmids with replicons ColpVC (n = 68; 100%), IncX1 (n = 66; 97%), IncA/C2 (n = 65; 95.5%), ColRNAI (n = 43; 63.2%), IncI1 (n = 32; 47%), ColMG828, Col156, IncHI2A, IncHI2, IncQ1, IncX4, IncY, and TrfA (each with n < 4; <4% each). Antibiotic resistance genes were found, with high frequencies of fosA7 (n = 68; 100%), mdf(A) (n = 68; 100%), tet(34) (n = 68; 100%), sul2 (n = 64; 94.1%), and blaCMY-2 (n = 56; 82.3%), along with an overall multidrug resistance (MDR) profile. Ten Salmonella pathogenicity islands (SPI1 to SPI5, SPI9, and SPI11 to SPI14) and 139 virulence genes were also detected. The SH-BPL profile was like those of other previous S. Heidelberg isolates from poultry around the world in the 1990s. In conclusion, the present study demonstrates the recent introduction (2004) and high level of dissemination of an MDR S. Heidelberg lineage in Brazilian poultry operations. IMPORTANCES. Heidelberg is the most frequent serovar in several broiler farms from the main Brazilian poultry-producing regions. Therefore, avian-source foods (mainly chicken carcasses) commercialized in the country and exported to other continents are contaminated with this foodborne pathogen, generating several national and international economic losses. In addition, isolates of this serovar are usually resistant to antibiotics and can cause human invasive and septicemic infection, representing a public health concern. This study demonstrates the use of whole-genome sequencing (WGS) to obtain epidemiological information for one S. Heidelberg lineage highly spread among Brazilian poultry farms. This information will help to define biosecurity measures to control this important Salmonella serovar in Brazilian and worldwide poultry operations.