Southern Region Produce Safety Alliance Grower Training: Using Pre- and Post-Training Knowledge Assessments to Understand Training Effectiveness.

The Produce Safety Alliance (PSA) grower training was introduced in 2016 as the standardized curriculum to meet the training requirements of the Food and Drug Administration's (FDA) Food Safety Modernization Act's (FSMA) Produce Safety Rule (PSR). The PSR states that at least one supervisor or responsible party from each farm must have successfully completed this food safety training or one equivalent to the standardized curriculum, as recognized by the FDA. This study evaluated the effectiveness of PSA trainings conducted between 2017 and 2019 in the Southern United States by the Southern Regional Center for Food Safety Training, Outreach, and Technical Assistance by analyzing pre- and posttest assessments. Effectiveness was based on a 25-question knowledge assessment administered to participants before (n = 2494) and after (n = 2460) each training. The knowledge assessment indicated the overall effectiveness of the training, with average scores increasing significantly from pretest (15.9/25, 63.4%) to posttest (20.3/25, 81.3%) (P < 0.001). The greatest knowledge gains were seen in the Postharvest Handling and Sanitation, How to Develop a Farm Food Safety Plan, and Agricultural Water modules. Notably, these modules had lower posttest scores compared to the other modules, indicating that the amount of knowledge gained did not necessarily correspond with a sufficient understanding of the material. To ensure that participants understand all aspects of the PSR and best practices to minimize food safety risks, additional or advanced trainings may be needed. Additionally, the current testing instrument (pre-/posttest) used for PSA grower training, while validated, may not be optimal, thus alternative methods to assess the training effectiveness are likely needed.

Modeling the formulation pH of elderberry syrup with multiple weak acids.

The objective of this work was to develop methods to assess the influence of the ingredients of an acidified elderberry syrup on product pH. A measure of total ingredient buffering (tBeta) was defined as the area under the buffer capacity curve of a food mixture or ingredient for pH 2-12. Citric acid (1% w/v), elderberry juice (75% v/v), and malic acid (0.75% w/v) had greater buffering (tBeta values of 15.33, 12.00, and 10.95, respectively) than ascorbic acid (0.75%) or lemon juice (3% v/v) (tBeta of 5.74 and 3.30, respectively). All other ingredients, including added spices (≤1% each) and honey (25% w/v), had tBeta values <2. The observed pH for the syrup mixture (pH 2.67) was within 0.11 pH units of the predicted pH based on combined buffer models of the acid and low acid ingredients (pH 2.78) using Matlab software. A total of 16 model syrup formulations containing elderberry juice with mixed acids (malic, acetic, and ascorbic) and having pH values between 3 and 4 were prepared. The pH values of the formulations were compared to predicted values from combined buffer models of the individual ingredients. Regression analysis indicated an excellent fit of the observed and predicted pH data, with a root mean square error of 0.076 pH units. The results indicated that buffer models may be useful for in silico estimates of how the ingredients in acid and acidified foods may influence pH, thus aiding in product development and safety assessments. PRACTICAL APPLICATION: Buffer models using recently developed titration methods for individual acid and low-acid food ingredients can be used to estimate the pH of formulations of these ingredients in silico. The total buffering (tBeta) for ingredients or mixtures, along with ingredient concentrations, may be a useful metric for helping to determine which ingredients will have the greatest impact on pH. Such models can aid product development efforts and safety assessments.

Microbial Load of Fresh Produce and Paired Equipment Surfaces in Packing Facilities Near the U.S. and Mexico Border.

Several produce-associated outbreaks have been linked to the packing facility. Equipment surfaces may be an important source of contamination. The goal was to assess whether the microbial load of packing facility surfaces is associated with the microbial load of produce. From November 2000 to December 2003, 487 matched produce (14 types) and equipment surfaces (six production steps) were sampled from eight packing facilities in the United States near the border with Mexico and enumerated for aerobic plate counts (APC), Escherichia coli , Enterococcus, and coliforms. Bivariate correlations were assessed by Spearman's ρ, and adjusted associations were assessed by multilevel mixed linear regression models. In general, the microbial load both increased and decreased on produce (0.2 to 1.0 log CFU/g) and equipment surfaces (0.5 to 3.0 log CFU/cm2) across production steps. Equipment surface and produce microbial loads were correlated, but correlations varied from none to high depending on the equipment surface. For example, significant correlations (P < 0.01) included APC (ρ = 0.386) and Enterococcus (ρ = 0.562) with the harvest bin, E. coli (ρ = 0.372) and Enterococcus (ρ = 0.355) with the merry-go-round, Enterococcus (ρ = 0.679) with rinse cycle equipment, APC (ρ = 0.542) with the conveyer belt, and for all indicators with the packing box (ρ = 0.310 to 0.657). After controlling for crop type, sample replicate group, and sample location, there were significant positive associations between the log concentration of Enterococcus on produce and the harvest bin (ß = 0.259, P < 0.01) and the rinse cycle (ß = 0.010, P = 0.01), and between the log concentration of all indicators on produce and the packing box (ß = 0.155 to 0.500, all P < 0.01). These statistically significant associations between microbial loads on packing facility surfaces and fresh produce confirm the importance of packing facility sanitation to protect produce quality and safety.

Associations between weather and microbial load on fresh produce prior to harvest.

Contaminated produce causes approximately 1 million cases of foodborne illness and 1 billion dollars in damages to the U.S. economy annually. The environmental conditions, especially weather, that influence the inoculation, proliferation, and dispersal of microbial load on produce are not well understood. Using a mixed models approach, we examined the relationship of temperature and precipitation to microbial indicators of contamination on fresh produce on the farm over a week-long period prior to harvest. Between 2000 and 2002, we assayed for four microbial indicators of contamination (aerobic plate count, Enterococcus, total coliforms, and Escherichia coli) on 10 produce types in 15 fields in the southern United States. The sample collection times varied, with most occurring between January and May. We collected hourly weather data for the corresponding time period and location. Our results indicated that there was a significant association between the average daily temperature (20°C) and both log aerobic plate count (e.g., an increase of 0.074 log CFU/g [standard error {SE}, 0.023] per °C increase in weekly average temperature) and log Enterococcus (e.g., an increase of 0.15 log CFU/g [SE, 0.031] per °C increase in weekly average temperature) for approximately 5 days prior to sample collection. Daily total precipitation was significantly associated with log coliforms on 2 days (∼0.11 log CFU/g [SE, 0.06] per mm of precipitation) during the week-long lag period prior to harvest. Our results suggest that microbial indicator concentrations may increase as the temperature increases. Precipitation may have a positive but complex relationship with microbial indicators, as precipitation may create moist conditions conducive to bacterial growth, spread contamination onto the field, or wash contamination off of the plant.

Microbial concentrations on fresh produce are affected by postharvest processing, importation, and season.

In the United States, the proportion of foodborne illness outbreaks associated with consumption of contaminated domestic and imported fresh fruits and vegetables (produce) has increased over the past several decades. To address this public health concern, the goal of this work was to identify and quantify factors associated with microbial contamination of produce in pre- and postharvest phases of the farm-to-fork continuum. From 2000 to 2003, we collected 923 samples of 14 types of produce (grown in the southern United States or in the northern border states of Mexico) from 15 farms and eight packing sheds located in the southern United States. To assess microbial quality, samples were enumerated for Escherichia coli, total aerobic bacteria, total coliforms, and total Enterococcus. Most produce types had significantly higher microbial concentrations when sampled at the packing shed than when sampled at the farm. In addition, we observed seasonal differences in the microbial concentrations on samples grown in the United States, with higher mean indicator concentrations detected in the fall (September, October, and November). We developed a predictive, multivariate logistic regression model to identify and quantify factors that were associated with detectable concentrations of E. coli contamination on produce. These factors included produce type (specifically, cabbage or cantaloupe), season of collection (harvested in the fall), and packing step (bin, box, conveyor belt, or turntable). These results can be used to identify specific mechanisms of produce contamination and propose interventions that may decrease the likelihood of produce-associated illness.

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA.

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

A field study of the microbiological quality of fresh produce of domestic and Mexican origin.

Produce is responsible for an increasingly larger proportion of foodborne disease outbreaks. In particular, the globalization of the food supply may introduce new food safety risks and allow widespread distribution of contaminated food, particularly produce. The objectives of this study were to: (i) compare the overall quality of domestic and Mexican produce throughout the packing process; (ii) examine changes in microbiological quality of both domestic and Mexican produce at each stage of production and processing; and (iii) evaluate the prevalence of select pathogens on fresh produce, including leafy green, herbs, melons, and vegetables. Furthermore, we also sought to characterize the antibiotic resistance profiles of Enterococcus faecium and Enterococcus faecalis strains isolated from fresh produce. A total of 466 produce and matching environmental swab samples was collected from various locations in packing sheds in the southern US from November 2002 through December 2003. These samples were assayed by enumerative tests for total aerobic bacteria (APC), total coliforms, total Enterococcus, and E. coli. Produce samples were also analyzed for the presence of Salmonella, Listeria monocytogenes, Shigella, and E. coli O157:H7. A total of 112 E. faecium and E. faecalis isolates were further screened for antibiotic resistance using a panel of seventeen antibiotics. Overall, the microbiological quality of fresh produce ranged from 4.0 to 7.9 log(10) CFU/g (APC); less than 1.0 log(10) to 4.5 log(10) CFU/g (coliforms); less than 1.0 log(10) to 4.0 log(10) CFU/g (E. coli); and less than 1.0 log(10) to 5.4 log(10) CFU/g (Enterococcus). No Salmonella, Shigella, or E. coli O157:H7 were detected from the 466 25-g produce samples tested. However, three domestic cabbage samples were found to be positive for L. monocytogenes. Of the Enterococcus isolates, E. faecium had a higher degree of resistance to antibiotics in general, while Enterococcus spp. isolated from Mexican produce had a higher degree of antibiotic resistance when compared to strains isolated from produce samples of domestic origin. Despite increased attention to the role of imported produce in foodborne disease, this study does not support the assumption that domestic produce is of higher microbial quality than Mexican produce.

A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR.

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.

A field study of the microbiological quality of fresh produce.

The Centers for Disease Control and Prevention has reported that foodborne disease outbreaks associated with fruits and vegetables increased during the past decade. This study was conducted to characterize the routes of microbial contamination in produce and to identify areas of potential contamination from production through postharvest handling. We report here the levels of bacterial indicator organisms and the prevalence of selected pathogens in produce samples collected from the southern United States. A total of 398 produce samples (leafy greens, herbs, and cantaloupe) were collected through production and the packing shed and assayed by enumerative tests for total aerobic bacteria, total coliforms, total Enterococcus, and Escherichia coli. These samples also were analyzed for Salmonella, Listeria monocytogenes, and E. coli O157:H7. Microbiological methods were based on methods recommended by the U.S. Food and Drug Administration. For all leafy greens and herbs, geometric mean indicator levels ranged from 4.5 to 6.2 log CFU/g (aerobic plate count); less than 1 to 4.3 log CFU/g (coliforms and Enterococcus); and less than 1 to 1.5 log CFU/g (E. coli). In many cases, indicator levels remained relatively constant throughout the packing shed, particularly for mustard greens. However, for cilantro and parsley, total coliform levels increased during the packing process. For cantaloupe, microbial levels significantly increased from field through packing, with ranges of 6.4 to 7.0 log CFU/g (aerobic plate count); 2.1 to 4.3 log CFU/g (coliforms); 3.5 to 5.2 log CFU/g (Enterococcus); and less than 1 to 2.5 log CFU/g (E. coli). The prevalence of pathogens for all samples was 0, 0, and 0.7% (3 of 398) for L. monocytogenes, E. coli O157:H7, and Salmonella, respectively. This study demonstrates that each step from production to consumption may affect the microbial load of produce and reinforces government recommendations for ensuring a high-quality product.

Antimicrobial resistance of Enterococcus species isolated from produce.

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.