Rain splash-mediated dispersal of Escherichia coli from fecal deposits to field-grown lettuce in the mid- and south Atlantic U.S. regions is affected by mulch type.

Introduction: Wildlife feces can contaminate vegetables when enteric bacteria are released by rain and splashed onto crops. Regulations require growers to identify and not harvest produce that is likely contaminated, but U.S. federal standards do not define dimensions for no-harvest zones. Moreover, mulching, used to retain soil moisture and maximize crop yield may impact rain-mediated bacterial dispersal from feces. Methods: To assess Escherichia coli dissemination from a fecal point source to lettuce grown on various mulches, lettuce cv. 'Magenta' was transplanted into raised beds with plastic, biodegradable plastic, straw, or left uncovered at field sites in Maryland and Georgia. Eleven days post-transplant, 10 g of rabbit manure spiked with ~8 log CFU g-1 E. coli were deposited in each bed. One day following natural or simulated rain events, lettuce was sampled along 1.5 m transects on either side of fecal deposits. Lettuce-associated E. coli was semi-quantified with an MPN assay and dependence on fecal age (stale or fresh), lettuce age (baby leaf or mature head), distance from point source, mulch and post-rain days were statistically evaluated. Results: Distance (p<0.001), fecal age (p<0.001) and mulch (p<0.01) were factors for E. coli transfer from point source to lettuce. The highest and lowest E. coli estimates were measured from lettuce grown on biodegradable plastic and straw, respectively, with a 2-log MPN difference (p<0.001). Mulch and distance were also significant factors in E. coli recovery 3 days post-rain (both p<0.001), where plastic mulches differed from bare ground and straw (p<0.01). For all treatments, fewer E. coli were retrieved from lettuce at 0.3 m, 3 days post-rain compared to 1 day (p<0.001). Fitting the data to a Weibull Model predicated that a 7-log reduction in E. coli from fecal levels would be achieved at 1.2-1.4 m from the point source on plastic mulches, 0.75 m on bare soil (p<0.05) and 0.43 m on straw (p<0.01). Discussion: Straw and bare ground limited rain-mediated E. coli dispersal from feces to lettuce compared to plastic mulches. Fecal age was negatively associated with E. coli dispersal. These findings can inform harvesting recommendations for measures related to animal intrusion in vegetable production areas.

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.

Longitudinal Survey of Aeromonas hydrophila and Foodborne Pathogens in a Commercial Aquaponics System.

Aquaponic production of fresh produce is a sustainable agricultural method becoming widely adopted, though few studies have investigated potential food safety hazards within commercial systems. A longitudinal study was conducted to isolate and quantify several foodborne pathogens from a commercial, aquaponic farm, and to elucidate their distribution throughout. The survey was conducted over 2 years on a controlled-environment farm containing Nile tilapia (Oreochromis niloticus) and lettuce (Lactuca sativa). Samples (N = 1,047) were collected bimonthly from three identical, independent systems, and included lettuce leaves, roots, fingerlings (7-126 d old), feces from mature fish (>126 d old), water, and sponge swabs collected from the tank interior surface. Most probable number of generic Escherichia coli were determined using IDEXX Colilert Quanti-Tray. Enumeration and enrichment were used to detect Shiga toxin-producing E. coli (STEC), Salmonella enterica, Listeria monocytogenes, Aeromonas spp., Aeromonas hydrophilia, and Pseudomonas aeruginosa. Generic E. coli, STEC, L. monocytogenes, and S. enterica were not detected in collected samples. P. aeruginosa was isolated from water (7/351; 1.99%), swabs (3/351; 0.85%), feces (2/108; 1.85%), and lettuce leaves (2/99; 2.02%). A. hydrophila was isolated from all sample types (623/1047; 59.50%). The incidence of A. hydrophila in water (X2 = 23.234, p < 0.001) and sponge samples (X2 = 21.352, p < 0.001) increased over time.

Prevalence and molecular characterization of Salmonella isolated from wild birds in fresh produce environments.

Wild birds pose a difficult food safety risk to manage because they can avoid traditional wildlife mitigation strategies, such as fences. Birds often use agricultural fields and structures as foraging and nesting areas, which can lead to defecation on crops and subsequent transfer of foodborne pathogens. To assess the food safety risk associated with these events, wild bird feces were collected from produce fields across the southeastern United States during the 2021 and 2022 growing seasons. In total 773 fecal samples were collected from 45 farms across Florida, Georgia, South Carolina, and Tennessee, and 2.1% (n = 16) of samples were Salmonella-positive. Importantly, 75% of Salmonella were isolated from moist feces, showing reduced Salmonella viability when feces dry out. 16S microbiome analysis showed that presence of culturable Salmonella in moist feces correlated to a higher proportion of the Enterobacteriaceae family. From the Salmonella-positive samples, 62.5% (10/16) contained multi-serovar Salmonella populations. Overall, 13 serovars were detected, including six most commonly attributed to human illness (Enteriditis, Newport, Typhimurium, Infantis, Saintpaul, and Muenchen). PCR screening identified an additional 59 Salmonella-positive fecal samples, which were distributed across moist (n = 44) and dried feces (n = 15). On-farm point counts and molecular identification from fecal samples identified 57 bird species, including for 10 Salmonella-positive fecal samples. Overall, there was a low prevalence of Salmonella in fecal samples, especially in dried feces, and we found no evidence of Salmonella transmission to proximal foliage or produce. Fecal samples collected in farms close together shared highly related isolates by whole genome sequencing and also had highly similar Salmonella populations with comparable relative frequencies of the same serovars, suggesting the birds acquired Salmonella from a common source.

16S microbiome analysis of microbial communities in distribution centers handling fresh produce.

Little is known about the microbial communities found in distribution centers (DCs), especially in those storing and handling food. As many foodborne bacteria are known to establish residence in food facilities, it is reasonable to assume that DCs handling foods are also susceptible to pathogen colonization. To investigate the microbial communities within DCs, 16S amplicon sequencing was completed on 317 environmental surface sponge swabs collected in DCs (n = 18) across the United States. An additional 317 swabs were collected in parallel to determine if any viable Listeria species were also present at each sampling site. There were significant differences in median diversity measures (observed, Shannon, and Chao1) across individual DCs, and top genera across all reads were Carnobacterium_A, Psychrobacter, Pseudomonas_E, Leaf454, and Staphylococcus based on taxonomic classifications using the Genome Taxonomy Database. Of the 39 16S samples containing Listeria ASVs, four of these samples had corresponding Listeria positive microbiological samples. Data indicated a predominance of ASVs identified as cold-tolerant bacteria in environmental samples collected in DCs. Differential abundance analysis identified Carnobacterium_A, Psychrobacter, and Pseudomonas_E present at a significantly greater abundance in Listeria positive microbiological compared to those negative for Listeria. Additionally, microbiome composition varied significantly across groupings within variables (e.g., DC, season, general sampling location).

Factors that predict Listeria prevalence in distribution centers handling fresh produce.

Listeria species prevalence has been investigated at nearly all stages of the fresh produce supply chain; however, it has not been examined in transportation and distribution center (DC) related environments. Between December 2019 and March 2021, 18 DCs handling fresh produce were environmentally sampled for Listeria. Swab samples were collected from a variety of surfaces (e.g., floors, pallets, forklifts) in several areas of each DC (e.g., cold storage, shipping and receiving docks). Impaction air samples, relative humidity, and temperature data were also collected. While no Listeria spp. were isolated from air samples (n = 170), they were isolated from 49 of 982 (ca. 5%) environmental samples. The proportion of Listeria spp. positive samples varied significantly across individual DCs (P < 0.01). Several facility characteristics were significantly associated with a Listeria spp. positive sample, such as zone, sampling site dryness, and cleaning regimen. A random forest model (sensitivity: 0.786, specificity: 0.874) identified geographical location and general sampling location (e.g., cold storage rooms, shipping docks) as the two most important variables associated with Listeria spp. detection. This study identified likely harborage sites (e.g., floors, cleaning equipment) of Listeria spp. in DCs across the US and emphasized the importance of sanitation operations in Listeria-prone areas.

The prevalence and concentration of Salmonella enterica in poultry litter in the southern United States.

Poultry litter is applied to crop production land in the southern United States as a waste management strategy as it is a nitrogen-rich fertilizer and plentiful throughout the region. While litter is a known reservoir for human enteric pathogens including Salmonella enterica, little is known regarding pathogen prevalence, concentration, and common serotypes within the material. Litter from thirteen farms across four southern states was examined for Salmonella. Samples (n = 490) from six of the thirteen (46.2%) farms tested positive. Thirty-three samples out of 490 (6.7%) were Salmonella positive. Salmonella was ca. 95% less likely to be collected from stacked litter piles than from the poultry house floor or pasture, and every day increase in litter age reduced the likelihood of recovering Salmonella by 5.1%. When present, concentrations of Salmonella in contaminated poultry litter were variable, ranging from <0.45 to >280,000 MPN/g. The most prevalent serotypes found were Kentucky (45.5%), Kiambu (18.2%), and Michigan (12.1%). Salmonella Kentucky also had the greatest distribution and was found on 4 of the 6 (66.7%) positive farms. Results from this survey demonstrated that Salmonella prevalence and concentration in poultry litter is highly variable, and good agricultural practices are critical to safely use poultry litter as a soil amendment on fresh produce fields.

Prevalence of Listeria monocytogenes and indicator microorganisms in Florida cantaloupe packinghouses, 2013-2014.

Prior to the 2013 cantaloupe season, the US Food and Drug Administration notified the industry that inspections of a subset of packinghouses would commence that year in response to the 2011 Listeria monocytogenes outbreak associated with cantaloupe. In May 2013, five Florida cantaloupe packinghouses participated in an environmental monitoring survey to evaluate their sanitary conditions prior to a potential FDA inspection. Two facilities participated again in 2014. Surface swabs (n = 374) were collected in each facility and included up to 60 food contact and non-food contact surfaces, including water. Samples were enumerated for total plate counts (TPC), generic Escherichia coli, and coliforms, and enriched for Listeria. Listeria were confirmed and speciated by sequencing of the partial sigB gene, and further characterized by pulsed field gel electrophoresis (AscI and Apal). In 2013, two zone 1 surfaces in same facility, were positive for L. monocytogenes (2/233). No L. monocytogenes was detected (n = 103) in the two facilities sampled the following year, including the previously L. monocytogenes-positive facility. Prevalence of L. monocytogenes in FL cantaloupe packinghouses was generally low (2/374), compared to other food environments. TPC, coliforms, E. coli and Listeria spp. were poor indicators of L. monocytogenes contamination in Florida packinghouses.

A Systematic Review of Listeria Species and Listeria monocytogenes Prevalence, Persistence, and Diversity throughout the Fresh Produce Supply Chain.

Listeria monocytogenes is an increasing food safety concern throughout the produce supply chain as it has been linked to produce associated outbreaks and recalls. To our knowledge, this is the first systematic literature review to investigate Listeria species and L. monocytogenes prevalence, persistence, and diversity at each stage along the supply chain. This review identified 64 articles of 4863 candidate articles obtained from four Boolean search queries in six databases. Included studies examined naturally detected/isolated Listeria species and L. monocytogenes in fresh produce-related environments, and/or from past fresh produce associated outbreaks or from produce directly. Listeria species and L. monocytogenes were detected in each stage of the fresh produce supply chain. The greatest prevalence of Listeria species was observed in natural environments and outdoor production, with prevalence generally decreasing with each progression of the supply chain (e.g., packinghouse to distribution to retail). L. monocytogenes prevalence ranged from 61.1% to not detected (0.00%) across the entire supply chain for included studies. Listeria persistence and diversity were also investigated more in natural, production, and processing environments, compared to other supply chain environments (e.g., retail). Data gaps were identified for future produce safety research, for example, in the transportation and distribution center environment.

Postharvest Reduction of Salmonella enterica on Tomatoes Using a Pelargonic Acid Emulsion.

A novel produce wash consisting of pelargonic acid (PEL) emulsions was tested on tomatoes contaminated with a five-serovar Salmonella enterica cocktail. Ability to reduce contamination on the inoculated tomato surface, as well as mitigation of subsequent cross-contamination to uninoculated tomatoes washed in re-used/spent wash water were examined. Sanitizer efficacy was also examined over 1 and 7 d storage time (8 °C, recommended for red ripe tomatoes) and in the presence of 0.5% (w/v) organic load. PEL performed statistically the same (p ≤ 0.05) at both 30 mM and 50 mM concentrations and resulted in greater than 1, 5 and 6 log CFU/g Salmonella reductions at 0 h, 1 d and 7 d, respectively, when compared to a water-only or no rinse (NR) treatment. This was also a significantly greater reduction than was observed due to chlorine (sodium hypochlorite) and peroxyacetic acid (PAA) at all time points (p ≤ 0.01). Organic load had no impact on sanitizer efficacy for all examined treatments. Finally, PEL had a deleterious impact on tomato texture. At 1 d, ca. 5 N and 7 N were required to achieve tomato skin penetration and compression, respectively, compared to >9 N and 15 N required by all other treatments (p ≤ 0.05). While PEL sanitizers effectively reduced inoculated Salmonella and subsequent transfer to uninoculated tomatoes, reformulation may be necessary to prevent deleterious quality impacts on produce.

Transcriptomic Behavior of Salmonella enterica Newport in Response to Oxidative Sanitizers.

ABSTRACT: Agricultural water is a known source of contamination to fresh produce and can contain foodborne pathogens including Salmonella enterica, pathogenic Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni. To mitigate such risks, antimicrobial agents such as hypochlorites and peroxyacetic acid (PAA) can be applied to in-line irrigation systems as well as to water used in postharvest washing. Although these compounds are effective and widely used, some pathogenic bacteria adapt to survive exposure. RNA sequencing was used to analyze the Salmonella Newport transcriptome after exposure to sodium hypochlorite (NaOCl) and PAA in a simulated agricultural water system. Overall cellular adaptive response was determined quantitatively as a function of overall gene expression of the >4,000 genes in the Salmonella Newport genome. Differentially expressed genes ranged from 11 due to 10-ppm NaOCl treatment, 316 due to 20-ppm NaOCl treatment, 1,719 due to 10-ppm PAA treatment, and 2,010 due to 20-ppm PAA treatment compared with that of the controls (water only). Differentially expressed transcripts included cellular functions such as biosynthesis, degradation, energy generation, and nonmetabolically linked functions. Oxidative exposure upregulated genes associated with key virulence, attachment, and gene transfer. Amino acid biosynthesis was upregulated due to NaOCl exposure but primarily downregulated when Salmonella Newport was exposed to PAA. Slight upregulation occurred in nucleoside and nucleotide biosynthesis, a known DNA repair mechanism seen during exposure to sanitizers. Our results indicate that Salmonella Newport reacts differently when exposed to NaOCl versus PAA, despite oxidative activity being the primary modes of antimicrobial action of both compounds.

Reduction of Escherichia coli, as a surrogate for Salmonella spp., on the surface of grapefruit during various packingline processes.

The US Produce Safety Rule allows for use of water that does not meet its microbial standards if corrective measures are employed. This research was initiated to determine the suitability of nonpathogenic Escherichia coli as a surrogate for Salmonella during citrus washing, and to evaluate the removal of E. coli from grapefruit on two pilot packinglines (CREC and IRREC) as corrective measures. Whole grapefruit were inoculated with either E. coli or Salmonella and dried, and exposed to a variety of treatments on a lab-scale brush wash system. Individual processes were evaluated on the pilot packinglines with E. coli only. In all lab-scale brush wash system treatments, bacterial population reductions between E. coli and Salmonella were not significantly different (P ≤ 0.05). On pilot packinglines, E. coli populations were reduced by 3.59 to >5.11 log CFU/grapefruit at the CREC packingline, and by 3.30 to >5.13 log CFU/grapefruit at the IRREC packingline. Treatment of fruit through complete packingline processing at both locations reduced E. coli populations to levels below the detection limit (<1 log CFU/grapefruit). The studies indicate E. coli is an appropriate surrogate for Salmonella under tested conditions, and that standard citrus packingline processes can be used as a corrective measure.

Antimicrobial Efficacy of an Array of Essential Oils Against Lactic Acid Bacteria.

The essential oils of clove bud, cinnamon bark and thyme, and their individual compounds including allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde, cinnamic acid, eugenol, and thymol were initially assessed for antimicrobial activity against 9 lactic acid bacteria (LAB) species. Carvacrol and thymol were the most inhibitory with MICs of 0.1% (v/v and w/v, respectively). Cinnamaldehyde, cinnamon bark oil, clove bud oil, eugenol, and thyme oil were moderately inhibitive (MICs = 0.2% v/v), while cinnamic acid required a concentration of 0.5% (w/v). AIT was not effective with MICs in excess of concentrations tested (0.75% v/v). The bactericidal capability of the oil components carvacrol, cinnamaldehyde, eugenol, and thymol were further examined against Pediococcus acidilactici, Lactobacillus buchneri, and Leuconostoc citrovorum. Thymol at 0.1% (w/v) was bactericidal against L. citrovorum (>4-log reduction), but resulted in a 2-log CFU/mL reduction against L. buchneri and P. acidilactici. Cinnamaldehyde at 0.2% to 0.25% (v/v) was effective against L. citrovorum, L. buchneri, and P. acidilactici, resulting in a >2-log reduction. All 3 organisms were susceptible to 0.2% carvacrol with >3-log reduction observed after exposure for 6 h. Eugenol was the least effective. Concentrations of 0.2% and 0.25% (v/v) were needed to achieve an initial reduction in population, >3-log CFU/mL after 6 h exposure. However, at 0.2%, P. acidilactici and L. buchneri recovered to initial populations in 48 to 72 h. Results indicate essential oils have the capacity to inactivate LAB that are commonly associated with spoilage of shelf stable low-acid foods.