Methodological differences between studies confound one-size-fits-all approaches to managing surface waterways for food and water safety.

Even though differences in methodology (e.g., sample volume and detection method) have been shown to affect observed microbial water quality, multiple sampling and laboratory protocols continue to be used for water quality monitoring. Research is needed to determine how these differences impact the comparability of findings to generate best management practices and the ability to perform meta-analyses. This study addresses this knowledge gap by compiling and analyzing a data set representing 2,429,990 unique data points on at least one microbial water quality target (e.g., Salmonella presence and Escherichia coli concentration). Variance partitioning analysis was used to quantify the variance in likelihood of detecting each pathogenic target that was uniquely and jointly attributable to non-methodological versus methodological factors. The strength of the association between microbial water quality and select methodological and non-methodological factors was quantified using conditional forest and regression analysis. Fecal indicator bacteria concentrations were more strongly associated with non-methodological factors than methodological factors based on conditional forest analysis. Variance partitioning analysis could not disentangle non-methodological and methodological signals for pathogenic Escherichia coli, Salmonella, and Listeria. This suggests our current perceptions of foodborne pathogen ecology in water systems are confounded by methodological differences between studies. For example, 31% of total variance in likelihood of Salmonella detection was explained by methodological and/or non-methodological factors, 18% was jointly attributable to both methodological and non-methodological factors. Only 13% of total variance was uniquely attributable to non-methodological factors for Salmonella, highlighting the need for standardization of methods for microbiological water quality testing for comparison across studies.IMPORTANCEThe microbial ecology of water is already complex, without the added complications of methodological differences between studies. This study highlights the difficulty in comparing water quality data from projects that used different sampling or laboratory methods. These findings have direct implications for end users as there is no clear way to generalize findings in order to characterize broad-scale ecological phenomenon and develop science-based guidance. To best support development of risk assessments and guidance for monitoring and managing waters, data collection and methods need to be standardized across studies. A minimum set of data attributes that all studies should collect and report in a standardized way is needed. Given the diversity of methods used within applied and environmental microbiology, similar studies are needed for other microbiology subfields to ensure that guidance and policy are based on a robust interpretation of the literature.

Assessment of Contamination Risk from Fecal Matter Presence on Fruit and Mulch in the tomato fields based on generic Escherichia coli population.

The purpose of this study was to evaluate the fate of generic Escherichia coli in fecal pats under different field conditions and to predict Salmonella and Shiga toxin-producing E. coli (STEC) survival dynamics based on developed models. Eight trials were conducted during spring and fall in both North and Central Florida. Fresh cattle feces (1g) was placed on mature green round tomatoes located inside (TIP) and outside (TOP) of the plant canopy. Fecal pats (10 g) were placed under (MUP) and distant (MDP) to tomato plants on plastic mulch. Pathogen populations were predicted based on developed models. Declines in generic E. coli populations over 7 days (Log CFU/g) in fecal pats were between 0.9 and 2.7 on TIP, 1.2 and 3.0 on TOP, 0.2 and 1.2 on MUP, and 0.4 and 1.5 on MDP in the Central Florida fall and spring trials, respectively. E. coli populations remained stable at ≤4.2 and ≤ 6.5 Log CFU/g during all North Florida trials. The concentration changes in predicted Salmonella and STEC population were less than 2.1 Log CFU/g in fecal pats for all conditions. Developed models predicted similar pathogen survival trends to generic E. coli with no dramatical impact under the field conditions.

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.

Parsimonious Mechanistic Modeling of Bacterial Runoff into Irrigation Ponds To Inform Food Safety Management of Agricultural Water Quality.

Pond irrigation water comprises a major pathway of pathogenic bacteria to fresh produce. Current regulatory methods have been shown to be ineffective in assessing this risk when variability of bacterial concentrations is large. This paper proposes using mechanistic modeling of bacterial transport as a way to identify improved strategies for mitigating this risk pathway. If the mechanistic model is successfully tested against observed data, global sensitivity analysis (GSA) can identify important mechanisms to inform alternative, preventive bacterial control practices. Model development favored parsimony and prediction of peak bacterial concentration events. Data from two highly variable surface water irrigation ponds showed that the model performance was similar or superior to that of existing pathogen transport models, with a Nash-Sutcliffe efficiency of 0.48 and 0.18 for the two ponds. GSA quantified bacterial sourcing and hydrology as the most important processes driving pond bacterial contamination events. Model analysis has two main implications for improved regulatory methods: that peak concentration events are associated with runoff-producing rainfall events and that intercepting bacterial runoff transport may be the best option to prevent bacterial contamination of surface water irrigation ponds and thus fresh produce. This research suggests the need for temporal management strategies. IMPORTANCE Preventive management of agricultural waters requires understanding of the drivers of bacterial contamination events. We propose mechanistic modeling as a way forward to understand and predict such events and have developed and tested a parsimonious model for rain-driven surface runoff contributing to generic Escherichia coli contamination of irrigation ponds in Central Florida. While the model was able to predict the timing of peak events reasonably well, the highly variable magnitude of the peaks was less well predicted. This indicates the need to collect more data on the fecal contamination inputs of these ponds and the use of mechanistic modeling and global sensitivity analysis to identify the most important data needs.

Assessment of the Effect of Thermotherapy on 'Candidatus Liberibacter asiaticus' Viability in Woody Tissue of Citrus via Graft-Based Assays and RNA Assays.

In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Thermotherapy for HLB-affected trees was proposed as a short-term management solution to maintain field productivity. It was hypothesized that thermotherapy could eliminate HLB from affected branches; therefore, the study objectives were to show which time-temperature combinations eliminated CLas from woody tissues. Hardening, rounded Valencia twigs collected from HLB-affected field trees were treated in a steam chamber at different time-temperature combinations (50°C for 60 s; 55°C for 0, 30, 60, 90, and 120 s; 60°C for 30 s; and an untreated control). Three independent repetitions of 13 branches per treatment were grafted onto healthy rootstocks and tested to detect CLas after 6, 9, and 12 months. For the RNA-based CLas viability assay, three branches per treatment were treated and bark samples were peeled for RNA extraction and subsequent gene expression analyses. During the grafting study, at 12 months after grafting, a very low frequency of trees grafted with twigs treated at 55°C for 90 s and 55°C for 120 s had detectable CLas DNA. In the few individuals with CLas, titers were significantly lower (P ≤ 0.0001) and could have been remnants of degrading DNA. Additionally, there was a significant decrease (P ≤ 0.0001) in CLas 16S rRNA expression at 55°C for 90 s, 55°C for 120 s, and 60°C for 30 s (3.4-fold change, 3.4-fold change, and 2.3-fold change, respectively) in samples 5 days after treatment. Heat injury, not total CLas kill, could explain the limited changes in transcriptional activity; however, failed recovery and eventual death of CLas resulted in no CLas detection in most of the grafted trees treated with the highest temperatures or longest durations.

Planar Interdigitated Aptasensor for Flow-Through Detection of Listeria spp. in Hydroponic Lettuce Growth Media.

Irrigation water is a primary source of fresh produce contamination by bacteria during the preharvest, particularly in hydroponic systems where the control of pests and pathogens is a major challenge. In this work, we demonstrate the development of a Listeria biosensor using platinum interdigitated microelectrodes (Pt-IME). The sensor is incorporated into a particle/sediment trap for the real-time analysis of irrigation water in a hydroponic lettuce system. We demonstrate the application of this system using a smartphone-based potentiostat for rapid on-site analysis of water quality. A detailed characterization of the electrochemical behavior was conducted in the presence/absence of DNA and Listeria spp., which was followed by calibration in various solutions with and without flow. In flow conditions (100 mL samples), the aptasensor had a sensitivity of 3.37 ± 0.21 k log-CFU-1 mL, and the LOD was 48 ± 12 CFU mL-1 with a linear range of 102 to 104 CFU mL-1. In stagnant solution with no flow, the aptasensor performance was significantly improved in buffer, vegetable broth, and hydroponic media. Sensor hysteresis ranged from 2 to 16% after rinsing in a strong basic solution (direct reuse) and was insignificant after removing the aptamer via washing in Piranha solution (reuse after adsorption with fresh aptamer). This is the first demonstration of an aptasensor used to monitor microbial water quality for hydroponic lettuce in real time using a smartphone-based acquisition system for volumes that conform with the regulatory standards. The aptasensor demonstrated a recovery of 90% and may be reused a limited number of times with minor washing steps.

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.

Modeling the risk of salmonellosis from consumption of pistachios produced and consumed in the United States.

The risk of salmonellosis from consumption of pistachios produced and consumed in the U.S. was assessed through quantitative microbial risk assessment. Data on Salmonella prevalence and concentration on pistachios, nut crop volume, storage times and temperatures during processing and handling, and reductions during storage or from roasting were derived from laboratory experiments, published literature, and industry expert opinion. Uncertainty was analyzed via what-if scenarios for Salmonella prevalence, concentration, storage reduction, treatment variability, portion of crop treated, and increased consumption. The estimated U.S. incidence of salmonellosis when 100% of pistachios were exposed to a 4 ± 0 log reduction treatment averaged 1.4 cases per billion servings, or <1 case/year, without considering Salmonella decline during storage. Including Salmonella decline during storage reduced the salmonellosis estimates approximately 10-fold. The predicted arithmetic mean number of cases associated with individual 500,000-kg storage silos, contaminated at the highest observed levels, ranged from 5 to 530 when the product was consumed untreated, but was reduced to below 1 case per silo when a 4 ± 0 log reduction treatment was applied. Assuming a uniform 4-log reduction treatment is applied to 100% of the crop and there is no decline of Salmonella during storage, the assessment indicates the following: 10-fold increases in either Salmonella prevalence or concentration, 2-fold increases in both prevalence and concentration, or consumption of >0.05% of untreated product volume yield an arithmetic mean risk of >1 case/year.

Development of an Avirulent Salmonella Surrogate for Modeling Pathogen Behavior in Pre- and Postharvest Environments.

UNLABELLED: Recurrent outbreaks of bacterial gastroenteritis linked to the consumption of fresh fruits and vegetables highlight the paucity of understanding of the ecology of Salmonella enterica under crop production and postharvest conditions. These gaps in knowledge are due, at least in part, to the lack of suitable surrogate organisms for studies for which biosafety level 2 is problematic. Therefore, we constructed and validated an avirulent strain of Salmonella enterica serovar Typhimurium. The strain lacks major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 as well as the virulence plasmid pSLT. Deletions and the absence of genomic rearrangements were confirmed by genomic sequencing, and the surrogate behaved like the parental wild-type strain on selective media. A loss-of-function (phoN) selective marker allowed the differentiation of this strain from wild-type strains on a medium containing a chromogenic substrate for alkaline phosphatase. Lack of virulence was confirmed by oral infection of female BALB/c mice. The strain persisted in tomatoes, cantaloupes, leafy greens, and soil with the same kinetics as the parental wild-type and selected outbreak strains, and it reached similar final population levels. The responses of this strain to heat treatment and disinfectants were similar to those of the wild type, supporting its potential as a surrogate for future studies on the ecology and survival of Salmonella in production and processing environments. IMPORTANCE: There is significant interest in understanding the ecology of human pathogens in environments outside of their animal hosts, including the crop production environment. However, manipulative field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks. Therefore, we constructed an avirulent strain of S. enterica serovar Typhimurium and characterized it extensively.

Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States.

Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.

Evaluation of aqueous and alcohol-based quaternary ammonium sanitizers for inactivating Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes on peanut and pistachio shells.

This study evaluated the efficacy of aqueous (aQUAT) and isopropyl alcohol-based quaternary ammonium (ipQUAT) sanitizers for reducing Salmonella spp., Escherichia coli O157:H7, or Listeria monocytogenes populations on peanut and pistachio shell pieces. Inoculated nutshells were mixed with QUAT sanitizers, water, or 70% ethanol and enumerated immediately or after incubation at 30 °C for 48 h. None of the treatments had any immediate effect on Salmonella or E. coli O157:H7 populations on the peanut or pistachio shells. L. monocytogenes populations declined immediately on the peanut and pistachio shells treated with aQUAT or ipQUAT. After incubation, Salmonella and E. coli O157:H7 populations increased significantly on the water- or aQUAT-treated peanut and pistachio shells. L. monocytogenes populations also increased significantly on the water- or aQUAT-treated peanut shells, but levels did not change on the water-treated pistachio shells and levels were just above the limit of detection on the aQUAT-treated pistachio shells. After treatment with ipQUAT and 48-h incubation, Salmonella and E. coli O157:H7 populations decreased to or below the limit of detection on both shell types; L. monocytogenes populations remained at or below the limit of detection on both shell types.

Cross contamination of Escherichia coli O157:H7 between lettuce and wash water during home-scale washing.

Lettuce and leafy greens have been implicated in multiple foodborne disease outbreaks. This study quantifies cross contamination between lettuce pieces in a small-scale home environment. A five-strain cocktail of relevant Escherichia coli O157:H7 strains was used. Bacterial transfer between single inoculated lettuce leaf pieces to 10 non-inoculated lettuce leaf pieces that were washed in a stainless steel bowl of water for 30 s, 1 min, 2 min, and 5 min was quantified. Regardless of washing time, the wash water became contaminated with 90-99% of bacteria originally present on the inoculated lettuce leaf piece. The E. coli O157:H7 concentration on initially inoculated leaf pieces was reduced ∼ 2 log CFU. Each initially uncontaminated lettuce leaf piece had ∼ 1% of the E. coli O157:H7 from the inoculated lettuce piece transferred to it after washing, with more transfer occurring during the shortest (30 s) and longest (5 min) wash times. In all cases the log percent transfer rates were essentially normally distributed. In all scenarios, most of the E. coli O157:H7 (90-99%) transferred from the inoculated lettuce pieces to the wash water. Washing with plain tap water reduces levels of E. coli O157:H7 on the inoculated lettuce leaf pieces, but also spreads contamination to previously uncontaminated leaf pieces.

Diversity of Salmonella isolates from central Florida surface waters.

Identification of Salmonella serotypes is important for understanding the environmental diversity of the genus Salmonella. This study evaluates the diversity of Salmonella isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental Salmonella isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608-3615, 2006, http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562 Salmonella isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three Salmonella enterica serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:-. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella serotype.

Distributions of Salmonella subtypes differ between two U.S. produce-growing regions.

Salmonella accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify Salmonella diversity and aid in identification of Salmonella contamination sources, we characterized Salmonella isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the Salmonella serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were Salmonella positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional Salmonella diversity associated with production environments, we collected a smaller set of samples (n = 65) from South Florida (SFL) production environments and compared the Salmonella diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were Salmonella positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were Salmonella enterica serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were Salmonella serovars Saphra and Newport and S. enterica subsp. diarizonae serovar 50:r:z. High PFGE type diversity (Simpson's diversity index, 0.90 ± 0.02) was observed among Salmonella isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was <0.000001; therefore, Salmonella isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of Salmonella in production environments.

Modeling the growth of Listeria monocytogenes on cut cantaloupe, honeydew and watermelon.

A recent outbreak linked to whole cantaloupes underscores the importance of understanding growth kinetics of Listeria monocytogenes in cut melons at different temperatures. Whole cantaloupe, watermelon, and honeydew purchased from a local supermarket were cut into 10 ± 1 g cubes. A four-strain cocktail of L. monocytogenes from food related outbreaks was used to inoculate fruit, resulting in ~10(3) CFU/10 g. Samples were stored at 4, 10, 15, 20, or 25 °C and L. monocytogenes were enumerated at appropriate time intervals. The square root model was used to describe L. monocytogenes growth rate as a function of temperature. The model was compared to prior models for Salmonella and Escherichia coli O157:H7 growth on cut melon, as well as models for L. monocytogenes on cantaloupe and L. monocytogenes ComBase models. The current model predicts faster growth of L. monocytogenes vs. Salmonella and E. coli O157:H7 at temperatures below 20 °C, and agrees with estimates from ComBase Predictor, and a corrected published model for L. monocytogenes on cut cantaloupe. The model predicts ~4 log CFU increase following 15 days at 5 °C, and ∼1 log CFU increase following 6 days at 4 °C. The model can also be used in subsequent quantitative microbial risk assessments.

Salmonella and Listeria monocytogenes survival on Field Packed Cantaloupe Contact Surfaces.

Field-packing of cantaloupes involves numerous food contact surfaces that can contamination melons with foodborne pathogens; the soil on these surfaces increases throughout the harvest day. Data is lacking on the cross-contamination risk from contaminated food contact surfaces under the dry conditions typical of cantaloupe field-packing operations. This study sought to evaluate the survival of Salmonella and Listeria monocytogenes on cantaloupe field-pack food contact surfaces using both a wet and dry inoculum to provide insights into managing foodborne pathogen contamination risks. Five clean or fouled materials (cotton gloves, nitrile gloves, rubber gloves, cotton rags, and stainless steel) were inoculated with a cocktail of either Salmonella or L. monocytogenes. A wet inoculum was spot inoculated (100 µL) onto coupons. A dry inoculum was prepared by mixing wet inoculum with 100 g of sterile sand, and shaking the coupons with the inoculated sand for 2min. Coupons were held at 35°C (35% RH) and enumerated at 0, 2, 4, 6 and 8 h. Significant differences in pathogen concentrations over time were calculated and the GInaFiT add-in tool for Excel was used to build Log-linear, Weibull, and Biphasic die-off models. Depending on the material type, coupon condition, and inoculum type, Salmonella and L. monocytogenes reductions over 8 h ranged from 0.3-3.3 and -0.4-4.2 log10 CFU/coupon, respectively. For all material types, Salmonella reductions were highest on wet-inoculated clean coupons; L. monocytogenes varied by material type. Weibull and biphasic models were a better fit of respective pathogen die-off curves than linear models. Overall, faster die-off rates were seen for wet inoculated and clean materials. Since pathogen populations remained viable over the study duration and both inoculum type and coupon condition impacted survival, frequent sanitation or replacement of food contact surfaces during the operational day is needed to reduce the risk of cross-contamination.

Factors Associated with the Prevalence of Salmonella, Generic Escherichia coli, and Coliforms in Florida's Agricultural Soils.

Limited data exist on the environmental factors that impact pathogen prevalence in the soil. The prevalence of foodborne pathogens, Salmonella and Listeria monocytogenes, and the prevalence and concentration of generic E. coli in Florida's agricultural soils were evaluated to understand the potential risk of microbial contamination at the preharvest level. For all organisms but L. monocytogenes, a longitudinal field study was performed in three geographically distributed agricultural areas across Florida. At each location, 20 unique 5 by 5 m field sampling sites were selected, and soil was collected and evaluated for Salmonella presence (25 g) and E. coli and coliform concentrations (5 g). Complementary data collected from October 2021 to April 2022 included: weather; adjacent land use; soil properties, including macro- and micro-nutrients; and field management practices. The overall Salmonella and generic E. coli prevalence was 0.418% (1/239) and 11.3% (27/239), respectively; with mean E. coli concentrations in positive samples of 1.56 log CFU/g. Farm A had the highest prevalence of generic E. coli, 22.8% (18/79); followed by Farm B, 10% (8/80); and Farm C 1.25% (1/80). A significant relationship (p < 0.05) was observed between generic E. coli and coliforms, and farm and sampling trip. Variation in the prevalence of generic E. coli and changes in coliform concentrations between farms suggest environmental factors (e.g. soil properties) at the three farms were different. While Salmonella was only detected once, generic E. coli was detected in Florida soils throughout the duration of the growing season meaning activities that limit contact between soil and horticultural crops should continue to be emphasized. Samples collected during an independent sampling trip were evaluated for L. monocytogenes, which was not detected. The influence of local environmental factors on the prevalence of indicator organisms in the soil presents a unique challenge when evaluating the applicability of more global models to predict pathogen prevalence in preharvest produce environments.

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.

Predicting Salmonella populations from biological, chemical, and physical indicators in Florida surface waters.

Coliforms, Escherichia coli, and various physicochemical water characteristics have been suggested as indicators of microbial water quality or index organisms for pathogen populations. The relationship between the presence and/or concentration of Salmonella and biological, physical, or chemical indicators in Central Florida surface water samples over 12 consecutive months was explored. Samples were taken monthly for 12 months from 18 locations throughout Central Florida (n = 202). Air and water temperature, pH, oxidation-reduction potential (ORP), turbidity, and conductivity were measured. Weather data were obtained from nearby weather stations. Aerobic plate counts and most probable numbers (MPN) for Salmonella, E. coli, and coliforms were performed. Weak linear relationships existed between biological indicators (E. coli/coliforms) and Salmonella levels (R(2) < 0.1) and between physicochemical indicators and Salmonella levels (R(2) < 0.1). The average rainfall (previous day, week, and month) before sampling did not correlate well with bacterial levels. Logistic regression analysis showed that E. coli concentration can predict the probability of enumerating selected Salmonella levels. The lack of good correlations between biological indicators and Salmonella levels and between physicochemical indicators and Salmonella levels shows that the relationship between pathogens and indicators is complex. However, Escherichia coli provides a reasonable way to predict Salmonella levels in Central Florida surface water through logistic regression.

Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery.

Illnesses from Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella have been associated with the consumption of numerous produce items. Little is known about the effect of consumer handling practices on the fate of these pathogens on celery. The objective of this study was to determine pathogen behavior at different temperatures under different storage conditions. Commercial fresh-cut celery was inoculated at ca. 3logCFU/g onto either freshly cut or outer uncut surfaces and stored in either sealed polyethylene bags or closed containers. Samples were enumerated following storage for 0, 1, 3, 5, and 7 days when held at 4 °C or 12 °C, and after 0, 8, and 17 h, and 1, and 2 days when held at 22 °C. At 4 °C, all populations declined by 0.5-1.0logCFU/g over 7 days. At 12 °C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased by ca. 0.5logCFU/g over 7 days. At 22 °C, E. coli O157:H7, Salmonella, and L. monocytogenes populations increased by ca. 1, 2, or 0.3logCFU/g, respectively, with the majority of growth occurring during the first 17 h. On occasion, populations on cut surfaces were significantly higher than those on uncut surfaces. Results indicate that populations are reduced under refrigeration, but survive and may grow at elevated temperatures.