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.

Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species.

Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I-VI toxin-antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods.

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.

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.

Ranking Food Safety Priorities of the Fresh Produce Industry in the United States.

A broad understanding of community member food safety priorities in the fresh produce supply chain does not currently exist. This information is essential to improve food safety knowledge and practices effectively and efficiently throughout the fresh produce industry; therefore, the goal of this study was to identify and rank community produce safety priorities in the United States. Survey questions were designed and approved by food safety experts for participants to rank 24 fresh produce safety priorities. The anonymous survey was distributed online via Qualtrics™ to fresh produce community members from November 2020 to May 2021. A score was calculated for each priority by summing weighted ranking scores across responses. Descriptive statistics and logistic regression were used to determine frequencies and distribution of response and identify factors (e.g., role in produce safety, size/location of organization/operation) that influenced rankings. A total of 281 respondents represented fourteen different roles in the fresh produce industry, with most identified as growers (39.5%). Produce operations were distributed across the U.S. and annual produce sales ranged from below $25,000 to over $5,000,000. Health and hygiene, training, postharvest sanitation, traceability, and harvest sanitation were ranked as the top five food safety priorities. These findings provide insight into community member priorities in fresh produce safety and can be used to inform intervention efforts, ranging from specialized training for produce growers and packers, industry-driven research projects, and gaps in risk communication strategies.

Fate and Growth Kinetics of Salmonella and Listeria monocytogenes on Mangoes During Storage.

Imported mangoes have been linked to outbreaks of salmonellosis in the USA. The purpose of this research was to evaluate the persistence and growth kinetics of Salmonella and Listeria monocytogenes on the intact surface of whole 'Ataulfo', 'Kent', and 'Tommy Atkins' mangoes stored at three different temperatures. L. monocytogenes was also evaluated on fresh-cut 'Tommy Atkins' mangoes stored at 4, 12, 20 ± 2°C. Whole mangoes were spot inoculated with rifampicin-resistant pathogen cocktails (6 log CFU/mango) onto the midsection of whole fruit (n = 6). Fruit was stored at 12, 20, or 30 ± 2°C and sampled for up to 28 days. The specific growth rates derived from DMFit models as a function of time were used to develop secondary models. On 'Kent' mangoes, Salmonella had a population increase from 0.3 to 1.1 log CFU/mango with a linear growth rate of ∼0.004, 0.01, and 0.06 log CFU/mango/h at 12, 20, and 30°C, respectively. At 20 and 30°C, Salmonella growth rates were significantly higher than 12°C (P < 0.05). No clear Salmonella growth trend was observed; populations decreased up to 1.6 log CFU/mango on 'Tommy Atkins' and 'Ataulfo' at 12°C. Populations of L. monocytogenes on whole and fresh-cut mangoes declined regardless of temperature and storage period. Food safety during storage should be the top priority for fresh-cut tropical fruit processors.

Salmonella transfer potential between tomatoes and cartons used for distribution.

Corrugated fiberboard boxes (cartons) can be reused during fresh market tomato packing and repacking. The fate of Salmonella on the new, used, and dirty tomato packaging cartons, and Salmonella transfer between tomatoes and new, used, and dirty packaging cartons was assessed. Mature green tomatoes or blank cartons were spot inoculated with cocktail of rifampicin-resistant Salmonella strains before touching cartons/tomatoes at 0, 1, or 24 h postinoculation. Tomatoes were placed on new, used, and dirty carton squares (5 by 5 cm) for 0, 1, and 7 days of contact at 12°C and 25°C with a relative humidity value of 85%. Transfer coefficients (TCs) were calculated for all conditions. Salmonella populations decreased following inoculation by 2-3 log units during 24 h drying regardless of storage temperature; the presence of debris enhanced survival at 12°C. In general, the highest transfer rates occurred with wet inoculum. The highest Salmonella transfer was calculated for wet inoculated tomatoes with 7 days of contact time at 25°C (TC = 14.7). Increasing contact time decreased TCs for new cartons, but increased TCs for used and dirty cartons. Regardless of carton condition or storage temperature, a greater population of Salmonella was transferred from tomatoes to cartons than from cartons to tomatoes. Salmonella transfer between tomatoes and cartons is highly dependent on moisture, with increased levels of moisture increasing transfer, highlighting the importance of harvesting and packing dry tomatoes.

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.

Identification and Subtyping of Salmonella Isolates Using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF).

Subtyping of bacterial isolates of the same genus and species is an important tool in epidemiological investigations. A number of phenotypic and genotypic subtyping methods are available; however, most of these methods are labor-intensive and time-consuming and require considerable operator skill and a wealth of reagents. Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF), an alternative to conventional subtyping methods, offers a rapid, reproducible method for bacterial identification with a high sensitivity and specificity and at minimal cost. The purpose of this study was to determine the feasibility of using MALDI-TOF to differentiate between six Salmonella serovars recovered from experimental microcosms inoculated with known strains of Salmonella. Following the establishment of a MALDI-TOF reference library for this project, the identity of 843 Salmonella isolates recovered from these microcosms was assessed using both MALDI-TOF and conventional methods (serotyping/PCR). All 843 isolates were identified as being Salmonella species. Overall, 803/843 (95%) of these isolates were identified similarly using the two different methods. Positive percent agreement at the serovar level ranged from 79 to 100%, and negative percent agreement for all serovars was greater than 98%. Cohen's kappa ranged from 0.85 to 0.98 for the different serovars. This study demonstrates that MALDI-TOF is a viable alternative for the rapid identification and differentiation of Salmonella serovars.

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.

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.

Application of Chitosan Microparticles against Human Norovirus.

ABSTRACT: Human norovirus (HuNoV) is the leading cause of foodborne illness outbreaks and the second most common cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CMs) against HuNoV GII.4 Sydney and its cultivable surrogate Tulane virus (TuV) in suspensions mimicking fecally contaminated water. CMs were prepared by cross-linking chitosan molecules with sodium sulfate, and the antiviral activity of CMs was assessed with an infectivity assay on TuV and by quantitative reverse transcription PCR on TuV and HuNoV. A 3% CM suspension in phosphate-buffered saline (pH 7.2) bound to TuV particles but had a negligible impact on virus infectivity (P > 0.05). A 10-min contact time resulted in a 1.5-log reduction in genomic copies per mL of TuV and HuNoV in fecal suspensions (P < 0.05). Despite the negligible impact on viral infectivity, CMs can moderately bind to infectious virus particles and help purify environmental water by removing these particles. In this study, TuV was a suitable surrogate for HuNoV with similar log reductions in fecal suspension. These findings highlight the potential application of CM as a novel treatment to minimize the spread of waterborne viral pathogens.

Big Data Impacting Dynamic Food Safety Risk Management in the Food Chain.

Foodborne pathogens are a major contributor to foodborne illness worldwide. The adaptation of a more quantitative risk-based approach, with metrics such as Food safety Objectives (FSO) and Performance Objectives (PO) necessitates quantitative inputs from all stages of the food value chain. The potential exists for utilization of big data, generated through digital transformational technologies, as inputs to a dynamic risk management concept for food safety microbiology. The industrial revolution in Internet of Things (IoT) will leverage data inputs from precision agriculture, connected factories/logistics, precision healthcare, and precision food safety, to improve the dynamism of microbial risk management. Furthermore, interconnectivity of public health databases, social media, and e-commerce tools as well as technologies such as blockchain will enhance traceability for retrospective and real-time management of foodborne cases. Despite the enormous potential of data volume and velocity, some challenges remain, including data ownership, interoperability, and accessibility. This paper gives insight to the prospective use of big data for dynamic risk management from a microbiological safety perspective in the context of the International Commission on Microbiological Specifications for Foods (ICMSF) conceptual equation, and describes examples of how a dynamic risk management system (DRMS) could be used in real-time to identify hazards and control Shiga toxin-producing Escherichia coli risks related to leafy greens.

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.

Survival of Shiga Toxin-Producing Escherichia coli in Various Wild Animal Feces That May Contaminate Produce.

ABSTRACT: Domestic and wild animal intrusions are identified as a food safety risk during fresh produce production. The purpose of this study was to evaluate the survival of Shiga toxin-producing Escherichia coli (STEC) in cattle, feral pig, waterfowl, deer, and raccoon feces from sources in California, Delaware, Florida, and Ohio. Fecal samples were inoculated with a cocktail of rifampin-resistant STEC serotypes (O103, O104, O111, O145, and O157) (104 to 106 CFU/g of feces). Inoculated feces were held at ambient temperature. Populations of surviving cells were monitored throughout 1 year (364 days), with viable populations being enumerated by spread plating and enrichment when the bacteria were no longer detected by plating. Representative colonies were collected at various time intervals based on availability from different locations to determine the persistence of surviving STEC serotypes. Over the 364-day storage period, similar survival trends were observed for each type of animal feces from all states except for cattle and deer feces from Ohio. STEC populations remained the highest in cattle and deer feces from all states between days 28 and 364, except for those from Ohio. Feral pig, waterfowl, and raccoon feces had populations of STEC of <1.0 log CFU/g starting from day 112 in feces from all states. E. coli O103 and O104 were the predominant serotypes throughout the entire storage period in feces from all animals and from all states. The survival of both O157 and non-O157 STEC strains in domesticated and wild animal feces indicates a potential risk of contamination from animal intrusion.

Survival of Salmonella in Various Wild Animal Feces That May Contaminate Produce.

ABSTRACT: Heightened concerns about wildlife on produce farms and possible introduction of pathogens to the food supply have resulted in required actions following intrusion events. The purpose of this study was to evaluate the survival of Salmonella in feces from cattle and various wild animals (feral pigs, waterfowl, deer, and raccoons) in California, Delaware, Florida, and Ohio. Feces were inoculated with rifampin-resistant Salmonella enterica cocktails that included six serotypes: Typhimurium, Montevideo, Anatum, Javiana, Braenderup, and Newport (104 to 106 CFU/g). Fecal samples were stored at ambient temperature. Populations were enumerated for up to 1 year (364 days) by spread plating onto tryptic soy agar supplemented with rifampin. When no colonies were detected, samples were enriched. Colonies were banked on various sampling days based on availability of serotyping in each state. During the 364-day storage period, Salmonella populations decreased to ≤2.0 log CFU/g by day 84 in pig, waterfowl, and raccoon feces from all states. Salmonella populations in cattle and deer feces were 3.3 to 6.1 log CFU/g on day 336 or 364; however, in Ohio Salmonella was not detected after 120 days. Salmonella serotypes Anatum, Braenderup, and Javiana were the predominant serotypes throughout the storage period in all animal feces and states. Determination of appropriate risk mitigation strategies following animal intrusions can improve our understanding of pathogen survival in animal feces.

Quantification of Salmonella enterica transfer between tomatoes, soil, and plastic mulch.

Tomatoes have been linked to Salmonella outbreaks in the United States (US). Plasticulture systems, that combine raised beds, plastic mulch, drip irrigation and fumigation, are common in commercial staked fresh tomato production in the US. The US FDA Produce Safety Rule prohibits the distribution of any produce covered by the rule (including fresh market tomatoes) that drops to the ground before harvest. This research was undertaken to better characterize the risks posed by tomatoes that touch plastic mulch or soil immediately before or during harvest. Research was conducted in three states (Florida, Maryland, and Ohio). Each state utilized tomatoes from their state at the point of harvest maturity most common in that state. Each state used indigenous soil and plastic mulch for transfer scenarios. New plastic mulch obtained directly from the application roll and used plastic mulch that had been present on beds for a growing season were evaluated. A five-strain cocktail of Salmonella enterica isolates obtained from tomato outbreaks was used. Mulch (new or used), soil, or tomatoes were spot inoculated with 100 µl of inoculum to obtain a final population of ~6 log CFU/surface. Items were either touched to each other immediately (1-2 s) after inoculation (wet contact) or allowed to dry at ambient temperature for 1 h or 24 h (dry contact). All surfaces remained in brief (1-5 s) or extended (24 h) contact at ambient temperature. Transfer of Salmonella between a tomato and plastic mulch or soil is dependent on contact time, dryness of the inoculum, type of soil, and contact surface. Transfer of Salmonella to and from the mulch and tomatoes for wet and 1 h dry inocula were similar with mean log % transfers varying from 0.7 ± 0.2 to 1.9 ± 0.1. The transfer of Salmonella between soil or plastic mulch to and from tomatoes was dependent on moisture with wet and 1 h dry inocula generally yielding significantly (p < 0.05) higher transfer than the 24 h dry inoculum. Results indicate that harvesting dry tomatoes significantly (p < 0.05) reduces the risk of contamination from soil or mulch contact. Transfer to tomatoes was generally significantly greater (p < 0.05) from new and used plastic mulch than from soil. If contamination and moisture levels are equivalent and contact times are equal to or <24 h before harvest, significantly (p < 0.05) more Salmonella transfers to tomatoes from mulch than from soil. Our findings support that harvesting tomatoes from soil has similar or lower risk than harvesting from plastic mulch.