Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves.

Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.

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

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

Biofilm-forming Ability of Bacillus thuringiensis Strains from Biopesticides on Polystyrene and their Attachment on Spinach.

Bacillus thuringiensis-based commercial products as a biopesticide have been used for more than 60 years in agriculture. However, as one of the species in B. cereus group, B. thuringiensis has been considered as an emerging hazard with the potential to cause food toxico-infections. The present study aimed to evaluate the biofilm-forming ability of B. thuringiensis biopesticide strains and their attachment on spinach, compared to foodborne B. cereus strains. Biofilm formations of tested strains were found to be strain-specific and affected by the nutrient conditions more than the incubation time. Nutrient starvation conditions generally reduced the biofilm formation of tested B. thuringiensis and B. cereus strains, particularly B. thuringiensis ABTS-1857 strain was found as the nonbiofilm former in starvation conditions. It is worth mentioning that B. thuringiensis SA-11 strain showed stronger biofilm-forming ability with more air-liquid interface biofilm than the other two B. thuringiensis biopesticide strains, but no such higher attachment of B. thuringiensis SA-11 to spinach was observed. These results indicate that B. thuringiensis SA-11 strain can enter the food processing lines by the attachment on spinach leaves, and it has the potential to form biofilms throughout the processing lines or the production environment when sufficient nutrients are available. However, more biofilm tests of B. thuringiensis biopesticide strains in the vegetable production chain should be performed. The dry formulation of commercial B. thuringiensis biopesticides enhanced their adhesion on spinach leaves, whereas the strength of adhesion was not improved by the formulation. In addition, 1-2 log reductions of spores after the intensive washing of spinach leaves in the lab were detected. However, the log reduction due to the actual washing done by the food processing companies in large-volume washing baths or by consumers at home would be limited and less than this lab simulation.

A Rapid Total Bacterial Count Method for Food Samples using Syringe Filters and Lectin-Conjugated Semiconductor Nanorods.

Total bacterial count in food is one of important food safety criteria. The current plate count method (Heterotrophic Plate Count) for food analysis requires microbiology lab facilities and at least 2 days turnover time. We developed a rapid fluorescence-based total bacterial count method that utilises semiconductor nanorods (SNRs) conjugated with a lectin Griffonia simplicifolia II (GSII-SNRs) to stain bacterial cells captured on syringe filters, via the common N-acetylglucosamine molecules on bacterial cell wall. This "Filter-and-Stain" detection method has a rapid turnover time of 20 min. The fluorescence emission can be seen under UV light with minimum interference from food sample background. The fluorescence intensity quantified through image analysis is proportional to the bacterial concentration with a limit of detection of 1000 CFU/mL, for total bacterial count assessment in food safety. Moreover, the GSII-SNRs do not bind to heat inactivated bacterial cells, and thus can differentiate live and dead bacteria. Our method has been validated with representative food (coffee powder, raw spinach leaves, and ready-to-eat tomato salsa) to demonstrate its high potential for on-site food safety assessment, especially in places with no immediate access to microbiology labs.

Cold-adapted Exiguobacterium sibiricum K1 as a potential bioinoculant in cold regions: Physiological and genomic elucidation of biocontrol and plant growth promotion.

The scarcity of soil nutrient availability under cold conditions of Himalayan regions needs a sustainable approach for better crop yields. The cold-adapted bacteria, Exiguobacterium sibiricum K1, with the potential to produce several plant growth-promoting (PGP) attributes, nitrogen fixation, indole acetic acid production, phosphate and potassium solubilization at 10 °C can provide an opportunity to promote crop yield improvement in an eco-friendly way under cold conditions. The bacterium also exhibited biocontrol activity against two phytopathogens and produced siderophore (53.0 ± 0.5 % psu). The strain's PGP properties were investigated using a spinach-based bioassay under controlled conditions. The bacterized seeds showed a notable increase in germination rate (23.2 %), shoot length (65.3 %), root length (56.6 %), leaf area (73.7 %), number of leaflets (65.2 %), and dry matter (65.2 %). Additionally, the leaf analysis indicated elevated chlorophyll pigments, i.e., chlorophyll a (55.5 %), chlorophyll b (42.8 %), carotenoids (35.2 %), percentage radical scavenging activity (47.4 %), and leaf nutrient uptake such as nitrogen (23.4 %), calcium (60.8 %), potassium (62.3 %), and magnesium (28.9 %). Moreover, the whole-genome sequencing and genome mining endorsed various biofertilisation-related genes, including genes for potassium and phosphate solubilization, iron and nitrogen acquisition, carbon dioxide fixation, and biocontrol ability of Exiguobacterium sibiricum K1. Overall, this study highlights the role of Exiguobacterium sibiricum K1 as a potential bioinoculant for improving crop yield under cold environments.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium, is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 µg/ml) than that of S. vesicarium (mean of 14.17 µg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium, including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola. The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola.

Strain-specific Growth Parameters are Important to Accurately Model Bacterial Growth on Baby Spinach in Simulation Models.

Digital tools to predict produce shelf life have the potential to reduce food waste and improve consumer satisfaction. To address this need, we (i) performed an observational study on the microbial quality of baby spinach, (ii) completed growth experiments of bacteria that are representative of the baby spinach microbiota, and (iii) developed an initial simulation model of bacterial growth on baby spinach. Our observational data showed that the predominant genera found on baby spinach were Pseudomonas, Pantoea and Exiguobacterium. Rifampicin-resistant mutants (rifR mutants) of representative bacterial subtypes were subsequently generated to obtain strain-specific growth parameters on baby spinach. These experiments showed that: (i) it is difficult to select rifR mutants that do not have fitness costs affecting growth (9 of 15 rifR mutants showed substantial differences in growth, compared to their corresponding wild-type strain) and (ii) based on estimates from primary growth models, the mean (geometric) maximum population of rifR mutants on baby spinach (7.6 log10 CFU/g, at 6°C) appears lower than that of the spinach microbiota (9.6 log10 CFU/g, at 6°C), even if rifR mutants did not have substantial growth-related fitness costs. Thus, a simulation model, parameterized with the data obtained here as well as literature data on home refrigeration temperatures, underestimated bacterial growth on baby spinach. The root mean square error of the simulation's output, compared against data from the observational study, was 1.11 log10 CFU/g. Sensitivity analysis was used to identify key parameters (e.g., strain maximum population) that impact the simulation model's output, allowing for prioritization of future data collection to improve the simulation model. Overall, this study provides a roadmap for the development of models to predict bacterial growth on leafy vegetables with strain-specific parameters and suggests that additional data are required to improve these models.

Fates of attached E. coli o157:h7 on intact leaf surfaces revealed leafy green susceptibility.

Leafy greens, especially lettuce, are repeatedly linked to foodborne outbreaks. This paper studied the susceptibility of different leafy greens to human pathogens. Five commonly consumed leafy greens, including romaine lettuce, green-leaf lettuce, baby spinach, kale, and collard, were selected by their outbreak frequencies. The behavior of E. coli O157:H7 87-23 on intact leaf surfaces and in their lysates was investigated. Bacterial attachment was positively correlated with leaf surface roughness and affected by the epicuticular wax composition. At room temperature, E. coli O157:H7 had the best growth potentials on romaine and green-leaf lettuce surfaces. The bacterial growth was positively correlated with stomata size and affected by epicuticular wax compositions. At 37 °C, E. coli O157:H7 87-23 was largely inhibited by spinach and collard lysates, and it became undetectable in kale lysate after 24 h of incubation. Kale and collard lysates also delayed or partially inhibited the bacterial growth in TSB and lettuce lysate at 37 °C, and they sharply reduced the E. coli O157:H7 population on green leaf lettuce at 4 °C. In summary, the susceptibility of leafy greens to E. coli O157:H7 is determined by a produce-specific combination of physiochemical properties and temperature.

Lytic Escherichia phage OSYSP acts additively and synergistically with gaseous ozone against Escherichia coli O157:H7 on spinach leaves.

Bacteriophage and gaseous ozone are evolving as meritorious alternatives to conventional sanitizers in food postharvest applications. Here, we investigated the efficacy of sequential treatments of a lytic bacteriophage and gaseous ozone, during vacuum cooling of fresh produce, against Escherichia coli O157:H7. Spinach leaves were spot-inoculated with 105-107 CFU g-1 E. coli O157:H7 B6-914 and treated with Escherichia phage OSYSP spray (109 PFU g-1), gaseous ozone, or their combination. Vacuum cooling, which preceded or followed phage application but ran concomitantly with ozone treatment, was performed in a custom-made vessel at the following process sequence: vacuum to 28.5 in. Hg, vessel pressurization to 10 psig with gas containing 1.5 g ozone/kg gas-mix, holding for 30 min, and vessel depressurization to ambient pressure. Bacteriophage or gaseous ozone inactivated E. coli O157:H7, applied at different initial populations on spinach leaves, by 1.7-2.0 or 1.8-3.5 log CFU g-1, respectively. At the high inoculum levels tested (7.1 log CFU g-1), sequential treatments of phage and ozone reduced E. coli O157:H7 population by 4.0 log CFU g-1, but when treatment order was reversed (i.e., ozone followed by bacteriophage), the combination synergistically decreased pathogen's population on spinach leaves by 5.2 log CFU g-1. Regardless the antibacterial application order, E. coli O157:H7 populations, applied initially at ~ 105 CFU g-1, were reduced below the enumeration method's detection level (i.e., < 101 CFU g-1). The study proved that bacteriophage-ozone combination, applied in conjunction with vacuum cooling, is a potent pathogen intervention strategy in fresh produce post-harvest applications.

Disinfection of Clostridioides difficile on spinach with epigallocatechin-based antimicrobial solutions and sodium hypochlorite.

The removal of C. difficile inoculated on fresh spinach leaves washed with antimicrobial solutions was investigated. In addition, the effect of washing solutions on the total aerobic mesophilic bacteria (TAMB) and Enterobacteriaceae in the fresh spinach was examined. The fresh spinach was washed through immersion in different concentrations (MIC, 2xMIC, and 4xMIC) of the natural disinfectant solution (NDS) consisting of EDTA, borax, and epigallocatechin gallate (EGCG) content developed in our laboratory and green tea extract-acetic acid (GTE-AA) for varying contact times (5 and 15 min). Different concentrations (50, 100, and 200 ppm) of sodium hypochlorite (NaOCl) and tap water as the control group were used to compare the effectiveness of the NDS. In addition, the effects of washing on the color, texture, and total phenol content of the spinach were determined. No statistical difference was observed in the washing of the spinach leaves with NDS prepared at 2xMIC and 4xMIC concentrations, while inhibition of C. difficile ranged between 2.11 and 2.32 logs. The highest inhibition was observed in the application of 50 ppm NaOCl for 15 min with a decrease of 2.88 logs in C. difficile spores. The GTE-AA and NDS decreased the number of TAMB by 2.27-3.08 log and, 3.21-3.66 log, respectively. Washing spinach leaves with natural disinfectant for 5 min caused a decrease of 2.58 logs in Enterobacteriaceae load, while washing with 50 ppm NaOCl for 15 min reduced Enterobacteriaceae load by 4 logs. Tap water was ineffective in reducing any microbial load. No difference was detected in the color parameters of the spinach through all washes. Although all antimicrobial washes made a difference in the texture of the spinach, the greatest loss in firmness was observed in the spinach washed with NaOCl. Washing spinach with epigallocatechin-based wash solutions can remove C. difficile in possible C. difficile contamination, thereby reducing the environmental load of C. difficile. Epigallocatechin-based disinfectants can be an alternative to chlorine-based disinfectants in improving the microbial quality of vegetables.

Pre-heated blades for harvesting baby-leaves reduce the risk of Escherichia coli internalization in leaves.

BACKGROUND: Pathogenic enterobacteria can travel through the plant vascular bundles by penetrating from cuts and persisting into ready-to-eat leafy greens. Because the cutting site is the main point of entrance and uptake, we tested how different cutting strategies can reduce bacterial internalization in leaves. Horizontal cuts at the base of the leaves were performed with two different types of tools: the first with a scalpel (by pulling the blade) and the second with a scissor-action that has blades that cuts by gliding against a thicker blade. Scissor-action generally makes closer border cuts. Blades of both types of tools have worked at 25 °C and 200 °C. The present study aimed to determine how these different types of cuts and temperatures affected bacterial uptake in leaves. Experiments were repeated on different plant genotypes and at different wilting stages. RESULTS: Our findings showed that cutting baby-leaves with a scissor action at 200 °C significantly reduced the bacterial uptake compared to the not heated (which simulates a mechanized lettuce harvester). The most effective cutting treatments for reducing bacterial uptake were in the order: scissor 200 °C > scissor 25 °C > scalpel 200 °C > scalpel 25 °C. The scissor heated at 200 °C also prevented bacterial uptake on wilted baby-leaves. CONCLUSION: The findings of the present study could provide a further contribution in terms of safety during harvest and suggest that a pre-heated blade supports safety during harvest of leafy greens. © 2022 Society of Chemical Industry.

Pre-harvest biocontrol of Listeria and Escherichia coli O157 on lettuce and spinach by lactic acid bacteria.

Recent outbreaks linked to contaminated leafy greens underline the need for identifying effective natural approaches to improve produce safety at pre-harvest level. Lactic acid bacteria (LAB) have been evaluated as biocontrol agents in food products. In this study, the efficacy of a cocktail of LAB including Lactococcus lactis, Lactiplantibacillus plantarum, Lactobacillus johnsonii, and Lactobacillus acidophilus as pre-harvest biocontrol agents against Listeria and Escherichia coli O157 on lettuce and spinach was investigated. Bacterial pathogens L. monocytogenes and E. coli O157:H7 and the non-pathogenic surrogates L. innocua and E. coli O157:H12 were used to spray-inoculate cultivars of lettuce and spinach grown in growth chamber and in field, respectively. Inoculated plants were spray-treated with water or a cocktail of LAB. On day 0, 3, and 5 post-inoculation, four samples from each group were collected and bacterial populations were determined by serial dilution and spiral plating on selective agars. LAB treatment exhibited an immediate antimicrobial efficacy against L. monocytogenes and E. coli O157:H7 on "Green Star" lettuce by ~2 and ~ 1 log reductions under growth chamber conditions, respectively (P < 0.05). The effect of LAB against E. coli O157:H7 on "New Red Fire" lettuce remained effective during the 5-day period in growth chamber (P < 0.05). Treatment of LAB delivered an effective bactericidal effect against E. coli O157:H12 immediately after application on the field-grown lettuce plants (P < 0.05). Approximately 1 log L. innocua reduction was observed on "Matador" and "Palco" spinach on day 5 (P < 0.05). Results of this study support that LAB could potentially be applied as biocontrol agents for controlling Listeria and E. coli O157 contamination on leafy greens at the pre-harvest level.

trans-Cinnamaldehyde-encapsulated zeolitic imidazolate framework-8 nanoparticle complex solutions to inactivate Escherichia coli O157:H7 on fresh spinach leaves.

This study synthesized and characterized ZIF-8 nanoparticles encapsulated with trans-cinnamaldehyde oil (TC) and evaluated their antimicrobial effectiveness against Escherichia coli O157:H7 on fresh spinach leaves. The antimicrobial activity of different mass ratios of TC-encapsulated ZIF-8 against E. coli O157:H7 (ATCC 43895) strain was assessed and the best mass ratio of 1:2 TC to ZIF-8 identified. Spinach leaves were treated with (1) 0.5TC@ZIF-8_PL nanoparticle complexes solution, (2) 200 ppm chlorine, (3) free TC, and (4) sterilized distilled water (control). All sample groups were rinsed for 1 min, dried in a biosafety cabinet, weighted, and packed in sterilized Whirl-pkTM Stand-Up sampling bags, and stored at 4°C for 15 days for shelf life studies. Samples were dipped into a solution of nanoparticles and another group was sprayed. The quality of spinach samples was assessed by monitoring changes in moisture content (MC), water activity (Aw), color, pH, texture (firmness and work), vitamin C content, total carotenoid, and chlorophyll content. Spinach leaves treated with 0.5TC@ZIF-8_PL had less (p < 0.05) water, total chlorophyll, and total carotenoid losses, with minimal changes in pH. However, treatment did not prevent the color degradation (p > 0.05) and adversely affected spinach firmness. The spinach samples treated with 200 ppm chlorine and free TC had higher (p < 0.05) total chlorophyll degradation than the samples treated with the nanoparticles. The mass ratio of TC-encapsulated ZIF-8 must be readjusted to reduce potential toxicity issues while maintaining the antimicrobial properties. PRACTICAL APPLICATION: Zeolitic imidazolate framework-8 (ZIF-8) nanoparticle complex can be used to encapsulate natural antimicrobials to inhibit growth of pathogens on fresh produce. A 2-log reduction in populations of Escherichia coli O157:H7 on fresh spinach leaves was achieved using trans-cinnamaldehyde at low concentrations. The results can be used to embed the compounds into polymeric films for antimicrobial packaging applications.

Rapid detection of Salmonella enterica in leafy greens by a novel DNA microarray-based PathogenDx system.

The diverse matrices pose great challenges for rapid detection of low Salmonella level (<10 CFU) in fresh produce. The applicability of microarray-based PathogenDx system for detecting low contamination of Salmonella Newport from leafy greens was evaluated. A pre-PCR preparation protocol including enrichment in universal pre-enrichment broth for 3 h followed by sample concentration using an InnovaPrep bio-concentrator or 6 h enrichment without a concentration step was used for detecting S. Newport from leafy greens with initial inoculum level at ∼6 CFU/25 g. Among 205 samples tested, 98%, 93%, 76%, and 60% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were tested positive after 3 h of enrichment with sample concentration. After 6 h of enrichment, 100%, 98%, 90%, and 82% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were positive. The samples were parallelly tested by the FDA bacterial analytical manual (BAM) method and 100% of spiked produce samples were tested positive. The overall analysis time of this methodology was between 8 and 11 h, including all pre-enrichment and concentration steps, in contrast to 4-5 days required for BAM method. The system correctly differentiated all 108 Salmonella strains and 35 non-Salmonella strains used in the study. This novel microarray approach provides a rapid method for detecting Salmonella in leafy greens.

Metagenetic characterization of bacterial communities associated with ready-to-eat leafy vegetables and study of temperature effect on their composition during storage.

Ready-to-eat (RTE) and fresh-cut vegetables meet the current needs for healthy and easy-to-prepare food. However, raw vegetables are widely known to harbor large and diverse bacterial communities promoting spoilage and reducing their shelf-life. A better understanding of their bacterial community and the impact of various environmental factors on its composition is essential to ensure the production of high-quality fresh-cut produce. Therefore, a metagenetic amplicon approach, based on gyrB sequencing, was applied for deciphering the bacterial communities associated with the spoilage of RTE rocket and baby spinach and monitoring the changes occurring in their composition during storage at different temperatures. Our results indicated that Pseudomonas genus was the main spoilage group for both leafy vegetables. Specifically, Pseudomonas viridiflava was dominant in most samples of rocket, while a new Pseudomonas species as well as, Pseudomonas fluorescens and/or Pseudomonas fragi were highly abundant in baby spinach. A significant variability on bacterial species composition among different batches of each vegetable type was observed. In the case of baby spinach, the impact of temperature and/or storage time on bacterial microbiota was not explicitly revealed at batch-level. Concerning rocket, the storage time was the most influential factor resulting in the reduction of Pseudomonas species' abundances and the parallel increase of lactic acid bacteria abundances. The results suggest that a large-scale sampling and further investigation of the various environmental factors shaping the microbiota are needed for gaining deeper knowledge of the diverse bacterial communities on RTE leafy vegetables and thus, enhance the quality of these products.

Seed inoculation with antagonistic bacteria limits occurrence of E. coli O157:H7gfp + on baby spinach leaves.

BACKROUND: During the last decades, outbreaks of foodborne illnesses have increasingly been linked to fresh and/or minimally processed fruit and vegetables. Enterohemorrhagic Escherichia coli was the causal agent for major outbreaks in Europe with leafy green vegetables and sprouts. To improve food safety, microbial antagonism has received attention during recent years and could be one of the solution to prevent contamination of food borne pathogens on fresh produce. Here we investigate the antagonistic effect of three bacterial strains (Pseudomonas orientalis, P. flavescens and Rhodococcus sp.) isolated from spinach leaves against E. coli O157:H7gfp + under laboratory and greenhouse conditions. RESULTS: Our results shows that significantly less culturable E.coli O157:H7gfp + were retrieved from the spinach canopy subjected to antagonist seed treatment than canopy inoculation. Seeds inoculated with Rhodococcus sp. significantly reduced growth of E. coli O157:H7gfp + compared with the other antagonists. The result from the in vitro study shows a significant reduction of growth of E. coli O157:H7gfp+, but only after 44 h when E. coli O157:H7gfp + was propagated in a mixture of spent media from all three antagonists. CONCLUSIONS: The antagonistic effect on phyllospheric E.coli O157:H7gfp + observed after seed inoculation with Rhodococcus sp. might be an indication of induced resistance mechanism in the crop. In addition, there was a small reduction of culturable E.coli O157:H7gfp + when propagated in spent media from all three antagonists. Nutritional conditions rather than metabolites formed by the three chosen organisms appear to be critical for controlling E. coli O157:H7gfp+.

Growth and survival of aerobic and Gram-negative bacteria on fresh spinach in a Chinese supply chain from harvest through distribution and refrigerated storage.

Spinach is a highly perishable product that degrades over time, including due to bacteria contaminating the product prior to packaging, yet the dynamics of bacterial spoilage and factors that affect it are not well understood. Notably, while China is the top producer of spinach globally, there is limited available microbiological data in the literature for spinach supply chains in China. The overall goal of this foundational study was to establish a baseline understanding of bacterial population dynamics on spinach from harvest to 10 days postprocessing for a Chinese supply chain that includes distribution via traditional grocery (a local physical store) and eCommerce (an online store). To this end, organic spinach samples were collected at different stages in a Chinese supply chain by following the same 3 lots, starting at point-of-harvest through processing and distribution via a local grocery store and eCommerce. After distribution, the same 3 lots were stored at 4 °C with microbiological testing performed on multiple days up to day 10 postprocessing, simulating storage at the point-of-consumer. Results showed aerobic plate counts and total Gram-negative counts did not significantly differ across stages in the supply chain from harvest through processing. However, packaged spinach from the same processing facility and lots, exhibited different patterns in bacterial levels across 0 to 10 days postprocessing, depending on whether it was distributed via the local grocery store or eCommerce. Evaluation of bacterial populations performed on a subset of the packaged spinach samples indicated Gram-negative bacteria, in particular Pseudomonas, were predominant across all days of testing (days 0, 3, and 10 postprocessing), with populations differing at the genus level by day. Overall, this study improves our understanding of the dynamics of bacterial populations on spinach and provides baseline data needed for future studies.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched foods. The stx assay does not differentiate between stx1 and stx2 but detects the presence of stx1 and/or stx2. OBJECTIVE: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated for AOAC®  Performance Tested MethodsSM certification. METHODS: Matrix studies, inclusivity/exclusivity, robustness testing, product stability, and lot-to-lot variability testing were conducted to assess the method's performance. RESULTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) demonstrated equivalent results to the United States Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 5C.00 reference method for fresh raw ground beef, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A reference method for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) detected all STEC E. coli strains (E. coli strains with stx1 and/or stx2 genes) and did not detect any of the 45 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay's performance. Product consistency and stability testing demonstrated no differences between the lots evaluated. CONCLUSION: The data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of Shiga toxin-producing E. coli in raw ground beef and spinach. HIGHLIGHTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method is suitable for the rapid and specific detection of Shiga toxin-producing E. coli in fresh raw ground beef, and spinach.

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterization of isolated multidrug-resistant Escherichia coli.

AIM: To investigate the microbiological quality, potential foodborne pathogen presence, and to phenotypically (antimicrobial resistance [AMR] profiles) and genotypically (DNA fingerprints and diarrhoeagenic genes) characterize Escherichia coli isolated throughout spinach production systems from farm-to-sale. METHODS AND RESULTS: Samples (n = 288) were collected from two commercial supply chains using either river or borehole irrigation water. E. coli was enumerated throughout the chain where river water was directly used for overhead irrigation at levels between 0.00 and 3.22 log colony forming unit (CFU) g-1 . Following enrichment, isolation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification, E. coli was isolated from 22.57% (n = 65/288) of all samples. Salmonella spp. were isolated from 3% (n = 9/288) of river and irrigation water samples on one farm, and no Listeria monocytogenes was detected throughout the study. Of the 80 characterized E. coli isolates, one harboured the stx2 virulence gene, while 43.75% (n = 35) were multidrug resistant. Overall, 26.30% of the multidrug-resistant E. coli isolates were from production scenario one that used river irrigation water, and 17.50% from the second production scenario that used borehole irrigation water. A greater percentage of resistance phenotypes were from water E. coli isolates (52.50%), than isolates from spinach (37.50%). E. coli isolates from spinach and irrigation water clustered together at high similarity values (>90%) using enterobacterial repetitive intergenic consensus-polymerase chan reaction analysis. CONCLUSIONS: This study reported the presence of multidrug-resistant environmental E. coli throughout spinach production from farm, during processing and up to retail. Furthermore, the similarity of multi-drug resistant E. coli isolates suggests transfer from irrigation water to spinach in both scenarios, reiterating that irrigation water for vegetables consumed raw, should comply with standardized microbiological safety guidelines. SIGNIFICANCE AND IMPACT OF STUDY: Multidrug-resistant E. coli presence throughout spinach production emphasizes the necessity of increased surveillance of AMR in fresh produce and the production environment within a One Health paradigm to develop AMR mitigation strategies.

Lysinibacillus xylanilyticus Strain GIC41 as a Potential Plant Biostimulant.

To identify Lysinibacillus strains with the potential to function as plant biostimulants, we screened 10 previously isolated Lysinibacillus strains from the rhizosphere and soil for their plant growth-promoting (PGP) effects. In vitro tests showed that all strains produced indole-3-acetic acid. In primary screening, the PGP effects of these strains were assessed on spinach seedlings grown on Jiffy-7 pellets; strains GIC31, GIC41, and GIC51 markedly promoted shoot growth. In secondary screening, the PGP efficacies of these three strains were examined using spinach seedlings grown in pots under controlled conditions. Only GIC41 exerted consistent and significant PGP effects; therefore, it was selected for subsequent experiments. The results of 6-week glasshouse experiments revealed that GIC41 markedly increased shoot dry weight by ca. 12-49% over that of the control. The impact of fertilization levels on the PGP efficacy of GIC41 was investigated using pot experiments. The application of a specific level of fertilizer was required for the induction of sufficient PGP effects by this strain. The phylogenetic ana-lysis based on the 16S rDNA sequence identified GIC41 as L. xylanilyticus. Collectively, these results show the potential of strain GIC41 to function as a plant biostimulant.