Antibiotic resistance plasmids in Enterobacteriaceae isolated from fresh produce in northern Germany.

In this study, the genomes of 22 Enterobacteriaceae isolates from fresh produce and herbs obtained from retail markets in northern Germany were completely sequenced with MiSeq short-read and MinION long-read sequencing and assembled using a Unicycler hybrid assembly. The data showed that 17 of the strains harbored between one and five plasmids, whereas in five strains, only the circular chromosomal DNA was detected. In total, 38 plasmids were identified. The size of the plasmids detected varied between ca. 2,000 and 326,000 bp, and heavy metal resistance genes were found on seven (18.4%) of the plasmids. Eleven plasmids (28.9%) showed the presence of antibiotic resistance genes. Among large plasmids (>32,000 bp), IncF plasmids (specifically, IncFIB and IncFII) were the most abundant replicon types, while all small plasmids were Col-replicons. Six plasmids harbored unit and composite transposons carrying antibiotic resistance genes, with IS26 identified as the primary insertion sequence. Class 1 integrons carrying antibiotic resistance genes were also detected on chromosomes of two Citrobacter isolates and on four plasmids. Mob-suite analysis revealed that 36.8% of plasmids in this study were found to be conjugative, while 28.9% were identified as mobilizable. Overall, our study showed that Enterobacteriaceae from fresh produce possess antibiotic resistance genes on both chromosome and plasmid, some of which are considered to be transferable. This indicates the potential for Enterobacteriaceae from fresh produce that is usually eaten in the raw state to contribute to the transfer of resistance genes to bacteria of the human gastrointestinal system. IMPORTANCE: This study showed that Enterobacteriaceae from raw vegetables carried plasmids ranging in size from 2,715 to 326,286 bp, of which about less than one-third carried antibiotic resistance genes encoding resistance toward antibiotics such as tetracyclines, aminoglycosides, fosfomycins, sulfonamides, quinolones, and ß-lactam antibiotics. Some strains encoded multiple resistances, and some encoded extended-spectrum ß-lactamases. The study highlights the potential of produce, which may be eaten raw, as a potential vehicle for the transfer of antibiotic-resistant bacteria.

Unde venis? Bacterial resistance from environmental reservoirs to lettuce: tracking microbiome and resistome over a growth period.

Fresh produce is suggested to contribute highly to shaping the gut resistome. We investigated the impact of pig manure and irrigation water quality on microbiome and resistome of field-grown lettuce over an entire growth period. Lettuce was grown under four regimes, combining soil amendment with manure (with/without) with sprinkler irrigation using river water with an upstream wastewater input, disinfected by UV (with/without). Lettuce leaves, soil, and water samples were collected weekly and analysed by bacterial cultivation, 16S rRNA gene amplicon sequencing, and shotgun metagenomics from total community DNA. Cultivation yielded only few clinically relevant antibiotic-resistant bacteria (ARB), but numbers of ARB on lettuce increased over time, while no treatment-dependent changes were observed. Microbiome analysis confirmed a temporal trend. Antibiotic resistance genes (ARGs) unique to lettuce and water included multidrug and ß-lactam ARGs, whereas lettuce and soil uniquely shared mainly glycopeptide and tetracycline ARGs. Surface water carried clinically relevant ARB (e.g. ESBL-producing Escherichia coli or Serratia fonticola) without affecting the overall lettuce resistome significantly. Resistance markers including biocide and metal resistance were increased in lettuce grown with manure, especially young lettuce (increased soil contact). Overall, while all investigated environments had their share as sources of the lettuce resistome, manure was the main source especially on young plants. We therefore suggest minimizing soil-vegetable contact to minimize resistance markers on fresh produce.

Developing an Agent-Based Model that Predicts Listeria spp. Transmission to Assess Listeria Control Strategies in Retail Stores.

Contamination of fresh produce with Listeria monocytogenes can occur throughout the supply chain, including at retail, where Listeria spp., including L. monocytogenes, may be introduced and spread via various routes. However, limited tools are available for retailers to assess practices that can enhance control of Listeria transmission to fresh produce. Therefore, we developed an agent-based model that can simulate Listeria transmission in retail produce sections to optimize environmental sampling programs and evaluate control strategies. A single retail store was used as a model environment, in which various routes of Listeria introduction into and transmission between environmental surfaces were modeled. Model prediction (i.e., Listeria prevalence) was validated using a published longitudinal study for all surfaces that were included in both the model and the validation data. Sensitivity analysis using the Partial Rank Correlation Coefficient showed that (i) initial Listeria concentration from incoming produce, (ii) transfer coefficient from produce to employee's hands, and (iii) transfer coefficient from consumer to produce were the top three parameters that were significantly (p < 0.0018) associated with the mean Listeria prevalence across all agents, suggesting that the accuracy of these parameters are important for prediction of overall Listeria prevalence at retail. Cluster analysis grouped agents with similar contamination patterns into six unique clusters; this information can be used to optimize the sampling plans for retail environments. Scenario analysis suggested that (i) more stringent supplier control as well as (ii) practices reducing Listeria transmission via consumer's hands may have the largest impact on reducing finished product contamination. Overall, we show that an agent-based model can serve as a foundational tool to help with decision-making on Listeria control strategies at retail.

Staphylococcus spp. in Salad Vegetables: Biodiversity, Antimicrobial Resistance, and First Identification of Methicillin-Resistant Strains in the United Arab Emirates Food Supply.

Contamination of leafy greens with Staphylococcus spp. can occur at various supply chain stages, from farm to table. This study comprehensively analyzes the species diversity, antimicrobial resistance, and virulence factors of Staphylococci in salad vegetables from markets in the United Arab Emirates (UAE). A total of 343 salad items were sampled from three major cities in the UAE from May 2022 to February 2023 and tested for the presence of Staphylococcus spp. using standard culture-based methods. Species-level identification was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was conducted using the VITEK-2 system with AST-P592 cards. Additionally, whole genome sequencing (WGS) of ten selected isolates was performed to characterize antimicrobial resistance determinants and toxin-related virulence factors. Nine Staphylococcus species were identified in 37.6% (129/343) of the tested salad items, with coagulase-negative staphylococci (CoNS) dominating (87.6% [113/129]) and S. xylosus being the most prevalent (89.4% [101/113]). S. aureus was found in 4.6% (14/343) of the salad samples, averaging 1.7 log10 CFU/g. One isolate was confirmed as methicillin-resistant S. aureus, harboring the mecA gene. It belonged to multi-locus sequence type ST-672 and spa type t384 and was isolated from imported fresh dill. Among the characterized S. xylosus (n = 45), 13.3% tested positive in the cefoxitin screen test, and 6.6% were non-susceptible to oxacillin. WGS analysis revealed that the cytolysin gene (cylR2) was the only toxin-associated factor found in S. xylosus, while a methicillin-sensitive S. aureus isolate harbored the Panton-Valentine Leukocidin (LukSF/PVL) gene. This research is the first to document the presence of methicillin-resistant S. aureus in the UAE food chain. Furthermore, S. xylosus (a coagulase-negative staphylococcus not commonly screened in food) has demonstrated phenotypic resistance to clinically relevant antimicrobials. This underscores the need for vigilant monitoring of antimicrobial resistance in bacterial contaminants, whether pathogenic or commensal, at the human-food interface.

Towards a risk-based food safety management system in the fresh produce supply chain in Da Nang, Viet Nam.

Food safety has emerged as a paramount concern for both Vietnamese consumers and the government. However, limited data are available on food safety management systems in Viet Nam. This study identified significant gaps in good agricultural and hygienic practices along the fresh produce chain (farmers and traditional wholesalers/market sellers) in the region of Da Nang, Viet Nam. This was achieved through a survey on good agricultural and hygienic practices for farmers (n = 100) and sellers (n = 100), which researchers further supplemented by microbiological analysis for E. coli, Salmonella spp., and Listeria monocytogenes on leafy greens, water in contact with produce and contact surfaces (hands). The results indicated that 86.0 % of farmers and 54.0 % of sellers received food safety training in the last 3 years; and women dominated both vegetable cultivation but also trading. Farm-level deficiencies included inadequate handwashing practices, lack of documentation for manure application schedules, improper washing and drying of harvest tools, failure to keep containers elevated off the ground, improper storage of vegetables, and inadequate covering of containers, with respectively 34.0 %, 30.3 %, 12.1 %, 41.7 % and 7.9 % of farmers executing the practice as prescribed by the WHO/FAO '5 keys of growing safer fruits and vegetables'. As for sellers, the most dominant gaps (<50.0 % compliance) were the way of handwashing and the practice of keeping containers elevated off the ground before, during, and after harvesting. The microbiological analysis confirmed that, in a total of 36 fresh produce samples including mustard greens, cucumber, lettuce, and crown daisy, the number of samples positive for E. coli, Salmonella spp., and L. monocytogenes were 12, 2, and 10 respectively. Samples of hands and the irrigation water showed high contamination with E. coli. Based on identified gaps, risk communication tools were developed and distributed amongst farmers, sellers, and Da Nang food safety management authority (governmental organisation performing inspections in the traditional food markets). As intervention, two farmers and two sellers were trained in safe agricultural practices for the cultivation of fresh vegetables (managerial intervention) and instructed to use tap water as irrigation water instead of uncontrolled surface water (technological intervention). A post-assessment was conducted, including redoing the survey on good practices and microbiological analysis. The outcome of these interventions showed positive results in terms of good agricultural and hygienic practices resulting in improved hygiene levels and safety of the fresh produce. The findings from this research have the potential to provide a model for the development of a science-based risk management strategy in alternative food chains or geographic areas in emerging countries.

Identification of pathways required for Salmonella to colonize alfalfa using TraDIS-Xpress.

Enteropathogenic bacteria, such as Salmonella, have been linked to numerous fresh produce outbreaks, posing a significant public health threat. The ability of Salmonella to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which pose a challenge to food decontamination and can increase pathogenic bacterial load in the food chain. Preventing Salmonella colonization of food products and food processing environments is crucial for reducing the incidence of foodborne outbreaks. Understanding the mechanisms of establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-Directed Insertion site Sequencing (TraDIS-Xpress) to investigate the mechanisms used by Salmonella enterica serovar Typhimurium to colonize and establish on fresh produce over time. We established an alfalfa colonization model and compared the findings to those obtained from glass surfaces. Our research identified distinct mechanisms required for Salmonella establishment on alfalfa compared with glass surfaces over time. These include the type III secretion system (sirC), Fe-S cluster assembly (iscA), curcumin degradation (curA), and copper tolerance (cueR). Shared pathways across surfaces included NADH hydrogenase synthesis (nuoA and nuoB), fimbrial regulation (fimA and fimZ), stress response (rpoS), LPS O-antigen synthesis (rfbJ), iron acquisition (ybaN), and ethanolamine utilization (eutT and eutQ). Notably, flagellum biosynthesis differentially impacted the colonization of biotic and abiotic environments over time. Understanding the genetic underpinnings of Salmonella establishment on both biotic and abiotic surfaces over time offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. IMPORTANCE: Salmonella is the second most costly foodborne illness in the United Kingdom, accounting for £0.2 billion annually, with numerous outbreaks linked to fresh produce, such as leafy greens, cucumbers, tomatoes, and alfalfa sprouts. The ability of Salmonella to colonize and establish itself in fresh produce poses a significant challenge, hindering decontamination efforts and increasing the risk of illness. Understanding the key mechanisms of Salmonella to colonize plants over time is key to finding new ways to prevent and control contamination of fresh produce. This study identified genes and pathways important for Salmonella colonization of alfalfa and compared those with colonization of glass using a genome-wide screen. Genes with roles in flagellum biosynthesis, lipopolysaccharide production, and stringent response regulation varied in their significance between plants and glass. This work deepens our understanding of the requirements for plant colonization by Salmonella, revealing how gene essentiality changes over time and in different environments. This knowledge is key to developing effective strategies to reduce the risk of foodborne disease.

Wet steam method for reducing attached cells of Salmonella and Listeria monocytogenes on Hass avocado.

The consumption of avocados and their products has been linked to outbreaks of illness caused by Salmonella enterica and Listeria monocytogenes. These pathogens have been isolated from avocados collected from farms and markets. After contact with the avocado epicarp, the cells of Salmonella and L. monocytogenes can become loosely attached (LA) by suspension in a film of water and attraction by electrostatic forces, or strongly attached (SA) by physical and irreversible attachment mechanisms. Attached cells may have greater resistance to agents used to decontaminate the fruit. The effect of applying wet steam (WS) to the epicarp of Hass avocados on the reduction LA and SA counts of Salmonella and L. monocytogenes was evaluated as a function of the exposure time. The inoculated avocados were washed and exposed to WS for 30, 45, and 60 s inside a treatment chamber. Salmonella was found to be more susceptible to WS than L. monocytogenes. The efficacy of steam in reducing LA and SA cell numbers was similar for both pathogens. Steaming avocados for 60 s reduced LA Salmonella and L. monocytogenes cells by 4.6 and 4.8 log CFU/avocado, whereas SA cells were decreased by 5.2 and 4.4 log CFU/avocado, respectively.•Steaming the avocados for 60 s produced the greatest reduction in loosely and strongly attached cells for both pathogens.•Wet steam treatment efficiently eliminated the loosely and strongly attached cells of both pathogens.•The Listeria monocytogenes attached cells showed greater resistance to steam treatment than Salmonella.

The fate of Salmonella enterica and Listeria monocytogenes in the pulp of eight native Brazilian and exotic fruits.

Despite the wide variety of native and exotic fruits in Brazil, there is limited understanding of their ability to support pathogens during storage. This study aimed to evaluate the behavior of Salmonella enterica and Listeria monocytogenes inoculated into the pulp of eight fruits native and exotic to Brazil: Jenipapo (Genipa americana L.), Umbu (Spondias tuberosa Arruda), Maná (Solanum sessiliflorum), Cajá-manga (Spondias dulcis), Physalis (Physalis angulata L.), Feijoa (Acca sellowiana), Cupuaçu (Theobroma grandiflorum) (average pH < 3.3) and in a low acidy fruit: Abiu (Pouteria caimito) (pH 6.11). The pathogens were inoculated into the different fruits and stored at 10, 20, 30 and 37 °C for up to 12 h and 6 days, respectively. Among the fruits evaluated, Abiu was the only one that allowed Salmonella growth, showing higher δ-values at 20 and 30 °C (5.6 log CFU/g for both temperatures). For Physalis and Feijoa, there was a small reduction in the pathogen concentration (<1 log-cycle), mainly at 10 and 20 °C, indicating its ability to remain in the matrices. For the other fruits, notable negative δ-values were obtained, indicating a tendency towards microbial inactivation. The survival potential was significantly affected by temperature in Abiu, Maná, Cupuaçu, and Cajá-manga (p < 0.05). The same phenomena regarding δ-value were observed for L. monocytogenes population, with the greatest survival potential observed at 20 °C in Abiu (3.3 log CFU/g). Regarding the exponential growth rates in Abiu, the highest values were observed at 30 and 37 °C, both for Salmonella (4.6 and 4.9 log (CFU/g)/day, respectively) and for L. monocytogenes (2.8 and 2.7 log (CFU/g)/day, respectively), with no significant difference between both temperatures. Regarding microbial inactivation, L. monocytogenes showed greater resistance than Salmonella in practically all matrices. Jenipapo and Umbu were the pulps that, in general, had the greatest effect on reducing the population of pathogens. Furthermore, the increase in storage temperature seems to favor the increase on inactivation rates. In conclusion, Salmonella and L. monocytogenes can grow only in Abiu pulp, although they can survive in some acidic tropical fruits kept at refrigeration and abusive temperatures.

Draft genome sequences of antibiotic-resistant Serratia and Enterobacter species isolated from imported fresh produce in Georgia, USA.

Imported foods play an essential role in food security and in fulfilling consumer demand. However, these foods can also carry antibiotic-resistant bacteria, which might be introduced into the country of importation. Here, we report the draft genomes of antibiotic-resistant bacteria that were isolated from imported fresh produce in Georgia, USA.

A portable, easy-to-use paper-based biosensor for rapid in-field detection of fecal contamination on fresh produce farms.

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations are towards clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

Development and characterization of edible and active coating based on xanthan gum nanoemulsion incorporating betel leaf extract for fresh produce preservation.

Developing an edible and active coating, incorporating environmentally-friendly antimicrobial agents into edible polymers, provides an eco-friendly alternative to conventional packaging and exhibits significant potential in preserving the quality of postharvest food. Herein, we aim to develop a novel edible and active coating based on xanthan gum (XG) nanoemulsion (NE) incorporating betel leaf extract (BLE) for the preservation of fresh produce. The total phenolic content, total flavonoid content, and antioxidant capacity of the methanol extract of BLE at various concentrations were characterized. Further development of the active coating at different formulations of Tween 80 (1 % and 3 % w/v), XG (0.1 % to 0.5 % w/v), and BLE (1 % to 5 % w/v) was characterized by physical stability, viscosity, and antimicrobial properties. Results showed that the active coating at 1 % BLE showed significant antimicrobial properties against diverse bacterial and fungal foodborne pathogens (e.g., B. cereus, S. aureus) and fungal cultures (e.g., C. albicans). The study also examined the shelf-life of tomatoes coated with the BLE-XG NE solution, stored at 4 °C for 27 days. Analyses of weight retention, soluble solids, pH, texture, sensory attributes, and microbial populations showed that the coating effectively preserved tomato quality, highlighting its potential to preserve fresh produce and enhance food security.

Characterization and efficacy of Salmonella phage cocktail PHA46 in the control of Salmonella Newport and Typhimurium internalized into cherry tomatoes.

Non-typhoid Salmonella enterica causes salmonellosis illness, and this bacterium can contaminate food throughout the production chain, including those that are consumed as raw products. Salmonella enterica can adhere to and internalize into fresh produce such as cherry tomatoes. It has been reported that lytic bacteriophages (phages) can be used as a biocontrol agent in the agricultural field, being an alternative for the control of Salmonella in red meat, fish, lettuce, and cabbage. The aim of this study was to characterize the two phages present in the PHA46 cocktail to determine their morphology, genome, host range, and resistance to different temperatures and pHs values; and later evaluate their lytic activity to reduce the adherence to and internalization of Salmonella enterica serovars Newport and Typhimurium into cherry tomatoes. In addition, in this work, we also explored the effect of the PHA46 cocktail on the virulence of S. Newport-45 and S. Typhimurium SL1344, recovered from the interior of cherry tomatoes, on the lifespan of the animal model Caenorhabditis elegans. The nematode C. elegans, recently has been used to test the virulence of Salmonella and it is easy to maintain and work with in the laboratory. The results revealed that the morphology obtained by Transmission Electron Microscopy of two phages from the PHA46 cocktail correspond to a myovirus, the analyses of their genomes sequences did not report virulence or antimicrobial resistance genes. The PHA46 sample is specific for 33 different serovars from different Salmonella strains and shows stability at 7 °C and pH 6. Also, the PHA46 cocktail was effective in reducing the adherence of S. Newport-45 and S. Typhimurium SL1344 to cherry tomatoes, at an average of 0.9 log10, respectively. Regarding internalized bacteria, the reduction was at an average of 1.2 log10, of the serovars mentioned above. The lifespan experiments in C. elegans showed by itself, that the PHA46 cocktail was harmless to the nematode, and the virulence from both Salmonella strains grown in vitro is diminished in the presence of the PHA46 cocktail. In conclusion, these results showed that the PHA46 cocktail could be a good candidate to be used as a biocontrol agent against Salmonella enterica.

Evaluating the efficacy of peroxyacetic acid in preventing Salmonella cross-contamination on tomatoes in a model flume system.

The use of flume tanks for tomato processing has been identified as a potential source of cross-contamination, which could result in foodborne illness. This study's objective was to assess the efficacy of peroxyacetic acid (PAA) at a concentration of ≤80 mg/L in preventing Salmonella enterica cross-contamination under various organic loads in a benchtop model tomato flume tank. The stability of 80 mg/L PAA at different chemical oxygen demand (COD) levels was also tested. Tomatoes were spot inoculated with a five-serovar rifampin-resistant (rif+) Salmonella cocktail (106 or 108 colony forming unit (CFU)/tomato). Inoculated (n = 3) and uninoculated (n = 9) tomatoes were introduced into the flume system containing 0-80 mg/L PAA and 0 or 300 mg/L COD. After washing for 30, 60, or 120 s, uninoculated tomatoes were sampled and analyzed for cross-contamination. All experiments were conducted in triplicate. Increasing the organic load (measured as COD) affected the stability of PAA in water with significantly faster dissociation when exposed to 300 mg/L COD. The concentration of PAA, inoculum level, COD levels, and time intervals were all significant factors that affected cross-contamination. Cross-contamination occurred at the high inoculum level (108 CFU/tomato) even when 80 mg/L PAA was present in the model flume tank, regardless of the organic load level. When the tomatoes were contaminated at a level of 106 CFU/tomato, concentrations as low as 5 mg/L of PAA were effective in preventing cross-contamination at 0 mg/L COD; however, 100 % tomatoes (9/9) were positive when the organic load increased to 300 mg/L COD. When the PAA concentration was increased to 10 mg/L, it effectively prevented cross-contamination in the tank, regardless of the presence of organic load. These results suggest that using PAA at concentrations below the maximum limit remains effective in limiting bacterial cross-contamination and offers a more environment-friendly option for tomato packinghouse operators.

The influence of pH on the efficacy of oxidation-reduction potential (ORP) to predict chlorine disinfection of surrogate bacteria, Escherichia coli O157:H7 and Listeria monocytogenes in oxidant demand free conditions and fresh produce wash water.

Oxidation-reduction potential (ORP) is commonly used as a rapid measurement of the antimicrobial potential of free chlorine during industrial fresh produce washing. The current study tested the hypothesis that ORP can act as a "single variable" measurement of bacterial (vegetative and endospores) inactivation effectiveness with free chlorine irrespective of the water pH value. This situation has on occasion been assumed but never confirmed nor disproven. Chlorine-dosed pH 6.5 and 8.5 phosphate buffer solutions were inoculated with Escherichia coli (E. coli), Listeria innocua (L. innocua), or Bacillus subtilis (B. subtilis) endospores. ORP, free chlorine (FC), and log reduction were monitored after 5 s (for E. coli and L. innocua) and up to 30 min (for B. subtilis spores) of disinfection. Logistic and exponential models were developed to describe how bacteria reduction varied as a function of ORP at different pH levels. Validation tests were performed in phosphate buffered pH 6.5 and 8.5 cabbage wash water periodically dosed with FC, cabbage extract and a cocktail of Escherichia coli O157:H7 (E. coli O157:H7) and Listeria monocytogenes (L. monocytogenes). The built logistic and exponential models confirmed that at equal ORP values, the inactivation of the surrogate strains was not consistent across pH 6.5 and pH 8.5, with higher reductions at higher pH. This is the opposite of the well-known free chlorine-controlled bacterial inactivation, where the antibacterial effect is higher at lower pH. The validation test results indicated that in the cabbage wash water, the relationship between disinfection efficiency and ORP was consistent with the oxidant demand free systems. The study suggests that ORP cannot serve as a reliable single variable measurement to predict bacterial disinfection in buffered systems. When using ORP to monitor and control the antibacterial effectiveness of the chlorinated wash water, it is crucial to take into account (and control) the pH.

Dynamics of Listeriamonocytogenes and Salmonella enterica on Cooked Vegetables During Storage.

Fresh vegetables have been linked to multiple foodborne outbreaks in the U.S., with Listeria monocytogenes and Salmonella enterica identified as leading causes. Beyond raw vegetables, cooked vegetables can also pose food safety concerns due to improper cooking temperature and time combinations or postcooking contamination. Cooked vegetables, having had their native microbiota reduced through heat inactivation, might provide an environment that favors the growth of pathogens due to diminished microbial competition. While the risks associated with raw vegetables are recognized, the survival and growth of pathogens on cooked vegetables remain inadequately studied. This study investigated the growth kinetics of both L. monocytogenes and S. enterica on various cooked vegetables (carrot, corn, onions, green bell pepper, and potato). Vegetables were cooked at 177°C until the internal temperature reached 90°C and then cooled to 5°C. Cooled vegetables were inoculated with a four-strain cocktail of either L. monocytogenes or S. enterica at 3 log CFU/g, then stored at different temperatures (5, 10, or 25°C) for up to 7 days. Both pathogens survived on all vegetables when stored at 5°C. At 10°C, both pathogens proliferated on all vegetables, with the exception of L. monocytogenes on pepper. At 25°C, the highest growth rates were observed by both pathogens on carrot (5.55 ± 0.22 and 6.42 ± 0.23 log CFU/g/d for L. monocytogenes and S. enterica, respectively). S. enterica displayed higher growth rates at 25°C compared to L. monocytogenes on all vegetables. Overall, these results bridge the knowledge gap concerning the growth kinetics of both S. enterica and L. monocytogenes on various cooked vegetables, offering insights to further enhance food safety.

A comprehensive systematic review and meta-analysis of Listeria monocytogenes prevalence in food products in South Korea.

Listeria monocytogenes is a foodborne pathogen that can cause deadly severe listeriosis. While systematic review and meta-analysis are powerful tools for comprehensive analysis by pooling every related study, these approaches to L.monocytogenes contamination food have yet to be studied in South Korea. We aimed to identify high-risk L.monocytogenes foods in South Korea through a prevalence survey of retail food products for the first time. A total of 13,684 samples of 59 articles were used for meta-analysis through the systematic review, and the results were synthesized using a random-effects model considering the heterogeneity. The overall pooled prevalence was 2.26 % (95 % CI: 1.44-3.52 %). Among nine food categories, meat exhibited the highest prevalence at 8.32 % (95 % CI: 4.42-12.14 %) after sample size restriction. Specifically, a post-hoc sensitivity analysis was conducted to identify the prevalence difference among subgroups and the source of heterogeneity. Intriguingly, the analysis revealed chicken as the primary contributor to the elevated prevalence of L.monocytogenes, a key factor deriving the observed heterogeneity. This study carries significant implications for public health and food safety in Korea. Furthermore, knowledge of differences in prevalence levels in various foods will be able to be used as a predictive guideline for foodborne outbreaks.

Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Natural Polysaccharide Film-Based Triboelectric Sensor for Fruit Transportation Collision Monitoring.

Transportation-induced damage to fresh produce is a big challenge in logistics. Current acceleration and pressure sensors for collision monitoring face issues of power dependency, high cost, and environmental concerns. Here, a self-powered and environmentally friendly triboelectric sensor has been developed to monitor fruit collisions in transportation packaging. Microcrystalline cellulose/chitosan and sodium alginate films were prepared as positive and negative tribo-layers to assemble a natural polysaccharide film-based triboelectric nanogenerator (NP-TENG). The NP-TENG's electrical output was proportional to the structure parameters (contact surface roughness and separation gap of the tribo-layers) and the vibration factors (force and frequency) and exhibited excellent stability and durability (over 100,000 cycles under 13 N at 10 Hz). The high mechanical-to-electrical conversion efficiency (instantaneous areal power density of 9.6 mW/m2) and force sensitivity (2.2 V/N) enabled the NP-TENG to be a potential sensor for monitoring fresh produce collisions in packaging during logistics. Transportation simulation measurements of kiwifruits verified that the sensor's electrical outputs increased with the vibration frequency and stacking layer while varying at different packaging locations. This study suggests that the NP-TENG can effectively monitor collision damage during fruit transportation, providing new insights into developing intelligent food packaging systems to reduce postharvest supply chain losses.

Biomimetic leaves with immobilized catalase for machine learning-enabled validating fresh produce sanitation processes.

Washing and sanitation are vital steps during the postharvest processing of fresh produce to reduce the microbial load on the produce surface. Although current process control and validation tools effectively predict sanitizer concentrations in wash water, they have significant limitations in assessing sanitizer effectiveness for reducing microbial counts on produce surfaces. These challenges highlight the urgent need to improve the validation of sanitation processes, especially considering the presence of dynamic organic contaminants and complex surface topographies. This study aims to provide the fresh produce industry with a novel, reliable, and highly accurate method for validating the sanitation efficacy on the produce surface. Our results demonstrate the feasibility of using a food-grade, catalase (CAT)-immobilized biomimetic leaf in combination with vibrational spectroscopy and machine learning to predict microbial inactivation on microgreen surfaces. This was tested using two sanitizers: sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2). The developed CAT-immobilized leaf-replicated PDMS (CAT@L-PDMS) effectively mimics the microscale topographies and bacterial distribution on the leaf surface. Alterations in the FTIR spectra of CAT@L-PDMS, following simulated sanitation processes, indicate chemical changes due to CAT oxidation induced by NaClO or H2O2 treatments, facilitating the subsequent machine learning modeling. Among the five algorithms tested, the competitive adaptive reweighted sampling partial least squares discriminant analysis (CARS-PLSDA) algorithm was the most effective for classifying the inactivation efficacy of E. coli on microgreen leaf surfaces. It predicted bacterial reduction on microgreen surfaces with 100% accuracy in both training and prediction sets for NaClO, and 95% in the training set and 86% in the prediction set for H2O2. This approach can improve the validation of fresh produce sanitation processes and pave the way for future research.