Machine Learning and Imputation to Characterize Human Norovirus Genotype Susceptibility to Sodium Hypochlorite.

Human norovirus (HuNoV) is the leading cause of foodborne illness in the developed world and a major contributor to gastroenteritis globally. Its low infectious dose and environmental persistence necessitate effective disinfection protocols. Sodium hypochlorite (NaOCl) bleach is a widely used disinfectant for controlling HuNoV transmission via contaminated fomites. This study aimed to evaluate the susceptibility of HuNoV genotypes (n = 11) from genogroups I, II, and IV to NaOCl in suspension. HuNoV was incubated for 1 and 5 min in diethyl pyrocarbonate (DEPC) treated water containing 50 ppm, 100 ppm, or 150 ppm NaOCl, buffered to maintain a pH between 7.0 and 7.5. Neutralization was achieved by a tenfold dilution into 100% fetal bovine serum. RNase pre-treatment followed by RT-qPCR was used to distinguish between infectious and non-infectious HuNoV. Statistical methods, including imputation, machine learning, and generalized linear models, were applied to process and analyze the data. Results showed that NaOCl reduced viral loads across all genotypes, though efficacy varied. Genotypes GI.1, GII.4 New Orleans, and GII.4 Sydney were the least susceptible, while GII.6 and GII.13 were the most susceptible. All NaOCl concentrations above 0 ppm were statistically indistinguishable, and exposure duration did not significantly affect HuNoV reduction, suggesting rapid inactivation at effective concentrations. For instance, some genotypes were completely inactivated within 1 min, rendering extended exposure unnecessary, while other genotypes maintained the initial concentration at both 1 and 5 min, indicating a need for longer contact times. These findings underscore the critical role of HuNoV genotype selection in testing disinfection protocols and optimizing NaOCl concentrations. Understanding HuNoV susceptibility to NaOCl bleach informs better disinfection strategies, aiding public health and food safety authorities in reducing HuNoV transmission and outbreaks.

Salmonella Prevalence in Raw Cocoa Beans and a Microbiological Risk Assessment to Evaluate the Impact of Cocoa Liquor Processing on the Reduction of Salmonella.

Salmonella in raw cocoa beans (n = 870) from main sourcing areas over nine months was analyzed. It was detected in 71 (ca. 8.2%) samples, with a contamination level of 0.3-46 MPN/g except for one sample (4.1 × 104 CFU/g). Using prevalence and concentration data as input, the impact of thermal treatment in cocoa processing on the risk estimate of acquiring salmonellosis by a random Belgian chocolate consumer was calculated by a quantitative microbiological risk assessment (QMRA) approach. A modular process risk model from raw cocoa beans to cocoa liquor up to a hypothetical final product (70-90% dark chocolate tablet) was set up to understand changes in Salmonella concentrations following the production process. Different thermal treatments during bean or nib steam, nib roasting, or liquor sterilization (achieving a 0-6 log reduction of Salmonella) were simulated. Based on the generic FAO/WHO Salmonella dose-response model and the chocolate consumption data in Belgium, salmonellosis risk per serving and cases per year at population level were estimated. When a 5 log reduction of Salmonella was achieved, the estimated mean risk per serving was 3.35 × 10-8 (95% CI: 3.27 × 10-10-1.59 × 10-7), and estimated salmonellosis cases per year (11.7 million population) was 88 (95% CI: <1-418). The estimated mean risk per serving was 3.35 × 10-9 (95% CI: 3.27 × 10-11-1.59 × 10-8), and the estimated salmonellosis cases per year was 9 (95% CI: <1-42), for a 6 log reduction. The current QMRA model solely considered Salmonella reduction in a single-step thermal treatment in the cocoa process. Inactivation obtained during other process steps (e.g. grinding) might occur but was not considered. As the purpose was to use QMRA as a tool to evaluate the log reduction in the cocoa processing, no postcontamination from the processing environment and ingredients was included. A minimum of 5 log reduction of Salmonella in the single-step thermal treatment of cocoa process was considered to be adequate.

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.

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.

Prevalence and growth potential of Listeria monocytogenes in innovative, pre-packed, plant-based ready-to-eat food products on the Belgian market.

In recent years, pre-packed ready-to-eat (RTE) food products on the Belgian market have shifted to a more plant-based composition due to a variety of reasons, including consumer concerns about health, animal welfare, and sustainability. However, similar to animal-based RTE foods, plant-based RTE foods can be susceptible to the presence and outgrowth of Listeria monocytogenes (L. monocytogenes). Three innovative, pre-packed, plant-based RTE food product categories on the Belgian market were identified based upon data gaps regarding the prevalence and growth potential of this pathogen. These were vegetarian and vegan deli sandwich slices (category 1), fresh-cut (mixes of) leafy vegetables (category 2), and multi-ingredient salad bowls (category 3). Reports on associated listeriosis outbreaks and recalls were collected and a comprehensive literature review on the prevalence of L. monocytogenes (i.e. detection in 25 g food) was performed. In addition, the prevalence of L. monocytogenes was also determined through an exploratory retail survey of ca. 50 different RTE products of each category. A batch was considered positive if L. monocytogenes was detected in a food item, either on the day of purchase, at the end of shelf life, or both. During the retail survey, L. monocytogenes was not detected in category 2 (0 out of 51 batches), while 1 out of 51 and 6 out of 48 batches were found positive for respectively category 1 and 3. The observed L. monocytogenes concentration did not exceed 10 CFU/g at any point in time in any batch. Furthermore, challenge tests were performed to determine the growth potential of L. monocytogenes in nine pre-packed, plant-based RTE food products (two to four different products of each category, and three different batches per product). After inoculation, products were stored for half of their shelf life at 7 °C and half of their shelf life at 9 °C (simulation of resp. retail and consumer storage). In six of the nine challenge tests executed, growth of L. monocytogenes was supported (i.e. growth potential ≥0.50 log10 CFU/g during shelf life). The highest growth potential was observed for fresh-cut iceberg lettuce (3.60 log10 CFU/g in 9 days), but a large variation regarding the growth potential of L. monocytogenes was noted both between and within the three studied pre-packed, plant-based RTE food product categories. This variation was mainly caused by differences in product composition, physicochemical product characteristics, present (competitive) microbiota such as lactic acid bacteria, applied preservation techniques, and shelf life.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores.

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1-79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5-8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

Microbiological profiling and knowledge of food preservation technology to support guidance on a neutropenic diet for immunocompromised patients.

The current society consists of an increasing number of people vulnerable to infections. For certain people with severe immunodeficiency, a neutropenic or low-microbial diet is being prescribed, which substitutes high-risk foods that are more likely to contain human (opportunistic) pathogens with lower-risk alternatives. These neutropenic dietary guidelines are typically set up from a clinical and nutritional perspective, rather than from a food processing and food preservation perspective. In this study, the current guidelines in use by the Ghent University Hospital were evaluated based on the current knowledge of food processing and preservation technologies and the scientific evidence on microbiological quality, safety, and hygiene of processed foods. Three criteria are identified to be important: (1) the microbial contamination level and composition; (2) the potential presence of established foodborne pathogens such as Salmonella spp. (to which a zero-tolerance policy is recommended); and (3) an increased vigilance for L. monocytogenes as an opportunistic foodborne pathogen with a high mortality rate in immunocompromised individuals (to which a zero-tolerance policy should apply). A combination of these three criteria was used as a framework for the evaluation of the suitability of foodstuffs to be included in a low-microbial diet. Differences in processing technologies, initial contamination of products, etc., however, lead to a high degree of variability in microbial contamination and make it difficult to unambiguously accept or reject a certain type of foodstuff without prior knowledge of the ingredients and the processing and preservation technologies applied during manufacturing and subsequent storage conditions. A restricted screening on a selection of (minimally processed) plant-based foodstuffs on the retail market in Flanders, Belgium supported decision-making on the inclusion of these food types in a low-microbial diet. Still, when determining the suitability of a foodstuff to be included in a low-microbial diet, not only the microbiological status but also nutritional and sensorial properties should be assessed, which requires multidisciplinary communication and collaboration.

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment.

Chitin soil amendment is known to improve soil quality, plant growth and stress resilience, but the underlying mechanisms are not well understood. In this study, we monitored chitin's effect on lettuce physiology every two weeks through an eight-week growth period, analyzed the early transcriptional reprogramming and related metabolomic changes of lettuce, in response to crab chitin treatment in peat-based potting soil. In commercial growth conditions, chitin amendment still promoted lettuce growth, increased chlorophyll content, the number of leaves and crop head weight from week six. The flavonoid content in lettuce leaves was altered as well, showing an increase at week two but a decrease from week six. Transcriptomic analysis showed that over 300 genes in lettuce root were significantly differentially expressed after chitin soil treatment. Gene Ontology-term (GO) enrichment analysis revealed statistical overrepresentation of GO terms linked to photosynthesis, pigment metabolic process and phenylpropanoid metabolic process. Further analysis of the differentially expressed genes (DEGs) showed that the flavonoid pathway was mostly upregulated whereas the bifurcation of upstream phenylpropanoid pathway towards lignin biosynthesis was mostly downregulated. Metabolomic analysis revealed the upregulation of salicylic acid, chlorogenic acid, ferulic acid, and p-coumaric acid in chitin-treated lettuce seedlings. These phenolic compounds (PCs) mainly influence the phenylpropanoid biosynthesis pathway and may play important roles in plant defense reactions. Our results suggest that chitin soil amendments might activate induced resistance by priming lettuce plants and promote lettuce growth via transcriptional changes.

Prevalence, attachment ability and strength of the biological control agent Bacillus thuringiensis on tomato.

Bacillus thuringiensis (Bt) is commonly used as a biological control agent (BCA) to control insect pests in edible plant production and can as such be introduced into the food chain of fresh produce. Using standard food diagnostics Bt will be detected and reported as presumptive B. cereus. Tomato plants are often sprayed with Bt biopesticides for insect control, thus these Bt BCAs can also reach the tomato fruits and persist until consumption. In this study, vine tomatoes from the retail in Belgium (Flanders) were investigated for the occurrence and residual numbers of presumptive B. cereus and Bt. Of 109 tomato samples, 61 (56%) were tested positive for presumptive B. cereus. Of the presumptive B. cereus isolates (n = 213) recovered from these samples, 98% were identified as Bt by the production of parasporal crystals. Further quantitative real-time PCR assays on a subselection of Bt isolates (n = 61) showed that 95% of Bt isolates were indistinguishable from Bt biopesticide strains that are approved to be used on crops in the EU. Furthermore, the attachment strength of tested Bt biopesticide strains showed easier wash-off properties if using the commercial Bt granule formulation than the unformulated lab-cultured Bt or B. cereus spore suspensions.

Identification and characterization of Bacillus thuringiensis and other Bacillus cereus group isolates from spinach by whole genome sequencing.

Bacillus thuringiensis (Bt), used as a biological control agent (BCA), can persist on plants, and from there can be introduced into the final food product. In routine food safety diagnostics, these Bt residues cannot be distinguished from natural populations of Bacillus cereus present in plants and all are enumerated as "presumptive B. cereus." In this study, information on eventual use of Bt biopesticides, brand, application times and intervals provided by three food processing companies in Belgium, were integrated with quantitative data on presumptive B. cereus measured from fresh to frozen food products. This information together with data on genomic similarity obtained via whole genome sequencing (WGS) and cry gene profiling using a quantitative real-time PCR (qPCR) assay, confirmed that six out of 11 Bt isolates originated from the applied Bt biocontrol products. These identified Bt strains were shown to carry enterotoxin genes (nhe, hbl, cytK-2) and express Hbl enterotoxin in vitro. It was also noted that these Bt biopesticide strains showed no growth at standard refrigeration temperatures and a low or moderate biofilm-forming ability and cytotoxic activity. Our results also showed that the use of Bt as a BCA on spinach plants in the field led to higher residual counts of Bt in spinach (fresh or frozen) in the food supply chain, but the residual counts exceeding at present commonly assumed safety limit of 105 CFU/g was only found in one fresh spinach sample. It is therefore recommended to establish a pre-harvest interval for Bt biopesticide application in the field to lower the likelihood of noncompliance to the generic B. cereus safety limit. Furthermore, WGS was found to be the best way to identify Bt biopesticide isolates at the strain level for foodborne outbreaks and clinical surveillance. The developed qPCR assay for screening on the presence of cry genes in presumptive B. cereus can be applied as a rapid routine test as an amendment to the already existing test on Bt crystal proteins determined via phase-contrast microscopy.

Behavior of the Biological Control Agent Bacillus thuringiensis subsp. aizawai ABTS-1857 and Salmonella enterica on Spinach Plants and Cut Leaves.

Fresh produce has been identified as an important vehicle for the transmission of foodborne pathogens. This study evaluated the behavior of vegetative cells and spores of Bacillus thuringiensis, one of the main biological control agents (BCAs) used in the world, and Salmonella enterica on spinach plants (pre-harvest) and spinach cut leaves (post-harvest) at 12°C, experimentally inoculated as single or co-cultures. The results evidenced that spray-inoculated commercial BCA containing Bacillus thuringiensis subsp. aizawai ABTS-1857 (BTa ABTS-1857) spores persisted well on spinach leaves in both pre- and post-harvest simulations. However, when BTa ABTS-1857 vegetative cells were spray-inoculated, more than 2 log reductions in the counts of B. thuringiensis were observed during 20 days pre- and 5 days post-harvest simulations, respectively. The counts of S. Montevideo on the spinach cut leaves during post-harvest storage at 12°C for 5 days remained unchanged, whereas 1 log reduction was noted during pre-harvest. Moreover, during pre-harvest simulation, when co-inoculated with BTa ABTS-1857 vegetative cells or spores, additional 0.5 or 1.0 log reductions were detected on the counts of S. Montevideo in the spinach leaves on the 10th day. These results were obtained under laboratory conditions, and further findings in longitudinal studies from farm (in the agricultural field) to retail (end of shelf life) will contribute to understanding of the role of B. thuringiensis as a BCA on growth/survival of Salmonella spp. in fresh produce.

Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Robustness of fermented carrot juice against Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7.

Artisanal vegetable fermentations are regaining popularity in industrialized countries, but they could be prone to contamination with foodborne pathogens. By simulating home or small-scale restaurant fermentations, we evaluated the microbiological safety of spontaneous carrot juice fermentations. Raw carrot juice was spiked with Listeria monocytogenes, Salmonella enterica subsp. enterica Typhimurium and Escherichia coli O157:H7, and the microbial dynamics were followed throughout the entire fermentation process by cultivation and amplicon sequencing. In addition, the behavior of these pathogens was also monitored after addition of raw cucumber juice and storage under refrigerated conditions to mimic post-contamination issues. Although the numbers of the pathogens increased during the first phase of the fermentation, the pathogens were not able to persist throughout the fermentation. Their numbers fell below the detection limit after 8 days of fermentation at 20 °C. Further investigation using amplicon sequencing also showed that there was no major impact on the general microbial dynamics of the spontaneous carrot juice fermentation. This indicates that the artisanal carrot juice fermentation is a robust process which resists the persistence of pathogens. More caution is needed however when mixing the final fermented product with a raw juice. When simulating pathogen post-contamination, both Salmonella enterica and Escherichia coli were able to survive in the refrigerated fermented juice up to 10 days after the fermentation. Listeria monocytogenes was detected up to 8 days in the refrigerated juice. Pasteurization of the raw juice before adding it to the fermented product is thus recommended.

Potential of Human Norovirus Surrogates and Salmonella enterica Contamination of Pre-harvest Basil (Ocimum basilicum) via Leaf Surface and Plant Substrate.

Fresh produce has been identified as an important vehicle for foodborne pathogen transmission and fresh culinary herbs have occasionally been associated with human pathogens and illness. In this study, the fate of human NoV surrogates [murine norovirus 1 (MNV-1) and Tulane virus (TV)] and three strains of Salmonella enterica on pre-harvest basil (Ocimum basilicum) was investigated. The persistence after contamination via either leaf surface or plant substrate was tested respectively. After 3 days, both MNV-1 and TV on pre-harvest leaves were at non-detectable levels (>5.5-log reduction for MNV-1 and >3.3-log reduction for TV). The three Salmonella strains showed consistent reductions of 3- to 4-log. At day 6 and 9, all the tested samples showed low levels of infectivity which were close or below the detection limits (1.7-log PFU/sample leaf for MNV-1 and TV, 0.7-log CFU/sample leaf for Salmonella) except for S. Thompson FMFP 899, one out of three samples showed to maintain present at exceptional high levels (day 6: 5.5-log CFU/sample leaf; day 9: 6.7-log CFU/sample leaf). Possibilities of microbial internalization into the edible parts of basil via the roots was demonstrated with both MNV-1 and S. enterica Thompson FMFP 899. The infectivity of internalized MNV-1 and S. enterica both decreased to non-detectable levels within 9 days after inoculation. Moreover, it should be noticed that very high microbial inoculation was used in the experimental set-up (8.46-log PFU/ml of MNV-1, 8.60-log CFU/ml of S. enterica), which is abnormal in the real-life expected contamination scenario. Within the tested scenarios in this study, S. enterica contaminated on the adaxial leaf surface of basil plants while in growth, and remained/reached a high population of over 6-log CFU/sample leaf after 9 days in one out of three samples, thus showed the highest potential for causing foodborne infection.

Inactivation of viruses and bacteria on strawberries using a levulinic acid plus sodium dodecyl sulfate based sanitizer, taking sensorial and chemical food safety aspects into account.

The efficacy of levulinic acid (LVA) in combination with sodium dodecyl sulfate (SDS) in removal of foodborne viruses, enteric bacterial pathogens and their surrogates on fresh strawberries was investigated. Inoculated strawberries were treated with potable water, sodium hypochlorite solution (50ppm), 0.5% LVA plus 0.5% SDS solution, and 5% LVA plus 2% SDS solution respectively for 2min, followed by spray-rinsing with potable water. Water washing removed at least 1.0-log of the tested viral and bacterial strains from the strawberries' surfaces. The 50ppm chlorine wash induced 3.4, 1.5 and 2.1-log reductions for hepatitis A virus (HAV), murine norovirus-1 (MNV-1) and MS2 bacteriophage, respectively. In comparison, the tested bacterial strains showed uniform reductions around 1.6-log CFU/ml. The 0.5% LVA plus 0.5% SDS wash induced 2.7, 1.4 and 2.4-log reductions for HAV, MNV-1 and MS2, which were comparable with the reductions induced by chlorine (P>0.05). For bacteria, over 2.0-log reductions were obtained for Enterococcus faecium, Listeria monocytogenes and Salmonella, while Escherichia coli O157:H7 and Escherichia coli P1 showed reductions of 1.9 and 1.8-log CFU/ml. Higher concentration of LVA plus SDS showed no significantly higher reductions (P>0.05). Sensory tests of washed strawberries and chemical residue analysis of LVA on strawberries after washing were also performed. In conclusion, this study demonstrates good performance of 0.5% LVA plus 0.5% SDS to reduce the levels of enteric pathogens if present on strawberries without altering taste and introducing chemical safety issues.

Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results.

Quantitative contamination assessment of Escherichia coli in baby spinach primary production in Spain: Effects of weather conditions and agricultural practices.

A quantitative microbial contamination model of Escherichia coli during primary production of baby spinach was developed. The model included only systematic contamination routes (e.g. soil and irrigation water) and it was used to evaluate the potential impact of weather conditions, agricultural practices as well as bacterial fitness in soil on the E. coli levels present in the crop at harvest. The model can be used to estimate E. coli contamination of baby spinach via irrigation water, via soil splashing due to irrigation water or rain events, and also including the inactivation of E. coli on plants due to solar radiation during a variable time of culturing before harvest. Seasonality, solar radiation and rainfall were predicted to have an important impact on the E. coli contamination. Winter conditions increased E. coli prevalence and levels when compared to spring conditions. As regards agricultural practices, both water quality and irrigation system slightly influenced E. coli levels on baby spinach. The good microbiological quality of the irrigation water (average E. coli counts in positive water samples below 1 log/100mL) could have influenced the differences observed among the tested agricultural practices (water treatment and irrigation system). This quantitative microbial contamination model represents a preliminary framework that assesses the potential impact of different factors and intervention strategies affecting E. coli concentrations at field level. Taking into account that E. coli strains may serve as a surrogate organism for enteric bacterial pathogens, obtained results on E. coli levels on baby spinach may be indicative of the potential behaviour of these pathogens under defined conditions.

Analysis of domestic refrigerator temperatures and home storage time distributions for shelf-life studies and food safety risk assessment.

In the framework of food safety, when mimicking the consumer phase, the storage time and temperature used are mainly considered as single point estimates instead of probability distributions. This singlepoint approach does not take into account the variability within a population and could lead to an overestimation of the parameters. Therefore, the aim of this study was to analyse data on domestic refrigerator temperatures and storage times of chilled food in European countries in order to draw general rules which could be used either in shelf-life testing or risk assessment. In relation to domestic refrigerator temperatures, 15 studies provided pertinent data. Twelve studies presented normal distributions, according to the authors or from the data fitted into distributions. Analysis of temperature distributions revealed that the countries were separated into two groups: northern European countries and southern European countries. The overall variability of European domestic refrigerators is described by a normal distribution: N (7.0, 2.7)°C for southern countries, and, N (6.1, 2.8)°C for the northern countries. Concerning storage times, seven papers were pertinent. Analysis indicated that the storage time was likely to end in the first days or weeks (depending on the product use-by-date) after purchase. Data fitting showed the exponential distribution was the most appropriate distribution to describe the time that food spent at consumer's place. The storage time was described by an exponential distribution corresponding to the use-by date period divided by 4. In conclusion, knowing that collecting data is time and money consuming, in the absence of data, and at least for the European market and for refrigerated products, building a domestic refrigerator temperature distribution using a Normal law and a time-to-consumption distribution using an Exponential law would be appropriate.