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The new European regulation on minimum quality requirements (MQR) for water reuse (EU, 2020/741) was launched in May 2020 and describes the directives for the use of reclaimed water for agricultural irrigation. This Regulation will be directly applicable in all Member States from 26 June 2023. Since its publication in 2020, concerns have raised about potential non-compliance situations in water reuse systems. The present study represents a case study where three different water reuse systems have been monitored to establish their compliance with the MQR. Each water reuse system includes a wastewater treatment plant (WWTP), a distribution/storage system and an end-user point, where water is used for irrigation of leafy greens. The selected water reuse systems allowed us to compare the efficacy of water treatments implemented in two WWTPs as well as the impact of three different irrigation systems (drip, furrow and overhead irrigation). The presence and concentration of indicator microorganisms (Escherichia coli and C. perfringens spores) as well as pathogenic bacteria (Shiga toxin-producing, E. coli (STEC), E. coli O157:H7, and Salmonella spp.) were monitored in different sampling points (influent and effluent of the WWTPs, water reservoirs located at the distribution system and the end-user point at the irrigation system as well as in the leafy greens during their growing cycle. Average levels of E. coli (0.73 ± 1.20 log cfu E. coli/100 mL) obtained at the point where the WWTP operator delivers reclaimed water to the next actor in the chain, defined in the European regulation as the ‘point of compliance’, were within the established MQR (

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.

Growth of L. monocytogenes is among the most important factors affecting the risk of human listeriosis. In ready to eat leafy greens, the use of anti-Listeria treatments represents a good alternative to inhibit growth during storage. Several commercially available antimicrobial agents have been suggested as effective intervention strategies. Among them, phage preparations and bacteriocin-producing strains have shown promising results against L. monocytogenes. In this study, we investigate the efficacy of two commercially available surface treatments, the bacteriophage formulation PhageGuard Listex (Micreos Food Safety B.V., NL) and the bacteriocin-producing culture SafePro® (CHR Hansen, DK) to inactivate L. monocytogenes in fresh-cut curly endive after processing and during storage. Fresh-cut endive was inoculated with a cold-adapted L. monocytogenes cocktail of 6 strains (4.4 ± 0.0 log cfu/g) and treated with the anti-Listeria treatments. The treatments were applied using a spray system at two different places within the processing line, on the conveyor belt and in the centrifuge. A total of 5 different treatments were applied: i) Untreated (CT); ii) PhageGuard Listex on the conveyor belt (Listex_Conveyor); iii) PhageGuard Listex during centrifugation (Listex_Centrifuge); iv) SafePro on the conveyor belt (SafePro_Conveyor); and v) SafePro during centrifugation (SafePro_Centrifuge). Samples were stored 3 days at 5 °C plus 5 days at 8 °C. PhageGuard Listex treatment reduced L. monocytogenes in fresh-cut endive by 2.5 logs, regardless of the place of treatment application (conveyor belt or centrifuge). On the other hand, SafePro only reduced L. monocytogenes by 0.2 and 0.4 logs, at the conveyor belt and centrifuge, respectively. Maximum L. monocytogenes reductions of about 3.5 log units were observed in fresh-cut endive treated with PhageGuard Listex after 3 days of storage. At the end of the shelf life (8 days), the initial trends were maintained and the fresh-cut curly endive treated with PhageGuard Listex showed the lowest L. monocytogenes concentration. However, by the end of the shelf-life, L. monocytogenes showed higher levels (1.3-fold) than immediately after the application of the treatment. One hypothesis could be that L. monocytogenes cells, which were able to survive the anti-Listeria treatments, were also able to proliferate under the specific storage conditions. Based on the obtained results, PhageGuard Listex seems to be a promising decontamination agent for leafy greens aiming to reduce growth of the bacteria but further work is needed.

The purpose of this study was to evaluate and model the growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to a neutral electrolyzed water (NEW) treatment, packaged in passive modified atmosphere and subsequently stored at different temperatures (4, 8, 13, 16°C) for a maximum of 27 days. Results indicated that E. coli O157:H7 was able to grow at 8, 13, and 16°C, and declined at 4°C. However at 8°C, the lag time lasted 19 days, above the typical shelf-life time for this type of products. A secondary model predicting growth rate as a function of temperature was developed based on a square-root function. A comparison with literature data indicated that the growth predicted by the model for E. coli O157:H7 was again lower than those observed with other disinfection treatments or packaging conditions (chlorinated water, untreated product, NEW, etc.). The specific models here developed might be applied to predict growth in products treated with NEW and to improve existing quantitative risk assessments.

The impact of reclaimed and surface water on the microbiological safety of hydroponic tomatoes was assessed. Greenhouse tomatoes were irrigated with reclaimed and surface water and grown on two hydroponic substrates (coconut fiber and rock wool). Water samples (n=208) were taken from irrigation water, with and without the addition of fertilizers and drainage water, and hydroponic tomatoes (n=72). Samples were analyzed for indicator microorganisms, generic Escherichia coli and Listeria spp., and pathogenic bacteria such as Salmonella spp. and Shiga-toxigenic E. coli (STEC), using multiplex real-time PCR (RT-PCR) after enrichment. The correlation between climatological parameters such as temperature and the levels of microorganisms in water samples was also determined. In irrigation water, generic E. coli counts were higher in reclaimed than in surface water whereas Listeria spp. numbers increased after adding the fertilizers in both water sources. In drainage water, no clear differences in E. coli and Listeria numbers were observed between reclaimed and surface water. No positive samples for STEC were found in irrigation water. Presumptive positives for Salmonella spp. were found in 7.7% of the water samples and 62.5% of these samples were reclaimed water. Salmonella-positive samples by RT-PCR could not be confirmed by conventional methods. Higher concentrations of E. coli were associated with Salmonella-presumptive positive samples. Climatological parameters, such as temperature, were not correlated with the E. coli and Listeria spp. counts. Tomato samples were negative for bacterial pathogens, while generic E. coli and Listeria spp. counts were below the detection limit. The prevalence of presumptive Salmonella spp. found in irrigation water (reclaimed and surface water) was high, which might present a risk of contamination. The absence of pathogens on greenhouse hydroponic tomatoes indicates that good agricultural practices (GAP) were in place, avoiding the microbial contamination of the fruit.

9 páginas, 2 figuras, 3 tablas.-- Review., It is well known that fresh-cut processors usually rely on wash water sanitizers to reduce microbial counts in order to maintain quality and extend shelf-life of the end product. Water is a useful tool for reducing potential contamination but it can also transfer pathogenic microorganisms. Washing with sanitizers is important in fresh-cut produce hygiene, particularly removing soil and debris, but especially in water disinfection to avoid cross-contamination between clean and contaminated product. Most of the sanitizing solutions induce higher microbial reduction after washing when compared to water washing, but after storage, epiphytic microorganisms grow rapidly, reaching similar levels. In fact, despite the general idea that sanitizers are used to reduce the microbial population on the produce, their main effect is maintaining the microbial quality of the water. The use of potable water instead of water containing chemical disinfection agents for washing fresh-cut vegetables is being advocated in some European countries. However, the problems of using an inadequate sanitizer or even none are considered in this manuscript. The need for a standardized approach to evaluate and compare the efficiency of sanitizing agents is also presented. Most new alternative techniques accentuate the problems with chlorine suggesting that the industry should move away from this traditional disinfection agent. However, the use of chlorine based sanitizers are presented as belonging to the most effective and efficient sanitizers when adequate doses are used. In this review improvements in water disinfection and sanitation strategies, including a shower pre-washing step and a final rinse of the produce, are suggested., The authors are grateful to CICYT (project AGL2007-65056) for financial support. M.V. Selma and F. López-Gálvez are holders of grants funded by CSIC (I3PDR-7-01 Contract and I3P grant). Ana Allende was the recipient of a Ramon y Cajal Grant from the Spanish Ministry of Science and Research.