The Gamma concept approach as a tool to predict fresh produce supporting or not the growth of L. monocytogenes.

Challenge tests are commonly employed to evaluate the growth behavior of L. monocytogenes in food matrices; they are known for being expensive and time-consuming. An alternative could be the use of predictive models to forecast microbial behavior under different conditions. In this study, the growth behavior of L. monocytogenes in different fresh produce was evaluated using a predictive model based on the Gamma concept considering pH, water activity (aw), and temperature as input factors. An extensive literature search resulted in a total of 105 research articles selected to collect growth/no growth behavior data of L. monocytogenes. Up to 808 L. monocytogenes behavior values and physicochemical characteristics were extracted for different fruits and vegetables. The predictive performance of the model as a tool for identifying the produce commodities supporting the growth of L. monocytogenes was proved by comparing with the experimental data collected from the literature. The model provided satisfactory predictions on the behavior of L. monocytogenes in vegetables (>80% agreement with experimental observations). For leafy greens, a 90% agreement was achieved. In contrast, the performance of the Gamma model was less satisfactory for fruits, as it tends to overestimate the potential of acid commodities to inhibit the growth of L. monocytogenes.

Bioactive compounds in lettuce: Highlighting the benefits to human health and impacts of preharvest and postharvest practices.

Lettuce is one of the most commonly consumed leafy vegetables worldwide and is available throughout the entire year. Lettuce is also a significant source of natural phytochemicals. These compounds, including glycosylated flavonoids, phenolic acids, carotenoids, the vitamin B groups, ascorbic acid, tocopherols, and sesquiterpene lactones, are essential nutritional bioactive compounds. This review aims to provide a comprehensive understanding of the composition of health-promoting compounds in different types of lettuce, the potential health benefits of lettuce in reducing the risks of chronic diseases, and the effect of preharvest and postharvest practices on the biosynthesis and accumulation of health-promoting compounds in lettuce.

Peroxyacetic acid and chlorine dioxide unlike chlorine induce viable but non-culturable (VBNC) stage of Listeria monocytogenes and Escherichia coli O157:H7 in wash water.

The elaboration of guidelines for the industry to establish minimum concentration to prevent cross-contamination during washing practices based on operational limits is the core of the recommended criteria for the use of sanitizers. Several studies have evidenced that sanitizers reduced the levels of foodborne pathogens. However, they might lead to the progress into a viable but non-culturable (VBNC) state of the cells. This evidence has raised concerns regarding the effectiveness of the recommended washing practices for the inactivation of microbial cells present in the process wash water (PWW). The present study evaluated if the most commonly used sanitizers, including sodium hypochlorite (chlorine), peroxyacetic acid (PAA) and chlorine dioxide (ClO2) at established operational limits induced the VBNC stage of Listeria monocytogenes and Escherichia coli O157:H7. Prevention of cross-contamination was examined in four different types of PWW from washing shredded lettuce and cabbage, diced onions, and baby spinach under simulated commercial conditions of high organic matter and 1 min contact time. The results obtained for chlorine showed that recommended operational limits (20-25 mg/L free chlorine) were effective in inactivating L. monocytogenes and E. coli O157:H7 in the different PWWs. However, the operational limits established for PAA (80 mg/L) and ClO2 (3 mg/L) reduced the levels of culturable pathogenic bacteria but induced the VBNC state of the remaining cells. Consequently, the operational limits for chlorine are satisfactory to inactivate foodborne pathogens present in PWW and prevent cross-contamination but higher concentrations or longer contact times should be needed for PAA and ClO2 to reduce the likelihood of the induction of VBNC bacteria cells, as it represents a hazard.

Critical points affecting the microbiological safety of bell peppers washed with peroxyacetic acid in a commercial packinghouse.

The washing stage from a bell pepper commercial packinghouse was assessed to study some of the critical control points related to bacterial cross-contamination. The washing line comprised two overhead spray bars applications: a pre-wash step without peroxyacetic acid (PAA), and a wash step with PAA. The physicochemical characteristics of the wash water and the bacterial quality and safety of the wash water and bell peppers (including aerobic mesophilic bacteria (AMB), Salmonella spp., and Shiga-toxigenic E. coli (STEC)) were studied. Additionally, the performance of commercial test methods (reflectometry, amperometric probe, chronoamperometric sensor) for measuring the residual concentration of PAA was examined. The bacterial load of the pre-wash water (8.7 ± 1.3 log cfu/100 mL AMB) was very high and thus peppers after the pre-wash showed a significantly higher bacterial load (4.9 ± 0.9 log cfu/g AMB) than the unwashed (3.8 ± 0.7 log cfu/g AMB) or the washed peppers (3.3 ± 0.8 log cfu/g AMB) (p < 0.05). However, no pathogenic bacteria were detected in bell pepper samples (n = 40), and only one water sample was confirmed positive for STEC (n = 64, 1.6% prevalence). The chronoamperometric sensor (PAASense) and the online amperometric probe showed similar results, while the reflectometry (Quantofix) significantly sub estimated (p < 0.05) PAA concentration. The results obtained highlight the need for interventions to improve hygiene in the washing line to ensure the microbiological quality and safety of bell peppers. The maintenance of optimal PAA concentrations in all the washing steps is critical for reducing the chance of water-mediated cross-contamination.

Demonstration tests of irrigation water disinfection with chlorine dioxide in open field cultivation of baby spinach.

BACKGROUND: Treatments for the disinfection of irrigation water have to be evaluated by demonstration tests carried out under commercial settings taking into account not only their antimicrobial activity but also the potential phytotoxic effects on the crop. The consequences of the treatment of irrigation water with chlorine dioxide (ClO2 ) used for sprinkler irrigation of baby spinach in two commercial agricultural fields was assessed. RESULTS: Residual ClO2 levels at the sprinklers in the treated field were always below 1 mg L-1 . ClO2 treatment provoked limited but statistically significant reductions in culturable Escherichia coli counts (0.2-0.3 log reductions), but not in the viable E. coli counts in water, suggesting the presence of viable but non-culturable cells (VBNC). Although disinfected irrigation water did not have an impact on the microbial loads of Enterobacteriaceae nor on the quality characteristics of baby spinach, it caused the accumulation of chlorates (up to 0.99 mg kg-1 in plants) and the reduction of the photosynthetic efficiency of baby spinach. CONCLUSION: Low concentrations of ClO2 are effective in reducing the culturable E. coli present in irrigation water but it might induce the VBNC state. Presence of disinfection by-products and their accumulation in the crop must be considered to adjust doses in order to avoid crop damage and chemical safety risks. © 2017 Society of Chemical Industry.

Disinfection by-products in baby lettuce irrigated with electrolysed water.

BACKGROUND: Irrigation water disinfection reduces the microbial load but it might lead to the formation and accumulation of disinfection by-products (DBPs) in the crop. If DBPs are present in the irrigation water, they can accumulate in the crop, particularly after the regrowth, and be affected by the postharvest handling such as washing and storage. To evaluate the potential accumulation of DBPs, baby lettuce was grown using irrigation water treated with electrolysed water (EW) in a commercial greenhouse over three consecutive harvests and regrowths. The impact of postharvest practices such as washing and storage on DBP content was also assessed. RESULTS: Use of EW caused the accumulation of chlorates in irrigation water (0.02-0.14 mg L-1 ), and in the fresh produce (0.05-0.10 mg kg-1 ). On the other hand, the disinfection treatment had minor impact regarding the presence of trihalomethanes (THMs) in water (0.3-8.7 µg L-1 max), and in baby lettuce (0.3-2.9 µg kg-1 max). CONCLUSIONS: Disinfection of irrigation water with EW caused the accumulation of chlorates in the crop reaching levels higher than the current maximum residual limit established in the EU legislation for leafy greens. © 2017 Society of Chemical Industry.

Pre- and postharvest preventive measures and intervention strategies to control microbial food safety hazards of fresh leafy vegetables.

This review includes an overview of the most important preventive measures along the farm to fork chain to prevent microbial contamination of leafy greens. It also includes the technological and managerial interventions related to primary production, postharvest handling, processing practices, distribution, and consumer handling to eliminate pathogens in leafy greens. When the microbiological risk is already present, preventive measures to limit actual contamination events or pathogen survival are considered intervention strategies. In codes of practice the focus is mainly put on explaining preventive measures. However, it is also important to establish more focused intervention strategies. This review is centered mainly on leafy vegetables as the commodity identified as the highest priority in terms of fresh produce microbial safety from a global perspective. There is no unique preventive measure or intervention strategy that could be applied at one point of the food chain. We should encourage growers of leafy greens to establish procedures based on the HACCP principles at the level of primary production. The traceability of leafy vegetables along the chain is an essential element in ensuring food safety. Thus, in dealing with the food safety issues associated with fresh produce it is clear that a multidisciplinary farm to fork strategy is required.

Cross-contamination of Escherichia coli O157:H7 is inhibited by electrolyzed water combined with salt under dynamic conditions of increasing organic matter.

Water can be a vector for foodborne pathogen cross-contamination during washing of vegetables if an efficient method of water disinfection is not used. Chlorination is the disinfection method most widely used, but it generates disinfection by-products such as trihalomethanes (THMs). Therefore, alternative disinfection methods are sought. In this study, a dynamic system was used to simulate the commercial conditions of a washing tank. Organic matter and the inoculum of Escherichia coli O157:H7 were progressively added to the wash water in the washing tank. We evaluated the effectiveness of the electrolyzed water (EW) when combining with the addition of salt (1, 0.5 and 0.15 g/L NaCl) on the pathogenic inactivation, organic matter depletion and THM generation. Results indicated that electrolysis of vegetable wash water with addition of salt (0.5 g/L NaCl) was able to eliminate E. coli O157:H7 population build-up and decrease COD accumulation while low levels of THMs were produced.

Effects of oxygen-depleted atmospheres on survival and growth of Listeria monocytogenes on fresh-cut Iceberg lettuce stored at mild abuse commercial temperatures.

The effects of oxygen-depleted atmospheres, 0.25% O2+12% CO2 (balance N2) and 2% O2 + 6% CO2 (balance N2), on growth of Listeria monocytogenes on fresh-cut Iceberg lettuce were determined. The study was carried out at mild abuse temperatures using controlled atmosphere chambers. During storage at a constant temperature of 7 °C, growth was enhanced at the lower oxygen level of 0.25% O2 by Day 10. Over 17 days of storage at temperatures designed to mimic mild abuse commercial conditions, there were again significantly higher counts under 0.25% O2 from Day 10 onwards. These were 0.9 and 0.7 log cycles higher on Days 14 and 17, respectively. When a model lettuce agar medium was used to eliminate possible interactions with competing flora the direct effects of the atmosphere enhancing the growth of L. monocytogenes was also observed. It is concluded that use of very O2-depleted atmospheres for control of enzymatic browning of fresh-cut Iceberg lettuce may introduce a potential hazard under some commercial conditions. There is a need for greater vigilance and possibly additional measures to ensure consumer safety.

Physiological, phytochemical and structural changes of multi-leaf lettuce caused by salt stress.

BACKGROUND: Environmental stress due to salt has been described to enhance lettuce processability, shelf life and consumer acceptability. Moderate salinity causes altered leaf carotenoid, lignin, phenolic and flavonol levels without noticeable changes in the green colour and morphology of lettuce. The aim of this study was to understand the improvements in processability, due to salt stress, related to textural properties and structural characteristics. RESULTS: Physiological, phytochemical and structural changes were observed that were of significant relevance to salt stress (50, 100 and 150 mmol L(-1) NaCl). Fresh weight per plant, fresh weight per leaf, leaf area, water content, colour saturation, chlorophyll a and b and the area of the intercellular spaces decreased when the concentration of salt was increased. Solute concentration, elasticity, total and individual phenolic acids and the areas occupied by the palisade and spongy parenchyma cells increased when the concentration of salt was increased. CONCLUSION: These data illustrate that salt stress can have a positive impact on certain structural parameters, especially tissue elasticity, that can be closely linked to a higher number of cells, of lower size and high leaf strength, explaining the postharvest longevity of lettuce. However, leaf growth and visual quality could be negatively affected by salt stress.

Modelling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: chlorine washing and modified atmosphere packaging.

Fresh-cut iceberg lettuce inoculated with Escherichia coli O157:H7 was submitted to chlorine washing (150 mg/mL) and modified atmosphere packaging on laboratory scale. Populations of E. coli O157:H7 were assessed in fresh-cut lettuce stored at 4, 8, 13 and 16 °C using 6-8 replicates in each analysis point in order to capture experimental variability. The pathogen was able to grow at temperatures ≥8 °C, although at low temperatures, growth data presented a high variability between replicates. Indeed, at 8 °C after 15 days, some replicates did not show growth while other replicates did present an increase. A growth primary model was fitted to the raw growth data to estimate lag time and maximum growth rate. The prediction and confidence bands for the fitted growth models were estimated based on Monte-Carlo method. The estimated maximum growth rates (log cfu/day) corresponded to 0.14 (95% CI: 0.06-0.31), 0.55 (95% CI: 0.17-1.20) and 1.43 (95% CI: 0.82-2.15) for 8, 13 and 16 °C, respectively. A square-root secondary model was satisfactorily derived from the estimated growth rates (R(2) > 0.80; Bf = 0.97; Af = 1.46). Predictive models and data obtained in this study are intended to improve quantitative risk assessment studies for E. coli O157:H7 in leafy green products.

Influence of nutrient solutions in an open-field soilless system on the quality characteristics and shelf life of fresh-cut red and green lettuces (Lactuca sativa L.) in different seasons.

BACKGROUND: Little information is available about the impact of nutrient solution ion concentration on quality characteristics and shelf life of fresh-cut lettuce grown in soilless systems in open field. Three lettuce genotypes, lollo rosso and red oak leaf as red-leafed genotypes and butterhead as green-leafed genotype, were studied. The influence of three nutrient solutions with low, medium and high ion concentrations, which varied in the macroanion (NO3⁻) and macrocations (K⁺, Ca²âº and NH4⁺), were compared in summer and winter. RESULTS: The nutrient solutions evaluated in this study for the production of lettuce in a soilless system did not strongly influence the quality characteristics of the raw material. When the ion concentration of the nutrient solution was increased, fresh weight decreased, although it depended on the genotype and season. Maturity index and dry matter content varied with the season but independently of the nutrient solution. In summer, maturity index was higher and dry matter lower than in winter. Initial texture and visual quality were not influenced by the nutrient solution. Medium ion concentration provided the highest content of vitamin C and phenolic compounds. Our observations pointed out that the genotype had a strong influence on the shelf life of the fresh-cut product with minor differences among nutrient solutions. In general, red-leafed lettuces showed the highest antioxidant content, helping the maintenance of sensory characteristics throughout storage. CONCLUSION: The combination of optimal nutrient solution ion concentration and suitable cultivar is considered essential to ensure lettuce post-cutting life.

Optimization of a ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry method for monitoring haloacetic acids as chlorinated disinfection by-products in the fresh-cut industry.

Haloacetic acids (HAAs) are one of the most important chlorinated disinfection by-products generated during water disinfection in the fresh-cut industry, and they can remain in the product, resulting in a consumer health risk. In this study, ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM) analysis used for drinking water was optimized and applied for the quantification of nine HAAs (HAA9) in fresh-cut lettuce and process water samples, with the complex matrix interferences for separation, and quantification problems. The method showed good selectivity, specificity and linearity, satisfactory values for trueness (recoveries of 80-116 %), precision (<22 %), and uncertainty (<55 %). Quantification limits varied from 1 to 5 µg L-1 or µg kg-1. The matrix effect for tribromoacetic, bromochloroacetic and chlorodibromoacetic acid was corrected by matrix-matched calibration and standard addition. After storage at -20 °C, only monobromoacetic acid was the HAA which loss happened after 7 days. The application of the methodology in lettuce and process water samples from the industry was successfully implemented. Therefore, this method could be employed for the quality control and regulatory analysis of HAAs in fresh products and process water from the fruit and vegetable industry.

Optimization of the Use of a Commercial Phage-Based Product as a Control Strategy of Listeria monocytogenes in the Fresh-Cut Industry.

A commercial phage biocontrol for reducing Listeria monocytogenes has been described as an effective tool for improving fresh produce safety. Critical challenges in the phage application must be overcome for the industrial application. The validation studies were performed in two processing lines of two industry collaborators in Spain and Denmark, using shredded iceberg lettuce as the ready-to-eat (RTE), high process volume product. The biocontrol treatment optimized in lab-scale trials for the application of PhageGuard ListexTM was confirmed in industrial settings by four tests, two in Spain and two in Denmark. Results showed that the method of application that included the device and the processing operation step was appropriate for the proper application. The proper dose of Phage Guard ListexTM was reached in shredded iceberg lettuce and the surface was adequately covered for the successful application of phages. There was no impact on the headspace gas composition (CO2 and O2 levels), nor on the color when untreated and treated samples were compared. The post-process treatment with PhageGuard ListexTM did not cause any detrimental impact on the sensory quality, including flavor, texture, browning, spoilage, and visual appearance over the shelf-life as the phage solution was applied as a fine, mist solution.

Surveillance on ESBL-Escherichia coli and Indicator ARG in Wastewater and Reclaimed Water of Four Regions of Spain: Impact of Different Disinfection Treatments.

In the present study, the occurrence of indicator antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) both in the influent and the effluent of four Spanish wastewater treatment plants (WWTPs) was monitored for 12 months, and the susceptibility profiles of 89 recovered extended spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates were obtained against a wide range of antimicrobials. The aim of the study was to better understand whether the current wastewater treatment practices allow us to obtain safe reclaimed water mitigating the spread of ARB and ARGs to the environment. Results showed high concentrations of ESBL-producing E. coli as well as a high prevalence of a range of ARGs in the influent samples. The reclamation treatments implemented in the WWTPs were effective in reducing both the occurrence of ESBL E. coli and ARGs, although significant differences were observed among WWTPs. Despite these reductions in occurrence observed upon wastewater treatment, our findings suggest that WWTP effluents may represent an important source of ARGs, which could be transferred among environmental bacteria and disseminate antimicrobial resistance through the food chain. Remarkably, no major differences were observed in the susceptibility profiles of the ESBL E. coli isolated from influent and effluent waters, indicating that water treatments do not give rise to the emergence of new resistance phenotypes.

Electrochemical disinfection: an efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter.

The efficacy of an electrochemical treatment in water disinfection, using boron-doped diamond electrodes, was studied and its suitability for the fresh-cut produce industry analyzed. Tap water (TW), and tap water supplemented with NaCl (NaClW) containing different levels of organic matter (Chemical Oxygen Demand (COD) around 60, 300, 550 ± 50 and 750 ± 50 mg/L) obtained from lettuce, were inoculated with a cocktail of Escherichia coli O157:H7 at 105 cfu/mL. Changes in levels of E. coli O157:H7, free, combined and total chlorine, pH, oxidation-reduction potential, COD and temperature were monitored during the treatments. In NaClW, free chlorine was produced more rapidly than in TW and, as a consequence, reductions of 5 log units of E. coli O157:H7 were achieved faster (0.17, 4, 15 and 24 min for water with 60, 300, 500 and 750 mg/L of COD, respectively) than in TW alone (0.9, 25, 60 min and 90 min for water with 60, 300, 600 and 800 mg/L of COD, respectively). Nonetheless, the equipment showed potential for water disinfection and organic matter reduction even without adding NaCl. Additionally, different mathematical models were assessed to account for microbial inactivation curves obtained from the electrochemical treatments.

Frozen Vegetable Processing Plants Can Harbour Diverse Listeria monocytogenes Populations: Identification of Critical Operations by WGS.

Frozen vegetables have emerged as a concern due to their association with foodborne outbreaks such as the multi-country outbreak of Listeria monocytogenes serogroup IVb linked to frozen corn. The capacity of L. monocytogenes to colonize food-processing environments is well-known, making the bacteria a real problem for consumers. However, the significance of the processing environment in the contamination of frozen foods is not well established. This study aimed to identify potential contamination niches of L. monocytogenes in a frozen processing plant and characterize the recovered isolates. A frozen vegetable processing plant was monitored before cleaning activities. A total of 78 points were sampled, including frozen vegetables. Environmental samples belonged to food-contact surfaces (FCS); and non-food-contact surfaces (n-FCS). Positive L. monocytogenes samples were found in FCS (n = 4), n-FCS (n = 9), and the final product (n = 1). A whole-genome sequencing (WGS) analysis revealed two clusters belonging to serotypes 1/2a-3a and 1/2b-3b). The genetic characterization revealed the presence of four different sequence types previously detected in the food industry. The isolate obtained from the final product was the same as one isolate found in n-FCS. A multi-virulence-locus sequence typing (MVLST) analysis showed four different virulence types (VT). The results obtained highlight the relevant role that n-FCS such as floors and drains can play in spreading L. monocytogenes contamination to the final product.

Strategies for mitigating chlorinated disinfection byproducts in wastewater treatment plants.

A case study of 15 wastewater treatment plants (WWTPs) at a full-scale was assessed for the risks of disinfection byproduct (DBP) formation, mainly the regulated trihalomethanes (THMs) and haloacetic acids (HAAs) and chlorate as an inorganic byproduct regulated recently in the EU. Raw wastewater from large, medium/small urban areas were treated with single or combined disinfection processes (i.e., chlorine, peracetic acid (PAA) and ultraviolet (UV) radiation). Sampling was executed once a month over seven months for the medium/small WWTPs and twice a month for the large ones. Due to the potential risk of SARS-CoV-2 contaminated wastewater, several inactivation methods were examined before the DBP analysis. Due to the inactivation step, the stability of THM4 and HAA9 suffered reductions, monitoring their presence only in the effluents after the disinfection treatments. In contrast, chlorate levels remained unchanged after the inactivation treatment; thus both raw wastewater and effluents were examined for their occurrence before disinfection treatments. Results showed that chlorate residues in the raw wastewater varied greatly from undetected levels to as high as 42.2 mg L-1. As the continuous monitoring of DBPs was performed, a positive correlation with chlorine or chlorine/UV was found. Changes in the physicochemical parameters indicated that the quality of the raw wastewater varied considerably depending on the WWTPs, and it influenced byproduct formation. In all WWTPs, chlorine alone or combined with UV significantly increased the presence of THMs, HAAs, and chlorate levels in the treated effluents. When the same WWTPs changed to PAA or PAA/UV, DBPs were diminished completely. This study highlights the risk of chlorate residues in raw wastewater during the pandemic. It also showed how the chemical risks of DBP formation could be reduced by changing the chlorinated disinfection technologies to PAA or PAA/UV, particularly if reclaimed water is intended for agricultural irrigation to minimize DBP residues.

Monitoring of human enteric virus and coliphages throughout water reuse system of wastewater treatment plants to irrigation endpoint of leafy greens.

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5-6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20-25%) negative for total coliphages showed moderate norovirus counts (1-3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses.

New standards at European Union level on water reuse for agricultural irrigation: Are the Spanish wastewater treatment plants ready to produce and distribute reclaimed water within the minimum quality requirements?

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 (<1 log cfu/100 mL) (EU, 2020/741). On the other hand, average levels of E. coli at the end-user point (1.0 ± 1.2 log cfu/100 mL) were below the recommended threshold (2 log cfu E. coli/100 mL) for irrigation water based on the guidance document on microbiological risks in fresh fruits and vegetables at primary production (EC, 2017/C_163/01). However, several outlier points were observed among the samples taken at the irrigation point, which were linked to a specific cross-contamination event within the distribution/storage system. Regarding pathogenic bacteria, water samples from the influent of the WWTPs showed a 100% prevalence, while only 5% of the effluent samples were positive for any of the monitored pathogenic bacteria. Obtained results indicate that reclaimed water produced in the selected water reuse system is suitable to be used as irrigation water. However, efforts are necessary not only in the establishment of advance disinfection treatments but also in the maintenance of the distribution/storage systems.