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.

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.

Fluence requirements in existing UV disinfection facilities to comply with EU validation performance targets for reclaimed water: a case study.

Since 2020, there is a new European Regulation (EU, 2020/741) on minimum requirements for water reuse, where routine and validation monitoring requirements (log reductions of indicator microorganisms and reference pathogens) have been established. Many reclamation facilities that are already in operation might have difficulties to comply with these performance targets. Existing disinfection systems must be expanded and upgraded. In the case of UV disinfection systems, fluence requirements must be determined to properly design with a focus on the safety and economic-environmental viability of reclaimed water. This study can be used as a reference to develop fluence-response curves for Clostridium perfringens spores, Escherichia coli, and total and F-specific coliphages, indicator microorganisms referred to in the new European Regulation. Eight UV-LED collimated beam tests were performed. Samples were obtained from filtered effluent of secondary treatment from two wastewater treatment plants (WWTPs) which ranged between 30 and 54%. Results showed UV sensitivity of 33.46 mJ/cm2 log I for C. perfringens spores and 2.86 mJ/cm2 log I for E. coli, both from environmental origin. Coliphages were inactivated below the limit of quantitation. The non-dominance of MS2 phages in environmental F-specific coliphages was observed.

Wastewater-Based Epidemiology to Describe the Evolution of SARS-CoV-2 in the South-East of Spain, and Application of Phylogenetic Analysis and a Machine Learning Approach.

The COVID-19 pandemic has posed a significant global threat, leading to several initiatives for its control and management. One such initiative involves wastewater-based epidemiology, which has gained attention for its potential to provide early warning of virus outbreaks and real-time information on its spread. In this study, wastewater samples from two wastewater treatment plants (WWTPs) located in the southeast of Spain (region of Murcia), namely Murcia, and Cartagena, were analyzed using RT-qPCR and high-throughput sequencing techniques to describe the evolution of SARS-CoV-2 in the South-East of Spain. Additionally, phylogenetic analysis and machine learning approaches were applied to develop a pre-screening tool for the identification of differences among the variant composition of different wastewater samples. The results confirmed that the levels of SARS-CoV-2 in these wastewater samples changed concerning the number of SARS-CoV-2 cases detected in the population, and variant occurrences were in line with clinical reported data. The sequence analyses helped to describe how the different SARS-CoV-2 variants have been replaced over time. Additionally, the phylogenetic analysis showed that samples obtained at close sampling times exhibited a higher similarity than those obtained more distantly in time. A second analysis using a machine learning approach based on the mutations found in the SARS-CoV-2 spike protein was also conducted. Hierarchical clustering (HC) was used as an efficient unsupervised approach for data analysis. Results indicated that samples obtained in October 2022 in Murcia and Cartagena were significantly different, which corresponded well with the different virus variants circulating in the two locations. The proposed methods in this study are adequate for comparing consensus sequence types of the SARS-CoV-2 sequences as a preliminary evaluation of potential changes in the variants that are circulating in a given population at a specific time point.

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.

Spanish wastewater reveals the current spread of Monkeypox virus.

Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n = 312) from 24 different wastewater treatment plants were obtained between May 9 (week 19 of 2022) and August 4 (week 31 of 2022). Following concentration of viral particles by a validated aluminum adsorption-precipitation method, a qPCR procedure allowed us to detect MPXV DNA in 56 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 × 103 to 8.7 × 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

Cross-contamination of Escherichia coli O157:H7 and Listeria monocytogenes in the viable but non-culturable (VBNC) state during washing of leafy greens and the revival during shelf-life.

Some water disinfection treatments, such as chlorine and chlorine dioxide, used in the fresh-cut industry to maintain the microbiological quality of process water (PW), inactivate bacterial cells in the water but they also lead to the induction of an intermediate state between viable and non-viable known as viable but non-culturable (VBNC) state. Viable cells can participate in cross-contamination events but the significance of VBNC cells in PW, transfer to the product and potential resuscitation capacity during storage is unclear. The present study aims to determine first, if VBNC cells present in PW can cross-contaminate leafy greens during washing and secondly its potential revival during shelf-life. Process water characterized by a high chemical oxygen demand, due to the presence of high levels of organic matter, was inoculated with Listeria monocytogenes or Escherichia coli O157:H7. Inoculated PW was then treated for 1 min with chlorine dioxide (3 mg/L) or chlorine (5 mg/L) to generate VBNC cells. Absence of culturable cells was confirmed by plate count and VBNC cells by viability quantitative polymerase chain reaction (v-qPCR) complemented with two dyes, ethidium (EMA) and propidium (PMAxx) monoazide. Cross-contamination of shredded lettuce was demonstrated by monitoring the VBNC cells after washing the product for 1 min in the contaminated PW and during shelf life (15 days at 7 °C). In the case of L. monocytogenes, considering the total concentration of L. monocytogenes VBNC cells present in the PW, only a low proportion of cells were able to cross-contaminate the product during washing. VBNC L. monocytogenes cells were able to resuscitate on the product during shelf life, although levels of cultivable bacteria, close to the limit if detection (0.7 ± 0.0 log CFU/g), were only detected at the end of storage. On the other hand, VBNC cells of E. coli O157:H7 present in PW were not able to cross-contaminate shredded lettuce during washing. Moreover, when shredded lettuce was artificially inoculated with VBNC E. coli O157:H7, resuscitation of the VBNC cells during storage (15 days at 7 °C) was not observed. Based on the results obtained, injured L. monocytogenes cells present in the PW are able to be transferred to the product during washing. If VBNC L. monocytogenes cells present in leafy greens (shredded lettuce and baby spinach), they can resuscitate, although cultivable numbers remained very low. Taking all the results together, it could be concluded that under industrial conditions, VBNC cells can be transferred from water to product during washing, but their capacity to resuscitate in the leafy greens during storage is low.

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.

New Challenges for Detection and Control of Foodborne Pathogens: From Tools to People.

Contamination of foods by human pathogenic microorganisms is a major concern to both food safety and public health [...].

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.

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.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Occurrence and Accumulation of Human Enteric Viruses and Phages in Process Water from the Fresh Produce Industry.

The virological quality of process water (PW) used by the produce industry has received limited attention. As a first step to overcoming technical limitations in monitoring viruses in PW, the analytical performance of ultrafiltration was assessed to concentrate viral particles from 20 L of spiked PW. The selected method used for sample concentration of PW was carefully validated, thus enabling the accurate quantification and estimation of viral titers of human enteric viruses and phages. PW from the produce industry was collected periodically from the washing tanks of commercial facilities. The analysis of coliphages was performed by plaque assay, while the occurrence of enteric viruses and crAssphage was determined by molecular techniques. Significant differences in the physicochemical composition of PW, mostly due to the different nature of fresh produce types and differences in the sanitizer used in commercial operation, were observed. Accumulation of crAssphage and coliphages was observed in PW, but correlation with human enteric viruses was not possible due to the low prevalence of these pathogens in the PW analyzed. The obtained results showed that depending on the type of product washed, the product/water ratio and the residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly.

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.

Effect of Weather on the Die-Off of Escherichia coli and Attenuated Salmonella enterica Serovar Typhimurium on Preharvest Leafy Greens following Irrigation with Contaminated Water.

The Food Safety Modernization Act (FSMA) includes a time-to-harvest interval following the application of noncompliant water to preharvest produce to allow for microbial die-off. However, additional scientific evidence is needed to support this rule. This study aimed to determine the impact of weather on the die-off rate of Escherichia coli and Salmonella on spinach and lettuce under field conditions. Standardized, replicated field trials were conducted in California, New York, and Spain over 2 years. Baby spinach and lettuce were grown and inoculated with an ∼104-CFU/ml cocktail of E. coli and attenuated Salmonella Leaf samples were collected at 7 time points (0 to 96 h) following inoculation; E. coli and Salmonella were enumerated. The associations of die-off with study design factors (location, produce type, and bacteria) and weather were assessed using log-linear and biphasic segmented log-linear regression. A segmented log-linear model best fit die-off on inoculated leaves in most cases, with a greater variation in the segment 1 die-off rate across trials (-0.46 [95% confidence interval {95% CI}, -0.52, -0.41] to -6.99 [95% CI, -7.38, -6.59] log10 die-off/day) than in the segment 2 die-off rate (0.28 [95% CI, -0.20, 0.77] to -1.00 [95% CI, -1.16, -0.85] log10 die-off/day). A lower relative humidity was associated with a faster segment 1 die-off and an earlier breakpoint (the time when segment 1 die-off rate switches to the segment 2 rate). Relative humidity was also found to be associated with whether die-off would comply with FSMA's specified die-off rate of -0.5 log10 die-off/day.IMPORTANCE The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off rates of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella, and die-off tended to become faster as relative humidity decreased. Thus, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation.

The significance of viable but non-culturable (VBNC) cells in the food industry is not well known, mainly because of the lack of suitable detection methodologies to distinguish them from dead cells. The study aimed at the selection of the method to differentiate dead and VBNC cells of Listeria monocytogenes in process wash water (PWW) from the fruit and vegetable industry. Different methodologies were examined including (i) flow cytometry, (ii) viability quantitative polymerase chain reaction (v-qPCR) using an improved version of the propidium monoazide (PMAxx) dye as DNA amplificatory inhibitor, and (iii) v-qPCR combining ethidium monoazide (EMA) and PMAxx. The results showed that the flow cytometry, although previously recommended, was not a suitable methodology to differentiate between dead and VBNC cells in PWW, probably because of the complex composition of the water, causing interferences and leading to an overestimation of the dead cells. Based on results obtained, the v-qPCR combined with EMA and PMAxx was the most suitable technique for the detection and quantification of VBNC cells in PWW. Concentrations of 10 µM EMA and 75 µM PMAxx incubated at 40°C for 40 min followed by a 15-min light exposure inhibited most of the qPCR amplification from dead cells. For the first time, this methodology was validated in an industrial processing line for shredded lettuce washed with chlorine (10 mg/L). The analysis of PWW samples allowed the differentiation of dead and VBNC cells. Therefore, this method can be considered as a rapid and reliable one recommended for the detection of VBNC cells in complex water matrixes such as those of the food industry. However, the complete discrimination of dead and VBNC cells was not achieved, which led to a slight overestimation of the percentage of VBNC cells in PWW, mostly, due to the complex composition of this type of water. More studies are needed to determine the significance of VBNC cells in case of potential cross-contamination of fresh produce during washing.

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), the area with the lowest COVID-19 prevalence within Iberian Peninsula. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was validated using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10 ± 3.5% and 10 ± 2.1% in influent water (n = 2) and 3.3 ± 1.6% and 6.2 ± 1.0% in effluent water (n = 2) samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater samples of 5.4 ± 0.2 log10 genomic copies/L on average. Two secondary water samples resulted positive (2 out of 18) and all tertiary water samples tested as negative (0 out 12). This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that members of the community were shedding SARS-CoV-2 RNA in their stool even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, this environmental surveillance could be implemented by municipalities right away as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown.

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.

Post-process treatments are effective strategies to reduce Listeria monocytogenes on the surface of leafy greens: A pilot study.

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 ±â€¯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.