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.

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.

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.

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.

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.

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.

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.

Impact of weather conditions, leaf age and irrigation water disinfection on the major epiphytic bacterial genera of baby spinach grown in an open field.

The effects of factors such as weather conditions, leaf age and irrigation water disinfection on the main bacterial genera (total bacterial, Enterobacteriaceae and Pseudomonas) of baby spinach were studied. Culture-dependent and independent quantification techniques were compared. Cultivation was carried out over two consecutive trials in commercial open field divided in two plots: 1) baby spinach irrigated with untreated surface water and 2) baby spinach irrigated with chlorine dioxide (ClO2) treated water. In all the cases, higher concentrations of bacteria were detected using molecular quantification in comparison with culture dependent techniques. Based on the obtained results, wind speed, solar radiation and relative humidity seem to have an impact on the levels of total bacterial, Enterobacteriaceae and Pseudomonas during cultivation of baby spinach. However, further studies would be needed to confirm this tendency. Water disinfection treatments (ClO2), when applied to irrigation water, impacted differently the bacterial genera evaluated in the present study. Thus, although no significant effects were observed in total bacterial enumerations of baby spinach irrigated with ClO2 treated water; significant reductions were detected in Enterobacteriaceae (19%) and Pseudomonas spp. (14%) levels. These results were also confirmed using specific culture-dependent methods. On the other hand, leaf age did not influence the levels of the main bacterial genera of baby spinach. Considering that, a large proportion of foodborne and pathogenic bacteria associated to fresh produce belong to the Enterobacteriaceae family and Pseudomonas genera, reductions in these bacterial groups could be beneficial. However, these groups are very diverse, making difficult to link the measurement of Enterobacteriaceae and Pseudomonas levels with the presence/abundance of potential pathogenic and spoilage microorganisms.

Quantitative microbial exposure modelling as a tool to evaluate the impact of contamination level of surface irrigation water and seasonality on fecal hygiene indicator E. coli in leafy green production.

The use of Quantitative Microbial Exposure Assessment (QMEA) modelling of faecal hygiene indicator microorganisms (e.g. E. coli), is proposed as an alternative approach to the use of Quantitative Microbiological Risk Assessment (QMRA) models of enteric pathogenic microorganisms in the fresh produce chain. As more field data and quantitative microbial models become available, the potential use of QMEA models as a tool to assess the impact of different risk mitigation strategies increases helping growers to make the right decisions. This paper focuses on the pros and cons of previously published QMRAs as well as on the proposal of an alternative approach based on the use of a quantitative microbial contamination modelling to investigate how the selection of the irrigation water sources affect the E. coli loads in leafy greens at harvest. The modified model describes the final E. coli levels of baby spinach when different water sources with different contamination levels are applied and the impact of seasonality. Substantial differences were observed between the irrigation water sources while seasonality only had small effects on the simulated levels of E. coli in the harvested baby spinach. Based on the results, the produce grown using irrigation water from drainage ditches show E. coli levels above threshold levels (2 log CFU/g) while less than 1% of baby spinach irrigated with water obtained from water reservoirs where above this limit. The use of this QMEA model will help growers in the decision-making process to reduce microbial contamination of leafy greens.

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.

Impact of solar radiation exposure on phyllosphere bacterial community of red-pigmented baby leaf lettuce.

Solar radiation has been identified as a stress factor affecting phyllosphere associated bacteria colonization and survival during primary production. In the present study, the impact of different solar radiation doses on the phyllosphere microbiota of red-pigmented baby leaf lettuce cultivated in open field under commercial conditions was evaluated. Four weeks before harvest, the growing field was divided into four plots; each one was consecutively covered with one-week-interval with a light-excluding plastic to reduce the sunlight exposure. Four different solar radiation treatments were generated and cumulative photosynthetically active radiation (PAR) was used to differentiate treatments as follows: 4889 ± 428 µmol/m2/s (uncovered), 4265 ± 356 µmol/m2/s (covered for 1 week), 3602 ± 225 µmol/m2/s (covered for 2 weeks) and 3115 ± 313 µmol/m2/s (covered for 3 weeks). The size and composition of the phyllosphere bacterial community were determined by cultivation-depended (plate count) and independent (qPCR) techniques. Exposure to decreased levels of cumulative PAR did not produce significant differences in total bacterial community size, regardless of the chosen quantification techniques. However, total bacteria size quantified by qPCR was around 3.5 orders of magnitude higher than those obtained by plate count. The observed differences between cultivation-depended and independent techniques could be attributed to the presence of non-viable or viable but non-culturable (VBNC) bacteria. The bacterial community structure was analyzed using temperature gradient gel electrophoresis (TGGE), and significant differences were detected when the four solar treatment were compared. A qPCR approach was applied to the quantification of specific bacterial phyla and classes, previously identified in the phyllosphere of plants available literature, confirming that Proteobacteria, Bacteroidetes, Actinobacterias and Firmicutes were the most abundantly represented phyla in lettuce. Treatment comparison revealed higher proportions of Gammaproteobacteria as opposed to the Betaproteobacteria on the lettuce exposed to the lowest cumulative PAR dose (3115 ± 313 µmol/m2/s). The obtained results demonstrated that the solar radiation is a relevant environmental factor influencing the relative abundance of specific-groups of phyllosphere-associated bacteria in pigmented baby leaf lettuce.

Antimicrobial activity of hydroxytyrosol: a current controversy.

This study focus on the main factors that affect the antimicrobial capacity of hydroxytyrosol, including the concentration (200, 400, and 1000 µg/mL), target strains, and the culture media (nutrient-rich and less-rich culture media). The potential HT degradation was also evaluated by HPLC-PAD. Kinetic parameters from growth curves showed that HT concentrations produced a doses-dependent shift when compared to the untreated control. In most of the cases, the highest tested dose (1000 µg/mL) was needed to inhibit growth of the selected strains. However, all the strains were able to grow even at the highest HT dose when cultivated in nutrient-rich culture media. It was observed that HT concentrations were reduced by about 15%, except for Escherichia coli 533 and 679 in Muller Hinton broth, where HT was reduced up to 35%. The results showed a limited antimicrobial activity, contrary to information previously published in some research papers.

Determination of interglycosidic linkages in O-glycosyl flavones by high-performance liquid chromatography/photodiode-array detection coupled to electrospray ionization ion trap mass spectrometry. Its application to Tetragonula carbonaria honey from Australia.

RATIONALE: Tetragonula carbonaria pot-honeys are highly valued as a food source and for their biological activities in Australia, and there is a growing interest to know its composition. Phenolic metabolites, which could be related to their beneficial properties, have not been studied in depth yet. METHODS: Mass spectrometry (MS) coupled to liquid chromatography (LC) is an advanced technique for the study of complex flavonoids present in difficult food matrices that hampers their isolation and purification. This allows the tentative characterization of diglycosides/triglycosides establishing the position of the O-glycosylation on the sugar moiety by the study of the MS data in T. carbonaria pot-honeys from Australia. RESULTS: Their spectra obtained by high-performance liquid chromatography/photodiode-array detection/electrospray ionization ion trap mass spectrometry (HPLC/DAD/ESI-MS(n) ) revealed for the first time 19 quercetin, kaempferol and isorhamnetin O-glycosides. These compounds were clustered in flavonoid triglycosides, diglycosides and monoglycosides. The first cluster contained one flavonoid trihexoside, two -3-O-(2-hexosyl, 6-rhamnosyl)hexosides and their isomers and two -3-O-(2,6-di-rhamnosyl)hexosides. In the second cluster, eleven flavonoid diglycosides such as three -3-O-(2-hexosyl)hexosides, four -3-O-(2-rhamnosyl)hexosides and one -3-O-(6-rhamnosyl)hexoside as well as two -3-O-(2-pentosyl)hexosides and one tentative -3-O-(3-pentosyl)hexoside were detected. In the monoglycoside group, only one flavonoid -3-O-hexoside was identified. CONCLUSIONS: The occurrence of this large number of flavonoid glycosides could be due to the low glucosidase activity previously reported in stingless bee honey.

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.

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.

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.

Evaluation of Pseudomonas aeruginosa (PAO1) adhesion to human alveolar epithelial cells A549 using SYTO 9 dye.

Pseudomonas aeruginosa is an aerobic Gram-negative bacterium characterized by a natural resistance to several antibiotics. It is a major cause of nosocomial infections in patients with compromised host defence mechanisms mainly related to the respiratory tract. P. aeruginosa infection first step is the adhesion of the bacteria to the host cells and thus, the development of techniques that can easily assess adhesion of bacteria strains and of bacteria isolated from biological samples is fundamental. The aim of our work was to develop a fast and effective method to evaluate the adhesion of P. aeruginosa to bronchial epithelial cells. To meet our goal we optimized a staining protocol using the vital dye SYTO 9 and P. aeruginosa PAO1. We established the appropriate dying conditions as well as the stability of the stained bacteria. Adhesion was first measured using the traditional plate counting method and then, adhesion values were compared to those obtained using a fluorescence microplate reader and epifluorescence microscopy. Our results show that the use of SYTO 9 does not interfere with the bacteria viability, bacteria cell growth, and adhesion of P. aeruginosa to A549 epithelial cells. Both the fluorescence microplate reader and the epifluorescence microscopy gave similar results to those attained with the plate counting method, however, the epifluorescence microscopy also allowed for simultaneous discrimination of damaging effects on the human cells. Overall, our data indicate that the use of SYTO 9 combined with a fluorescence microplate reader or an epifluorescence microscope provides a rapid method to evaluate the adhesion of P. aeruginosa to human epithelial cells. However, to show unequivocally that a specific drug or compound has a truly inhibitory effect on the bacterial adhesion without affecting the number of human cells, the epifluorescence microscopy is recommended.

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 [...].

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.