Identification of pathways required for Salmonella to colonize alfalfa using TraDIS-Xpress.

Enteropathogenic bacteria, such as Salmonella, have been linked to numerous fresh produce outbreaks, posing a significant public health threat. The ability of Salmonella to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which pose a challenge to food decontamination and can increase pathogenic bacterial load in the food chain. Preventing Salmonella colonization of food products and food processing environments is crucial for reducing the incidence of foodborne outbreaks. Understanding the mechanisms of establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-Directed Insertion site Sequencing (TraDIS-Xpress) to investigate the mechanisms used by Salmonella enterica serovar Typhimurium to colonize and establish on fresh produce over time. We established an alfalfa colonization model and compared the findings to those obtained from glass surfaces. Our research identified distinct mechanisms required for Salmonella establishment on alfalfa compared with glass surfaces over time. These include the type III secretion system (sirC), Fe-S cluster assembly (iscA), curcumin degradation (curA), and copper tolerance (cueR). Shared pathways across surfaces included NADH hydrogenase synthesis (nuoA and nuoB), fimbrial regulation (fimA and fimZ), stress response (rpoS), LPS O-antigen synthesis (rfbJ), iron acquisition (ybaN), and ethanolamine utilization (eutT and eutQ). Notably, flagellum biosynthesis differentially impacted the colonization of biotic and abiotic environments over time. Understanding the genetic underpinnings of Salmonella establishment on both biotic and abiotic surfaces over time offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. IMPORTANCE: Salmonella is the second most costly foodborne illness in the United Kingdom, accounting for £0.2 billion annually, with numerous outbreaks linked to fresh produce, such as leafy greens, cucumbers, tomatoes, and alfalfa sprouts. The ability of Salmonella to colonize and establish itself in fresh produce poses a significant challenge, hindering decontamination efforts and increasing the risk of illness. Understanding the key mechanisms of Salmonella to colonize plants over time is key to finding new ways to prevent and control contamination of fresh produce. This study identified genes and pathways important for Salmonella colonization of alfalfa and compared those with colonization of glass using a genome-wide screen. Genes with roles in flagellum biosynthesis, lipopolysaccharide production, and stringent response regulation varied in their significance between plants and glass. This work deepens our understanding of the requirements for plant colonization by Salmonella, revealing how gene essentiality changes over time and in different environments. This knowledge is key to developing effective strategies to reduce the risk of foodborne disease.

The influence of pH on the efficacy of oxidation-reduction potential (ORP) to predict chlorine disinfection of surrogate bacteria, Escherichia coli O157:H7 and Listeria monocytogenes in oxidant demand free conditions and fresh produce wash water.

Oxidation-reduction potential (ORP) is commonly used as a rapid measurement of the antimicrobial potential of free chlorine during industrial fresh produce washing. The current study tested the hypothesis that ORP can act as a "single variable" measurement of bacterial (vegetative and endospores) inactivation effectiveness with free chlorine irrespective of the water pH value. This situation has on occasion been assumed but never confirmed nor disproven. Chlorine-dosed pH 6.5 and 8.5 phosphate buffer solutions were inoculated with Escherichia coli (E. coli), Listeria innocua (L. innocua), or Bacillus subtilis (B. subtilis) endospores. ORP, free chlorine (FC), and log reduction were monitored after 5 s (for E. coli and L. innocua) and up to 30 min (for B. subtilis spores) of disinfection. Logistic and exponential models were developed to describe how bacteria reduction varied as a function of ORP at different pH levels. Validation tests were performed in phosphate buffered pH 6.5 and 8.5 cabbage wash water periodically dosed with FC, cabbage extract and a cocktail of Escherichia coli O157:H7 (E. coli O157:H7) and Listeria monocytogenes (L. monocytogenes). The built logistic and exponential models confirmed that at equal ORP values, the inactivation of the surrogate strains was not consistent across pH 6.5 and pH 8.5, with higher reductions at higher pH. This is the opposite of the well-known free chlorine-controlled bacterial inactivation, where the antibacterial effect is higher at lower pH. The validation test results indicated that in the cabbage wash water, the relationship between disinfection efficiency and ORP was consistent with the oxidant demand free systems. The study suggests that ORP cannot serve as a reliable single variable measurement to predict bacterial disinfection in buffered systems. When using ORP to monitor and control the antibacterial effectiveness of the chlorinated wash water, it is crucial to take into account (and control) the pH.

First Report of Alternaria alternata Causing Decay in Exported Sweet Basil During Freight from Israel to Europe.

Sweet basil (Ocimum basilicum) is an important spice herb grown in Israel for local markets and export. The crop is used as a fresh culinary herb or spice, and the essential oils are used in cosmetics and food flavorings. Due to increased demand, the production area of basil has increased in Israel. Postharvest losses due to fungal disease are a major economic concern for growers. In the summer of 2019, a leaf spot was observed in postharvest shipments of sweet basil destined for Europe; in late winter of 2022, leaf spots were observed on greenhouse-grown sweet basil. Fungal isolates from infected leaves were characterized by morphology in culture as Alternaria spp. PCR amplification of the Alternaria major allergen Alt a1, ITS, and gdp gene regions of the recovered isolates confirmed the presence of A. alternata, a common pathogen of numerous herbs and spice plants. In vitro growth tests demonstrated that 25°C was the optimum temperature for growth of the isolates. The isolates were tested for pathogenicity and found to infect a commonly grown cultivar of basil, cultivar Eli (previously cultivar Perrie). Foliar symptoms in pathogenicity tests were identical to those observed in commercial shipments and in the field, which completed Koch's postulates. Control of the nascent disease by applying fungicides to the plants may be necessary to reduce postharvest losses.

Bacteroidales as a fecal contamination indicator in fresh produce industry: A baseline measurement.

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in California's Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The quantification of Bacteroidales concentration was performed using qPCR, revealing a notably low concentration (0-2.00 copies/cm2) in the commercial fields. To further enhance the applicability of our findings, we developed a user-friendly method for real-world fecal contamination risk assessment that seamlessly integrates with industry practices. Through the generation of heatmaps that visually illustrate varying risk levels across fields, this approach can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their land. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.

Protective Effect of Select Bacterial Species Representative of Fresh Produce on Human Norovirus Surrogates Exposed to Disinfecting Pulsed Light.

Contamination of berries and leafy greens with human norovirus (HuNoV) is a major cause of outbreaks of epidemic gastroenteritis worldwide. Using murine norovirus type 1 (MNV-1) and Tulane virus, we studied the possible extension of HuNoV persistence by biofilm-producing epiphytic bacteria on fresh produce. Nine bacterial species frequently found on the surface of berries and leafy greens (Bacillus cereus, Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Lactobacillus plantarum, Pantoea agglomerans, Pseudomonas fluorescens, Raoultella terrigena, and Xanthomonas campestris) were evaluated for the ability to form biofilms in the MBEC Assay Biofilm Inoculator and in 96-well microplates. The biofilm-forming bacteria were further tested for binding MNV-1 and Tulane virus and the ability to protect them against loss of capsid integrity upon exposure to disinfecting pulsed light at a fluence of 11.52 J/cm2. Based on viral reductions, MNV-1 did not benefit from attachment to biofilm whereas Tulane virus was significantly more resistant than the control when attached to biofilms of E. cloacae (P ≤ 0.01), E. coli (P ≤ 0.01), K. kristinae (P ≤ 0.01), P. agglomerans (P ≤ 0.05), or P. fluorescens (P ≤ 0.0001). Enzymatic dispersion of biofilm and microscopic observations suggest that the biofilm matrix composition may contribute to the virus resistance. Our results indicate that direct virus-biofilm interaction protects Tulane virus against disinfecting pulsed light, and that HuNoV on fresh produce therefore might resist such treatment more than suggested by laboratory tests so far. IMPORTANCE Recent studies have shown that bacteria may be involved in the attachment of HuNoV to the surface of fresh produce. Because these foods are difficult to disinfect by conventional methods without compromising product quality, nonthermal nonchemical disinfectants such as pulsed light are being investigated. We seek to understand how HuNoV interacts with epiphytic bacteria, particularly with biofilms formed by bacterial epiphytes, with cells and extracellular polymeric substances, and to determine if it thus escapes inactivation by pulsed light. The results of this study should advance understanding of the effects of epiphytic biofilms on the persistence of HuNoV particle integrity after pulsed light treatment and thus guide the design of novel pathogen control strategies in the food industry.

Impact of water reuse on agricultural practices and human health.

Climate change is altering the habits of the population. Extensive drought periods and overuse of potable water led to significant water shortages in many different places. Therefore, new water sources are necessary for usage in applications where the microbiological and chemical water quality demands are less stringent, as for agriculture. In this study, we planted, germinated, and grew vegetables/fruits (cherry tomato, lettuce, and carrot) using three types of potential waters for irrigation: secondary-treated wastewater, chlorine-treated wastewater, and green wall-treated greywater, to observe potential health risks of foodstuff consumption. In this study the waters and crops were analyzed for three taxonomic groups: bacteria, enteric viruses, and protozoa. Enteric viruses, human Norovirus I (hNoVGI) and Enterovirus (EntV), were detected in tomato and carrots irrigated with secondary-treated and chlorine-treated wastewater, in concentrations as high as 2.63 log genome units (GU)/g. On the other hand, Aichi viruses were detected in lettuce. Bacteria and protozoa remained undetected in all fresh produce although being detected in both types of wastewaters. Fresh produce irrigated with green wall-treated greywater were free from the chosen pathogens. This suggests that green wall-treated greywater may be a valuable option for crop irrigation, directly impacting the cities of the future vision, and the circular and green economy concepts. On the other hand, this work demonstrates that further advancement is still necessary to improve reclaimed water to the point where it no longer constitutes risk of foodborne diseases and to human health.

Vacuolar localisation of anthocyanin pigmentation in microgreen cotyledons of basil, cabbage and mustard greens does not impact on colonisation by Shiga-toxigenic Escherichia coli O157:H7.

Microgreens, the immature plants harvested after a few weeks of growth, are perceived as a heathy, nutritious food ingredient but may be susceptible to colonisation by human pathogens including Shiga-toxigenic Escherichia coli (STEC). Some microgreen cultivars accumulate anthocyanins or secrete essential oils which, when extracted or purified, have been reported to inhibit bacterial growth. Therefore, the impact of anthocyanins on bacterial colonisation by STEC (Sakai) was compared for three species that have pigmented cultivars: basil (Ocimum basilicum L.), cabbage (Brassica oleracea L.) and mustard greens (Brassica juncea L.). Inoculation with low concentrations of STEC (Sakai) (3 log10 colony forming units/ml (CFU/ml)) during seed germination resulted in extensive colonisation at the point of harvest, accumulating to âˆ¼ 8 log10 CFU/g FW in all cultivars. Bacterial colonies frequently aligned with anticlinal walls on the surface of epidermal cells of the cotyledons and, in basil, associated with peltate and capitate gland cells. Crude lysates of pigmented and non-pigmented basil cultivars had no impact on STEC (Sakai) growth rates, viability status or biofilm formation. Anthocyanins are located within plant vacuoles of these microgreen cultivars and did not affect colonisation by STEC (Sakai) and pigmentation therefore cannot be considered as a controlling factor in bacterial interactions.

Antimicrobial function of yeast against pathogenic and spoilage microorganisms via either antagonism or encapsulation: A review.

Contaminations of pathogenic and spoilage microbes on foods are threatening food safety and quality, highlighting the importance of developing antimicrobial agents. According to different working mechanisms, the antimicrobial activities of yeast-based agents were summarized from two aspects: antagonism and encapsulation. Antagonistic yeasts are usually applied as biocontrol agents for the preservation of fruits and vegetables via inactivating spoilage microbes, usually phytopathogens. This review systematically summarized various species of antagonistic yeasts, potential combinations to improve the antimicrobial efficiency, and the antagonistic mechanisms. The wide applications of the antagonistic yeasts are significantly limited by undesirable antimicrobial efficiency, poor environmental resistance, and a narrow antimicrobial spectrum. Another strategy for achieving effective antimicrobial activity is to encapsulate various chemical antimicrobial agents into a yeast-based carrier that has been previously inactivated. This is accomplished by immersing the dead yeast cells with porous structure in an antimicrobial suspension and applying high vacuum pressure to allow the agents to diffuse inside the yeast cells. Typical antimicrobial agents encapsulated in the yeast carriers have been reviewed, including chlorine-based biocides, antimicrobial essential oils, and photosensitizers. Benefiting from the existence of the inactive yeast carrier, the antimicrobial efficiencies and functional durability of the encapsulated antimicrobial agents, such as chlorine-based agents, essential oils, and photosensitizers, are significantly improved compared with the unencapsulated ones.

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.

Adhesion of Escherichia coli O157:H7 during sublethal injury and resuscitation: Importance of pili and surface properties.

Escherichia coli O157:H7 can recover from sublethally injured (SI) state, which causes threat of foodborne illness. Adhesion plays a key role in the carriage of pathogens in food. In this study, we investigated the adhesion ability of SI and recovered E. coli O157:H7 wildtype and its three pili-deficient mutants (curli, type 1 fimbriae, and type IV pili) on six food-related surfaces. Plate counting was used to determine adhesion population after washing and oscillating the surfaces. Spinach exhibited the stronger adhesion population of E. coli O157:H7 than the other fresh produces (p < 0.05). In addition, at least one key pili dominated adhesion on these surfaces, and curli was always included. The adhesion population and contribution of different types of pili were jointly affected by surface and physiological state. This can be attributed to high hydrophobicity and positive charge density on surface and different expression levels of csgB, fimA, fimC and ppdD in SI and recovered cells. Among glucose, mannose, maltose, fructose, lactose, and sucrose, addition of 0.5% mannose could reduce adhesion of cells at all physiological states on stainless steel. Overall, this research will provide support for controlling adhesion of SI and recovered E. coli O157:H7.

A loop-mediated isothermal amplification assay to detect Bacteroidales and assess risk of fecal contamination.

Fecal contamination of fresh produce from human and animal sources is a public health concern due to the risk of foodborne illnesses. The current standard laboratory procedures for microbiological analyses usually require an enrichment step that involves several hours. Molecular techniques such as polymerase chain reaction (PCR) have been used to directly detect pathogens from the samples, however, due to the low quantity of pathogen present and small volumes used for PCR, enrichment is usually required. Additionally, the need for specialized equipment and experienced workers hinders the use of these molecular techniques for field testing. Here, we developed a rapid risk-assessment assay for fecal contamination by targeting Bacteroidales using loop-mediated isothermal amplification (LAMP). The assay allows for naked-eye observation of reactions with as few as ∼8 copies of Bacteroidales per cm2 of the surface in the field. We evaluated this assay with complex field samples as well as on-site field studies. Our on-field studies demonstrated that the Bacteroidales LAMP assay enables us to easily and quickly (<50 min) assess the risk of fecal contamination from animal operations, with a concordance of 85.3% when compared to lab-based qPCR. These results were obtained without expensive equipment (when compared to standard laboratory procedures). These assays could be used to determine site-specific risk and help the decision-making process of fresh produce growers.

Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Use of bentonite-coated activated carbon for improving the sensitivity of RT-qPCR detection of norovirus from vegetables and fruits: The ISO 15216-1:2017 standard method extension.

Produce-related foodborne outbreaks are becoming increasingly prevalent worldwide. In plant tissues, various compounds, including polysaccharides, phenolic compounds, and chlorophyll, can inhibit RT-PCR detection of viruses. In this study, we developed a highly sensitive RT-qPCR in combination with the bentonite-coated activated carbon (BCAC) assay for detection of norovirus from fruits and vegetables, which could be completed within 7 h and was about 10-100 fold more sensitive than the standard procedures (ISO 15216-1:2017). The extraction efficiencies of three surrogate viruses (MS2, MNV-1, and TV) from five fresh produce (lettuce, cherry tomato, blueberry, strawberry, and spinach) were higher with BCAC treatment than those of control groups, ranging from 17.82% to 98.60%. The average detection limit of these viruses using the BCAC-RT-qPCR method was stable at an average of 102 PFU/g or GC/g. Finally, this BCAC-RT-qPCR method was applied for detection of human norovirus GII.4 spiked onto lettuce and cherry tomato. The viral extraction efficiencies were up to 53.43% and 95.56%, respectively, which is almost four and seven times better than those without BCAC. Therefore, the BCAC-RT-qPCR method can be used to detect low levels of foodborne viruses from produce.

A review of solar and solar-assisted drying of fresh produce: state of the art, drying kinetics, and product qualities.

Global demand exists for high-quality fresh produce. Nevertheless, the quality of fresh produce is severely impacted by its perishability due to its high moisture content. Therefore, fresh produces are preserved using artificial dryers (hot-air dryers, catalytic infrared dryers, etc.) driven by electricity or natural fuels. Nonetheless, the exorbitant cost of power has heightened the need for sustainable resources, notably solar energy, for drying. Hence, this article is a review of how solar dryers and solar-assisted dryers have affected the drying kinetics and quality of fresh produce in the last 5 years. The review showed that solar drying modeling technology (thin-layer modeling, computational fluid dynamics, adaptive-network-based fuzzy interference system, artificial neural network) helps examine fresh produce drying characteristics using various simulation tools before developing any procedure. Solar-assisted drying shortens drying times and increases drying rates. Besides, the quality of the dried fresh produce (color, aroma, appearance, rehydration, etc.) should always be considered. Hybrid solar drying produces higher drying rates and product quality than other solar dryers. However, energy analysis needs to be done as several studies have recognized energy efficiency and product quality. In addition, fresh produce must be pre-treated before solar drying to maintain the final product quality. Therefore, future studies should focus on creating other pretreatment techniques to produce the needed chemical and physical changes and enhance mass and heat transfer. Finally, the influence of solar drying on the final products' nutrient retention or loss, functionalities, or sensory characteristics needs further investigation and comparison to other non-solar drying technologies. © 2023 Society of Chemical Industry.

Population dynamics of Listeria spp., Salmonella spp., and Escherichia coli on fresh produce: A scoping review.

Collation of the current scope of literature related to population dynamics (i.e., growth, die-off, survival) of foodborne pathogens on fresh produce can aid in informing future research directions and help stakeholders identify relevant research literature. A scoping review was conducted to gather and synthesize literature that investigates population dynamics of pathogenic and non-pathogenic Listeria spp., Salmonella spp., and Escherichia coli on whole unprocessed fresh produce (defined as produce not having undergone chopping, cutting, homogenization, irradiation, or pasteurization). Literature sources were identified using an exhaustive search of research and industry reports published prior to September 23, 2021, followed by screening for relevance based on strict, a priori eligibility criteria. A total of 277 studies that met all eligibility criteria were subjected to an in-depth qualitative review of various factors (e.g., produce commodities, study settings, inoculation methodologies) that affect population dynamics. Included studies represent investigations of population dynamics on produce before (i.e., pre-harvest; n = 143) and after (i.e., post-harvest; n = 144) harvest. Several knowledge gaps were identified, including the limited representation of (i) pre-harvest studies that investigated population dynamics of Listeria spp. on produce (n = 13, 9% of pre-harvest studies), (ii) pre-harvest studies that were carried out on non-sprouts produce types grown using hydroponic cultivation practices (n = 7, 5% of pre-harvest studies), and (iii) post-harvest studies that reported the relative humidity conditions under which experiments were carried out (n = 56, 39% of post-harvest studies). These and other knowledge gaps summarized in this scoping review represent areas of research that can be investigated in future studies.

Internalization of Salmonella in Leafy Greens and Impact on Acid Tolerance.

Salmonella colonizes the surface or the inner part of leafy greens, while the ability of internalized bacteria to evade common disinfection practices may pose a considerable risk. Hereby, we aimed to assess how the colonization and internalization of Salmonella spp. (i) vary with the type of leafy green, the storage conditions (temperature, time), and Salmonella serovar at phenotypic and gene transcriptional level (regarding stress- and virulence- or type III secretion system [T3SS]-associated genes) and (ii) potentially impact the survival of the pathogen against subsequent exposure at lethal pH (2.7), mimicking the gastric acidity. Internalized Salmonella reached 3.0 to 5.0 log CFU/g depending on storage conditions and vegetable, with spinach and chicory allowing the highest (P < 0.05) internalization. Prolonged storage (48 h) at 20°C increased the recovery of internalized Salmonella in spinach and green amaranth by 1.0 to 1.5 log units. Colonization of Salmonella on/in leafy vegetables induced the transcription (maximum fold change [FCmax], ∼2,000) of T3SS-related genes. Interserovar variation regarding the internalization ability of Salmonella was observed only in lettuce and green amaranth in a time- and temperature-dependent manner. Attached cells exhibited higher survival rates against low pH than the internalized subpopulation; however, habituation at 20°C in lettuce and amaranth induced acid tolerance to internalized cells, manifested by the 1.5 to 2.0 log CFU/g survivors after 75 min at pH 2.7. Habituation of Salmonella in vegetable extracts sensitized it toward acid, while indigenous microbiota had limited impact on acid resistance of the organism. These findings reveal physiological aspects of Salmonella colonizing leafy vegetables that could be useful in fresh produce microbial risk assessment. IMPORTANCE Consumption of leafy greens has been increasingly associated with foodborne illnesses, and their contamination could occur at pre- and/or postharvest level. Human pathogens may become passively or actively internalized in plant tissues, thereby escaping decontamination procedures. Plant colonization may impact bacterial physiology such as stress resistance and virulence. In this study, it was demonstrated that internalization of Salmonella spp., at the postharvest level, varied with type of vegetable, serovar, and storage conditions. Attached and internalized subpopulations of Salmonella on/in leafy greens showed distinct physiological responses regarding transcriptional changes of stress- and virulence-associated genes, as well as survival capacity against subsequent exposure to lethal pH (2.7). These findings could contribute to a better understanding and potential (re)definition of the risk of enteric pathogens colonizing leafy greens, as well as to the design of intervention strategies aiming to improve the microbiological safety of fresh produce.

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterization of isolated multidrug-resistant Escherichia coli.

AIM: To investigate the microbiological quality, potential foodborne pathogen presence, and to phenotypically (antimicrobial resistance [AMR] profiles) and genotypically (DNA fingerprints and diarrhoeagenic genes) characterize Escherichia coli isolated throughout spinach production systems from farm-to-sale. METHODS AND RESULTS: Samples (n = 288) were collected from two commercial supply chains using either river or borehole irrigation water. E. coli was enumerated throughout the chain where river water was directly used for overhead irrigation at levels between 0.00 and 3.22 log colony forming unit (CFU) g-1 . Following enrichment, isolation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification, E. coli was isolated from 22.57% (n = 65/288) of all samples. Salmonella spp. were isolated from 3% (n = 9/288) of river and irrigation water samples on one farm, and no Listeria monocytogenes was detected throughout the study. Of the 80 characterized E. coli isolates, one harboured the stx2 virulence gene, while 43.75% (n = 35) were multidrug resistant. Overall, 26.30% of the multidrug-resistant E. coli isolates were from production scenario one that used river irrigation water, and 17.50% from the second production scenario that used borehole irrigation water. A greater percentage of resistance phenotypes were from water E. coli isolates (52.50%), than isolates from spinach (37.50%). E. coli isolates from spinach and irrigation water clustered together at high similarity values (>90%) using enterobacterial repetitive intergenic consensus-polymerase chan reaction analysis. CONCLUSIONS: This study reported the presence of multidrug-resistant environmental E. coli throughout spinach production from farm, during processing and up to retail. Furthermore, the similarity of multi-drug resistant E. coli isolates suggests transfer from irrigation water to spinach in both scenarios, reiterating that irrigation water for vegetables consumed raw, should comply with standardized microbiological safety guidelines. SIGNIFICANCE AND IMPACT OF STUDY: Multidrug-resistant E. coli presence throughout spinach production emphasizes the necessity of increased surveillance of AMR in fresh produce and the production environment within a One Health paradigm to develop AMR mitigation strategies.

Agricultural practices in Brazilian organic farms and microbiological characteristics of samples collected along the production chain.

AIMS: To gather data on agricultural practices in organic farms in Sao Paulo, Brazil, and evaluate their relationship with the microbiological characteristics of samples collected along the production chain. METHODS AND RESULTS: Practices data were based on field observations and interviews with farmers in 10 selected organic lettuce producing farms. Counts of Enterobacteriaceae and surveys for Salmonella were performed in samples of lettuce (before and after washing), fertilizers, irrigation and washing water, all collected in the same farm. Water samples were also tested for total coliforms and generic Escherichia coli. Isolated Enterobacteriaceae were identified by MALDI-TOF MS. Contamination of lettuce was influenced by some agricultural practices: chicken manure-based fertilization resulted in higher Enterobacteriaceae counts in lettuce when compared to other types of manure, whereas pre-washed lettuces presented lower microbial counts than non-pre-washed samples. Salmonella was detected in one lettuce sample by qPCR. Escherichia coli was detected in all irrigation water samples. All sample types contained Enterobacteriaceae species commonly reported as opportunistic human pathogens. CONCLUSIONS: The data highlight the need for improvement in the good agricultural practices in the studied farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on agricultural practices and microbiological characteristics of organic lettuce, contributing to the development of more accurate risk assessments.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Rapid detection of Salmonella enterica in leafy greens by a novel DNA microarray-based PathogenDx system.

The diverse matrices pose great challenges for rapid detection of low Salmonella level (<10 CFU) in fresh produce. The applicability of microarray-based PathogenDx system for detecting low contamination of Salmonella Newport from leafy greens was evaluated. A pre-PCR preparation protocol including enrichment in universal pre-enrichment broth for 3 h followed by sample concentration using an InnovaPrep bio-concentrator or 6 h enrichment without a concentration step was used for detecting S. Newport from leafy greens with initial inoculum level at ∼6 CFU/25 g. Among 205 samples tested, 98%, 93%, 76%, and 60% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were tested positive after 3 h of enrichment with sample concentration. After 6 h of enrichment, 100%, 98%, 90%, and 82% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were positive. The samples were parallelly tested by the FDA bacterial analytical manual (BAM) method and 100% of spiked produce samples were tested positive. The overall analysis time of this methodology was between 8 and 11 h, including all pre-enrichment and concentration steps, in contrast to 4-5 days required for BAM method. The system correctly differentiated all 108 Salmonella strains and 35 non-Salmonella strains used in the study. This novel microarray approach provides a rapid method for detecting Salmonella in leafy greens.