Prevalence of Salmonella spp. in meat, seafood, and leafy green vegetables from local markets and vegetable farms in Phnom Penh, Cambodia.

Salmonella is a major bacterial concern for public health globally. Although there are limited documentation on the prevalence of Salmonella species in Cambodia's food chain, some reports indicate that salmonellosis is a severe gastrointestinal infection in its population and especially in children. To investigate the presence of Salmonella spp., 285 food samples (75 meat, 50 seafood, and 160 leafy green vegetable samples) were randomly collected from various local markets in Phnom Penh capital and nearby farms in Cambodia. Concurrently, field observations were conducted to collect data on food hygiene and practices among the relevant actors. All food samples were analyzed using bacterial culture and plate counts, and the findings were confirmed serially with biochemical, serological, and PCR tests. The observational data on food hygiene and practices from farm to market revealed that the spread of Salmonella in the food-value chain from farm to market could pose health risks to consumers. The overall prevalence of Salmonella spp. was 48.4% (138/285), while the prevalence in meat, seafood, and vegetables was 71% (53/75), 64% (32/50), and 33% (53/160), respectively. Mean Salmonella plate count ranged from 1.2 to 7.40 log10 CFU/g, and there was no significant difference in bacterial counts between meat, seafood, and vegetable samples (p > 0.05). The most common serogroups among the isolated Salmonella spp. were B and C. These results suggest that a large proportion of meat, seafood, and vegetable products sold at local markets in Phnom Penh are contaminated with Salmonella spp. This is likely linked to inadequate hygiene and sanitation practices, including handling, storage, and preservation conditions. Observations on farms suggested that the prevalence of Salmonella in vegetables sold at the market could be linked to contamination relating to agricultural practices. Thus, controlling the spread of foodborne salmonellosis through the food-value chain from farms and retailers to consumers is warranted to enhance food safety in Cambodia.

The Effect of Dietary Nitrate on the Oral Microbiome and Salivary Biomarkers in Individuals with High Blood Pressure.

BACKGROUND: Green leafy vegetables (GLV) contain inorganic nitrate, an anion with potential prebiotic effects on the oral microbiome. However, it remains unclear whether GLV and pharmacological supplementation [potassium nitrate (PN)] with a nitrate salt induce similar effects on the oral microbiome. OBJECTIVES: This study aimed to compare the effect of GLV with PN supplementation on the oral microbiome composition and salivary biomarkers in individuals with high blood pressure. METHODS: Seventy individuals were randomly allocated to 3 different groups to follow a 5-wk dietary intervention. Group 1 consumed 300 mg/d of nitrate in form of GLV. Group 2 consumed pills with 300 mg/d of PN and low-nitrate vegetables. Group 3 consumed pills with potassium chloride (placebo: PLAC) and low-nitrate vegetables. The oral microbiome composition and salivary biomarkers of oral health were analyzed before and after the dietary intervention. RESULTS: The GLV and PN groups showed similar microbial changes, probably nitrate-dependent, including an increase in the abundance of Neisseria, Capnocytophaga, Campylobacter species, and a decrease in Veillonella, Megasphaera, Actinomyces, and Eubacterium species after the treatment. Increased abundance of Rothia species, and reduced abundance of Streptococcus, Prevotella, Actinomyces, and Mogibacterium species were observed in the GLV group, which could be nitrate-independent. GLV and PN treatments increased salivary pH, but only GLV treatment showed an increase in the salivary buffering capacity and a reduction of lactate. CONCLUSION: The combination of nitrate-dependent and nitrate-independent microbial changes in the GLV group has a stronger effect to potentially improve oral health biomarkers compared with PN.

Pesticides residues in leafy green vegetables and irrigation waters in Accra, Ghana.

Pesticides are used in vegetable farming to control pests and diseases, reduce crop losses and improve yield. The study examined pesticide residues in irrigation waters and leafy green vegetables grown in some farming areas in Accra, Ghana. Three types of irrigation water sources (n = 23) and two exotic and four indigenous Ghanaian leafy vegetables (n = 34) from 10 farming areas in Accra, Ghana were collected and examined for 15 organochlorines, 13 organophosphorus and 9 synthetic pyrethroids pesticide residues using the modified QuEChERS procedure. Pesticide residues were detected on 50% (17/34) of the leafy vegetable and 52% (12/23) of the irrigation water samples analysed. Chlorpyrifos and deltamethrin were the most detected pesticide residues in the vegetables and irrigation water. About 26.5% of the vegetables contained pesticide residues exceeding the EU maximum residue limits, so vegetable farmers should be encouraged to comply with appropriate measures on pesticide use to enhance food safety.

Intraspecies competition among Salmonella enterica isolates in the lettuce leaf apoplast.

Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.

Parasitic Contamination of Fresh Leafy Green Vegetables Sold in Northern Lebanon.

Contaminated, raw or undercooked vegetables can transmit parasitic infections. Here, we investigated parasitic contamination of leafy green vegetables sold in local markets in the Tripoli district, Lebanon, during two consecutive autumn seasons (2020-2021). The study involved the microscopic examination of 300 samples of five different types of vegetables (60 samples per type) and used standardized qualitative parasitological techniques for some protozoa and helminths. The results showed that 16.7% (95% interval for p: 12.6%, 21.4%) (50/300) of the vegetable samples were contaminated with at least one parasite. The most frequently detected parasite was Blastocystis spp. (8.7%; 26/300); this was followed in frequency by Ascaris spp. (3.7%; 11/300). Among the different vegetable types, lettuce (23.3%; 14/60) was the most contaminated, while arugula was the least contaminated (11.7%; 7/60). The statistical analysis did not reveal any significant association between the prevalence of parasitic contamination and the investigated risk factors, which included collection date, vegetable type, market storage status, and wetness of vegetables at the time of purchase (p > 0.05). The high prevalence of parasitic contamination also suggested the potential presence of other microbial pathogens. These findings are important because leafy green vegetables are preferentially and heavily consumed raw in Lebanon. Thus, implementing effective measures that target the farm-to-fork continuum is recommended in order to reduce the spread of intestinal pathogens.

Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the 'mustard bomb' reaction in reducing pungency upon the mastication of fresh tissue-a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Sensory Evaluation of Common Ice Plant (Mesembryanthemum crystallinum L.) in Response to Sodium Chloride Concentration in Hydroponic Nutrient Solution.

Common ice plant (Mesembryanthemum crystallinum L.) is a novel edible plant with a succulent and savory flavor. The plants display prominent epidermal bladder cells (EBCs) on the surface of the leaves that store water and sodium chloride (NaCl). The plants have high nutritional value and are adapted to saline soils. Previous research has determined the impact of NaCl on the growth and mineral content of ice plant, but as NaCl has an impact on a food's sensory properties, an interesting question is whether saline growth media can affect the plant's taste and texture, and if this alters consumers' sensory response to ice plant. The objective of this study was to evaluate the sensory aspects of ice plant, as well as consumer liking in response to increasing NaCl concentration in hydroponic nutrient solution. Four-week-old seedlings of ice plant were transplanted into deep water culture (DWC) hydroponic systems and treated with five NaCl concentrations (0 M [control], 0.05 M, 0.10 M, 0.20 M, and 0.40 M NaCl). Eight-week-old plants (after four weeks of NaCl treatment) were harvested, and the middle leaves of each plant were sampled for consumer testing. A total of 115 participants evaluated various flavor, texture, and appearance aspects of ice plant and provided their liking ratings. The consumers were able to discriminate differences in salt intensity from the plants based on NaCl treatment in the hydroponic nutrient solution. Low NaCl concentrations (0.05-0.10 M) did not have obvious adverse effect on consumer liking, which aligns with the result of previous research that 0.05-0.10 M NaCl could largely stimulate the growth of ice plant. NaCl concentrations higher than 0.20 M are not recommended from both a production and consumer perspective. With increased NaCl level in plant samples, the consumers detected more saltiness, sourness, and fishiness, less green flavor, and similar levels of bitterness and sweetness. NaCl treatment had no effects on leaf appearance and texture, and the consumers' overall liking was mainly determined by flavor. Overall, ice plant presents some unique attributes (salty and juicy) compared to other edible salad greens; however, consumer awareness of ice plant is very low, and purchase intent is relatively low as well. Consumers picture ice plant being used mainly in salads and in restaurants.

The live bacterial load and microbiota composition of prepacked "ready-to-eat" leafy greens during household conditions, with special reference to E. coli.

Ready-to-eat (RTE) leafy greens are popular products that unfortunately have been associated with numerous foodborne illness outbreaks. Since the influence of consumer practices is essential for their quality and safety, the objective of this study was to analyze the microbiota of RTE products throughout shelf life during simulated household conditions. Products from different companies were analyzed in terms of plate counts, and resealed and unopened packages were compared. High bacterial loads were found, up to a total plate count of 9.6 log10 CFU/g, and Enterobacteriaceae plate counts up to 6.0 CFU/g on the expiration date. The effect of consumer practice varied, thus no conclusions regarding resealed or unopened bags could be drawn. The tested products contained opportunistic pathogens, such as Enterobacter homaechei, Hafnia paralvei and Pantoea agglomerans. Amplicon sequencing revealed that the relative abundance of major taxonomic groups changed during shelf life; Pseudomonadaceae and Xanthomonadaceae decreased, while Flavobacteriaceae and Marinomonadaceae inceased. Inoculation with E. coli CCUG 29300T showed that the relative abundance of Escherichia-Shigella was lower on rocket than on other tested leafy greens. Inoculation with E. coli strain 921 indicate growth at the beginning of shelf-life time, while E. coli 731 increases at the end, seemingly able to adapt to cold storage conditions. The high levels of live microorganisms, the detection of opportunistic pathogens, and the ability of E. coli strains to grow at refrigeration temperature raise concerns and indicate that the shelf life may be shortened to achieve a safer product. Due to variations between products, further studies are needed to define how long the shelf-life of these products should be, to ensure a safe product even at the end of the shelf-life period.

Behavior of Silver Nanoparticles in Chlorinated Lettuce Wash Water.

ABSTRACT: Use of silver nanoparticles (Ag NPs) in pesticides may lead to residual levels in food crops, thus raising food safety and environmental concerns. Because little is known about Ag NP behavior in wash water during typical commercial washing of fresh produce, this study assessed the temporal changes in Ag NP behavior when exposed to 2 to 100 mg/L free chlorine (Cl2) in simulated lettuce wash water for up to 10 days. Aggregate size and zeta potential of Ag NPs (5 mg/L) were evaluated in the presence and absence of dissolved lettuce extract (DLE, 0.1%), with Ag NPs in deionized water serving as the control treatment. In the presence of chlorine, greater aggregation of Ag NPs occurred over time (49 to 431 nm) compared with the control treatment (P < 0.05). Lower zeta potentials (-39 to -95 mV) were observed in the chlorine-only treatments, likely due to the formation of AgCl particles. Larger aggregates and lower zeta potentials were also observed in DLE (84 to 273 nm and -28 to -32 mV, respectively), as compared with the control treatment. After 7 to 10 days, larger aggregates were seen in the chlorine-only treatments as compared with the DLE treatments, despite lower zeta potentials, probably facilitated by nucleation and crystal growth of AgCl. Transmission electron microscopy with energy dispersive spectroscopy confirmed the formation of AgCl-Ag NP composite particles with chlorine and the embedding of AgCl and Ag NPs in the DLE matrix. Thus, DLE might stabilize and protect Ag NPs from chlorine. These findings indicate that chlorine and plant-released organic material can substantially change the behavior of Ag NPs, which may, in turn, impact both removal from fresh-cut produce during washing and their environmental fate.

Reduced Abundance of Nitrate-Reducing Bacteria in the Oral Microbiota of Women with Future Preeclampsia.

The oral microbiota can contribute to the regulation of blood pressure by increasing the availability of nitric oxide through the reduction of nitrate to nitrite, which can be converted into nitric oxide in the stomach and then enter the circulation. It is unclear if the composition of the oral microbiota is different between women who do and do not develop preeclampsia. This study aimed to compare the composition of the buccal microbiota just prior to the development of symptoms at 36 weeks gestation in 12 women who developed late-onset preeclampsia and 24 matched women who remained normotensive throughout pregnancy by 16S rRNA gene amplicon sequencing. The abundance of the nitrate-reducing Veillonella spp V. parvula and V. dispar and a subunit of nitrate reductase narH was compared using real-time PCR. The abundance of bacteria was correlated with maternal blood pressure and dietary intake of nitrate-containing vegetables. The results showed that the abundance of nitrate-reducing bacteria including Veillonella, specifically V. parvula, and Prevotella was reduced in women who developed preeclampsia. Veillonella but not Prevotella abundance was negatively correlated with maternal blood pressure. The dietary intake of nitrate-containing vegetables did not differ between the groups and was not correlated with the abundance of Veillonella. There was no difference in the abundance of the nitrate reductase subunit narH between the groups. These results suggest that the abundance of nitrate-reducing bacteria is reduced in the oral microbiota of women who later develop preeclampsia, indicating a potential pathway for prevention.

A microfluidic approach for studying microcolonization of Escherichia coli O157:H7 on leaf trichome-mimicking surfaces under fluid shear stress.

Escherichia coli O157:H7 have previously been associated with disease outbreaks associated with leafy green vegetables. However, the physical mechanisms that determine the spatial organization of bacteria onto leafy greens are still not clear. Microfluidics with embedded trichome-mimicking microposts were employed to investigate the role of shear flow and configuration of trichomes on E. coli O157:H7 microcolonization. We characterized the three-dimensional microcolonization of green fluorescent protein (GFP)-tagged E. coli O157:H7 using multiphoton fluorescence microscopy and compared their differences under static (no flow; incubated for 36 h at 37°C) and fluid shear conditions (750 nl/min for 36 h at 37°C). For micropatterned trichome arrays, we demonstrated that natural wax-mixed polydimethylsiloxane retains similar topographies and contact angles to the surface of trichome-bearing leafy greens. Our results showed that E. coli O157:H7 under fluid shear stress aligned their colonization parallel to the direction of flow. In a static condition, their colonization had no preferential alignment, with statistically similar angular distributions in all directions. In addition, depending on dimensions of the trichome arrays and flow conditions, different bacterial microcolonization patterns grew radially from initial attachment; they formed into filamentous structures and developed into bridges by surface hydrophobicity and flow-induced shear with a nutrient-rich medium. Collectively, these results demonstrate how the consequences of bacterial colonization in response to shear flow can affect pathogenic bacterial contamination of leafy greens and biofilm architectures.

Characterization of Multidrug-Resistant Escherichia coli Isolated from Two Commercial Lettuce and Spinach Supply Chains.

ABSTRACT: Leafy green vegetables have increasingly been reported as a reservoir of multidrug-resistant pathogenic Enterobacteriaceae, with Shiga toxin-producing Escherichia coli frequently implicated in disease outbreaks worldwide. This study examined the presence and characteristics of antibiotic resistance, diarrheagenic virulence genes, and phylogenetic groupings of E. coli isolates (n = 51) from commercially produced lettuce and spinach from farms, through processing, and at the point of sale. Multidrug resistance was observed in 33 (64.7%) of the 51 E. coli isolates, with 35.7% (10 of 28) being generic and 100% (23 of 23) being extended-spectrum ß-lactamase/AmpC producing. Resistance of E. coli isolates was observed against neomycin (51 of 51, 100%), ampicillin (36 of 51, 70.6%), amoxicillin (35 of 51, 68.6%), tetracycline (23 of 51, 45%), trimethoprim-sulfamethoxazole (22 of 51, 43%), chloramphenicol (13 of 51, 25.5%), Augmentin (6 of 51, 11.8%), and gentamicin (4 of 51, 7.8%), with 100% (51 of 51) susceptibility to imipenem. Virulence gene eae was detected in two E. coli isolates from irrigation water sources only, whereas none of the other virulence genes for which we tested were detected. Most of the E. coli strains belonged to phylogenetic group B2 (25.5%; n = 13), B1 (19.6%; n = 10), and A (17.6%; n = 9), with D (5.9%; n = 3) less distributed. Although diarrheagenic E. coli was not detected, antibiotic resistance in E. coli prevalent in the supply chain was evident. In addition, a clear link between E. coli isolates from irrigation water sources and leafy green vegetables through DNA fingerprinting was established, indicating the potential transfer of E. coli from irrigation water to minimally processed leafy green vegetables.

Effect of Selenium Application and Growth Stage at Harvest on Hydrophilic and Lipophilic Antioxidants in Lamb's Lettuce (Valerianella locusta L. Laterr.).

Lamb's lettuce (Valerianella locusta L. Laterr.) is a leafy green vegetable that is rich in various biological active compounds and is widely used in ready-to-eat salads. The cultivation conditions and growth stage could affect the secondary metabolism in plants and thereby modify their food value. In the present study, the effect of selenium (Se) application in various concentrations (5.0, 10.0, and 20.0 µM) on the contents of Se, phenolic compounds, vitamin C, carotenoids, chlorophylls, and antioxidant activity of hydrophilic and lipophilic extracts of lamb's lettuce harvested at three growth stages (38, 52, and 66 days after sowing (DAS)) was studied. Se application significantly increased the Se concentration in the shoots (up to 124.4 µg g-1 dry weight), as well as the contents of chlorogenic acid, total flavonoids, total phenolics, ascorbic acid, chlorophyll b, and the antioxidant activity of hydrophilic and lipophilic extracts. A higher content of phenolic compounds and higher antioxidant activity of hydrophilic extracts was observed at the first growth stage (38 DAS). On the contrary, higher contents of lipophilic compounds (chlorophyll a, chlorophyll b, lutein, ß-carotene) and higher antioxidant activity of lipophilic extracts were found for shoots harvested at later stages (52 and 66 DAS).

Persistence of Salmonella enterica and Enterococcus faecium NRRL B-2354 on Baby Spinach Subjected to Temperature Abuse after Exposure to Sub-Lethal Stresses.

The exposure of foodborne pathogens such as Salmonella enterica to a sub-lethal stress may protect bacterial cells against distinct stresses during the production of leafy greens, which can constitute potential health hazards to consumers. In this study, we evaluated how the prior exposure of S. enterica to sub-lethal food processing-related stresses influenced its subsequent persistence on baby spinach under cold (4 °C for 7 days) and temperature abuse (37 °C for 2 h + 4 °C for 7 days) conditions. We also compared the survival characteristics of pre-stressed S. enterica and Enterococcus faecium NRRL B-2354 as its surrogate on baby spinach. A cocktail of three S. enterica serovars, as well as S. Typhimurium ATCC 14028 wild type and its ΔrpoS mutant, and E. faecium NRRL B-2354, was first exposed to sub-lethal desiccation, oxidation, heat shock, and acid stresses. Afterward, baby spinach was inoculated with unstressed or pre-stressed cells at 7.0 log CFU/sample unit, followed by 7-day storage under cold and temperature abuse conditions. The unstressed S. enterica (fresh cells in sterile 0.85% saline) decreased rapidly within the first day and thereafter persisted around 5.5 log CFU/sample unit under both conditions. The desiccation-stressed S. enterica showed the highest bacterial counts (p < 0.05) compared to other conditions. The unstressed S. enterica survived better (p < 0.05) than the oxidation- and acid-stressed S. enterica, while there were no significant differences (p > 0.05) between the unstressed and heat-shocked S. enterica. Unlike the wild type, temperature abuse did not lead to the enhanced survival of the ΔrpoS mutant after exposure to desiccation stress, indicating that the rpoS gene could play a critical role in the persistence of desiccation-stressed S. enterica subjected to temperature abuse. E. faecium NRRL B-2354 was more persistent (p < 0.05) than the pre-stressed S. enterica under both conditions, suggesting its use as a suitable surrogate for pre-stressed S. enterica by providing a sufficient safety margin. Our results demonstrate the merit of considering the prior exposure of foodborne pathogens to sub-lethal stresses when validating the storage conditions for leafy greens.

Hydrothermal Processing and In Vitro Simulated Human Digestion Affects the Bioaccessibility and Bioactivity of Phenolic Compounds in African Pumpkin (Momordica balsamina) Leaves.

The African pumpkin (Momordica balsamina) contains bioactive phenolic compounds that may assist in reducing oxidative stress in the human body. The leaves are mainly consumed after boiling in water for a specific time; this hydrothermal process and conditions of the gastrointestinal tract may affect the presence and bioactivity of phenolics either positively or negatively. In this study, the effects of hydrothermal processing (boiling) and in vitro simulated human digestion on the phenolic composition, bioaccessibility and bioactivity in African pumpkin were investigated in comparison with those of spinach (Spinacia oleracea). A high-resolution ultra-performance liquid chromatography, coupled with diode array detection, quadrupole time-of-flight and mass spectrometer (UPLC-DAD-QTOF-MS) was used to profile phenolic metabolites. Metabolites such as 3-caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were highly concentrated in the boiled vegetable extracts compared to the raw undigested and all digested samples. The majority of African pumpkin and spinach extracts (non-digested and digested) protected Deoxyribonucleic acid (DNA), (mouse fibroblast) L929 and human epithelial colorectal adenocarcinoma (Caco-2) cells from 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative damage. From these results, the consumption of boiled African pumpkin leaves, as well as spinach, could be encouraged, as bioactive metabolites present may reduce oxidative stress in the body.

Evaluation of PhageDX Salmonella Assay for Salmonella Detection in Hydroponic Curly Lettuce.

Lettuce is one of the most consumed leafy vegetables worldwide and has been involved in multiple foodborne outbreaks. Salmonella is one of the most prevalent etiological agents of foodborne disease (FBD) in lettuces, and its detection may take several days depending on the chosen method. This study evaluates a new rapid method that uses recombinant bacteriophages to detect Salmonella in hydroponic curly lettuce. First, the ability of the assay to detect six Salmonella serovars at three different concentrations (1, 10, and 100 CFU/well) was tested. Second, the detection of Salmonella was tested in lettuces using a cocktail of the same Salmonella serovars and concentrations after a 7 h enrichment. The results of these experiments showed that the detection limit was dependent on the serovar tested. Most serovars were detected in only 2 h when the concentration was 100 CFU/well. Salmonella was detected in 9 h (7 h enrichment + 2 h bioluminescence assay) in all lettuce samples with 10 CFU/25 g or more. Salmonella detection was not influenced by natural microbiota of lettuces. This study demonstrated that the phage assay was sensitive and faster than other detection methods, indicating that it is a better alternative for Salmonella detection on lettuces.

Comparison of droplet digital PCR vs real-time PCR for Yersinia enterocolitica detection in vegetables.

Yersiniosis - the 4th most commonly reported zoonosis in the European Union - is caused by the consumption of food contaminated with the bacterium Yersinia enterocolitica. The number of human cases and contaminated food samples is probably underestimated since conventional molecular methods currently proposed for Yersinia enterocolitica detection proved to have several limitations. Critical issues associated with the detection of Yersinia enterocolitica in meat and/or meat product has already been investigated, whereas data on the possible limits of the molecular methods for Yersinia enterocolitica detection in vegetables are still lacking. According to ISO method (ISO 18867:2015), real-time polymerase chain reaction (rtPCR) should be adopted for Yersinia enterocolitica detection, even if it proved to be affected by some biases. Recently, Droplet Digital PCR (ddPCR) has been introduced as a useful tool to detect and quantify different pathogenic bacteria in complex food matrices. However, its potential application for Yersinia enterocolitica detection in vegetables has never been investigated before. In the present study two molecular platforms (rtPCR and ddPCR) were used to evaluate the pathogen's behaviour in experimentally contaminated leafy greens (Lactuca sativa L.) and to assess the rate of detection achievable after the incubation for eleven days at different temperatures. By comparing, noticeable differences emerged between the two technical approaches: only ddPCR allowed the detection of the pathogen in leafy greens when contaminated at low levels. Moreover, results of the present work highlighted the importance of length and temperature of incubation on the survival and/or the growth of Yersinia enterocolitica in vegetables: at 18 and 25 °C the concentration of the pathogen considerably decreases along incubation. Based on data, the use of rtPCR leads to an underestimation of the true prevalence of pathogenic Y. enterocolitica in vegetables, while temperature and time currently proposed for Y. enterocolitica (25 °C for 24 h), allow optimizing detection. To conclude, ddPCR may be undoubtedly proposed as a reliable alternative strategy for the quick detection of the pathogen in food samples.

Antibiotic Resistance Profile of Salmonella enterica Isolated from Exotic and Indigenous Leafy Green Vegetables in Accra, Ghana.

ABSTRACT: Fresh produce-borne enteric bacterial pathogens that are resistant to antibiotics have posed serious challenges to food safety and public health worldwide. This study was conducted to evaluate the antibiotic resistance profiles of Salmonella enterica isolates (n = 33) recovered from exotic and indigenous leafy green vegetable samples (n = 328) collected from 50 vegetable farms in 12 farming areas and 37 vegetable sellers in four market centers in Accra, Ghana, from March 2016 to March 2017 and to determine the distribution of integrons among antibiotic-resistant isolates. The susceptibility of the Salmonella isolates to 12 antibiotics was assayed with the standard disk diffusion assay. The MICs of the five most resisted antibiotics were determined with a twofold macrodilution method. A PCR assay was used to detect the presence of integrons in Salmonella isolates, and PCR products with the amplified integron gene cassette were purified and sequenced with the Sanger sequencing technology. The Salmonella isolates used in the study were resistant to at least one tested antibiotic, and 30.3% (10 of 33) of the isolates were multidrug resistant. Most isolates (81.8%) were resistant to sulfisoxazole. The MICs of tetracycline, cefoxitin, streptomycin, ampicillin, and sulfisoxazole were 16, 32, 64, 64, and >1,024 µg/mL, respectively. Five patterns of multidrug resistance were observed among the Salmonella isolates, and the most common patterns were AAuFox (30.3%) and AAuFoxSSu (18.1%). One (3.0%) of the 33 Salmonella isolates tested positive for the class 1 integron, with a gene cassette of about 800 bp. Nucleotide sequencing revealed that this class 1 integron carried a single gene, dfrA7. Further studies are needed to determine whether the consumption of contaminated leafy green vegetables is a route for acquiring antibiotic-resistant Salmonella in Accra, Ghana.

Dispersive solid-phase extraction using microporous metal-organic framework UiO-66: Improving the matrix compounds removal for assaying pesticide residues in organic and conventional vegetables.

An efficient dispersive solid-phase extraction method was developed to trace pesticide residues in commonly consumed vegetables. In this method, UiO-66 with uniform micropores was used as sorbent, and gas chromatography-mass spectrometry was applied to detect the pesticides. Thanks to the size sieving action of uniform micropores, UiO-66 directly extracted the target pesticides from vegetable matrices and excluded the relatively large matrix compounds. This well eliminated the matrix effect. The important experimental conditions were evaluated by orthogonal array experimental design. In optimized conditions, good linearity (R2 ≥ 0.99), detection limits (0.4-2.0 ng/g), recoveries (60.9-117.5%) and precision (relativestandarddeviations < 14.6%) were achieved. Moreover, the sorbent UiO-66 can be reused more than 20 times. These demonstrate a simple, reliable and robust method to screen the pesticide residues in vegetables. Furthermore, the validated method was applied to detect the pesticides in various organic and conventional vegetables.

Photocatalytically Enhanced Inactivation of Internalized Pathogenic Bacteria in Fresh Produce Using UV Irradiation with Nano-Titanium Dioxide.

ABSTRACT: Once pathogens are internalized in fresh produce, they pose a challenging food safety issue because they are not effectively inactivated by conventional rinsing or sanitization. To protect food safety and public health, the objectives of this study were to examine internalized levels of foodborne pathogens in different types of fresh produce and to investigate the effectiveness of photocatalytically enhanced inactivation of internalized pathogens in fresh produce using UV irradiation with titanium dioxide (TiO2). For this, green fluorescent protein-labeled Salmonella Typhimurium and Escherichia coli O157:H7 were inoculated on the leaf surface of four types of fresh produce (∼108 CFU per leaf), and varying concentrations of TiO2 suspension (0.50, 0.75, 1.00, 1.25, and 1.50 µg/mL) were applied to the surface of contaminated leaves. Depending on the nature of each vegetable, the internalized bacterial level differed (2 to 5 log CFU/g of leaf). When UV irradiation (6,000 J/m2) was applied, the internalized Salmonella Typhimurium and E. coli levels were reduced by 0.8 to 2.4 log CFU per leaf; with TiO2, the reduction was 1.1 to 3.7 log CFU per leaf. The inactivation efficiency increased as the TiO2 concentration increased (up to 1.50 µg per leaf). These results indicate that TiO2 application enhanced the photocatalytic inactivation of internalized foodborne pathogens. The application of TiO2 would be most practical before UV irradiation and before distributing the produce. This study established a platform for future research on the inactivation of various internalized pathogens for protecting public health and scaling up fresh produce treatments by the food industry.