Mixed Salad Research Articles

Comparative Life Cycle Assessment of alternative winter salad value chains supplying the United Kingdom

Lettuce is an established food commodity in the UK increasingly facing supply challenges in winter due to adverse weather events and rising energy costs. We investigate whether an agroecologically grown salad mix of lettuce and underutilised leafy greens produced in the Azores, Portugal, could be part of a sustainable solution. We performed a Life Cycle Assessment to compare the environmental impacts of this salad mix with four other value chains for winter salad supply to the UK: conventional open-field lettuce production chains in Spain using (1) current irrigation practices; (2) 100 % desalinated irrigation water; or hydroponic controlled environment agriculture within the UK powered by (3) the national electricity mix; (4) 100 % wind-generated electricity. Results indicated that the leafy-greens agroecological value chain incurred the smallest environmental burdens across 7 to 11 of 16 impact categories studied. Substituting Spanish winter salad supply with agroecological leafy green production in the Azores, if well managed, could reduce many environmental burdens whilst diversifying leafy greens intake. Nevertheless, all winter value chains were associated with larger environmental burdens than conventional open-field production of lettuce in the UK summer, pointing to the importance of seasonal consumption and wider adoption of agroecological techniques to effectively reduce environmental impact.

In vitro bioactivities and shelf life protective properties of sumac extracts against foodborne pathogens and computational analysis of sumac compounds

The aim of this study was to investigate the antibacterial, antibiofilm, antioxidant and shelf life protective potentials of infusion, decoction, and room temperature samples of sumac fruits prepared as used by the local people against three foodborne pathogens including Bacillus cereus, Escherichia coli O157:H7, and Staphylococcus aureus isolated from food samples. The room temperature samples of sumac showed a remarkable antibacterial activity (81.25%) against S. aureus. Infusion and decoction samples of sumac fruit showed an effective antibacterial activity (∼70%) against E. coli O157:H7. Moderate efficacy against B. cereus was observed in all treatment groups. Antibiofilm effects of sumac were observed against B. cereus and S. aureus, but not against E. coli O157:H7. The total antioxidant activity was found to be close to the reference compounds. The twenty-two phenolic and flavonoid chemical compounds (gallic acid, gentisic acid, protocatechuic acid, protocatechualdehyde, 4-OH-benzoic acid, salicylic acid, chlorogenic acid, cafeic acid, p-coumaric acid, ferulic acid, rosmarinic acid, sinapic acid, trans-cinnamic acid, rutin, resveratrol, myricetin, naringenin, quercetin, luteolin, kaempferol, apigenin, and chrysin) were identified by Liquid Chromatography Mass Spectrometer (LC-MS/MS) and these molecules were analyzed by computational analyses using SwissADME, admetSAR 3.0 and the AntiBac-Pred web tool. In addition, the antibacterial efficacy of sumac fruit extracts was tested on mixed Mediterranean salad samples to evaluate the shelf-life enhancing effect. The efficacy of sumac fruit against common foodborne pathogens suggests that the ethnobotanical use of sumac may provide protection against bacterial diseases and can be utilized as an additive and/or ingredient in the food industry.

Shelf-life extension of Thai green papaya salad dressing by hurdle technology

Green papaya salad or Som Tum is the most popular spicy mixed salads in Thailand due to its unique rich flavor. Green papaya salad dressing (GPSD) is made from various ingredients such as fresh chili pepper, fresh garlic, rind tamarind, fish sauce and lime oil, including the limitation in controlling the taste and flavor of salad dressing and its poor shelf-life. In this study, a convenient ready-to-eat GPSD was developed. Hurdle technology was applied to extend shelf-life of the GPSD based on monitoring of microbial contamination and food pathogens throughout the process. Hurdle technology able to decrease total plate count (TPC) from 5.6 ± 0.2 to 1 ± 0.3 log CFU/g and yeast and mold (Y&M) from 4.2 ± 0.3 to <1 log CFU/g. After 12 weeks of storage at 5 ± 2 °C, slightly increase of TPC was detected as 1.5 ± 0.2 log CFU/g and no changes were found for Y&M and other pathogens. At week 12, GPSD stored at 32 ± 2 °C was found to have higher TPC and Y&M (3.7 ± 0.3 and 2.4 ± 0.3 log CFU/g, respectively). Therefore, a combination of hurdles that combines low aw, low pH, heat treatment, low temperature after hot filling, and chilled storage could extend the shelf-life of GPSD with satisfy sensorial test result and be suitable for minimally processed salad dressing.

Characterizing the US Market for Salad Mixes through the Lens of Environmental Preferences

The consumption of salad mixes has increased because of their convenience and nutritional value, resulting in significant sales increases during the past decade. Conversely, the uses of pest-control chemicals, long-distance transportation of salad mixes, and plastic packaging have raised environmental concerns among “green consumers.” Because proenvironmental products are becoming more widespread, this study delved into market segments of salad mix consumers based on their preferences for proenvironmental labels. Data for this study were collected via a 2020 web-based survey of 2100 salad mix consumers in the United States. We performed a comprehensive two-stage cluster analysis that integrated both hierarchical and partitioning methods. This analysis was based on consumers’ preferences and evaluations of production (low energy use, low fertilizer use, low greenhouse gas emissions, low water use, and pollinator-friendly) and marketing-related (biodegradable packaging, low carbon footprint, and low food miles) proenvironmental labels. Three segments were identified. We used ordered probit regression to assess the impact of consumer demographic characteristics, market preferences, and environmental perceptions on cluster membership. The deep-rooted segment, which represented 36% of the sample, highly valued all proenvironmental labels related to salad mixes and had a particular preference for labels that included low fertilizer use, pollinator-friendly production methods, and low greenhouse gas emissions. The indecisive segment comprised 40% of the sample and moderately valued all proenvironmental labels; this group mainly comprised individuals with lower income levels and those living in rural areas. The skeptic segment represented 23% of the sample and valued environmental labels less than the deep-rooted and indecisive segments did; additionally, they reported the lowest consumption of salad mixes. These findings can help retailers and policymakers communicate information about proenvironmental labels more effectively to each segment of salad mix consumers.

Anthony Vidler's IAS Mystery

Abstract The honking of the geese going south was beginning to get on his nerves. How to work with that going on. And the library was getting colder. The day had been grey, the soil damp, the air raw and feeling like snow. It might snow tonight. Not a long walk home though—and the apartment was at least warm. If it wasn't for the people upstairs, creaking, stamping (or that's what it sounded like), and shouting—but maybe just talking given the thin walls. Maybe they were using Skype—his own had long since ceased to connect. Needs a new password, or better, someone to call. He had felt the walls this morning—damp and a little soft— old plasterboard, wooden panels slightly buckled from heat and damp in that order. How could they let the housing get so run-down? But that was how monasteries worked, and this, all said and done, was a sort of monastery. Without the religion of course. But religion was there all right—under the radar, couched in academic formulae, a kind of ersatz nonbelief in belief. He had heard it at lunch—the endless parroting of rehashed research, of half-baked theories, of platitudes about how good the food was, or how much better the food was yesterday, or how interesting the mixed cold vegetarian salad was, or that you couldn't count on anything these days, or you are new here aren't you? Kindly inquiries, sensitive overtures, only half-listening to the replies.

Outbreak of Salmonella Typhimurium linked to Swedish pre-washed rocket salad, Sweden, September to November 2022.

In September 2022, the Public Health Agency of Sweden observed an increase in domestic Salmonella Typhimurium cases through the Swedish electronic notification system, and an outbreak strain was identified with whole genome sequencing. Overall, 109 cases with symptom onset between 17 September and 24 November 2022 were reported from 20 of 21 Swedish regions. The median age of cases was 52 years (range 4-87 years) and 62% were female. A case-control study found cases to be associated with consumption of rocket salad (adjusted odds ratio (aOR) = 4.9; 95% confidence interval (CI): 2.4-10, p value < 0.001) and bagged mixed salad (aOR = 4.0; 95% CI: 1.9-8.1, p value < 0.001). Trace-back, supported by Finnish authorities who identified the Swedish outbreak strain in a Finnish cluster during the same time period, identified rocket salad, cultivated, pre-washed and pre-packed in Sweden as the likely source of the outbreak. No microbiological analyses of rocket salad were performed. Our investigation indicates that bagged leafy greens such as rocket salad, regardless of pre-washing procedures in the production chain, may contain Salmonella and cause outbreaks, posing a health risk to consumers. We emphasise the need for primary producers of leafy greens to identify possible contamination points to prevent outbreaks.

Effect of Different Pre-Growth Temperatures on the Survival Kinetics of Salmonella enterica and Listeria monocytogenes in Fresh-Cut Salad during Refrigerated Storage.

The effect of the pre-growth temperature of bacterial cultures on their subsequent survival kinetics in fresh-cut produce during refrigerated storage was investigated in this study. Three-strain cocktails of Listeria monocytogenes and Salmonella enterica, cultured at different growth temperatures (4, 21, and 37 °C) were inoculated on fresh-cut mixed salad and on individual produce in the mixed salad. The inoculated samples were stored at 4 °C and 80 ± 2% relative humidity (RH) for up to 72 h and the growth, survival, or death kinetics were determined at regular intervals. The results indicate that depending upon the type of pathogen tested, the pre-growth temperature(s) and the type of produce showed a significant (p ≤ 0.05) effect on the survival kinetics. Among the tested produce, mixed salad showed the highest reduction in L. monocytogenes pre-grown at 37 °C (1.33 log CFU/g) followed by red cabbage (0.56 log CFU/g), iceberg lettuce (0.52 log CFU/g), and carrot (-0.62 log CFU/g), after 72 h, respectively. In the case of Salmonella, carrot showed the highest reduction (1.07 log CFU/g for 37 °C pre-grown culture) followed by mixed salad (0.78 log CFU/g for 37 °C pre-grown culture), cabbage (0.76 log CFU/g for 21 °C pre-grown culture), and lettuce (0.65 log CFU/g for 4 °C pre-grown culture), respectively. Among the tested ComBase predictive models, the Baranyi-Roberts model better fitted the experimental data. These findings indicate that the appropriate selection of pre-growth environmental conditions is critical to better understand the kinetics of foodborne pathogens.

Environmental Consequences of Shelf Life Extension: Conventional versus Active Packaging for Fresh-Cut Salads

The use of active coatings in fresh food packaging is an innovative technique that optimizes the functional properties of films, resulting in a longer product shelf life and reduced food waste. But, which is more sustainable, active packaging (AP) or conventional packaging (CP) for the packaging of fresh-cut products? To answer this research question, this study analyzes the environmental performance of AP during its life cycle for packaging a minimally processed fresh salad mix compared with CP, in terms of its manufacture and use. The AP is a bag that includes a bioactive component, oregano essential oil (OEO), which is an inhibitor of microbial growth, incorporated into an ethylene vinyl alcohol copolymer (EVOH) coating on a conventional polypropylene (PP) film. To this end, a Life Cycle Assessment (LCA) was carried out based on ISO 14040 and 14044, using the ReCiPe methodology. The results showed that using active packaging has a beneficial affect, reducing the amount of produced food by 30% compared with conventional packaging over the same period. The reductions in the studied impact categories were greater than 50% in most of them, with a 62% reduction in global warming. The proposed sensitivity analysis showed the difference between the disposal or treatment of waste generated by the packaging production process and the packaged product, indicating that this step is of great importance for the environmental impacts and sustainability of this process. In 80% of the scenarios analyzed, the AP achieved better results than the CP in terms of damage categories.

ADANA İLİNDE PERAKENDE SATILAN MEYVE VE SEBZELERDEN İZOLE EDİLEN GRAM-NEGATİF BAKTERİLERİN YAYGINLIĞI

In this study, 65 samples of fresh products (leafy vegetables, ready-to-eat mixed salads, and fruits) retailed in Adana province of Turkiye were investigated for presence of Gram-negative bacteria. Salmonella spp. was undetected in any samples. However, Klebsiella pneumoniae (22%), Stenotrophomonas maltophilia (15%), Citrobacter freundii (11%), E. coli (11%), Klebsiella oxytoca (11%), Acinetobacter spp. [A. baumannii, A. pittii, (7%)], Pseudomonas putida (7%), were the predominant microorganisms and were detected in (19/65) 29.2% of samples by MALDI-TOF system. Their bacterial counts were between 3.6 and 8.3 log CFU/g. Strains of Enterobacter ludwigii (4%), Delftia lacustris (4%), Burkholderia cenocepacia (4%), and Burkholderia vietnamiensis (4%) were less detected. Our results revealed the fruits and vegetables produced in Adana province generally comply with microbiological criteria of the Turkish Food Codex interms of Salmonella spp., but higher bacterial counst of E. coli (between 5.8 and 6.3 log CFU/g) was found. Besides, Escherichia coli, Acinetobacter spp., Klebsiella pneumoniae, Citrobacter freundii, and Stenotrophomonas maltophilia species could be risky to human health.

Assessing the profitability of scaling up for retail access: Lessons from local salad mix in Southeast Michigan

Changes to the supermarket supply chain in recent decades have “squeezed out” local and small farmers in exchange for more consolidated and global suppliers. As a result, these small-scale farmers have turned to more direct-to-consumer markets, which capture a higher price point but also bear higher marketing costs. Previous research indicates potential saturation and lack of profita­bility in this market type. Researchers have explored strategies for “scaling up” local farmers into intermediary supply chains, such as grocery retail, and have tested the profitability of hybrid marketing strategies with positive results. However, there are very few studies that utilize production costs to test market feasibility, and even fewer that include retailer willingness-to-pay estimates. To assess strategies from the perspectives of both producers and buyers, this study uses salad mix in Southeast Michigan as a pilot case. Farmer-generated production costs incurred for strategies and production types were estimated in focus groups, and retailer willingness-to-pay estimates were obtained in interviews. The analysis suggests that a combination of more efficient harvest technology and central processing would have the greatest impact on increasing profitability, but the dramatic effect that central processing has on output price makes it the most feasible strategy for small-scale farmers. In addition, the minimal costs of organic certification for small farmers are likely to be justified by the price premium that grocery retailers are willing to pay. Hydroponic production may be challenging to break even at a smaller scale but could potentially meet retailers’ price prefer­ences at larger scales. Pairing production cost estimates with buyer willingness-to-pay estimates may generate more comprehensive assessments of the relative profitability of potential scaling-up strategies. This method could be applied to other crops, regions, and produce buyers by cooperative extension, nonprofit, or local government person­nel working with small farmers on their market development plans.

Development of a targeted amplicon sequencing method for genotyping Cyclospora cayetanensis from fresh produce and clinical samples with enhanced genomic resolution and sensitivity.

Outbreaks of cyclosporiasis, an enteric illness caused by the parasite Cyclospora cayetanensis, have been associated with consumption of various types of fresh produce. Although a method is in use for genotyping C. cayetanensis from clinical specimens, the very low abundance of C. cayetanensis in food and environmental samples presents a greater challenge. To complement epidemiological investigations, a molecular surveillance tool is needed for use in genetic linkage of food vehicles to cyclosporiasis illnesses, estimation of the scope of outbreaks or clusters of illness, and determination of geographical areas involved. We developed a targeted amplicon sequencing (TAS) assay that incorporates a further enrichment step to gain the requisite sensitivity for genotyping C. cayetanensis contaminating fresh produce samples. The TAS assay targets 52 loci, 49 of which are located in the nuclear genome, and encompasses 396 currently known SNP sites. The performance of the TAS assay was evaluated using lettuce, basil, cilantro, salad mix, and blackberries inoculated with C. cayetanensis oocysts. A minimum of 24 markers were haplotyped even at low contamination levels of 10 oocysts in 25 g leafy greens. The artificially contaminated fresh produce samples were included in a genetic distance analysis based on haplotype presence/absence with publicly available C. cayetanensis whole genome sequence assemblies. Oocysts from two different sources were used for inoculation, and samples receiving the same oocyst preparation clustered together, but separately from the other group, demonstrating the utility of the assay for genetically linking samples. Clinical fecal samples with low parasite loads were also successfully genotyped. This work represents a significant advance in the ability to genotype C. cayetanensis contaminating fresh produce along with greatly expanding the genomic diversity included for genetic clustering of clinical specimens.

The Occurrence of Serotypes and Virulence Genes of Listeria monocytogenes in Various Food Products

Background: Given that controlling Listeria contamination is very important in food chain system, the knowledge of their prevalence in food is very important. Therefore, this study aims to examine the prevalence of important Listeria species in various food types and evaluate serotype distribution, as well as the study of the virulence factors of L. monocytogenes.&#x0D; Methods: During July 2018 and January 2020, 900 food samples were collected in the North of Iran, including beef, chicken, fish, shrimp, milk and yogurt, green vegetables, mixed vegetable salad, Olivier salad, and cottage cheese. After isolation and identification steps, each bacterial DNA was extracted. Then, using specific primers, species, serotypes, and virulence genes of Listeria isolates were evaluated in the samples by Polymerase Chain Reaction (PCR) method.&#x0D; Results: The test results of 136 samples (15.1%) were positive for Listeria spp. and the most contaminated food was beef (35%) followed by chicken (29%) and green vegetables (23%). The most isolated Listeria spp. was L. monocytogenes and L. ivanovii. Among L. monocytogenes isolates, the dominant serogroups were 1/2a and 4b; furthermore, all of the isolates of this species harbored four virulence genes, including hlyA, plc, iap, and actA.&#x0D; Conclusion: These reports highlighted the importance of food safety in various food products, particularly raw meats and vegetables. Moreover, contamination of healthy foods such as fish and vegetables with Listeria is an indicator of public health.

The Listeria monocytogenes exopolysaccharide significantly enhances colonization and survival on fresh produce.

Fresh produce contaminated with Listeria monocytogenes has caused major listeriosis outbreaks in the last decades. Our knowledge about components of the listerial biofilms formed on fresh produce and their roles in causing foodborne illness remains incomplete. Here, we investigated, for the first time, the role of the listerial Pss exopolysaccharide (EPS) in plant surface colonization and stress tolerance. Pss is the main component of L. monocytogenes biofilms synthesized at elevated levels of the second messenger c-di-GMP. We developed a new biofilm model, whereby L. monocytogenes EGD-e and its derivatives are grown in the liquid minimal medium in the presence of pieces of wood or fresh produce. After 48-h incubation, the numbers of colony forming units of the Pss-synthesizing strain on pieces of wood, cantaloupe, celery and mixed salads were 2-12-fold higher, compared to the wild-type strain. Colonization of manmade materials, metals and plastics, was largely unaffected by the presence of Pss. The biofilms formed by the EPS-synthesizing strain on cantaloupe rind were 6-16-fold more tolerant of desiccation, which resembles conditions of whole cantaloupe storage and transportation. Further, listeria in the EPS-biofilms survived exposure to low pH, a condition encountered by bacteria on the contaminated produce during passage through the stomach, by 11-116-fold better than the wild-type strain. We surmise that L. monocytogenes strains synthesizing Pss EPS have an enormous, 102-104-fold, advantage over the non-synthesizing strains in colonizing fresh produce, surviving during storage and reaching small intestines of consumers where they may cause disease. The magnitude of the EPS effect calls for better understanding of factors inducing Pss synthesis and suggests that prevention of listerial EPS-biofilms may significantly enhance fresh produce safety.

Salad Days: Pairwise Gene Edits Food to Topple Nutrition Barriers

Salad Days: Pairwise Gene Edits Food to Topple Nutrition Barriers

Effectiveness of a Phage Cocktail as a Potential Biocontrol Agent against Saprophytic Bacteria in Ready-To-Eat Plant-Based Food.

This study aimed to evaluate the effectiveness of the phage cocktail to improve the microbiological quality of five different mixed-leaf salads: rucola, mixed-leaf salad with carrot, mixed-leaf salad with beetroot, washed and unwashed spinach, during storage in refrigerated conditions. Enterobacterales rods constituted a significant group of bacteria in the tested products. Selected bacteria were tested for antibiotic resistance profiles and then used to search for specific bacteriophages. Forty-three phages targeting bacteria dominant in mixed-leaf salads were isolated from sewage. Their titer was determined, and lytic activity was assessed using the Bioscreen C Pro automated growth analyzer. Two methods of phage cocktail application including spraying, and an absorption pad were effective for rucola, mixed leaf salad with carrot, and mixed leaf salad with beetroot. The maximum reduction level after 48 h of incubation reached 99.9% compared to the control sample. In washed and unwashed spinach, attempts to reduce the number of microorganisms did not bring the desired effect. The decrease in bacteria count in the lettuce mixes depended on the composition of the autochthonous saprophytic bacteria species. Both phage cocktail application methods effectively improved the microbiological quality of minimally processed products. Whole-spectral phage cocktail application may constitute an alternative food microbiological quality improvement method without affecting food properties.

Ready-to-eat foods as a possible source of Helicobacter pylori

Ready-to-eat (RTE) foods can be risky for consumers as they are not usually cooked or heated before consumption. We set out to assess RTE foods available in the Czech market network as a possible source of Helicobacter pylori. Attention was paid to RTE foods containing at least one ingredient of animal origin. A total of 50 food samples were chosen and divided into 3 categories. The “composite foods” category (n = 29) included sandwiches (n = 5), baguettes (n = 11), tortillas (n = 6), buns (n = 2), toast bread (n = 1), croissant (n = 1), bagels (n = 2), and pretzel (n = 1). The “salads” category included commercially produced mixed salads (n = 13). The “other” category (n = 8) included smoked salmon (n = 2), smoked mackerel (n = 2), smoked herring (n = 1), marinated salmon (n = 1), surimi (n = 1) and carpaccio (n = 1). The samples were analysed using nested-PCR. The glmM gene of H. pylori was detected in 50% (n = 25) of all samples. The “composite foods” category had the highest number of positive samples 58.6% (n = 21), followed by “other” with 50% (n = 4), and “salads” with 30.8% (n = 4). The results of our study showed that consumers eating RTE foods may be exposed to a risk of infection with H. pylori.

Microbiological Quality of Ready-to-Eat Salad Products Collected from Retail and Catering Settings in England during 2020 to 2021

Salad and other fresh produce were collected in England from retail and catering businesses during 2020 to 2021 and were tested for Salmonella, Shiga toxin-producing Escherichia coli (STEC), Listeria, Bacillus cereus, and E. coli. Of the 604 samples collected, 57% were from retail settings and 43% were from catering settings; 61% were either salad leaves or salad leaves mixed with other products. Equal numbers of samples were prepacked or loose, and 50% were refrigerated at the time of sampling. Combining results for all microbiological parameters, 84% were interpreted as satisfactory, 12% were interpreted as borderline, and 4% were interpreted as unsatisfactory. One sample (prepacked leaves, cucumber, and tomato from a caterer) was categorized as unacceptable and potentially injurious because of detection of STEC O76; no STEC from human infections in the United Kingdom matched this isolate. No Salmonella enterica was detected, but Listeria monocytogenes was recovered from 11 samples: 1 at 20 CFU/g and the remainder at <20 CFU/g. B. cereus was detected at borderline levels (103 to ≤105 CFU/g) in 9% of samples and at an unsatisfactory level (>105 CFU/g) in one sample. E. coli was detected in 3% of samples at borderline levels (20 to ≤102 CFU/g) and in 4% at unsatisfactory levels (>102 CFU/g). There was a significant association between detection of L. monocytogenes and borderline or unsatisfactory levels of E. coli. There were no specific risk profiles associated with products with the higher levels of B. cereus, STEC, or Listeria, but elevated levels of E. coli were predominantly confined to loose products from the United Kingdom collected from caterers in summer or autumn 2021 and may have resulted from relaxation of COVID-19 restrictions. Among the L. monocytogenes isolates, only one matched those from human cases and was recovered from a prepacked mixed salad from a catering business in 2021. This isolate was the same strain as that responsible for a multicountry outbreak (2015 to 2018) associated with Hungarian-produced frozen sweet corn; no link to the outbreak food chain was established.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers

Backgrounds: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. ResultsIn this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. ConclusionThis study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

Isolation, identification, and antibiogram studies of Escherichia coli from ready-to-eat foods in Mymensingh, Bangladesh.

Background and Aim:Ready-to-eat (RTE) foods are widely used at home, restaurants, and during festivals in Bangladesh. So it is very important to investigate possible microbial contamination in RTE foods. Therefore, this study aimed to determine the total coliform count (TCC), isolate, identify, and characterize the Escherichia coli in RTE foods. The antimicrobial sensitivity of E. coli obtained from RTE foods was also performed using 12 commonly used antibiotics.Materials and Methods:A total of 100 RTE food samples were collected aseptically and comprised of ten samples each: Burger, pizza, sandwich, chicken roll, chicken meat loaf, chicken fry, salad vegetable, ice-cream, yogurt, and milkshake sold in Mymensingh City. Samples were inoculated onto Eosin methylene blue agar and incubated at 37°C for 24 h. Isolation and identification of bacteria were performed based on cultural, staining, and biochemical properties, followed by a polymerase chain reaction.Results:The TCC in Chicken meat loaf, burger, pizza, sandwich, salad vegetable ice-cream, and yogurt samples were 3.57 ± 0.96, 3.69 ± 0.08, 3.50 ± 0.60, 2.60 ± 0.20, 4.09 ± 0.29, 4.44 ± 0.25, and 3.14 ± 0.30 mean log colony-forming units ± standard deviation/mL, respectively. The study found a higher prevalence of E. coli in RTE salad vegetable products than in RTE meat and milk products. Forty percent of the mixed vegetable salad samples showed positive results for E. coli. Whereas E. coli prevalence in RTE meat and milk products was 20% and 16.7%, respectively. All the 21 isolates were subjected to antibiotic susceptibility test against 12 different antibiotics. It was observed that 46.1% were susceptible, 16.6% were intermediate, 46.1% were resistant, and 47.6% were multidrug-resistant (MDR) among seven different antibiotic classes. E. coli isolates were resistant to cephalexin, ceftazidime, oxytetracycline, and ampicillin and sensitive to gentamycin, followed by kanamycin, ceftriaxone, colistin, and enrofloxacin.Conclusion:The study revealed that RTE foods are a serious issue from a public health point of view. To achieve a safer level of E. coli in RTE foods sold for human consumption, public food outlets must improve hygienic and good production procedures. Moreover, MDR E. coli in these foods pose serious public health threats.

Ready-to-eat salads and berry fruits purchased in Italy contaminated by Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica

Ready-to-eat (RTE) salads and berries are increasingly consumed in industrialized countries. These products can be contaminated by pathogenic parasites that have been responsible for foodborne outbreaks worldwide. In Italy, there are few data on contamination of RTE salads and berries with parasite transmission stages and this requires more-in-depth investigations. To estimate the prevalence of contamination with Cryptosporidium spp. and Giardia duodenalis in these fresh products, a total of 324 packages of local RTE mixed salads - belonging to three different industrial brands – and 324 packages of berries - blueberries from Peru, blackberries from Mexico, raspberries from Italy - were bought from supermarkets located in the Provinces of Bari and Foggia, Apulia, Italy. A pool size of nine packages was chosen and a total of 72 pools were processed in the whole year. After washing, the pellets were examined by microscopy (FLOTAC) and tested using conventional simplex PCR, targeting Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp., and sequencing. Several Cryptosporidium species and Giardia duodenalis assemblages, some of which are of potential zoonotic relevance, as well as Entamoeba spp., were identified in both matrices. By microscopy, Giardia-like cysts in local raspberries and Entamoeba-like cysts in imported blueberries were detected. Giardia duodenalis (Assemblages A, B and E) and Entamoeba histolytica were molecularly confirmed with overall prevalences of 4.6% (95% C.I. 3.0–6.8) and 1% (95% C.I. 0.3–2.1), respectively. Molecular methods identified Cryptosporidium ryanae, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum in both matrices, with a prevalence of 5.1% (95% C.I. 3.3–7.3). A distinct seasonality in prevalence was observed for G. duodenalis, with most positives occurring in spring, whereas Cryptosporidium showed no significant seasonal variations. These results highlight that inadequate management of fresh produce, both locally produced and imported, along the food chain may have the potential for consequences on human health.