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Escherichia albertii is an emerging enteropathogen. Although E. albertii -specific detection and isolation methods have been developed, their efficiency on food samples have not yet been systematically studied. To establish a series of effective methods for detecting E. albertii in food, an interlaboratory study was conducted in 11 laboratories using enrichment with modified E. coli broth supplemented with cefixime and tellurite (CT-mEC), real-time PCR assay, and plating on four kinds of selective agars. This study focused on the detection efficiency of an E. albertii -specific real-time PCR assay (EA-rtPCR) and plating on deoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), DHL supplemented with rhamnose and xylose (RX-DHL), and MAC supplemented with rhamnose and xylose (RX-MAC). Chicken and bean sprout samples were inoculated with E. albertii either at 17.7 CFU/25 g (low inoculation level) or 88.5 CFU/25 g (high inoculation level), and uninoculated samples were used as controls. The sensitivity of EA-rtPCR was 1.000 for chicken and bean sprout samples inoculated with E. albertii at low and high inoculation levels. The Ct values of bean sprout samples were higher than those of the chicken samples. Analysis of microbial distribution by 16S rRNA gene amplicon sequencing in enriched cultures of bean sprout samples showed that approximately >96 % of the population comprised unidentified genus of family Enterobacteriaceae and genus Acinetobacter in samples which E. albertii was not isolated. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a high inoculation level of E. albertii was 1.000 and 0.848–0.970, respectively. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a low inoculation level of E. albertii was 0.939–1.000 and 0.515–0.727, respectively. The E. albertii -positive rate in all colonies isolated in this study was 89–90 % in RX-DHL and RX-MAC, and 64 and 44 % in DHL and MAC, respectively. Therefore, the sensitivity of RX-supplemented agar was higher than that of the agars without these sugars. Using a combination of enrichment in CT-mEC and E. albertii isolation on selective agars supplemented with RX, E. albertii at an inoculation level of over 17.5 CFU/25 g of food was detected with a sensitivity of 1.000 and 0.667–0.727 in chicken and bean sprouts, respectively. Therefore, screening for E. albertii -specific genes using EA-rtPCR followed by isolation with RX-DHL or RX-MAC is an efficient method for E. albertii detection in food. • An interlaboratory study with E. albertii was conducted with 11 laboratories. • Sensitivities of >0.667 were detected in E. albertii (17.5 CFU)-inoculated foods. • Food matrix might influence the isolation of E. albertii. • Selective agars of RX-DHL and RX-MAC were superior than DHL and MAC. • EA-rtPCR-screening followed by isolation is efficient for E. albertii detection. [ABSTRACT FROM AUTHOR]

Sprouts and spent sprout irrigation water (SSIW) present unique challenges for the development of a Salmonella detection method in food matrices. This study aimed to compare universal preenrichment broth (UPB) and lactose broth (LB) as preenrichment media for cultural and rapid screening methods and to compare their abilities to recover Salmonella in SSIW samples from different sprout varieties (i.e., alfalfa, broccoli, and mung bean sprouts). The associated co-enriched microbiota with different sprout varieties using different preenrichment media were also examined using a quasi-metagenomic approach. The performance of media and detection methods was compared using the relative level of detection (RLOD) value, as recommended by ISO 16140-2:2016. The level of detection (LOD) for Salmonella culture method with UPB was similar to that with LB in low aerobic plate count (APC) background samples (the relative LOD, i.e., RLOD, was nearly 1 after adjusting for the effects of SSIW variety and serovar), but significantly lower than that with LB in high APC background samples (RLOD = 0.32). The LOD for Salmonella with selected rapid methods was comparable to each other (RLOD from 0.97 to 1.50) and to the culture method (RLOD from 0.69 to 1.03), and no significant difference was detected between preenrichment broths in low APC background samples with RLOD values between 0.76 and 1.04. In samples with a high APC background, however, a drastic difference in LOD was observed between methods and between preenrichment broths for each method. The RLOD ranged from 0.03 to 0.32 when UPB was compared to LB as preenrichment broth. The composition and relative abundance (RA) of co-enriched microbiota was affected by multiple factors including food matrices, preenrichment media and Salmonella contamination. Altogether, this study validated UPB as a better preenrichment broth than LB for the detection of Salmonella enterica from SSIW. This study also suggested UPB may also be an optimal preenrichment medium for rapid screening methods when APC level is high. The observation of potential exclusion of Salmonella in preenrichment through the overgrowth of competitive microflora from the quasi-metagenomic study provided novel information that may be used to further optimize preenrichment formulations. • Universal preenrichment broth (UPB) is a significantly better preenrichment medium than lactose broth (LB) for Salmonella detection from sprout spent irrigation water (SSIW). • When a high aerobic plate count was present in SSIW, UPB performed better with downstream rapid screening methods than LB. • APC level significantly affected Salmonella relative level of detection between methods and between preenrichment broths. [ABSTRACT FROM AUTHOR]

Abstract Seeds are usual source of contamination and their sprouts are commonly associated foodborne illness. Therefore, the aim of this study was to evaluate the antibacterial vapor phase efficiency of allspice, thyme and rosemary essential oils on two foodborne pathogens in in vitro and on alfalfa seeds, including the chemical profile of the tested EOs and their effect on the sensory characteristics of the sprouts. Antibacterial activity was determined through the minimal inhibitory concentration (MIC) of EOs in vapor phase to inhibit the growth of Listeria monocytogenes and Salmonella Typhimurium in culture media and on alfalfa seeds. Also, the germination and the effect on sensory characteristics of the sprouts were determined. Thyme EO was the most effective of the tested EOs on culture media and on alfalfa seeds, against both bacteria. When rosemary EO was tested against L. monocytogenes in alfalfa seeds, the MIC (4.0 mL/L air) was higher, compared to the one obtained in culture media (2.7 mL/L air). But when this EO was tested against S. Typhimurium, the MIC in alfalfa seeds was lower than in culture media (11.7 vs 13.3 mL/L air). Allspice EO resulted more effective against both bacteria in alfalfa seeds (6.0 mL/L air for L. monocytogenes and 6.7 mL/L air for S. Typhimurium), compared to culture media (12.0 mL/L air for L. monocytogenes and 13.3 mL/L air for S. Typhimurium). Vapor phase EOs MICs resulted in significant (p ≤ 0.05) decreases of L. monocytogenes and S. Typhimurium counts compared to the control. There also was a significant (p ≤ 0.05) difference between systems (in vitro or on alfalfa seeds) despite the microorganism or the evaluated EO. Treatment alfalfa seed with vapor phase EOs, did not affect the seed germination. Sensory acceptability of the sprouts, obtained of treated seeds, did not were significant (p ≥ 0.05) different of the sprouts obtained from the non-treated seeds. Highlights • Antibacterial activity of essential oils (EOs) applied in vapor phase was assessed. • Allspice, thyme, and rosemary EOs in vapor phase exhibited antibacterial activities. • Studied EOs inhibited Listeria and Salmonella in culture media and alfalfa seeds. • Treatment of alfalfa seeds with EOs did not affect sensory acceptability of sprouts. • Studied vapor phase EOs did not affect germination of alfalfa seeds. [ABSTRACT FROM AUTHOR]

Abstract In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting agg R/ aai C, stx / eae , and wzx O104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75–80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the agg R and aai C genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts. Graphical abstract Unlabelled Image Highlights • Background bacterial population of sprouts grows during the storage phase at 4 °C. • The PCR assay was specific for detection of the O104 strains. • The detection limit for the PCR assays was 1 CFU/25 g in sprouts. • Microbiological and PCR-methods were comparable in terms of performance. [ABSTRACT FROM AUTHOR]

Abstract The food safety implications of microgreens, an emerging salad crop, have been studied only minimally. The farm to fork continuum of microgreens and sprouts has some overlap in terms of production, physical characteristics, and consumption. This review describes the food safety risk of microgreens as compared to sprouts, potential control points for microgreen production, what is known to date about pathogen transfer in the microgreen production environment, and where microgreens differ from sprouts and their mature vegetable counterparts. The synthesis of published research to date may help to inform Good Agricultural Practices (GAPs) and Good Handling Practices (GHPs) for the emerging microgreen industry. Highlights • Microgreen production shares traits with both sprout and vegetable production. • Only eight studies since 2009 addressed microgreen food safety. • Contamination sources include growth media, irrigation water, and seeds. • Hydroponically grown microgreens are more susceptible to contamination. • Microgreen variety and contaminant type likely influence food safety risk. [ABSTRACT FROM AUTHOR]

Abstract Several outbreaks of illness have been associated with consumption of alfalfa sprouts contaminated with Shiga toxin-producing Escherichia coli (STEC) and Salmonella. The ozone application was investigated as an intervention. Alfalfa seeds were inoculated with cocktails of 3 Salmonella strains, including serotypes Typhimurium, Agona and Saintpaul, and 3 strains of Shiga toxin-producing Escherichia coli (STEC) including serotypes O104:H4, O157:H7 and O121:H19 with a final load of 7.0 log CFU/ml. Then, the inoculated seeds, and the sprouts obtained from these seeds were separately subjected to aqueous ozone treatment containing (5 mg/L) ozone for varied times of exposure. The mean log reductions for Salmonella achieved on seeds after 10, 15, and 20 min of ozone exposure were 1.6 ± 0.5, 1.7 ± 0.3, 2.1 ± 0.5, respectively and 1.5 ± 0.4, 1.6 ± 0.4, 2.1 ± 0.5 for STEC, respectively. For sprouts obtained from the inoculated seed, the mean log reductions for Salmonella after 10, 15, and 20 min exposure times were 0.7 ± 0.2, 1.1 ± 0.4, 3.6 ± 0.2, respectively, whereas the mean log reductions for STEC were 0.7 ± 0.1, 1.2 ± 0.3 and 1.8 ± 0.2, respectively. At each contact time, there were no differences in log reductions between pathogens on seeds (P > 0.05), whereas on sprouts, the reductions obtained at 20 min were significantly greater (P < 0.05) for Salmonella than for STEC. On both seeds and sprouts, the exposure time had significant (P < 0.05) effects on log reductions of Salmonella and STEC. The weight, color properties and shelf life of ozonated sprouts were also tested. The ozonation did not have negative effects on germination (%), color and mass of sprouts in comparison with the controls. This study confirmed that it is possible to substantially reduce Salmonella and STEC by using a low ozone concentration (5 mg/L) and reduce food safety risk with less concern about the safety for processing workers of this treatment, this without affecting seed germination. This procedure may be a promising intervention to reduce Salmonella and STEC from alfalfa seeds and sprouts. Highlights • Effectiveness of ozonated water treatments was affected by the time of contact. • Salmonella and STEC were reduced by 2.1 log cycles on alfalfa seed by ozone treatment. • Salmonella and STEC reduced by 3.6 and 1.8 log cycles on alfalfa sprouts by ozone treatment • Ozone treatment of sprouts did not affect sprout yield or color. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the antimicrobial activities of gaseous essential oils (EO gases) against Listeria monocytogenes on the surfaces of a laboratory medium and radish sprouts. We determined the minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) values of EO gases from eight EOs extracted from basil leaves, carrot seed, cinnamon bark, cinnamon leaves, clove flower buds, oregano leaves, thyme flowers (linalool), and thyme leaves (thymol) against L . monocytogenes on a nutrient agar supplemented with 1% glucose and 0.025% bromocresol purple (NGBA). Oregano, thyme thymol, and cinnamon bark EO gases showed the strongest antilisterial activities (MIC and MLC, 78.1 μL/L). We also investigated the inhibitory and lethal activities of these gases against L . monocytogenes on the surface of radish sprouts. The number of L . monocytogenes after exposure to EO gases at ≥ 156 μL/L was significantly ( P ≤ 0.05) lower than that of untreated L . monocytogenes . For example, the initial number of L . monocytogenes on the surface of radish sprouts (ca. 6.3 log CFU/g) decreased by 1.4 log CFU/g within 24 h at 30 °C and 43% relative humidity (RH) without EO gas treatment, whereas the number of L . monocytogenes after exposure to oregano, thyme thymol, and cinnamon bark EO gases at 156 μL/L decreased by 2.1, 2.1, and 1.8 log CFU/g, respectively, after 24 h. Although EO gases exerted greater lethal activities at higher concentrations (312 and 625 μL/L), L . monocytogenes on the surface of radish sprouts was not completely inactivated. The number of L . monocytogenes on sprouts treated with oregano, thyme thymol, and cinnamon bark EO gases at 625 μL/L decreased by 2.7–3.0 log CFU/g after 24 h at 30 °C and 43% RH. Results indicate that EO gases that showed antilisterial activities on a laboratory medium also exhibited reduced lethal activity on the surface of radish sprouts. These findings will be useful when developing strategies to inactivate L . monocytogenes and possibly other foodborne pathogens on sprouts and perhaps other foods using EO gases. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica , Y. pseudotuberculosis and Y. pestis ) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica , 22 Y. pestis , 24 Y. pseudotuberculosis and a diverse selection of 17 other non- Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40 bp in length and had 94–100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis . Both salads (n = 2) and 35% of sprout products (n = 46) including 7.1% of alfalfa sprouts (n = 14), 81% of bean sprouts (n = 16), 12% of mixed sprouts (n = 8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n = 5), onion sprouts (n = 1), and pea sprouts (n = 2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1 × 10 2 to 1 × 10 6 Y. enterocolitica per 1 mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples. [ABSTRACT FROM AUTHOR]

This study investigated the effects of cold plasma treatment (CPT) on the inhibition of Salmonella typhimurium on radish sprouts and the quality attributes of the sprouts. Radish sprouts were treated with nitrogen (N 2 )-cold plasma at 900 W and 667 Pa for 0, 2, 5, 10, and 20 min using a microwave-powered CPT system. The sensory attributes of the radish sprouts, appearance and odor, were evaluated before and after the treatment. The effects of N 2 -CPT for 10 min on microbial growth and the quality attributes of the radish sprouts were evaluated during storage for 12 days at 4 and 10 °C. N 2 -CPT at 900 W and 667 Pa for 20 min reduced the number of S . typhimurium by 2.6 ± 0.4 log CFU/g. The moisture content of the radish sprouts decreased with treatment time. The appearance and odor of the radish sprouts were not altered by CPT ( p > 0.05) and this treatment did not affect the quality attributes of the sprouts in terms of color, ascorbic acid concentration, or antioxidant activity during storage at both 4 and 10 °C. These findings suggest that CPT has the potential to improve the microbiological safety of radish sprouts with reference to S . typhimurium during cold storage without significant detriment to its quality properties. [ABSTRACT FROM AUTHOR]

Microgreens are niche salad greens which have increased in popularity among consumers in recent years. Due to similarities with sprouts and leafy greens—both attributed to numerous foodborne disease outbreaks—characterization of the food safety risks associated with microgreen production is warranted. The present study aimed to determine the fate and persistence of a human norovirus (HuNoV) surrogate, Tulane virus (TV), within a microgreen production system. Initially, the persistence of TV in two types of microgreen soil-free cultivation matrix (SFCM)—BioStrate® (biostrate) and peat—was determined. On day 0, water containing 7.6 log PFU of TV was applied to SFCM in growing trays, and the trays were maintained under microgreen growth conditions. TV persisted throughout the 10-day observation in biostrate and peat with overall reductions of 3.04 and 1.76 log plaque forming units (PFU) per tray, respectively. Subsequently, the transfer of TV to microgreen edible tissue was determined when planted on contaminated SFCM. Trays containing each type of SFCM were pre-inoculated with 7.6 log PFU of TV and equally divided into two areas. On day 0, sunflower (SF) or pea shoot (PS) seeds were planted on one-half of each tray, while the other half was left unplanted to serve as a control. The microgreens were harvested on day 10, and SFCM samples were collected from planted and unplanted areas of each tray. No TV were detected from the edible portion of either type of microgreen, yet TV were still present in the SFCM. TV concentrations were significantly lower in the root-containing planted area compared with the unplanted area for both biostrate (P = 0.0282) and peat (P = 0.0054). The mean differences of TV concentrations between unplanted and planted areas were 1.22 and 0.51 log PFU/g for biostrate and peat, respectively. In a subsequent investigation, TV transfer from day 7 inoculated SFCM to microgreens edible portion was not detected either. Overall, this study characterized the viral risk in a microgreen production system, which will help to understand the potential food safety risk related to HuNoV and to develop preventive measures. • Tulane virus persists in soilless substrates for at least 10 days. • TV Internalization within edible tissue of sunflower and pea shoot was not observed. • TV concentrations in soilless substrates were impacted by microgreen rhizosphere. [ABSTRACT FROM AUTHOR]

Fresh produce is frequently contaminated by microorganisms, which may lead to spoilage or even pose a threat to human health. In particular sprouts are considered to be among the most risky foods sold at retail since they are grown in an environment practically ideal for growth of bacteria and usually consumed raw. Because heat treatment has a detrimental effect on the germination abilities of sprout seeds, alternative treatment technologies need to be developed for microbial inactivation purposes. In this study, non-thermal plasma decontamination of sprout seeds is evaluated as a promising option to enhance food safety while maintaining the seed germination capabilities. In detail, investigations focus on understanding the efficiency of non-thermal plasma inactivation of microorganisms as influenced by the type of microbial contamination, substrate surface properties and moisture content, as well as variations in the power input to the plasma device. To evaluate the impact of these parameters, we studied the reduction of native microbiota or artificially applied E. coli on alfalfa, onion, radish and cress seeds exposed to non-thermal plasma in an atmospheric pressure pulsed dielectric barrier discharge streamed with argon. Plasma treatment resulted in a maximum reduction of 3.4 logarithmic units for E. coli on cress seeds. A major challenge in plasma decontamination of granular food products turned out to be the complex surface topology, where the rough surface with cracks and crevices can shield microorganisms from plasma-generated reactive species, thus reducing the treatment efficiency. However, improvement of the inactivation efficiency was possible by optimizing substrate characteristics such as the moisture level and by tuning the power supply settings (voltage, frequency) to increase the production of reactive species. While the germination ability of alfalfa seeds was considerably decreased by harsh plasma treatment, enhanced germination was observed under mild conditions. In conclusion, the results from this study indicate that cold plasma treatment represents a promising technology for inactivation of bacteria on seeds used for sprout production while preserving their germination properties. [ABSTRACT FROM AUTHOR]

Fresh mung bean sprouts have been identified as a source of many Salmonella outbreaks worldwide. The aim of this study was to develop a rapid and accurate detection methodology for low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts using real-time PCR coupled with either immunomagnetic separation (PCR-IMS) or centrifugation (PCR-cen). Initially, three parameters of IMS; specificity/sensitivity, bacterial concentration and bead incubation time were optimized. Secondly, limit of detection (LOD) was determined for the optimized PCR-IMS and PCR-cen. These two methods were compared against PCR alone (PCR) and the standard culture method (ISO) for their ability to detect Salmonella using inoculated and uninoculated sprouts. Under optimum IMS conditions (10 5 CFU/ml for 30 min), capture efficiency of Salmonella in sprout suspensions was lower than 40%, most probably due to the non-specific binding of the background microbiota. PCR-IMS and PCR-cen had a similar LOD at 10 3 CFU/ml, which was one log unit lower than PCR. Enrichment of 10 h was sufficient to detect 100% of the inoculated sprouts with both PCR-IMS and PCR-cen, which was significantly faster compared to PCR and the ISO method. Moreover, the validation study using uninoculated sprouts revealed that PCR-IMS and PCR-cen were equally effective on Salmonella detection, showing 98.3% accuracy. These results suggest that PCR-cen would be the effective and less costly method for the detection of both healthy and sanitizer-injured Salmonella on mung bean sprouts. [ABSTRACT FROM AUTHOR]

Escherichia coli O104:H4 strain 11-3088 encoding Stx2a is epidemiologically related to the foodborne outbreak associated with sprouts in Germany, 2011. Sprouting provides suitable conditions for bacterial growth and may lead to transduction of non-pathogenic strains of E. coli with Stx phages. Although transduction of E. coli by Stx phages in food has been documented, data on the phages from E. coli O104:H4 is limited. This study determined the host range of the bacteriophage Φ11-3088 from E. coli O104:H4 using E. coli O104:H4 ∆stx2::gfp::amp r and demonstrated phage transduction during sprouting. The Φ11-3088 ∆stx transduced 5/45 strains, including generic E. coli , pap -positive E. coli O103:H2, ETEC, and S. sonnei. The expression level of Φ11-3088 ∆stx differed among lysogens upon induction. Of the 3 highly induced lysogens, the lytic cycle was induced in E. coli O104:H4 ∆stx2::gfp::amp r and O103:H2 but not in S. sonnei. E. coli DH5α was the only strain susceptible to lytic infection by Φ11-3088 ∆stx. To explore the effect of drying and rehydration during seed storage and sprouting on phage induction and transduction, mung beans inoculated with the phage donor E. coli O104:H4 ∆stx2::gfp::amp r (8 log CFU/g) were dried, rehydrated, and incubated with the phage recipient E. coli DH5α (7 log CFU/g) for 96 h. Sprouted seeds harbored about 3 log CFU/g of putative lysogens that acquired ampicillin resistance. At the end of sprouting, 71 % of putative lysogens encoded gfp , confirming phage transduction. Overall, stx transfer by phages may increase the cell counts of STEC during sprouting by converting generic E. coli to STEC. • The stx2 -encoding Φ11-3088 has a narrow host range (5/45) for lysogenic infection. • The phage Φ11-3088 transmitted stx2 to ETEC and Shigella by transduction. • The induction of the Stx2 prophage differed in different lysogens. • STEC on dry seeds mediated the spread of stx2 during sprouting. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the antioxidant and antibacterial activities (AOA and ABA) of broccoli sprout extract (BSE) nanoliposomes co-encapsulated into basil seeds gum (BSG). The characteristics of the BSE-loaded nanoliposomes and nano-capsules were firstly determined. Their functional (antioxidant and antibacterial) properties were tested in vitro , and their anti- Listeria effect (at 0.4 and 0.8% w/w) on ricotta cheese was evaluated. The produced nanoliposomes and nano-capsules were spherical in shape and did not tend to accumulate. The mean particle size, polydispersity index (PDI) and encapsulation efficiency (EE) were observed about 39.60 and 69.00 nm, 0.279 and 0.496, 85.73 and 88.46% for nano-capsules and nanoliposomes, respectively. The zeta potential (ζ) values were observed at −65.73 and −71.16 mV and therefore the nanoparticles had good stability and uniform particle size distribution. Encapsulation had no significant effect on total phenol and flavonoids content of the BSE. The amounts of these active compounds were in the range of 25.12–26.97 mg GAE/g dw and 6.84–6.95 mg QE/g dw, respectively. The free and encapsulated BSE displaying good AOA in the DPPH, ABTS and FRAP assays. Results of the ABA measured by inhibition zone diameter and the minimum inhibitory concentration (MIC) demonstrated that the free BSE had antibacterial action against the tested Gram-positive and Gram-negative bacteria, and the nano-encapsulation process led to improved ABA of this extract. The organic acids in BES indicated the presence of high levels of citric, malic and oxalic acids at 613, 98 and, 45 (mg/g dw), respectively. The BSE-loaded nanoparticles showed remarkable anti- Listeria activity in ricotta cheese, which their activity increased with increasing their concentration. In conclusion, BSE-loaded nanoliposomes and nano-capsules have potential interest to be used as natural antioxidants and preservatives for food applications. [Display omitted] • Co-encapsulation of nano-liposomes BSE into basil seed gum BSG was successfully achieved. • The co-encapsulated nano-liposomes had good stability and size distribution. • The liposomes were able to maintain the functional properties of the core. • Co-encapsulation of BSE as nano-liposomes showed anti- Listeria properties in Ricotta cheese. [ABSTRACT FROM AUTHOR]

Outbreaks of foodborne illness from consumption of sprouts have been linked to contaminated seeds prior to germination. Due to the long sprouting period at ambient temperatures and high humidity, germinating seeds contaminated with low pathogen levels (0.1 log CFU/g) can result in sprouts with high numbers (≥ 108 CFU/g) of pathogens. Currently, the recommended treatment method involves soaking seeds in 20,000 ppm (2%) calcium hypochlorite prior to germination. In this study, an alternative treatment involving soaking seeds in a carvacrol nanoemulsion was tested for its efficacy against Salmonella enterica subspecies enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing E. coli O157:H7 (ATCC 42895) contaminated mung bean and alfalfa seeds. The antimicrobial treatment was performed by soaking inoculated seed batches in the spontaneous nanoemulsion (4000 or 8000 ppm) for 30 or 60 min. The spontaneous nanoemulsion was formed by titrating the oil phase (carvacrol and medium chain triglycerides) and water-soluble surfactant (Tween 80®) into sodium citrate buffer. Following treatment, the numbers of surviving cells were determined by suspending the seeds in TSB and performing plate counts and/or Most Probable Number (MPN) enumeration. Treated seeds were sprouted and tested for the presence of the appropriate pathogen. This treatment successfully inactivated low levels (2 and 3 log CFU/g) of S. Enteritidis and E. coli on either seed types when soaked for either 30 or 60 min at nanoemulsion concentrations corresponding to 4000 (0.4%) or 8000 (0.8%) ppm carvacrol. Inoculated alfalfa seeds treated with 4000 ppm nanoemulsion, required a 60 min treatment time to show a similar 2-3 log reduction. Complete inactivation was confirmed by germinating treated seeds and performing microbiological testing. Total sprout yield was not compromised by any of the tested treatments. These results show that carvacrol nanoemulsions may be an alternative antimicrobial treatment method for mung bean and alfalfa seeds. [ABSTRACT FROM AUTHOR]

Abstract: Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O104 have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. An outbreak that occurred in Europe in 2011 linked to fenugreek sprouts was caused by E. coli O104:H4 that had characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that encoded for Shiga toxin 2. In this study, methods were developed for detection of this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR assays for enteroaggregative STEC O104:H4 targeted the stx 2, aggR, and wzx 104 genes, and for STEC O104 targeted the stx 1-2, ehxA, and wzx 104 genes. After incubating artificially contaminated sprouts at 4°C for 48h and overnight enrichment in modified buffered peptone water with pyruvate supplemented with three antibiotics (mBPWp), the pathogens were detected in all samples inoculated at a level of ca. 100CFU/25g. Several samples inoculated at lower concentrations of ca. 10CFU/25g were negative by the PCR assays, and this could have been due to cells not surviving or not being able to recover after the stress treatment at 4°C for 48h. For isolation of the pathogens, immunomagnetic separation (IMS) using magnetic beads coated with antibodies against O104 were employed, and this was followed by plating the beads onto mRBA and CHROMagar STEC O104 for isolation of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7. Presumptive colonies were confirmed by agglutination using latex particles attached to antibodies against serogroup O104 and by the multiplex PCR assays. The methodologies described in this study for detection of enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104, and they enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well. [Copyright &y& Elsevier]

The following reports on the generation of hydroxyl-radical activated water prepared by passing a hydrogen peroxide solution containing Fe(III) catalyst through a UV-C reactor. The activated water was subsequently evaluated for antimicrobial activity against Escherichia coli O157:H7 in suspension or when inoculated onto mung beans. Hydroxyl-radical generation was assessed through the oxidation of methylene blue when reacted with activated water prepared from solutions of different pH (4–10), UV-C dose (32–128 mJ/cm2), hydrogen peroxide (0–1000 mg/L) and Fe(III) concentration (0–100 mg/L). Methylene blue oxidation was associated with high concentrations of each reactant with a positive correlation with Fe(III) concentration. Inactivation curves of E. coli O157:H7 in activated water were diphasic with an initial slow rate that increased after 15 min contact time. In contrast to the methylene blue assay, the antimicrobial action of activated water was associated with high hydrogen peroxide (500 mg/mL) and low Fe(III) catalyst (1 mg/L) with no significant interaction with UV-C dose. Evidence would suggest that the mode-of-inactivation was through a radical propagation reaction that is rate-limited by the reduction of Fe (III) to Fe (II). Here, the initial activation process via UV-C illumination results in photo-reduction of Fe(III) and propagates the formation of hydroxyl-radicals. Fe(III) to Fe(II) cycling continues with oxidation of cell structures that ultimately leads to loss of viability due to accumulation of cellular damage. When activated water was used to soak mung beans inoculated with E. coli O157:H7 a 1 log reduction was obtained with a 19% increase in germinated beans and 8.5% higher sprout yield relative to controls. The oxidation reduction potential decreased from 477 mV to 288 mV and pH increased from 3.97 to 5.47, over the 24 h mung bean soak period. The reduction of Salmonella and Listeria monocytogenes on mung beans soaked in activated water was <1 log CFU/g with all three pathogens growing back over the sprouting period. From the results it can be concluded that activated water can enhance the germination of mung beans along with sprout yield but has limited capacity when applied alone as a seed disinfection method. • UV-C treatment of a hydrogen peroxide solution containing Fe(III) generated activated water. • Methylene blue assay confirmed the generation of hydroxyl-radicals in activated water. • Kill curves for E. coli O157:H7 in activated water exhibited diphasic inactivation. • Mode-of-inactivation appeared to be through a chain-reaction initiated by Fe(III). • Activated water increased bean germination and sprout yield with pathogen reduction. [ABSTRACT FROM AUTHOR]

Alfalfa sprouts have high nutritional values when consumed raw, but are easily contaminated by food pathogens. This study aimed to evaluate effects of essential oil (EO) vapors, RF heating and their combinations on microbial inactivation, germination rate and generated sprouts' quality of alfalfa seeds at moisture contents of 7.52%, 9.53% and 11.45% wet basis (w.b.). Results showed that cinnamon oil and oregano oil vapors both had antimicrobial effects against Salmonella on alfalfa seeds and cinnamon oil vapor had a little better activity. Weibull model well fitted the Salmonella inactivation curves under RF heating. The rate of Salmonella inactivation increased but germination rate decreased with increasing temperature and moisture content of seeds. The intermittent RF treatments improved the heating uniformity and germination rate of the whole batch of seeds as compared to the continuous RF heating. The combination of intermittent RF heating and cinnamon oil vapor exhibited additive antimicrobial effects, up to 3.99–4.12 log CFU/g Salmonella reductions, and maintained the germination rate above 90%. However, for natural microbial decontamination, combined treatments only caused 0.56–0.82 log CFU/g reductions of total bacterial counts. The fresh weight, length, flavor, the content of phenolics and ascorbic acid, and antioxidant capacity of generated sprouts were not significantly impacted. The study provided an effective method for microbial control on sprouting seeds. • Salmonella inactivation rate on alfalfa seeds increased with increasing temperature and moisture under RF heating • Germination rate decreased as increasing temperature and moisture • Intermittent RF heating improved seed germination rate compared to continuous heating • RF combined with cinnamon oil effectively inactivated Salmonella on seeds and guaranteed seeds' quality [ABSTRACT FROM AUTHOR]

The behavior of enteroaggregative Escherichia coli (EAEC), non-O157 shiga toxin-producing E. coli (non-O157-STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) on mung bean seeds at 25±2°C and during germination and sprouting of mung bean seeds at 20±2° and 30±2°C and on mung bean sprouts at 3±2°C was determined. When mung bean seeds were inoculated with EAEC, non-O157 STEC, EIEC, EPEC or ETEC strains, all these diarrheagenic E. coli pathotypes (DEPs) survived at least 90days on mung bean seeds at 25±2°C. All DEPs grew during germination and sprouting of seeds, reaching counts of approximately 5 Log and 7 Log CFU/g after 2days at 20±2° and 30±2°C, respectively. However, when the sprouts were inoculated after 1day of seeds germination and stored at 20±2° or 30±2°C, no growth was observed for any DEPs during sprouting at 20±2°C per 9 d; however, a significant increase in the concentration of DEPs of approximately 0.7 log CFU/g was observed during sprouting at 30±2°C after 1day of sprout contamination. Refrigeration reduced the number of viable DEPs strains on sprouts after 10days in storage; nevertheless, these decreases have no practical significance in the safety of the sprouts. [ABSTRACT FROM AUTHOR]

Abstract: The use of propidium monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing concentrations were permeable to PMA which was confirmed by LIVE/DEAD-FCM. However, the PMA-qPCR assay allows specific quantification among the autochthonous microbiota of food products. Therefore, the PMA-qPCR assay was used to evaluate its applicability in artificially contaminated iceberg lettuce and soya sprouts. Amplification signal was negative for the spiking tests performed with any of the EO-killed E. coli cells. It demonstrates that the PMA-qPCR assay is a suitable technique for monitoring E. coli O157:H7 inactivation by essential oils in fresh-cut vegetables. [Copyright &y& Elsevier]

Abstract: Salmonella enterica subsp. enterica serovar Weltevreden is a dominant serovar associated with foodborne gastroenteritis in South-East Asia and emerging in Europe associated with fresh vegetables. Here we compared the genome of strain 2007-60-3289-1 linked to an alfalfa sprout outbreak in Scandinavia with a S. Weltevreden strain isolated from scallops in the USA and with other S. enterica serovars. A unique plasmid pSW82 was identified for S. Weltevreden carrying a two-component type II non-ribosomal peptide synthase/polyketide synthase. Analysis of all available complete S. enterica genomes identified differences for presence of type VI secretion systems and carbohydrate metabolic pathways. Differential transcription thereof was observed when S. Weltevreden strains were grown in vitro or on sprouts. [Copyright &y& Elsevier]

Abstract: Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37°C for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix. [Copyright &y& Elsevier]

Abstract: Two Salmonella bacteriophages (SSP5 and SSP6) were isolated and characterized based on their morphology and host range, and evaluated for their potential to control Salmonella Oranienburg in vitro and on experimentally contaminated alfalfa seeds. Phages SSP5 and SSP6 were classified as members of the Myoviridae and Siphoviridae families, respectively. Both phages had a broad host range of over 65% of the 41 Salmonella strains tested. During in vitro trials, the phages resulted in incomplete lysis of Salmonella cultures, in spite of high levels of phage remaining in the system. Phage SSP5 was more effective in reducing Salmonella populations. Addition of phage SSP6 to alfalfa seeds previously contaminated with S. Oranienburg caused an approximately 1 log10 CFU g−1 reduction of viable Salmonella, which was achieved 3 h after phage application. Thereafter the phage had no inhibitory effect on Salmonella population growth. A second addition of the same (SSP6) or different (SSP5) phage to a Salmonella culture treated with phage SSP6, did not affect Salmonella populations. It was further shown that development of Salmonella permanently resistant to phage was not evident in either seed or in vitro challenge trials, suggesting the existence of a temporary, acquired, non-specific phage resistance phenomenon. These factors may complicate the use of phages for biocontrol. [Copyright &y& Elsevier]

Abstract: Sprouts eaten raw are increasingly being perceived as hazardous foods as they have been implicated in Escherichia coli O157:H7 outbreaks where the seeds were found to be the likely source of contamination. The objective of our study was to evaluate the potential of using high hydrostatic pressure (HHP) technology for alfalfa seed decontamination. Alfalfa seeds inoculated with a cocktail of five strains of E. coli O157:H7 were subjected to pressures of 500 and 600 MPa for 2 min at 20 °C in a dry or wet (immersed in water) state. Immersing seeds in water during pressurization considerably enhanced inactivation of E. coli O157:H7 achieving reductions of 3.5 log and 5.7 log at 500 and 600 MPa, respectively. When dry seeds were pressurized, both pressure levels reduced the counts by <0.7 log. To test the efficacy of HHP to completely decontaminate seeds whilst meeting the FDA requirement of 5 log reductions, seeds inoculated with a ~5 log CFU/g of E. coli O157:H7 were pressure-treated at 600 and 650 MPa at 20 °C for holding times of 2 to 20 min. A >5 log reduction in the population was achieved when 600 MPa was applied for durations of ≥6 min although survivors were still detected by enrichment. When the pressure was stepped up to 650 MPa, the threshold time required to achieve complete elimination was 15 min. Un-inoculated seeds pressure-treated at 650 MPa for 15 min at 20 °C successfully sprouted achieving a germination rate identical to untreated seeds after eight days of sprouting. These results therefore demonstrate the promising application of HHP on alfalfa seeds to eliminate the risk of E. coli O157:H7 infections associated with consumption of raw alfalfa sprouts. [Copyright &y& Elsevier]

Abstract: A survey of fresh and minimally-processed fruit and vegetables, and sprouts was conducted in several retail establishments in the Lleida area (Catalonia, Spain) during 2005–2006 to determine whether microbial contamination, and in particular potentially pathogenic bacteria, was present under these commodities. A total of 300 samples—including 21 ready-to-eat fruits, 28 whole fresh vegetables, 15 sprout samples and 237 ready-to-eat salads containing from one to six vegetables—were purchased from 4 supermarkets. They were tested for mesophilic and psychrotrophic aerobic counts, yeasts and moulds, lactic acid bacteria, Enterobacteriaceae, presumptive E. coli and Listeria monocytogenes counts as well as for the presence of Salmonella, E. coli O157:H7, Yersinia enterocolitica and thermotolerant Campylobacter. Results for the fresh-cut vegetables that we analyzed showed that, in general, the highest microorganism counts were associated with grated carrot, arugula and spinach (7.8, 7.5 and 7.4 log cfu g−1 of aerobic mesophilic microorganisms; 6.1, 5.8 and 5.2 log cfu g−1 of yeast and moulds; 5.9, 4.0 and 5.1 log cfu g−1 lactic acid bacteria and 6.2, 5.3 and 6.0 log cfu g−1 of Enterobacteriaceae). The lowest counts were generally associated with fresh-cut endive and lettuce (6.2 and 6.3 log cfu g−1 of aerobic mesophilic microorganisms; 4.4 and 4.6 log cfu g−1 of yeast and moulds; 2.7 and 3.8 log cfu g−1 lactic acid bacteria and 4.8 and 4.4 log cfu g−1 of Enterobacteriaceae). Counts of psychrotrophic microorganisms were as high as those of mesophilic microorganisms. Microbiological counts for fresh-cut fruit were very low. Sprouts were highly contaminated with mesophilic (7.9 log cfu g−1), psychrotrophic microorganisms (7.3 log cfu g−1) and Enterobacteriaceae (7.2 log cfu g−1) and showed a high incidence of E. coli (40% of samples). Of the samples analyzed, four (1.3%) were Salmonella positive and two (0.7%) harboured L. monocytogenes. None of the samples was positive for E. coli O157:H7, pathogenic Y. enterocolitica or thermotolerant Campylobacter. [Copyright &y& Elsevier]

Abstract: A limited survey of fresh and minimally processed vegetables, and sprouts was conducted in the Washington, DC area to determine if potentially toxigenic and pathogenic fungi were present in these commodities. Thirty-nine ready-to-eat salads, 29 whole fresh vegetables and 116 sprout samples (bean, alfalfa, broccoli, crunchy, garlic, spicy, onion, clover, lentil and multi-seed sprouts) were purchased from 13 local supermarkets and tested for yeast and mould counts as well as the presence of toxigenic moulds. Yeasts were the most prevalent organisms found in these samples, at levels ranging from less than 100 to 4.0×108 cfu/g. Mould counts generally ranged from less than 100 to 4.0×104 cfu/g. Two crunchy sprout samples, however, contained unusually high numbers of Penicillium (1.1×108 and 1.3×108 cfu/g), two alfalfa sprout samples contained Geotrichum populations about 106 cfu/g, and two alfalfa sprout samples had Cladosporium counts higher than 2.5×105 cfu/g. The most common moulds found in fresh and minimally processed vegetables were Cladosporium, Alternaria and Penicillium; less common was Geotrichum. The most frequently isolated moulds from sprouts were Alternaria, Cladosporium, Penicillium, and Phoma. Phoma was especially common in alfalfa sprouts. Fusarium, Rhizopus, Mucor, and Geotrichum were isolated less often. [Copyright &y& Elsevier]

As part of larger survey of microbial contamination of fruits and vegetables in Norway, four different sprouted seed products were analysed for bacterial and parasitic contaminants (n=300 for bacterial analyses and n=from 17 to 171 for parasite analyses, depending on parasite).Escherichia coli O157, Salmonella, Listeria monocytogenes, Cyclospora oocysts, Ascaris eggs and other helminth parasites were not detected in any of the sprout samples. Thermotolerant coliform bacteria (TCB) were isolated from approximately 25% of the sprout samples, with the highest percentage of TCB positive samples in alfalfa sprouts. Most TCB were Enterobacter spp. and Klebsiella. E. coli was isolated from 8 of 62 TCB positive mung bean sprout samples. Cryptosporidium oocysts were detected in 8% of the sprout samples and Giardia cysts were detected in 2% of the samples. All the Cryptosporidium positive samples, and most of the Giardia positive samples, were mung bean sprouts. Parasite concentrations in positive samples were low (between 1 and 3 oocysts/cysts per 50 g sprouts).Sprout irrigation water was also analysed for microbial contaminants. E. coli O157 and L. monocytogenes were not detected. TCB were isolated from approximately 40% of the water samples. Salmonella reading was isolated from three samples of spent irrigation water on 3 consecutive days. Cryptosporidium and Giardia were also isolated from spent irrigation water.Additionally, eight samples of unsprouted mung bean seed were analysed for Cryptosporidium oocysts and Giardia cysts. One or both of these parasites were detected in six of the unsprouted seed samples at concentrations of between 1 and 5 oocysts/cysts per 100 g unsprouted seed.Whilst our results support spent irrigation water as the most suitable matrix for testing for bacteria, unsprouted seed is considered the more useful matrix for analysing for parasite contaminants. [ABSTRACT FROM AUTHOR]

Alfalfa and other types of sprouts are known to harbor large populations of native microorganisms. As some of these microbes may be causes of reduced shelf life of the product (plant pathogens and other spoilage organisms) and sprouts may, on occasion, harbor bacteria pathogenic towards humans, the addition of antimicrobial compounds to the irrigation water may be warranted. In this study, we tested the efficacy of several antimicrobial compounds for reducing the native microbial populations on alfalfa sprouts during propagation. These compounds included H2O2, peroxyacetic acid+hydrogen peroxide (Tsunami 100™), acidified NaClO2, NaClO2 (Aquatize™), EDTA, Na3PO4 and NaOCl. When added to the irrigation water at various concentrations, none of the antimicrobial compounds reduced the levels of any class of native microflora by more than 1 log10 without evidence of phytotoxicity. [Copyright &y& Elsevier]

The germination process causes changes in the chemical composition of seeds that improves the nutritional value of sprouts, while decreasing their microbiological safety, since the germination conditions are ideal for bacterial growth as well. This review explores the bacteriological safety of sprouts and their involvement in foodborne illness outbreaks, worldwide. Additionally, approaches to improve the shelf-life and microbiological safety of sprouts are discussed. According to the literature, sprout consumption is associated with more than 60 outbreaks of foodborne illness worldwide, since 1988. Alfalfa sprouts were most commonly involved in outbreaks and the most commonly implicated pathogens were Salmonella and pathogenic Escherichia coli (especially, Shiga toxin producing E. coli). In the pre-harvest stage, the implementation of good agricultural practices is an important tool for producing high-quality seeds. In the post-harvest stage, several methods of seed decontamination are used commercially, or have been investigated by researchers. After germination, seedlings should be kept under refrigeration and, if possible, cooked before consumption. Finally, microbiological analyses should be performed at all stages to monitor the hygiene of the sprout production process. • Sprouts are good sources of micronutrients, macronutrients and bioactive compounds. • At least 64 disease outbreaks implicating sprouts have been reported. • Seed germination conditions are ideal for microbial growth. • Safety measures can reduce sprout contamination. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) in sprouts have caused large scale outbreaks in the past involving severe illness. The combination of this very diverse pathogen and a food matrix with high numbers of background microbiota poses a particular challenge for detection and isolation. An acid treatment of the enrichment before plating on agar has been shown to improve the recovery of STEC from sprouts. After enrichment in buffered peptone water (BPW) at 37 °C we applied an acid treatment, followed by plating on tryptone bile x-glucuronide (TBX) agar (acid bile method). An inter-laboratory study was organized with 21 laboratories taking part to evaluate the performance parameters and applicability of the acid bile method. A sample set of six sprout samples was prepared consisting of two uninoculated samples and four spiked samples, each containing one of two STEC strains at one of two concentrations (low and high). Analyzing a set of six samples at the National Reference Laboratory (NRL E. coli), we determined the relative abundance of STEC without, after acid-, after bile- and after acid-bile treatment using real-time PCR. The participating laboratories successfully applied the acid bile method and were better able to detect (sensitivity 92.9% vs. 70.0%) and isolate (sensitivity 87.5% vs. 31.3%) STEC from positive samples using the acid bile method compared to non-acid methods. The relative limit of detection (RLOD) after isolation using the acid bile method (vs. non-acid method) was <1 for both STEC strains used, BfR-EC-14434 O133:H25 (0.146) and BfR-EC-16015 O26:H11 (0.073). A collection of STEC (n = 71) of diverse type and characteristics was assessed for their resistance towards the acid bile treatment selection. The majority (n = 65) of STEC strains could be recovered after acid treatment on TBX plates. However, a few strains (n = 6), among them clinical isolates were (partly) sensitive. These results suggest that an acid bile method is a rapid and reasonable approach to improve the recovery of STEC from sprouts when used in combination with methods targeting other selection markers. • Optimized enrichment with acid bile selection for STEC detection and isolation from sprouts • The acid bile method was compared to non-acid methods in an inter-laboratory study. • Laboratories clearly improved STEC analysis from sprouts using the acid bile method. • The majority of STEC strains tested could be recovered after acid bile selection assay. [ABSTRACT FROM AUTHOR]

Salmonella enterica outbreaks in sprouts originate from contaminated seeds; conventional prevention technologies have been reported from many research institutes. In this study, we applied a biological control approach to inhibit S. enterica growth using the seed-dwelling non-antagonistic bacteria. We isolated non-antibacterial seed-dwelling bacteria from vegetable sprouts. A total of 206 bacteria exhibiting non-antibacterial activity against S. enterica were subjected to alfalfa sprout development tests. Eight isolates exhibiting no deleterious effect on the growth of alfalfa sprouts were tested for S. enterica growth inhibition on alfalfa seeds and sprouts, and an isolate EUS78 was finally selected for further investigation. Based on 16S rRNA, gyrB , and rpoB gene sequence analyses, strain EUS78 was identified as Erwinia persicina. In population competition, the S. enterica population increased by >3 log CFU/g after 6 days of alfalfa sprout growth, whereas S. enterica growth was significantly inhibited by treatment with EUS78 (P <.05). This effect of S. enterica growth inhibition by EUS78 was sustained until the end of the alfalfa sprout harvest. Overall, bacterial strain EUS78 significantly reduced S. enterica growth on alfalfa sprouts in a manner consistent with competitive exclusion. These findings led us to monitor EUS78 behavior on seeds during early sprout development using fluorescence and scanning electron microscopy. Strain EUS78 initially colonized alfalfa sprout seed coat edges, cotyledons, and finally root surfaces during early sprout germination. As alfalfa sprouts grew, EUS78 bacterial cells established colonies on newly emerged plant tissues such as root tips. The results of this study suggest that strain EUS78 has potential as a biological control agent to inhibit S. enterica contamination in the sprout food industry. • Non-antagonistic Erwinia persicina EUS78 inhibited Salmonella enterica growth on alfalfa sprouts. • Salmonella enterica growth on alfalfa sprouts was reduced by competitive exclusion. • This is the first study to use a non-antagonistic bacterium to influence Salmonella contamination. • Erwinia persicina has potential as a sprout food industry biological control agent. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the effectiveness of a commercial Salmonella bacteriophage mixture (SalmoFresh™ 6-phage strains) and to compare its effectiveness with a chlorinated water treatment to reduce Salmonella on produce and seeds at different temperatures and storage times. Two sets of experiments were designed to test phage and chlorinated water effectiveness on produce at 2, 10 and 25 °C at different storage times (1, 24, 48 and 72 h). First, SalmoFresh™ was applied to the surface of lettuce, mung bean sprouts and mung bean seeds that were spot-inoculated with a five Salmonella strain mixture (Newport, Braenderup, Typhimurium, Kentucky, and Heidelberg, 105 CFU/mL) by spraying phages onto lettuce (n = 48 pieces, 3×3 cm2 per treatment) and sprouts (n = 48 pieces per treatment). A second set of experiments (scaled-up) consisted in the application of phages by immersion to Salmonella adulterated lettuce (600 g), 300 g sprouts (300 g) or mung bean seeds (30 g) in a phage cocktail (108 PFU/mL) for 15 min (lettuce and sprouts) or 1 h (seeds). Another group of samples was washed with chlorinated water and yet another group was treated with a combination of chlorinated water followed by phage cocktail. Each experiment was repeated three times by quadruplicates. After the treatments for spot-inoculated and scaled-up experiments, lettuce and sprouts were separated into different lots (10 g/lot) and stored at 2, 10 and 25 °C; Salmonella was enumerated after 1, 24, 48 and 72 h. Adulterated phage-treated seeds were packaged and stored dry at 25 °C. Salmonella was enumerated after 72 h of storage. Groups of phage treated mung bean seeds (720 g) were germinated, and the reduction in Salmonella determined. Results of microplate virulence assays indicated that SalmoFresh™ reduced (P = 0.007) Salmonella by an average of 5.34 logs CFU/mL after 5 h at 25 °C. Spraying SalmoFresh™ onto lettuce and sprouts reduced Salmonella by 0.76 and 0.83 log 10 CFU/g, respectively (P < 0.01). Immersion of produce in a phage solution was better at killing Salmonella P < 0.05) than spraying it onto the surface, reducing Salmonella by 2.43 and 2.16 log 10 CFU/g on lettuce and sprouts, respectively. SalmoFresh™ was an effective biocontrol intervention to reduce Salmonella on lettuce and sprouts. On seeds, although a reduction was observed, Salmonella was able to grow exponentially during germination; therefore, the phage cocktail was not effective on mung bean seeds or sprouts obtained from adulterated seeds. The combination of hurdles, chlorination fallowed by the phage cocktail was the most effective treatment to reduce Salmonella on lettuce and sprouts. • Bacteriophage could be an effective method of controlling pathogens on produce • SalmoFresh™ reduced Salmonella on lettuce and sprouts by 2 to 3 log 10 CFU/g • SalmoFresh™ wasn't effective at reducing Salmonella on mung bean seeds and germinated sprouts • SalmoFresh™ was more effective at reducing Salmonella on produce at 2 and 10 °C than 25 °C • The combination of hurdles, chlorination fallowed by the phage cocktail was the most effective treatment to reduce Salmonella on lettuce and sprouts [ABSTRACT FROM AUTHOR]