Fate of Escherichia coli O157:H7 in field-inoculated lettuce

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.     

Detection of E. coli O157:H7 from Ground Beef Using Fourier Transform Infrared (FT-IR) Spectroscopy and Chemometrics

Abstract: FT-IR spectroscopy methods for detection, differentiation, and quantification of E. coli O157:H7 strains separated from ground beef were developed. Filtration and immunomagnetic separation (IMS) were used to extract live and dead E. coli O157:H7 cells from contaminated ground beef prior to spectral acquisition. Spectra were analyzed using chemometric techniques in OPUS, TQ Analyst, and WinDAS software programs. Standard plate counts were used for development and validation of spectral analyses. The detection limit based on a selectivity value using the OPUS ident test was 10⁵ CFU/g for both Filtration-FT-IR and IMS-FT-IR methods. Experiments using ground beef inoculated with fewer cells (10¹ to 10² CFU/g) reached the detection limit at 6 h incubation. Partial least squares (PLS) models with cross validation were used to establish relationships between plate counts and FT-IR spectra. Better PLS predictions were obtained for quantifying live E. coli O157:H7 strains (R²≥ 0.9955, RMSEE ≤ 0.17, RPD ≥ 14) and different ratios of live and dead E. coli O157:H7 cells (R²= 0.9945, RMSEE = 2.75, RPD = 13.43) from ground beef using Filtration-FT-IR than IMS-FT-IR methods. Discriminant analysis and canonical variate analysis (CVA) of the spectra differentiated various strains of E. coli O157:H7 from an apathogenic control strain. CVA also separated spectra of 100% dead cells separated from ground beef from spectra of 0.5% live cells in the presence of 99.5% dead cells of E. coli O157:H7. These combined separation and FT-IR methods could be useful for rapid detection and differentiation of pathogens in complex foods.     

Effectiveness of Active Packaging on Control of Escherichia Coli O157:H7 and Total Aerobic Bacteria on Iceberg Lettuce

Contaminated leafy green vegetables have been linked to several outbreaks of human gastrointestinal infections. Antimicrobial interventions that are adoptable by the fresh produce industry for control of pathogen contamination are in great demand. This study was undertaken to evaluate the efficacy of sustained active packaging on control of Escherichia coli O157:H7 and total aerobic bacteria on lettuce. Commercial Iceberg lettuce was inoculated with a 3‐strain mixture of E. coli O157:H7 at 10² or 10⁴ CFU/g. The contaminated lettuce and un‐inoculated controls were placed respectively in 5 different active packaging structures. Traditional, nonactive packaging structure was included as controls. Packaged lettuce was stored at 4, 10, or 22 °C for 3 wk and sampled weekly for the population of E. coli O157:H7 and total aerobic bacteria. Results showed that packaging structures with ClO₂ generator, CO₂ generator, or one of the O₂ scavengers effectively controlled the growth of E. coli O157:H7 and total aerobic bacteria under all storage conditions. Packaging structure with the ClO₂ generator was most effective and no E. coli O157:H7 was detected in samples packaged in this structure except for those that were inoculated with 4 log CFU/g of E. coli O157:H7 and stored at 22 °C. Packaging structures with an oxygen scavenger and the allyl isothiocyanate generator were mostly ineffective in control of the growth of the bacteria on Iceberg lettuce. The research suggests that some of the packaging structures evaluated in the study can be used to control the presence of foodborne pathogens on leafy green vegetables.     

Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx ₁, stx ₂, and wzy _O157 genes in combination with probes and primers targeting either the fliC _h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx ₁, stx ₂, wzy _O157, and eae genes; however, using the assay targeting the stx ₁, stx ₂, wzy _O157, and fliC _h7 gene combination, a positive result was always obtained for the fliC _h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC _h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx ₁, stx ₂, eae, and wzy _O157 or the fliC _h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC _h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.     

Switchgrass extractives to mitigate Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium contamination of romaine lettuce at pre- and postharvest

The antimicrobial potential of switchgrass extractives (SE) was evaluated on cut lettuce leaves and romaine lettuce in planta, using rifampicin-resistant Escherichia coli O157:H7 and Salmonella Typhimurium strain LT2 as model pathogens. Cut lettuce leaves were swabbed with E. coli O157:H7 or S. Typhimurium followed by surface treatment with 0.8% SE, 0.6% sodium hypochlorite, or water for 1 to 45 min. For in planta studies, SE was swabbed on demarcated leaf surfaces either prior to or after inoculation of greenhouse-grown lettuce with E. coli O157:H7 or S. Typhimurium; the leaf samples were collected after 0, 24, and 48 h of treatment. Bacteria from inoculated leaves were enumerated on tryptic soy agar plates (and also on MacConkey's and XLT4 agar plates), and the recovered counts were statistically analyzed. Cut lettuce leaves showed E. coli O157:H7 reduction between 3.25 and 6.17 log CFU/leaf, whereas S. Typhimurium reductions were between 2.94 log CFU/leaf and 5.47 log CFU/leaf depending on the SE treatment durations, from initial levels of ∼7 log CFU/leaf. SE treatment of lettuce in planta, before bacterial inoculation, reduced E. coli O157:H7 and S. Typhimurium populations by 1.88 and 2.49 log CFU after 24 h and 3 h, respectively. However, SE treatment after bacterial inoculation of lettuce plants decreased E. coli O157:H7 populations by 3.04 log CFU (after 0 h) with negligible reduction of S. Typhimurium populations. Our findings demonstrate the potential of SE as a plant-based method for decontaminating E. coli O157:H7 on lettuce during pre- and postharvest stages in hurdle approaches.     

Activity of citrus essential oils against Escherichia coli O157:H7 and Salmonella spp. and effects on beef subprimal cuts under refrigeration

Abstract: Escherichia coli O157:H7 and Salmonella spp. are bacterial pathogens often associated with beef, and cause many cases of foodborne illness each year in the United States. During beef slaughter and processing, these bacteria may spread from the hide or intestines to the carcass. The objective of this research was to investigate the use of naturally occurring compounds citrus essential oils (CEOs) extracted from orange peel to reduce or eliminate these pathogens at the chilling stage of processing, or during fabrication. Brisket flats (used to simulate beef subprimals) were spot inoculated with approximately 6 log of surrogate generic E. coli cocktail (previously shown to be identical in growth and survival parameters to E. coli O157:H7 and Salmonella spp.). Following drying, CEOs were applied by spraying at concentrations of 3% and 6% to the surface of different pieces of meat. Treatments were applied using a custom built spray cabinet at 2.07 bar and applied at a rate of 3.79 L/min to replicate commercial practices. The CEOs significantly reduced (P < 0.05) the concentration of E. coli on the brisket flats in comparison to inoculated no spray or water sprayed controls over a period of 90 d, while causing an initial reduction of approximately 1.4 log units. Total aerobic bacteria and psychrotrophic counts were also reduced on uninoculated briskets following treatment. These results indicate that 3% cold‐pressed terpeneless Valencia orange oil could be used as an additional intervention against E. coli O157:H7 and Salmonella spp. at the refrigerated storage stage of processing. Practical Application: CEOs are natural compounds that have been designated as Generally Recognized as Safe (GRAS). They can be used to control Salmonella spp. and E. coli O157:H7 on beef carcasses at the chilling stage.     

Review of recent advances in improved lateral flow immunoassay for the detection of pathogenic Escherichia coli O157:H7 in foods

The incidence of foodborne diseases has continuingly increased over the years and resulted in public health problem globally. Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7) is a human pathogen that causes diarrhea and hemorrhagic colitis. E. coli O157:H7 can be found in various foods. It is important to detect this foodborne pathogen to provide safe food supply. A lot of methods, for example, culture and PCR‐based test, used to detect foodborne pathogens are laborious and time consuming. Hence, a variety of methods have been developed for rapid, simple and reliable detection of E. coli O157:H7 as it is required in many food analyses. Lateral flow immunoassay (LFIA) are advantageous over conventional detection methods in terms of their rapidity and simplicity for end user, especially the LFIA can be developed as the strip test for on‐site point‐of‐care test (POCT) products. Gold nanoparticles (AuNPs; colloid gold) are the most commonly used labels in the LFIA for the visual analysis, however, there are still several limitations that restrict their applications of traditional LFIA. Therefore, recent reports on improved LFIA for E. coli O157:H7 detection in foods are continuously reported. This review intends to provide these recent advances in improved LFIA methods for the detection of E. coli O157:H7 in foods.     

Efficacy of Slightly Acidic Electrolyzed Water and UV‐Ozonated Water Combination for Inactivating Escherichia Coli O157:H7 on Romaine and Iceberg Lettuce during Spray Washing Process

Spray washing is a common sanitizing method for the fresh produce industry. The purpose of this research was to investigate the antimicrobial effect of spraying slightly acidic electrolyzed water (SAEW) and a combination of ozonated water with ultraviolet (UV) in reducing Escherichia coli O157:H7 on romaine and iceberg lettuces. Both romaine and iceberg lettuces were spot inoculated with 100 μL of a 3 strain mixture of E. coli O157:H7 to achieve an inoculum of 6 log CFU/g on lettuce. A strong antimicrobial effect was observed for the UV‐ozonated water combination, which reduced the population of E. coli by 5 log CFU/g of E. coli O157:H7 on both lettuces. SAEW achieved about 5 log CFU/g reductions in the bacterial counts on romaine lettuce. However, less than 2.5 log CFU/g in the population of E. coli O157:H7 was reduced on iceberg lettuce. The difference may be due to bacteria aggregation near and within stomata for iceberg lettuce but not for romaine lettuce. The UV light treatment may stimulate the opening of the stomata for the UV‐ozonated water treatment and hence achieve better bacterial inactivation than the SAEW treatment for iceberg lettuce. Our results demonstrated that the combined treatment of SAEW and UV‐ozonated water in the spray washing process could more effectively reduce E. coli O157:H7 on lettuce, which in turn may help reduce incidences of E. coli O157:H7 outbreaks.     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Efficacy of Neutral pH Electrolyzed Water in Reducing Escherichia coli O157:H7 and Salmonella Typhimurium DT 104 on Fresh Produce Items using an Automated Washer at Simulated Food Service Conditions

The objective of this study was to determine the efficacy of neutral pH electrolyzed (NEO) water (155 mg/L free chlorine, pH 7.5) in reducing Escherichia coli O157:H7 and Salmonella Typhimurium DT 104 on romaine lettuce, iceberg lettuce, and tomatoes washed in an automated produce washer for different times and washing speeds. Tomatoes and lettuce leaves were spot inoculated with 100 μL of a 5 strain cocktail mixture of either pathogen and washed with 10 or 8 L of NEO water, respectively. Washing lettuce for 30 min at 65 rpm led to the greatest reductions, with 4.2 and 5.9 log CFU/g reductions achieved for E. coli O157:H7 and S. Typhimurium respectively on romaine, whereas iceberg lettuce reductions were 3.2 and 4.6 log CFU/g for E. coli O157:H7 and S. Typhimurium respectively. Washing tomatoes for 10 min at 65 rpm achieved reductions greater than 8 and 6 log CFU/tomato on S. Typhimurium and E. coli O157:H7 respectively. All pathogens were completely inactivated in NEO water wash solutions. No detrimental effects on the visual quality of the produce studied were observed under all treatment conditions. Results show the adoption of this washing procedure in food service operations could be useful in ensuring produce safety.     

Reduction of Escherichia coli O157:H7 in Biofilms Using Bacteriophage BPECO 19

Biofilm formation is a growing concern in the food industry. Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persists in food and food‐related environments and subsequently produce biofilms. The efficacy of bacteriophage BPECO 19 was evaluated against three E. coli O157:H7 strains in biofilms. Biofilms of the three E. coli O157:H7 strains were grown on abiotic (stainless steel, rubber, and minimum biofilm eradication concentration [MBEC^TM] device) and biotic (lettuce) surfaces at different temperatures. The effectiveness of bacteriophage BPECO 19 in reducing preformed biofilms on these surfaces was further evaluated by treating the surfaces with a phage suspension (10⁸ PFU/mL) for 2 h. The results indicated that the phage treatment significantly reduced (P  < 0.05) the number of adhered cells in all the surfaces. Following phage treatment, the viability of adhered cells was reduced by ≥3 log CFU/cm², 2.4 log CFU/cm², and 3.1 log CFU/peg in biofilms grown on stainless steel, rubber, and the MBEC^TM device, respectively. Likewise, the phage treatment reduced cell viability by ≥2 log CFU/cm² in biofilms grown on lettuce. Overall, these results suggested that bacteriophages such as BPECO 19 could be effective in reducing the viability of biofilm‐adhered cells.     

CONDITIONS AFFECTING AUTOINDUCER‐2‐BASED QUORUM‐SENSING OF ESCHERICHIA COLI O157:H7 ON FRESH BEEF

ABSTRACT

This study evaluated whether inoculated (none, 1, 5 log colony‐forming units [cfu]/cm²) Escherichia coli O157:H7 would result in detection of autoinducer (AI)‐2‐like activity on beef. Inoculated fresh beef, containing low (LNB) or high (HNB) initial levels of natural flora, was analyzed for bacterial populations and AI‐2‐like activity during aerobic or vacuum‐packaged storage (4, 10, 25C). As expected, no growth of E. coli O157:H7 was detected at 4C, while at 10C, growth was detected only on LNB samples stored aerobically; AI‐2‐like activity was minimal (P ≥ 0.05) at both temperatures. E. coli O157:H7 showed more growth in LNB than HNB, and in aerobically than vacuum‐packaged samples inoculated with 1 log cfu/cm² of the pathogen during storage at 25C. AI‐2‐like activity was generally higher in LNB than HNB samples stored aerobically at 25C, while no significant AI‐2‐like activity was detected in samples stored in vacuum packages. The results indicated that E. coli O157:H7 may exhibit AI‐2‐like activity on aerobically stored beef in the presence of lower initial levels of natural flora, and at temperatures allowing prolific growth of the pathogen. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be of importance in beef stored at low temperatures.

PRACTICAL APPLICATIONS

This study presents evidence that Escherichia coli O157:H7 showed autoinducer (AI)‐2 activity and involved in quorum‐sensing on fresh beefcontaining low initial levels of natural flora during aerobic storage at abusive storage temperatures. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be important in beef stored at recommended low temperatures.     

Survival and High‐Hydrostatic Pressure Inactivation of Foodborne Pathogens in Salmorejo,a Traditional Ready‐to‐Eat Food

Salmorejo is a traditional tomato‐based creamy product. Because salmorejo is not heat‐processed, there is a risk of contamination with foodborne pathogens from raw materials. Even though bacterial growth in salmorejo is strongly inhibited because of its acidic pH (close to 3.9), the growth and survival of 3 foodborne pathogens in this food has not been studied before. In this study, 3 cocktails consisting of Escherichia coli O157, Salmonella enterica serovar Enteritidis, and Listeria monocytogenes strains were inoculated in freshly prepared salmorejo. The food was treated by high hydrostatic pressure (HHP) at 400, 500, or 600 MPa for 8 min, or left untreated, and stored at 4 °C for 30 d. Viable cell counts were determined on selective media and also by the triple‐layer agar method in order to detect sublethally injured cells. In control samples, L. monocytogenes viable cells decreased by 2.4 log cycles at day 7 and were undetectable by day 15. S. enterica cells decreased by 0.5 or 2.4 log cycles at days 7 and 15 respectively, but still were detectable at day 30. E. coli O157 cells survived much better in salmorejo, decreasing only by 1.5 log cycles at day 30. Treatments at pressures of 400 MPa or higher reduced viable counts of L. monocytogenes and S. enterica to undetectable levels. HHP treatments significantly (P < 0.05) reduced E. coli counts by approximately 5.2 to 5.4 log cycles, but also yielded surviving cells that apparently were sublethally injured. Only samples treated at 600 MPA for 8 min were devoid of detectable E. coli cells during storage.     

The prevalence of verocytotoxin-producingEscherichia coli(VTEC) andE. coliO157:H7 in beef in Sweden determined by PCR assays and an immuno-magnetic separation (IMS) method

The number of cases of enterohaemorrhagicEscherichia coli(EHEC) in Sweden has increased dramatically since 1995, but no source of the infections has been identified. The prevalence of verocytotoxin-producingE. coli(VTEC), includingE. coliO157:H7 in imported and domestic raw beef in Sweden, was determined by PCR assays and immuno-magnetic separation (IMS, Dynal). VTEC was detected by a multiplex PCR directed at sequences on the vt1 and vt2 genes, and the prevalence ofE. coliO157:H7 was estimated by the IMS assay and a SZ-PCR which detectsE. coliO157:H7 (and some strains of O157:NM, O55:H7 and O55:NM). VTEC was detected in 15.055% (57/368) and 1.051% (6/543) of the imported and domestic beef samples, respectively, by the multiplex PCR. E. coliO157:H7 was detected in 0.053% and 2.054% of the imported beef samples by the IMS and SZ-PCR assays, respectively. In domestic beefE. coliO157:H7 was not detected by either method, indicating that the occurrence in Swedish beef was less than 0.056% with a 95% certainty. The overall prevalence of VTEC was estimated to be 4.050%, by adjusting for the relative proportion of imported and domestic beef in Sweden and, similarly, the overall prevalence ofE. coliO157:H7 was 0.0506% (IMS) or 0.055% (SZ-PCR). We propose that a useful strategy to analyse a large number of food samples for VTEC andE. coliO157:H7 is an initial screening of samples using the multiplex VT-PCR in conjunction with enrichment and buoyant density centrifugation, followed by further analyses on the fraction of VT-positive samples using either the IMS method or SZ-PCR.     

Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads

Ready-to-eat salads using baby-leaf and multi-leaf mixes are one of the most promising developments in the fresh-cut food industry. There is great interest in developing novel decontamination treatments, which are both safe for consumers and more efficient against foodborne pathogens. In this study, emulsions of essential oils (EOs) from Origanum compactum (oregano), Eugenia caryophyllus (clove), and Zataria multiflora Boiss (zataria) were applied by spray (0.8 ml) after the sanitizing washing step. The aim was to investigate their ability to control the growth of potentially cross-contaminating pathogens and endogenous microbiota in commercial baby leaves, processed in a fresh-cut produce company. Zataria EO emulsions of 3%, 5% and 10% reduced Escherichia coli O157:H7 by 1.7, 2.2 and 3.5 log cfu/g in baby-leaf salads after 5 days of storage at 7 °C. By contrast, reductions in E. coli O157:H7 counts remained the same when clove was applied at concentrations of 5% and 10% (2.5 log cfu/g reduction). Oregano (10%) reduced inoculated E. coli O157:H7 counts in baby-leaf salads by a maximum of 0.5 log cfu/g after 5 days of storage. Zataria showed strong antimicrobial efficacy against E. coli O157:H7 and also against the endogenous microbiota of baby-leaf salads stored for 9 days. Feline calicivirus (FCV), a norovirus surrogate, survived on inoculated baby-leaf salads during refrigerated storage (9 days at 7 °C) regardless of treatment. Refrigeration temperatures completely annulled the effectiveness of the EOs against FCV inoculated in baby-leaf salads as occurred in FCV cultures. This study shows that EOs, and zataria in particular, have great potential use as an additional barrier to reduce contamination-related risks in baby-leaf salads. However, further research should be done into foodborne viruses in order to improve food safety.     

Comparison of a real-time PCR and an IMS/culture method to detect Escherichia coli O26 and O111 in minced beef in the Republic of Ireland

This study compared an immuno-magnetic separation (IMS)/culture method and a real-time PCR method to detect Verocytotoxigenic Escherichia coli (VTEC) serovar O26 and/or O111 in minced beef. A total of 65 samples were examined, 40 of which were frozen beef samples previously established as containing E. coli O157, and 25 were samples of fresh minced beef, purchased from butcher shops in the Dublin area. After selective enrichment, all samples were (a) subjected to IMS, plated on differential media and identified as E. coli O26 or O111 using biochemical and immuno-logical methods; and (b) subjected to DNA extraction and real-time PCR analysis using primers and probes against E. coli O111 and O26 serovar specific genes, and verotoxin genes. Overall, from the 65 minced beef samples collected, three were positive for E. coli O26 by real-time PCR, with only one of these samples positive for E. coli O26 by the culture method. One sample was positive for E. coli O111 by both real-time PCR and the culture method. The two samples found positive for E. coli O26 by real-time PCR method but not by culture method belonged to the group of frozen beef samples, indicating that the previously developed culture method for the detection of E. coli O26 may not be suitable for the detection of freeze injured cells. In conclusion, this study highlights the role of beef meat in the transmission of non-O157 VTEC. The results of the study emphasize that the analyses for emergent pathogens should be included in food safety surveillance systems and that the development of standard methods for the detection of E. coli O26 and O111 in routine food testing is needed in order to reduce the consumer exposure to contaminated food.     

The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS^® and DuPont Qualicon BAX^®-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C_T) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C_T values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10⁶ cells/sample or higher, which are levels not typically seen in ground beef.     

Survival and growth of foodborne pathogens in minimally processed vegetables at 4 and 15 °C

Abstract: We conducted this study to investigate the survival and growth of pathogens on fresh vegetables stored at 4 and 15 °C. Vegetables (romaine lettuce, iceberg lettuce, perilla leaves, and sprouts) were inoculated with 4 pathogens (Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157:H7) and stored at 2 different temperatures for different periods of time (3, 6, 9, 12, and 15 d at 4 °C and 1, 2, 3, 5, and 7 d at 15 °C). Populations of the 4 pathogens tended to increase on all vegetables stored at 15 °C for 7 d. Populations of E. coli O157:H7 and S. Typhimurium increased significantly, by approximately 2 log₁₀CFU/g, on loose and head lettuce stored at 15 °C for 1 d. No significant differences were observed in the growth of different pathogens on vegetables stored at 4 °C for 15 d. E. coli O157:H7 did not survive on sprouts stored at 15 or 4 °C. The survival and growth of food pathogens on fresh vegetables were very different depending on the pathogen type and storage temperature. Practical Application: Survivals and growth of pathogens on various vegetables at 4 and 15 °C were observed in this study. Survivals and growth of pathogens on vegetables were different depending on the pathogen type and storage temperature. Therefore, vegetables should be stored under refrigerated conditions (below 4 °C) prior to consumption. This recommendation may vary depending on the type of vegetable.     

Development of oscillation method for reducing foodborne pathogens on lettuce and spinach

In this study, the efficacy of an oscillator for reducing the numbers of foodborne pathogens on lettuce and spinach was tested. A cocktail of three strains each of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes cells and of Bacillus cereus spores was inoculated onto lettuce and spinach leaves and followed by oscillation at 10 Hz and 20 Hz for up to 30 s. After treatment of inoculated lettuce leaf with an oscillator at 20 Hz for 30 s, 2.58, 2.82, 2.21 and 2.22 Log₁₀ CFU/g reductions were obtained with the cells of S. typhimurium, E. coli O157:H7 and L. monocytogenes and the spores of B. cereus, respectively. In the case of the oscillation treatment of spinach leaf, 2.89, 3.73, 2.46 and 2.25 Log₁₀ CFU/g reductions of those pathogens were achieved under the same condition. Statistically significant reductions were observed after oscillation treatment at 20 Hz for 5-10 s. The oscillation treatment at 10 Hz led to slightly less reductions of the pathogens tested as compared to the treatment at 20 Hz. In conclusion, the oscillation method developed shows to be highly efficacious in reducing foodborne pathogens on lettuce and spinach leaves.     

Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment

A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10² CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal’s ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10⁰ to 1.0 × 10⁴ CFU/g by using Rti-PCR.