Production of biofilm and quorum sensing by Escherichia coli O157:H7 and its transfer from contact surfaces to meat,poultry, ready-to-eat deli,and produce products

Multistate outbreaks of Escherichia coli O157:H7 infections through consumption of contaminated foods including produce products have brought a great safety concern. The objectives of this study were to determine the effect of biofilm and quorum sensing production on the attachment of E. coli O157:H7 on food contact surfaces and to evaluate the transfer of the pathogen from the food contact to various food products. E. coli O157:H7 produced maximum levels of AI-2 signals in 12 h of incubation in tested meat, poultry, and produce broths and subsequently formed strong biofilm in 24 h of incubation. In general, E. coli O157:H7 formed stronger biofilm on stainless steel than glass. Furthermore, E. coli O157:H7 that had attached on the surface of stainless steel was able to transfer to meat, poultry, ready-to-eat deli, and produce products. Strong attachment of the transferred pathogen on produce products (cantaloupe, lettuce, carrot, and spinach) was detected (>10³ CFU/cm²) even after washing these products with water. Our findings suggest that biofilm formation by E. coli O157:H7 on food contact surfaces can be a concern for efficient control of the pathogen particularly in produce products that require no heating or cooking prior to consumption.     

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.     

Role of O-antigen on the Escherichia coli O157:H7 cells hydrophobicity, charge and ability to attach to lettuce

Environmental factors encountered during growing and harvesting may contribute to Escherichia coli O157:H7 contamination of lettuce. Limited nutrients and extended exposure to water may cause E. coli O157:H7 to shed its O antigen. Absence of the O157-polysaccharide antigen could affect the cell's physicochemical properties (hydrophobicity and cell charge) and ultimately influence its attachment to surfaces. The objectives of this study were to evaluate the effect of the E. coli O157:H7 O-antigen on the cell's overall hydrophobicity, charge and ability to attach to cut edge and whole leaf iceberg lettuce surfaces. Three strains of E. coli O157:H7 (86-24 wild type; F-12, mutant lacking the O-antigen and pRFBE, plasmid for O157 gene reintroduced) were examined for their hydrophobicity, overall charge and ability to attach to lettuce. Overall, E. coli O157:H7 attached at higher levels to cut surfaces over whole leaf surfaces (P = 0.008) for all strains and treatments. Additionally, the strain lacking the O-antigen (F12) — attached significantly less to lettuce (P = 0.015) than the strains expressing the antigen (WT and pRFBE). Cells lacking the O antigen (strain F-12) were also significantly more hydrophobic than strains 86-24 or pRFBE (P ≤ 0.05). Surface charge differed among the strains tested (P ≤ 0.05); however, it did not appear to influence bacterial attachment to lettuce surfaces. The charge was not fully restored in the pRFBE strain (expression of O-antigen reintroduced), therefore, no conclusions can be made pertaining to the effect of charge on attachment in this study. Results indicate that E. coli O157:H7 cells which lack the O-antigen have greater hydrophobicity and attach at lower concentrations than cells expressing the O-antigen, to iceberg lettuce surfaces.     

Effects of X-ray radiation on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on shredded iceberg lettuce

The main goal of this investigation was to study the efficacy of X-ray doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) on inoculated Escherichia coli O157: H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on shredded iceberg lettuce. The second goal was to study the effect of X-ray on the inherent microflora counts and visual color of shredded iceberg lettuce during storage at 4 °C for 30 days. Treatment with 1.0 kGy X-ray significantly reduced the population of E. coli O157: H7, L. monocytogenes, Salmonella enterica and S. flexneri on shredded iceberg lettuce by 4.4, 4.1, 4.8 and 4.4-log CFU 5 cm⁻², respectively. Furthermore, more than a 5 log CFU reduction of E. coli O157: H7, L. monocytogenes, S. enterica and S. flexneri was achieved with 2.0 kGy X-ray. Treatment with X-ray reduced the initial microflora on iceberg lettuce and kept them significantly (p < 0.05) lower than the control during storage at 4 °C and 90% RH for 30 days. Treatment with X-ray did not significantly (p > 0.05) change the green color of iceberg lettuce leaves. Treatment with X-ray significantly reduced selected pathogens and inherent microorganisms on shredded iceberg lettuce leaves, which could be a good alternative to other technologies for produce (lettuce) industry.     

Efficacy of Chlorine Dioxide, Ozone, and Thyme Essential Oil or a Sequential Washing in Killing Escherichia coli O157:H7 on Lettuce and Baby Carrots

Chlorine dioxide (ClO₂), ozone, and thyme essential oil has been found to be effective in reducing pathogens, including Escherichia coli O157:H7, on selected produce. The efficacy of these sanitizers was evaluated, alone or through their sequential washing to achieve a 3 or more log reduction of mixed strains of E. coli O157:H7 on shredded lettuce and baby carrots. Samples sprinkle inoculated with mixed strains of E. coli O157:H7 were air-dried for 1 h at 22±2°C in a biosafety cabinet, stored at 4°C for 24 h, and then treated with different concentrations of disinfectants and exposure time. Sterile deionized water washing resulted in approximately 1log reduction ofE. coli O157:H7 after 10 min washing of lettuce and baby carrots. Gaseous treatments resulted in higher log reductions in comparison to aqueous washing. However, decolorization of lettuce leaves was observed during long exposure time. A logarithmic reduction of 1.48-1.97log₁₀ cfu/g was obtained using aqueous ClO₂ (10.0 mg/L for 10 min) ozonated water (9.7 mg/L for 10 min) or thyme oil suspension (1.0 mL/L for 5 min) on lettuce and baby carrots. Of the three sequential washing treatments used in this study, thyme oil followed by aqueous ClO₂/ozonated water, or ozonated water/aqueous ClO₂ were significantly (P<0.05) more effective in reducing E. coli O157:H7 (3.75 and 3.99log, and 3.83 and 4.34 log reduction) on lettuce and baby carrots, respectively. The results obtained from this study indicate that sequential washing treatments could achieve 3-4log reduction of E. coli O157:H7 on shredded lettuce and baby carrots.     

Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24 h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24 h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1 h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments.     

A mathematical risk model for Escherichia coli O157:H7 cross-contamination of lettuce during processing

A stochastic simulation modelling approach was taken to determine the extent of Escherichia coli O157:H7 contamination in fresh-cut bagged lettuce leaving the processing plant. A probabilistic model was constructed in Excel to account for E. coli O157:H7 cross contamination when contaminated lettuce enters the processing line. Simulation of the model was performed using @Risk Palisade© Software, providing an estimate of concentration and prevalence in the final bags of product. Three different scenarios, named S1, S2, and S3, were considered to represent the initial concentration on the contaminated batch entering the processing line which corresponded to 0.01, 1 and 100 cfu/g, respectively. The model was satisfactorily validated based on Standard Error of Prediction (SEP), which ranged from 0.00-35%. ANOVA analysis performed on simulated data revealed that the initial concentration in the contaminated batch (i.e., S1, S2, and S3) did not influence significantly (p = 0.4) the E. coli O157:H7 levels in bags derived from cross contamination. In addition, significantly different (p < 0.001) prevalence was observed at the different levels simulated (S1; S2 and S3). At the lowest contamination level (0.01 cfu/g), bags were cross-contaminated sporadically, resulting in very low E. coli O157:H7 populations (mean: ≤2 cfu/bag) and prevalence levels (<1%). In contrast, higher average prevalence levels were obtained for S2 and S3 corresponding to 3.05 and 13.39%, respectively. Furthermore, the impact of different interventions on E. coli O157:H7 cross-contamination (e.g., pathogen testing, chlorination, irradiation, and cleaning and disinfection procedures) was evaluated. Model showed that the pathogen was able to survive and be present in the final bags in all simulated interventions scenarios although irradiation (0.5 KGy) was a more effective decontamination step in reducing prevalence than chlorination or pathogen testing under the same simulated conditions.     

Mathematical modeling the cross-contamination of Escherichia coli O157:H7 on the surface of ready-to-eat meat product while slicing

Microbial cross-contamination either at home or production site is one of the major factors of causing contamination of foods and leading to the foodborne illness. The knowledge regarding Escherichia coli O157:H7 surface transfer on ready-to-eat (RTE) deli meat and the slicer used for slicing different RTE products are needed to ensure RTE food safety. The objectives of this study were to investigate and to model the surface cross-contamination of E. coli O157:H7 during slicing operation. A five-strain cocktail of E. coli O157:H7 was inoculated directly onto a slicer's round blade rim area at an initial level of ca. 4, 5, 6, 7 or 8 log CFU/blade (ca. 3, 4, 5, 6 or 7 log CFU/cm² of the blade edge area), and then the RTE deli meat (ham) was sliced to a thickness of 1–2 mm. For another cross-contamination scenario, a clean blade was initially used to slice ham which was pre-surface-inoculated with E. coli O157:H7 (ca. 4, 5, 6, 7 or 8 log CFU/100 cm² area), then, followed by slicing un-inoculated ham. Results showed that the developed empirical models were reasonably accurate in describing the transfer trend/pattern of E. coli O157:H7 between the blade and ham slices when the total inoculum level was ≥5 log CFU on the ham or blade. With an initial inoculum level at ≤4 log CFU, the experimental data showed a rather random microbial surface transfer pattern. The models, i.e., a power equation for direct-blade-surface-inoculation, and an exponential equation for ham-surface-inoculation are microbial load and sequential slice index dependent. The surface cross-contamination prediction of E. coli O157:H7 for sliced deli meat (ham) using the developed models were demonstrated. The empirical models may provide a useful tool in developing the RTE meat risk assessment.     

Alterations of the phylloepiphytic bacterial community associated with interactions of Escherichia coli O157:H7 during storage of packaged spinach at refrigeration temperatures

This study investigated the effects of packaging and storage temperature on the spinach phylloepiphytic bacterial community and fate of Escherichia coli O157:H7. Freshly harvested spinach was rinsed and/or disinfected, packaged and stored under typical retail conditions (4 °C) or under temperature abuse conditions (10 °C) for a period of 15 days. The final population size of culturable epiphytic bacteria after 15 days of storage was not affected by the temperature of storage or the presence of E. coli O157:H7. However, analysis of the bacterial community using denaturing gradient gel electrophoresis of 16s rDNA revealed changes with time of storage and the presence of E. coli O157:H7. Excision and sequencing of prominent DGGE bands identified that the majority of sequences belonged to the phyla Actinobacteria, Bacteroidetes, Firmicutes and Alphaprotebacteria. After 10 days of storage at 4 °C or 10 °C the population became more dominated by psychrotrophic bacteria. Removal of the epiphytic bacteria resulted in significant increases in numbers of E coli O157:H7 at 10 °C and was associated with decreased expression of E. coli O157:H7 virulence (stxA, curli, eaeA) and stress response (rpoS, sodB) genes. In conclusion, storage temperature and time of storage of packaged spinach affected the diversity of the epiphytic spinach microbiota which influenced the growth, establishment, physiology and potentially virulence of E. coli O157:H7.     

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.     

Efficacy of adding detergents to sanitizer solutions for inactivation of Escherichia coli O157:H7 on Romaine lettuce

Numerous Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh lettuce. The development of effective and easily implemented wash treatment could reduce such incidents. The purpose of this study was to evaluate the addition of food-grade detergents to sanitizer solutions for inactivation of E. coli O157:H7 on Romaine lettuce. Freshly-cut leaves of Romaine lettuce were dip-inoculated to achieve a final cell concentration of 7.8 ± 0.2 log CFU/g, air-dried for 2 h, and stored overnight at 4 °C. Leaves were then washed for 2 min in an experimental short chain fatty acid formulation (SCFA) or in one of the following solutions with or without 0.2% dodecylbenzenesulfonic acid or 0.2% sodium 2-ethyl hexyl sulfate: 1) deionized water; 2) 100 ppm chlorine dioxide; 3) 100 ppm chlorine; and 4) 200 ppm chlorine. Following wash treatment, samples were blended in neutralizing buffer (1:3) and surface plated on the selective media CT-SMAC. The efficacy of wash treatments, with or without the detergents, in inactivating E. coli O157:H7 cells on lettuce leaves were not significantly different. The most effective wash solution was SCFA, which was capable of reducing E. coli O157:H7 populations by more than 5 log CFU/g. The rest of the wash treatments resulted in a population reduction of less than 1 log CFU/g. The effectiveness of SCFA surpasses that of other sanitizer treatments tested in this study and requires further research to optimize treatments to preserve lettuce quality. Conventional detergents did not enhance the efficacy of any of the wash treatments tested during this study.     

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.     

Effects of packaging type and storage temperature on the growth of foodborne pathogens on shredded ‘Romaine’ lettuce

Fresh produce can be a vehicle for the transmission of pathogens capable of causing human illnesses and some of them can grow on fresh-cut vegetables. The survival and growth of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes inoculated onto shredded lettuce was determined under modified atmosphere packaging conditions, at various storage temperatures. We also monitored changes in pH and gas atmospheres within the packages and the growth of psychrotrophic and mesophilic microorganisms. After pathogen inoculation, shredded lettuce was packaged in films of different permeability and stored at 5 and 25 °C. After 10 days at 5 °C populations of E. coli O157:H7 and Salmonella decreased approximately 1.00 log unit while L. monocytogenes increased about 1.00 log unit, in all package films. Moreover, the pathogens level increased between 2.44 and 4.19 log units after 3 days at 25 °C. Psychrotrophic and mesophilic bacteria had similar growth at both temperatures with higher populations in air than in the other atmospheres. The composition of the storage atmosphere within the packaging of lettuce had no significant effect on the survival and growth of the pathogens used in this study at refrigeration temperatures. The results obtained can be considered as a warning indicator, which reinforces the necessity for corrective measures to avoid contamination of vegetables.     

Prevalence and characterization of Escherichia coli O157:H7 isolates from meat and meat products sold in Amathole District,Eastern Cape Province of South Africa

Meat and meat products have been implicated in outbreaks of Escherichia coli O157:H7 in most parts of the world. In the Amathole District Municipality of the Eastern Cape Province of South Africa, a large number of households consume meat and meat products daily, although the microbiological quality of these types of food is questionable. The present study investigated the prevalence of E. coli O157:H7 isolated from selected meat and meat products (45 samples each of biltong, cold meat, mincemeat, and polony) sold in this area. Strains of E. coli O157:H7 were isolated by enrichment culture and confirmed by polymerase chain reaction (PCR). Also investigated were the antibiogram profiles of the E. coli O157:H7 isolates. Five (2.8%) out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the fliC_H7, rfbE_O157, and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all the eight antibiotics tested. To prevent E. coli O157:H7 infections, meat and meat products such as biltong, cold meat, mincemeat and polony should be properly handled, and packed in sterile polyvinyl wrappers.     

Modelling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging

Fresh-cut iceberg lettuce inoculated with Escherichia coli O157:H7 was submitted to chlorine washing (150 mg/mL) and modified atmosphere packaging on laboratory scale. Populations of E. coli O157:H7 were assessed in fresh-cut lettuce stored at 4, 8, 13 and 16 °C using 6–8 replicates in each analysis point in order to capture experimental variability. The pathogen was able to grow at temperatures ≥8 °C, although at low temperatures, growth data presented a high variability between replicates. Indeed, at 8 °C after 15 days, some replicates did not show growth while other replicates did present an increase. A growth primary model was fitted to the raw growth data to estimate lag time and maximum growth rate. The prediction and confidence bands for the fitted growth models were estimated based on Monte-Carlo method. The estimated maximum growth rates (log cfu/day) corresponded to 0.14 (95% CI: 0.06–0.31), 0.55 (95% CI: 0.17–1.20) and 1.43 (95% CI: 0.82–2.15) for 8, 13 and 16 °C, respectively. A square-root secondary model was satisfactorily derived from the estimated growth rates (R² > 0.80; Bf = 0.97; Af = 1.46). Predictive models and data obtained in this study are intended to improve quantitative risk assessment studies for E. coli O157:H7 in leafy green products.     

Antibacterial activity of oregano and thyme essential oils against Listeria monocytogenes and Escherichia coli O157:H7 in feta cheese packaged under modified atmosphere

The antibacterial activity of the essential oils (EO) of oregano and thyme added at doses of 0.1 or 0.2 and 0.1 ml/100 g, respectively, to feta cheese inoculated with Escherichia coli O157:H7 or Listeria monocytogenes was investigated during cheese storage under modified atmosphere packaging (MAP) of 50% CO₂ and 50% N₂ at 4 °C. Compositional analysis showed that the predominant phenols were carvacrol and thymol for both EO. In control feta inoculated with the pathogens and stored under MAP, results showed that E. coli O157:H7 and L. monocytogenes strains survived up to 32 and 28 days of storage. However, in feta cheese treated with oregano EO at the dose of 0.1 ml/100 g, E. coli O157:H7 or L. monocytogenes survived up to 22 and 18 days, respectively, whereas at the dose of 0.2 ml/100 g up to16 or 14 days, respectively. Feta cheese treated with thyme EO at 0.1 ml/100 g showed populations of E. coli O157:H7 or L. monocytogenes not significantly different (P > 0.05) than those of feta cheese treated with oregano at 0.1 ml/100 g. Although both essential oils exhibited equal antibacterial activity against both pathogens, the populations of L. monocytogenes decreased faster (P < 0.05) than those of E. coli O157:H7 during the refrigerated storage, indicating a stronger antibacterial activity of both essential oils against the former pathogen.     

Survival of Escherichia coli O157:H7 in channel catfish pond and holding tank water

Survival of Escherichia coli O157:H7 in channel catfish (Ictalurus punctatus), pond and holding tank water was investigated. Water from three channel catfish ponds was inoculated with ampicillin/nalidixic acid-resistant E. coli O157:H7 transformed with a plasmid encoding for green fluorescent protein at 10⁵, 10⁶, and 10⁷ CFU/ml. Samples were taken from surface, internal organs, and skin scrape of fish and pond water for E. coli O157:H7 enumeration on brain heart infusion (BHI) agar containing ampicillin and nalidixic acid. To determine the survival of E. coli O157:H7 in catfish holding tank water from two farmers markets, the water was inoculated with 10⁷E. coli O157:H7 CFU/ml. E. coli O157:H7 were detected by direct plating for 33 and 69 d in pond and holding tank water, respectively. A rapid decrease of the pathogen was observed in the first 2 weeks to reach 2 log CFU/ml. When E. coli O157:H7 was not recovered by direct plating, the pathogen was isolated by enrichment in TSB for approximately another 30 d from pond and holding tank water. The populations of E. coli O157:H7 found in the internal organs and skin scrape were 5.5 log and 2.5 log CFU/ml, respectively. E. coli O157:H7 from internal organs and water were recovered for at least 12 d. Results suggest that E. coli O157:H7 can survive in channel catfish pond and holding tank water and channel catfish may become a potential carrier of the pathogen.     

Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 μg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.     

The prevalence of Escherichia coli O157 and O157:H7 in ground beef and raw meatball by immunomagnetic separation and the detection of virulence genes using multiplex PCR

The present study was conducted to investigate the presence of Escherichia coli O157 and O157:H7 strains and to detect the presence of the stx1, stx2, and eaeA genes in isolates derived from 200 samples (100 samples from fresh ground beef and 100 samples from raw meatball). The samples were purchased from the Samsun Province in Turkey, over a period of 1 year. Enrichment-based immunomagnetic separation and multiplex polymerase chain reaction were applied for these analyses. E. coli O157 was detected in five of the 200 (2.5%) samples tested (one isolated from ground beef and four from meatball samples), whereas E. coli O157: H7 was not detected in any sample. During the analysis, eight strains of E. coli O157 were obtained. The genes stx1, stx2, and eaeA were detected in two E. coli O157 isolates obtained from two meatball samples, whereas only the eaeA and the stx2 genes were detected in four E. coli O157 strains that were isolated from one meatball sample. None of the stx1, stx2, and eaeA was detected in the E. coli O157 isolates obtained from the ground beef and the one meatball samples.     

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.