Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Prevalence of Escherichia coli O157 and Salmonella spp. in solid bovine manure in California using real-time quantitative PCR.

The occurrence of Escherichia coli O157 and Salmonella spp. in solid bovine manure was investigated through a multi-county survey in California. Solid bovine manure samples (n = 91) were collected from 13 dairy farms located in multiple counties in California between June 2016 and August 2017. To quantify pathogens, DNA was extracted from bacteria in manure samples. Afterwards, the prevalence and levels of E. coli O157 and Salmonella spp. in solid bovine manure were determined by real-time quantitative PCR (qPCR). The prevalence of E. coli O157 and Salmonella spp. in solid bovine manure was 15·4 and 6·6% respectively. Escherichia coli O157 and Salmonella spp. levels in positive samples ranged from 3·1 to 5·3 log CFU per g and from positive (the population was <3 log CFU per g) to 5·2 log CFU per g respectively. Surface samples of manure piles had higher prevalence and levels of E. coli O157 and Salmonella spp. than subsurface samples, while no seasonal effects on pathogen occurrence were observed. Our results indicated that solid bovine manure is a source of E. coli O157 and Salmonella spp. and the application of untreated manure as biological soil amendments may pose potential risks to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggested that the presence of Escherichia coli O157 and Salmonella spp. in solid bovine manure may pose potential risks if untreated manure is applied as biological soil amendments. Considering the large-scale sampling used in this study, the observations provide a holistic assessment in terms of pathogen prevalence in solid bovine manure.

Increased Iron Availability Aggravates the Infection of Escherichia coli O157:H7 in Mice.

Iron plays an important role both in bacterial pathogenicity and in host defense mechanisms, which has frequently been underestimated. The primary purpose of this study was to investigate the influence of iron supplementation on the progression of bacterial infection. We used mice as an experimental model to supplement iron after Escherichia coli (E. coli) O157:H7 infection and found that iron supplementation exacerbated clinical symptoms of bacterial infection by increasing mortality and reducing body weight. Iron supplementation promoted the colonization of bacteria and enhanced inflammatory responses by increasing C-reaction protein level and the phagocytic capacity of PBMCs, as well as upregulating the expression of TNF-α and IL-1ß in E. coli O157:H7-challenged mice. In vitro cell experiment confirmed that an excess of iron would enhance the growth of E. coli O157:H7 and worsen the outcome of bacterial infection. Therefore, it is certainly plausible that iron supplementation in bacterial infection may worsen rather than improve host outcome.

Preweaned heifer management on US dairy operations: Part IV. Factors associated with the presence of Escherichia coli O157 in preweaned dairy heifers.

Dairy calves shed pathogenic Escherichia coli O157 (O157) in feces and are a potential route of exposure for human infections. As part of the National Animal Health Monitoring System's (NAHMS) Dairy 2014 study, we evaluated farm, animal, and environmental factors associated with O157 presence in dairy heifer calves. For this O157 study, calves were enrolled from 100 dairy operations in 13 states. Each operation collected data from calves from birth to weaning over an 18-mo period. A single fecal sample was collected from 487 calves in western states and from 871 calves in eastern states (n = 1,358 total), and O157 was detected in 2.5% (n = 34) of fecal samples. Descriptive statistics and univariable screening were used to determine which farm practices, environmental factors, and calf health measures were associated with O157 detection. Multilevel logistic models, controlling for dairy operation, were created using backward elimination of screened variables. The final O157 main effects model included variables for source of colostrum, temperature-humidity index (THI), and serum IgG concentration. Higher serum IgG was associated with lower odds of O157 shedding, whereas calves fed colostrum from their own dam had higher odds of O157 shedding than calves fed colostrum from pooled sources. Interaction models showed that THI level modified the effect of colostrum source on O157 shedding; calves with a THI indicative of heat stress had a significantly increased presence of O157 when fed colostrum from a first-lactation dam. The THI level also modified the effects of serum IgG. Calves with thermoneutral or heat stress THI values had increased presence of O157 with poor (<10 g/L) or adequate (10-15 g/L) serum IgG levels compared with those having excellent (≥15 g/L) serum IgG levels. These results highlight factors that influence the presence of O157 in preweaned dairy heifer calves and may be used to guide practices that mitigate shedding through improved animal husbandry.

Combination of garlic essential oil, allyl isothiocyanate, and nisin Z as bio-preservatives in fresh sausage.

The effects of natural antimicrobial compounds (garlic essential oil [GO], allyl isothiocyanate [AITC], and nisin Z [NI]) on microbiological, physicochemical and sensory characteristics of fresh sausage were assessed. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) towards Escherichia coli O157:H7 and Lactobacillus plantarum were determined in vitro. Sausages inoculated with E. coli O157:H7, were treated with different combinations of antimicrobials and assessed for microbiological and physicochemical parameters during storage (6C for 20 d). Treatments that presented the greatest antimicrobial effects were subjected to sensory evaluation. Combinations of 20 mg/kg NI + 125 µL/kg GO + 62.5 µL/kg AITC or 20 mg/kg NI + 62.5 µL/kg GO + 125 µL/kg AITC were effective in reducing E. coli O157H7 and spoilage lactic acid bacteria, and maintained the physicochemical characteristics of fresh sausage. Combinations of NI, GO and AITC were effective to improve the safety and the shelf life of fresh sausage, with no impact on its sensory acceptance.

Growth kinetics of Escherichia coli O157: H7 on the epicarp of fresh vegetables and fruits

ABSTRACT Despite the increasing reports on the incidence of fresh vegetables and fruits as a possible vehicle for human pathogens, there is currently limited knowledge on the growth potential of Escherichia coli O157:H7 on different plant substrates. This study analyzed the selective adhesion and growth of E. coli O157:H7 on chili habanero (Capsicum chinense L.), cucumber (Cucumis sativus), radish (Raphanus sativus), tomato (Lycopersicon esculentum), beet (Beta vulgaris subsp. vulgaris), and onion (Allium cepa L.) under laboratory conditions. The Gompertz parameters were used to determine the growth kinetics. Scanning electron microscopy was used to visualize the adhesion of E. coli O157:H7 on the epicarp of the samples. Predictive models were constructed to compare the growth of E. coli O157:H7 on the samples with different intrinsic factors and to demonstrate the low selectivity of the pathogen. No significant difference was observed in the lag-phase duration (LPD), generation time (GT), and exponential growth rate (EGR) of the pathogen adhered to the samples. The interaction between the microorganism and the substrate was less supportive to the growth of E. coli O157:H7 for onion, whereas for tomato and cucumber, the time for the microorganism to attain the maximum growth rate (M) was significantly longer than that recorded for other samples.

Viability of and Escherichia coli O157:H7 and Listeria monocytogenes in a delicatessen appetizer (yogurt-based) salad as affected by citrus extract (Citrox©) and storage temperature.

The antimicrobial effect of citrus extract (at 1 mL/kg [C1] and 2 mL/kg [C2]) on naturally occurring microbiota and inoculated pathogens (E. coli O157:H7 and L. monocytogenes at ca. 6 log cfu/g) in the traditional Greek yogurt-based salad Tzatziki stored at 4, 10, or 21 °C, was examined. Lactic acid bacteria (LAB) were high (8.0-8.5 log cfu/g) and varied only minimally for both the control (untreated) and the citrus extract-treated salad samples, whereas the higher citrus extract concentration yielded the lowest yeast populations, irrespective of temperature, during the entire storage period. Populations of inoculated E. coli (6 log cfu/g) declined in both untreated and citrus extract-treated samples from day 0-70, 35, and 15 at 4, 10, and 21 °C, respectively. Citrus extract had a significant effect on the survival of the inoculated E. coli O157:H7, with reductions of 2.8-4.8 log cfu/g in the citrus extract-treated samples at the end of the storage period. Our data show that L. monocytogenes survived in both untreated and citrus extract-treated samples during the entire storage period, irrespective of the storage temperature. The higher concentration of citrus extract had a significant effect on the survival of L. monocytogenes in the treated samples, and reductions of 1.5-3.0 logs were noted on final day 70, 35 and 15 at 4, 10 and 21 °C, respectively. The results of our study demonstrated the potential of citrus extract as a natural compound that can control the growth of food-borne pathogenic bacteria, such as E. coli O157:H7 and L. monocytogenes in Tzatziki, a yogurt-based salad.

Growth kinetics of Escherichia coli O157:H7 on the epicarp of fresh vegetables and fruits.

Despite the increasing reports on the incidence of fresh vegetables and fruits as a possible vehicle for human pathogens, there is currently limited knowledge on the growth potential of Escherichia coli O157:H7 on different plant substrates. This study analyzed the selective adhesion and growth of E. coli O157:H7 on chili habanero (Capsicum chinense L.), cucumber (Cucumis sativus), radish (Raphanus sativus), tomato (Lycopersicon esculentum), beet (Beta vulgaris subsp. vulgaris), and onion (Allium cepa L.) under laboratory conditions. The Gompertz parameters were used to determine the growth kinetics. Scanning electron microscopy was used to visualize the adhesion of E. coli O157:H7 on the epicarp of the samples. Predictive models were constructed to compare the growth of E. coli O157:H7 on the samples with different intrinsic factors and to demonstrate the low selectivity of the pathogen. No significant difference was observed in the lag-phase duration (LPD), generation time (GT), and exponential growth rate (EGR) of the pathogen adhered to the samples. The interaction between the microorganism and the substrate was less supportive to the growth of E. coli O157:H7 for onion, whereas for tomato and cucumber, the time for the microorganism to attain the maximum growth rate (M) was significantly longer than that recorded for other samples.

Variations of Escherichia coli O157:H7 Survival in Purple Soils.

Escherichia coli O157:H7 is a well-recognized cause of human illness. Survival of Escherichia coli O157:H7 in five purple soils from Sichuan Province was investigated. The dynamics of E. coli O157:H7 survival in purple soils were described by the Weibull model. Results showed that this model is suitable to fit survival curves of E. coli O157:H7 in purple soils, with the calculated td value (survival time needed to reach the detection limit of 100 CFU·g-1) ranging from 2.99 days to 26.36 days. The longest survival time of E. coli O157:H7 was observed in neutral purple soils (24.49 days), followed by alkalescent purple soil (18.62 days) and acid purple soil (3.48 days). The redundancy analysis (RDA) revealed that td values were significantly enhanced by soil nutrition (total organic carbon (OC), total nitrogen (TN), available potassium (AK) and the ratio of humic acid to fulvic acid (Ha/Fa)), but were significantly suppressed by iron and aluminum oxide.

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest, after washing and simulated wholesale and retail distribution.

AIMS: To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0-25°C, dependent on season). METHODS AND RESULTS: Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near-harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g-1 . Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure-contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g-1 after processing and retail storage. Manure-contaminated leeks harboured 0·73-1·55 cfu E. coli O145 g-1 after washing and storage. There was no cross-contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure-contaminated carrots. There were five post-distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest. CONCLUSIONS: Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce. SIGNIFICANCE AND IMPACT: Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.

Impacts of pulsed electric field and heat treatment on quality and sensory properties and microbial inactivation of pomegranate juice.

Synergistic effects of pulsed electric field+mild heat on quality properties of pomegranate juice were modeled using the best-fit multiple (non-) linear regression models with inactivation kinetics parameters of Escherichia coli O157:H7 and Staphylococcus aureus. No significant difference was detected between the control and the treated samples in terms of pH; °Brix; total antioxidant capacity; total monomeric anthocyanin content; total ascorbic acid concentration; and the sensory properties of flavor, taste, aftertaste, and overall acceptance ( p > 0.05). An exposure of 65.3 J and 40 ℃ caused an increase on conductivity; titratable acidity; L*, a*, and b* values; and a decrease of browning index, total phenolic content, total antioxidant capacity, total monomeric anthocyanin content, total ascorbic acid concentration, and in the sensory properties of color and sourness in pomegranate juice. The goodness-of-fit for the best-fit multiple (non-) linear regression models in descending order belonged to E. coli O157:H7 (92.98%), S. aureus (84.06%), color a* (83.9%), titratable acidity (81.3%), color L* (78.5%), color b* (78.3%), conductivity (74.8%), total phenolic content (74.1%), and total ascorbic acid concentration (64.74%), respectively. De and ze values for E. coli O157:H7 and S. aureus ranged from 105.64 to 1093.25 and from 79.18 to 1057.73 µs with 27.39 and 30.80 J, consequently.

Combined Treatment with Low Concentrations of Aqueous and Gaseous Chlorine Dioxide Inactivates Escherichia coli O157:H7 and Salmonella Typhimurium Inoculated on Paprika.

Combined treatment with gaseous and aqueous chlorine dioxide (ClO2) was performed to improve the microbiological safety and quality of paprika. A single treatment of 50 ppmv ClO2 gas for 30 min decreased the populations of Escherichia coli O157:H7 and Salmonella Typhimurium by 2.33 and 2.91 log CFU/g, respectively. In addition, a single treatment of aqueous ClO2 (50 ppm) for 5 min decreased these populations by 1.86 and 1.37, respectively. The most dramatic effects were achieved by combined treatment of 50 ppm aqueous and gaseous ClO2 for 30 min, which decreased populations of E. coli O157:H7 and S. Typhimurium by 4.11 and 3.61 log CFU/g, respectively. With regard to the qualities of paprika, no adverse effects were elicited by the combined treatment. Thus, combined treatment with aqueous and gaseous ClO2 is a suitable approach that can be used to improve the microbial safety and quality of paprika.

Highly sensitive Escherichia coli shear horizontal surface acoustic wave biosensor with silicon dioxide nanostructures.

Surface acoustic wave mediated transductions have been widely used in the sensors and actuators applications. In this study, a shear horizontal surface acoustic wave (SHSAW) was used for the detection of food pathogenic Escherichia coli O157:H7 (E.coli O157:H7), a dangerous strain among 225 E. coli unique serotypes. A few cells of this bacterium are able to cause young children to be most vulnerable to serious complications. Presence of higher than 1cfu E.coli O157:H7 in 25g of food has been considered as a dangerous level. The SHSAW biosensor was fabricated on 64° YX LiNbO3 substrate. Its sensitivity was enhanced by depositing 130.5nm thin layer of SiO2 nanostructures with particle size lesser than 70nm. The nanostructures act both as a waveguide as well as a physical surface modification of the sensor prior to biomolecular immobilization. A specific DNA sequence from E. coli O157:H7 having 22 mers as an amine-terminated probe ssDNA was immobilized on the thin film sensing area through chemical functionalization [(CHO-(CH2)3-CHO) and APTES; NH2-(CH2)3-Si(OC2H5)3]. The high-performance of sensor was shown with the specific oligonucleotide target and attained the sensitivity of 0.6439nM/0.1kHz and detection limit was down to 1.8femto-molar (1.8×10-15M). Further evidence was provided by specificity analysis using single mismatched and complementary oligonucleotide sequences.

Assessing the growth of Escherichia coli O157:H7 and Salmonella in spinach, lettuce, parsley and chard extracts at different storage temperatures.

AIMS: The objective of this work was to study the growth potential of Escherichia coli O157:H7 and Salmonella spp. in leafy vegetable extracts at different temperature conditions. METHODS AND RESULTS: Cocktails of five strains of E. coli O157:H7 and of Salmonella enterica were used. Inoculated aqueous vegetable extracts were incubated at 8, 10, 16 and 20°C during 21 days. Microbial growth was monitored using Bioscreen C(®) . In spinach extract, results showed that for E. coli O157:H7 and Salmonella significant differences (P < 0·05) for µabs (maximum absorbance rate) were obtained. For both pathogens, growth in chard was slightly lower. In contrast, iceberg lettuce and parsley showed the lowest values of µabs , below 0·008 h(-1) . The coefficients of variance (CoV) calculated for the different replicates evidenced that at low temperature (8°C) a more variable behaviour of both pathogens is expected (CoV > 180%). CONCLUSIONS: This study provides evidence that aqueous extracts from vegetable tissues can result in distinct growth niche producing different response in various types of vegetables. SIGNIFICANCE AND IMPACT OF THE STUDY: Finally, these results can be used as basis to establish risk rankings of pathogens and leafy vegetable matrices with relation to their potential growth.

A large Great Britain-wide outbreak of STEC O157 phage type 8 linked to handling of raw leeks and potatoes.

Between December 2010 and July 2011, 252 cases of STEC O157 PT8 stx1 + 2 infection were reported in England, Scotland and Wales. This was the largest outbreak of STEC reported in England and the second largest in the UK to date. Eighty cases were hospitalized, with two cases of haemolytic uraemic syndrome and one death reported. Routine investigative data were used to generate a hypothesis but the subsequent case-control study was inconclusive. A second, more detailed, hypothesis generation exercise identified consumption or handling of vegetables as a potential mode of transmission. A second case-control study demonstrated that cases were more likely than controls to live in households whose members handled or prepared leeks bought unwrapped [odds ratio (OR) 40, 95% confidence interval (CI) 2·08-769·4], and potatoes bought in sacks (OR 13·13, 95% CI 1·19-145·3). This appears to be the first outbreak of STEC O157 infection linked to the handling of leeks.

Determining the potential link between irrigation water quality and the microbiological quality of onions by phenotypic and genotypic characterization of Escherichia coli isolates.

The potential transfer of human pathogenic bacteria present in irrigation water onto fresh produce was investigated, because surface water sources used for irrigation purposes in South Africa have increasingly been reported to be contaminated with enteric bacterial pathogens. A microbiological analysis was performed of a selected river in Limpopo Province, South Africa, that is often contaminated with raw sewage from municipal sewage works and overhead irrigated onions produced on a commercial farm. Counts of Escherichia coli, coliforms, aerobic bacteria, fungi, and yeasts and the prevalence of E. coli O157:H7, Salmonella, and Listeria monocytogenes were determined. Identities of bacterial isolates from irrigation water and onions were confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry, PCR, and biochemical tests. To establish a potential link between the microbiological quality of the irrigation source and the onions, the E. coli isolates from both were subjected to antibiotic resistance, virulence gene, and enterobacterial repetitive intergenic consensus PCR analyses. River water E. coli counts exceeded South African Department of Water Affairs and World Health Organization irrigation water guidelines. Counts of aerobic bacteria, coliforms, fungi, and yeasts of onions from the market were acceptable according to Department of Health Directorate, Food Control, South Africa, microbiological guidelines for ready-to-eat fresh fruits and vegetables. E. coli O157:H7, Salmonella, and L. monocytogenes were not detected in onions, whereas only Salmonella was detected in 22% of water samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and PCR identification of E. coli isolates from water and onions correlated. Of the 45 E. coli isolates from water and onions, 42.2% were resistant to multiple antibiotics. Virulence genes eae, stx1, and stx2 were detected in 2.2, 6.6, and 2.2% of the E. coli isolates, respectively. Phenotypic (antimicrobial) and genotypic (virulence gene prevalence, DNA fingerprinting) analyses showed a link between river, dam, irrigation pivot point, and onion E. coli isolates.

Comparison of the microbiological quality of environmentally friendly and conventionally grown vegetables sold at retail markets in Korea.

Fresh produce is usually eaten raw without cooking or heating, which may increase the probability of foodborne infection. The microbiological quality of 11 types of fresh, raw vegetables (romaine lettuce, sesame leaves, crown daisy, garlic chives, iceberg lettuce, cabbage, broccoli, leek, chili pepper, capsicum, and zucchini) purchased at retail markets in Iksan, Korea as affected by cultivation method (environmentally friendly vegetables [organic, pesticide-free, and low-pesticide vegetables] and conventionally grown vegetables) and harvest season was determined. Escherichia coli O157:H7 and Salmonella were not detected in all samples of vegetables tested. Aerobic mesophiles (>6 log cfu/g) were detected in environmentally friendly romaine lettuce and crown daisy and environmentally friendly and conventionally grown garlic chives, which also contained coliforms (>3 log cfu/g). Sesame leaf and crown daisy (regardless of cultivation method), as well as conventionally grown romaine lettuce and leek, contained >1 log cfu/g of E. coli. The overall microbiological quality of environmentally friendly and conventionally grown vegetables was not significantly different (P > 0.05). However, there were seasonal effects on populations of coliforms and generic E. coli on vegetables. The greatest numbers of microorganisms were isolated from environmentally friendly or conventionally grown vegetables purchased in winter. The vegetables, regardless of cultivation method or season, should be subjected to appropriate antimicrobial treatment to enhance their microbial safety.

Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products.

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10(2) CFU/ml (1.2 × 10(2) CFU/ml for S. Typhimurium, 4.0 × 10(2) CFU/ml for E. coli O157:H7 and 5.4 × 10(2) CFU/ml for L. monocytogenes) in pure culture and 10(3) CFU/g (5.1 × 10(3) CFU/g for S. Typhimurium, 7.5 × 10(3) CFU/g for E. coli O157:H7 and 8.4 × 10(3) CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use.

A nanoporous membrane-based impedimetric immunosensor for label-free detection of pathogenic bacteria in whole milk.

We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody-pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1 kHz versus concentration of E. coli O157:H7 (10-10(5)cfu/ml) was obtained, and the detection limit found to be as low as 10 cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7 cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics.

Salmonella and Escherichia coli O157:H7 survival in soil and translocation into leeks (Allium porrum) as influenced by an arbuscular mycorrhizal fungus (Glomus intraradices).

A study was conducted to determine the influence of arbuscular mycorrhizal (AM) fungi on Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) in autoclaved soil and translocation into leek plants. Six-week-old leek plants (with [Myc+] or without [Myc-] AM fungi) were inoculated with composite suspensions of Salmonella or EHEC at ca. 8.2 log CFU/plant into soil. Soil, root, and shoot samples were analyzed for pathogens on days 1, 8, 15, and 22 postinoculation. Initial populations (day 1) were ca. 3.1 and 2.1 log CFU/root, ca. 2.0 and 1.5 log CFU/shoot, and ca. 5.5 and 5.1 CFU/g of soil for Salmonella and EHEC, respectively. Enrichments indicated that at days 8 and 22, only 31% of root samples were positive for EHEC, versus 73% positive for Salmonella. The mean Salmonella level in soil was 3.4 log CFU/g at day 22, while EHEC populations dropped to ≤ 0.75 log CFU/g by day 15. Overall, Salmonella survived in a greater number of shoot, root, and soil samples, compared with the survival of EHEC. EHEC was not present in Myc- shoots after day 8 (0/16 samples positive); however, EHEC persisted in higher numbers (P = 0.05) in Myc+ shoots (4/16 positive) at days 15 and 22. Salmonella, likewise, survived in statistically higher numbers of Myc+ shoot samples (8/8) at day 8, compared with survival in Myc- shoots (i.e., only 4/8). These results suggest that AM fungi may potentially enhance the survival of E. coli O157:H7 and Salmonella in the stems of growing leek plants.