Inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium in manure-amended soils studied in outdoor lysimeters

The inactivation of Escherichia coli O157:H7 (CCUG 44857) and Salmonella enterica serovar Typhimurium was investigated in two agricultural soils (sandy loam and silty clay) amended with poultry manure, cattle manure slurry or human urine. The study was performed in soil lysimeters placed outdoors, and was repeated over two consecutive years. The amendments, inoculated with E. coli O157:H7 and Salmonella Typhimurium, were mixed with soil on the top of the lysimeters. Samples were collected from the top 5-cm layer of each lysimeter at regular intervals, and the inactivation was monitored over 6 months, by the plate spread method and by enrichment. The inactivation was modelled by fitting a non-linear model to the data, and pathogen reduction times were calculated (90 and 99% reduction). The results showed that the inactivation of E. coli O157:H7 and Salmonella Typhimurium varied depending on the manure type used and its carbon content. The longest inactivation time occurred in samples amended with poultry manure, in which both E. coli O157:H7 and Salmonella Typhimurium were detected up to day 90 with the spread plate method. The most rapid inactivation for both pathogens occurred in soil amended with urine. However, low amounts of culturable E. coli O157:H7 and Salmonella Typhimurium were detected by enrichment throughout the study period (180 days), regardless of manure type.     

Volatile compounds from Escherichia coli O157:H7 and their absorption by strawberry fruit

Volatile compounds emitted by cultures of two strains of the pathogenic bacterium Escherichia coliO157:H7 and a nonpathogenic strain of E. coli were trapped on Super-Q porous polymer and identified by GC-MS. The predominant compound produced by all three strains was indole with lesser amounts of other components including methyl ketones, 2-heptanone, 2-nonanone, 2-undecanone, and 2-tridecanone. The vapor-phase profiles of these strains were similar for most chemicals identified but differed with regard to ketones. Strawberry fruit was shown to be a suitable host for E. coli O157:H7 with the population of the bacterium either increasing or remaining stable after 3 days depending on inoculation level. Headspace analysis of the volatile compounds from inoculated fruit yielded no detectable quantity of indole. Strawberry fruit readily absorbed indole and other volatile compounds produced by the bacteria and in some cases metabolized the compounds to new volatile products. Thus, headspace "marker" compounds indicating possible bacterial contamination of fruit were largely removed from the vapor phase by the strawberries.     

实时荧光定量PCR方法检测大肠杆菌O157:H7

针对大肠杆菌(Escherichia coli )O157:H7的致病基因eae设计了特异性引物，并用荧光染料SYBR GreenⅠ 进行了实时定量聚合酶链式反应（Real-time PCR）。熔解曲线显示产物特异性较强，无非目的条带和二聚体产生。并对反应程序进行了优化，确定了最佳的反应程序，最终在合适的模板浓度内得出了标准曲线。结果显示Real-time PCR 比普通PCR灵敏1 000倍。     

Storage stability and antibacterial activity against Escherichia coli O157:H7 of carvacrol in edible apple films made by two different casting methods

The antimicrobial activities against Escherichia coli O157:H7 as well as the stability of carvacrol, the main constituent of oregano oil, were evaluated during the preparation and storage of apple-based edible films made by two different casting methods, continuous casting and batch casting. Antimicrobial assays of films and high-performance liquid chromatography (HPLC) analysis of film extracts following storage up to 49 days at 5 and 25 degrees C revealed that (a) optimum antimicrobial effects were apparent with carvacrol levels of approximately 1.0% added to the purees prior to film preparation, (b) carvacrol in the films and film weights remained unchanged over the storage period of up to 7 weeks, and (c) casting methods affected carvacrol concentration, bactericidal activity, physicochemical properties, and colors of the apple films. Carvacrol addition to the purees used to prepare the films reduced water vapor and oxygen permeability of apple films. The results indicate that carvacrol has a dual benefit. It can be used to both impart antimicrobial activities and enhance barrier properties of edible films. The cited observations facilitate relating compositional and physicochemical properties of apple puree films containing volatile plant antimicrobials to their use in foods.     

Fate of chlorate present in cattle wastes and its impact on Salmonella Typhimurium and Escherichia coli O157:H7

Chlorate salts are being developed as a feed additive to reduce the numbers of pathogens in feedlot cattle. A series of studies was conducted to determine whether chlorate, at concentrations expected to be excreted in urine of dosed cattle, would also reduce the populations of pathogens in cattle wastes (a mixture of urine and feces) and to determine the fate of chlorate in cattle wastes. Chlorate salts present in a urine-manure-soil mixture at 0, 17, 33, and 67 ppm had no significant effect on the rates of Escherichia coli O157:H7 or Salmonella Typhimurium inactivation from batch cultures. Chlorate was rapidly degraded when incubated at 20 and 30 degrees C with half-lives of 0.1 to 4 days. Chlorate degradation in batch cultures was slowest at 5 degrees C with half-lives of 2.9 to 30 days. The half-life of 100 ppm chlorate in an artificial lagoon system charged with slurry from a feedlot lagoon was 88 h. From an environmental standpoint, chlorate use in feedlot cattle would likely have minimal impacts because any chlorate that escaped degradation on the feedlot floor would be degraded in lagoon systems. Collectively, these results suggest that chlorate administered to cattle and excreted in wastes would have no significant secondary effects on pathogens present in mixed wastes on pen floors. Lack of chlorate efficacy was likely due to low chlorate concentrations in mixed wastes relative to chlorate levels shown to be active in live animals, and the rapid degradation of chlorate to chloride at temperatures of 20 degrees C and above.     

Plant extracts, spices, and essential oils inactivate Escherichia coli O157:H7 and reduce formation of potentially carcinogenic heterocyclic amines in cooked beef patties

Meats need to be heated to inactivate foodborne pathogens such as Escherichia coli O157:H7. High-temperature treatment used to prepare well-done meats increases the formation of carcinogenic heterocyclic amines (HCAs). We evaluated the ability of plant extracts, spices, and essential oils to simultaneously inactivate E. coli O157:H7 and suppress HCA formation in heated hamburger patties. Ground beef with added antimicrobials was inoculated with E. coli O157:H7 (10(7) CFU/g). Patties were cooked to reach 45 °C at the geometric center, flipped, and cooked for 5 min. Samples were then taken for microbiological and mass spectrometry analysis of HCAs. Some compounds were inhibitory only against E. coli or HCA formation, while some others inhibited both. Addition of 5% olive or apple skin extracts reduced E. coli O157:H7 populations to below the detection limit and by 1.6 log CFU/g, respectively. Similarly, 1% lemongrass oil reduced E. coli O157:H7 to below detection limits, while clove bud oil reduced the pathogen by 1.6 log CFU/g. The major heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were concurrently reduced with the addition of olive extract by 79.5% and 84.3% and with apple extract by 76.1% and 82.1%, respectively. Similar results were observed with clove bud oil: MeIQx and PhIP were reduced by 35% and 52.1%, respectively. Addition of onion powder decreased formation of PhIP by 94.3%. These results suggest that edible natural plant compounds have the potential to prevent foodborne infections as well as carcinogenesis in humans consuming heat-processed meat products.     

Response of Escherichia coli O157:H7 survival to pH of cultivated soils

Purpose

The Escherichia coli (E. coli) O157:H7 survival dynamics in original and pH-modified agricultural soils were investigated to determinate how E. coli O157:H7 survival responded to the pH values of different soils, identify the relationships between E. coli O157:H7 survival time (t _d ) and soil properties, and assess the potential pathogen contamination after soil pH changed.

Materials and methods

The six soil samples were collected from different provinces of China, and 18 pH-modified soil samples were obtained from original soils by treating the original soils with direct electric current. The E. coli O157:H7 cells were inoculated into 24 soils and incubated at soil moisture of ?33 kPa and 25 °C. The soils were sampled for determining the numbers of E. coli O157:H7 at given time intervals over the incubation. The effects of soil pH change and other properties on the t _d values were analyzed.

Results and discussion

The t _d values in the test soils were between 7.1—24.7 days. Results indicate that soil pH, texture, and free Fe₂O₃ (Fe_d) were the most important factors impacting the t _d values in the test soils. Further, the response of E. coli O157:H7 survival to pH change varied with different soils. In the acidic soils (shorter t _d values), the t _d values decreased as the pH decreased and Fe_d increased, while in the neutral or alkaline soils (pH?≥?6.45, longer t _d values), the t _d values did not change significantly with pH.

Conclusions

The changes of amorphous and free sesquioxides induced by pH change might strengthen the response of E. coli O157:H7 survival to soil pH. Closer attention should be paid to E. coli O157:H7 long survival in soils and its potential environmental contamination risk.     

Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Humic acids are ubiquitous and abundant in terrestrial environments; therefore, they are often co-extracted with nucleic acids and interfere with quantitative PCR (qPCR) assays. In this study a recently developed NanoGene assay that is resistant to interference by humic acids was evaluated for gene detection in soil samples. The NanoGene assay utilizes a combination of magnetic beads, dual quantum dots labels, and DNA hybridization in solution. Seven soil samples containing different amounts of organic matter were tested to compare NanoGene and qPCR assays for their respective ability to detect a bacterial pathogen. We spiked the soils with Escherichia coli O157:H7, extracted genomic DNA, and conducted NanoGene and qPCR assays targeting the E. coli O157:H7-specific eaeA gene. To prevent the inhibition of PCR that is common when using DNA extracted from soils, we used a range of template DNA concentrations and BSA addition in the qPCR assay. Compared to the qPCR assay the NanoGene assay was significantly more resistant to the inhibitory effect of humic acids, successfully quantifying the eaeA gene within a linear (R² = 0.99) range of 10⁵ through 10⁸ CFU/g soil for all seven soil samples tested. In contrast, the qPCR assay was significantly inhibited using the same template DNA isolated from soils containing a range of organic content (2.0%–12%). Interestingly, the qPCR assay was still inhibited despite additional purification steps, suggesting that humic acids were still associated with DNA at a level that was inhibitory to qPCR. This study demonstrated that the NanoGene assay is suitable for quantitative gene detection in diverse soil types and is not susceptible to inhibition by humic acids and other organic compounds that commonly lead to false negative results in qPCR assays.     

Effect of Molasses on Regrowth of E. coli O157:H7 And Salmonella in Compost Teas

Compost water extracts (compost teas) are gaining popularity among organic growers, largely because of their disease suppressive activity when applied to foliage or soil. Production methods often include addition of supplemental constituents, particularly molasses, to stimulate plant-beneficial microbial populations. We have found that molasses amendments also favor regrowth of human pathogenic bacteria, raising public health concerns about potential contamination of treated crops, particularly produce intended for fresh consumption. Using disease outbreak strains marked with green fluorescent protein (GFP) and spontaneous antibiotic-resistance, we found that regrowth of Salmonella enterica serovar Thompson and Escherichia coli O157:H7 was positively correlated with molasses concentration. For Salmonella, regrowth was also dependent on the type of starter compost material used. Salmonella populations increased from 1 at time 0 to over 1000 CFU ml^?1 in dairy manure compost tea with 1% molasses, and from 1 at time 0 to over 350,000 CFU ml^?1 in chicken manure compost tea by 72 h. E. coli populations increased from 1 at time 0 to approximately 1000 CFU ml^?1 in both types of tea by 72 h. Pathogen regrowth did not occur when molasses was eliminated or kept to 0.2%.     

Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl

Thirty-four kinds of citrus essential oils and their components were investigated for radical-scavenging activities by the HPLC method using 1,1-diphenyl-2-picrylhydrazyl (DPPH). To examine the oils' relative radical-scavenging activities compared with that of a standard antioxidant, Trolox was employed. All of the essential oils were found to have scavenging effects on DPPH in the range of 17. 7-64.0%. The radical-scavenging activities of 31 kinds of citrus essential oils were comparable with or stronger than that of Trolox (p < 0.05). The oils of Ichang lemon (64.0%, 172.2 mg of Trolox equiv/mL), Tahiti lime (63.2%, 170.2 mg of Trolox equiv/mL), and Eureka lemon (61.8%, 166.2 mg of Trolox equiv/mL) were stronger radical scavengers than other citrus oils. Citrus volatile components such as geraniol (87.7%, 235.9 mg of Trolox equiv/mL), terpinolene (87.4%, 235.2 mg of Trolox equiv/mL), and gamma-terpinene (84.7%, 227.9 mg of Trolox equiv/mL) showed marked scavenging activities on DPPH (p < 0.05).     

Glycopeptide derived from hen egg ovomucin has the ability to bind enterohemorrhagic Escherichia coli O157:H7

Ovomucin glycopeptide (OGP) was prepared by size exclusion chromatography after Pronase digestion of hen egg ovomucin, and the binding of OGP to foodborne pathogens (Bacillus cereus,Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, and Staphylococcus aureus) was investigaed. Binding assays with biotinylated bacteria as probes in microtiter plates showed that OGP bound to only E. coli O157:H7 among these foodborne pathogens. Periodate treatment markedly reduced the binding ability, indicating that E. coli O157:H7 bound to carbohydrate moieties of OGP. Lectin blot analysis with Maackia amurensis (MAA) and Sambucus nigra (SNA), which are specific for oligosaccharides containing sialic acid, revealed their binding sites in OGP were similar to the E. coli O157:H7 binding sites that were probed with biotinylated E. coli O157:H7 after Western blotting of OGP. Sialydase treatment of OGP abolished its ability to bind E. coli O157:H7, demonstrating that sialic acid played an important role in the binding. These results suggest that OGP has E. coli O157:H7-specific binding sites that consist of sialic acid. On the basis of these properties, OGP has the potential to be an ingredient with a protective effect against E. coli O157:H7 infection and to be a novel probe for the detection of E. coli O157:H7 in the food hygiene field.     

Sherlock MIS系统鉴定土壤中大肠杆菌O157∶H7的研究

<正>污水灌溉和人畜粪肥施用一直是国内外农业中有机废弃物资源化利用的重要途径,这种现象在我国尤为普遍。然而,生活污水含有大量细菌,工业和医院废水更是富含各种病原体,污泥、垃圾和粪肥均可能携带大量病原微生物和寄生虫卵。显而易见的是,这些有害生物种群可随污水灌溉和粪肥施用进入环境中,并通过污染土壤、水源、大气和农畜产品等途径传播或"引燃"疫情的爆发,从而对     

A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria

A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.     

Physiological response and protein expression under acid stress of Escherichia coli O157:H7 TWC01 isolated from Taiwan

Escherichia coli O157:H7 has an unusually high resistance to acidic environments. Some research has revealed that acid-adapted cells, by exposure to moderately acidic conditions, are more resistant to a subsequent strong acidic challenge or other stress. This study was conducted to understand the protein expression regulation of acid tolerance response (ATR) of a local isolated E. coli O157:H7 TWC01 (TWC01) induced by an acidic environment. TWC01 cells were acid adapted by using hydrochloric acid (HCl) or lactic acid as acidifier to induce ATR. The total proteins of adapted cells were extracted for proteomic analysis and protein identification by matrix-assisted laser desorption ionization quadrupole time-of-flight tandem mass spectrometry (MALDI-Q-TOF MS/MS). Furthermore, the effects of acid adaptation on shiga-like toxin (stx) secretion were examined. Results revealed that acid adaptation depressed stx production of E. coli O157:H7 TWC01 during adaptation and did not improve post-stress toxin production. Image analysis of the gel indicated that numerous proteins were up-regulated and that lactic acid had a greater effect than HCl did (percentages of up-regulated proteins were 57.64 and 35.47%, respectively). Analysis of proteins by mass spectrometry revealed that most of the up-regulated proteins were metabolism-related, including phosphoglycerate kinase (PGK), glutamate decarboxylases alpha and beta (GadA, GadB), adenine phosphoribosyltransferase (APRT), and dihydrodipicolinate synthase (DHDPS). Others were related to translation (e.g., elongation factor Tu, elongation factor G), protein folding (e.g., alkyl hydroperoxide reductase), and membrane proteins (e.g., ompA precursor and ompR). The variation of protein expression showed that acid resistance was induced in TWC01 and was primarily manifested via expression of up-regulated proteins that contribute to increased energy conservation and polypeptide synthesis.     

蚯蚓粪中乳酸菌的分离及其在大肠杆菌O157：H7中的抗菌活性

本研究利用SL培养基从蚯蚓粪中分离到54株具有产酸性能的菌株,并以E.coli O157：H7（EDL933株）作为指示菌株,采用点种法检测分离菌株的抑菌活性。结果表明其中6个菌株对指示菌具有拮抗作用,通过形态特征,结合16S rDNA序列分析,初步鉴定该6个菌株分别为食物魏斯特菌（Listeria welshimeri）、乳酸片球菌（Pediococcus acidilactici）、短乳杆菌（Lactobacillus brevis）和格氏乳球菌（Lactococcus garvieae）。分离到的乳酸菌对E.coli O157：H7（EDL933株）具有显著的抑制作用,发酵温度和初始pH值影响发酵液的抑菌作用,优化环境因子可以促进拮抗菌对E.coli O157：H7的抑制作用。本研究为进一步分离抗菌产物用于人畜共患病的预防和治疗提供了理论依据。     

Acaricidal activity of pine essential oils and their main components against Tyrophagus putrescentiae,a stored food mite

Some essential oils obtained from the branches of four Pinus species (P. pinea L., P. halepensis Mill., P. pinaster Soil in Ait., and P. nigra Arnold) have been evaluated for their acaricidal activity by aerial diffusion against the stored food mite Tyrophagus putrescentiae (L.). All the essential oils showed a good efficacy, but P. pinea oil and its two constituents 1,8-cineole and limonene were the most effective compounds, showing 100% acaricidal activity at 8 microL; 1,8-cineole showed the same activity at 6 microL.     

S. typhimurium and E. coli O157:H7 retention and transport in agricultural soil during irrigation practices

During animal waste agricultural applications, the major concern is pathogen spreading, which may contaminate surface water and groundwater. Among the pathogenic microorganisms found in animal waste, Salmonella typhimurium and Escherichia coli O157:H7 are of particular concern. When transported in sub‐surface agricultural soil, S. typhimurium and E. coli O157:H7 are captured at the air–water–sediment interfaces through physical interactions. Because in situ colloids contribute to the formation of air–water–sediment, their mobilization affects the transport of S. typhimurium and E. coli O157:H7. The impact of irrigation rates on in situ colloid mobilization and S. typhimurium and E. coli O157:H7 transport was investigated in intact soil columns collected from an agricultural site in Gadsden County of Florida, USA. The columns were irrigated with sterilized nano‐pure deionized water to mobilize the colloids in the soil by stepwise increases in flow rate. For each flow rate, after colloids were mobilized and steady state was reached, S. typhimurium and E. coli O157:H7 were introduced. The cumulative amount of released in situ colloids increased linearly with the irrigation rates (R² = 0.986–0.996) and transport of the bacteria was enhanced after colloid mobilization. Interactions of the bacteria with the sediments and the air‐water interface were characterized: these played an important role in controlling S. typhimurium and E. coli O157:H7 retention in soil.     

Mechanical, barrier, and antimicrobial properties of apple puree edible films containing plant essential oils

Edible films, as carriers of antimicrobial compounds, constitute an approach for incorporating plant essential oils (EOs) onto fresh-cut fruit surfaces. The effect against Escherichia coli O157:H7 of oregano, cinnamon, and lemongrass oils in apple puree film-forming solution (APFFS) and in an edible film made from the apple puree solution (APEF) was investigated along with the mechanical and physical properties of the films. Bactericidal activities of APFFS, expressed as BA50 values (BA50 values are defined as the percentage of antimicrobial that killed 50% of the bacteria under the test conditions) ranged from 0.019% for oregano oil to 0.094% for cinnamon oil. Oregano oil in the apple puree and in the film was highly effective against E. coli O157:H7. The data show that (a) the order of antimicrobial activities was oregano oil > lemongrass oil > cinnamon oil and (b) addition of the essential oils into film-forming solution decreased water vapor permeability and increased oxygen permeability, but did not significantly alter the tensile properties of the films. These results show that plant-derived essential oils can be used to prepare apple-based antimicrobial edible films for various food applications.     

Inhibitory effects of citrus essential oils and their components on the formation of N-nitrosodimethylamine

Twenty-eight kinds of citrus essential oils and their components were studied for inhibitory effects on the formation of N-nitrosodimethylamine (NDMA). The reaction mixture consisted of dimethylamine and sodium nitrite adjusted at pH 3.6, in addition to essential oils and an emulsifying agent. The quantification was determined by high-performance liquid chromatography monitored at 220 nm. All of the essential oils inhibited the formation of NDMA in the range of 20-85%. The oils of ujukitsu (Citrus ujukitsu Hort. ex Shirai), yuzu (C. junos Tanaka), mochiyu (C. inflata Hort. ex Tanaka), and ponkan (C. reticulata Blanco cv. F-2426) inhibited the formation of NDMA much more effectively than other citrus oils. The inhibitory proportions of components of citrus essential oils such as myrcene, alpha-terpinene, and terpinolene were as high as 80%.     

Fate of Escherichia coli and Escherichia coli O157 in soils and drainage water following cattle slurry application at 3 sites in southern Scotland

Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 10⁴ to 5.7 × 10⁵ colony forming units per mL (c.f.u. mL^–1) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (10³ to 10⁴ c.f.u. mL^–1). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 10⁵) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL^–1 for much of the sampling period after slurry application (1–3 months).