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。     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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。     

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.     

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.     

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%.     

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.     

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.     

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.     

Potential health risks associated with the persistence of Escherichia coli O157 in agricultural environments

Abstract. Escherichia coli serotype O157 is a virulent human pathogen the global incidence of which has increased. It has been demonstrated that cattle are the primary reservoir of this pathogen. This has serious implications for the land-based disposal of organic wastes such as cattle manure, cattle slurry and abattoir waste. Further, it also has serious ramifications for the protection of surface and groundwater drinking supplies and public access to pasture land. However, while soil and vegetation can be expected to directly influence the survival of this pathogen, there is a paucity of information concerning the behaviour and survival of E. coli O157 in agricultural environments. It appears that E. coli O157 presently contaminates between 1 to 15% of UK cattle herds, depending on region, and that faecal excretion of the bacterium shows a distinct seasonality which also reflects the incidence of human infections. E. coli O157 can remain viable in soil for greater than 4 months and appears to be a highly resilient pathogen possessing the capability to adapt easily to environmental stresses. While most human cases of E. coli O157 related food poisoning have been associated with the consumption of contaminated meat and dairy products, there is also evidence that human infection has occurred through the ingestion of contaminated soil, fruit and vegetables and drinking water. In this review the potential threat to human health posed by the application of contaminated organic wastes to soil and possible strategies for reducing the amount of pathogen entering the food chain are highlighted.     

Survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in various soil particles: importance of the attached bacterial phenotype

The risk of enteropathogens to food and water is highly dependent on their survival in soil environments. Here, the effects of soil type, particle size, the presence of natural organic matter (NOM) or Fe/Al (hydro)oxides on pathogenic Escherichia coli O157:H7 survival in sterilized soil particles were assessed through survival, attachment, metabolic activity, and qRT-PCR analyses. The abundance of inoculated E. coli O157:H7 in Brown soil (Alfisol) particles increased 0.6–1.4 log₁₀ CFU/g within 3 days (except for NOM-stripped clay), while that in Red soil (Ultisol) particles decreased rapidly in 8 days post-inoculation. Additionally, survival of bacteria was significantly enhanced when Fe/Al (hydro)oxides had been removed from Red soil particles. For the two soils, E. coli O157:H7 survived the longest in NOM-present clays and the bacterial adenosine 5′-triphosphate (ATP) levels were 0.7–2.0 times greater in clays than in sands and silts on day 8. Moreover, clays were more effective than silts and sands in binding cells and changing the expressions of acetate pathway-associated genes (pta and ackA). For silts and sands, E. coli O157:H7 decayed more rapidly in the presence of NOM and similar trends of bacterial ATP levels were observed between NOM-stripped and NOM-present soil particles, indicating that the primary role of NOM was not as a nutrient supply. These findings indicate that soil particles function mainly through attachment to change the metabolic pathway of E. coli O157:H7 and ultimately impact the survival of bacterial pathogens in soils.     

苏云金芽胞杆菌类ABC transporter基因zwa-FEG能提高对双效菌素的抗性

双效菌素（Zwittermicin A,ZwA）是一种氨基多元醇类新型广谱抗生素,最初是从蜡状芽胞杆菌(Bacillus cereus) UW85中发现的,zmaR为它的抗性基因。在本实验室已测得的苏云金芽胞杆菌（Bacillus thuringiensis）YBT-1520菌株全基因组序列中有ZwA合成基因簇完整序列,分析发现在其末端存在一个类似ABC transporter的序列（命名为zwa-FEG）。将zwa-EFG在大肠杆菌（Escherichia coli）中表达,大肠杆菌能获得对ZwA的抗性;将zwa-EFG转移到产ZwA的菌株UW85中,能使其ZwA产量显著提高。推测zwa-EFG是一种ZwA专一性的ABC transporter,能将ZwA分泌到胞外,从而增强ZwA的合成和对ZwA的抗性。     

大肠杆菌猪水肿毒素B亚单位基因的克隆与鉴定

根据已知大肠杆菌志贺氏菌样毒素Ⅱ型变异体Ｂ亚单位（ＳＬＴ－ＩＩｖＢ）基因序列,设计并合成３条２对引物,以大肠杆菌Ｏ１３８基因组为模板,通过聚合酶链式反应（ＰＣＲ）分别扩增出２０４ ｂｐ的基因序列及包含一段信号肽的２６１ ｂｐ基因序列。将这两段ＤＮＡ分别克隆进表达性载体质粒ＰＹＡ３３３４的ＥｃｏＲⅠ和ＢａｍＨⅠ位点之间,经地高辛标记探针进行Ｓｏｕｔｈｅｒｎ杂交和酶切分析鉴定,并对两条扩增片段进行序列