特异性抗肠出血性大肠杆菌O157:H7内毒素鸡卵黄抗体的制备

目的:比较研究LPS和O-SP的免疫原性,制备出特异性抗内毒素(LPS)鸡卵黄免疫球蛋白(eggyolkimmunoglobulin,IgY),用于对E.coliO157:H7的免疫预防及检测。方法:分别用灭活E.coliO157:H7菌体、不同浓度内毒素(LPS)及O-特异性多糖链(O-SP)与不完全弗氏佐剂充分乳化后作抗原免疫临产蛋母鸡,以水稀释法从卵黄中提取抗体,阴离子交换色谱法实现对抗体的纯化,间接ELISA及双向免疫扩散检测抗体产生效价与活性。结果与结论:所制内毒素(LPS)和O-SP均有较好的免疫原性,刺激鸡体后可产生高效价抗体,灭活菌体、600μg/mlLPS、1200μg/mlLPS、2000μg/mlLPS、O-SP抗体产生效价最高可分别达1:32000、1:28000、1:32000、1:12000、1:40000。结合离子交换色谱,用0.185mol/L的离子强度可实现一步洗脱纯化抗体,制备出高纯度、高活性、特异性强的鸡卵黄免疫球蛋白IgY,为大肠杆菌O157:H7的感染防治提供了可靠的保证,在此实验基础之上可进一步探讨应用特异性IgY对大肠杆菌O157:H7的检测。     

Efficacy of Ozone Against Escherichia coli O157:H7 on Apples

Apples were inoculated with Escherichia coli O157:H7 and treated with ozone. Sanitization treatments were more effective when ozone was bubbled during apple washing than by dipping apples in pre‐ozonated water. The corresponding decreases in counts of E. coli O157:H7 during 3‐min treatments were 3.7 and 2.6 log₁₀ CFU on apple surface, respectively, compared to < 1 log₁₀ CFU decrease in the stem‐calyx region in both delivery methods. Optimum conditions for decontamination of whole apples with ozone included a pretreatment with a wetting agent, followed by bubbling ozone for 3 min in the wash water, which decreased the count of E. coli O157:H7 by 3.3 log₁₀CFU/g.     

食品中大肠杆菌O157:H7控制新技术研究进展

论述了杀菌剂清洗技术、高压脉冲电场技术、高密度CO2技术、超高压技术和辐照技术等新技术的原理及其在控制食品中大肠杆菌O157:H7应用方面的研究进展。展望了未来该研究领域的发展趋势。     

Escherichia coli O157: H7 Growth in Laboratory Media as Affected by Phenolic Antioxidants

Five strains of Escherichia coli O157:H7 with ATCC 11775 E. coli were grown in brain heart infusion (BHI) broth (pH 5.8, adjusted with citric acid) and treated with butylated hydroxyanisole (BHA), butylated hy-droxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate (PG) individually or combined. Additives ranged from 100–400 ppm with inocula levels between 5 and 104 CFU/mL in tissue culture plates or in flasks; samples were incubated at 4°C or 37°C for 24 hr. Additive antimicrobial efficacy varied with inoculum level and incubation temperature. BHA at <200 ppm was bactericidal on all strains. Poly-hydroxyl additives (TBHQ, PG) were less effective at 4°C. BHA-BHT combinations were synergistic at 4°C.     

Inactivation of E. coli O157:H7 Using a Pulsed Nonthermal Plasma System

ABSTRACT: The use of elevated temperatures to pasteurize food products leads to a loss of food quality, which stimulates research into novel nonthermal processes for food preservation. Using nonthermal plasma discharges, applied directly into the food product, proved effective in reducing the number of Escherichia coli O157:H7 cells in apple juice, by up to 7 log units. The pulsed nonthermal plasma system was developed and used to study the effects of different process parameters on the inactivation. The process parameters considered were discharge voltage, pulse number, and pulse frequency. The apple juice used had a pH of 4 and a conductivity of 2.29 mS/cm.     

生物传感器检测食源性大肠杆菌O157:H7研究新进展

近年来, 生物传感器因具有快速、简便、灵敏度高、低成本等优势被广泛应用到临床检测、环境监测等领域。该技术在食品安全领域也逐步得到重视, 尤其在病原微生物的快速检测方面。本文从免疫识别和核酸识别两方面简要介绍生物传感器技术检测食源性大肠杆菌O157:H7研究的最新进展, 对生物传感器技术存在的问题及未来的研究方向进行了总结及展望。     

A note on Escherichia coli O157:H7 serotype in Turkish meat products

255 minced beef, 101 soudjouk and 50 uncooked hamburger samples were analyzed for the presence of Escherichia coli O157:H7 serotype. m-EC and LST broths were used as selective enrichment media and SMAC agar was used as a selective isolation medium. A total of 3 E. coli O157 were isolated by conventional culture techniques; one from each of minced beef, uncooked hamburger and soudjouk but none were identified as the H7 serotype. For determination of selective media-growing cohabitant bacteria, 2645 isolates were obtained from SMAC agar. Results showed that E. coli type 1, Hafnia alvei and Citrobacter freundii were dominant competitive flora. As selective enrichment broth-growing cohabitant microflora existed at higher levels, it was too difficult to isolate E. coli O157 from these mixed flora. Therefore, conventional methods are not suitable for these types of products, because of isolation difficulties and failure to confirm.     

Survival and Growth of Vibrio cholerae O1, Salmonella typhi, and Escherichia coli O157:H7 in Alfalfa Sprouts

The survival and growth of Vibrio cholerae O1, Salmonella typhi , and Escherichia coli O157:H7 during germination and sprouting of disinfected alfalfa seeds and alfalfa sprouts was determined. All pathogens showed ability to grow during germination and sprouting, reaching counts of approximately 6.0 log₁₀ CFU/g after 24 h. No growth was observed for any pathogen when the sprouts were inoculated after 24 h of seed germination. At this time, the background microflora was abundant. Numbers of pathogens inoculated on alfalfa sprouts decreased less than 1 log10 CFU/g over 15 d of refrigeration. Alfalfa sprouts can be an important factor contributing to the endemicity for typhoid fever and cholera in México.     

Cold plasma reduction of Salmonella and Escherichia coli O157:H7 on almonds using ambient pressure gases

Contamination of raw nuts, including almonds, is a food safety concern. Cold plasma is a novel antimicrobial intervention that can eliminate foodborne pathogens. The objective of this work was to evaluate the efficacy of rapid cold plasma treatments in eliminating Salmonella and Escherichia coli O157:H7 from dry almonds. Three isolates of Salmonella (S. Anatum F4317, S. Stanley H0558, and S. Enteritidis PT30) and 3 isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894) were separately grown and spot-inoculated (10 μL) onto whole almonds and allowed to dry for 10 min. Inoculated almonds were treated with a cold plasma jet, with treatment variables evaluated in a factorial design for each isolate: time, distance, and feed gas. Treatment time was 0 s (control), 10 s, or 20 s. Distance from the emitter was 2, 4, or 6 cm. Feed gas was dry air or nitrogen. After treatment, the almonds were sampled using swabs. Survivors were enumerated on tryptic soy agar (TSA) plates. Cold plasma significantly reduced both pathogens on almonds. The greatest reduction observed was 1.34 log cfu/mL reduction of E. coli O157:H7 C9490 after 20 s treatment at 6 cm spacing. The interaction of treatment time with distance from plasma emitter head was complex, and isolate-dependent. Longer duration of treatment did not always result in enhanced reductions. In general, nitrogen as a feed gas resulted in a reduced antimicrobial efficacy compared to dry air. These results indicate that short pulses of atmospheric pressure cold plasma can significantly reduce Salmonella and E. coli O157:H7 on almonds.     

Models of the behavior of Escherichia coli O157:H7 in raw sterile ground beef stored at 5 to 46 degrees C

Escherichia coli O157:H7 can contaminate raw ground beef and cause serious human foodborne illness. Previous reports describe the behavior of E. coli O157:H7 in ground beef under different storage conditions; however, models are lacking for the pathogen's behavior in raw ground beef stored over a broad range of temperature. Using sterile irradiated raw ground beef, the behavioral kinetics of 10 individual E. coli O157:H7 strains and/or a 5- or 10-strain cocktail were measured at storage temperatures from 5° to 46 °C. Growth occurred from 6 to 45 °C. Although lag phase duration (LPD) decreased from 10.5 to 45 °C, no lag phase was observed at 6, 8, or 10 °C. The specific growth rate (SGR) increased from 6 to 42 °C then declined up to 45 °C. In contrast to these profiles, the maximum population density (MPD) declined with increasing temperature, from approximately 9.7 to 8.2 log cfu/g. Bias (B_f) and accuracy (A_f) factors for an E. coli O157:H7 broth-based aerobic growth model (10 to 42 °C) applied to the observations in ground beef were 1.05, 2.70, 1.00 and 1.29, 2.87, 1.03, for SGR, LPD and MPD, respectively. New secondary models increased the accuracy of predictions (5 to 45 °C), with B_f and A_f for SGR, LPD, and MPD of 1.00, 1.06, and 1.00 and 1.14, 1.33, and 1.02, respectively. These new models offer improved tools for designing and implementing food safety systems and assessing the impact of E. coli O157:H7 disease.     

Impact of Changes in Sugar Exudate Created by Biological Damage to Tomato Plants on the Persistence of Escherichia coli O157:H7

ABSTRACT: The survival of enteric pathogens on vegetable leaves improves due to presence of phytopathogens. Phytopathogen damage alters the microenvironment on the leaf surface. The objective of this study was to identify differences in sugar concentrations in tomato leaves damaged by biotropic plant pathogens and determine if these differences affect Escherichia coli O157:H7 survival. E. coli O157:H7 survived better on tomato plants damaged by Xanthomonas campestris than on healthy plants (P = 0.012). The most common sugars and sugar alcohols in the damaged leaf exudate were glucose, fructose, inositol, and sucrose. The abundance of sucrose and inositol differed between the healthy and infected plants (P < 0.05). In this study, it was found that phytopathogen damaged plants increased sugar availability on the leaf surface for E. coli O157:H7 to proliferate. Keeping plants free from biological damage can limit the amount of leaching of sugars that could allow human pathogens to proliferate. There is the possibility of increasing food safety of vegetable products by limiting phytopathogenic damage to plants.     

Survival of Escherichia coli O157:H7 in cucumber fermentation brines

Abstract: Bacterial pathogens have been reported on fresh cucumbers and other vegetables used for commercial fermentation. The Food and Drug Administration currently has a 5‐log reduction standard for E. coli O157:H7 and other vegetative pathogens in acidified pickle products. For fermented vegetables, which are acid foods, there is little data documenting the conditions needed to kill acid resistant pathogens. To address this knowledge gap, we obtained 10 different cucumber fermentation brines at different stages of fermentation from 5 domestic commercial plants. Cucumber brines were used to represent vegetable fermentations because cabbage and other vegetables may have inhibitory compounds that may affect survival. The 5‐log reduction times for E. coli O157:H7 strains in the commercial brines were found to be positively correlated with brine pH, and ranged from 3 to 24 d for pH values of 3.2 to 4.6, respectively. In a laboratory cucumber juice medium that had been previously fermented with Lactobacillus plantarum or Leuconostoc mesenteroides (pH 3.9), a 5‐log reduction was achieved within 1 to 16 d depending on pH, acid concentration, and temperature. During competitive growth at 30 °C in the presence of L. plantarum or L. mesenteroides in cucumber juice, E. coli O157:H7 cell numbers were reduced to below the level of detection within 2 to 3 d. These data may be used to aid manufacturers of fermented vegetable products determine safe production practices based on fermentation pH and temperature. Practical Application: Disease causing strains of the bacterium E. coli may be present on fresh vegetables. Our investigation determined the time needed to kill E. coli in cucumber fermentation brines and how E. coli strains are killed in competition with naturally present lactic acid bacteria. Our results showed how brine pH and other brine conditions affected the killing of E. coli strains. These data can be used by producers of fermented vegetable products to help assure consumer safety.     

大肠杆菌O157:H7快速培养的初步研究

本研究将Oxyrase酶加入到接种了大肠杆菌O157:H7的培养基中以促进这种兼性厌氧微生物的生长。与不加Oxyrase酶的对照组相比,添加了Oxyrase酶的培养基中的大肠杆菌O157:H7的浓度明显提高。实验结果表明,Oxyrase酶在大肠杆菌O157:H7 快速培养中具有潜在的利用价值。     

Growth Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Escherichia coli O157:H7

Escherichia coli O157:H7‐specific antibodies (immunoglobulin Y [IgY]) were isolated by the water‐dilution method from the egg yolk of chickens that were immunized with E. coli O157:H7 whole cells. The specific‐binding activity of IgY against E. coli O157:H7 as determined by the enzyme immuno assay showed high levels of activity against bacterial whole cells. IgY binding activity was further demonstrated to have an inhibitory effect on E. coli O157:H7 growth in a liquid medium. The antibacterial function of IgY appeared to result from the interaction of IgY with surface components of E. coli O157:H7, as proven from observation of immunofluorescence and immunoelectron microscopy.     

Comparison of Decontamination Efficacy of Antimicrobial Treatments for Beef Trimmings against Escherichia coli O157:H7 and 6 Non-O157 Shiga Toxin-Producing E. coli Serogroups

Abstract: The decontamination efficacy of 6 chemical treatments for beef trimmings were evaluated against Escherichia coli O157:H7 and 6 non‐O157 Shiga toxin‐producing E. coli (nSTEC) serogroups. Rifampicin‐resistant 4‐strain mixtures of E. coli O157:H7 and nSTEC serogroups O26, O45, O103, O111, O121, and O145 were separately inoculated (3 to 4 log CFU/cm²) onto trimmings (10 × 5 × 1 cm; approximately 100 g) fabricated from beef chuck rolls, and were immersed for 30 s in solutions of acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.5), Bromitize^® Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or for 5 s in SYNTRx 3300 (pH 1.0). Each antimicrobial was tested independently together with an untreated control. Results showed that all tested decontamination treatments were similarly effective against the 6 nSTEC serogroups as they were against E. coli O157:H7. Irrespective of pathogen inoculum, treatment of beef trimmings with acidified sodium chlorite, peroxyacetic acid, or sodium metasilicate effectively (P < 0.05) reduced initial pathogen counts (3.4 to 3.9 log CFU/cm²) by 0.7 to 1.0, 0.6 to 1.0, and 1.3 to 1.5 log CFU/cm², respectively. Reductions of pathogen counts (3.1 to 3.2 log CFU/cm²) by Bromitize Plus, AFTEC 3000, and SYNTRx 3300 were 0.1 to 0.4 log CFU/cm², depending on treatment. Findings of this study should be useful to regulatory authorities and the meat industry as they consider nSTEC contamination in beef trimmings. Practical Applications: Findings of this study should be useful to: (i) meat processors as they design and conduct studies to validate the efficacy of antimicrobial treatments to control pathogen contamination on fresh beef products; and (ii) regulatory agencies as they consider approaches for better control of the studied pathogens.     

Antimicrobial Effects of Quillaja saponaria Extract Against Escherichia coli O157:H7 and the Emerging Non‐O157 Shiga Toxin‐Producing E. coli

Natural alternate methods to control the spread of Shiga toxin‐producing Escherichia coli (STEC) are important to prevent foodborne outbreaks. Quillaja saponaria aqueous bark extracts (QE), cleared by the U.S. Food and Drug Administration as a natural flavorant, contain bioactive polyphenols, tannins, and tri‐terpenoid saponins with anti‐inflammatory and antimicrobial activity. The objective of this study was to determine the effects of commercial QE against E. coli O157:H7 and non‐O157 strains over 16 h at 37 °C and RT. Overnight cultures of 4 E. coli O157:H7 strains and 6 non‐O157 STECs in Tryptic Soy Broth (TSB) were washed and resuspended in phosphate‐buffered saline (PBS, pH 7.2), and treated with QE and controls including citric acid (pH 3.75), sodium benzoate (0.1% w/w), acidified sodium benzoate (pH 3.75) or PBS for 6 h or 16 h. Recovered bacteria were enumerated after plating on Tryptic Soy Agar, from duplicate treatments, replicated thrice and the data were statistically analyzed. The 4 QE‐treated E. coli O157:H7 strains from initial ～7.5 log CFU had remaining counts between 6.79 and 3.5 log CFU after 16 h at RT. QE‐treated non‐O157 STECs showed lower reductions with remaining counts ranging from 6.81 to 4.55 log CFU after 16 h at RT.  Incubation at 37 °C caused reduction to nondetectable levels within 1 h, without any significant reduction in controls. Scanning electron microscopy studies revealed damaged cell membranes of treated bacteria after 1 h at 37 °C. QE shows potential to control the spread of STECs, and further research in model food systems is needed.     

应用DPO-PCR技术检测肠出血性大肠杆菌O157∶H7

利用双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）聚合酶链式反应（polymerase chain reaction,PCR）技术检测肠出血性大肠杆菌O157∶H7。根据DPO引物设计原则,以肠出血性大肠杆菌O157∶H7 rfbE 基因为靶基因设计一对DPO引物,经过反应体系的优化,建立了肠出血性大肠杆菌O157∶H7 DPO-PCR检测方法, 其检测灵敏度约为94 CFU/mL。与常规PCR方法相比,所建立的DPO-PCR方法对退火温度不敏感,在引物设计和实 验过程中不需要对引物及其退火温度反复优化,同时基于DPO引物的特殊结构又增强了其特异性。DPO-PCR方法 设计简易、特异性强,为致病性微生物的快速准确检测提供了新途径。     

大肠杆菌O157:H7量子点免疫层析试纸条的研制

研制一种大肠杆菌O157:H7量子点免疫层析试纸。利用自制水溶性量子点静电偶联大肠杆菌O157:H7单克隆抗体,将大肠杆菌O157:H7单克隆抗体和羊抗兔二抗划线于硝酸纤维素膜分别作为检测线和质控线,制备双抗体夹心法检测大肠杆菌O157:H7的量子点免疫层析试纸。该试纸条能在5min内完成检测,检测限制为1×104 CFU/mL,对常见的8种食源菌无交叉反应。基于量子点的大肠杆菌O157:H7免疫层析试纸操作简便,灵敏度和特异性较好,可用于食品快速检测。     

抗大肠杆菌O157:H7的卵黄特异性IgY的研究

以大肠杆菌O157:H7为抗原免疫产蛋母鸡,从鸡卵黄中提取免疫球蛋白,建立抗大肠杆菌O157:H7的特异性IgY的效价检测方法,并研究母鸡的免疫应答性,以及抗体的提取方法和体外抑菌效果.研究结果表明,初次免疫后第6d,在卵黄中可以检测到抗大肠杆菌O157:H7 IgY,效价为1:7200;经加强免疫后效价迅速上升,至第44d达到最高效价1:230400;免疫后360 d,效价仍维持在1:7200.用水稀释法、硫酸铵分级盐析和Sephadex G-25凝胶过滤以提取IgY,提纯后IgY的效价是之前的4倍.SDS-PAGE鉴定抗体的纯度,电泳图谱中出现抗体的轻链和重链两条带.体外抑菌实验表明,IgY能抑制大肠杆菌O157:H7的生长.