复合乳酸菌对肠出血性大肠杆菌O157∶H7抑制作用的研究

本研究利用实验室保存的7株常见乳酸菌进行抑制大肠杆菌O157∶H7实验,其中4株乳酸菌对大肠杆菌O157∶H7有明显抑制效果,分别为嗜酸乳杆菌(SL1)、鼠李糖乳杆菌(SL2)、干酪乳杆菌(SL3)、植物乳杆菌(SL4)。将4株乳酸菌混合后不但抑菌效果强于4株任意单独菌株,其发酵滤液pH相比4株单独菌株下降速度最快,且pH最低。说明混合后的乳酸菌产酸能力也明显增强。将SL1、SL2、SL3、SL4通过排列组合(C(n,m))可分成11组混合菌液,然后通过牛津杯抑菌方法进行抑菌实验,结果显示乳酸菌组合(SL2、SL3、SL4)具有最佳抑菌效果。再将SL2、SL3、SL4按正交设计方案以不同比例的接种量混合成复合菌剂进行抑菌实验,结果表明:乳酸菌组合(SL2、SL3、SL4)以3∶1∶3比例制备复合菌剂时,对大肠杆菌O157∶H7的抑制效果最好。      

肠出血性大肠杆菌O157∶H7的检测方法进展

肠出血性大肠杆菌(EHEC)O157:H7为新近报道的食源性强致病菌,对病原菌快速、简便、准确、灵敏的检测方法是预防控制的关键,本文对国内外常用的鉴别培养、免疫检测以及PCR等检测方法进行了系统的综述。     

食品中肠出血性大肠杆菌O157∶H7的检测技术研究进展

近年来,因食源性致病菌造成的食品中毒正在不断增加,严重威胁了人类的健康,大肠杆菌（E.coli） O157∶H7由于其低感染剂量和高致病性等特点引起了研究人员和世界卫生组织的高度重视。因此精准快速的检测食品中的E.coli O157∶H7对食品安全问题有着重要的意义,是预防和控制食源性疾病传播的重要技术,为此本文对E.coli O157∶H7的常规检测方法、免疫学法、分子生物学法和其他方法进行论述,介绍了这些方法的优缺点,对各种方法进行了整体比较,以期为有关工作者在选择检测方法或研发新方法提供参考。     

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。     

肠出血性大肠杆菌O157:H7的分子生物学检测及PCR检测

大肠杆菌一些特殊的血清型具有致病性,肠出血性大肠杆菌是大肠杆菌的一个亚型,主要致病菌株为O157:H7,可引起感染性腹泻,因能引起人类的出血性肠炎而得名。本文综述了分子生物学检测肠出血性大肠杆菌O157:H7的研究进展。分子生物学检测是利用抗原抗体特异性结合反应检测各种物质的分析方法,主要包括酶联免疫吸附法(ELISA)、胶体免疫金层析法以及免疫磁珠分离法(IMS)。PCR技术检测肠出血性大肠杆菌O157:H7,主要包括常规PCR检测、多重PCR检测以及实时荧光定量PCR检测。这两种方法灵敏度高、特异性强、操作简便、结果准确等优点,是检测肠出血性大肠杆菌O157:H7的常用方法。     

武汉肉类食品中大肠杆菌O157∶H7分离株stx亚型和毒力特征分析

从22 家市场销售的117 份肉类食品中分离出4 株大肠杆菌O157∶H7菌株,经PCR检测这4 株菌的stx、hly、 eae毒力基因均为阳性。采用聚合酶链式反应（polymerase chain reaction,PCR）法对这4 株菌的stx亚型进行鉴定。 3 株菌同时携带stx1、stx2基因,且均为stx1a、stx2a亚型。菌株EC5.11仅携带stx2基因,但在所有的stx2分型PCR反 应中都为阴性。用PCR扩增该菌株stx2基因全长并克隆测序,序列分析结果表明EC5.11志贺毒素基因为stx2c亚型。 采用Vero细胞毒性实验检测这4 株菌产志贺毒素的状况,结果显示这些菌株都具有一定的Vero细胞毒性。     

基于可视化抗体宏阵列技术的出血性大肠杆菌O157∶H7定量检测

目的：探索建立一种新的出血性大肠杆菌O157∶H7（Escherichia coli O157∶H7）的快速定量检测技术。方法：以硝酸纤维素膜为基片,用生物芯片点样仪制作抗体宏阵列,采用“双抗夹心”原理,对出血性大肠杆菌O157∶H7进行定量检测研究。结果：本方法对出血性大肠杆菌O157∶H7的检出限为3.4×105 CFU/mL,在105～107CFU/mL细菌浓度范围内,宏阵列的灰度值与细菌浓度之间有较好的线性关系。该方法可在2.5 h内同步检测多个样品中出血性大肠杆菌O157∶H7的浓度。结论：通过用标准菌株、模拟带菌等研究显示,用宏阵列技术快速定量检测出血性大肠杆菌O157∶H7,结果肉眼可见,稳定准确,同时操作简便、成本低廉、无需大型设备,制作好的抗体宏阵列可用于快速评估食品中出血性大肠杆菌O157∶H7的污染状况,尤其适合于基层实验室进行快速高通量样品筛查。     

出血性大肠杆菌O157∶H7单克隆抗体制备及免疫胶体金试纸条的研制

将骨髓瘤细胞SP2/0与出血性大肠杆菌O157∶H7免疫小鼠得到的脾细胞融合,通过筛选得到3株稳定分泌抗出血性大肠杆菌O157∶H7单克隆抗体的杂交瘤细胞株D3、E7、B9,D3和B9亚类为IgG1,E7亚类为IgG2a,轻链亚型均为κ。交叉反应结果显示这3株单抗仅结合出血性大肠杆菌O157∶H7,对15株其他细菌无反应,测得胶体金标记单抗最佳pH、最佳结合量分别为D3:pH 7.5⁸.0、24μg/mL;B9:pH 7.5、12μg/mL;E7∶pH7.5、18μg/mL,抗体配对选择胶体金结合D3喷涂金标垫、E7以1 mg/mL喷涂NC膜作T线为最优组合,试纸条检测出血性大肠杆菌O157∶H7灵敏度为2.1×106CFU/mL,且仅可检出出血性大肠杆菌O157∶H7,对25株其他细菌均无交叉反应,模拟带菌实验表明,对市售面包、牛奶、果冻各25 g(mL)均添加约200 CFU出血性大肠杆菌O157∶H7,增菌培养8、10、10 h即可检出。研究表明,实验自主制备的抗体性能良好,试纸条特异性、灵敏度达到国外同类产品,可在政府食源性致病菌监管部门、食品企业推广运用。     

温州市食品中肠出血性大肠杆菌O157∶H7污染状况调查

目的调查温州市食品中肠出血性大肠杆菌O157∶H7的污染状况,了解肠出血性大肠杆菌O157∶H7毒力因子的携带与耐药情况。方法采用分层随机抽样的方法,对2008—2011年抽取的231份食品样品,用免疫磁珠富集法对大肠杆菌O157∶H7进行分离,对该分离株进行生化、血清学鉴定,以纸片法(K-B法)进行药敏试验;采用PCR方法检测O、H抗原基因和stx1、stx2、eaeA、hlyA等4种毒力基因。结果 231份样品中,分离出1株肠出血性大肠杆菌O157∶H7,检出率为0.43%;O157抗原和H7抗原的核酸检测结果阳性,但未检测到stx1、stx2、eaeA、hlyA等4种毒力基因,菌株对红霉素、利福平、150μg/片和10μg/片两种浓度的0/129(二氨基二异丙基喋啶磷酸盐)耐药。结论温州市食品中存在大肠杆菌O157∶H7的污染,检出率较低,但提示要加强主动监测,通过有效干预,防范肠出血性大肠杆菌O157∶H7所致食源性疾病的发生。     

大肠杆菌O157的志贺毒素基因克隆和测序

将一株大肠杆菌O157PCR扩增stx2基因全长并克隆测序。该菌株stx2基因与GenBank数据库收录的stx2基因最高同源性为98%,在3个核苷酸位点存在基因突变。采用邻位相连法构建进化树,序列分析结果表明O157为stx2C基因亚型。了解大肠杆菌O157的基因突变情况,并为开发大肠杆菌分子检测方法提供了参考。     

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.     

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.     

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.     

Effect of free-SH containing compounds on allyl isothiocyanate antimicrobial activity against Escherichia coli O157:H7

ABSTRACT:  Escherichia coli O157:H7 contamination is a significant meat safety issue in many countries. Allyl isothiocyanate (AIT) is a natural compound found to limit the survival of E. coli O157:H7 and other pathogens in meat and meat products. In the present study, it was found that glutathione and cysteine naturally present in meat can interfere with AIT antimicrobial activity. Spectroscopy, HPLC, and LC-MS were used to confirm that glutathione was able to react with AIT and formed a conjugate with no or low bactericidal activity against the tested organisms. The same reaction also occurred at pH values of 4.9 and 5.8 at 25 and 4 °C, respectively, which broadly represent storage conditions in raw beef (pH 5.8) and during fermented sausage (pH 4.9) manufacture. Reactions observed help to explain reduction in antimicrobial potency of AIT in food (meat) systems.     

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.     

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.     

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.