Antibacterial mechanism of kojic acid and tea polyphenols against Escherichia coli O157:H7 through transcriptomic analysis

Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans.The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E.coli O157:H7 than using either alone.This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level.The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E.coli O157:H7 found that acid resistance systems in kojic acid were activated,and the cell membrane and genomic DNA were destructed in the cells,resulting in“oxygen starvation”.The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP,which affected the bacteria's life metabolic process.Interestingly,we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA,respiration,tricarboxylic acid cycle.The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E.coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.

基于紫胶红素的靶标有色化免疫层析试纸条联检大肠杆菌O157∶H7和沙门氏菌

为摆脱免疫层析试纸条对配对抗体的依赖,文章设计了一种新型靶标有色化免疫层析试纸条,使用紫胶红素和十二合水硫酸铝钾媒染剂,通过先媒后染的方法,实现了免疫层析试纸条的“可视化”。验证结果显示,该试纸条对大肠杆菌O157∶H7和沙门氏菌的检测限均达到了106 CFU/mL;同时,与常见食源性致病菌间不存在交叉反应,在鸡胸肉、鸡蛋、牛奶等食品基质中能够在增菌9 h内检出。文章成功设计了一种操作简单、检测成本低,更易国产化的快速检测免疫层析试纸条,为后续免疫层析试纸条的设计提供了新思路。

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。

大肠杆菌O157:H7烈性噬菌体SF08的分离、表征及在食品中的应用

本研究通过双层平板法从湖水中分离、纯化出一株大肠杆菌O157:H7烈性噬菌体,并命名为SF08。对其生物学特性、基因组序列及在即食鸡爪中的抑菌效果进行分析。结果表明,SF08由长约124 nm的可收缩尾巴和直径约76 nm的多面体头部组成。最佳感染复数(Multiplicity of Infection,MOI)为0.01;潜伏期为20 min,裂解期为20~110 min,爆发量为66 PFU/cell;在pH 3~12和30~70℃条件下能保持较高的活性。基因组全长59704 bp,G+C含量为56.52%,共有72个开放阅读框(ORF)。SF08在4℃下可以极显著地抑制即食鸡爪中模拟污染的大肠杆菌O157:H7(P<0.01)。本研究表明SF08具有较好的生物学特性及安全性,为后续研发和制备天然杀菌剂提供理论基础和参考。

一种可快速检测大肠杆菌O157:H7的纸基传感器的制备与应用

大肠杆菌O157:H7是一种致病性较强的食源性细菌,通常在生制食品中被检出,一旦被人体摄入会引发肠道疾病,严重危害公众健康。本研究制备了功能化的长余辉探针,并结合适配体的特异性识别功能构建了能快速检测大肠杆菌O157:H7的纸基传感器,检出限达7.5×10^(3) CFU·mL^(-1)。该传感器可同时检测3个样品,具有特异性好、操作简便和环境友好的特点,可应用于大量样品中大肠杆菌O157:H7的快速筛查。

不同方法检测食品中大肠埃希氏菌O157:H7/HM能力验证结果比较与分析

为提高实验室对食品中大肠埃希氏菌O157∶H7的检验检测能力,笔者单位参加了由中国检验检疫科学研究院测试评价中心组织的食品中大肠埃希氏菌O157∶H7检测能力验证计划。此次实验采用国家标准《食品安全国家标准食品微生物学检验大肠埃希氏菌O157∶H7/NM检验》(GB 4789.36—2016)中的第一法和实时荧光PCR方法,分别对能力验证样品进行检测。结果显示,样品23-G520中未检出大肠埃希氏菌O157∶H7;样品23-F568中检出大肠埃希氏菌O157∶H7,结果反馈为满意。在能力验证实验中,不同检测方法同时使用、综合判断,能有效提高检测能力和结果的准确性。

基于dsDNA-CuNCs的荧光ELISA检测牛奶中的E.coli O157:H7

目的:建立了一种灵敏检测牛奶中大肠杆菌O157:H7的新型荧光酶联免疫吸附方法。方法:以碱性磷酸酶水解焦磷酸盐-Cu^(2+)配位复合物,释放出Cu^(2+)形成铜纳米簇作为信号报告探针,通过对关键因素Cu^(2+)浓度、焦磷酸盐浓度、DNA模板的浓度和长度进行优化。结果:在最优条件下,大肠杆菌O157:H7的菌数为5×10^(4)~1×10^(8)CFU/mL时,与荧光信号值有较好的线性关系(R^(2)=0.9808),最低检出限为2.4×10^(4)CFU/mL。结论:该方法成功地应用于低脂牛奶和脱脂牛奶样本中大肠杆菌O157:H7的测定,平均回收率为92.2%~10^(8).5%,相对标准偏差为4.9%~10.4%。

牛源性Escherichia coli O157:H7分离鉴定、耐药性及耐药基因分析

了解某牛场犊牛群发腹泻后继发胸膜炎、腹膜炎并造成犊牛大量死亡的主要病原菌及其耐药性情况。采集病死犊牛肺、腹膜和肠系膜等脏器,进行病原菌分离、鉴定,并对分离的主要致病菌株进行药敏试验及耐药基因检测。结果表明,引起此次犊牛腹泻及胸膜炎、腹膜炎的主要致病菌为E.coli O157:H7;药敏试验结果表明分离菌对青霉素、四环素和氨苄西林等12种抗生素耐药,对头孢哌酮、头孢他啶、丁胺卡那等8种抗生素敏感,表现为多重耐药。该分离菌同时携带tet B、str B、aad B、aph A、flo R和TEM等耐药基因,耐药基因型和耐药表型不完全相同。研究可为E.coli O157:H7的防治和耐药性控制提供依据。

用于Escherichia coli O157∶H7直接快速检测的倏逝波荧光核酸适配体传感器研究

通过融合倏逝波荧光光纤传感器和特异性核酸适配体的优势,提出了一种基于倏逝波荧光原理及其与病原菌尺寸效应的Escherichia coli O157∶H7(E.coli O157∶H7)直接快速检测方法。基本原理是当一定浓度荧光标记E.coli O157∶H7核酸适配体加入样品检测池时,倏逝波激发荧光分子发出荧光,利用倏逝波全光纤生物传感器即可实现荧光信号的定量检测;当荧光标记的核酸适配体与E.coli O157∶H7混合后加入样品检测池,因倏逝波渗入深度仅为100 nm,导致特异性结合E.coli O157∶H7的核酸适配体标记荧光分子不能被激发,从而使得检测荧光信号降低;利用荧光信号强度与E.coli O157∶H7浓度的比例关系即可实现其定量检测。结果表明:该方法检测E.coli O157∶H7的检测限可达610 CFU/mL,线性检测区间为1.1×10^3-1.4×10^7 CFU/mL。实际水样加标回收率在40%-180%之间,相对标准偏差在10%之内,水样基质对E.coli O157∶H7的检测没有明显影响。本研究建立基于倏逝波荧光原理及其与病原菌尺寸效应的生物传感分析方法具有普适性,仅需使用不同荧光标记的生物识别分子即可实现其他病原菌的直接快速检测。

食品中Escherichia coli O157:H7微滴数字PCR绝对定量检测方法的建立

为建立食品中Escherichia coli O157:H7的微滴数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)快速定量检测方法,针对E.coli O157:H7的特异性单拷贝基因hlyA设计引物探针,并进行特异性、灵敏度和重复性实验,同时通过人工污染三文鱼样品的检测,比较平板计数法、real-time PCR和ddPCR方法的测定值效果。结果表明,所建立的E.coli O157:H7 ddPCR检测方法具有良好的特异性、灵敏性和重复性。细菌纯培养液中定量限为105 CFU/mL,检出限为25 CFU/mL,人工污染三文鱼样品中检出限为110 CFU/g。对不同人工污染水平的三文鱼样品,ddPCR与平板计数的测定值结果无显著性差异(P>0.05),比real-time PCR方法的测定值结果更加稳定、准确。因此,本研究建立的ddPCR方法能够更加快速、准确、灵敏、特异地绝对定量检测食品中E.coli O157:H7。

纳米金与纳米金花标记的免疫层析法的建立及快速检测大肠杆菌O157∶H7的比较研究

为实现大肠杆菌O157∶H7早期现场的快速检测,该试验通过化学还原法与介导生长法制备了AuNPs与AuNFs作为标记探针,建立2种用于大肠杆菌O157∶H7现场快速检测的免疫层析试纸条,并对2种试纸条的的灵敏度、特异性和准确性进行检测。结果显示,所建立的2种免疫层析方法均可在15 min内检出食品中的大肠杆菌O157∶H7,其中AuNP试纸条最低检出限为3.2×10^(5)CFU/mL,AuNFs试纸条最低检出限为3.2×10^(4)CFU/mL,与单增李斯特菌、沙门氏菌、坂崎肠杆菌等常见食源性致病菌没有交叉反应。对人工污染果冻、牛奶和牛肉等样品AuNPs试纸条分别在前增菌5、6、5 h时检出,AuNFs试纸条分别在前增菌5、5、4 h时检出,且不受食品复杂机制的影响。结果表明,所建立的2种免疫层析试纸条灵敏度高、特异性强、操作简便,适用于大肠杆菌O157∶H7现场快速检测。

Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

To prepare monoclonal antibodies （MAb） and antisera specific for Escherichia coli （E.coli） O157, and to develop a sandwich enzyme-linked immunosorbent assay （ELISA） to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157：H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157：H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113：H21 belonged to subtype IgM. The ascetic titers of the antibody was 1：1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1： 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157：H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113：H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157：H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

Prevalence of shiga toxins(stx_1,stx_2),eaeA and hly genes of Escherichia coli O157:H7 strains among children with acute gastroenteritis in southern of Iran

Objective:To survey the prevalence severe diarrhea arising from these bacteria in children under 5 years old in Marvdasht.Methods:In this study faecal sample from 615 children aged <5years old who were hospitalized lor gastroenteritis in Fars hospitals in Iran were collected and then enriched in Escherichia coli(E.coli) broth and modified tryplone soy broth with novobiocin media,fermentation of sorbitol,lactose and β— glucoronidase activity of isolated strains was examined by CT—SMAC,VRBA and chromogenic media respectively.Then isolation of E.coli O157:H7 have been confirmed with the use of specific antisera and with multiplex PCR method presence of virulence genes including:xtx_1.stx_2,eae.A.hly has been analyzed.Results:E.coli O157:H7 was detected in 7(1.14%) stool specimens.A significanl difference was seen between detection rale of isolated bacteria from age groups 18-23 months and other age groups(P=0.004).Out of considered virulence genes.only 1 of the isolated strains(0.16%)he stx,and eaeA genes were seen and also all isolated hacleria had resistance to penicillin,ampicillin and erythromycin antibiotics.Conclusions:We found thai children < 2 years of age were at highest risk of infection with E.coli O157:H7.Regarding severity of E.coli O157:H7 pathogenesis,low infectious dose and lack of routine assay for detection ol these bacleria in clinical laboratory,further and completed studies on diagnosis and genolyping of this E.coli O157:H7 strain has been recommended.

In Vitro Assessment of Probiotic Potential of Lactobacilus acidophilus and Antagonistic Activity Against Escherichia coli O157:H7

The antagonistic activity of Lactobacillus acidophilus KLDS1.0901, KLDS1.0902, KLDSI.1003 and NCFM against Escherichia coli O157 ： H7 were investigated in this study. The culture supematants of all the L. acidophilus stains showed high bacteriostatic activities against E. coli O 157 ： H7 and the bacteriostatic substances of their Cell-Free Supernatants （CFS） were preliminarily determined from organic acids. The bacteriostatic activity from CFS or viable L. acidophilus against E. coli O157 ： H7 was also assessed by using co-incubation methods, CFS had high bactericidal activity against E. coli O157 ： H7, no viable E. coli O157 ： H7 was detected when 5×10^7 CfU ofE. coli O157 ： H7 was added to 5 mL of CFS and incubated at 37℃ for 2 h. However, L. acidophilus themselves had no bacteriostatic activity after directly contacted with E. coli O157 ： H7. The inhibition E. coli O157 ： H7 adhesion and colonization of L. acidophilus were also investigated based on competition, exclusion and displacement assays. L. acidophilus KLDS1.0901, KLDSI.1003 and NCFM strains were effective to displace E. coli O157 ： H7 from a Caco-2 cell layer in competition and exclusion assays. However, in displacement assay, all of the strains showed no significant antagonistic activities. Meanwhile, the probiotic potential of L. acidophilus strains was investigated based on adhesion assay to Caco-2 cells and anti- inflammatory effects by IL-8 produced in Caco-2 cells. The adhesion ability and anti-inflammatory effects of L. acidophilus strains showed a strain-dependent manner. In general, L. acidophilus KLDS 1.0901 and NCFM showed better probiotic potential than KLDS1.0902 and KLDSI.1003. Thus, the use ofL. acidophilus KLDS1.0901 and NCFM to prevent or treat of diseases associated induced E. coli O157 ： H7 in vivo was suggested.

The intervention effects of Lactobacillus casei LC2W on Escherichia coli O157:H7-induced mouse colitis

This study investigated the intervention effects of Lactobacillus casei LC2W in murine(SPF C57BL/c)challenge infection models induced by Escherichia coli O157:H7.Mice were fed streptomycin with water for 3 days prior to intragastric gavage by E.coli O157:H7(Control)or L.casei LC2W together with E.coli O157:H7(Intervention)to explore the role of L.casei LC2W by biochemical indicators,histological evaluation,expression of colonic pro-inflammatory and intestinal barrier factors related to enteritis.Results showed that the administration of L.casei LC2W was able to alleviate the symptoms of colitis induced by E.coli O157:H7,exhibiting lower weight loss as well as more intact colon tissue.Furthermore,L.casei LC2W could down-regulate the expression of pro-inflammatory cytokines(TNF-α,IL-6,and IL-1β)and protect the intestinal barrier function by improving the level of MUC2,ZO-1 and E-cadherin 1 compared to the control group.These results demonstrate that L.casei LC2W can reduce the severity of E.coli O157:H7 infection,and suggest L.casei LC2W may maintain the immune balance and intestinal barrier to reduce colitis.In addition,we found the effect of intervention is similar to that of prevention,which is better than that of treatment.

Effects of Sodium Lactate on the Survival of <i>Listeria Monocytogenes</i>, <i>Escherichia coli</i>O157:H7, and <i>Salmonella</i>spp. in Cooked Ham at Refrigerated and Abuse Temperatures

The objective of this study was to determine the effect of sodium lactate on the survival of Listeria monocytogenes, Escherichia coli O157: H7, and Salmonella spp. in cooked ham during storage at refrigerated and abuse temperatures. Cooked ham was added with 0% - 3% lactate, inoculated with a multiple-strain mixture of L. monocytogenes, E. coli O157: H7, or Salmonella spp. and stored at 4oC - 15oC for up to 35 day. The growth of the three pathogens was inhibited in ham containing 3% lactate, and no growth of E. coli O157: H7 and Salmonella spp. occurred at the lowest storage tem- peratures of 6 and 8oC, respectively. In ham containing no lactate, the average growth rates were 0.256 - 0.380 log CFU/day for L. monocytogenes at 4oC - 8oC, 0.242 - 0.315 log CFU/day for E. coli O157: H7 at 8oC - 15oC, and 0.249 - 0.328 log CFU/day for Salmonella spp. at 10oC - 15oC. The addition of 1% or 2% lactate significantly (P < 0.05) reduced the growth rates of the three pathogens, and the effect was more profound at lower temperatures. Salmonella spp. were more sensitive to the effect of lactate than L. monocytogenes and E. coli O157: H7. Polynomial models were developed to describe the growth rates of the three pathogens as affected by the lactate concentration and storage tem- perature. Results from this study demonstrate the effect of lactate on the growth of L. monocytogenes, E. coli O157: H7, and Salmonella spp. in cooked ham and indicate the effective lactate concentrations and storage temperatures that can be used to enhance the microbiological safety of ready-to-eat ham products.

基于抗体-适配体夹心生物传感器检测大肠杆菌O157:H7

大肠杆菌O157:H7(Escherichia coli O157:H7)是一种重要的与公共卫生相关的食源性病原体。抗体分子是体液免疫应答中最重要的效应分子,具有多种生物学活性,最主要的生物学功能是与相应抗原特异性结合。适配体是体外合成的较短DNA序列,可通过识别特定区域和靶标特异性结合。利用抗体和适配体的特异性识别功能及免疫磁珠的捕获作用和磁分离,并通过多克隆抗体和裁剪得到的适配体搭建生物传感器,使用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR)可以实现对靶标在(8×10^(3))-(8×10^(6))CFU/mL范围内的定量检测,检测限为800 CFU/mL。

Microarray analysis of Escherichia coli0157:H7

瞄准：为检测 O157 建立快速、特定、敏感的方法: 有 DNA 微芯片的 H7。方法：遗传因子的细菌的反和 E 的剧毒的基因的特定的 oligonucleotide 探针(26-28 nt ) 。关口 i O157 : H7 和另外的相关病原体基因被预先综合并且在稳固的支持上使不能调动做微芯片。编码 O157 菌体抗原(rfbE ) 的四基因， H7 flagellar (警察) 和毒素(SLT1， SLT2 ) 被复合 PCR 与四份特定的教材监视。为杂交标记样品的 Fluorescence-Cy3 被 PCR 与标记 Cy3 的单个素数产生。杂交在 45 度为 60 min 被执行。微芯片图象用一台共焦的荧光灯的扫描仪被拿。结果：12 不同细菌的紧张与四剧毒的基因的各种各样的联合被检测。所有 O157 : H7 紧张由复合 PCR 产出阳性结果。与这些教材产生的 PCR 产品的尺寸从 210 ～ 678 bp 变化了。所有 rfbE/fliC/SLT1/SLT2 探针明确地从 O157 认出了标记 Cy3 的荧光灯的样品: 包含 O157 和 H7 基因的 H7 紧张，或紧张。O157 的没有生气杂交: H7 荧光灯的样品发生在另外的探针。Non-O157 : H7 病原体没能在可比较的条件下面产出任何信号。如果 O157 单个 PCR 的标记 Cy3 的荧光灯的产品是冲淡的 50 褶层，没有信号在统帅玫瑰胶化电气泳动被发现，但是一个积极信号在微数组杂交被发现。结论：O157 的 Microarray 分析: H7 是为细菌的病原体的鉴定和察觉的一个快速、特定、有效的方法。

PCR-DGGE analysis of earthworm gut bacteria diversity in stress of <i>Escherichia coli</i>O157:H7

In order to test if the intestinal bacteria play an important role in antibacterial ability of earthworm, we chose Escherichia coli O157:H7, an anthropozoonosis pathogen, as a biological stressor and studied the change of intestinal bacteria community of earthworm by PCR-DGGE analysis. Results showed that the pathogen merely existed 1 - 3 days, then almost disappeared after through the earthworm’s gut. In this period, the diversity and abundance index of intestinal bacteria increased first, then decreased, and finally kept stably after 7 days. The result demonstrated that the intestinal bacteria of earthworm had ability of adjust community structure to eliminate the pathogen E. coli O157:H7, and the amount of bacteria Bacillus increased significantly, which might be the positive antagonism to E. coli O157:H7.