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.

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。

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.

Analysis on the Epidemiological Characteristics of Escherichia coli O157:H7 Infection in Xuzhou,Jiangsu,China,1999

Objective： To determine epidemiologic features of an Escherichia coli O157：H7 outbreak occurred in Xuzhou, Jiangsu Province, China in 1999, and assess the incidence of E. coli O157：H7 in diarrhea patients and host animals and its relationship with disease onset, and provide a scientific basis for establishing prevention and control strategies. Methods： Epidemiological, microbiological, and molecular methods were performed to identify risk factors and describe the ecology of E. coli O157：H7 in the enviromnent. Results： From May to September, in 1999, 99 cases of E. coli O157：H7 infection were confirmed. Fifty-six patients were enrolled in the case-control study. Bad personal health habits and poor sanitary conditions in the kitchen were associated with increased risks of infection, whereas hand washing was protective. The household survey indicated that residents in the epidemic region during the outbreak had higher than expected rates of diarrhea. The total E. coli O157：H7 carrier rate in the livestock was 12.36%（22/178）, specifically 19.15% in cattle, 12.50% in goat, and 11.11% in swine. Numerical analysis of pulsed-field gel electrophoresis（PFGE） profiles divided strains into two clusters with 77.5% homology. One cluster contained 11 strains isolated from diarrheal patients, foods, and animals. The other cluster comprised 10 strains from patients and environment. Conclusion： In a large outbreak of E. coli O157：H7 infection among predominantly elderly residents in Xuzhou, high rates of carriage of E. coli O157：H7 among host animals most likely resulted in contamination of the environment, thereby leading to the outbreak. Effective and preventive control measures should be taken to avoid contamination, including environmental and family health improvement, good personal hygiene, and safe food handling practices.

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。

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.

Sensing Escherichia coli O157:H7 via frequency shift through a self-assembled monolayer based QCM immunosensor

By means of the specific immuno-recognition and ultra-sensitive mass detection, a quartz crystal microbalance (QCM) biosensor for Escherichia coli O157:H7 detection was developed in this work. As a suitable surfactant, 16-mercaptohexadecanoic acid (MHDA) was introduced onto the Au surface of QCM, and then self-assembled with N-hydroxysuccinimide (NHS) raster as a reactive intermediate to provide an active interface for the specific antibody immobilization. The binding of target bacteria with the immobilized antibodies decreased the sensor’s resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of the electrochemical techniques. Using the immersion-dry-immersion procedure, this QCM biosensor could detect 2.0×102 colony forming units (CFU)/ml E. coli O157:H7. In order to reduce the fabrication time, a polyelectrolyte layer-by-layer self-assembly (LBL-SA) method was adopted for fast construction. Finally, the reproducibility of this biosensor was discussed.

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.

Electronic Detection of Escherichia coli O157︰H7 Using Single-Walled Carbon Nanotubes Field-Effect Transistor Biosensor

Field effect transistors (FET) based on Single-Walled Carbon Nanotubes (SWNTs) become the hot topic in fields of nano-electronic, clinical diagnostics, environmental testing etc. in recent years. In this paper, we reported a simple, scalable way to enrich semiconducting SWNTs by using HNO3/H2SO4. Then carbon nanotube field-effect transistors (CNTFET) biosensor was fabricated with the enrichment SWNTs for Escherichia coli O157︰H7 detection. The response of each CNTFET was monitored in real time before and after introduction of the Escherichia coli O157︰H7 at various concentrations. The results show that CNT-FET biosensors we fabricated are sensitive to change of concentration of solution and response time is really short.

Current epidemiological status of enterohaemorrhagic Escherichia coli O157:H7 in Africa

Enterohaemorrhagic E. coil （EHEC） has been isolated from human diarrhoeal disease,hemorrhagic colitis （HC）, and haemolytic uremic syndrome （HUS）. After 20 years since the first description of E. coli O157：H7 in USA, EHEC has become an important public health problem worldwide.1＇2 Given the magnitude and severity of recent outbreaks of E. coli O157：H7 infection, there is an urgent need to reduce the human hazard caused by this pathogen. Cattle are considered as major reservoir of E. coli O157：H7 because consumption of undercooked meat products from cattle has been often associated with many human infections.

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.

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.

Serotypes of Non-O157 Shigatoxigenic <i>Escherichia coli</i>(STEC)

Non-O157 STEC has been shown to have a diverse ecological distribution among food-animals. It has been associated with both outbreaks and individual cases of severe illness. This group of the organisms is now considered as a major contributor to human disease. The clinical description of the diseases caused by these organisms is reviewed. The host specificity of these pathogens is described and discussed. These organisms appear widespread among food animals like cattle and sheep, and can therefore affect a range of foods directly from the meat and excretions of these animals being used in farming practices. This article reviews the origins, diversity and pathogenesis of non-O157 STEC.

Survival and Growth Characteristics of <i>Escherichia coli </i>O157:H7 in Pomegranate-Carrot and Pomegranate-Apple Blend Juices

Unpasteurized fruit juice has been implicated in outbreaks of Escherichia coli O157:H7 (E. coli O157:H7). Meanwhile, certain fruit, such as pomegranate, contains antimicrobial components. The objective of this study was to investigate the survival and growth characteristics of E. coli O157:H7 on pomegranate juice in laboratory medium and in pomegranate-carrot and pomegranate-apple blend juices at different concentrations. Single strain of E. coli O157:H7 (E0019, H1730, and Cider) was inoculated into brain heart infusion (BHI) broth or a mixture of three strains was inoculated into the blended juices containing pomegranate juice. Our results showed that the addition of pomegranate juice inhibited the growth of tested E. coli O157:H7 in both laboratory medium and blended juices.The antimicrobial activity increased with increased concentrations of pomegranate juice (P < 0.001) and incubation times.The bacterial population was reduced by at least 2 log CFU/ml in BHI broth and juice blend samples in the presence of 20% and 40% Pomegranate juice respectively. Sensory evaluations performed using a 9 point hedonic scale showed significant satisfaction on using 40% pomegranate juice blend with carrot and apple juices. Our study suggests that pomegranate juice could be used as a natural antimicrobial in different food systems including juices to inhibit the growth of E. coli O157:H7.

Evaluation of Organic Acid-Based Sanitizers for Reduction of <i>Escherichia coli</i>O157:H7 during Flume-Washing of Organic Leafy Greens

Antimicrobial efficacy of three novel organic sanitizers, CHICO WashTM, C8C10 and CG100, was evaluated for the reduction of Escherichia coli O157:H7 during flume-washing of organic leafy greens. Organic formulations at various concentrations: CHICO (C3H8I0C3O7) WashTM (1:20 ratio) and C8C10 and CG100 (0.2 and 0.4%), along with the controls: hydrogen-peroxide and water, were used for washing organic baby and mature spinach and romaine and iceberg lettuce. Leafy greens were inoculated with a 2-strain cocktail of E. coli O157:H7 (6 logs CFU/mL) and washed in each treatment for 1 or 2 minutes. The treated leafy greens were stored at 4°C and surviving pathogen populations determined on days 0, 1, and 3 of storage. Organic sanitizers, for both treatment times, significantly (P E. coli O157:H7 on all the leafy greens during storage. Highest reduction (3.4 logs CFU/g) was observed after treatment with CG100 (0.4%) in romaine lettuce, while CHICO WashTM showed greater than 2 logs CFU/g reduction on all the leafy greens, by day 3. This study demonstrates the potential application of organic sanitizers in flume-washing of organic leafy greens for the reduction of E. coli O157:H7.

A Survey of <i>Escherichia coli</i>O157:H7 Virulence Factors: The First 25 Years and 13 Genomes

Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak in 1982, and in the 25 years that followed, many new strains were identified and emerged in numerous outbreaks of human disease. Extensive research has been conducted to identify virulence factor genes involved in the pathogenesis of E. coli O157:H7 and many genome sequences of E. coli O157:H7 strains have become available to the scientific community. Here, we provide a comprehensive overview of the research that has been conducted over the first 25 years to identify 394 known or putative virulence factor genes present in the genomes of E. coli O157:H7 strains. Finally, an examination of the conservation of these 394 virulence factor genes across additional genomes of E. coli O157:H7 is provided which summarizes the first 25 years and 13 genomes of this human pathogen.

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。

Effect of Direct-Fed Microbial Supplementation on Pathogenic <i>Escherichia coli</i>Fecal Shedding, Live Performance, and Carcass Characteristics in Feedlot Steers

Three experiments were conducted to evaluate direct-fed microbial (DFM) supplementation on live performance, carcass characteristics, and fecal shedding of E. coli in feedlot steers. In Exp. 1, 400 steers (BW = 348 kg) were assigned to treatments: CON = lactose carrier only, BOV = P. freudenreichii (NP24) + L. acidophilus (NP51), BOVD = P. freudenreichii (NP24) + L. acidophilus (NP51), and COMB = BOV fed for the first 101 d on feed, followed by BOVD for the final 28 d prior to harvest. In Exp. 2 (n = 1800;BW = 354 kg) and Exp. 3 (n = 112;BW = 397 kg), steers were utilized in a randomized complete block design and assigned to DFM treatments using low dose and high dose, respectively. Fecal samples were collected prior to harvest and analyzed for E. coli serogroups. In Exp. 1, DFM reduced (P < 0.01) the concentration of E. coli O157. Prevalence of O157 was reduced by BOVD supplementation in Exp. 2 and 3 (P < 0.01 and P = 0.08, respectively), and concentration of E. coli O157 in positive samples was reduced in both experiments where enumeration was performed (P ≤ 0.02). Weighted mean differences across the three experiments were equal to a 33% reduction in the prevalence of E. coli O157:H7 in BOVD treated cattle. A significant reduction in prevalence of O26, O45, O103, and O121 was observed in Exp. 2 (P ≤ 0.03). These results indicate that high levels of L. acidophilus (NP51) may represent an effective pre-harvest food safety intervention to reduce fecal shedding of several E. coli serogroups.