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。

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.

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.

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.

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.

EHEC O_(157)∶H_7肠粘附力增强突变株的构建

〔目的〕构建对肠上皮细胞粘附力增强的肠出血性大肠埃希菌O157∶H7(EHECO157)突变株 ,为体内、体外研究其粘附及其粘附机制提供可行的手段。〔方法〕分别将pSC10 1和mini -Tn5Km2通过电穿孔转化和结合导入O157∶H7Sakai菌株中 ,筛选粘附力增强的转化子感染Caco -2细胞株和实验小鼠 ,观察Caco -2单层细胞中的微菌落数量和小鼠粪便中突变株脱落的时程。〔结果〕经诱变 ,共分离筛选出 8株突变株 ,其形成微菌落的数量大于野毒株至少 3倍以上 ;8个突变株中有 6株转座子的插入位置为E .coliK -12同源区的yhiE基因处 ,其突变株在小鼠粪便中的脱落时程明显高于野毒株 ,P <0 .0 1。〔结论〕O157Sakai中的yhiE基因可能具有调节O157粘附肠道上皮细胞能力的作用 ,尽管其调节机制尚有待于进一步阐明 ,但本文结果显示其可能具有负调节的作用。另外 。

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.

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.

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.

上海市动物及其产品中大肠埃希菌O157:H7带菌情况的调查

为了解上海市动物及动物产品中大肠埃希菌O 157∶H 7带菌情况及其毒力采集上海市猪、牛等动物粪便以及市场和超市猪肉、牛肉、牛奶、虾仁等样本568 份,应用免疫胶体金技术、分离培养、形态特征观察生化特性鉴定、血清学试验、多重引物PCR进行大肠埃希菌O 157∶H 7的分离、鉴定及毒力基因分析。结果表明,上海市动物及动物产品中存在大肠埃希菌O 157∶H 7,检出率为2.05%。其中牛粪、猪粪中检出率较高,分别为9.76%和8.57%。

Comparison between <i>E. coli</i>O157:H7 and <i>Bifidobacterium</i>spp. Activity in Almond Pudding Infant Supplemental Food

Almond pudding is a common traditional Iranian complementary food for infants after starting solid foods. Escherichia coli O157:H7 is one of the leading pathogenic microorganisms that cause serious foodborne disease in different populations including infants. The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. The study objective of this research was to screen the survival characteristics of E. coli O157:H7 as well as four strains of Bifidobacterium subspecies (spp.) in almond pudding. The bacterial strains were studied after three and six hours of incubation at 37℃ in-vitro. Luria-Bertani (LB) broth was used as a basic medium for both Bifidobacterium spp. and E. coli experiments in anaerobic and aerobic conditions, respectively. The viability of Bifidobacterium spp. increased from 2.46 ± 0.2 to 6.57 ±1.3 log10 CFU/ml in low inoculum and from 4.53 ± 0.7 to 7.2 ± 0.4 in high inoculum experiments in 6 hours. However, the growth of E. coli O157:H7 from 3.12 ± 0.2 to 4.99 ± 0.1 log10 CFU/ml was significantly (P < 0.05) lower compared to Bifidobacterium spp. The results illus- trate impaired growth of E. coli O157:H7 and enhanced growth of Bifidobacterium spp. in almond pudding. The finding demonstrated that almond pudding in infant’s diet may indirectly enhance the protection against survival and growth of E. coli O157:H7 by increasing the Bifidobacterium spp. populations in infant’s gastrointestinal system.

肠出血性大肠杆菌O157∶H7 eae基因原核表达及间接ELISA的初步建立

对肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)O157∶H7 LEE毒力岛上的eae基因进行克隆与原核表达,以表达产物紧密素(intimin)为包被抗原,初步建立O157∶H7血清抗体间接ELISA。结果表明,抗原最佳包被浓度为0.88μg/mL,血清最佳稀释倍数为200;与鸡大肠杆菌其他血清型O1、O2、O11、O18、O35、O45、O78、O86、O88、O147、鸡白痢以及新城疫等血清均无交叉反应,该方法特异性较好;血清稀释倍数为1 600倍时仍能检测到O157∶H7 intimin特异性抗体,该方法灵敏度较高。

大肠埃希菌O_(157):H_7在8种食品中存活力观察

目的 探讨肠出血性大肠埃希菌 (EHEC)O157:H7在不同食品中的存活力。方法 采用EHECO157:H7933第 5代菌株人工污染 8种食品 ,染菌量为 7log10cfu/ml·g ;对该菌在 4、2 5℃条件下的存活力进行实验观察。 结果 EHECO157:H7在全奶、奶粉、鱼肉香肠及猪肉松 4种动物性食品中存活较长时间 ( 70d以上 ) ,且存活菌量呈先升后降的变化趋势 ,在 2种温度条件下均能增殖 1～ 2log10cfu/ (ml·g) ,到达高峰后呈对数衰减趋势 ,4 2d后仍有 3～ 6log10cfu/ml·g活菌量 ;在橙汁汽水和乳酸菌饮料中存活时间相对较短 ,分别为 2～ 5d和 10～ 2 7d ,4℃橙汁汽水和 2 5℃乳酸菌饮料在衰减至 4log10cfu/ (ml·g)后急剧下降 :在草莓酱和苹果酱中 ,两种温度条件下EHECO157:H7均呈间断性检出。结论 EHECO157:H7在常见食品中的存活能力强。

应用免疫磁珠法首次在宝山区检出大肠杆菌O_(157):H_7

[目的 ] 了解宝山区是否存在大肠杆菌O157∶H7以及动物带菌和食品、水源的污染情况。 [方法 ] 对采集的肉、乳制品、家禽、家畜粪便 ,快餐原料、各类水源等标本应用免疫磁珠法集菌进行E .coliO157∶H7分离和鉴定。 [结果 ] 共采集家禽、家畜、肉乳制品、快餐原料、各类水源等标本 610份 ,检出E .coliO157∶H73株 ,总检出率为 0 .49%。其中以肉类制品检出率最高 ,为 1.3 7% ;其次为其它标本 ,检出率为 1.0 2 % ,家禽、家畜粪便 ,检出率为 0 .3 8%。 [结论

应用斑点免疫层析、免疫磁珠法快速分离大肠杆菌O_(157)∶H_7

目的 :探讨斑点免疫层析法 (DICA)和免疫磁珠法 (IMS)在分离大肠杆菌 O1 57∶ H7(E.Coli O1 57∶ H7)上的快速性和敏感性。方法 :先用 DICA法对腹泻病人、家禽 (畜 )粪便增菌液进行快速筛检 ,再用 IMS法对所有标本做进一步检测 ,并和直接分离法作对照。结果 :DICA法阳性率为 10 .34% (43/ 4 16 ) ,IMS法检出率 1.92 % (8/ 4 16 ) ,DICA法假阳性率为 8.4 1% (35 / 4 16 ) ,无一例 DICA法假阴性 ,而直接法检出率为 0 .2 4 % (1/ 4 16 ) ,IMS法比直接法检出率有显著性提高 (P<0 .0 5 )。结论 :两法结合应用于检测 E.Coli O1 57∶ H7具有快速、省时、灵敏度高等特点 ,是开展 E.Coli O1 57∶ H7病原学检索值得推广应用的方法。