Sequence Analysis of Type III Effector tccP and tccP2 Genes in Escherichia coli O157:H7 from Chinese Water-chestnut

[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.

肠出血性大肠杆菌O157P:H7紧密黏附素的基因克隆、表达及功能研究

目的获得高纯度的eae基因表达蛋白紧密黏附素(Intimin),研究其黏附作用。方法从肠出血性大肠杆菌(EHEC)O157:H7全基因组中扩增出eae基因,T-A克隆后,将eae插入载体pET28a(+),并转化至E.coli BL21(DE3)中表达;用Ni2+-NTA琼脂糖柱纯化出重组蛋白;SDS-PAGE检测目的蛋白相对分子质量,免疫印记分析其免疫反应性,免疫荧光检测其黏附性。结果获得了大小约2805bp的eae片段;构建了重组载体pET28a(+)-eae,并在E.coli BL21(DE3)中以包涵体形式表达Intimin,Mr约97000;Ni2+-NTA琼脂糖柱纯化出Intimin;大肠杆菌O157:H7多抗血清在Mr约97000处检测出一条特异性Intimin带;Intimin可黏附在HEp-2细胞表面。结论高纯度的重组蛋白Intimin具有一定的免疫反应性,能与HEp-2细胞黏附,为进一步研究Intimin蛋白与宿主受体蛋白的相互作用奠定基础。

冷等离子体技术对黄瓜表面大肠杆菌O157∶H7生物膜的清除作用研究

利用冷源氮气等离子体杀菌技术清除新鲜黄瓜表面的大肠杆菌O157∶H7生物膜。试管实验结果表明,等离子体对大肠杆菌O157∶H7生物膜有较好的清除作用,在作用功率为500 W,连续作用4 min后对生物膜清除率达到99.99%。当等离子体应用于黄瓜表面时,作用4 min时,杀菌率达到99.21%。激光共聚焦显微镜结果显示:等离子体作用后,菌落数量和细菌生物膜厚度明显小于对照组。场发射扫描电子显微镜结果显示:与对照组比较,等离子组4 min处理后细菌群落附着数量显著变少。总之,等离子体技术在基本维持食品感官的前提下提高了新鲜黄瓜的微生物安全性。

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.

Microarray analysis of Escherichia coli0157:H7

AIM： To establish the rapid, specific, and sensitive method for detecting O157：H7 with DNA microchips. METHODS： Specific oligonucleotide probes （26-28 nt） of bacterial antigenic and virulent genes of E. coliO157：H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen （rfbE）, H7 flagellar antigen （fliC） and toxins （SLT1, SLT2） were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-1abeled single prime. Hybridization was performed for 60 min at 45 ℃. Microchip images were taken using a confocal fluorescent scanner. RESULTS： Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157：H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-1abeled fluorescent samples from O157：H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157：H7 fluorescent samples occurred in other probes. Non-O157：H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-1abeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION： Microarray analysis of O157：H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.

Computational prediction and experimental validation of novel markers for detection of STEC O157:H7

AIM:To identify and assess the novel makers for detection of Shiga toxin producing Escherichia coli (STEC) O157:H7 with an integrated computational and experimental approach. METHODS:High-throughput NCBI blast (E-value cutoff e-5) was used to search homologous genes among all sequenced prokaryotic genomes of each gene encoded in each of the three strains of STEC O157:H7 with complete genomes,aiming to find unique genes in O157:H7 as its potential markers. To ensure that the identified markers from the three strains of STEC O157:H7 can serve as general markers for all the STEC O157:H7 strains,a genomic barcode approach was used to select the markers to minimize the possibility of choosing a marker gene as part of a transposable element. Effectiveness of the markers predicted was then validated by running polymerase chain reaction (PCR) on 18 strains of O157:H7 with 5 additional genomes used as negative controls. RESULTS:The blast search identified 20,16 and 20 genes,respectively,in the three sequenced strains of STEC O157:H7,which had no homologs in any of the other prokaryotic genomes. Three genes,wzy,Z0372 and Z0344,common to the three gene lists,were selected based on the genomic barcode approach. PCR showed an identification accuracy of 100% on the 18 tested strains and the 5 controls. CONCLUSION:The three identified novel markers,wzy,Z0372 and Z0344,are highly promising for the detection of STEC O157:H7,in complementary to the known markers.

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.

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.

Construction and prokaryotic expression of the fusion protein Stx2B-IntiminC300 of EHEC O157: H7 and its immunoprophylactic potential

To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.

A Primary Test of EHEC O104:H4 and EHEC O157:H7 in Certain Kinds of Food in Wuhan,China

Objective:This paper provided preliminary description of food contamination derived from Enterohemorrhagic Escherichia coli(EHEC)O104:H4 and EHEC O157:H7 in Wuhan in June 2011.Methods:47 food samples,including vegetables and meat,were subjectively sampled from some restaurants.PCR assays were used to detect EHEC O104:H4 and EHEC O157:H7.Results: The PCR results showed that none of the samples were positive for either EHEC O104:H4 or EHEC O157:H7.Conclusion:Although large outbreaks of gastroenteritis and the hemolytic uremic syndrome caused by EHEC O104:H4 had occurred in some European countries,China has had few outbreaks associated with EHEC O104:H4.This shows that food supply is relatively safe in China.Nevertheless,many ongoing problems of food safety in China are still not solved showing the necessity of further studies on food safety.

The Use of Str Mutations for Enhancement of Hydrogen Peroxide Formation by Lactobacillus Delbrueckii MH-IO at Refrigeration

The strains ofLactobacillus delbrueckii subsp, lactis widely used in food preservation due to ability produce high amount of hydrogen peroxide at refrigerator temperatures to inhibit food-borne pathogens and psychrophilic spoilage microorganisms. In order to improve of bio-preservation efficacy ofL. delbrueckii MH 10 mutations causing resistance to streptomycin （str） were used. Among UV-mutagenized population of L. delbruecla＇i three str mutants producing high amounts of H2O2 were selected. Sir mutants produced significant amounts of hydrogen peroxide 50-60 μg/ml in sodium phosphate buffer （0.2 M, pH 6.5） and in beef broth （BB） at 5 ℃ for 5 days submerged cultivation without of growth. Evaluation mutants antibacterialactivity at refrigeration temperatures against food-borne pathogen Escherichia coli O157：H7 revealed elimination of pathogen total number up to practically undetectable amount for 3 days. In case of solid-state cultivation on agar-based medium, disks soaked by mutant cells suspensions formed larger inhibitory zones on E. coli O157：H7 lawn for one-day cold exposition. The size of inhibition zone depends on concentration of LAB cells. Str mutants L. delbrueckii reduced initial amount 2 - 105 of E. coil O 157：H7 in ground beef up to 3 log for 3 days of solid-state cocultivation when the wild strain reduced only 2 log. The application ofL. delbrueckii mutants did not cause any changes in sensory characteristics of ground beef, moreover promotes expanding of shelf-life due to inhibition of psychrophilic spoilage microorganisms.

Prevalence of Antibiotic Resistant Bacteria on Tomato Surfaces and Effectiveness of Disinfectants in Reducing the Microbial Load

This study was conducted to isolate and identify antibiotic resistant bacteria in fresh tomato sold in three different local markets of Dhaka city. On an average 〉 5.0 log CFU/g of natural microbial populations and coliform bacteria was present in the samples. Prevalence of Salmonella spp., E. coli, Listeria spp., and Yersinia spp., were recorded through cultivation dependent assay of tomato samples of different markets. Almost all of the isolates were uniformly resistant to rifampicin, erythromycin, clindamycin and oxacillin （100%）, vancomycin （93%）, amoxicillin （87%）, whereas 60% of the isolates were found to be resistant to ampicillin and cephalexin. Antibiotic sensitivity test of the presumptive E. coli and Salmonella spp. and corresponding plasmid profiling indicated a correlation of plasmid mediated multidrug resistance （MDR） of these environmental bacteria. Washing with sanitizing agents such as acidified sodium chlorite （ASC） and 200 ppm chlorinated water was able to reduce _〈 2.0 log CFU/g of natural microflora and coliform bacteria. In contrast, ASC was able to reduce 〈 2.0 log CFU/g of other food borne pathogens and, chlorinated water was able to reduce up to undetectable level. Therefore, washing of tomato with 200 ppm chlorinated water could be useful in reducing the pathogen populations on tomatoes.

纳豆菌对致病者生长抑制作用的初步研究

目的 探讨纳豆菌与O157∶H7、金黄色葡萄球菌、沙门菌这 3种致病菌分别共同培养时细菌数的变化情况。方法 分别培养纳豆菌和 3种病菌并进行纯化。①选取培养纳豆菌与致病菌同时培养 ;②先培养纳豆菌 8h ,再加致病菌。分别观察抑制结果。结果 纳豆菌对金黄色葡萄球菌的抑制作用较为明显 ;对O157∶H7也有一定的抑制作用 ;而沙门菌的抑制作用不明显。如果将纳豆菌先培养 6～ 8h ,再将O157∶H7、金黄色葡萄球接种进去共同培养 ,则抑菌作用更为明显。

分子对接比较随机RNA片段与同源建模蛋白及其模板的相互作用

为了促进开发大肠杆菌O157∶H7快速检测适体生物传感器,对其鞭毛蛋白紧密黏附素以肠致病性大肠杆菌紧密黏附素为模板,进行空间结构同源建模并作为目标抗原,用不同对接方法模拟预测研究比较其与随机RNA序列的相互作用。方法可行性通过模拟研究已知亲和性同一RNA适体与不同物种凝血酶相互作用论证。结果表明,不同随机RNA序列与不同菌株的紧密黏附素空间相互作用的位点预测有一定差异,说明针对不同蛋白质进行高亲和适体筛选具有一定的可行性。分子对接研究表明因不同长度RNA所形成空间结构不同,而与大肠杆菌O157∶H7紧密黏附素结合位点存在一定差异、与PRIdictor预测位点结果接近;具有不同RNA结构模体的片段对紧密黏附素的亲和力不同,有从不同片段中筛选高亲和力的RNA适体的可能。分子模拟进一步研究RNA-紧密黏附素相互作用的方法具有一定的可行性,将有助于进一步通过设计合成RNA改进适体筛选、研发的相关生物技术推广,以及应用创新。