Green Tea Extract (Camellia sinensis) Fermented by Lactobacillus fermentum Attenuates Alcohol-Induced Liver Damage

The preventive effects of glycomacropeptide (GMP) against intestinal infection were investigated, and conjugates of GMP with xylooligosaccharide (XOS) and carboxymethyldextran (CMD) were prepared by the Maillard reaction to enhance the effect of GMP. The binding ability of GMP to intestinal pathogenic bacteria was evaluated by a binding assay with biotinylated bacteria. GMP showed the ability to bind to Salmonella enteritidis and enterohemorrhagic Escherichia coli O157:H7 (EHEC O157). This binding ability was decreased by a sialidase treatment and completely eliminated by periodate oxidation. These results indicate that such carbohydrate moieties as sialic acid in GMP are involved in binding to S. enteritidis and EHEC O157. The preventive effect of GMP on the adhesion of pathogenic bacteria to Caco-2 cells was also investigated. GMP showed an inhibitory effect on the adhesion of EHEC O157 in a dose-dependent manner, although it was not a potent inhibitor of the adhesion of Salmonella infection. However, in the case of Salmonella infection, GMP–XOS and GMP–CMD significantly suppressed IL-8 production which was the index of infection. Our results indicate GMP to be a promising agent for preventing intestinal infection.     

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:H7 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC O157:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C. butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:H7 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C. butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:H7 infection in gnotobiotic mice.     

Heparin blocks the adhesion of E. coli O157:H7 to human colonic epithelial cells

Adhesion of Shiga toxin-producing Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to human colonic epithelium is a critical step for infection by this type of bacteria. Here, we demonstrate that adherence of EHEC O157:H7 to cultured human colonic T84 epithelial monolayers can be blocked by heparin and heparan sulfate in a dose-dependent fashion. In doing this, heparin and heparan sulfate also prevent dysfunction of the T84 barrier and disorganization of epithelial tight junction protein ZO-1 caused by EHEC O157:H7. This inhibition by heparin and heparan sulfate seems to result from a block in the binding interactions of bacteria intimin with epithelial β₁ integrins. This study provides evidence, for the first time, that heparin and heparan sulfate can serve as novel effective blockers in preventing EHEC O157:H7 infection.     

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.     

EHEC O157:H7 z3276基因缺失株构建及其生物学特性

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。     

Lipopolysaccharide O-antigen of enterohemorrhagic Escherichia coli O157:H7 is required for killing both insects and mammals

Studies of enterohemorrhagic Escherichia coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals.     

EHEC 的新型粘附因子研究进展*

摘要：肠出血性大肠杆菌（enterohemorrhage ，EHEC）是一种重要的人畜共患病，世界各地包括中国都有不同规模的暴发流 行。EHEC有多种血清型，其中毒力最强血清型是O157：H7。EHEC O157：H7 感染除可使人发生常规腹泻外，还可在5%-10%的病 例中引发严重并发症，甚至死亡。该菌是重要的食源性致病菌，危害严重，缺乏有效的防治手段，而抗生素治疗可能会加剧溶血性 尿毒症（haemolutic uraemic syndrome，HUS）。由于以上特点EHEC O157：H7 成为世界公共卫生问题，引起微生物学家和公共卫生 工作者的广泛关注。目前，临床针对EHEC 感染只是对症治疗和适当的抗菌治疗。粘附是EHEC感染宿主细胞的第一步，没有这 一步，细菌和宿主肠道细胞之间不会发生任何的相互作用，而且对于许多病原菌来说，粘附具有宿主特异性。本文概述了EHEC 的流行病学及粘附机理，并对近年在EHEC 研究中的发现一些新型粘附因子和一些假设的定植因子的研究背景及作用机理作一 综述。     

EHEC的新型粘附因子研究进展

肠出血性大肠杆菌（enterohemorrhage E-coli,EHEC）是一种重要的人畜共患病,世界各地包括中国都有不同规模的暴发流行。EHEC有多种血清型,其中毒力最强血清型是0157：H7。EHEC0157：H7感染除可使人发生常规腹泻外,还可在5％-10％的病例中引发严重并发症,甚至死亡。该菌是重要的食源性致病菌,危害严重,缺乏有效的防治手段,而抗生素治疗可能会加剧溶血性尿毒症（haemoluticuraemicsyndrome,HUS）。由于以上特点EHEC0157：H7成为世界公共卫生问题,引起微生物学家和公共卫生工作者的广泛关注。目前,临床针对EHEC感染只是对症治疗和适当的抗茵治疗。粘附是EHEC感染宿主细胞的第一步,没有这一步,细菌和宿主肠道细胞之间不会发生任何的相互作用,而且对于许多病原菌来说,粘附具有宿主特异性。本文概述了EHEC的流行病学及粘附机理,并对近年在EHEC研究中的发现一些新型粘附因子和一些假设的定植因子的研究背詈及作用机理作一综述。     

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S. enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S. enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40-43 degrees C) or to graded numbers (10(1)-10(8)) of bacteria and in villus-like cells exposed to S. enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S. enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S. enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.     

Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli.

Two types of pathogenic Escherichia coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), cause diarrheal disease by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia. The intimin (eae) gene is part of a pathogenicity island, a 35-kb segment of DNA that has been acquired independently in different groups of pathogens. Nucleotide sequences of eae of three EPEC and four EHEC strains representing distinct clonal lineages revealed an exceptionally high level of divergence (15%) in the amino acid sequences of alpha, beta, and gamma intimin molecules, most of which is concentrated in the C-terminal region. The gamma intimin sequences from E. coli strains with serotypes O157:H7, O55:H7, and O157:H- are virtually identical, supporting the hypothesis that these bacteria belong to a single clonal lineage. Sequences of beta intimin of EPEC strains of serotypes O111:H2 and O128:H2 show substantial differences from alpha and gamma intimins, indicating that these strains have evolved independently. Strong nonrandom clustering of polymorphic sites indicates that the intimin genes are mosaics, suggesting that protein divergence has been accelerated by recombination and diversifying selection.     

Effect of Lactobacillus isolates on the adhesion of pathogens to chicken intestinal mucus in vitro

AIMS: The aims of this study were to investigate in vitro the effects of Lactobacillus isolates from a chicken on adhesion of pathogenic Salmonella and Escherichia coli to chicken intestinal mucus obtained from different intestinal regions. METHODS AND RESULTS: Bacteria were labelled by using methyl-1,2-[(3)H]-thymidine. The bacterial adhesion was assessed by measuring the radioactivity of bacteria adhered to the mucus. The results showed that the abilities of Lactobacillus spp. to bind to the same intestinal mucus were higher than those of pathogenic Salmonella and E. coli. Pretreatment of intestinal mucus with Lactobacillus fermentum and Lactobacillus acidophilus, alone or in combination, reduced the adhesion of the tested pathogens, but the reductive extent of pathogenic adhesion by Lactobacillus spp. in combination was relatively high. CONCLUSIONS: The tested bacteria had different adhesions to mucus glycoproteins isolated from different intestinal regions of chicken. Lactobacillus acidophilus and Lact. fermentum in combination revealed a better ability to inhibit attachments of Salmonella and E. coli to chicken intestinal mucus than Lactobacillus sp. alone. SIGNIFICANCE AND IMPACT OF THE STUDY: A mixture of intestinal Lactobacillus spp. from a chicken may play a protective role in excluding pathogenic Salmonella and E. coli from the intestine of chicken.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

Encoded protein from ycbR gene of enterohemorrhagic Escherichia coli O157:H7 associated with adherence to HEp-2 cells

Adhesion is one of the significant virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. It is regulated by specific loci in the genome sequence. This study mainly focused on investigating the influence of ycbR gene and its encoded YCBR protein on the adhesion of EHEC O157:H7 to HEp-2 cells. In the first part, mutants of EHEC O157:H7 were constructed through TnphoA mutagenesis and assayed for adherent ability. Six mutant strains with lost adherence to HEp-2 cells were isolated and then sequenced using a primer that hybridized to phoA sequence downstream of the fusion joint. The sequencing results indicated that the gene of eae and ycbR, between the initiation codon and the −10 sequence of Z4182, yciI, ARAC-type regulator protein, and high-affinity gluconate transporter of EHEC were all possibly related to adhesion. Of the six genes, the ycbR gene was cloned to the pET28a vector to analyze its function further. Recombinant YCBR protein fused to a His tag (YCBR-His) was expressed under IPTG induction and purified by Ni-NTA column. The purified protein was subcutaneously injected to rabbits to prepare antisera. The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of EHEC O157:H7 to HEp-2 cells. These observations suggested that ycbR encoded a novel adherence-associated determinant of EHEC O157:H7, which could contribute to the adhesive capacity of the bacteria.     

Protective effect of bifidus milk on the experimental infection with Salmonella enteritidis subsp. typhimurium in conventional and gnotobiotic mice

The ability of Bifidobacterium bifidum from a commercial bifidus milk to antagonize Salmonella enteritidis subsp. typhimurium in vivo, and to reduce the pathological consequences for the host, was determined using conventional and gnotobiotic mice. Conventional animals received daily, by gavage, 0.1 ml bifidus milk containing about 10(9) cfu B. bifidum and germ-free animals received a single 0.1 ml dose. The conventional and gnotobiotic groups were challenged orally with 10(2) cfu of the pathogenic bacteria 5 and/or 10 d after the beginning of treatment. Control groups were treated with milk. Bifidus milk protected both animal models against the challenge with the pathogenic bacteria, as demonstrated by survival and histopathological data. However, to obtain the protective effect in gnotobiotic animals, the treatment had to be initiated 10 d before the challenge. In experimental and control gnotobiotic mice, Salm. enteritidis subsp. typhimurium became similarly established at levels ranging from 10(8) to 10(9) viable cells g-1 of faeces and remained at these high levels until the animals died or were sacrificed. It was concluded that the protection against Salm. enteritidis subsp. typhimurium observed in conventional and gnotobiotic mice treated with bifidus milk was not due to the reduction of the intestinal populations of the pathogenic bacteria.     

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.     

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Antibacterial abietane-type diterpenoid, taxodone from Metasequoia glyptostroboides Miki ex Hu

In an attempt to isolate bioactive constituents, ethyl acetate cone extract of Metasequoia glyptostroboides was subjected to a column chromatographic analysis that resulted in isolation of an abietane-type diterpenoid, taxodone. Its structure was elucidated by spectroscopic means. Further, taxodone showed potential antibacterial effect as diameters of zones of inhibition against foodborne pathogenic bacteria, such as Listeria monocytogenes ATCC 19166, Salmonella typhimurium KCTC 2515, S. enteritidis KCTC 2021, Escherichia coli ATCC 8739, E. coli O157:H7 ATCC 43888, Enterobacter aerogenes KCTC 2190, Staphylococcus aureus ATCC 6538 and S. aureus KCTC 1916, were found in the range of 9.4 to 14.2 mm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of taxodone against the employed bacterial pathogens were found in the range of 250 to 1000 and 250 to less than 2000 microg/ml, respectively. Also the compound had a strong antibacterial effect on the viable counts of the tested bacteria. These findings indicate that the bioactive compound taxodone present in M. glyptostroboides could be used as an antibacterial agent in food industry to inhibit the growth of certain important foodborne pathogens.