• Journal of Dairy Science and Biotechnology
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• v.33 no.1
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• pp.59-67
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• 2015

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is well-characterized as an important food-borne pathogen worldwide and causes human diseases such as diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) by producing shiga-like toxin (Stx). It has been reported that a number of dairy foods, including cheese, can act as the source of EHEC O157:H7 infections. In addition to the toxicity of Stx, recently it has been indicated that EHEC O157:H7 possesses virulence factors related to attachment, quorum sensing, and biofilms. Moreover, these novel virulence factors might become critical points to be considered in the future production of food derived from animals. Here, we review the evidences that support these insights on new virulence factors and discuss the potential mechanisms mediating the pathogenesis of EHEC O157:H7 in the dairy food industry.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.495-502
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• 2004

Intimin, the product of eae gene in EHEC O157:H7, is required for intimate adherence. In this study, the C-terminaI region(281 amino acids) of the EHEC OI57:H7 intimin were expressed as a protein fusion with (His)$_6$ which was used to raise antiserum in rabbits. The antiserum reacted in western blot with a 94kDa outer membrane protein of EHEC O157:H7. It was observed that the antibody titers both in egg yolk and serum appeared in 2${\sim}$4 weeks after immunization with fusion protein. At the time of 8 weeks, the titre of egg yolk was found to be higher than that of sera. According to the results of neutralization test, chicken egg-yolk antibody(lgY) against the recombinant intimin strongly reacted to EHEC O157:H7. We conclude that a truncated recombinant intimin could be used as an immunogen to elicit antibody(lgY) against O157:H7.

• Biomedical Science Letters
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• v.4 no.1
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• pp.43-56
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• 1998

A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

• Food Science of Animal Resources
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• v.24 no.1
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• pp.86-91
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• 2004

The ability of probiotics containing Lactobacillus acidophilus to adhere to the intestinal epithelium may play an important role in colonization of the gastrointestinal tract and preventing enteric pathogen such as enterohemorrhagic E. coli(EHEC O157:H7. In the study, we investigated the adhesion to human intestinal epithelial cells(HT-29) of strains of L. acidophilus(3 from human, 2 from pig, and 1 from calf). All of the tested strains of L. acidophilus were highly observed adhesion ability(from 10$\^$6/ to 10$\^$7/ cfu/mL), compared to L. rhamnosus GG as control. Also, adhered strains of L. acidophilus were significantly preserved in serial wash-out steps. However, no correlation could be observed between cell surface hydrophobicity and adhesion abilities of the tested strains of L. acidophilus. Inhibition of adhesion of EHEC O157:H7 was also examined, a 2 log cycle reduction was observed by all of the tested strains of L. acidophilus. These results suggest that the strains of L. acidophilus with high adhesion ability are resistant to wash-out and adhesion ability inhibition by selected strains of L. acidophilus helps to prevent adhesion of EHEC O157:H7 to intestinal epithelial cells.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.401-407
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• 2014

Quorum sensing and the stringent response are well-known regulation systems for the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). However, how these two systems interact is not well known. E. coli strains with mutations in two regulation systems, ${\Delta}luxS$ (ECM101) and ${\Delta}luxS{\Delta}relA{\Delta}spoT$ (ECM201), and the ${\Delta}luxS$ complement strain to ECM201 (ECM202) were created from EHEC O157:H7 EDL933 to investigate how the regulatory systems interact. The phenotypic changes of the mutant strains were characterized and compared with the wild type. The mutant strains exhibited no obvious growth defects, although acid resistance and cellular cytotoxicity were decreased significantly in all the mutant strains. Phenotypic characterization revealed that mutations in the stringent response system (ECM201 and ECM202) influenced the metabolic (defective utilization of arabinose and L-sorbose) and enzymatic activities (decreased trypsin activity, and increased ${\alpha}$-glucosidase activity). In contrast, the quorum sensing system mutant (ECM101) did not display these phenotypes. The motility of the quorum sensing system mutant (ECM101) was unchanged, but mutation in the stringent response system influenced the motility. Our results suggest that quorum sensing interacts with the stringent response regulation system.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• Journal of Food Hygiene and Safety
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• v.18 no.3
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• pp.118-124
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• 2003

For the rapid detection of various pathogenic microorganisms from food sample, various kinds of kits have been developed and commercially available in the markets. With the advantages of speed, accuracy and easiness, the market of these kits has gradually increased for the QC and QA field of food company as well as testing facilities or laboratories. In this study, the characteristics such as the detection limit and the sensitivity of immunochromatographic type of rapid detection kit (Donga Co, Korea, D-kit) for E. coli 0157:H7 developed by monoclonal antibody were examined and also the possibility of application of the kit to food samples was evaluated. The reference kits used for comparison study were Reveal E. coli 0157:H7 (Neogen Co., USA, R-kit) and VIP EHEC kit (Biocontrol Inc., USA, V-kit) occupying major market share. In the detection limit test with the E. coli 0157:H7 reference, both R-kit and D-kit showed a distinct positive reaction in $10^4$/ml and weak positive reaction in $10^3$/ml, whereas V-kit showed a same reaction in 105/ml. Also, it was identified that the culture treated with heat showed more sensitivity than no heat treated culture. The sensitivity test was conducted against 22 isolates of E. coli 0157:H7, 7 strains of non-O157:H7 verotoxin-producing E. coli, 40 strains of E. coli with different O and H antigen type, and 38 strains of non-E. coli Enterobacteriaceae, and all of the test strains except three were showed exactly three were showed exactly the same reaction against three kinds of the tested kits. All the three kinds of kits showed a positive reaction against E. coli O157:H19, E. coli O148:H18 and Salmonella galinarium. We suppose that there might be a similarity in serological property between these three strains and O157:H7. From the test results, it can be concluded that there is (was) no difference between the D-kit developed in this study and R-kit or V-kit based on the detection limit and sensitivity.

• Journal of Dairy Science and Biotechnology
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• v.28 no.2
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• pp.7-11
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• 2010

In recent times, antimicrobial resistance has been a concern because of its relation to national health and food safety. In this study, we reviewed the management of antibiotics and antimicrobial-resistant microorganisms in domestic and foreign countries and analyzed microorganisms and antimicrobial resistance in dairy farms and raw milk. The isolates from dairy farms were Micrococcus spp., Staphylococcus chromogens, Escherichia coli, Bacillus licheniformis, Escherichia coli O157:H7, Pseudomonas plecoglossicida, Enterobacter spp., and Kluyvera intermedia. Rummeliibacillus stabekisii, Paenibacillus badius, Pantoea agglomerans, B. oleronius, B. fusiformis, and B. badius were isolated from feed E. coli and Kurthia gibsonii, from feces and S. pasteuri, S. aureus, S. chromogenes, and Salmonella spp., from raw milk. Pathogens isolated from dairy farms and raw milk were tested for susceptibility to 20 types of antibiotics. E. coli (EAEC) and E. coli O157:H7 (EHEC) isolated from dairy farms, E. coli (EAEC) isolated from feces, and Salmonella spp. isolated from raw milk showed resistance to multiple antibiotics. These results show that antimicrobial-resistant microorganisms should be more effectively managed to improve the safety of dairy farms.

• Clinical and Experimental Pediatrics
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• v.57 no.2
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• pp.96-99
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• 2014

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in childhood and is primarily diagnosed in up to 4.5% of children who undergo chronic renal replacement therapy. Escherichia coli serotype O157:H7 is the predominant bacterial strain identified in patients with HUS; more than 100 types of Shiga toxin-producing enterohemorrhagic E. coli (EHEC) subtypes have also been isolated. The typical HUS manifestations are microangiopathic hemolytic anemia, thrombocytopenia, and renal insufficiency. In typical HUS cases, more serious EHEC manifestations include severe hemorrhagic colitis, bowel necrosis and perforation, rectal prolapse, peritonitis, and intussusceptions. Colonic perforation, which has an incidence of 1%-2%, can be a fatal complication. In this study, we report a typical Shiga toxin-associated HUS case complicated by small intestinal perforation with refractory peritonitis that was possibly because of ischemic enteritis. Although the degree of renal damage is the main concern in HUS, extrarenal complications should also be considered in severe cases, as presented in our case.