• Korean Journal of Veterinary Research
• /
• v.36 no.2
• /
• pp.371-378
• /
• 1996

Three and 2 strains of E coli O157 were isolated from fecal materials of cattle (390) and pigs (420) in Korea, respectively. One strain of O157:H7 isolated from cattle and 2 strains of O157:H7 isolated from pigs were identified as verotoxin-1 (VT-1) produing strains and 2 strains (O157:H7 and O157:H-) isolated from cattle were identified as verotoxin-2 (VT-2) producing strains by neutralization test on HeLa and Vero cells. Culture supernatants of the isolates were cytotoxic to HeLa and Vero cells. The levels of cytotoxin produced by isolates were $10^2{\sim}10^4$ cytotoxic dose($CD_{50}$)/ml. Also, VT-2-converting bacteriophage was isolated from KSC109 strain which had been isolated from cattle. Molecular weight of the phage DNA was determined as approximately 45 Kb in 0.8% agarose gel electrophoresis, and morphology of the phage stained with phosphotungstic acid was observed by transmissible electron microscopy.

• Korean Journal of Veterinary Research
• /
• v.36 no.2
• /
• pp.379-387
• /
• 1996

The objects of the present study were to establish the method of purification, subunit dissociation of verotoxin-2 (VT2) produced by Escherichia coli O157:H7, and to investigate the characteristics of purified verotoxin-2 such as molecular weight and composition of amino acid. The results were summerized as follows; Verotoxin-2 was extracted by addition of polymyxin B sulfate into bacterial cell lysate prepared from Escherichia coli O157:H7(KSC109). As an initial step, the bacterial cell lysate was precipitated with 30% saturated ammonium sulfate. The precipitated crude toxin was then subjected to anion-exchange, chromatofocusing and cation-exchange chromatography. Using this scheme, we obtained highly purified toxin with a specific activity of $1.1{\times}10^9$ $CD_{50}/mg$. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) for purified VT2 showed two protein bands. The upper band, approximately 32 Kd, was supposed as A subunit and the lower band, approximately 7.7 Kd, was supposed as B subunit. When the toxin was separated in the subunit-dissociating solution, two peaks emerged with retention times of 15 and 28 min by HPLC. These peaks represented A subunit and B subunit, respectively. The amino acid composition of purified VT2 were made up in order of glutamic acid, histamine, asparaginic acid, histidine, lysine, alanine and leucine etc. The largest amount among the amino acid composing VT2 was methionine.

• Microbiology and Biotechnology Letters
• /
• v.50 no.3
• /
• pp.387-394
• /
• 2022

Verotoxin-1 (VT-1) or Shiga-like toxin 1 (Stx-1) is produced by enterohemorrhagic Escherichia coli (EHEC) and is an AB5 holotoxin with a strong inhibitor of protein synthesis. VT-1 is a type 2 ribosome-inactivating protein (RIP) that has been shown to have cytotoxic and anticancer potential by inducing necrosis, apoptosis, and cell cycle arrest, making it a promising antitumor candidate. Here, we tested the cytotoxicity of VT-1 on CaCo2 and NCM425 cell lines and the results showed that VT-1 was more potent on CaCo2. Morphological changes were also evaluated on the cellular level and the results showed that VT-1 caused a decrease in viable cell count, altered cell membrane permeability, and an increase in total nuclear intensity. On the other hand, VT-1 displayed a lesser impact on mitochondrial membrane potential (MMP) and cytochrome c release. On the expression of caspases 3 and 9, VT-1 exhibited an insignificant effect on both which alongside the mitochondrial membrane potential (MMP) and cytochrome c results, might indicate that CaCo2 suffered from the necrosis process as a mechanism of cell death after exposure to VT-1.

• Korean Journal of Veterinary Research
• /
• v.50 no.3
• /
• pp.231-237
• /
• 2010

This study was conducted to investigate characteristics and antimicrobial susceptibility of Escherichia (E.) coli isolates isolated from vaginal mucosa and clitorial fossa of 105 Thoroughbred mares suspicious of the genital disease in Korea during the period from March 2006 to July 2007. Ninety six E. coli isolates were identified as standard biochemical properties and using BIOLOG system. Fifty three isolates (55.2%) could be classified into a total of 21 O serotypes and forty three isolates (44.8%) were non-typeable with 51 O antisera used in this study. The verotoxin 1 (VT 1) and verotoxin 2 genes were analyzed by multiplex PCR. Among them, one isolate was detected VT 1 gene (130 bp). Most of isolates showed a high susceptibility in ciprofloxacin (100%), enrofloxacin (100%), norfloxacin (100%), cefoxitin (96.9%), gentamicin (96.9%), sulphamethoxazole (96.9%), nitrofurantoin (94.8%), amikacin (93.8%), nalidixic acid (92.7%) and tetracycline (90.6%). These results may provide the basic information to establish strategies for the treatment and prevention of reproductive disease in Thoroughbred mares in Korea.

• Journal of Food Hygiene and Safety
• /
• v.15 no.3
• /
• pp.241-247
• /
• 2000

The incidence of verotoxin-producing Escherichia coli(VTEC) was surveyed in domestic foods including hamburger, raw meats and vegetables from 1997 to 1999. The molecular biological characteristics of the isolates were analyzed. Three VTEC strain were isolated from 1,700 samples. Serotypes of those isolates were 0157 : H7, 026 H4, and 056 : Hl2, respectively. Serotype O26 : H4 produced VT I and VT II, and 055 Hl2 isolate produced VT I, however the 60 MDa plasmid DNA and eae gene were not found from both strains. One 0157 : H7 isolate produced VT II and harbour 60 MDa plasmid DNA, however eae gene was not found in the strain. Although they produced VT, it seemed that the virulence of two strains were relatively weak because of the lack of the eae gene. In addition, the serotype O157 : H7 isolate resistant to ampicillin and streptomycin, while isolates of serotype O26 : H4 and O55 : Hl2 were multi-resistant to antibiotics including ampicillin, carbenicillin , cephalothin, trimethoprim/sulfamethoxazole and tetracycline. Supernatants of cultures of all three isolates were showed cytotoxic effect to vero and HeLa cell

• Pediatric Gastroenterology, Hepatology & Nutrition
• /
• v.20 no.2
• /
• pp.107-113
• /
• 2017

Purpose: This study clarified the bacterial pathogens currently causing acute infectious enterocolitis (AIE) in children and evaluated the clinical characteristics and ultrasonographic findings according to the different pathogens. Methods: Medical records regarding age, sex, clinical symptoms, laboratory data, identified enteropathogens, ultrasonographic findings, treatment, and outcome of 34 patients who were diagnosed with AIE via stool examination using multiplex polymerase chain reaction (PCR) or culture, were retrospectively reviewed. Results: Twenty-four patients (70.6%) were male. The mean age of the patients was $8.5{\pm}6.2$ (range, 1.1-17.1) years. Six bacterial pathogens were isolated: Salmonella species (spp.) (32.4%), Campylobacter spp. (20.6%), verotoxin-producing Escherichia coli (14.7%), Staphylococcus aureus (11.8%), Clostridium difficile (8.8%), and Shigella spp. (2.9%). Abdominal pain occurred in all patients regardless of pathogen. The patients infected with Salmonella were older than those infected with verotoxin-producing E. coli (p<0.05). C-reactive protein levels were higher in patients with Salmonella and Campylobacter infections than in those with verotoxin-producing E. coli infection (p< 0.05), the other clinical and laboratory data were indistinguishable between pathogens. Ultrasonography demonstrated diverse involvement of bowel segments according to pathogen. Wall thickening of both the ileum and the entire colon was the most common lesion site regardless of pathogen. Conclusion: Various bacterial agents cause AIE and the symptoms are diverse symptoms, however, all most children recovered spontaneously. Use of multiplex PCR on stool samples warrants improvement of its sensitivity for diagnosis of enteropathogenic bacteria. Ultrasonographic examination is useful for diagnosis of AIE; it can also detect the disease extent and severity.

• Korean Journal of Veterinary Research
• /
• v.37 no.3
• /
• pp.541-546
• /
• 1997

There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

• Journal of Microbiology
• /
• v.37 no.3
• /
• pp.168-174
• /
• 1999

Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

• Journal of Food Hygiene and Safety
• /
• v.12 no.1
• /
• pp.78-82
• /
• 1997

Surface swab samples from beef (188), pork (240) and chicken (95) carcasses were collected from slaughterhouse in Kangwon and Kyunggi areas from March through July 1996. The samples were examined on the level of E. coli biotype I relevant to fecal contamination due to unsanitary processing control and the existence of verocytotoxin-producing E. coli (VTEC). E. coli biotype I were confirmed from 38.8% of beef, 40.0% of pork, and 69.5% of chicken carcasses. Little variation was noted among three sampling points; rump, flank and neck of beef, ham, belly and jowls of pork. coli O157:H7 was only confirmed from 2 of 188 beef carcasses. E. coli biotype I. All the isolated E. coli O157 showed positive for vero cell cytotoxicity test. Isolation rate of E. coli O157 in summer was higher than in spring. In case of pork and chicken carcasses, E. coli O157 was isolated in summer only.

• Childhood Kidney Diseases
• /
• v.23 no.1
• /
• pp.43-47
• /
• 2019

Atypical hemolytic uremic syndrome (aHUS), a rare form of thrombotic microangiopathy, is distinguished from the typical form by the absence of a preceding verotoxin-producing Escherichia coli infection. Notably, aHUS occurs in association with genetic or acquired disorders causing dysregulation of the alternative complement pathway. Patients with aHUS may show the presence of anti-complement factor H (CFH) autoantibodies. This acquired form of aHUS (antiCFH-aHUS) primarily affects children aged 9-13 years. We report a case of a 13-year-old Lao girl with clinical features of aHUS (most likely anti-CFH-aHUS). The initial presentation of the patient met the classical clinical triad of thrombotic microangiopathy (microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury) without preceding diarrheal illness. Low serum levels of complement 3 and normal levels of complement 4 indicated abnormal activation of the alternative complement pathway. Plasma infusion and high-dose corticosteroid therapy resulted in improvement of the renal function and hematological profile, although the patient subsequently died of infectious complications. This is the first case report that describes aHUS (possibly anti-CFH-aHUS) in Laos.