Fecal isolates of Escherichia coli which were collected from diarrheal patients and HUS patient in Pusan National University Hospital between 1990 and 1996, were serotyped and analyzed for plasmid DNA profile, biotype, HEp-2 cell adherence ability, reactivity to eae probe and for production of verotoxins (VT). In order to ease the diagnosis of EHEC infection, a LPS- based solid phase enzyme linked immunosorbent assay was utilized to detect serological diagnosis of EHEC infection. The following results were obtained. Among 150 EPEC isolates and HUS patient's stool, 7 EHEC were found. The 7 EHEC belonged to 5 different serotypes 0157:H7, 0143:H-, 0166:H-, 0128:H2, 026:H-, and 0111:H 21 previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Biochemical cheracteristic analysis indicated 7 strains were biotype 1 and was found to have siderophilins suggesting their advantagous growth in vivo. For plasmid profiles, all strains had 60 MDa plasmid and several smaller sizes of plasmids. Three strains of Escherichia coli serotype 0157:H7, 0128:H2, and 026:H- showed one pattern of adherence in the HEp-2 cell assay namely, localized adherence and were positive for eae probe when tested by colony blot hybridization assay. PCR using specific primers for VT1, VT2 was tested, and all 7 strains carried VT1 gene only. PCR products of 130-bp (VT1) and 346-bp (VT2) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC. The serum obtained from HUS patient of enterohemorrhagic E. coli were analyzed for rises in titer of intibody to somatic 02, 026, 0111, 0128, 0143, 0145, 0157, and 0165. Although response to the somatic 0 correlated significantly with response to the 026 rises of antibody titer to somatic 0 in acute stage of disease and anti-VT had not so many relation to that of VT. These results suggest that ELISA can be used to detect somatic 0 in serum and it is a useful method to diagnose the infection caused by EHEC rapidly.
Antibodies* ; Busan ; Colitis ; Diagnosis* ; DNA ; Enterohemorrhagic Escherichia coli* ; Enteropathogenic Escherichia coli ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Multiplex Polymerase Chain Reaction ; Plasmids ; Polymerase Chain Reaction* ; Shiga Toxins ; Transferrin

Cholera enterotoxin (CT) is a major virulence determinant of Vibrio cholerae 01. CI' is known to be the major virulence factor of Vibrio cholerae 01 and in accordance with the recent report showing which V. cholerae non-01 has ctx gene, we performed the molecular genetic study for the detection of ctx gene related to the production of CT at the subject Vibrio spp. except for V. cholerae non-01 and V. cholerae non-01 stock cultured in the laboratory of microbiology, College of Medicine, Pusan National University and the Vibrio spp. isolated from the marine products of Pusan General Fish Market and the sea water, and then its results are as follows: 1. PCR for the detection of ctx gene at the subject of V. cholerae 01:61H-151 having the ctx gene of which the denaturation is 1 rninute at 95'C, annealing to 1min, 30 sec at 60'C, the extension to be 1min. 30 sec at 72'C and 30 or 40 cycles. ctx gene was detected from 4 strains of V. cholera non-01 derived from the environment isolates. 2. Adjusting the quantity of chromosomal DNA used as template DNA to be from 0.1 pg to 1 ng, in order to know the PCR conditions for the effective search of ctx gene, and the detection limit of the system was 10 pg of chromosomal DNA. 3. The broth culture was used for template DNA, ctx gene of 302 bp was detected from 4 V. cholerae non-01, as in the case of chromosomal DNA, and the cell number was possible to be detected to 3 * 10.4. We attempted the confirmation of ctx gene through Southern blot hybridization, labeling with P and then it was confirmed only from 4 V. cholerae non-01 as like PCR results. 5. As the result of the sensitivity of PCR and Southern blot hybridization, it was shown to be possible which 10 pg was detected in case of chromosomal DNA and in case of cultured broth, the cell number was detected until 10 at PCR and Southern blot hybridization, and thus it was examed which its sensitivity was same.
Blotting, Southern ; Busan ; Cell Count ; Cholera Toxin* ; Cholera* ; DNA* ; Enterotoxins ; Limit of Detection ; Molecular Biology ; Operon* ; Polymerase Chain Reaction* ; Seawater ; Vibrio cholerae* ; Vibrio* ; Virulence

No abstract available.
Animals ; Interleukin-2 ; Mice*

No abstract available.
Transposases*

No Abstract Available.
Methicillin Resistance* ; Methicillin* ; Methicillin-Resistant Staphylococcus aureus* ; Staphylococcus aureus* ; Staphylococcus*

No Abstract Available.
Vibrio parahaemolyticus* ; Vibrio* ; Virulence Factors* ; Virulence*

No Abstract Available.
Methicillin Resistance* ; Methicillin* ; Methicillin-Resistant Staphylococcus aureus* ; Staphylococcus aureus* ; Staphylococcus*

No Abstract Available.
Vibrio parahaemolyticus* ; Vibrio* ; Virulence Factors* ; Virulence*

No abstract available.
Escherichia coli* ; Escherichia*

A total of 100 Vibrio spp. strains were examined for production of various extracellular enzyme and for plasmid content plasmid were subjected to digestion with restriction enzymes. Most of them produced extracellular enzyme more than one, especially V. parahaemolyticus and V. cholerae non-01 strains were showed production of various extracellular enzymes. About the 55% Vibrio spp. have the plasmid more than one, but a lot of Vibrio spp. (about 45%) did not possess any plasmid. Most of these plasmid various derivatives ranged from 2.4 kb-23 kb, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between 70 kb-100 kb). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH, CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 6 strains of environmental isolates. Nine strains of 11 strains, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 4 strains of V. parahaemolyticus from environmental isolates, could be successfully amplified in 400 bp by PCR, no amplification products were obtained from TDH-negative strains. The PCR results were consistent with DNA hybridization. In the experiments of ctx gene detection, in all, 3 strains of V. cholerae non-01 from clinical isolate and 1 strains of V. cholerae non-01 from environmental isolate were observed CT- positive. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.
Cholera ; Digestion ; DNA ; Plasmids ; Polymerase Chain Reaction ; Vibrio* ; Virulence Factors* ; Virulence*