Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

A Molecular Epidemiologic Study by Plasmid DNA and Random Amplified Polymorphic DNA Analyses of Salmonella enterica serotype Enteritidis Isolated in Food Borne Outbreaks in Gwangju, 1997 / 감염과화학요법

BACKGROUND: During the past 10 years, there has been an increased incidence of gastrointestinal infections caused by salmonellae in Korea. In 1999, there were several outbreaks and sporadic occurrences of food borne infections due to Salmonella enterica serotype Enteritidis in Gwangju. Thus, there is a need for careful monitoring of its occurrence. METHODS: Salmonella Enteritidis were isolated from feces samples of patients with foodborne diarrhea in Gwangju, 1999. We performed antigen typing, examination of biochemical properties, anibiotic susceptibility test, plasmid typing and RAPD analysis to characterize of S. Enteritidis isolates. RESULTS: There were three Salmonella outbreaks (April, July, October), and 203 isolates of S. Enteritidis were isolated from the 286 patients. Eighteen isolates were obtained from the patients of sporadic occurrences. Antigenic types of the isolates were O antigen; D1 (1, 9, 12), H antigen phase 1; (g, m), serotype; Enteritidis. The isolates were susceptible to most of the antibiotics. We performed plasmid DNA analysis of the isolates, and the results showed 3 plasmids (8, 6, 3.8 kb) in 14 of 14 strains from outbreaks; 3 plasmids (8, 6, 3.8 kb) in 2 isolates from sporadic cases; 4 plasmids (8, 6, 3.8, 2 kb) in 10 isolates from sporadic occurrences, and 2 isolates from food specimens. However, 1 isolate from patients and 2 isolates from Ham-Yang, Kyung Nam, did not contain plasmids. RAPD analysis showed that all isolates from Gwangju in 1999 showed relatively uniform characteristics which were different from those derived from Ham-Yang, Kyung-Nam. CONCLUSION: The present data demonstrated that most food poisoning cases by S. Enteritidis in Gwangju, 1999, were originated from the same Salmonella Enteritidis strains.

A Molecular Epidemiologic Study by Plasmid DNA and Random Amplified Polymorphic DNA Analyses of Salmonella enterica serotype Enteritidis Isolated in Food Borne Outbreaks in Gwangju, 1997 / 감염과화학요법

BACKGROUND: During the past 10 years, there has been an increased incidence of gastrointestinal infections caused by salmonellae in Korea. In 1999, there were several outbreaks and sporadic occurrences of food borne infections due to Salmonella enterica serotype Enteritidis in Gwangju. Thus, there is a need for careful monitoring of its occurrence. METHODS: Salmonella Enteritidis were isolated from feces samples of patients with foodborne diarrhea in Gwangju, 1999. We performed antigen typing, examination of biochemical properties, anibiotic susceptibility test, plasmid typing and RAPD analysis to characterize of S. Enteritidis isolates. RESULTS: There were three Salmonella outbreaks (April, July, October), and 203 isolates of S. Enteritidis were isolated from the 286 patients. Eighteen isolates were obtained from the patients of sporadic occurrences. Antigenic types of the isolates were O antigen; D1 (1, 9, 12), H antigen phase 1; (g, m), serotype; Enteritidis. The isolates were susceptible to most of the antibiotics. We performed plasmid DNA analysis of the isolates, and the results showed 3 plasmids (8, 6, 3.8 kb) in 14 of 14 strains from outbreaks; 3 plasmids (8, 6, 3.8 kb) in 2 isolates from sporadic cases; 4 plasmids (8, 6, 3.8, 2 kb) in 10 isolates from sporadic occurrences, and 2 isolates from food specimens. However, 1 isolate from patients and 2 isolates from Ham-Yang, Kyung Nam, did not contain plasmids. RAPD analysis showed that all isolates from Gwangju in 1999 showed relatively uniform characteristics which were different from those derived from Ham-Yang, Kyung-Nam. CONCLUSION: The present data demonstrated that most food poisoning cases by S. Enteritidis in Gwangju, 1999, were originated from the same Salmonella Enteritidis strains.

Antisense Deoxyoligonucleotides Inhibit Activities of Matrix Metalloproteinase-2 in Human Fibrosarcoma HT1080 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.

Effect of Salinity, Temperature, and Glucose on the Production of Vibrio vulnificus Hemolysin

Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and 37 degrees C than in 2.5% and 25 degrees C. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and 25degrees C) was changed into that reflecting human body (0.9% salinity and 37 degrees C), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at 37 degrees C. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose- dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.

Phenolate Siderophore Stimulates Growth of Vibrio vulnificus: Application of CAS Agar Diffusion Assay - Comparison of Siderophore Production Among Strains

No abstract available.

The Role of Vibrio vulnificus Capsular Polysaccharide as a Virulence Factor

To evaluate the role of capsular polysaccharide (CPS) as a virulence factor, the interaction of V. vulnificus with mouse peritoneal macrophages and serum, which are involved in the clearance of bacteria from blood and other tissues, were examined. In this study, MO6-24/0 (wild strain; hemolysin- and capsule-positive), MO6-24/I' (acapsular spontaneous mutant), CVD 752 (acapsular transposon mutant), and CVD 707 (hemolysin-negative and capsule-positive mutant) were used. The strain with CPS (MO6-24/0 and CVD 707) were more resistant to phagocytosis by mouse peritoneal macrophages compared with acapsular strains (MO6-24/T and CVD 752), and the resistance to phagocytosis was not changed by serum opsonin in the capsular strains. Acapsular strains were more susceptible to serum bactericidal activity than the capsular strains through the classical complement pathway. MO6-24/0 strain were detected in blood, spleen, liver and lung at 4 hours after intraperitoneally infection, whereas CVD 752 were not detected. All tested strains could induced the transcription of inflammatory cytokine gene such as IL-1, IL-6, IL-10 and TNF-u, and their inductions were not decreased by cytochalasin B treatment. This results demonstrate that CPS of V. vulnificus plays an important role in V. vulnificus infection through interfering nonspecific host defense system such as blood clearance and phagocytosis.

A Basic Study for the Development of Effective Preventive Measure against Vibrio vulnificus Septicemia: Bactericidal Mechanism of Osmotic Shock

The establishment of effective preventive measure against V. vulnificus septicemia is urgently required. It was reported that V. vulnificus osmotically shocked by distilled water lost viability rapidly but regain viability after appropriate resuscitation (RS) procedure. But V. vulnificus was reported to be completely killed when osmotically shocked in the presence of ethylenediaminetetraacetic acid (EDTA). This study was carried out to uncover the bactericidal mechanism of osmotic shock and the mechanism of potentiation of osmotic shock by EDTA. When about 2.0 x 10(7) CFU/ml of V. vulnificus were inoculated in distilled water, the number of viable cells abruptly decreased to 2.5 x 10(3) CFU/ml in 1 min. and slowly thereafter to 1.0 x 10(1) CFU/ml in 5 min. After RS, there was a increase in the number of surviving bacteria by 10(3) to 10(4) fold. When the bacteria were inoculated in 1 mM EDTA solution, osmotic concentration of which is about 30 mEq./1, no colony could be observed even in 1 minute. The turbidity decreased abruptly as soon as the bacteria were inoculated in distilled water or in the 1 mM EDTA solution, but rather slowly thereafter. When V. vulnificus whose cellular constituents were labeled with 3H-L-amino acid mixture was inoculated in distilled water or in the 1 mM EDTA solution, about 35% of the whole cell radioactivity was released in the 1 mM EDTA solution in 30 sec while about 6% of the whole cell radioactivity was released to the supernatant in distilled water in 5 minutes. The cell surface hydrophilicity decreased significantly by osmotic shock. The decrease was more significant when the bacteria were inoculated in 1 mM EDTA solution than in distilled water. Bacterial cell volume analysis with a flow cytometer revealed that the osmotic shock balloons V. vulnificus. The increase in the cell volume was more prominent in 1 mM EDTA solution. When the cytoplasmic RNA content in the osmotically shocked bacteria was measured by a flow cytometer, the frequency of the cells with decreased RNA content increased after osmotic shock, and the degree of increase was more prominent in 1 mM EDTA solution. Number of non-staining cells also increased after osmotic shock, and the degree of increase was more prominent in the 1 mM EDTA solution. To see whether the susceptibility to osmotic shock is unique to V. vulnificus, bactericidal kinetic curves of other Vibrio species were observed after inoculating in distilled water. V. cholerae and V. mimicus were more resistant to the osmotic shock than V. vulnificus. V. parahaemolyticus, V. furnissii, V. fluvialis, V. damsela, and V. harveyi showed similar susceptibility to osmotic shock as V. vulnificus. V. alginolyticus and V. hollisae were more susceptible than V. vulnificus. The concentration of NaCl in culture media influenced the susceptibility of V. vulnificus to osmotic shock. V. vulnificus grown in 0.5% NaCl was more resistant to the osmotic shock than that grown in 2.5% NaCl. Taken together, it was concluded that osmotic shock causes leakage of the cytoplasmic contents(ribosomes etc.). And EDTA was supposed to quantitatively potentiate the bactericidal effect of the osmotic shock. Susceptibility to osmotic shock was influenced by the osmolarity of culture media and appeared to be a phenotypic property of V. vulnificus.

In vitro effect of ciprofloxacin on vibrio vulnificus

No abstract available.

Expression of cell surface antigens and oncogene of leukemic U-937 cells by differentiation inducers / 대한암학회지

No abstract available.

Effect of retinoic acid, beta-carotene, and ascorbic acid on the mutagenicity of some anticancer antibiotics / 대한암학회지

No abstract available.

In vitro ractericidal activity of quinolones against vibrio vulnificus

No abstract available.

Enhanced resistance of mice to listeria infection with nativesteroid dehydroepiandrosterone(DHEA) / 대한면역학회지

No abstract available.

Effect of Iron and Its Chelators on Vibrio vulnificus Septicemia Experimentally Induced in Mice / 대한피부과학회지

Infection with V. vulnificus resulting in septicemia accompanied with skin gangrene and high mortality of 50% or more freqently occurs in people with liver disenses. And it has also been demonstrated that serum iron, essential to the growth of microorganisms, has been elevated in liver damaged animals. In spite of many efforts to reveal the pathogenesis of this fatal disease, there is no clear conclusion so far. Significant increase or decrease in LD of V. vulnificus (CDC C7184) was observed when mice were treated with ferric arnmonium citrate (FAC) and a specific iron chelator, desferal(Df), originated from Streptomyces pilosus and a broad spectrurn cation chelator, calciurn disodium ethylenediaminetetraacetate (CaEDTA) widly used in heavy metal poisoning treated alone or in combination. The results were obtained as follows. FAC and Df lowered LD to approximately 1.96x 10(3) colony forming unit (CFU) and 9.77x10(2) CFU respectively from 4.46 x 10(5) CFU, LDso of the control group. However, CaEDTA elevated the I D to 4.97 X 10(7) CFU. The LD of the group administered FAC and Df simultaneously was about 9.28x10(1) CFU. Whereas, the LD of the group administered FAC and CaEDTA simultaneously was approximately 7.88 x 10(5), similar to that of the control group. This study demonstrates that there is a close association of the iron with V. vulnificus septicemia and Df lowers LD of the rnice. CaED7A, however, elevated the LD. The author hereby proposes carefully iron chelators such as CaEDTA as an agent for a new adjuvant therapy of the V. vulnificus septicernia.

In vitro activities of eight antibiotics against methicillin-resistant S. aureus and S. epidermidis strains isolated in Korea

Staphylococcus aureus and Staphylococcus epidermidis strains isolated at eight large medical centers in Korea were examined for methicillin resistance and resistance to eight other antibiotics; cefazolin, cefamandole, cefuroxime, cefoxitin, cefotaxime, moxalactam, penicillin G and vancomycin. Methicillin resistance was found in 296 of 1225 strains (24.2%) of S. aureus and 126 of 348 strains (36.2%) of S. epidermidis. Methicillinresistant strains were isolated from all sources with the frequency of isolation ranging from 11% to 60%. From pleural effusion, throat swab and blood, methicillin-resistant strains of S. aureus were more frequently isolated with statistical significance (Chi-squared test, 95% confidence). Almost all of Methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE) strains were multiply resistant to one or more tested eight antibiotics. However only 7(2.4%) of 296 MRSA strains and 2(1.6%) of 126 MRSE strains were resistant to vancomycin. Vancomycin was the most effective antibiotic against staphylococcal isolates as well as MRSA and MRSE.