Isolation and identification of Vibrio vulnificus and Vibrio parahaemolyticus from coast of Pusan and Daechon

This study was focused on the isolation of pathogenic Vibrio species, V. vulnificus and V. parahaemolyticus from marine environment from May to July of 1999. Isolation sites were coast near by Pusan and Daechon. The results obtained were as follows: 1. Seventy strains of V. parahaemolyticus and 19 strains of V. vulnificus were isolated from a total of 120 specimens. 2. Nineteen strains of V. vulnificus did not fermented arabinose and salicin but fermented lactose and cellobiose. All of V. parahaemolyticus isolates did not fermented lactose and cellobiose. 47 strains of V. parahaemolyticus fermented arabinose but 53 strains did not fermented salicin. 3. V. vulnificus and V. parahaemolyticus isolates showed three different API index numbers with 5046105 and 4346107 dominant. 4. V. vulnificus did not grow on 0% and 8% NaCl containing medium. V. parahaemolyticus grew on 8% NaCl containing medium. 5. V. vulnificus isolates and V. parahaemolyticus revealed different outer membrane protein p rofiles on SDS-PAGE.

The Effect of Iron Limmted Condition on Outer Membrane of Vibrio mimicus

Vibrio mimicus, marine bacteria pathogenic for fish, can causes acute gastroenteritis in human. Iron limmited condition like in human body, may change the surface structure of V. mimicus. In this study we obse'rved the effect of iron limmited condition on outer membrane protein of V. mimicus. Ethylenediamine-di (O-hydroxy-phenylacetic) acid (EDDA), an iron chelator, delayed the time to reach expotential growth of V. mimicus in brain heart infusion medium from 3 hours to 20 hours. Outer membrane protein of V. mimicus-CON (cultured in BHI) and V. mimicus-EDDA (cultured in BHI contain EDDA) were seperated by 1% sarcosine from total cell envelop. SDS-PAGE of V. mimicus-EDDA and V. mimicus-CON showed similar protein profiles contain 37 kDa major protein but 86 and 90 kDa protein were induced differently. Immunological properties of above protein were determined by ELISA and western blotting. 86 kDa EDDA- specific OMP was induced in V. mimicus (isolate 96-1), V. parahaemolyticus (serotype 09), V. alginolyticus (isolate 95-1), E. coli (human isolate) and V. vulnificus ATCC 27562 in iron limmited condition.

Characterization of a Vibrio parahaemolyticus Phage Isolated from Marine

A novel bacteriophage, designated as VPP97, that infects the strains of Vibiro parahaemolyticus (hallophilic, Gram-negative bacterium) isolated most commonly from marine environments, has been discovered, and several of its properties have been determined. The plaques were clear and sized 0.6-1.0 mm in diameter. The virion forms a single band on 70% sucrose gradient and p1.50 CsC1 gradient by sucrose gradient centrifugation and CsCI gradient centrifugation respectively. It has a hexagonal head and a relatively long tail, as shown by electron microscopy. Vibrio alginolyticus, Vibrio fluvialis and Vibrio furnissii were also sensitive to this phage It was almost totally inactivated at 70 degree C and at pH below 5 or over 10. The nucleic acid of VPP97 is composed of DNA. The VPP97 had 9 specific structural proteins sized between 21.5 kDa and 97.4 kDa on SDS-PAGE. When V. parahaemolyticus cultures were treated with either phage VPP97 or one of the several antibiotics for 2 hours, the viable number of V. parahaemolyticus treated with the phage VPP97 is lower than that treated with chloramphenicol, erythromycin or penicillin, but not lower than that treated with tetracycline. Mice that have responded to the phage treatment revealed the lower numbers of V. parahaemolyticus in small intestine and less damage on small intestine compared to the untreated mice. Therefore, we suggest that the phage treatment appears effective to the infection by V. parahaemolyticus.

Purification Siderophore from Vibrio mimicus ATCC 33653 and its Effect to Bacterial Pathogenecity

Growth under conditions of iron-restriction and the production of siderophore was examined in Vibrio mimicus ATCC 33653. This strain grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine-di (o-hydroxyphenylacetic acid). Chrorne azurol S (CAS) agar and solution were used to detect the production of siderophore under these condition. Siderophore could be detected in the iron-rcstricted culture supernatants. The siderophore was extracted from iron-restricted culture supernatants by phenol-chloroform-ether method and purified by Dowex ion-exchange and Sephadex G-25 gel filtracton chromatography. The purified siderophore was confirmed by paper chromatography and HPLC. The Purified siderophore enhanced the growth of V. mimicus when the bacterium was grown in iron limited medium. Injection of both the siderohore and the bacteria to mice resulted in more rapid death than that of the only bacteria. However, the siderophore did not show lethality to mice and any toxicity to cell line like HeLa and U937.

Isolation and Detection of Vibrio vulnificus from the Southern Sea of Korea for the Prevention of V. vulnificus Infection (The Characteristics of Vibrio vulnificus Isolates from the Southern Sea of Korea)

Vibrio vulnificus, which causes serious septicemia, has been isolated from the Southern Sea of Korea. Five strains were identified by Farmer's biochemical test and API 20E kit. V. vulnificus ATCC 27562 was used as the reference strain and V. parahaemolyticus was used as the comparative strain. Three of the five strains could grow at 37% and the others only at 30 degrees C. The proteins pattern of cell lysates from the isolates were analyzed by SDS-PAGE and densitometery. Distinct protein band pattern was observed with the reference strain and the isolates in comparison with V. parahaemolyticus. Antiserum made against V. vulnificus ATCC 27562, was used for ELISA and Western blotting analysis to test the isolates. In ELISA analysis, the three strains being able to grow at 37 degrees C showed significantly higher reactivity to the antiserum than that of V. parahaemolyticus, while the other two grown only at 30 degrees C showed no significant difference. By Western blotting analysis, distinctive 30 and 36 kDa bands were observed only in the reference strain and the isolates. Twenty six and 54 kDa bands were observed with only three of the five strains being able to grow at 37 degrees C. The SDS-PAGE profiles of the outer membrane proteins of the isolates shared common features with the reference strain but distinctive from V. parahaemolyticus.

Viral Disease of the Cultured Penaeus chinensis and Penaeus japonicus

From June to October in 1993~1995, cultured Penaeus chinesis and Penaeus japonicus occurred mass mortality at the farm in Western Sea of Korea. The disease was reproduced in healthy shrimp by injection of filtered (at 0.45 micromeger) homogenate of infected shrimp. So we concluded hat it was filterable agents like virus. Clinical symptoms were white spots on the carapace and reddish tail. Histopathological changes were characterized by hypertrophied nuclei at cuticular epidermis lymphoid organ, hematopoietic tissue. In negatively stained preparation, the virion revealed rod-shaped, enveloped, nonoccluded. Cytopathic effect (CPE) were not observed by virus in CHSE-214, RTG-2, EPC, FHM cell lines. Base on the above facts, the reason of mass mortality of penaied shrimp was baculovirus.

Detection of Rifampin-resistance in Mycobacterium tuberculosis

Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. Rifampin is a key component among therapeutic regimens for the tuberculosis; therefore, patients who have drug resistance do not convalesce satisfactorily. The molecular mechanism of resistance to rifampin in M. tuberulosis has been elucidated. Substitutions of a limited number of highly conserved amino acids encoded by the rpoB gene are responsible for the ""single-step"" high-level resistance of M. tuberculosis to rifampin. Currently, two genotype-based protocols allow drug test from minimally grown cultured materials: (i)mutation identification by direct sequencing of PCR-amplified material. and (ii)mutation screening by PCR-SSCP. The purpose of this study is to evaluate the usefulness of the both methods. A sample of 75 isolates of M. tuberculosis was studied, and it inculded 36 rifampin-resistant strains and 39 rifampin-sensitive strains by conventional methods. Mutaions were identified in 36 rifampin-resistant isolates but in none of 39 sensitive isolates. All mutations were clustered within a region of 23 amino acids. Both methods allow detection of rifampin resistance in 2 to 3 days and will thus help in the early management of infection by M. tuberculosis.

Identification of Vibrio vulnificus in Pusan and Southern Sea of Korea in 1996 using API 20E Kit

The halophilic bacterium, Vibrio vulnificus, causes acute fulminating wound infections and septicemia in human. Especially the septicemia shows high mortality above 50%. In Korea, septicemia by V. vulnificus was reported at westem and southern coast in every year. Here, we try to isolate this V. vulnipcus at Kyoung-nam area and coast of Pusan during 1996. Purposed sites were Dadaepo, Songjung, Chungsapo and Mipo of Pusan and Kijang, Ilkuang, Juksoung, Dongam, Waljun and Chilam of southern sea. Total 40 strains of V. vulnipcus were isolated from sea samples. Biochemical characteristics of isolated V. vulnificus were almost same with reference strain V. vulnificus ATCC 27562 on Farmer's tests and on API 20E kit test. V. vulnificus isolates in 1996, fermented cellobiose and salicin but arabinose. and had resistance to 7% sodium chloride.

Pathogenicitic Characterization of Purified beta-Hemolysin - Produced by Vibrio mimicus

In order to investigate the main factor of pathogenicity in V. mimicus, we have studied the toxic effects of j3-hemolysin produced by V. mimicus. The purified hemolysin of V. mimicus was active erythrocytes from three animal species including mouse, rabbit and rat, but the hemolysin was most active against rabbit erythrocyte. The hemolysin lysed cultured cell and killed mouse. Rapid death of mouse was observed with rather small doses of the toxin. Intravenous injection of 20 mg of the purified toxin killed mice within 25 sec. The hemolysin also had a lethal effect on intraperitoneal injection into mice although less than on intravenous injection. Purified hemolysin injected rabbits had large morphological change in jejunum. In electron micrograph of thin sections of the human erythrocytes, cells were threated with the hemolysin at 37 C for 5 min., significant changes were not observed. But after 10 min., hemolysis was observed and after 60 min., complete degradation of human erythrocyte was observed.

The Detection of Rifampin-Resistant Mycobacterium tuberculosis by Polymerase Chain Reaction and Single - Strand Conformation Polymorphism Analysis

Control of tuberculosis is threatened by widesread emergence of drug resistant Mycobacterium tuberculosis. Rifampin is a key component among therapeutic regimens for the tuberculosis; therefore patients in whom resistance to this drug develop have a poor outlook, particularly if rifampin resistance is associated with resistance to other tuberculosis drugs. The purpose of this study was to detect the mutation in rpoB gene of rifampin resistant M. tuberculosis in Korea and to evaluate the usefulness of the method in clinical aspects. A sample of 80 M. tuberculosis was studied, and it included 40 rifampin resistance isolates and 40 rifampin sensitive isolates by conventional methods. The detection method involved the amplification by polymerase chain reaction (PCR) of the Rif' region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products (157 bp). Mutation were identified in 39 of 40 rifampin resistant isolates, and in 1 of 40 rifampin sensitive isolates.

Characterization About Vibrio alginolyticus Phage Isolated from Marine Products

Two phages for the pathogenic V. alginolyticus were isolated from marine products. These 2 phages were examined temperature stability, pH stability, inactivation by UV irradiation, damage on restriction system of host cell, antibody production, structure protein analysis and western blotting assay. V. alginolyticus phages(VAPs) fomed plaques about 0.5 - 0.9mm in diameter and bands 50 - 60% in sucrose density gradient, VAPs were stable below 65'C, pH 5 - 10 and mostly inactivation by UV irradiation for 120sec. Latend period was 15 - 20 min. and burst size was 1.3 - 1.4 * 10 PFU/cell. Restriction system of V. alginolyticus isolated was mostly inactivated by 45C, 20min. heating. VAPs had 14 specific structural proteins and 5 proteins related to antibody production.

Electromicroscopic Characterization about Vibrio alginolyticus Phage Isolated from Marine Products

The study of bacteriophage began by F.W. Twort in 1915 and the lytic cycle recognized by d'Herellel in 1917. It repeated about the marine bacteriophage containing Vibrio phage by Smith, Spencer and Ju. Authors isolated 2 virulent phages for the pathogenic V. alginolyticus from marine products. These 2 phages were examined their ultrastructure & host-infection by elecron microscopy and in vivo test using skin of rats. V. alginolyticus phages(VAPs) fomed plaques about 0.5 - 0.9mm in diameter and bands 50 - 60% in sucrose density gradient. VAP had 50 - 120nm tail and 40 - 90nm head in diameter. In vivo test, using rat skin, as well as in vitro test VAP had the activity to V. alginolyticus isolated.