천연항균성 물질을 이용한 Vibrio vulnificus의 살균 및 독소생성 억제효과

조성환,서일원,최종덕,전상수,라택균,정수근,강동훈 國立統營水産專門大學 附設 水産科學硏究所 1993 수산과학연구소보고 Vol.4 No.-

생선회용 해산물에 오염되어, 생식한 경우 발생되는 패혈병의 원인균주인 Vibrio vulnificus의 생육 및 독성생성을 억제할 목적으로 천연 항균제인 Grapefruit종자추출물(GPSE)을 이용하여 그 항균효과를 검토하였다. In vitro 시험결과, Vibrio vulnificus에 대한 GFSE의 생육최소저해농도는 50~100 ppm 정도이었으며, Vibrio vulnificus 균체세포틀 100 ppm 농도의 GFSE 용액으로 처리하고 전자현미경 촬영시료로 조제하여 촬영한 후, 미생물형태을 무처리대조구와 비교, 검토한 결과, GFSE 처리로 세포막 기능이 파괴되어 세포내용물이 균체외부로 유출되어 균체의 생육이 억제되고 균체내부가 빈 ghost 형태의 균체수가 증대되어 GFSE의 항균효과를 확인할 수 있었다. 한편, 생선회용 해산물에 GFSE를 근육주사하고 Vibrio vulnificus를 접종한 수조내에서 사육하여 채취한 어육부를 마쇄하여 일정량씩을 표준사료에 첨가해서 통닭용 병아리를 실험동물로 하여 사양실험을 실시한 결과. 체중증가율, 단백질이용효율, 혈청내 효소활성, 혈청의 주요성분함량 등에 있어서, GFSE 처리수조내에서 사육한 해산물을 첨가한 시료시험구의 경우, 무첨가사료처리구와 비교할 때 뚜렷한 차이를 목격할 수 없었으며, GFSE 첨가에 따른 실험동물의 성장저해 또는 독성현상을 유발하지 않음을 알 수 있었다. 아울러, 각 처리구에서 사양한 해산물의 어육부에 오염된 Vibrio vulnificus의 균수를 측정한 결과, 250ppm 이상의 GFSE 처리구에서는 Vibrio vulnificus를 전혀 검출할 수 없었다. To prevent food-poisoning outbreaks by Vibrio vulnificus the antimicrobial efficacy of grapefruit seed extract (GFSE) was examined. Minimal inhibitory concentration (in vitro) for the microorganism was found to be 50~100 ppm. Transmission electron micrographs of Vibrio vulnificus showed the biocidal action of this natural antimicrobial agent would be related to specific respiratory effect coupled with the destruction of permeable function of microbial cell membrane. After Anguilla japonia GFSE-injected to the body was incubated in the seawater contaminated by Vibrio vulnficus the fish meats were taken up, mixed with control diet and used as a diet in the feeding experiment. In this experiment the effect of GFSE treated with fish muscle on body weight protein efficiency ratio, serum enzymes and serum blood component of broiler chicks was investigated. It is proved from this study that there is neither Vibriosis nor toxicity associated with GFSE itself and fish meats treated with it when it is injected to the fish body at a level of 250 ppm or less.

비브리오 패혈증균의 균체내독소 정제 및 특성에 관하여

신일식,정현정,김영만 동의대학교 생활과학연구소 1998 生活科學論集 Vol.2 No.-

Vibrio vulnificus의 균체내독소의 특성을 파악하여 비브리오 패혈증의 발병원인 구명을 위한 자료를 제공하고자 생균과 균체파쇄액의 치사독성과 내열성 및 혈관투과성항진작용을 실험한 결과는 다음과 같다. 1. 환자분리균(V. vulnificus CDC B3547)과 환경분리균(V. vulnificus B57)의 독력은 차이가 없었다. 2. V. vulnificus의 균수가 10 7/ml이상일 때 강한 치사독력이 나타났다. 3. 균체파쇄액의 독성은 80℃ 20분에 완전히 불활성화 되었다. 4. 균체파쇄액은 용혈성은 없었으나 세포독성은 인정되었다. 5. V. vulnificus의 새앙쥐에 대한 주 치사독소는 균체 내에 존재했으나 LPS와 LPS-protein complex는 기존의 방법으로 분리할 수 없었다. To determine the cause of Vibrio septicemia by understanding the characteristics of endotoxin from Vibrio vulnificus, lethal dose, heat resistance and vascular permeability enhancing activity were evaluated using vegetative cell and cell homogenate and the result is as follows: 1. Vibrio vulnificus CDC B3547 of patient origin did not exihibit any significant difference in toxicity compared to Vibrio vulnificus B57 of environment origin. 2. Strong toxicity was observed when viable cell count of Vibrio vulnificus CDC B3547 was more than 10 7/ml. 3. Toxicity of cell homogenate was completely inactivited upon heating at 80℃ for 20min. 4. Cell homogenate did not show hemolytic activity but was acknowledge to have cytotoxicity. 5. Major lethal toxin against mouse was existed in Vibrio vulnificus CDC B3547; however, separation of LPS and LPS-protein complex was not successful using the current technique.

Vibrio vulnificus의 인혈청살균력에 대한 감수성과 Vibrio 감염이 마우스의 Hematocrit치에 미치는 영향

하대유,임선영,전상남,김철기,Ha, Tai-You,Im, Suhn-Young,Chun, Sang-Nam,Kim, Chul-Kee 대한미생물학회 1986 大韓微生物學會誌 Vol.21 No.3

Vibrio vulnificus, a halophilic Vibrio has gained worldwide attention as a cause of severe and frequently fatal wound infections and life-threatening septicemia. For this reason V. vulnificus is thought to produce extreme hemoconcentration and rapid death after infection, and because V. vulnificus is thought to be less susceptible to bactericidal activity of normal human serum, the present study was undertaken to assess the susceptibility of V. vulnificus to human serum bactericidal activity with that of V. parahemolyticus and V. cholerae and to assess the effect of Vibrio species, Salmonella typhimurium and E. coli on hematocrit values in experimentally infected mice. Serum bactericidal activity against both V. vulnificus and V. cholerae was higher than against V. parahemoltyicus. Survival of the test strains in heat-inactivated human serum was greater than that in heat-uninactiveted serum. Both V. parahemolyticus and V. cholerae produced slight hemoconcentration within 2 to 6 hr after intraperitoneal injection of $10^7$ viable bacteria into mice. In contrast, V. vulnificus, S. typhimurium and E. coli produced hemodilution rather than hemoconcentration after 4 or 6 hr after infection. With these results the author can conclude that V. vulnificus is more susceptible to serum bactericidal activity than other Vibrio species, and V. vulnificus did not produce hemoconcentration.

Vibrio 패혈증 환자에서 V. vulnificus 균의 신속한 동정을 위한 새로운 선택 배지

유동오 ( Dong O You ),정상원 ( Sang Won Jeong ),박석돈 ( Seok Don Park ) 대한피부과학회 2003 대한피부과학회지 Vol.41 No.6

N/A Background : Vibrio(Ⅴ.) vulnificus is a halophilic, gram-negative bacillus that causes a fatal sepsis in patients with underlying chronic disease such as liver cirrhosis and alcoholic abuse. Because Ⅴ. vulnificus infection has a fulminant course and high mortality rate, early recognition and rapid diagnosis with prompt therapy are necessary to improve survival rate. Objective : The purpose of this study was to develop a new selective medium for rapid identification of Ⅴ. vulnificus through color change of medium according to pH from patients suspected of having Ⅴ. vulnificus sepsis. Methods : Rapid isolation and identification of Ⅴ. vulnificus can be possible by modifying the component of PNC(5% peptone, 1% NaCl, and 0.08% cellobiose [pH 8.0]) broth medium. From this PNC broth, a basal broth(5% peptone + 1% NaCl + cellobiose) was prepared and used to evaluate additional medium ,supplements(cellobiose concentration [0.08, 0.2, 0.1%], pH [6.8, 7.5, 8.0] and pH indicator dye [bromthymo] blue, thymol blue, phenol red, bromcresol purple, crystal violet, cresol red, and neutral red]). To examine the rapid identification and selectivity of this basal medium according to various conditions, Ⅴ. vulnificus was tested by using saline and normal human blood containing these bacteria(1,000 bacteria/㎖), respectively at 37℃. A positive reaction(Ⅴ. vulnificus growth) appeared as color change. The selectivity and identification capacity of this new broth was tested by using other 6 Vibrio species and 14 strains of other bacteria Results : Color change appeared only in the medium including bromthymol blue and thymol blue as a pH indicator dye. It was called the basal medium containing blue dyes as PNCB(peptone, NaCl, cellobiose and blue dye) medium. It took an average time of 4.Bhr for becoming aware of yellow color change in PNCB broth after cultivating with saline mixed with Ⅴ. vulnificus and 6hr in PNCB broth after cultivating with blood mixed with Ⅴ. vulnificus. One Vibrio and another 3 bacteria produced color change. So we confirmed that the final composition and pH of PNCB broth medium was 5% peptone, 1% NaCl, 0.2% cellebiose, 0.0004% bromthymol blue and 0.0004% thymol blue [pH 7.5] Conclusions : PNCB broth could be used as a selective and differential medium for rapid isolation and identification of Ⅴ. vulnificus in patients with Ⅴ. vulnificus sepsis. (Korean J Dermatol 2003;41(6) : 696-701)

Repetitive Sequence-based Genomic Fingerprinting을 이용한 Vibrio 속 균의 분자분류 및 동정

김규원 ( Gyu Won Kim ),정헌종 ( Hun Jong Chung ),박철민 ( Chul Min Park ),김기정 ( Kijeong Kim ),김원용 ( Won Yong Kim ),정상인 ( Sang In Chung ) 대한내과학회 2006 대한내과학회지 Vol.71 No.2

목적: 여러 rep-PCR genomic fingerprinting 방법들을 실시하여 비브리오 균종간의 유전적 상관성을 조사하고 이를 이용한 V. vulnificus의 신속한 분자 동정법을 개발하고자 하였다. 방법: 이 연구에서 사용한 균주는 48주로서 REP-, ERIC-, BOX- 및 SERE-PCR을 13종의 비브리오를 대상으로 수행하였다. 결과: ERIC-PCR은 서로 구별이 가능하였으며 약 320bp 크기의 특이 절편이 V. vulnificus에서 발견되었다. 그러나 REP-, BOX- 및 SERE-PCR은 V. vulnificus 균주들을 다른 비브리오균종들로부터 분류하는데 효과적이지 못하였다. 결론: ERIC-PCR은 V. vulnificus를 다른 Vibrio 균종들로부터 신속하게 분류하고 동정하는데 유용하게 이용될 수 있을 것으로 생각된다. Background: The aims of this study were to compare the suitability of repetitive-PCR genomic fingerprinting procedures to investigate genetic relatedness of the genus Vibrio and its applicability for the molecular identification of Vibrio vulnificus. Methods: Forty-eight Vibrio strains were included for this study. REP-, ERIC-, BOX- and SERE-PCR were compared with 13 members of the genus Vibrio. Results: REP-, BOX- and SERE-PCR showed V. vulnificus strains could not be separated well from other Vibrio species. However, approximately 320 bp of highly discriminatory specific fragments was recovered from V. vulnificus strains by ERIC-PCR. Conclusions: ERIC-PCR could be used as rapid classification and identification methods of V. vulnificus from other members of the genus Vibrio.(Korean J Med 71:189-197, 2006)

Vitek GNI+ Card를 이용한 Vibrio vulnificus의 동정

윤명,김광진,신종희,서순팔,양동욱 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.3

Vibrio vulnificus 감염증으로 확진된 92예의 임상적 고찰

유종선,문범,지미선,최성규,백강우,김기태,박창환,윤종만 대한감염학회 1995 감염 Vol.27 No.4

목적: V. vulnificus 감염증은 전남지방에서 매년 발생하고 있고 지속적인 계몽과 집중적인 치료에도 불구하고 아직도 높은 치사율을 나타내고 있어 1983년부터 1994년까지의 전남지방의 V. vulnificus 감염증의 임상상과 역학적인 특성을 분석 검토해보았다. 방법:1983년부터 1994년 9월 까지 전남대학병원에 내원한 환자중 균 배양검사로 V. vulnificus 감염증으로 확진된 92예를 대상으로 세균학적 검사소견, 역학적 특성, 임상소견을 분석 검토하였다. 혈액배양은 BACTEC blood culture system(Bekton Dickinson)을 이용하였고, 피부병변 조직배양은 혈액한천배지, MacConkey agar, Thioglycolate broth에 접종 배양한 후 배양된 세균을 ATB 32GN(biome-rieux. France)과 API 20E(biomerieux. France)로 동정하였다. 항생제 감수성은 Kirby-Bauer 디스크(BBL Sensi disc)확산법으로 검사하였다. 결과: 1) V. vulnificus는 혈액배양에서 20예(22%), 조직배양에서 43예(47%), 혈액과 조직에서 27예(29%), 혈액과 복수에서 1예(1%), 혈액과 뇌척수액에서 1예(1%)가 배양되었다. 원판 확산법에 의한 균 감수성검사에서는 tetracycline, chloramphenicol, ampicillin, gentamicin, piperacillin, cefoperazone, cefotaxime, ciprofloxacin등은 100%의 감수성을 나타냈고 amikacin, kanamycin, carbenicillin등은 약 85%, cephalothin은 77%의 감수성을 나타냈으며 colistin은 전 균주에서 내성을 나타냈다. 2) 연도별 발생빈도는 85년에 21예로 가장 많았고, 94년 12예, 89년 10예, 87년 9예, 91년 8예, 93년 8예의 순이었다. 월별로는 5월에서 10월 사이에 발생하였으며 특히 7월에서 9월 사이에 78예(85%)로 집중적으로 발생하였다. 지역별로는 해안지방과 인접 내륙지방에 주로 발생하는 경향을 보였다. 3) 간질환이나 중등도 이상의 음주력이 있는 40-50대의 남자에서 어패류를 생식 또는 불충분하게 조리하여 섭취했을때 많이 발생하였다. 환자 발생시기의 남서해안 근해 평균수온은 16.3℃-28.7℃였으며 특히 22.0℃-23.9℃시기의 발생이 전체의 54%를 차지하였고 해수온도가 상승함에 따라 발생빈도가 증가하는 경향을 보였다(r=0.43 p=0.006). 4) 대부분이 원발성 패혈증 증세를 보였고 하지의 부종과 수포등 피부병변만 나타나고 패혈증 증세가 없었던 창상감염증 1예와 뇌막염 증세를 동반한 패혈증이 1예 관찰되었다. 임상증상으로는 주로 피부병변(96%), 사지통(56%), 저혈압(52%), 발열(47%), 오한(43%), 설사(33%), 복통(26%), 구토(25%)등이 나타났고, 심한 경우에는 핍뇨(9%), 호흡곤란(7%), 혈변(2%)도 나타났다. 피부병변으로는 수포(56%), 부종(52%), 홍반(51%), 괴사(18%), 자반(11%), 구진(7%), 농포(2%), 등이 관찰되었고 주로 하지(73%)에 많이 발생하였다. 5) 사망률은 전체적으로는 57.6%이였고, 점차 감소화 하는 추세를 보였다. 사망군과 생존군간에는 생명징후, 잠복기에 따른 차이는 없었으나 백혈구수와 혈소판수가 감소할수록, AST가 높을수록 사망률이 높았다 결론:V. vulnificus 감염증은 일단 패혈증이 발생하면 병의 진행이 빠르고 감수성 있는 항생제로 집중적인 치료를 해주어도 사망률이 높기때문에 발병후 치료보다 예방이 훨씬 중요하리라 사료된다. Background:V. vulnificus infection is a highly fatal disease prevailing in Chonnam area. To increase understanding of V. vulnificus infection, we investigated clinical and epidemiological characteristics of V. vulnificus infection in Chonnam area. Methods:We retrospectively reviewed the clinical recores of 92 cases with V. vulnificus infection which admitted to Chonnam University Hospital from 1983 to 1994 and bacteriologically were confirmed. Results: 1) V. vulnificus was isolated from blood in 22%, tissue in 47%, blood and tissue in 29%, blood and peritoneal fluid in 1%, and blood and cerebrospinal fluid in 1% of the cases. All the isolates were susceptible to tetracycline, chloramphenicol, ampicillin, gentamicin, piperacillin, cefoperazone and cefotaxime. About eighty-five pervent of the isolates were susceptible to amikacin, kanamycin and carbenicillin and seventy-seven percent to cephalothin. All the isolates were resistant to colistin. 2) The yearly distribution was 21 cases in 1985, followed by 12 in 1994, 10 in 1989, 9 in 1987 in decreasing. The monthly distribution was each 27 cases in July and August, followed by 24 in September, 8 in June, 5 in October, 1 in May. The majority of cases occurred in coastal areas and nearly inlands of Chonnam province. 3) All of V. vulnificus infections occurred in association with ingestion of raw seafoods. The age and sex distribution ranged from the thirties to the eighties and occurred mainly in adult males in the fifties(48%) and the forties(24%). Of ninety-two cases, 73 cases had underlying liver diseases and 67 cases were heavy alcohol consumers. 4) Almost all cases showed primary septicemia except one which had wound infection without septicemia. One case showed septicemia with meningeal symptoms. Clinical manifestations included skin lesion(96%), pain in the extremities or myalgia (56%), hypotension(52%), fever(47%), chills(43%), diarrhea(33%), abdominal pain(26%), oliguria(9%), dyspnea(7%) and melena(2%). Skin lesions included vesicles or bullae(56%), edema(52%), erythema(51%), necrosis(18%), purpura(11%), macules(7%) and pustules(2%). Most of the skin lesions developed in the lower extremities. 5) The average sea water temperature ranged from 16.3℃ to 28.7℃ which was provided by National Fisheries Research & Developement Agency from 1983 to 1994, when V. vulnificus infection occurred. As sea water temperature rose, the incidence of V. vulnificus infection tended to be increased(r=0.43 p=0.006) 6) The overall case fatality rate was 57.6%. There were no significant differences between survivors and non-survivors in vital signs, incubation period, arrival time, and liver function test except aspartate aminotransferase. However, leukocyte and platelet counts were significanty lower in non-survivors (p<0.05). Conclusion:In spite of the susceptible antibiotics therapy and intensive care, V. vulnificus infection was highly fatal. Therefore, we stress that prevention is more important rather than treatment of the disease.

Vibrio vulnificus의 혈청형 : Vibrio vulnificus균의 신속한 동정 방법으로서의 이용과 예후적 가치

고명옥(Myoung Ok Koh),박석돈(Seok Don Park) 대한피부과학회 1992 大韓皮膚科學會誌 Vol.30 No.6

Vibrio vulnificus is a halophilic gram-negative, marine bacterium which causes fulminating and potentially fatal human disease. A new sensitive and specific serogrouping procedure was developed for identifying V. vulnificus. Reference antis:ra(01-014) to V. vulnificus were prepared by vaccinated rabbits with heat-killed bacteria, and the antisera were examined whether erogrouping test will be available for rapid and sensitive identification method of V. vulnifiius or not. All of thirty-seven clinical strains tested were agglutinated with 5 kinds of aritisera such as 04A, 01, 013, 03, and 014. Of these serovar 04A was predominant(75.7%). The author investigated the correlation among certain serovars, consumed seafoods, and nortality rate to verify whether the serovar is of pognostic value. There was no significant orrelation among them. These results suggest theart serogrouping procedure could be of value in rapidly identifying V. vulnificus. (Kor J Derrnatol 1992;30(6):787-793)

Vibrio parahaemolyticus와 Vibrio vulnificus 중복 감염에 의한 급성 위장관염 1예

이남용,기창석,이 혁,송재훈 대한감염학회 1998 감염 Vol.30 No.5

비브리오속의 중복 감염은 대부분의 병원성 비브리오속이 해수에서 잘 증식하는 호염성 세균이며 30℃ 이하의 낮은 온도에서 잘 자랄 뿐만 아니라 생굴과 어패류에서 자주 분리되는 등 동일한 생태를 보이는데 반해 매우 드물다. 그러나, 비브리오속은 여러 가지 질병 양상을 보일 수 있으며 인체 침습성, 병원성 및 항균제 감수성이 다르기 때문에 중복 감염을 진단하는 것은 환자 치료에 매우 중요하다. 저자들은 밴댕이(Herklotsichthys zunasi)회를 먹은 후 점액성 설사와 복통 및 구토를 보인 환자에서 Vibiro parahaemolyticus와 Vibrio vulnificus를 동시에 분리하였기에 증례와 함께 보고한다. Acute gastroenteritis caused by more than one Vibrio species has been rarely reported although most pathogenic Vibrio species share the same natural habitats. Recognition of multiple Vibrio species in one or more infectious processes is crucial in the management of patients because invasiveness, virulence, and resistance to antimicrobials can vary from species to species. We report a case of acute gastroenteritis by Vibrio parahaemolyticus and Vibrio vulnificus. The patient was a previously healthy 47-year old man who presented with watery mucoid diarrhea, abdominal cramps, and vomiting. On the previous day, he had eaten raw fish and developed the above symptoms about 10 hours later. The patient received only supportive therapy and was discharged on the same day with improvement. V. parahaemolyticus and V. vulnificus were isolated from stool specimen. The patient was fully recovered and subsequent stool culture demonstrated no pathogenic microorganisms.

염도와 수온의 변화가 Vibrio vulnificus의 생존에 미치는 영향

권지영,김영만 동의대학교 생활과학연구소 1998 生活科學論集 Vol.2 No.-

Vibrio vulnificus is a gram-negative, halophilic, oxidase-positive, lactose-positive, motile, rod shaped bacterium that has been associated with primary septicemia and wound infection. Elucidating the growth and survival of V. vulnificus in ecological conditions is of great importance to develop sanitary measure against this microorganism. Thus we simulated the ecological conditions and evaluated the effect. About 10 5 CFU/ml of V. vulnificus was inoculated to fresh water, brackish water (1% NaCl), sea water(3% NaCl), and bottom deposit solution. The same concentration of V. vulnificus was also inoculated to distilled water, 1% NaCl solution and 3% NaCl solution as controls. These were stored at 4, 15 and 25℃, respectively and were used to assess the effects of temperature and salinity on the survival of V. vulnificus. In fresh water V. vulnificus could not survive regardless of storage temperature. In case of brackish water and sea water survival time of V. vulnificus was the longest at 25℃, and the number of V. vulnificus was decreased most rapidly at 4?. V. vulnificus survived longer in brackish water than in any other conditions. In bottom deposit solution containing brackish water, the survival time of V. both biological and physicochemical factors such as temperature and salinity could affect survival of V. vulnificus V. vulnificus, damaged in normal fresh water, did not grow on TCBS agar of selective plating medium but grew on BHI agar plate; however, V. vulnificus was recovered by addition of salt and nutrient materials.