Journal of Applied Bacteriology 1992, 73, 197-202
Incidence of toxigenic vibrios in foods available in Taiwan Hin-Chung Wong, Shu-Hua Ting and Wan-Ru Shieh Department of Microbiology,Soochow University. Taipei, Taiwan,Republic of China 4063/12/91: accepted 20 March 1992
total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V.fluvialis I, V.juvialis 11, V . parahaemolyticus, V. mimicus, Aeromonas cuviue, A . hydrophila, A . sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. T h e Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V . cholerae, V. Juvialis I and V. juviulis I1 isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-01 V. cholerae (2.07%) and two V . parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods. H . C . W O N G , S . H . T I N G A N D W . R . S H I E H . 1992.A
INTRODUCTION
Vibrio cholerae is the causative organism of epidemic cholera and occurs mostly in tropical regions. Vibrio parahaemolyticus has been involved in about 50% or more of the food-borne bacteria1 infections in Taiwan and Japan where seafoods are widely consumed (Takeda 1983; Chiou et al. 1991). In addition, a number of other Vibrio species have been involved in diarrhoea1 diseases, e.g. V . mimicus, V . jluvialis I, V . jluvialis 11, and V . hollisae (Joseph et al. 1983; Morris & Black 1985). Vibrio vulnificus, V . alginolyticus and V . damsela have been involved in wound and other infections (Joseph et al. 1983; Morris & Black 1985). Cholera toxin (CT) and other enterotoxigenic factors have been purified and characterized in V. cholerae 0 1 , non-01 V. cholerae (Takao et al. 1985; Arita et al. 1986), V . mimicus (Spira & Fedorka-Cray 1984), and V . hollisae (Kothary & Richardson 1987). Enterotoxigenic activity was also demostrated in V. fluvialis I and V . fluvialis I1 (Lockwood et al. 1982; Morris & Black 1985). The ecology of vibrios has been studied in fresh, brackish, and seawater systems. Vibrios occur widely in the waters and sediments (Kaysner et al. 1987a,b; Venkateswaran et al. 1988a,b), and frequently associate with zooplanktons (Kaysner et al. 1987b). The incidence of vibrios other than V. cholerae and V . parahaemolyticus in foods, however, has not been clearly investigated. In Tawain food and clinical material associCorrespondence ro : Hin-Chung Wong, Department of Microbiology, Soochow University, Taipei, Taiwan 11102, Republic of China.
ated with cases of food poisoning are not, as a routine, screened for vibrios other than V. cholerae and V. parahaemolyticus. The roles of newly emerged pathogenic vibrios in local food poisoning have yet to be identified. We studied the incidence of various vibtios in the seafood and aquacultured foods available in Taiwan and further characterized them in terms of enzymatic and toxigenic activities. We found a high incidence of toxigenic vibrios in local food samples and that cell toxicity was significantly related to some enzyme activities. MATERIALS AND METHODS isolation and identification of vibrios
A total of 113 seafood and aquacultured samples obtained in the summer of 1990 were examined. The samples included the following major categories : oysters (Crassostrea gigas), hard clams (Meretrzx lusoria), freshwater clams (Corbicula Jlurninea), crabs (Portunus spp.), grass shrimps (Penaeus monodon) and other seawater shrimps (Penaeus spp.). Oysters and grass shrimps were aquacultured products. Samples (25 g) obtained from the markets were blended with 100 ml of 3% NaCl solution. Ten ml of the homogenate was enriched in 100 ml of alkaline peptone water and incubated at 37°C for 7 to 8 h (Sakazaki & Shimada 1982; West & Colwell 1984). A loopful of the enrichment culture was plated on thiosulfate citrate-bile salt-sucrose agar (TCBS, Difco) and incubated
198 HIN-CHUNG WONG E r A L .
at 37°C for 18 to 24 h. Ten yellow or blue-green colonies
for each sample were randomly sclected and cultured in 10% skim milk, then stored at - 70°C. Cultures that were susceptible to 0/129 (2,4-diamino-6,7-diisopropylpteridine) susceptible isolates were screened in Mueller Hinton broth (Difco) supplemented with 1% NaCl and 16 pg/ml 0/129 (Sigma). Susceptible isolates (vibrios or related species) as well as the haemolytic 0/129 nonsusceptible isolates were identified to species level. For the isolation of V. hollisae, the enrichment culture was also plated on special selective agar medium containing (g/l): phytone, 5; peptone, 5; beef extract, 5; wmannitol, 10; maltose, 10; NaCI, 20; agar, 13; bromthymol blue, 0.04; cresol red, 0.04 (Nishibuchi et al. 1988). The suspect colonies were simultaneously inoculated on marine agar (Difco) and TCBS agar media. Organisms grown on marine agar but not on TCBS agar were identified. The strains were identified by the Microbact 24E (Disposable Products PTY Ltd, Adelaide, South Australia) rapid identification kit. Additional characteristics such as gas production during glucose fermentation and the 0/129 disk susceptibility were done by the method of West & Colwell (1984). All the media used for identification contained 1% NaCI.
Assay of haemoiytic activity
The isolates were grown on Tryptic Soy Agar (TSA, Difco) supplemented with 1% NaCl and 5% of defibrinated sheep blood, incubated at 37°C overnight. The haemolysis was registered as: -, negative; weak; and + + +, moderate; strong.
+ + + +,
+,
++
Detection of cholera toxin and thermostable direct haemolysin
Presence of CT and thermostable direct haemolysin (TDH) in the culture supernatant fluids were detected by the reversed passive latex agglutination method (Denka Seiken) according to the manufacturer's procedures. Vibrio cholerae and V . mimicus isolates were cultured in syncase culture medium containing (g/l): casamino acid, 10; sucrose, 5; NazHPO,, 5; K,HPO,, 5 ; Na2S04, 0.089; MgCl, , 6H,O, 0.042; MnC12 . 4H20, 0,004; FeCI, . 6Hz0, 0.005. Other halophilic vibrios were cultured in a rich medium containing (g/l): NaCI, 30; peptone, 10; Na2HP04, 5; glucose, 5 ; pH 76-74, incubated at 30" or 37"C, respectively. Overnight cultured bacterial cells were pelleted by centrifugation and the supernatant fluids were assayed for the toxins. Detection of cytotoxic and cytotonic actlvities
Cytotoxicity (cytotoxic and cytotonic) of culture supernatant fluids were assayed by Chinese hamster ovary (CHO) cells (Wong et al. 1988). CHO cells were cultured in McCoy 5A medium (Gibco) supplemented with 10% fetal bovine serum, streptomycin 200 mg/l, kanamycin 50 mg/l, and fungizon 1-25 mg/l (Gibco) in 96-well microplates. Twenty pl of the supernatant fluid were added to each well of CHO cells containing 150 p1 animal culture medium, incubated overnight and the transformation of cells was observed. Destruction of monolayer or rounding of 30% or more of the cells was recognized as cytotoxic reaction, while elongation of 30% or more of the cells was recognized as cytotonic reaction. Statistical analysis
Assays of enzyme activities
Lipase activity was determined by inoculating the isolates on a lipase assay medium containing (g/l): peptone, 10; NaCI, 10; CaCIz , H20, 0.1; agar, 20, containing 1% Tween 80 and incubated at 37°C overnight. A white precipitation around the colonies indicated lipase activity. Protease activity was determined on a skim milk agar medium containing (g/l): skim milk, 15; NaCI, 10; agar, 15 incubated at 37°C overnight. Clear zones around the colonies indicated proteolytic activity.
Serotyping
The V. cholerae isolates grown on TSA containing 1% NaCl at 37°C overnight were serotyped with antiscra (Denka Seiken Co., Tokyo) according to the procedure recommended by the supplier.
Variations of data in relation to other factors were analysed by analysis of variance.
RESULTS incidence of vibrios In foods
The incidence of vibrios in these major food categories is summarized in Table 1. The incidence of V. cholerae, V . Juvialis I, V. mimicus, and V . parahaemolyticus was high in nearly all the seafoods or aquacultured foods sampled. There was a high incidence of Aeromonas species. A total of 1088 isolates were identified and 67.5% of these isolates were Vibrio species: V. alginolyticus, I/. cholerae, V .fluvialis I, V .juvialis 11, V. mamicus, and V. parahaemolyticus. Aeromonas caviae, A . hydrophila, A . sobria, and some other bacteria were identified (Table 2). Only one
TOXIGENIC VlBRlOS I N FOODS OF TAIWAN 199
Table 1 Incidence of Vibrio and
Incidence (%)
Aeromonas species in samples of foods
available in Taiwan Species
Oysters n=16
Hard clams n=20
Freshwater clams n = 14
Shrimps n=47
Crabs n = 12
Vibrio alginolyticus cholerae Jluvialis I Jluvialis I1 mimicus parahaemolyticus Aeromonas caviae hydrophiIa sobria
68.8 93.8 68.8 12.5 43.8 68.8 12.5 6.3 43.8
60.0 55.0 65.0 10.0 35.0 100.0 55.0 30.0 15.0
57-1 85.7 78.6 7.1 50.0 92.9 14.3 21.4 21.4
70.2 55.3 6.4 8.5 38.3 70.2 19.1 17.0 36.2
66.7 50.0 25.0 8.3 8.3 41.7 0
25.0 50.0
n, Number of samples.
isolate was identified as V , cholerae 01, and V . hollisae was not found. Analysis of variance showed that there was no significant variation in species distribution among the different kinds of shrimps, so all shrimp results were presented as one group. When all the major food categories were analysed, distribution of bacterial species was not homogenous (Table 2). Vibrio cholerae, V . parahaemolyticus and V. fluvialis I were the major species found in oysters and freshwater clams. Vibrio alginolyticus was the major species in shrimps and crabs. Vibrio parahaemolyticus was the major species in hard clams and it was also freely distributed in other food samples (Table 2). Haernolysls and enzyme actlvltles
Haemolytic activity of all the Vibrio and Aeromonas isolates are summarized in Table 3. Analysis of variance showed
that the distribution of haemolysis of different species was significantly different. Ninety-seven percent of V .fluvialis I strain showed haemolytic activity, while 65.5% of V . parahaemolyticus isolates were haemolytic (Table 3). Vibrto mimicus, V . parahaemolyticus, and the Aeromonas isolates were mostly weak haemolytic, while those of V. cholerae, V. fluvialis I and V. fluvialis I1 were comparatively strongly haemolytic. T h e majority of the Vibrio strains showed lipase and protease activities (Table 4).T h e magnitude of lipase and protease activity in terms of size of the activity zone was also recorded (results not shown). Strong lipase activity was found in most of these vibrios. By comparison, the majority of the Aeromonas isolates were lipase negative or showed weak activity and V . alginolyticus and V . parahaernolyticus isolates were most weakly proteolytic; those of V.fluvialis I and V .fluvialis I1 were most strongly proteolytic.
Table 2 Percentage of Vibrio and
Percentage of isolates from
Aeromonas species among isolates obtained
from food samples Species Vibrio alginolyticus cholerae fluvialis I fluvialis I1 mimicus parahaemolyticus Vibrio spp. Aeromonas caviae hydrophila sobria Pseudomonas spp.
Others*
No. of isolates
Oysters Hard clams n = 159 n = 227
174 162 77 12 45 206 58 52 30 53 165 54
11.3 27.7 14.5 1.3 5.7 13.2 3-1 1-9 0.6 6.9 10.1 3.7
10.1 7.0 10.1 0.9 4.8 26-9 1.8 11.9 2.6 3.1 15.9 4.8
Freshwater clams n = 127 8.7 22.8 11.8 0.8 7.9 22.8 9.4 1*6 3.1 2.4 3.1 5.5
Shrimps Crabs n = 79
n = 462
21.0 13.4 1.7 0.9 2.8 16-0 0.1
4-1 2-6 5.0 20-1 5.4
20.3 12.7 10.1 1.3 1.3 17-7 0 0 63 7-6 16.5 6.4
n, Number of isolates.
* Other species included Actinobacillus, Flavobacterium, Moraxella, Pasteurella. Forty-two isolates were unidentified.
200 HIN-CHUNG WONG ET A L .
Table 3 Haemolytic activity of Vibrio and Aeromonas species isolated from food
YOof isolates with haemolytic activity
Species
-
Vibrio alginolyticus cholerae juv i a l i s I fluvialis I1 mimicus parahaemolyticus L4eromonas caviae hydrop hila sobria
25-3 16.0 2.6 25.0 15.6 34.5 15.4
-, Negative;
10.0
11.3
+
++
+++
++++
39.7 25.9 15.6 25.0 51.1 53.4 75.0 36.7 34.0
14-9
9-8 25.9 36.4 33.3 8.9 3.9 0 16.7 13.2
10-3 22.0 3.9 0 2.2 3.4 0 3.3
9.9
41-6 16.7 22-2 4.9 9.6
33.3 34.0
samples
7.5
+, weak; + + and + + +, moderate; + + + +, strong.
Table 4 Lipase and protease activity of Vibrio and Aeromonas species isolated from food samples
Percentage positive Species
1.ipase
Protease
Vibrro alginolyticw cholerae juvialis I fluvialis I1 mimicus para haemolyticus Aeromonas caviae hy drop hila sobria
98-1 98.0 95.7 81.8 97.7 97-9 69.2
78.2 91.4 90.9 100 93.3 87.4 69.2 76.7 79.2
81.5
82.4
isolates) originating from oyster, freshwater clam and shrimp samples produced CT. Only two (1.65% of V. parahaemolyticus) which originated from the same hard clam sample produced TDH. Cytotoxicity of all these 448 vibrio strains was assayed by C H O cell culture. Twenty three (5.1%) of V. cholerae, V. fluvialis I, V . fluvialis 11, V. mimicus, V. parahaemolyticus, and Vibrio spp. showed cytotonic activity, while 48.7% showed cytotoxic activity (Table 5). Only 20.7% of V. parahaemolyticus isolates were cytotoxic, while over 50% of those of other species showed cytotoxic activity (Table 5). DISCUSSION
Toxlgenic activitles
A total of 448 vibrio strains (35 V. alginolyticus, 155 V . cholerae, 74 V. Juvialis I, 9 V . fluvialis 11, 41 V . mimicus, 121 V. parahaemolyticus, and 13 unidentified vibrios) were examined for the production of CT and all the V. parahaemolyticus isolates were examined for TDH. Only three of the non-01 V. cholerae isolates (2.07% of V. cholerae
T h e ecology of vibrios in aquatic systems has been well studied and distribution of Vibrio species is affected by salinity, nutrient eflluent, and other factors (Venkateswaran et a/. 1989a,b). However, the occurrence and populations of various Vibrio species in foods may differ from the ecological findings. In ecological studies, the incidence of halophilic vibrios is consistently high in seawater and seafood samples (Molitoris et al. 1985; Venkateswaran et a / . 1989b). I n our study the incidence and population of halophilic vibrios such as V. alginolyticus, V. fluvialis I, and V. paraTable 5 Cytotoxic and cytotonic activities of Vibrio isolates assayed by Chinese
Percentage of reaction Species
No. of isolates
Negative
Cytotoxic
Cytotonic
Vibrio alginolyticus cholerae fluvialis I fluvialis I1
20.0 43.2 33.8 33.3 39.0 72.8 0
80.0
parahaemolyticus Vibrio spp.
35 155 74 9 41 121 A3
0 5.2 6.8 11.1 2.5 5.8
Total
448
46.2
48.7
mimicus
51.6 58.1 55.5 58-5
20.7 84.6
7.7
5.1
hamster
Ovary
(cHo)
TOXIGENIC V l B R l O S
IN FOODS OF TAIWA N 201
~
huemolyticus were also high in freshwater clams (Tables 1 and 2). Vibrio parahuemolyticus was also isolated from freshwater samples in India and Japan (Joseph et al. 1983; Sarkar et al. 1985; Venkateswaran et al. 1989a). Unknown conditions may promote the survival of the halophilic vibrios in freshwater or in the freshwater clams (Venkateswaran et al. 1989a). Incidence and population of V . cholerae and V . mimicus in foods of seawater origin (oysters, hard clams, shrimps and crabs) were mostly high (Table 1 and 2). These vibrios have originated from the estuarine waters. IR Taiwan oysters are cultivated and hard clams harvested in the intertidal zones. River clams are harvested in the rivers and streams. Grass shrimps are aquacultured, and other shrimps and crabs are usually collected from marine environments. Non-01 V. cholerae are common in the coastal environments (Kaysner et al. 1987b;Venkateswaran et al. 1989b) and its abundance may be a result of riverine influence (Venkateswaran et al. 1989b). Sewage, agricultural and industrial effluents that have not been well monitored in this region may also have important impact on the Occurrence of vibrios in foods. Apart from the natural environmental factors, post-harvest processes may be also responsible for the distribution of those halophilic vibrios in freshwater products. The V. cholerae 0 1 serotype and the Kanagawa-positive V . parahaemolyticus have frequently been involved in cholera and gastroenteritis, respectively. Some strains of non-0 1 V . cholerae and Kanagawa-negative V . parahaemobticus are also involved in bacteremia (Wistrom 1989), wound infection and diarrhoea1 diseases (Kay et al. 1984; Johnson et al. 1984; Honda et al. 1989). In addition, a number of toxins were detected in the environmental strains of these species (non-01 V . cholerae or Kanagawanegative V. parahuemolyticus) (Takao et al. 1985; Arita et al. 1986; Ichinose et al. 1987; Sarkar et al. 1987; Honda et al. 1989). Determinations of serotype and Kanagawa phenomenon could not therefore discriminate all the pathogenic strains of V. cholerae and V. parahaemolyticus, respectively. Generally, only a proportion of the vibrios isolated from the environment are toxigenic; it is necessary, therefore, to determine the toxigenic characteristics of those isolated from foods to understand their roles in food-borne illnesses. In this report all the strains were examined for enzyme activities (protease and lipase) and haemolysis, and most of the vibrios for CT, TDH and cytotoxicities. Proteases have been isolated from various Vzbrzo species (Crowther et al. 1987; Kothary & Kreger 1987; Chowdhury et al. 1990). Different roles of proteases have been demonstrated; for example, haemagglutination (Booth & Finkelstein 1986), toxin processing (Hall & Drasar 1990), permeability enhancement (Crowther et al. 1987; Miyoshi & Shinoda
1988). About 78-100% of the Vibrio species isolated were protease-positive (Table 4) ; V.Jluvialis I and V.fluvialis I1 isolates were strong in protease activity (results not shown). Analysis of variance for the Vibrio species isolated showed that distribution of protease and lipase activities were significantly related to the cytotoxicity assayed by CHO cells. It may be possible, therefore, that protease or lipase of all these Vibrio species has a role in cytotoxicity. A number of haemolysins were produced by various Vibrio species and serotypes (Yamamoto et al. 1984; Nishibuchi et a/. 1988). These haemolysins have different toxicity (Takeda 1983; Ichinose et al. 1987). It is well documented that environmental non-0 1 V . cholerae strains usually have strong haemolytic activity (Venkateswaran et al. 1989a; Amaro et al. 1990) and the majority of the haemolytic environmental non-0 1 V . cholerae strains showed cytotoxicity (Venkateswaran et al. 1989a; Amaro et al. 1990). In this study, strong haemolytic activity was observed in V. cholerae, V. fluvialis I and V . fluvialis I1 (Table 3), and about half of them showed cytotoxicity (Table 5). Although a large number of the Vibrio isolates showed cytotoxicity, only a few of them showed C T or T D H activities. It is possible that a wide spectrum of cytotoxic or cytotonic toxins may be produced by different Vibrio species in foods. In conclusion, the incidence of the toxigenic Vibrio species was high in the seafood and aquacultured foods available in Taiwan and about half of them showed cytotoxicity and high haemolytic activity. These results should prompt us to pay more attention to the role of these vibrios in local food-borne diseases. ACKNOWLEDGEMENT
The study is supported by the Division of Food Hygiene, Department of Health of the Republic of China (FS80-14). REFERENCES AMARO, C., TORANZO A ,. E . , G O N Z A L E Z ,E . A . , B L A N C OJ ,. , P U J A L T EM, . J . , A Z N A RR. , & G A R A YE, . (1990) Surface and virulence properties of environmental Vibrio cholerae non-01 from Albufera Lake (Valencia, Spain). Applied
and Environrnentd Microbiology 56, 114g1147, A R I T A , M., T A K E D AT, . , H O N D AT , . & M I W A T A NT I ,. (1 986) Purification and characterization of Vibrio cholerae non-01 heat-stable enterotoxin. Infection and Immunity 52, 45-49. BOOTH,B . A . & F I N K E L S T E I R N ., A . (1986) Presence of hemagglutinin/protease and other potential virulence factors in 0 1 and non-01 Vibrio cholerae. Journal of Infectious Diseases 154, 183-186. CHIOIJ,A . , CHEN, L.-HI. & C H E N ,S . - K . (1991) Foodborne illness in Taiwan, 1981-1989. Food Auwafza 43,7&71.
202 HIN-CHUNG WONG E r A L .
C H O W D H U R M.A.R., Y, M I Y O S H I ,S . - I . & SHINODA, S. (1990) Purification and characterization of a protease produced by Vibrio mimicus. Infection and Immunity 58,41594162. C R O W T H E RR, . S . , R O O M I ,N . W . , F A H I MR, . E . & FORS T N E R , J . F . (1987) Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine. Biochimica et Biophysica Acta 924, 393-402. H A L L , R.H. & D R A S A RB.S. , (1990) Vibrio cholerae H I y A hemolysin is processed by proteolysis. Infection and Immunity 58,3375-3379. H O N D AT , . , N I , Y. & M I W A T A N T I , . (1989) Purification of a TDH-related hemolysin produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus 06 : K46. F E M S Microbiological Letters 48, 241-245. ICHINOSE, Y ., YAMAMOTO,K . , N A K A S O N E ,N . , T A N A B E ,M . J . , T A K E D A ,T., M I W A T A N I ,T. & I W A N A G A , M . (1987) Enterotoxicity of El Tor-like hemolysin of non-01 Vibrio cholerae. Infection and Immunity 55, 10901093. JOHNSON, D . E . , W E I N B E R G ,L . , C I A R K O W S K IJ,. , WEST, P . & COLWELL,R.R. (1984) Wound infection caused by Kanagawa-negative Vibrio parahaemolyticus. Journal of Clinical Microbiology 20, 81 1-812. JOSEPH,S . W . , COLWELL,R . R . & K A P E R ,J . B . (1983) Vibrio parahaemolyticus and related halophilic vibrios. CRC Crirical Revien, of Microbiology 10, 77-123. K A Y ,B.A., S A C K R , . B . , S P I R A ,W . M . , G U E R R AH, . E . , G U E R R E R OC.E., , C H A P A R R OE., , Y I , A.E., SALAZARL I N D O ,E . , C H E A ,E . , W A C H S M U T HI .,K . & D A V I E S , B. R . (1984) Vibrio cholerae non-01 isolated from five people with diarrhoea in Lima. Lancet 1, 218. K A Y S N E RC.A., , A B E Y T A , C.O., J R , W E K E L L ,M . M . , D E P A O L A ,A., J R , STOTT, R . F . & L E I T C H , J . M . (lY87a) Virulent strains of Vibrio uulnr&us isolated from estuaries of the United States West Coast. Applied and Environm e n d Microbiology 53, 1349-1351. K A Y S N E RC.A., , ABEYTA,C., J R , WEKELL, M.M., D E P A O L A A, . , J R , S T O T T , R . F . & L E I T C H , J . M . (1987b) Incidence of Vibrio cholerae from estuaries of the United States West Coast. Applied and Environmental Microbiology 53, 1344-1348. K O T H A R YM, . H . & K R E G E RA.S. , (1987) Purification and characterization of an elastolytic protease of Vibrio vulnifcus. Journal of General Microbiology 133, 1783-1791. KOTIIARY M, . H . & RICIiARDSON, S . H . (1987) Fluid accumulation in infant mice caused by Vibrio hollisae and its extracellular enterotoxin. Infection and Immunity 55, 626-630. I,OCKWOOD, D.E., K R E G E RA.S. , & RICHARDSON S ., H . (1982) Detection of toxins produced by Vibrio fluvialis. Infection and Immunity 35, 702-708. M I Y O S H IS, . & S H I N O D AS, . (1988) Role of the protease in the permeability enhancement by Vibrio vulnrjicus. Microbiology and Immunology 32, 1025-1032. M O L I T O R I SE, . , J O S E P H , S . W . , K R I C H E V S K YM, . I . , SINDHUHARDJA, w. & C O L W E L L , R.R. (1985) Characterization and distribution of Vibrio alginolyticus and Vibrio para-
haemolyticus isolated in Indonesia. Applied and Environmental Microbiology 50, 1388-1394. M O R R I SJ, . G . & BLACK,R . E . (1985) Cholera and other vibrioses in the United States. N e w England Journal of Medicine 312,343-350. N I S H I B U C H IM , . , D O K E , S . , T O I Z U M I S., , U M E D AT , ., YOH, M . & M I W A T A NT. I , (1988) Isolation from a coastal fish of Vibrio hollisae capable of producing a hemolysin of Vibrio parahaemolyticus. Applied and Environmental Microbiology 54, 2144-2146. S A K A Z AR K. I& , S H I M A DT. A , (1982) Vibrio species as causative agents of food-borne infection. Developments in Food Microbiology-2, ed. Robinson, R.K. pp. 123-151. London : Elsevier Applied Science Publishers. S A R K A RB,. L . , K U M A RR, . , D E , S.P. & PAL, S.C. (1987) Hemolytic activity of and lethal toxin production by environmental strains of Vibrio parahaemolyticus. Applied and Environmental Microbiology 53, 2696-2698. SARKAR, B.L., N A I R , G.B., B A N E R J E EA.K. , & PAL, S. C . (1985) Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta. Applied and Environmental Microbiology 49, 132-136. SPIRAW , . M . & F E D O R K A - C R AP.J. Y , (1984) Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin. Infection and Immunity 45, 679-684. TAKAO, T., S H I M O N I S H IY, . , KOBAYASHI, M., NISHIM U R A , O., A R I T A ,M . , T A K E D AT , . , H O N D AT, . & M I W A T A N IT. , (1985) Amino acid sequence of heat-stable enterotoxin produced by Vibrio cholerae non-01. F E B S Letters 193,250-254. T A K E DY. A , (1983) Thermostable direct hemolysin of Vibrio purahaemolyticus. Pharmaceutical Therapy 19, 123-146. V E N K A T E S W A R AKN.,, K I I Y U K I A ,C . , T A K A KM I ,. , N A K A N OH, . , M A T S U D AH., , K A W A K A MHI ,. & HASHI MOTO,H . (1989a) Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan. Applied and Environmental Microbiology 55, 2613-2618. V E N K A T E S W A R AKN.,, N A K A N O , H . , O K A B E , T . , K., H. TAKAYAM A , MATSUDA,0. & HASHIMOTO, (1989b) Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan. Applied and Environmental Microbiology 55, 55%567. W E S T , P.A. & C O L W E L L ,R . R . (1984) In Vibrios in the Environment ed. Colwell, R.R. Ch. 20, pp. 285-363. New York: John Wiley & Sons. WISTROM,J . (1989) A case of n o n 4 : 1 Vibrio cholerae bacteremia from northern Europe. Journal of Infectious Diseases 160, 732-735. WONG, H . C . , C H E N , Y . L . & C H E N , C . L . F . (1988) Growth, germination and toxigenic activity of Bacillus cereus in milk products. Journal of Food Protection 51, 707-710. Y A M A M O T OK , . , A L - O M A N IM , . , H O N D AT , ., TAKEDA, Y. & M I W A T A N IT, . (1984) Non-01 Vibrio cholerae hemolysin : purification, partial characterization, and immunological relatedness to El Tor hemolysin. Infection and Immunity 45, 192-196.
Incidence of toxigenic vibrios in foods available in Taiwan Hin-Chung Wong, Shu-Hua Ting and Wan-Ru Shieh Department of Microbiology,Soochow University. Taipei, Taiwan,Republic of China 4063/12/91: accepted 20 March 1992
total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V.fluvialis I, V.juvialis 11, V . parahaemolyticus, V. mimicus, Aeromonas cuviue, A . hydrophila, A . sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. T h e Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V . cholerae, V. Juvialis I and V. juviulis I1 isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-01 V. cholerae (2.07%) and two V . parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods. H . C . W O N G , S . H . T I N G A N D W . R . S H I E H . 1992.A
INTRODUCTION
Vibrio cholerae is the causative organism of epidemic cholera and occurs mostly in tropical regions. Vibrio parahaemolyticus has been involved in about 50% or more of the food-borne bacteria1 infections in Taiwan and Japan where seafoods are widely consumed (Takeda 1983; Chiou et al. 1991). In addition, a number of other Vibrio species have been involved in diarrhoea1 diseases, e.g. V . mimicus, V . jluvialis I, V . jluvialis 11, and V . hollisae (Joseph et al. 1983; Morris & Black 1985). Vibrio vulnificus, V . alginolyticus and V . damsela have been involved in wound and other infections (Joseph et al. 1983; Morris & Black 1985). Cholera toxin (CT) and other enterotoxigenic factors have been purified and characterized in V. cholerae 0 1 , non-01 V. cholerae (Takao et al. 1985; Arita et al. 1986), V . mimicus (Spira & Fedorka-Cray 1984), and V . hollisae (Kothary & Richardson 1987). Enterotoxigenic activity was also demostrated in V. fluvialis I and V . fluvialis I1 (Lockwood et al. 1982; Morris & Black 1985). The ecology of vibrios has been studied in fresh, brackish, and seawater systems. Vibrios occur widely in the waters and sediments (Kaysner et al. 1987a,b; Venkateswaran et al. 1988a,b), and frequently associate with zooplanktons (Kaysner et al. 1987b). The incidence of vibrios other than V. cholerae and V . parahaemolyticus in foods, however, has not been clearly investigated. In Tawain food and clinical material associCorrespondence ro : Hin-Chung Wong, Department of Microbiology, Soochow University, Taipei, Taiwan 11102, Republic of China.
ated with cases of food poisoning are not, as a routine, screened for vibrios other than V. cholerae and V. parahaemolyticus. The roles of newly emerged pathogenic vibrios in local food poisoning have yet to be identified. We studied the incidence of various vibtios in the seafood and aquacultured foods available in Taiwan and further characterized them in terms of enzymatic and toxigenic activities. We found a high incidence of toxigenic vibrios in local food samples and that cell toxicity was significantly related to some enzyme activities. MATERIALS AND METHODS isolation and identification of vibrios
A total of 113 seafood and aquacultured samples obtained in the summer of 1990 were examined. The samples included the following major categories : oysters (Crassostrea gigas), hard clams (Meretrzx lusoria), freshwater clams (Corbicula Jlurninea), crabs (Portunus spp.), grass shrimps (Penaeus monodon) and other seawater shrimps (Penaeus spp.). Oysters and grass shrimps were aquacultured products. Samples (25 g) obtained from the markets were blended with 100 ml of 3% NaCl solution. Ten ml of the homogenate was enriched in 100 ml of alkaline peptone water and incubated at 37°C for 7 to 8 h (Sakazaki & Shimada 1982; West & Colwell 1984). A loopful of the enrichment culture was plated on thiosulfate citrate-bile salt-sucrose agar (TCBS, Difco) and incubated
198 HIN-CHUNG WONG E r A L .
at 37°C for 18 to 24 h. Ten yellow or blue-green colonies
for each sample were randomly sclected and cultured in 10% skim milk, then stored at - 70°C. Cultures that were susceptible to 0/129 (2,4-diamino-6,7-diisopropylpteridine) susceptible isolates were screened in Mueller Hinton broth (Difco) supplemented with 1% NaCl and 16 pg/ml 0/129 (Sigma). Susceptible isolates (vibrios or related species) as well as the haemolytic 0/129 nonsusceptible isolates were identified to species level. For the isolation of V. hollisae, the enrichment culture was also plated on special selective agar medium containing (g/l): phytone, 5; peptone, 5; beef extract, 5; wmannitol, 10; maltose, 10; NaCI, 20; agar, 13; bromthymol blue, 0.04; cresol red, 0.04 (Nishibuchi et al. 1988). The suspect colonies were simultaneously inoculated on marine agar (Difco) and TCBS agar media. Organisms grown on marine agar but not on TCBS agar were identified. The strains were identified by the Microbact 24E (Disposable Products PTY Ltd, Adelaide, South Australia) rapid identification kit. Additional characteristics such as gas production during glucose fermentation and the 0/129 disk susceptibility were done by the method of West & Colwell (1984). All the media used for identification contained 1% NaCI.
Assay of haemoiytic activity
The isolates were grown on Tryptic Soy Agar (TSA, Difco) supplemented with 1% NaCl and 5% of defibrinated sheep blood, incubated at 37°C overnight. The haemolysis was registered as: -, negative; weak; and + + +, moderate; strong.
+ + + +,
+,
++
Detection of cholera toxin and thermostable direct haemolysin
Presence of CT and thermostable direct haemolysin (TDH) in the culture supernatant fluids were detected by the reversed passive latex agglutination method (Denka Seiken) according to the manufacturer's procedures. Vibrio cholerae and V . mimicus isolates were cultured in syncase culture medium containing (g/l): casamino acid, 10; sucrose, 5; NazHPO,, 5; K,HPO,, 5 ; Na2S04, 0.089; MgCl, , 6H,O, 0.042; MnC12 . 4H20, 0,004; FeCI, . 6Hz0, 0.005. Other halophilic vibrios were cultured in a rich medium containing (g/l): NaCI, 30; peptone, 10; Na2HP04, 5; glucose, 5 ; pH 76-74, incubated at 30" or 37"C, respectively. Overnight cultured bacterial cells were pelleted by centrifugation and the supernatant fluids were assayed for the toxins. Detection of cytotoxic and cytotonic actlvities
Cytotoxicity (cytotoxic and cytotonic) of culture supernatant fluids were assayed by Chinese hamster ovary (CHO) cells (Wong et al. 1988). CHO cells were cultured in McCoy 5A medium (Gibco) supplemented with 10% fetal bovine serum, streptomycin 200 mg/l, kanamycin 50 mg/l, and fungizon 1-25 mg/l (Gibco) in 96-well microplates. Twenty pl of the supernatant fluid were added to each well of CHO cells containing 150 p1 animal culture medium, incubated overnight and the transformation of cells was observed. Destruction of monolayer or rounding of 30% or more of the cells was recognized as cytotoxic reaction, while elongation of 30% or more of the cells was recognized as cytotonic reaction. Statistical analysis
Assays of enzyme activities
Lipase activity was determined by inoculating the isolates on a lipase assay medium containing (g/l): peptone, 10; NaCI, 10; CaCIz , H20, 0.1; agar, 20, containing 1% Tween 80 and incubated at 37°C overnight. A white precipitation around the colonies indicated lipase activity. Protease activity was determined on a skim milk agar medium containing (g/l): skim milk, 15; NaCI, 10; agar, 15 incubated at 37°C overnight. Clear zones around the colonies indicated proteolytic activity.
Serotyping
The V. cholerae isolates grown on TSA containing 1% NaCl at 37°C overnight were serotyped with antiscra (Denka Seiken Co., Tokyo) according to the procedure recommended by the supplier.
Variations of data in relation to other factors were analysed by analysis of variance.
RESULTS incidence of vibrios In foods
The incidence of vibrios in these major food categories is summarized in Table 1. The incidence of V. cholerae, V . Juvialis I, V. mimicus, and V . parahaemolyticus was high in nearly all the seafoods or aquacultured foods sampled. There was a high incidence of Aeromonas species. A total of 1088 isolates were identified and 67.5% of these isolates were Vibrio species: V. alginolyticus, I/. cholerae, V .fluvialis I, V .juvialis 11, V. mamicus, and V. parahaemolyticus. Aeromonas caviae, A . hydrophila, A . sobria, and some other bacteria were identified (Table 2). Only one
TOXIGENIC VlBRlOS I N FOODS OF TAIWAN 199
Table 1 Incidence of Vibrio and
Incidence (%)
Aeromonas species in samples of foods
available in Taiwan Species
Oysters n=16
Hard clams n=20
Freshwater clams n = 14
Shrimps n=47
Crabs n = 12
Vibrio alginolyticus cholerae Jluvialis I Jluvialis I1 mimicus parahaemolyticus Aeromonas caviae hydrophiIa sobria
68.8 93.8 68.8 12.5 43.8 68.8 12.5 6.3 43.8
60.0 55.0 65.0 10.0 35.0 100.0 55.0 30.0 15.0
57-1 85.7 78.6 7.1 50.0 92.9 14.3 21.4 21.4
70.2 55.3 6.4 8.5 38.3 70.2 19.1 17.0 36.2
66.7 50.0 25.0 8.3 8.3 41.7 0
25.0 50.0
n, Number of samples.
isolate was identified as V , cholerae 01, and V . hollisae was not found. Analysis of variance showed that there was no significant variation in species distribution among the different kinds of shrimps, so all shrimp results were presented as one group. When all the major food categories were analysed, distribution of bacterial species was not homogenous (Table 2). Vibrio cholerae, V . parahaemolyticus and V. fluvialis I were the major species found in oysters and freshwater clams. Vibrio alginolyticus was the major species in shrimps and crabs. Vibrio parahaemolyticus was the major species in hard clams and it was also freely distributed in other food samples (Table 2). Haernolysls and enzyme actlvltles
Haemolytic activity of all the Vibrio and Aeromonas isolates are summarized in Table 3. Analysis of variance showed
that the distribution of haemolysis of different species was significantly different. Ninety-seven percent of V .fluvialis I strain showed haemolytic activity, while 65.5% of V . parahaemolyticus isolates were haemolytic (Table 3). Vibrto mimicus, V . parahaemolyticus, and the Aeromonas isolates were mostly weak haemolytic, while those of V. cholerae, V. fluvialis I and V. fluvialis I1 were comparatively strongly haemolytic. T h e majority of the Vibrio strains showed lipase and protease activities (Table 4).T h e magnitude of lipase and protease activity in terms of size of the activity zone was also recorded (results not shown). Strong lipase activity was found in most of these vibrios. By comparison, the majority of the Aeromonas isolates were lipase negative or showed weak activity and V . alginolyticus and V . parahaernolyticus isolates were most weakly proteolytic; those of V.fluvialis I and V .fluvialis I1 were most strongly proteolytic.
Table 2 Percentage of Vibrio and
Percentage of isolates from
Aeromonas species among isolates obtained
from food samples Species Vibrio alginolyticus cholerae fluvialis I fluvialis I1 mimicus parahaemolyticus Vibrio spp. Aeromonas caviae hydrophila sobria Pseudomonas spp.
Others*
No. of isolates
Oysters Hard clams n = 159 n = 227
174 162 77 12 45 206 58 52 30 53 165 54
11.3 27.7 14.5 1.3 5.7 13.2 3-1 1-9 0.6 6.9 10.1 3.7
10.1 7.0 10.1 0.9 4.8 26-9 1.8 11.9 2.6 3.1 15.9 4.8
Freshwater clams n = 127 8.7 22.8 11.8 0.8 7.9 22.8 9.4 1*6 3.1 2.4 3.1 5.5
Shrimps Crabs n = 79
n = 462
21.0 13.4 1.7 0.9 2.8 16-0 0.1
4-1 2-6 5.0 20-1 5.4
20.3 12.7 10.1 1.3 1.3 17-7 0 0 63 7-6 16.5 6.4
n, Number of isolates.
* Other species included Actinobacillus, Flavobacterium, Moraxella, Pasteurella. Forty-two isolates were unidentified.
200 HIN-CHUNG WONG ET A L .
Table 3 Haemolytic activity of Vibrio and Aeromonas species isolated from food
YOof isolates with haemolytic activity
Species
-
Vibrio alginolyticus cholerae juv i a l i s I fluvialis I1 mimicus parahaemolyticus L4eromonas caviae hydrop hila sobria
25-3 16.0 2.6 25.0 15.6 34.5 15.4
-, Negative;
10.0
11.3
+
++
+++
++++
39.7 25.9 15.6 25.0 51.1 53.4 75.0 36.7 34.0
14-9
9-8 25.9 36.4 33.3 8.9 3.9 0 16.7 13.2
10-3 22.0 3.9 0 2.2 3.4 0 3.3
9.9
41-6 16.7 22-2 4.9 9.6
33.3 34.0
samples
7.5
+, weak; + + and + + +, moderate; + + + +, strong.
Table 4 Lipase and protease activity of Vibrio and Aeromonas species isolated from food samples
Percentage positive Species
1.ipase
Protease
Vibrro alginolyticw cholerae juvialis I fluvialis I1 mimicus para haemolyticus Aeromonas caviae hy drop hila sobria
98-1 98.0 95.7 81.8 97.7 97-9 69.2
78.2 91.4 90.9 100 93.3 87.4 69.2 76.7 79.2
81.5
82.4
isolates) originating from oyster, freshwater clam and shrimp samples produced CT. Only two (1.65% of V. parahaemolyticus) which originated from the same hard clam sample produced TDH. Cytotoxicity of all these 448 vibrio strains was assayed by C H O cell culture. Twenty three (5.1%) of V. cholerae, V. fluvialis I, V . fluvialis 11, V. mimicus, V. parahaemolyticus, and Vibrio spp. showed cytotonic activity, while 48.7% showed cytotoxic activity (Table 5). Only 20.7% of V. parahaemolyticus isolates were cytotoxic, while over 50% of those of other species showed cytotoxic activity (Table 5). DISCUSSION
Toxlgenic activitles
A total of 448 vibrio strains (35 V. alginolyticus, 155 V . cholerae, 74 V. Juvialis I, 9 V . fluvialis 11, 41 V . mimicus, 121 V. parahaemolyticus, and 13 unidentified vibrios) were examined for the production of CT and all the V. parahaemolyticus isolates were examined for TDH. Only three of the non-01 V. cholerae isolates (2.07% of V. cholerae
T h e ecology of vibrios in aquatic systems has been well studied and distribution of Vibrio species is affected by salinity, nutrient eflluent, and other factors (Venkateswaran et a/. 1989a,b). However, the occurrence and populations of various Vibrio species in foods may differ from the ecological findings. In ecological studies, the incidence of halophilic vibrios is consistently high in seawater and seafood samples (Molitoris et al. 1985; Venkateswaran et a / . 1989b). I n our study the incidence and population of halophilic vibrios such as V. alginolyticus, V. fluvialis I, and V. paraTable 5 Cytotoxic and cytotonic activities of Vibrio isolates assayed by Chinese
Percentage of reaction Species
No. of isolates
Negative
Cytotoxic
Cytotonic
Vibrio alginolyticus cholerae fluvialis I fluvialis I1
20.0 43.2 33.8 33.3 39.0 72.8 0
80.0
parahaemolyticus Vibrio spp.
35 155 74 9 41 121 A3
0 5.2 6.8 11.1 2.5 5.8
Total
448
46.2
48.7
mimicus
51.6 58.1 55.5 58-5
20.7 84.6
7.7
5.1
hamster
Ovary
(cHo)
TOXIGENIC V l B R l O S
IN FOODS OF TAIWA N 201
~
huemolyticus were also high in freshwater clams (Tables 1 and 2). Vibrio parahuemolyticus was also isolated from freshwater samples in India and Japan (Joseph et al. 1983; Sarkar et al. 1985; Venkateswaran et al. 1989a). Unknown conditions may promote the survival of the halophilic vibrios in freshwater or in the freshwater clams (Venkateswaran et al. 1989a). Incidence and population of V . cholerae and V . mimicus in foods of seawater origin (oysters, hard clams, shrimps and crabs) were mostly high (Table 1 and 2). These vibrios have originated from the estuarine waters. IR Taiwan oysters are cultivated and hard clams harvested in the intertidal zones. River clams are harvested in the rivers and streams. Grass shrimps are aquacultured, and other shrimps and crabs are usually collected from marine environments. Non-01 V. cholerae are common in the coastal environments (Kaysner et al. 1987b;Venkateswaran et al. 1989b) and its abundance may be a result of riverine influence (Venkateswaran et al. 1989b). Sewage, agricultural and industrial effluents that have not been well monitored in this region may also have important impact on the Occurrence of vibrios in foods. Apart from the natural environmental factors, post-harvest processes may be also responsible for the distribution of those halophilic vibrios in freshwater products. The V. cholerae 0 1 serotype and the Kanagawa-positive V . parahaemolyticus have frequently been involved in cholera and gastroenteritis, respectively. Some strains of non-0 1 V . cholerae and Kanagawa-negative V . parahaemobticus are also involved in bacteremia (Wistrom 1989), wound infection and diarrhoea1 diseases (Kay et al. 1984; Johnson et al. 1984; Honda et al. 1989). In addition, a number of toxins were detected in the environmental strains of these species (non-01 V . cholerae or Kanagawanegative V. parahuemolyticus) (Takao et al. 1985; Arita et al. 1986; Ichinose et al. 1987; Sarkar et al. 1987; Honda et al. 1989). Determinations of serotype and Kanagawa phenomenon could not therefore discriminate all the pathogenic strains of V. cholerae and V. parahaemolyticus, respectively. Generally, only a proportion of the vibrios isolated from the environment are toxigenic; it is necessary, therefore, to determine the toxigenic characteristics of those isolated from foods to understand their roles in food-borne illnesses. In this report all the strains were examined for enzyme activities (protease and lipase) and haemolysis, and most of the vibrios for CT, TDH and cytotoxicities. Proteases have been isolated from various Vzbrzo species (Crowther et al. 1987; Kothary & Kreger 1987; Chowdhury et al. 1990). Different roles of proteases have been demonstrated; for example, haemagglutination (Booth & Finkelstein 1986), toxin processing (Hall & Drasar 1990), permeability enhancement (Crowther et al. 1987; Miyoshi & Shinoda
1988). About 78-100% of the Vibrio species isolated were protease-positive (Table 4) ; V.Jluvialis I and V.fluvialis I1 isolates were strong in protease activity (results not shown). Analysis of variance for the Vibrio species isolated showed that distribution of protease and lipase activities were significantly related to the cytotoxicity assayed by CHO cells. It may be possible, therefore, that protease or lipase of all these Vibrio species has a role in cytotoxicity. A number of haemolysins were produced by various Vibrio species and serotypes (Yamamoto et al. 1984; Nishibuchi et a/. 1988). These haemolysins have different toxicity (Takeda 1983; Ichinose et al. 1987). It is well documented that environmental non-0 1 V . cholerae strains usually have strong haemolytic activity (Venkateswaran et al. 1989a; Amaro et al. 1990) and the majority of the haemolytic environmental non-0 1 V . cholerae strains showed cytotoxicity (Venkateswaran et al. 1989a; Amaro et al. 1990). In this study, strong haemolytic activity was observed in V. cholerae, V. fluvialis I and V . fluvialis I1 (Table 3), and about half of them showed cytotoxicity (Table 5). Although a large number of the Vibrio isolates showed cytotoxicity, only a few of them showed C T or T D H activities. It is possible that a wide spectrum of cytotoxic or cytotonic toxins may be produced by different Vibrio species in foods. In conclusion, the incidence of the toxigenic Vibrio species was high in the seafood and aquacultured foods available in Taiwan and about half of them showed cytotoxicity and high haemolytic activity. These results should prompt us to pay more attention to the role of these vibrios in local food-borne diseases. ACKNOWLEDGEMENT
The study is supported by the Division of Food Hygiene, Department of Health of the Republic of China (FS80-14). REFERENCES AMARO, C., TORANZO A ,. E . , G O N Z A L E Z ,E . A . , B L A N C OJ ,. , P U J A L T EM, . J . , A Z N A RR. , & G A R A YE, . (1990) Surface and virulence properties of environmental Vibrio cholerae non-01 from Albufera Lake (Valencia, Spain). Applied
and Environrnentd Microbiology 56, 114g1147, A R I T A , M., T A K E D AT, . , H O N D AT , . & M I W A T A NT I ,. (1 986) Purification and characterization of Vibrio cholerae non-01 heat-stable enterotoxin. Infection and Immunity 52, 45-49. BOOTH,B . A . & F I N K E L S T E I R N ., A . (1986) Presence of hemagglutinin/protease and other potential virulence factors in 0 1 and non-01 Vibrio cholerae. Journal of Infectious Diseases 154, 183-186. CHIOIJ,A . , CHEN, L.-HI. & C H E N ,S . - K . (1991) Foodborne illness in Taiwan, 1981-1989. Food Auwafza 43,7&71.
202 HIN-CHUNG WONG E r A L .
C H O W D H U R M.A.R., Y, M I Y O S H I ,S . - I . & SHINODA, S. (1990) Purification and characterization of a protease produced by Vibrio mimicus. Infection and Immunity 58,41594162. C R O W T H E RR, . S . , R O O M I ,N . W . , F A H I MR, . E . & FORS T N E R , J . F . (1987) Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine. Biochimica et Biophysica Acta 924, 393-402. H A L L , R.H. & D R A S A RB.S. , (1990) Vibrio cholerae H I y A hemolysin is processed by proteolysis. Infection and Immunity 58,3375-3379. H O N D AT , . , N I , Y. & M I W A T A N T I , . (1989) Purification of a TDH-related hemolysin produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus 06 : K46. F E M S Microbiological Letters 48, 241-245. ICHINOSE, Y ., YAMAMOTO,K . , N A K A S O N E ,N . , T A N A B E ,M . J . , T A K E D A ,T., M I W A T A N I ,T. & I W A N A G A , M . (1987) Enterotoxicity of El Tor-like hemolysin of non-01 Vibrio cholerae. Infection and Immunity 55, 10901093. JOHNSON, D . E . , W E I N B E R G ,L . , C I A R K O W S K IJ,. , WEST, P . & COLWELL,R.R. (1984) Wound infection caused by Kanagawa-negative Vibrio parahaemolyticus. Journal of Clinical Microbiology 20, 81 1-812. JOSEPH,S . W . , COLWELL,R . R . & K A P E R ,J . B . (1983) Vibrio parahaemolyticus and related halophilic vibrios. CRC Crirical Revien, of Microbiology 10, 77-123. K A Y ,B.A., S A C K R , . B . , S P I R A ,W . M . , G U E R R AH, . E . , G U E R R E R OC.E., , C H A P A R R OE., , Y I , A.E., SALAZARL I N D O ,E . , C H E A ,E . , W A C H S M U T HI .,K . & D A V I E S , B. R . (1984) Vibrio cholerae non-01 isolated from five people with diarrhoea in Lima. Lancet 1, 218. K A Y S N E RC.A., , A B E Y T A , C.O., J R , W E K E L L ,M . M . , D E P A O L A ,A., J R , STOTT, R . F . & L E I T C H , J . M . (lY87a) Virulent strains of Vibrio uulnr&us isolated from estuaries of the United States West Coast. Applied and Environm e n d Microbiology 53, 1349-1351. K A Y S N E RC.A., , ABEYTA,C., J R , WEKELL, M.M., D E P A O L A A, . , J R , S T O T T , R . F . & L E I T C H , J . M . (1987b) Incidence of Vibrio cholerae from estuaries of the United States West Coast. Applied and Environmental Microbiology 53, 1344-1348. K O T H A R YM, . H . & K R E G E RA.S. , (1987) Purification and characterization of an elastolytic protease of Vibrio vulnifcus. Journal of General Microbiology 133, 1783-1791. KOTIIARY M, . H . & RICIiARDSON, S . H . (1987) Fluid accumulation in infant mice caused by Vibrio hollisae and its extracellular enterotoxin. Infection and Immunity 55, 626-630. I,OCKWOOD, D.E., K R E G E RA.S. , & RICHARDSON S ., H . (1982) Detection of toxins produced by Vibrio fluvialis. Infection and Immunity 35, 702-708. M I Y O S H IS, . & S H I N O D AS, . (1988) Role of the protease in the permeability enhancement by Vibrio vulnrjicus. Microbiology and Immunology 32, 1025-1032. M O L I T O R I SE, . , J O S E P H , S . W . , K R I C H E V S K YM, . I . , SINDHUHARDJA, w. & C O L W E L L , R.R. (1985) Characterization and distribution of Vibrio alginolyticus and Vibrio para-
haemolyticus isolated in Indonesia. Applied and Environmental Microbiology 50, 1388-1394. M O R R I SJ, . G . & BLACK,R . E . (1985) Cholera and other vibrioses in the United States. N e w England Journal of Medicine 312,343-350. N I S H I B U C H IM , . , D O K E , S . , T O I Z U M I S., , U M E D AT , ., YOH, M . & M I W A T A NT. I , (1988) Isolation from a coastal fish of Vibrio hollisae capable of producing a hemolysin of Vibrio parahaemolyticus. Applied and Environmental Microbiology 54, 2144-2146. S A K A Z AR K. I& , S H I M A DT. A , (1982) Vibrio species as causative agents of food-borne infection. Developments in Food Microbiology-2, ed. Robinson, R.K. pp. 123-151. London : Elsevier Applied Science Publishers. S A R K A RB,. L . , K U M A RR, . , D E , S.P. & PAL, S.C. (1987) Hemolytic activity of and lethal toxin production by environmental strains of Vibrio parahaemolyticus. Applied and Environmental Microbiology 53, 2696-2698. SARKAR, B.L., N A I R , G.B., B A N E R J E EA.K. , & PAL, S. C . (1985) Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta. Applied and Environmental Microbiology 49, 132-136. SPIRAW , . M . & F E D O R K A - C R AP.J. Y , (1984) Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin. Infection and Immunity 45, 679-684. TAKAO, T., S H I M O N I S H IY, . , KOBAYASHI, M., NISHIM U R A , O., A R I T A ,M . , T A K E D AT , . , H O N D AT, . & M I W A T A N IT. , (1985) Amino acid sequence of heat-stable enterotoxin produced by Vibrio cholerae non-01. F E B S Letters 193,250-254. T A K E DY. A , (1983) Thermostable direct hemolysin of Vibrio purahaemolyticus. Pharmaceutical Therapy 19, 123-146. V E N K A T E S W A R AKN.,, K I I Y U K I A ,C . , T A K A KM I ,. , N A K A N OH, . , M A T S U D AH., , K A W A K A MHI ,. & HASHI MOTO,H . (1989a) Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan. Applied and Environmental Microbiology 55, 2613-2618. V E N K A T E S W A R AKN.,, N A K A N O , H . , O K A B E , T . , K., H. TAKAYAM A , MATSUDA,0. & HASHIMOTO, (1989b) Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan. Applied and Environmental Microbiology 55, 55%567. W E S T , P.A. & C O L W E L L ,R . R . (1984) In Vibrios in the Environment ed. Colwell, R.R. Ch. 20, pp. 285-363. New York: John Wiley & Sons. WISTROM,J . (1989) A case of n o n 4 : 1 Vibrio cholerae bacteremia from northern Europe. Journal of Infectious Diseases 160, 732-735. WONG, H . C . , C H E N , Y . L . & C H E N , C . L . F . (1988) Growth, germination and toxigenic activity of Bacillus cereus in milk products. Journal of Food Protection 51, 707-710. Y A M A M O T OK , . , A L - O M A N IM , . , H O N D AT , ., TAKEDA, Y. & M I W A T A N IT, . (1984) Non-01 Vibrio cholerae hemolysin : purification, partial characterization, and immunological relatedness to El Tor hemolysin. Infection and Immunity 45, 192-196.
