Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.     

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.     

Serovars of Vibrio parahaemolyticus

Seashore water samples collected along the coastline in Bulgaria and Rumania contained in large numbers OK serovars of V. parahaemolyticus; some of these had been isolated repeatedly over an extended time period: 01 K32, 03 K30, 03 K48, 04 K37, 04 K53, 05 K17, 05 K30. The serovar 05 K17 was virtually present in all water samples and was also isolated from a case of purulent ear infection in a child from Burgas. In contrast, strains recovered from Asian and African coastal water had different K antigens and were never identified in Europe. Two strains of V. parahaemolyticus (serovars 05 K15 and 07 K10) had positive swarming growth resembling that of V. alginolyticus. The first of these was Kanagawa-positive and was isolated from a case of severe diarrhea in Brazzaville. Vibrio parahaemolyticus isolates came from marine or brackish water specimens collected on sand banks, 3 strains were recovered from marine or brackish water in Africa. Vibrio harveyi, a sucrose-negative species important from differential diagnostic aspects, has been isolated from seashore water samples collected on coarse-sand or pebbly beaches.     

副溶血弧菌的致病机制

副溶血性弧菌是引发微生物食源性疾病的首要病原菌,其在临床上主要引起3种疾病,即胃肠炎、伤口感染和败血症。经过多年的研究,人们对副溶血性弧菌的致病机制有了一定的认识。我们着重介绍副溶血性弧菌的主要毒力因子、毒力基因表达调控及常用的毒力表型研究方法。     

Sublethal cold shock in Vibrio parahaemolyticus.

Exposing Vibrio parahaemolyticus populations to temperatures of 2 degrees C results in membrane damage, rendering cells incompetent to grow on media containing 5% NaCl.     

Two generalized transducing phages in Vibrio parahaemolyticus and Vibrio alginolyticus.

Two bacteriophages named phi VP253 and phi VP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2 x 10(-7) to 7.5 x 10(-5) per infected cell at the m.o.i. of approximately 1.0. The phage phi VP143, but not phi VP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.     

Occurrence of Vibrio parahaemolyticus in Dutch mussels.

A study was carried out on the occurrence of Vibrio parahaemolyticus in Dutch mussels originating from the East Schelde Estuary. In a totol of 79 10-g tissue samples, 3 (3.8%) were found to contain V. parahaemolyticus. In a second survey, 6 out of 23 bags of mussels (26%) contained one or more strains of V. parahaemolyticus in 5-g tissue samples. The many limitations of current methodology used in such surveys are stressed. Positive samples can be missed because viable cells may die during refrigerated transport. Surviving cells also may not be detected because they have been sublethally stressed. In addition, the unreliability of the identification criterion of no growth in 10% NaCl was demonstrated.     

Occurrence of Vibrio parahaemolyticus in Dutch mussels.

A study was carried out on the occurrence of Vibrio parahaemolyticus in Dutch mussels originating from the East Schelde Estuary. In a totol of 79 10-g tissue samples, 3 (3.8%) were found to contain V. parahaemolyticus. In a second survey, 6 out of 23 bags of mussels (26%) contained one or more strains of V. parahaemolyticus in 5-g tissue samples. The many limitations of current methodology used in such surveys are stressed. Positive samples can be missed because viable cells may die during refrigerated transport. Surviving cells also may not be detected because they have been sublethally stressed. In addition, the unreliability of the identification criterion of no growth in 10% NaCl was demonstrated.     

Membrane filter procedure for enumeration of Vibrio parahaemolyticus.

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.     

Effective ileal loop dose of Kanagawa-positive Vibrio parahaemolyticus.

Groups of 16 rabbits per strain were injected with broth culture dilutions of three Kanagawa-positive Vibrio parahaemolyticus strains. The effective dose required to produce ileal loop dilatation in 50% of rabbits for pure cultures of strains 10136-76 and 553-72 from patients stools and NY 477 from incriminated food was 1.1 x 10(6), 2.6 x 10(5), and 7.7 x 10(6) organisms, respectively. When each of these cultures was admixed with greater than or equal to 10(9) Vibrio alginolyticus cells, the 50% effective dose was 1.2 x 10(6), 1.1 x 10(7), and 1.3 x 10(8) cells, respectively. Although concomitant injection of large numbers of competitive nonvirulent cells did not affect the 50% effective dose for strain 10136-76, that for the remaining two was increased 20- to 40-fold. The initiation of ileal loop response as estimated from sigmoidal plots of proportion of positive loops versus cell concentrations was given by as few as 10(2) cells of strain 553-72. Strains NY 477 and 10136-76 required approximately 10(5) cells. Half of the maximal response from these plots corresponded well with the 50% effective dose for the strains. These results suggest that pathogenicity of Kanagawa-positive V. parahaemolyticus strains may involve the participation of some virulence mechanisms in addition to the Kanagawa hemolysin.     

Formation and function of Vibrio parahaemolyticus lateral flagella.

Formation of the lateral flagella (L-flagella) of Vibrio parahaemolyticus was studied immunologically, using specific antiserum against L-flagella. On solid medium, L-flagella were formed at both high (37 degrees C) and low (25 degrees C) temperatures, although at high temperatures they became dissociated from the cells and decomposed in the medium. L-flagella were not formed in liquid or soft-agar medium. Formation of L-flagella was decreased by lowering the pH of the medium and repressed by transferring the cells from solid medium to liquid medium. Mutants possessing L-flagella but not a polar monotrichous flagellum (M-flagellum) swarmed on solid medium, whereas mutants were grown on solid medium and then transfered to liquid medium, the cells oscillated until they lost L-flagella. It is postulated that L-flagella are locomotive organelles on solid medium and in some cases also in liquid medium, whereas M-flagella are locomotive organelles only in liquid medium.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of pili on Vibrio parahaemolyticus

Electron microscopic examination showed that all strains of Vibrio parahaemolyticus examined had pili on their surface when the organism was grown on marine agar at 28 degrees C for 6-12 h. The pili were morphologically stable on heat treatment at 60 degrees C for 10 min, but both the lateral and polar flagella possessed by this organism were labile. No immunological cross-reactivity between pili of enterotoxigenic Escherichia coli and Vibrio cholerae non-01 and those of V. parahaemolyticus was observed.