Adherence as a method of differentiating virulent and avirulent strains of Vibrio parahaemolyticus.

Usually only Kanagawa-positive strains of Vibrio parahaemolyticus are considered virulent; yet, a significant portion of V. parahaemolyticus food poisonings appear to be caused by Kanagawa-negative strains. Therefore, additional and more accurate measurements of a strain's food-poisoning potential are needed. Adherence of V. parahaemolyticus to human fetal intestinal (HFI) cells in vitro seems to offer this information. All strains of V. parahaemolyticus adhered to the HFI cells, but the degree of adherence was related to a number of factors. These included the age of the culture, the strain's Kanagawa reaction and source, the length of time the bacteria were exposed to the HFI cells, and the composition of the growth medium. Cells harvested during the late log phase of growth adhered more intensely than those harvested from the late stationary phase. Virulent strains, i.e., those involved in food poisoning, were observed to have a high adherence ability regardless of their Kanagawa reaction, whereas Kanagawa-negative strains isolated from seafood exhibited weak adherence intensities. Kanagawa-positive strains isolated from seafood adhered strongly to the HFI cells. The difference between the virulent and avirulent strains was quantitative in nature, and the greatest differences in adherence intensities were observed after short (10 to 15 min) exposure times. The presence of ferric iron in the growth medium was found to increase the adherence intensities of the virulent strains.     

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2

Abstract Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.     

Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

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)     

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)     

Serologic and molecular characterization of Vibrio parahaemolyticus strains isolated from seawater and fish products of the Gulf of Mexico

The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus. We isolated V. parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene. A total of 46 V. parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment. The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment. The most frequent serogroup was serogroup O3. This is the first report of the presence of KP-positive tdh-positive environmental V. parahaemolyticus strains in Mexico.     

Oral Immunization with Polyvalent Streptomycin-Dependent Escherichia coli Vaccine

Vibrio parahaemolyticus was the etiological agent in three food-related epidemics of gastroenteritis in Maryland, during August 1971. These outbreaks involved crab food products. Fifteen isolates of V. parahaemolyticus were made which included 11 from patients and 4 from foods. Serotype 04:K11 was the cause of the outbreaks. It was recovered from patients in each outbreak and gave a positive Kanagawa reaction, an indication of enteropathogenicity. Other patient isolates included types 03:K30, 03:K33, and an untypable isolate, all of which were Kanagawa negative. Food isolates included serotypes 03:K30, 02:K28, and two untypable isolates, all of which were Kanagawa negative. The outbreaks reported in this paper constitute the first confirmed foodborne epidemics due to V. parahaemolyticus in the United States. Methods for the isolation and identification of V. parahaemolyticus are presented, including a procedure for the simple conversion of conventional laboratory media into suitable culture media for this halophilic organism.     

The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.     

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.     

Incidence and level of Vibrio parahaemolyticus associated with freshwater plankton.

We isolated Vibrio parahaemolyticus from plankton samples collected from different freshwater areas in the vicinity of Calcutta, India. Levels of this organism in association with freshwater plankton were low (less than 90 organisms per g). All of the 16 strains of V. parahaemolyticus isolated in this study were Kanagawa negative, and all except 1 were untypable. The occurrence of V. parahaemolyticus in association with freshwater plankton suggests that adsorption onto plankton may aid the survival of this moderately halophilic organism in freshwater milieus.     

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.     

Occurrence of urease-positive Vibrio parahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable direct hemolysin (TDH) and the TDH-related-hemolysin genes.

A total of 132 strains of V. parahaemolyticus isolated from patients and from the suspected causal food items of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were examined for the ability to hydrolyze urea, with specific reference to the presence of the thermostable direct hemolysin gene (tdh) and the gene for thermostable direct hemolysin-related hemolysin (trh). Ten strains belonging to five different O-antigen serotypes were positive for urea hydrolysis (UH+), and four of these strains did not carry tdh. A total of 106 strains carried tdh, but less than 6% of them were UH+, whereas all trh-carrying strains were UH+. The evidence suggests that urea hydrolysis is not a reliable marker for identifying tdh-carrying V. parahaemolyticus strains in Japan (the Pacific Northeast) but may be a marker for trh-carrying strains.     

Enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum.

The enteropathogenicity of Vibrio parahaemolyticus was investigated by contrasting the effects of whole cells, cell fragments, cell-free preparations, and media constituents injected into rabbit ileal loops. Three of 20 cultures utilized were Kanagawa-negative strains from seawater and sea fish. The remaining 17 cultures included both Kanagawa-positive and -negative strains from Japanese victims of gastroenteritis. Broth culture filtrates concentrated 10-fold by dialysis against 30% Carbowax were unreactive, whereas lyophilized filtrates, regardless of Kanagawa type, as well as all sterile broth preparations containing greater than or equal to 5% NaCl gave positive reactions in the rabbit gut. In contrast, crude lysates derived from broth cultures of Kanagawa-positive strains caused loop dilatation; lysate supernatants were unreactive. Lysates of cells washed from brain heart infusion agar were more reactive than lysates from Trypticase soy agar-grown cells. When agar-grown cell lysates prepared by disruption in saline were dialyzed against distilled water, they were devoid of gut reactivity. Reactivity was restored in dialysands resuspended in saline and in dialysates concentrated 10-fold. The agar-grown cell lysates exhibited Kanagawa-type hemolysis. Our data support the conclusion that the rabbit loop reactivity observed with lyophilized, cell-free culture filtrates may result from excessively elevated NaCl concentrations, and that a toxic factor associated with large-cell particles may be dialyzable, depends on saline for expression, and resembles the Kanagawa hemolysin.     

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.     

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.     

The role of pili in colonization of the rabbit intestine by Vibrio parahaemolyticus Na2.

Vibrio parahaemolyticus Na2 and its pili were studied in relation to intestinal colonization. The isolated pili were adhesive to the intestinal epithelium. The adhesion of the organisms was blocked by masking the epithelial receptor with the purified pili, or by treating the organisms with anti-pilus antibody (Fab fraction). These results suggest that the pili of V. parahaemolyticus Na2 play an important role in the adhesion of the organisms to the rabbit intestine.     

Comparative phenotypic,molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.     

Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta.

The seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes was studied in 1982 and 1983. The occurrence of this organism in water and sediments at the three sites studied was very infrequent and was restricted to the summer months, although it was not always isolated during these months. The association of V. parahaemolyticus with plankton was chiefly confined to the summer months and progressively declined with the onset of monsoons, remaining below detectable levels during the postmonsoon and winter months. The incidence and counts of V. parahaemolyticus were consistently higher in association with plankton than with water and sediment samples. V. parahaemolyticus could be recovered throughout the period of investigation from freshly caught and market samples of freshwater fishes. The highest recovery rate of this halophile from fishes was invariably from fecal samples. Most of the strains isolated in this study were untypable, and those which could be typed were predominantly serotypes encountered in the environment. All the isolates were Kanagawa negative. From this study, it could be concluded that the survival of V. parahaemolyticus in freshwater ecosystems is transient and dependent on a biological host.     

Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta

The seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes was studied in 1982 and 1983. The occurrence of this organism in water and sediments at the three sites studied was very infrequent and was restricted to the summer months, although it was not always isolated during these months. The association of V. parahaemolyticus with plankton was chiefly confined to the summer months and progressively declined with the onset of monsoons, remaining below detectable levels during the postmonsoon and winter months. The incidence and counts of V. parahaemolyticus were consistently higher in association with plankton than with water and sediment samples. V. parahaemolyticus could be recovered throughout the period of investigation from freshly caught and market samples of freshwater fishes. The highest recovery rate of this halophile from fishes was invariably from fecal samples. Most of the strains isolated in this study were untypable, and those which could be typed were predominantly serotypes encountered in the environment. All the isolates were Kanagawa negative. From this study, it could be concluded that the survival of V. parahaemolyticus in freshwater ecosystems is transient and dependent on a biological host.     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.