A pulsed-field gel electrophoresis typing scheme for Vibrio parahaemolyticus isolates from fifteen countries.

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other maritime Asian countries where seafood is frequently consumed. A total of 535 strains of V. parahaemolyticus were recovered mostly (97%) from clinical samples obtained in Taiwan or in 14 other countries. These strains were typed by pulsed-field gel electrophoresis following SfiI digestion and a typing scheme was generated. The 115 different patterns identified were grouped into 13 types with dissimilarity values less than 15, plus 16 miscellaneous patterns not grouped into any of the types. Types I, A, D and J contained the most patterns, with the numbers of patterns being 17, 13, 12, and 11, respectively. However, types I, B, D, A, H and C contained the most strains, with the numbers of strains being 204, 73, 71, 54, 29 and 25, respectively. Type I consisted exclusively of the pandemic O3:K6 strains and genetically closely related strains. This PFGE typing scheme for V. parahaemolyticus could be used for the characterization of pathogenic isolates.

Rapid phenotypic characterization of Vibrio isolates by pyrolysis metastable atom bombardment mass spectrometry.

Pyrolysis mass spectrometry was investigated for rapid characterization of food-borne bacterial pathogens. Nine isolates of Vibrio parahaemolyticus and one isolate each of Vibrio fluvialis, Vibrio hollisae, and Vibrio vulnificus were analyzed. Pyrolysis mass spectra, generated via an alternative ionization method, metastable atom bombardment, were subject to principal component-discriminant analysis. The spectral patterns were used to distinguish Vibrio isolates differing in species, serotype and expression of the thermostable direct hemolysin gene. The patterns of similarity and dissimilarity amongst spectra in the Vibrio test set generally reflected those associated with species, serotype or hemolysin-producing genes, though the combined influence of these and other variables in the multi-dimensional data did not produce a simple clustering with respect to any one of these characteristics. These results suggested that with enough examples to model the most common combinations, the method should be able to characterize Vibrio isolates according to their phenotypic characteristics. Pyrolysis-mass spectrometry with metastable atom bombardment and pattern recognition appeared suitable for rapid infraspecific comparison of Vibrio isolates. This integrated analytical, pattern-recognition system should be examined further for potential utility in clinical and public health diagnostic contexts.

Molecular, serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental, food, and clinical sources in North America and Asia.

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.

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.

Comparison of Template Preparation Methods from Foods for Amplification of Escherichia coli O157 Shiga-Like Toxins Type I and II DNA by Multiplex Polymerase Chain Reaction.

Escherichia coli O157:H7 has been responsible for several recent food-borne outbreaks in the United States. To protect the public health, it is essential that rapid and sensitive methods be developed for detection of this pathogen in foods. Methods were compared for preparation of template DNA for the polymerase chain reaction (PCR) from enrichments of food homogenates seeded with E. coli O157:H7. Samples were enriched for 6 h at 37°C in modified tryptic soy broth supplemented with vancomycin, cefsulodin, and cefixime. Aliquots of the enrichments (10 ml or 1 ml) were analyzed by either washing twice with physiological saline or incubating with antibodies to O157 coupled to immunomagnetic beads (Dynal®) followed by resuspending and boiling the samples. A portion of the preparation was used in a multiplex PCR to amplify a 274-bp fragment from the sltI gene and a 364-bp fragment from the sltII gene. PCR amplification of 1-ml portions of enrichment broth was successful at inoculation levels of about 10 cells per g of food. Increasing the test sample volume to 10 ml and/or using an immunomagnetic separation step improved the PCR detection sensitivity to about 1 cell per g; the entire analysis can be completed within 12 h.

Enumeration and Differentiation of Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA Colony Hybridization Using the Hydrophobic Grid Membrane Filtration Technique for Isolation.

We have developed a means of differentiating and enumerating Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA colony hybridization directly on HGMF filters. V. parahaemolyticus can be detected by a tdh-3-radiolabeled gene probe and V. vuinificus detected by a specific cytotoxin-hemolysin-radiolabeled probe with enumeration directly from autoradiograms. This procedure is more rapid than current techniques allowing enumeration and identification of these two species in samples as diverse as seawater, oyster ( Crassostrea gigas ), and shrimp (Pandalidae family) within 4 d. Our method is based on a rapid technique (18 h) for isolation and enumeration of V. parahaemolyticus from food using a membrane filtration technique with hydrophobic grid filters (HGMF). With the HGMF method, however, it is not possible to differentiate V. parahaemolyticus from V. vuinificus since on the HGMF-sucrose-based agar used, the two species are indistinguishable as both species are unable to ferment sucrose. Using a combination of the HGMF and selective gene probes, these two species can be differentiated.

Campylobacter jejuni in a Washington State Shellfish Growing Bed Associated With Illness.

Consumption of raw Pacific oysters ( Crassotea gigas ) harvested from a Washington State recreational shellfish bed were associated with illness. Illness occurred within 2 d of ingestion of a half-dozen shellstock oysters. Each oyster consist of approximately 20 g of meat. The duration of illness lasted 2 d. Routinely, Campylobacter species have been found in several shellfish beds in the Puget Sound Bay. Its presence in the marine environment appears to be incidental and primarily, comes from wild birds, farm runoff, and sewage bypasses. This paper describes the first reported case of Campylobacter gastroenteritis associated with raw oyster consumption in the State of Washington.

Incidence of Motile Aeromonads From United States West Coast Shellfish Growing Estuaries.

The distribution of motile Aeromonas species in marine and tributary waters, sediment, and shellfish from 12 major estuarine areas in Washington, Oregon, and California with commercial or sport shellfish harvest was determined during the summer months. Aeromonas spp. were found in half of the total of 400 samples analyzed. Two enrichment broths, tryptic soy ampicillin broth (TSBA) and alkaline peptone water (APW), were compared for recovery of Aeromonas from Washington and Oregon samples. More Aeromonas were isolated using TSBA. For Washington and Oregon samples, recoveries using TSBA were 82 and 77% respectively compared to 31 and 50% using APW. For California samples, only APW was used with 28% samples positive. Of 767 isolates tested, 93.5% were positive for hemolysis, a trait reported to correlate with enterotoxin production and pathogenicity. Of the hemolysis positive strains, 59.5% were toxic to Y-1 adrenal cells.

Enumeration of Vibrio Species, Including V. cholerae , from Samples of an Oyster Growing Area, Grays Harbor, Washington.

Water, shellfish, and sediment samples from Grays Harbor, a major commercial oyster producing estuary in the State of Washington, were examined for levels of Vibrio species. Non-01 V. cholerae was found at low levels in 37.8% of the samples. While V. parahaemolyticus was found in all samples, levels were low. V. mimicus and V. fluvialis were found infrequently and at low levels. Potentially pathogenic strains of non-01 V. cholerae and Kanagawa positive V. parahaemolyticus were isolated from oysters suggesting a potential for human illness.

Microbiological Quality of Oysters ( Crassostrea gigas ) and Water of Live Holding Tanks in Seattle, WA Markets.

Oyster ( Crassostrea gigas ) and water samples from Live Holding Tanks at five different Seattle area retail markets were analyzed for microbiological quality indicators and for potential pathogens monthly from March to September, 1987. Aeromonas hydrophilia was the most frequently isolated potential pathogen in this study with a higher incidence in oysters (78%) compared to water (53%). Vibrio cholerae non 01 and V. fluvialis were isolated from oyster samples from two different markets but not from water. V. alginolyticus was isolated from 53% of the water samples but was not found in any of the oysters. One oyster sample had a non-pathogenic Yersinia entercolitica . Yersinia spp. were isolated from oyster samples from one tank at two sampling periods. Salmonella typhimurium was isolated from one oyster sample. Samples were examined for Listeria spp. during the August sampling period and none were detected. The aerobic plate count was similar for both oyster and water samples and averaged 2000 CFU/gm. Total coliform levels were significantly higher (P<.05) for oysters (525MPN/100gm) compared to water (11MPN/100ml). The degree of water turbidity, crowding and species diversity varied between markets and sampling periods.

Aeromonas hydrophila in Shellfish Growing Waters: Incidence and Media Evaluation.

Levels of Aeromonas hydrophila determined for the shellfish growing area of Grays Harbor, Washington, ranged from 3 to 4600/100 g in oysters and from 3 to 2400/100 ml in water. Of isolates tested, 80% produced a hemolysin, a trait reported to correlate with enterotoxin production and pathogenicity. Two enrichment broths, Tryptic Soy Broth with ampicillin (TSBA) and Modified Rimler Shotts Broth (MRSB) were compared in combination with three solid agar media: Rimler Shotts (RS), Peptone Beef Extract Glycogen (PBG), and MacConkey's (MCA) agars. TSBA was far superior to MRSB in isolating this species from the environmental samples tested.

Recovery of Aeromonas hydrophila from Oysters Implicated in an Outbreak of Foodborne Illness.

Potentially pathogenic Aeromonas hydrophila organism were isolated from oysters frozen at -72°C for 1-1/2 years. The oysters which had been associated with 472 cases of gastroenteritis in Louisiana in November 1982, were examined and found negative for Salmonella , pathogenic Vibrio parahaemolyticus , and diarrhetic shellfish poison. In 1983, oysters from the same shellfish growing area in Louisiana were implicated in seven cases of gastroenteritis caused by A. hydrophila . The oysters collected in 1982 were reexamined and found to contain A. hydrophila (MPN 9.3/100 g). Twenty-three of 28 strains identified by the MICRO-IS and API-20E systems were positive for at least one of the tests for virulence which included the suckling mouse test, the adrenal Y-1 mouse cell test, and hemolysin assays. Of five strains tested, all showed activity in the rabbit ileal loop. Although these results do not prove that A. hydrophila caused the outbreak in 1982, they suggest that in cases of foodborne illness involving oysters, A. hydrophila should be included in the screening tests.