Charles A. Kaysner

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

ABSTRACT Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g−1. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g−1). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g−1) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g−1 total V. parahaemolyticus or >10 g−1tdh- and/ortrh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.

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

ABSTRACT 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.

Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus.

The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour‐intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed. An alkaline phosphatase (AP)‐labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non‐vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API‐20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)‐labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP‐labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP‐ and DIG‐labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.

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.

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.

Vibrio parahaemolyticus gastroenteritis: An outbreak associated with raw oysters in the pacific northwest

During a 3-month period in the late summer and fall of 1981, six cases of gastroenteritis and one wound infection due to Vibrio parahaemolyticus were reported to public health agencies in Washington and Oregon. An investigation revealed that all of the gastroenteric illnesses were associated with eating raw oysters; that oysters eaten by five of the six patients were harvested at four divergent sites at Willapa Bay, Washington, a large commercial growing area; and that the V. parahaemolyticus isolates from those five patients were all Kanagawa positive, belonged to serotype 04:K12, and exhibited an atypical biochemical reaction, urea hydrolysis. No further cases linked to Willapa Bay oysters have been reported, and the infecting strain could not be found in sediment samples from the bay in February 1982. Thus, even though the origin of this self-limiting outbreak is obscure, the investigation demonstrated that the geographic distribution of V. parahaemolyticus infection in the United States includes the Pacific seacoast . Furthermore, oysters must be considered, along with crabs, shrimp, and lobster, as a vehicle of transmission of this infection in the United States.

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus.

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks

Aims: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north‐western United States between 1988 and 1997.

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.