Vibrio parahaemolyticus: A concern of seafood safety

Vibrio is a genus of ubiquitous bacteria found in a wide variety of aquatic and marine habitats; of the >100 described Vibrio spp., ~12 cause infections in humans. Vibrio cholerae can cause cholera, a severe diarrhoeal disease that can be quickly fatal if untreated and is typically transmitted via contaminated water and person-to-person contact. Non-cholera Vibrio spp. (for example, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus) cause vibriosis - infections normally acquired through exposure to sea water or through consumption of raw or undercooked contaminated seafood. Non-cholera bacteria can lead to several clinical manifestations, most commonly mild, self-limiting gastroenteritis, with the exception of V. vulnificus, an opportunistic pathogen with a high mortality that causes wound infections that can rapidly lead to septicaemia. Treatment for Vibrio spp. infection largely depends on the causative pathogen: for example, rehydration therapy for V. cholerae infection and debridement of infected tissues for V. vulnificus-associated wound infections, with antibiotic therapy for severe cholera and systemic infections. Although cholera is preventable and effective oral cholera vaccines are available, outbreaks can be triggered by natural or man-made events that contaminate drinking water or compromise access to safe water and sanitation. The incidence of vibriosis is rising, perhaps owing in part to the spread of Vibrio spp. favoured by climate change and rising sea water temperature.

Vibrio spp. infections.

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g(-1). Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh(+) V. parahaemolyticus than previously reported.

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environments. V. parahaemolyticus is the leading causal agent of human acute gastroenteritis following the consumption of raw, undercooked, or mishandled marine products. In rare cases, V. parahaemolyticus causes wound infection, ear infection or septicaemia in individuals with pre-existing medical conditions. V. parahaemolyticus has two hemolysins virulence factors that are thermostable direct hemolysin (tdh)-a pore-forming protein that contributes to the invasiveness of the bacterium in humans, and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. In addition, the bacterium is also encodes for adhesions and type III secretion systems (T3SS1 and T3SS2) to ensure its survival in the environment. This review aims at discussing the V. parahaemolyticus growth and characteristics, pathogenesis, prevalence and advances in molecular identification techniques.

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Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected 104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh+ and trh+ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.

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

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was </=10(1)-10(2) cells following a "double" multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneCombtrade mark (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneCombtrade mark assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneCombtrade mark DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

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Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Presence of E. coli is a useful indicator of fecal contamination in estuarine shellfish. Polymerase chain reaction using primers for uidA were used to detect DNA released from dead E. coli cells and from heatkilled cells in seeded oyster tissue homogenate. Two different methodologies were adopted for DNA extraction from the seeded oyster homogenate: Chelex™ 100 method and a modified cell-lysis method. Results showed PCR amplification of DNA purified by the modified cell-lysis method was able to eliminate detection of cell-free DNA or heat-killed non-viable cells at various concentrations tested as opposed to the Chelex™ 100 method. DNA-PCR detection was positive for uidA using the Chelex™ 100 DNA extraction method on oyster samples with high concentrations (6-100µg) of cell-free DNA or heat-killed cells (1 X 10 6 cells/ml). This suggests that a difference in DNA extraction techniques can eliminate false-positive results by PCR, thus making it a reliable and rapid diagnostic tool to differentiate viable and non-viable cells in shellfish samples. It would greatly enhance the microbiological safety of shellfish and the quality of risk-assessment involved in the detection of viable microbial pathogens in seafood samples.

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Cynthia W. Brasher

Papers

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

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Evaluation of PCR Amplification-based Detection of Heat-killed Escherichia coli and Cell-free DNA in Shellfish