Storage and preparation of samples
The majority of food samples examined for the presence of Vibrio species will be shellfish and other seafood. It is very unusual for other food categories to be routinely tested for these pathogens
Vibrio spp can grow very rapidly in seafood at ambient temperature and samples must be chilled to below 10oC immediately and then analysed as quickly as possible. However, the cells are easily damaged by rapid cooling and samples should not be cooled by direct contact with ice.
Sample preparation procedures for shellfish typically require pooling 10-12 individual animals. The pooled sample is then homogenised using a sterile high-speed blender. If sample dilutions are required they should be prepared with a diluent containing salt, such as phosphate buffered saline (PBS).
There are current ISO horizontal methods for the detection of potentially enteropathogenic Vibrio species in food. ISO/TS 21872-1:2007 is for the detection of V. cholerae and V. parahaemolyticus, while ISO/TS 21872-2:2007 is for the detection of other species, including V. vulnificus, V. fluvialis and V. mimicus, but not V. hollisae. Similar standard methods have been published elsewhere by other bodies, notably in the USFDA Bacteriological Analytical Manual (BAM).
The first stage in traditional detection methods exploits the ability of Vibrio spp to grow rapidly at relatively high pH values. Media containing sodium chloride and with a pH of about 8.6, such as alkaline saline peptone water (ASPW), are used for enrichment. Typically, a 6-hour preliminary enrichment (at 41.5oC for fresh products, or 37oC for frozen or salted products) is followed by a second enrichment in ASPW at 41.5oC (for V. cholerae and V. parahaemolyticus) or 37oC (for other species) for 18 hours.
The second enrichment culture is inoculated on to thiosulphate citrate bile salts sucrose (TCBS) agar and one other optional selective medium and incubated at 37oC for 24 hours. On TCBS agar, V. cholerae colonies are smooth and yellow, V. parahaemolyticus colonies appear blue-green and V. vulnificus colonies are green or yellow. Selective chromogenic agar media specifically designed for the differentiation of pathogenic Vibrio spp are also available. Examples include chromID™ Vibrio agar from bioMérieux and CHROMagar™ Vibrio.
A quantitative method for V. parahaemolyticus can be used for samples where significant numbers are expected. This applies a MPN technique based on enrichment of tenfold dilutions in ASPW, followed by plating onto selective agar.
A hydrophobic grid membrane filtration enumeration procedure (HGMF) has also been described.
There are very few commercially available rapid test kits for Vibrio spp. in foods – possibly because routine testing for these pathogens is largely confined to the seafood sector. However, the advent of molecular biology techniques has given rise to a number of PCR-based detection methods for Vibrio spp. targeting genes for cytotoxin and haemolysin production.
An example of a commercially available PCR-based method for pathogenic Vibrio detection is the BAX® System Real-Time PCR Assay from Dupont Qualicon. Part of the well established family of BAX® System assays, the Vibrio assay is able to detect the three most important species, V. cholerae, V. parahaemolyticus and V. vulnificus in the same sample within 24 hours, including an 18-20 hour enrichment. It is designed for testing seafood samples, including shrimp, oysters, crabs and tuna and is capable of detecting 104 CFU/ml.
Confirmation and identification
There are well established confirmation and identification procedures for pathogenic Vibrio spp., especially for V. cholerae. Preliminary identification based on colony appearance on TCBS agar is traditionally confirmed using classical biochemical tests.
Key tests are oxidase reaction and the presence or absence of lysine and ornithine decarboxylases and arginine dehydrolase. Media should be prepared with 2-3% sodium chloride to allow the growth of halophilic species.
V. cholerae isolates are further confirmed and characterised by serological agglutination testing, β haemolysis on blood agar and tests for cholera toxin production.
V. parahaemolyticus isolates can also be characterised by serological testing and by the Kanagawa test.
Although rapid confirmation and identification methods have been developed for Vibrios, few commercial products are available.
Biochemical confirmation can be accomplished using commercial identification systems such as the API 20E test strip from bioMérieux and Remel’s RapID™ NF PLUS System. However it is important to ensure that cultures are suspended in a saline medium to ensure the growth of halophilic species.
Immunological identification and confirmation tests based on enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) have been developed for pathogenic vibrios, but commercial tests kits are currently not widely available. A latex agglutination test, the VET-RPLA Kit (Oxoid), is available for cholera toxin detection in culture filtrates.
A number of molecular methods for confirmation have been developed, notably PCR assays for the identification of the three most important species. Labelled DNA probes can be used to confirm the pathogenicity of V. parahaemolyticus and V. vulnificus by detecting the genes for specific virulence factors, such as haemolysins, and a PCR method has been developed for cholera toxin, which is rarely present in V. cholerae isolates from food. Protocols for several of these methods are provided in the USFDA BAM.