Vibrio sp. Detection and Identification in Foods

Key Points

  • Three species, V. cholerae, V. parahaemolyticus and V. vulnificus, are important human pathogens and are potential contaminants in seafood.
  • Traditional detection and confirmation methods are well established and typically take 4-7 days for completion.
  • Comparatively few rapid methods are available, but a recently launched real-time PCR method can detect all three important species inside 24 hours.

Oysters Members of the genus Vibrio are all Gram-negative straight or curved rods, which do not form spores and are motile, usually by a single polar flagellum. Most are oxidase and catalase positive and ferment glucose without gas production. They occur naturally in fresh water and marine environments and some may be pathogenic to humans. Many of the pathogenic species, with the notable exception of Vibrio cholerae, are adapted to salt or brackish water habitats and are halophilic to some degree, being unable to grow in the absence of sodium chloride.

Three species are considered to be important human pathogens – V. cholerae, V. parahaemolyticus and V. vulnificus. All three have the potential to be foodborne, and are most often associated with the consumption of raw, or undercooked, shellfish. A number of other species have infrequently been isolated from the stools of people suffering from gastroenteritis and are considered to be occasional human pathogens. These include V. alginolyticus, V. fluvialis, V. furnissii, V. hollisae, V. metschnikovii and V. mimicus. These species are not generally regarded as significant foodborne pathogens.

Vibrio cholerae
V. cholerae is the cause of outbreaks and epidemics of cholera, a serious and potentially fatal gastrointestinal infection, which is still a major health problem in parts of the developing world. Humans are the principal reservoir for V. cholerae and cholera is usually associated with poor hygiene and polluted water supplies, but it may also be foodborne.

Contamination of fruits, vegetables and other foods usually occurs via an infected food handler, or by the use of polluted water in food preparation. Not all strains of V. cholerae cause cholera and the main virulence factor involved in the disease is the cholera enterotoxin (CT). Strains causing classic epidemic cholera generally belong to one of two serogroups, O1 or O139, although other strains have been reported to cause occasional isolated cases of cholera-like disease.

Non-O1/O139 strains can cause a less severe form of diarrhoeal disease and are more common clinical isolates in developed countries. These isolates are typically related to consumption of contaminated shellfish, especially raw oysters. Non-O1/O139 V. cholerae strains are not uncommon in certain estuarine waters and may contaminate shellfish harvested from polluted sites.

V. cholerae will grow rapidly in temperature-abused processed foods and will also survive for extended periods in chilled and even frozen foods, but it does not survive desiccation for more than 48 hours. The cells are not heat-resistant and are readily destroyed by cooking and pasteurisation.

Vibrio parahaemolyticus
V. parahaemolyticus is the species most likely to be associated with foodborne disease in humans. It can cause mild to moderate gastrointestinal infections, which are usually self limiting and rarely fatal. It is not normally involved in epidemics or large outbreaks, but is an important source of foodborne disease, especially in Japan and other Asian countries.

Infection is almost always associated with consumption of seafood and V. parahaemolyticus is very common in marine coastal waters in tropical regions and in temperate regions during the warmer months of the year. There is also evidence that it may be becoming more common in colder waters during the summer, possibly as a consequence of rising sea temperatures. As with V. cholerae, not all strains of V. paraheamolyticus are capable of causing human disease. Pathogenicity is associated with a thermostable haemolysin, called the Kanagawa phenomenon. Almost all isolates from cases of food poisoning are Kanagawa positive strains.

V. paraheamolyticus is part of the normal microflora of coastal and estuarine waters in almost all temperate regions and may be present in comparatively high numbers when the water temperature is at its highest during the summer. At these times, shellfish may easily become contaminated, but fortunately most environmental isolates are Kanagawa negative and do not cause illness. The possibility of infection means that shellfish from high-risk waters should not be eaten raw.

Most cases of V. paraheamolyticus infection in Europe can be traced to imported seafood, but there is evidence to suggest that it is becoming more common in domestically harvested shellfish. V. paraheamolyticus differs from V.cholerae in that it is an obligate halophile and will not grow unless a salt concentration of at least 0.5% is present. In fact it will grow at salt levels as high as 10%. Like V. cholerae it will grow rapidly in temperature abused foods and survives chilled and frozen storage, but not drying or mild heat processes.

Vibrio vulnificus
V. vulnificus is an occasional cause of serious infections, which may sometimes be foodborne. However, wound infection following contact with the marine environment is more likely. Foodborne infections can take the form of gastroenteritis in healthy adults, but in vulnerable individuals the pathogen can cause primary septicaemia, which is very serious and has a mortality rate of more than 50%. V. vulnificus is now the leading cause of death in the USA, related to consumption of seafood and is almost always associated with raw oysters from the Gulf of Mexico, which are thought to have a very high contamination rate . Only about 90 cases of V. vulnificus infection are reported each year in the USA and major outbreaks have not been recorded, but the high death rate means that it is regarded as a serious pathogen.

Elsewhere, sporadic cases have been identified in Europe, Korea and Taiwan. Like V. parahaemolyticus, V. vulnificus is a common part of the natural microflora of coastal waters in tropical and temperate regions worldwide, especially during the summer months. This being so, given the low number of cases of human infection, it is likely that only certain strains are pathogenic to man. V. vulnificus is a halophile requiring at least 0.5% salt to grow and will tolerate levels of up to 5%. It will multiply in live oysters, but not at temperatures of less than 13oC. Like other species it resists low temperatures for some time, but is destroyed by cooking and is not resistant to desiccation.

Methods

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).

Traditional methods
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.

Rapid methods
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

Traditional methods
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.

Rapid methods
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.

This guide has been prepared by Food Safety Info, scientific and technical information providers for the food industry. For more information, visit our web site at www.foodsafetywatch.com