Vibrio spp. represents a serious threat to human health. Three species in particular are linked to gastrointestinal issues and can lead to infections and septicemia: V. cholerae (VC), V. parahaemolyticus (VP), and V. vulnificus (VV). These pathogens are most commonly found in raw or undercooked seafood such as fish, squid, oyster, and shrimp. V. cholerae is the main factor that causes cholera, which is an important public health problem worldwide.
VP was first identified as a cause of foodborne illness in Japan in 1950 when 272 individuals became ill and 20 died after the consumption of semidried juvenile sardines. VP causes three major syndromes of clinical illness: gastroenteritis, wound infections, and septicemia. The most common syndrome is gastroenteritis. Symptoms of this syndrome include diarrhea with abdominal cramps, nausea, vomiting, headache, and low-grade fever. Strains from this pathogen that are isolated from diarrheal patients produce either the thermostable direct hemolysin (TDH), the TDH-related hemolysin (TRH), or both, while hardly any isolates from the environment have these properties.
In 2009, a Vibrio outbreak in Singapore was associated with consumption of Indian rojak (a traditional salad of fruits, vegetables, and seafood). The Singapore Ministry of Health concluded its investigations into the food poisoning cases and identified VP, traced to the cross-contamination of rojak and raw seafood ingredients harboring the bacteria as the source of the outbreak. Laboratory investigation confirmed 13 of the cases to be positive for VP, including the first fatal case.
The risk of these pathogens may only be getting worse. Scientists warn that, because climate change causes an increase in sea surface temperatures and a rise in sea levels, VP and VV infections will become more common. This is because warmer, rising waters create an even more welcoming environment for the deadly pathogen. Subsequentially, it is especially crucial that methods to efficiently detect Vibrio are developed.
Testing for Vibrio
FDA’s Bacteriological Analytical Manual (BAM) (Chapter 9) and the International Organization for Standardization (ISO) 21872-1:2017 are the two standard methods widely used for the detection of Vibrio. While these are the standard, there are still many issues that arise with these methods.
Neither of these methods provides a good selective enrichment medium for Vibrio species. Instead, different formulations of alkaline peptone water (APW) have been used as the preferred enrichment for certain Vibrio targets or food matrices. Still, no single enrichment procedure for classical isolation, by plating or selective media, has been validated by FDA or the ISO for all three strains.
Finding a single enrichment procedure that works for all three different strains is an important challenge faced by seasoned microbiologists today. The preferred enrichment temperature for VC is 42°C, but the preferred temperatures for VP and VV differ at 35-37°C. Furthermore, some food matrices containing high background flora or inhibitory compounds, such as bacterial growth or polymerase chain reaction (PCR) inhibitors, might require alternative enrichment schemes. In addition, the duration of enrichment and plating efficiencies of presumptive isolates could affect classical confirmation, making them difficult. Overgrowth of competing organisms might occur if enrichment duration exceeds 20 hours. This makes it difficult to isolate Vibrio on selective agar plates. Thiosulfate citrate bile salts sucrose (TCBS) agar is widely used as the main selective agar for isolation of the three target species by both the FDA-BAM and ISO methods.
Because climate change causes an increase in sea surface temperatures and a rise in sea levels, V. parahaemolyticus and V. vulnificus infections will become more common. This is because warmer, rising waters create an even more welcoming environment for the deadly pathogens.
Cultural confirmation is also a challenge. Not all isolates of the target species exhibit the same growth properties. Different isolates of the same species have shown as much as two logs differences in plate counts on TCBS plating efficiencies. This difference could be attributed to factors such as boiling time or depth of the poured media. Another challenge is that the Vibrio species might be subject to a biological phenomenon known as “viable but non-culturable.” When in this state, the pathogen is not able to be detected by traditional culture methods but is able to cause infection. A third challenge is that there are several atypical isolates of the target species, specifically for VP. Because of this issue, molecular-based methods, such as DNA sequencing, PCR-based methods, or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are good alternatives because they can confirm atypical Vibrio results, ultimately improving accuracy.
Standard methods are also labor intensive and rely on microbiological/biochemical identification. For seafood processors and inspections, current methods require at least three to five days for results and subjective interpretation for the screening of negative samples.
Food testing laboratories in the seafood industry are in need of a fast and accurate method to reliably detect the three main Vibrio species. An easy-to-use and rapid method that can reliably report results would allow seafood to safely get to market faster.
Real-Time PCR Detection
The advantages of real-time PCR are highlighted when used for the detection of Vibrio because of the challenges outlined above, such as the background flora naturally present in seafood matrices and the enrichment protocol challenges.
Since its invention, real-time PCR technology has been greatly improved so that it is more stable, accurate, and rapid for specific applications. As the technology evolved, new chemistries were developed based on fluorescence detection.
This evolution allowed for real-time PCR kits to achieve a high level of specificity and sensitivity when detecting Vibrio. Each test well can be used to detect all three important strains of the pathogen at the same time, decreasing the time it takes to get a result. A PCR kit may be able to detect the pathogen in 94 samples in less than two hours, rather than the typical three to five days. Further, the workflow is often optimized to be simple and user friendly.
While real-time PCR methods often offer quicker turnaround times than many of the standard methods, they can be prone to false positives due to free DNA from dead cells found in the sample. Emerging PCR-based methods should address this limitation.
Recently, Bio-Rad Laboratories received AOAC validation for its iQ-Check Vibrio assay. The assay uses a single-step enrichment followed by real-time PCR for the multiplex detection of VC, VP, and VV. This method provides rapid qualitative detection and differentiates among all three strains in seafood products. The solution also has an optional Free DNA Removal Solution that can address ambiguity caused by dead cell DNA by removing free DNA in the sample with a simple non-toxic protocol, while the intact DNA in living cells remains unaffected.
This method was evaluated and approved by the AOAC Performance Tested Methods (PTM 032002) program. Results of the AOAC-PTM validation study demonstrated no differences between the iQ-Check Vibrio method and the U.S. FDA BAM Vibrio reference method. The assay and the Free DNA Removal Solution were validated for use with 125-gram test portions of cooked and raw shrimp, raw mussels, raw oysters, and raw tuna. The assay was approved for use with Bio-Rad Vibrio Enrichment Broth (after a seven-to-nine hour enrichment period) and alkaline phosphate water (after a six- to 18-hour enrichment period), giving the user flexibility to optimize the method to their lab workflow, while significantly cutting down the traditional three to five days it takes to get results with standard methods.
Rapid methods like this one can greatly aid in outbreak investigation and management of public health concerns. The ability to obtain results in a shorter amount of time, particularly when it comes to pathogens such as Vibrio species, can be critical in reducing the impact from a food safety event.
Pastori is an international product manager at Bio-Rad Laboratories. Reach him at firstname.lastname@example.org. Wang is a field marketing specialist at Bio-Rad Laboratories. Reach him at email@example.com.