Vibrio parahaemolyticus, a Gram-negative, salt-loving bacterium common in marine environments, is the leading cause of acute hepatopancreatic necrosis, also known as “early death syndrome,” in aqua culture, and is responsible for a significant number of foodborne illnesses in humans.

Over the past two decades, the bacteria has led to a significant rise of infections in humans, more so than other foodborne pathogens. These infections primarily result from consuming raw fish and seafood, and particularly, shellfish.

Climate change, causing rising ocean temperatures and ocean acidification, has resulted in increased abundances of Vibrio parahaemolyticus in oceans worldwide. In fact, the most recent FoodNet annual report indicates that the overall incidence in 2021 rose by 45.5% when compared with the annual incidence from 2016 to 2018.

Traditional detection methods for bacteria are labor intensive and time consuming, falling short of the need for accurate, rapid, and convenient detection required by food safety supervision and food enterprises; however, researchers in Shanghai, China, have developed a point-of-care detection method that allows for the quick and sensitive identification of the bacteria in seafood.

This new method uses advanced techniques called recombinant polymerase amplification (RPA) and the CRISPR/Cas12a system, along with a test strip. The method provides a low-cost, simple, and visually clear way to quickly detect Vibrio parahaemolyticus in seafood.

The researchers note that RPA-CRISPR/Cas12a-ICS can detect Vibrio parahaemolyticus in salmon sashimi at extremely low levels, as little as 154 CFU/g, without needing to enrich the sample first. “Our innovative detection platform represents a significant advancement in the rapid and sensitive detection of Vibrio parahaemolyticus, proving especially valuable for ensuring seafood safety and preventing public health crises,” corresponding author Haijuan Zeng, leader of the Biotechnology Research Institute at the Shanghai Academy of Agricultural Sciences, said in a prepared statement.

Zeng, who designed and performed the experiments and analyzed the data, explained that by using this platform, Vibrio parahaemolyticus can be detected in approximately 30 minutes, with a limit of detection of 250 copies/μL for plasmid samples and 140 CFU/mL for bacteria. The platform has been validated with artificially contaminated food samples and various clinical isolates.

Furthermore, in the report, the researchers noted that adjusting the crRNA sequences could enable the identification of various other targets, allowing the optimized ssDNA concentration to be used for detecting different targets. Therefore, the RPA-CRISPR/Cas12a-ICS platform could be employed to detect foodborne pathogens linked to humans, adulterated foods, and even viruses.

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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 frederic_pastori@bio-rad.com. Wang is a field marketing specialist at Bio-Rad Laboratories. Reach him at weijia_wang@bio-rad.com.

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On Oct. 5, 2018, in a seemingly ordinary transaction, Eddie and Patti Clinton, retired Wake Co., North Carolina educators/administrators, purchased two 10-pound bags of raw whole fresh shrimp. The original source of the crustaceans was a fisherman who reportedly harvested the shrimp in or near the New River in the southeastern area of the Tarheel State.

Eddie placed the shrimp on ice in a cooler overnight. The next day he scooped the shrimp with his bare hands into smaller bags, then put them in his freezer. And, yes, he washed his hands after handling the raw shrimp.

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As of September 27, 2018, a total of 26 laboratory-confirmed cases of Vibrio parahaemolyticus infection were reported in people who ate fresh crab meat from Venezuela; the cases were reported by seven states and the District of Columbia. The U.S. FDA worked with federal, state, and local officials on the outbreak. This crab meat was labeled as “fresh” or “pre-cooked” and is a ready-to-eat product.

This outbreak investigation has ended. Processors and distributors should know that the FDA’s Bacterial Analytical Manual states that “A heat-processed product should not contain viable V. parahaemolyticus and if so, would indicate a significant problem in manufacturing practices or post-process contamination.” FDA has additional information for processors and distributors in the new section, “What should processors and distributors know?”

The FDA collaborated with state partners in conducting a traceback investigation. This investigation identified multiple Venezuelan processors that supplied multiple brands of crab meat during the outbreak. FDA’s traceback did not identify a single firm as the source of the outbreak.

As a result of the outbreak investigation, the FDA increased testing of fresh crab meat from Venezuela. The FDA did not identify Vibrio parahaemolyticus in any samples tested, but the FDA did identify Salmonella and Listeria monocytogenes in some crab meat samples collected at import. The affected products did not enter into U.S. commerce.

Consumers and restaurants may want to consider using pasteurized crab meat or fully recooking (bringing to an internal temperature of at least 165 degrees Fahrenheit) fresh crab meat, particularly for items that will be served cold.

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The southern Nordic fjords are heating up as Europe boils, and bacteria there are flourishing, infecting swimmers and seafood, including oysters that can take months until they are safe to eat again, Norway’s food safety authority said.

The warm waters in southern Norway and Sweden have accelerated the reproduction of the Vibrio bacteria, a species that can cause vibriosis, an illness with symptoms as simple as diarrhea and stomachache but which can also be fatal.

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For centuries, oyster lovers have slurped down raw shellfish, enjoying the taste plus the fact they are low in calories and high in protein, iron, and other nutrients. But as ocean temperatures have warmed, some of the shellfish have become incubators for various species of genus Vibrio bacteria, some of it harmless, some of it causing serious illness or even death in those with compromised immune systems.

According to the Molluscan Shellfish Institute of North America, Americans eat about 2.5 billion oysters each year, farmed rather than native-grown, as the latter populations have declined due to disease and other factors over the years.

Robert Tauxe, MD, MPH, deputy director at CDC’s Division of Foodborne, Waterborne, and Environmental Diseases.
Image Credit: CDC

Though Vibrio occur naturally in ocean saltwater and around estuaries and brackish water, their escalation in oysters, which filter feed and thus build up the bacteria, didn’t start until the 1970s when growers who had avoided selling the shellfish during the summer months to give them a chance to reproduce instead began selling them year-round, says Robert Tauxe, MD, MPH, deputy director, CDC’s Division of Foodborne, Waterborne, and Environmental Diseases based in Atlanta.

“The oyster harvests in the old days were suspended in the summer to let the oysters breed,” says Dr. Tauxe. “That changed in the 1970s and that is when Vibrio surfaced.”

He also notes while Vibrio occurs naturally, there is some suspicion that its movement from the Gulf of Mexico to northern U.S. oceans may be the result of its transfer in ballast water on ships, especially those in oil ports.

“Warm water increases the prevalence of Vibrio infections,” Dr. Tauxe says. Though infections are rare, they are most prevalent within the Gulf of Mexico coast from April to October. “It’s particularly important for people in the Gulf area during warm seasons to stay out of the water. Vibrio are champions among multipliers. They can multiply every 15-18 minutes.”

Up to 45,000 Vibrio cases occur a year, with most causing watery diarrhea, vomiting, abdominal pain, and even death. Of the 1,252 cases of vibriosis recorded in 2014, there were 326 hospitalizations and 27 deaths, according to the CDC’s Cholera and Other Vibrio Illness Surveillance system.

CDC surveillance epidemiologist Erin Burdette, MPH, adds that V. parahaemolyticus, the most common species of Vibrio, has been reported as far north as Maine in recent years. In 2004, an outbreak occurred in Alaska that was linked to oysters raised locally during one of the state’s warmest summers. In other cases, oysters are imported from other areas of the country and eaten elsewhere: therefore, harvest origins need to be traced if there’s a disease outbreak. During the winter, Vibrio cannot multiply but instead become dormant and drop to very low levels until the water warms again.

A Sentinel for a Changing World?

Dr. Tauxe likened Vibrio to a sentinel in the foodborne illness world. “There are other organisms that are impacted by an increase in heat to the water,” he says. “We expect to see new problems emerge as the landscape of foodborne infections changes.” This includes movement of pathogens like Vibrio into warming northern oceans and global food sourcing.

The three most frequently seen Vibrio are: 1) V. parahaemolyticus, which is the most common and is creeping up both U.S. coasts and their northern borders, 2) V. alginolyticus, which is the second-most common and whose infectiousness is associated with direct contact with sea water, and 3) V. vulnificus, which has the highest death rate, related to the consumption of raw shellfish and exposure to open wounds.

V. parahaemolyticus falls in the same family of bacteria that causes cholera. The bacteria do not change the taste, smell, or look of an oyster, so it’s hard for consumers to tell if the oyster has the bacteria or whether the strain it has is indeed infectious.

Species such as the potent V. vulnificus can enter the body through a wound. Those who have liver disease, chronic illnesses like diabetes, or are otherwise immune-compromised are particularly susceptible and should either stay out of the water or cover their wound with a waterproof bandage, according to Dr. Tauxe. That being said, V. vulnificus infections are uncommon—with just a couple 100 cases a year, half from wounds and half from raw oyster consumption.

Image Credit: CDC/Foodnet

Over the past 20 years, E. coli 0157, Yersinia, Campylobacter, and Listeria culture-confirmed infection rates have decreased significantly while Salmonella showed no change. Comparatively, Vibrio cases showed a sharp rise (see Chart 1), according to the CDC’s Foodborne Diseases Active Surveillance Network (FoodNet). In 2015, Vibrio cases were up 34 percent compared to 2006-2008. Relatively, E. coli 0157 was down 30 percent over the period.

Within the Vibrio types, V. Vulnificus has been decreasing in recent years, likely the result of increased control of post-harvest time and temperature requirements of the shellfish industry, Jason Strachman-Miller, an FDA spokesman, said in an email.

“A decrease of over 30 percent has been observed for 2013 and 2014,” he wrote. “While the illness records for 2015 have not been finalized, it is expected that this decrease will continue.”

However, annual illnesses for V. parahaemolyticus in 2013 and 2014 almost doubled, Strachman-Miller noted, likely because of the emergence of the Pacific Northwest type of V. parahaemolyticus in the Mid and North Atlantic. He stated preliminary numbers for 2015 indicate a return to the baseline, although illness data is not finalized.

Dr. Tauxe says the CDC supervises the human health aspects of Vibrio while the FDA supervises shellfish sanitation programs. State health departments send out alerts in an attempt to ensure consumer safety.

Testing the Waters

Currently, says Dr. Tauxe, the needed markers and tests for Vibrios are not in place.

Cheryl Whistler, PhD, researcher and associate professor of microbiology and genetics, University of New Hampshire, Durham, N.H., and her colleagues are working to change that. They are using whole genome analysis to assess genetic characteristics of distinct Vibrio populations in the Atlantic. The hope is that the information will help with more accurate trace-back. So far, their analyses are allowing identification of strain-specific genetic loci they can use to develop strain-specific quantitative polymerase chain reaction, or PCR, detection assays for rapid quantification of the strains causing the most concern.

“We can quantify the total degree of all strains [in the water],” Dr. Whistler says. “While we can detect the total number of Vibrio, we want to be able to quantify which of them are pathogens.” Not all Vibrio are pathenogenic. “It’s like looking at football spectators and trying to find only those with red hair, blue eyes, and no freckles.”

Dr. Whistler says she is interested in whether validating specific strains will be a better predictive tool in modeling the combination of conditions that increase risk and warns of cross-contamination. “The first case of Vibrio was a case of cooked oysters put back on the ice the raw product was on,” she says.

So while her tests can tell there is Vibrio in the water and its prevalence, ultimately it’s up to consumers to choose what they eat and how they eat it.

“Anybody who wants to eat an oyster should have the choice to cook it or not,” she says. “Raw oysters are a delicacy. Cooking loses the flavor and texture.”

Making Oysters Safe

There are some methods that have helped diminish harm from V. parahaemolyticus. For example, harvesters need to refrigerate oysters as soon as possible after catch to decrease the temperature, Dr. Tauxe explains. He says that approach has worked well in Japan, which had a big problem with the bacterium. The cooling doesn’t kill the oysters, which would alter their taste and texture, but prevents future growth of the bacterium.

Cooking also kills Vibrio, but the cooking or steaming must continue even after the shells open—five more minutes for boiling and nine more for steaming (see Reducing Risk of Vibriosis sidebar, below). Hard freezing will also reduce the bacterium count, but it kills the oyster and changes its taste and texture. Commercial pressure chambers are used as well.

In 2003, after an outbreak of V. Vulnificus killed five people, California instated a new requirement that oysters sourced from the Gulf of Mexico from April to October be cooked or treated, in some cases using high pressure and heat pasteurization, Dr. Tauxe states, adding that there now are no deaths.

“It [the program] was a pretty clear success,” he says. “The California strategy is worth thinking about. If it’s the warm months and the oysters are from warm water, people should be aware of the risk [of eating raw oysters].”

Valigra is a writer based in Harrison, Maine. Reach her at lvaligra@gmail.com.

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