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From: Food Quality & Safety magazine, December/January 2014

Vibrios and Human Health

by Cova Arias, PhD, and Jacque Kochak

Vibrios and Human Health

Most people can consume oysters by the dozens without ill effect, but people with liver disorders, diabetes, and immune-compromising conditions such as HIV/AIDS are at risk for the deadly vibriosis illness caused by a species of Vibrio. Individuals who take prescribed medication to decrease stomach acid or who have had gastric surgery are also at risk.

Vibrio vulnificus, a gram-negative bacteria that occurs naturally, especially in brackish, warm coastal Gulf of Mexico waters, causes a particularly horrific illness in vulnerable individuals—and the mortality rate hovers at 50 percent. Some 95 percent of all seafood-related deaths in the U.S. are caused by V. vulnificus, and the CDC reports that from 1996 till 2006, Vibrio infections increased by 78 percent.

The obvious solution to the problem would seem to be educating at-risk oyster eaters about the danger they’re courting. The Interstate Shellfish Sanitation Conference—a coalition of shellfish industry members and state and federal regulatory agencies—launched an expensive information campaign aimed at doctors and restaurants with the idea of educating high-risk individuals about the dangers of eating raw or undercooked oysters.

It didn’t work. Apparently, those who love the briny taste of raw live oysters are willing to take the risk.

In 2009, FDA proposed requiring all oyster producers to use a form of Post Harvest Processing (PHP) to sterilize raw oysters. The seafood industry pushed back, insisting that requiring PHP processes would drive many seafood companies out of business. That warning is not unrealistic because oyster harvesters are already under siege as a result of man’s degradation of the Gulf coastal environment and scourges like the oyster drill, a carnivorous marine snail that drills a hole in an oyster’s shell and sucks out the sweet innards.

Most oyster harvesters are small family operations, and they’ve already been decimated by oil spills in the Gulf. The nation’s richest oyster grounds have also been affected by a series of hurricanes lashing the region and flooding from the Mississippi River, which flushed a torrent of fresh water into the northern Gulf, reducing salinity. More recently, the Deepwater Horizon oil spill tainted the brand name “Gulf Seafood,” despite all the testing conducted by state and federal agencies that deemed seafood from the affected areas safe.

The FDA’s plan would have required all oysters harvested from the Gulf of Mexico between May and October to be processed. PHP, however, not only kills oysters—changing their taste and texture—it is expensive for small operations. Most don’t have the necessary capital to buy the equipment necessary to meet the proposed FDA regulations.

Avery Bates, vice president of the Organized Seafood Association-Alabama, told the Associated Press that two-thirds of Alabama’s 50 “mom-and-pop” oyster shops would close because of the costs associated with processing the oysters.

Is there a way to make eating oysters safer without decimating a struggling mom-and-pop oyster industry? Unexpectedly, research conducted in my lab at Auburn University could become central to the debate.

Depuration

Since 2007, Auburn has been studying a post-harvest process called depuration to eliminate Vibrio vulnificus from Gulf oysters. Depuration involves transferring shellfish from polluted waters to a controlled, cleaner aquatic environment, allowing then to “open” and eliminate contaminants themselves, thus reducing bacteria to low levels.

Mollusk depuration is common in Europe, where the process is used to eliminate microbes that proliferate in waters contaminated by fecal waste.

In the U.S., depuration systems must be approved by the FDA and are used only in Massachusetts (clams), Maine (clams and oysters), and Florida (clams). These depuration systems are utilized only in fecal-contaminated waters because depuration of pathogens that occur naturally, such as vibrios, has proven challenging.

Several studies have shown depuration’s potential for eliminating V. vulnificus. I did my undergraduate work with V. vulnificus in Spain, where the microbe is a problem for eel farmers but not a food safety issue. I realized depuration might be the only way to control V. vulnificus during summer months when it’s more prevalent, while also keeping oysters alive for raw consumption.

The research started out by constructing a flow-through tank system using seawater pumped in from the Gulf. The idea was that water flow would be uninterrupted and sufficient to remove feces and pseudo feces as well as prevent recontamination. Flow rate was maintained at 11 liter per minute for six days, and salinity and temperature were measured twice a day. V. vulnificus numbers in the oysters were enumerated at day zero, one, three, and six using the FDA Most Probable Number procedure.

We found depuration was successful—but only part of the time. Out of 11 depuration trials run in 2008 to 2009 using naturally infected oysters, we observed significant V. vulnificus reduction in only six.

During these preliminary trials, we modified some parameters to favor removal of the microbe while still maintaining optimum physiological activity of the oysters with salinity, temperature, and dissolved oxygen being the most significant parameters. For example, we tried cooling the incoming water to 15 degrees Celsius (59 degrees Fahrenheit) during depuration without observing a significant decrease in V. vulnificus numbers.

We also increased the water-flow rate and saw total clearance of V. vulnificus in oysters within six days. Unfortunately, this result could not be repeated consistently. All the oysters were collected from beds off Dauphin Island, a barrier island at the mouth of Mobile Bay. Why, we asked, was there such a high variability in depuration efficacy when oysters were collected from the same physical location with only a few months difference?

The answer appeared to be elegant in its simplicity. There was little variation in water temperature in our trials using seawater, but salinity fluctuated between 9.5 parts per thousand (ppt) and 30.1 ppt. Remember, the northern part of the Gulf of Mexico is really a giant, salty estuary fed by the Mobile and Mississippi Rivers and whipped by storms that regularly dump fresh water into the ocean. Salinity can drop from 20 ppt to 0 ppt in less than a day.

V. vulnificus thrives in brackish, but not too salty, water and the main difference in V. vulnificus numbers between the Atlantic Coast and the Gulf Coast during the warm summer months can be attributed to differences in salinity. Since we were using pumped-in seawater, we tried adding a brine solution to the water to keep salinity high. Adding brine to incoming saltwater is too expensive, however, to provide a long-term solution for oyster harvesters.

So we tried using artificial seawater instead, continuously circulating in the tank with a UV light sterilizer and an ammonia removal media filter to get rid of the toxic ammonia excreted by oysters as a waste product. We set up three tanks with different salinity levels for comparison and then went to work. For a post-harvest method to be approved by FDA, a 3-log difference has to be demonstrated. For example, 13,000 colony-forming units (CFUs) per gram of oysters would have to be brought down to less than 30. The higher the salinity we observed, the lower the number of CFUs.

The concept was then needed to prove to work consistently. We carried out four trials in 2012, using different salinities (15, 25, and 35 ppt). Our data showed that when salinity was at 35 ppt, the numbers of V. vulnificus decreased by at least three orders of magnitude in two (out of four) trials. Depuration at this salinity was able to reduce V. vulnificus levels below the FDA requirement of less than 30 most probable number per gram. Oysters tolerated the depuration conditions with very low mortality (less than 1 percent), although their condition index decreased during depuration (14 days); oysters were not fed during that time, but this is something that we can change in the future. In addition, depuration was effective at day 10, and prolonged times did not increase depuration efficacy. Hence, high salinity depuration is a promising method to reduce V. vulnificus in oysters while maintaining a live, fresh product.

Because depurated oysters are still alive, the taste differs only because they are slightly saltier. As noted, FDA-approved post-harvest techniques—such as freezing, heat-cool pasteurization, exposure to high hydrostatic pressure, and irradiation—kill the oyster and change the taste and texture, except for rarely used irradiation.

Cold Shock

The Gulf Oyster Industry Council estimates that only 10 percent of oysters currently undergo PHP, and oystermen usually use refrigeration to preserve live oysters. That is why a major focus of my lab also has been “cold shock,” the response of bacteria to cold. What mechanisms do V. vulnificus possess allowing adaptation to cold, and what risk does this pose to consumers?

The optimal temperature for V. vulnificus is 35 degrees Celsius (95 degrees Fahrenheit), not uncommon during summer on the Gulf coast. Most of these oystermen have small boats without refrigeration on board, so they go out for a short time and return to refrigerate their catch.

To study cold shock in V. vulnificus, we created a microarray to evaluate the expression of every one of V. vulnificus’ 4,488 genes at three different temperatures. We confirmed that when taking oysters down to 7 degrees Celsius (44.6 degrees Fahrenheit), the microbes’ proliferation is stopped. In fact, something major happens around 10 degrees Celsius (50 degrees Fahrenheit). That seems to be the threshold temperature where genes start turning on and the microbe gets into gear to handle cold.

When we lowered the temperature to 4 degrees Celsius (39.2 degrees Fahrenheit) instead of 7 degrees Celsius, the bacteria were no longer metabolically active, so our recommendation would be to keep oysters at 4 degrees Celsius.

We found, however, that if you take oysters down to 15 degrees Celsius (59 degrees Fahrenheit), leave them for several hours and then go down to 7 degrees Celsius, you can have problems. In that gap between 7 degrees Celsius and 15 degrees Celsius, the bacteria not only continue to grow, they adapt to the cold and can even proliferate.

HHP

Although oystermen who opt for a PHP method are in a decided minority, one of the most popular PHP methods is hydrostatic high-pressure (HHP) treatment. At the behest of a commercial seafood processor and distributor, we compared HHP-treated oysters to flash-frozen oysters and oysters kept raw at 4 degrees Celsius. We repeated the testing in the winter, the summer, and the fall.

HHP-treated oysters are supposed to have a shelf life of 21 days, and we found HHP treatment indeed eliminated the majority of human pathogens in oysters, at first reducing them to non-detectable levels. The bacteria that remained adapted rapidly and thrived under refrigeration, however, and after one week the HHP-treated oysters had more bacteria than the week-old raw oysters. In fact, I have rarely seen bacteria levels so high—but the good news is that oysters aren’t kept that long. HHP-treated oysters are certainly very safe, but they don’t seem to have a very long shelf life.

Another species of Vibrio, V. parahaemolyticus, infects oysters in the Gulf as well as in the cooler northern waters of the Pacific, from California to Alaska. Depending on the year, and on the salinity and temperature of the water, there may be more V. parahaemolyticus than V. vulnificus in oysters from Alabama’s Dauphin Island.

The CDC estimates vibriosis caused by V. parahaemolyticus causes some 4,500 illnesses annually. We have run a few depuration trials to see if increased salinity affects V. parahaemolyticus. As expected, high salinity depuration was not as effective in removing V. parahaemolyticus as it was in reducing V. vulnificus but an average of 2-log reduction was observed, which makes us optimistic about using depuration to reduce both pathogens.

Development of a high-salinity depuration system to reduce pathogenic vibrios in Gulf oysters will promote a struggling industry and reassure millions of oyster-eaters that they can eat the raw delicacy with confidence. There is regional interest in developing intensive oyster aquaculture, so oyster farmers also would benefit tremendously. Most importantly, a reliable, economical oyster depuration system would save lives.


Dr. Arias is a professor in fisheries and allied aquacultures at Auburn University and part of the multidisciplinary Auburn University Food Systems Initiative, which recently was awarded an FDA grant to help develop a food safety training program for food inspectors. Reach Dr. Arias at ariascr@auburn.edu. Kochak is the communications specialist at Auburn University Food Systems Institute.

 

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