Vibrios and Human Health

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.