Veni, Vidi, Vibrio June 18, 2019

This morning we performed DNA extraction and real time PCR looking for bacterial pathogens in clams! In Italy, shellfish represent around 70% of the aquaculture production, and also provide more than 90% of Europe's clams. Because of the high consumption of shellfish, performing DNA analysis on the shellfish is an important part of public health and food safety. This process keeps bacterial, fungal, and other harmful pathogens from being consumed.

Bivalve mollusks (clams, mussels, etc.) are considered high risk foods in Italy and are under strict regulations. Even though they go through a filtration system, the mollusks can maintain the following pathogens in their body: Salmonella, Hepatitis virus, Norovirus, Vibrio spp., Giardia, and biotoxins from algae. In particular, today we were focusing on searching for E. coli and Vibrio bacterial genes present in the clams. E. coli presence is usually indicative of fecal contamination, while Vibrio, if ingested by humans, can cause gastroenteritis and/or bacteremia (bacteria in the blood). During the filtration process, or depuration, impurities such as E. coli are removed. However, depuration does not remove Vibrio. This is creating a worrisome issue, as there is currently no legislation addressing this issue. While not all Vibrio species are high risk, both Vibrio vulnificus and Vibrio parahaemolyticus are considered microbial hazards.

To sum it up:

"Criteria for pathogenic viruses in live bivalve mollusks should be established when the analytical methods are developed sufficiently. There is a need for development of reliable methods for microbial hazards. "

Preparing the sample! We opened the clams and used a filter to collect a liquid sample, on which we performed DNA extraction and PCR. A filter was used to make sure only very small impurities (like bacteria) were included in the sample.

Receiving the lab instructions!

What We Did: DNA Extraction and Real Time PCR

There were 2 groups of clams that we worked with. Half of the groups used pre-depuration clams, and the other half used post-depuration clams.

Step 1. Lysis- Here we add chemicals to the homogenate (liquid sample) we collected, in order to break down cells and release their contents.

Step 2. Eliminate the Inhibitors

Step 3. Purification - To get rid of the cellular material we are not interested in, such as sugars, proteins, etc.

Step 4. Real Time PCR on extracted DNA- We pipetted our DNA into tubes with either Vibrio primers, E. coli primers, or universal bacterial primers.

** Primers are specific DNA sequences that look for, bind to, and indicate the presence of specific genes. For example, Vibrio primers will bind to a gene in Vibrio DNA, and will indicate the presence of Vibrio in the sample.

The Results:

What we found was that the pre-depurated clams contained both E. coli and Vibrio bacterial species. While the post-depurated clams contained the same level of Vibrio pathogens as the pre-depurated clams, and minimal to no E. coli.

Special thanks to Professor Cardazzo and Professor Fasolato from the University of Padova for working with and teaching us about the the importance of food safety analysis.

After our long and rewarding day, we explored the city of Padova and enjoyed its beautiful views. Not a bad end to the day!