Detecting pathogens in seafood

The food of choice at end-of year festivities and wedding breakfasts, seafood is an integral part of festive occasions. But in order to ensure that the party does not end badly, the quality and freshness of this tempting food must be guaranteed. Indeed, seafood can be a carrier of different pathogenic microorganisms. In Europe, the marketing of seafood is fortunately well controlled in order to minimize health risks to consumers. Georges Daube and his team at the Food Microbiology Laboratory of the University of Liege have developed a method of molecular biology which makes it possible to amplify specific genes of microorganisms in order not only to detect pathogens, but also to estimate their quantity.

From 1995 to 2009, the Food Microbiology Laboratory of the University of Liege, directed by Professor Georges Daube, has been the stand out laboratory in this area as far as the ministry of public health is concerned. "In this context, a large part of our activities consisted of managing approved laboratories but also to develop and validate efficient methods for assessing and managing the microbiological quality of food at a national level", explains Georges Daube, of the Food Science Department of the Faculty of Veterinary Medicine of the University of Liege. "We were more particularly specialized in the bacterial and virological examination of live bivalve molluscs", explains the Professor.

Oysters, mussels and other shellfish are particularly problematic from a food hygiene point of view because these organisms filter large quantities of water in order to feed. Particles suspended in the water carry bacteria and viruses, which come from human or animal feces and which are then concentrated in molluscs. "The role of our laboratory was to perfect methods for detecting these types of pathogens", continues the researcher.

Developing an efficient method for the AFSCA

Apart from the “filtering” activity of bivalve molluscs, the way we eat them, either raw or lightly cooked, represents an extra health risk. While oysters do not generally make it to the saucepan, the cooking of mussels and shellfish stops as soon as the bivalves open. "They are therefore not subjected to temperatures that are high enough to remove the microorganisms present in the water ", continues Georges Daube. " And as far as crustaceans are concerned, there are often cases or recontamination after cooking, when they are de-shelled for example. The bacteria and viruses present on the hands or on the raw shrimps handled just before can also make their way to our plates as well".

Although there is only one producer of oysters and one producer of mussels in Belgium, we consume seafood from France, Spain and other neighboring or other overseas countries. In order to ensure the safety of Belgian consumers, the Ministry of Health, and more particularly, the Federal Agency for Safety of the Food Chain (FASFC), needs efficient methods for carrying out these checks. This is exactly what Georges Daube and his colleagues suggest in a recent study published in Food Control (1). "At the beginning of the 2000s, we only had rudimentary methods for detecting the presence of microorganisms in food", indicates the scientist. "It was necessary to crush the food, place it in solution, and isolate the microorganisms by placing them in Petri dish cultures in order to allow them to multiply. These methods were very laborious".

A minimum infectious dose under control

Thanks to the support of Wallonia, Georges Daube worked in collaboration with Alain Vanderplasschen, Professor of immunology and vaccinology at ULg, and with Professor José Remacle of the University of Namur, in order to develop technologies to improve these methods. "The University of Namur has concentrated on the screening of pathogens in order to identify which ones are present in a given sample by the DNA microarrays  method", explains Georges Daube. "For our part we concentrated on another method of molecular biology, real-time PCR , which makes it possible to amplify specific  genes of microorganisms in order to detect pathogens and estimate their quantity". This quantitative aspect is crucial because, for each pathogen, there is a minimal infectious dose. Below this dose there is no health risk to humans because the pathogens will be destroyed in the stomach or the digestive system. "The method we have perfected and which has been the subject of an article in Food Control makes it possible therefore, not only to detect pathogens but to quantify them", explains the researcher.

Once the pathogens have been detected by the DNA microarrays or real-time PCR method, it is necessary to isolate the pathogens in order to be able to characterize them and to determine their virulence factors. "With Professor Vanderplasschen, we have developed antibodies coupled with magnetic particles which could be used to capture the microorganisms and enable us to specifically isolate them", continues Georges Daube.

An efficient method for seafood

Carried out between 2006 and 2008, this work led to the creation of an efficient method for the detection of six bacteria in seafood products: Campylobacter jejuni, Campylobacter coli, enterohaemorrhagic Escherichia coli O157, Salmonella spp, Vibrio parahaemolyticus, and Vibrio vulnificus.  "Commercial kits based on the PCR method have been on the market since 2008. These are used mainly for the qualitative method because the quantitative method requires the use of a reproducible method of DNA extraction. This stage is not always carried out in an optimal fashion and can therefore result in a false estimate of the quantity of pathogens present in a sample", explains the scientist.  despite the availability of commercial kits on the market, the recent publication of Professor Georges Daube's team confirms that the real-time PCR method can be considered as an optimal method in food quality control. "The advantage of our method is that it has been validated more specifically for the detection of pathogens on the matrices of live bivalve molluscs and on raw and cooked shrimps. Even though current marketing methods are standard for all foodstuffs, it is necessary to ensure that they perform well in terms of the matrix being studied. Otherwise there are risks of false negatives which can have very serious consequences", points out the researcher.

Rare but serious complications

The checks strongly limit the risk of contamination of consumers by seafood through the pathogens that these foods carry. However, it can happen that some pathogens escape the vigilance of authorities, producers or consumers themselves. What consequences can these pathogens have for human health?  "These are mainly bacteria and viruses that cattle, humans and poultry excrete in their feces which ends up in the sea", explains Georges Daube. "In most cases, these are bacteria which adhere to the cells that form the wall of the intestine thus causing diarrhoea, vomiting and fever. The most dangerous pathogens enter the cells of the intestine and destroy them which causes blood loss in stools. In rare cases, they can also enter the blood system and cause septicaemia, that is to say, a general infection of the organism. The immune system is them overcome and this can sometimes lead to death".

Among the six bacteria targeted by the above-mentioned detection method, enterohaemorrhagic Escherichia coli O157  is the most worrying. Indeed this causes haemorrhagic colitis but also releases toxins which destroy the inner layer of blood vessels. These are serious illnesses which can lead to nerve and/or kidney failure. 
It is clear then, the complications of a contamination are rare but can have dramatic consequences, especially on the health of individuals at risk (babies, elderly people, pregnant women and immunodeficient  people).

Escherichia coli, a sentinel bacteria for the environment

The icing on the cake with regards to seafood, molluscs and crustaceans can also contain concentrations of  marine biotoxins from microalgae known as dinoflagellates. "It must therefore be remembered that when we speak about symptoms following the ingestion of seafood, this can be due either to biotoxins or microorganisms", continues Georges Daube. To minimize the risks linked to biotoxins, the authorities are monitoring the quality of seawater and the quantity of dinoflagellates. "It is particularly due to these particular microalgae that, each summer,  oyster harvesting is forbidden in the Arcachon basin, for example", explains the scientist. Controlling the levels of microorganisms is currently done in the presence of an excessively high concentration of a bacteria, Escherichia coli (not to be confused with its pathogenic enterohaemorrhagic variant O157 mentioned above!), naturally present in the intestinal flora of humans and animals.  An excessive quantity of these bacteria reflects fecal contamination of water and an increased risk of finding some of the above-mentioned pathogens in molluscs.

"I can't recall there ever have been a large-scale epidemic in Belgium, but in France, where there are more seafood farms and a greater consumption of seafood, the germs mentioned in our article have already caused several epidemics", indicates Georges Daube.

There is no need for panic or a ban on oysters, lobsters or other kinds of seafood. It is simply a matter of noting that it is better to choose the option of products that are checked in-keeping with the law applied in the European Community.

Since 2009, the public health ministry has taken charge of the microbiology of foodstuffs. Georges Daube's laboratory has been specializing in microbial ecosystems, that is to say, all the microorganisms present in a biological sample. "This still covers food but also, more broadly speaking, water, the digestive apparatus, the environment etc. We use more efficient methods of high-throughput sequencing, thanks to which we can identify all the bacteria and viruses in a given biological sample", explains the researcher.

(1) B. Taminiau, N. Korsak, C. Lemaire, V. Delcenserie, G. Daube. Validation of real-time PCR for detection of six major pathogens in seafood products. Food Control. Volume 44, October 2014, Pages 130–137