Contamination of bathing water and shellfish by faeces: how to identify the sources

Some of these micro-organisms can be pathogenic for humans and therefore sicken not only swimmers but also shellfish consumers. Indeed, bivalves - oysters, mussels or clams - feed on plankton: they filter and thus concentrate the bacteria and viruses present in the water.

The majority of these pathogenic microorganisms are of faecal origin. They come from the digestive tract of humans and animals, like salmonella, Campylobacter, certain Escherichia coli (E. coli) that may be pathogenic, or human enteric viruses such as noroviruses which are known to be causative agents of infections or food poisoning.

In 2021, 8,3% of reported collective foodborne illnesses (TIAC) ​​were due to the consumption of shellfish. Enteric viruses have been suspected to be responsible for 75% of these.

These pathogenic micro-organisms are often brought to coastal waters by upstream anthropogenic discharges. They have, for example, been found in coastal areas and their watersheds in France.

The risk of contamination of coastal waters by these pathogenic micro-organisms cannot be estimated by their direct research due to their great diversity, their often low numbers and the cost and time required for these analyses.

In search of bacteria

Also, at the international level, an alternative has been adopted to assess the sanitary quality of surface waters and batches of shellfish. It consists in researching and quantifying bacterial indicators of faecal contamination: E. coli and intestinal enterococci by culture, which indicate the presence of faecal pollution, and therefore the possible presence of pathogenic microorganisms.

To preserve human health, according to European regulations, the classification of bathing areas (during the bathing season), growing areas (shellfish farming areas, monitored throughout the year) and shore fishing areas is based on research and quantification of these indicators in waters or shellfish.

High concentrations of these indicators can lead to downgrading or closure of these different areas and require purification of shellfish from shellfish-growing areas or relaying of these before their marketing.

The classification of bathing waters in 2022 in Europe shows that the France is ranked in 21ᵉ position if we take into account the parameter "proportion of water classified as excellent quality". Of the 2074 bathing areas in sea water and the 1296 in fresh water monitored in France, 77,9% and 73,2% of the bathing areas were respectively classified as excellent quality and 97,0% and 89,3% of sufficient quality, with a slight deterioration in the sanitary quality of water since 2019.

Identification of sources of faecal contamination

In order to improve the sanitary quality of water and shellfish, it is necessary to implement preventive and curative actions to limit pollution of faecal origin. Also, discrimination and prioritization of sources of contamination have become priorities.

To identify microbiological pollution and its origin, the first step is to assess the contamination by E. coli water and shellfish in the area concerned but also further upstream in the river waters of the watersheds that could impact them. The second step is to identify the origin of this indicator of faecal contamination.

To identify the sources of these bacteria, we use methods grouped under the term Microbial Source Tracking (MST or microbial source tracing, TSM).

At Ifremer, since 2005, we have adopted the approach based on the search for microbiological markers targeting the DNA of bacteria by gene amplification (the now famous quantitative PCR) and developed or used bacterial markers associated with a source of contamination precise: for example Pig, Ruminant (cattle and sheep), Wild birds and even more recently Seal markers.

For example, here is how the pig marker, referred to as Pig2Bac, was developed. He was internationally validated and has been used in numerous studies both in France and in other countries.

We first collected a large number of pig faeces and slurries from pig farms in France. We extracted the bacterial DNA from it and then amplified the DNA sequences corresponding to bacteria of the order Bacteroidales, selected to be the majority anaerobic bacteria of the intestinal flora of warm-blooded animals and humans, for some of them. they are specific to a host (here, porcine) and do not multiply in the environment.

We then compared these sequences to other sequences of bacteria of the same order but identified in other sources: human, bovine and avian.

Sequences of bacteria present specifically in pigs have thus been identified. PCR primers that target these sequences were then designed. Then, we verified that the Pork marker gave positive results in samples of porcine origin (evaluation of sensitivity), and negative in samples of other origins (evaluation of specificity). We have thus shown that this marker has a sensitivity of 100% and a specificity of 99%. These very good results therefore made it possible to validate this marker of porcine contamination.

However, for a marker to be relevant, it must show persistence in waters, similar to that of the indicator E. coli. We therefore monitored in the laboratory for several days the concentrations of the Pig2Bac marker and of E. coli in fresh water and sea water artificially contaminated with pig manure. This marker and others we also tested generally persisted a little shorter in the waters. which E. coli counted by culture : it would therefore rather target recent faecal contamination. However, it is important to consider that the variable conditions of temperature, salinity, oxygenation of the water have an influence on the persistence of these bacteria and that it is difficult to extrapolate these results to research in the natural environment.

This marker as well as those from other sources are used in scientific studies and routinely by water analysis laboratories to meet the demands of water managers.

To be more confident in the source identification results, we can only recommend searching several targets in parallel from the same source and from different sources; in this case we speak of the use of a "TSM toolbox" or MST-toolbox. Bacteroidales markers and chemical compounds, faecal stanols, have thus given interesting results. when tracking across multiple sites.

German mitochondrial markers which directly target the host's DNA, enteric viruses and certain bacteriophages or viruses of bacteria, as well as ingested chemical compounds such as caffeine and drugs in human or veterinary health can also be used.

In addition, we can compare bacterial communities in sources and waters by high-throughput sequencing methods.

The approach Microbial Source Tracking continues to be enriched by the description and validation of new markers, as well as by the integration of the latest technological developments for better target wanted agents. These developments and the integration of new methods can only improve the identification of sources of faecal contamination in the environment and therefore ultimately the quality of water and shellfish.

In conclusion, for proper use of markers and correct identification of sources, it is essential: to know the study area in which they are sought; to take into account the influence of factors such as rainfall, which can aggravate pollution; to go back to the rivers identified as being able to impact the bathing site or the shellfish area studied; to associate if possible several complementary markers and to search for them in several samples taken from the same site and in the waters upstream in contrasting conditions, rather than carrying out a simple one-off search with little discrimination.

Michele Gourmelon, Microbiologist, Ifremer

This article is republished from The Conversation under Creative Commons license. Read theoriginal article.

Image credit: Shutterstock/ Javier Ruiz