The aim of detection is to distinguish between pathogenic E. coli strains, responsible for digestive infections, and non-pathogenic strains. Indeed, E. coli is a natural part of the intestinal flora of mammals. While most strains are harmless for humans, some specific enterohaemorrhagic strains can cause bloody diarrhoea. These pathogenic strains produce a toxin responsible for haemolytic-uraemic syndrome (HUS) which, in the most severe cases, can lead to chronic kidney failure or even death for the most vulnerable individuals (young children and the elderly).
Two genes characteristic of pathogenic E. coli
The pathogenicity of enterohaemorrhagic E. coli is related to two genes. One determines its ability to produce powerful toxins, called Shiga toxins, and the other its potential for strong adherence to the intestinal walls. In order for a bacterium to be pathogenic, it must have both these characteristics.
These two genes are detected by PCR when testing for pathogenic E. coli in food. The problem is that in a sample of food, for example a piece of cheese or raw meat, there can be several different E. coli strains. “It is entirely possible to have one strain that has one of the genes and another strain with the other responsible gene. These two strains will be harmless because they each contain only one of the two genes, but we will still have a warning signal because the two genes were detected in the same food sample” explains Patrick Fach, Head of the COLiPATH Unit (pathogenic Escherichia coli) at ANSES's Laboratory for Food Safety.
Additional genetic markers to refine the result
With the ISO 13136:2012 reference detection method, it is necessary to isolate the strains to determine whether both genes are contained in the same bacterium. This process is time-consuming, expensive and not always fruitful, because it is difficult to distinguish between E. coli strains based on microbial cultures in Petri dishes.
ANSES therefore researched other genetic markers that might help determine whether the same E. coli strain contains both high-risk genes. The scientific team assessed these markers in collaboration with the French Interprofessional Centre for the Dairy Economy (CNIEL). The results showed that compared with the reference test, the number of presumptive positive samples decreased by 26.5% in unpasteurised goat's milk, by 51.9% in raw ewe's milk cheese, and by 29.7% in raw cow's milk cheese. This means there were fewer “false positives” during quality controls at the production stage, i.e. samples wrongly considered as containing pathogenic E. coli and requiring additional confirmatory tests.
Thanks to these new genetic markers, the test developed by ANSES is able to detect truly pathogenic Escherichia coli strains within 24 hours versus four days previously. This work has been published in the International Journal of Food Microbiology.
Better management of possible contamination risks
“Our test will enable risks of contamination to be managed more precisely, while maintaining a high level of safety” considers Patrick Fach. ANSES has protected this test with a European patent (EP2861762 B1) with an international extension (WO2013/186754) and has reached out to manufacturers so that new detection kits may be produced and sold to food-processing companies and health authorities in Europe and the United States.