TOX-Detect: Development and harmonisation of innovative methods for comprehensive analysis of foodborne toxigenic bacteria, ie. Staphylococci, Bacillus cereus and Clostridium perfringens

Foodborne disease outbreaks (FBOs) occur when people are exposed to unsafe levels of contaminants in food products, causing illnesses (most frequently gastrointestinal) and in some cases more serious disease or even death. According to the European Food Safety Authority (EFSA), a foodborne disease outbreak is defined as “an incident during which at least two people contract the same illness from the same contaminated food or drink” (EFSA Newsroom 2019). In the European Union (EU), the most frequent causative agents are bacteria, bacterial toxins, viruses, and parasites (EFSA Monitoring of foodborne diseases).

In recent years, thousands of foodborne outbreaks have been reported in the EU (5,175 in 2019 and 3,086 in 2020), with tens of thousands human cases, thousands of hospitalizations and less than one hundred deaths (The European Union One Health 2020 Zoonoses Report, The European Union One Health 2019 Zoonoses Report). Among the known causative agents, bacteria were reported to have caused the most outbreaks (35.4% in 2020 and 26.4% in 2019) followed by bacterial toxins (17.1% in 2020 and 19.3% in 2019), viruses (5.0% in 2020 and 10.7% in 2019), other causative agents (2.2% in 2020 and 3.0% in 2019), and parasites (0.5% in 2020 and 0.6% in 2019) (The European Union One Health 2020 Zoonoses Report, The European Union One Health 2019 Zoonoses Report). It should be noted that the causative agents of a large proportion of foodborne outbreaks remain unidentified.

Outbreaks caused by bacterial toxins represent a major proportion of reported FBOs (thousands of cases each year). Bacterial toxins can cause tissue damage and disable immune responses when ingested, triggering acute food-poisoning symptoms (such as vomiting, diarrhoea and light-headedness). At EU level, the most frequently reported causes of food-poisoning outbreaks (FPOs) are bacterial toxins produced by Bacillus spp. (Bacillus cereus), Clostridium spp. (C. perfringens and C. botulinum) and Staphylococcus spp. (S. aureus). The true incidence of FPOs caused by these toxigenic bacteria is underestimated for many reasons, including misdiagnosis, under-reporting (particularly of minor outbreaks), and improper sample collection or laboratory examination. The complexity and diversity of food matrices involved in FPOs caused by bacterial toxins add to the difficulty. Moreover, FPOs caused by toxigenic bacteria share similar symptoms, making it difficult to clearly identify the causative agent behind the outbreak during investigations. All this may also explain the high proportion of “weak evidence” FPOs reported by EFSA when bacterial toxins are the causative agent.

The scientific community involved in monitoring foodborne outbreaks caused by bacterial toxins needs harmonised and innovative detection methods for this underestimated One Health issue.

What is the TOX-Detect Project?

The TOX-Detect project involves 6 partners across Europe: The French Agency for Food, Environmental and Occupational Health & Safety (ANSES), the French National Research Institute for Agriculture, Food and Environment (INRAe) and the Institut Pasteur (IP) in France, Sciensano in Belgium, the German Federal Institute for Risk Assessment (BfR) in Germany, and the Norwegian Veterinary Institute (NVI) in Norway.

With these partnerships between human, animal and plant health, food safety, and environmental protection institutes, the TOX-Detect project was designed to develop and harmonise methods for detecting toxin expression and production by toxigenic bacteria across all One Health pillars.

At the start of the project, several important knowledge gaps were identified, including the need to:

• Implement a relevant collection of pathogenic strains including strains from various sources (animal, environmental, food and human samples) and different geographic locations.
• Develop tools not reliant on genome sequencing (non-NGS), but rather using protein/toxin detection and identification, such as mass spectrometry (MALDI-ToF, LC-MS) and enzymatic assays (ELISAs) for strain characterization.
• Establish a set of Standard Operating Protocols (SOPs) and evaluate the transferability of the methods thus developed through several inter-laboratory studies.

The TOX-Detect project aimed to address these knowledge gaps by:

• Collating and fully characterising a large number of toxin-producing bacterial isolates from environmental, clinical, and food samples provided by all the partner institutes.
• Developing dedicated ELISA and mass spectrometry methods, and libraries for the identification of the targeted pathogens.
• Harmonising Standard Operating Procedures (SOPs) for all the targeted virulence factors and methods.

It is crucial to adopt a One Health approach so as to detect and monitor toxigenic bacteria across all One Health pillars to prevent foodborne outbreaks and further improve food safety.

The Project Impact Brochure is available on Zenodo.