Microbial challenge testing against botulinum neurotoxin-producing clostridia: improve food safety or guarantee National Security?

Microbial challenge testing is a widely accepted tool for enhancing food safety. This approach assesses the behaviour of a specific microorganism in a food product to validate the product or its production process. Microbial challenge testing can be conducted in accordance with the UNI EN ISO 20976 series; however, for microorganisms covered by Regulation (EC) 2073/2005, such as Listeria monocytogenes, the EU reference laboratory has developed specific guidelines and policy documents. Botulinum neurotoxin-producing clostridia are not covered by the aforementioned regulation, and no specific EU-level rules have been published to date due to the absence of reference diagnostic methods and the rarity of botulism. However, given changes in consumer behaviour towards minimally processed foods and the increased availability of foods potentially at risk of botulism, the disease poses new concerns. The main objective of this work is to highlight this weakness in food legislation, propose guidelines for conducting microbial challenge tests against botulinum neurotoxin- producing clostridia and their toxins, and suggest an audit plan to control private laboratories and prevent the voluntary or accidental release of these agents. Based on published documents and procedures for setting specific shelf-life studies in relation to Clostridium botulinum, and on peer-reviewed literature, the paramount issues included in the guideline are: • Studies assessing the inactivation of neurotoxic spores can be conducted using a non- pathogenic surrogate; conversely, in other types of challenge testing, strains capable of producing the neurotoxins cannot be replaced, and toxin detection is mandatory, because the neurotoxins could be released into the food if the relative enumeration decreases compared to the inoculum. • Studies require a cocktail of strains that cover all toxinotypes of interest, including both proteolytic and non-proteolytic strains. • Strains have to be characterised and studied for their ability to grow in co-culture with the other component of the cocktail. The amount of toxins they can produce within the food matrix of interest should be evaluated prior to the study. • The strains cannot be inoculated at the production facility; however, if the food product permits, they may be inoculated after packaging using the double-barrier technique to avoid altering the packaging atmosphere. Because botulinum neurotoxins are the most dangerous poisons known to humans, laboratories must be accredited to UNI EN ISO IEC 17025, participate in interlaboratory exercises, maintain high biosecurity standards, and undergo biosecurity audits conducted by the national reference laboratory.