As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.

Unexpected contaminations of unauthorized genetically modified microorganisms (GMM) harbouring antimicrobial resistance (AMR) genes in food and feed enzymes, additives and flavourings commercialized on the European market have recently alerted the competent authorities regarding the food and feed safety. At the control level, we have therefore proposed a PCR-based strategy as first line screening targeting GMM carrying AMR genes in order to help enforcement laboratories. The potential presence of frequently used AMR genes is first investigated, using real-time PCR. In case of a suspicious matrix, the full-length of the detected AMR genes is then determined, using conventional PCR followed by Sanger sequencing, allowing to support the competent authorities in their evaluation related to potential health risks. In this study, PCR methods targeting an additional key AMR gene, being the tet-L gene (GenBank: D00946.1) conferring a resistance to tetracycline, were developed and successfully assessed in terms of specificity, sensitivity and applicability. In integrating these PCR methods, the proposed PCR-based strategy, initially targeting two key AMR genes conferring a resistance to chloramphenicol (GenBank: NC_002013.1) and kanamycin (GenBank: M19465.1), is consequently strengthened, allowing the coverage of a larger spectrum of potential GMM contaminations in microbial fermentation products.