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.

由于食品和饲料工业通常使用转基因微生物（GMM）来生产添加剂，酶和调味剂，因此它们经常带有抗微生物耐药性（AMR）基因作为选择标记，因此引起了人们对健康和环境的关注。因此，主管当局对控制此类微生物发酵产物的兴趣已大大提高，尤其是由于最近在意向用于欧洲食品和饲料链的细菌发酵产物中意外污染了未经授权的GMM或相关的重组DNA。然而，目前在执法实验室中尚无用于评估携带AMR基因的GMM和/或全长AMR基因的全球筛查策略。此外，相关GMM档案的保密性严重阻碍了进行此类控制的方法的开发。为了克服这个问题，在这项研究中对相关的公开可用专利进行了分析，以鉴定所有报告的AMR基因。在此基础上，氨基糖苷腺苷酸转移酶（赋予对卡那霉素和新霉素都有抗性的aadD）基因被确定为覆盖大范围GM细菌的关键靶标。针对此aadD，开发了一种实时PCR方法来筛选其潜在存在，以及与测序分析相关的巢式PCR方法以评估其全长。基因。这些新方法的性能已在特异性，敏感性和适用性方面进行了成功评估，从而使它们易于在执法实验室中实施。此外，将这些新开发的方法整合到我们最近提出的策略中（最初针对的是带有氯霉素抗性基因的GMM），可以大大提高生产发酵食品和饲料产品的转基因细菌的检测范围。因此，拟议战略产生的数据为主管当局提供了至关重要的支持，特别是评估食品和饲料安全的潜在风险。