Development of a Taxon-Specific Real-Time PCR Method Targeting the Bacillus subtilis Group to Strengthen the Control of Genetically Modified Bacteria in Fermentation Products

Abstract

Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilis, B. licheniformis, B. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), reinforcing control over the food and feed chain in order to guarantee its safety and traceability.

Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products

“…Four FE products from different brands were selected from previous studies [2,4,[6][7][8]13], based on their level of contamination with GMM alpha-amylase1 observed with qPCR (Table 1). Genomic DNA was extracted using the Quick-DNA™ HMW MagBead Kit (ZymoResearch) according to the manufacturer's instructions.…”

“…DNA extracted from isolated bacteria was analyzed by the GMM protease1 qPCR method as described in Section 2.1.2, and the BSG qPCR method specific to the Bacillus subtilis group developed previously [13]. DNA from isolates being both positive to the BSG marker and negative to the GMM protease1 marker was extracted as described previously [8,9] to avoid selecting protease GMM1 isolates, which were already extensively characterized [8].…”

Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase

“…DNA from the control plasmid (Genecust, France), artificially synthetized to carry one copy of the sequence targeted by the developed GMM alpha-amylase2 qPCR method, was used. DNA from microbial, human, plant and commercial FE materials ( Table 1 , Table 2 , Table 3 , Table 4 ) were obtained as previously described ( D’aes et al, 2022 , Deckers et al, 2021a , Fraiture et al, 2020a , Fraiture et al, 2020b , Fraiture et al, 2020c , Fraiture et al, 2020d , Fraiture et al, 2021b , Fraiture et al, 2021c , Fraiture et al, 2021d , Fraiture et al, 2022 ). For each commercial FE sample, the DNA extraction was carried out independently.…”

“…In addition to the European legislation, considering that antimicrobial resistance (AMR) genes are often harboured by such GMM contaminations, public health concerns, associated with the potential risk of AMR horizontal transfer to gut microbiota and pathogens, were raised ( Florez-Cuadrado et al, 2018 , Nadeem et al, 2020 , Tóth et al, 2020 , von Wintersdorff et al, 2016 , von Wrighta and Bruce, 2003 ). Consequently, a GMM detection strategy based on qPCR technology was developed for enforcement laboratories to support the Competent Authorities to ensure the food safety and traceability ( Fraiture et al, 2020a , Fraiture et al, 2020b , Fraiture et al, 2020c , Fraiture et al, 2020d , Fraiture et al, 2021a , Fraiture et al, 2021b , Fraiture et al, 2021c , Fraiture et al, 2021d , Fraiture et al, 2022 ). This GMM detection strategy is currently used by several European enforcement laboratories to control commercial microbial fermentation products for which sample analyses are regularly requested by the Competent Authorities.…”

“…This GMM detection strategy is currently used by several European enforcement laboratories to control commercial microbial fermentation products for which sample analyses are regularly requested by the Competent Authorities. The presence of GMM is firstly screened by targeting key genetic elements commonly found in genetically modified (GM) bacteria used for the production of fermentation products, including the Bacillus subtilis group (16S-23S region), three AMR genes conferring resistance to chloramphenicol ( cat ), tetracycline ( tet-l ) or kanamycin ( aadD ), as well as the pUB110 shuttle vector carrying aadD ( Fraiture et al, 2020a , Fraiture et al, 2020b , Fraiture et al, 2020c , Fraiture et al, 2021b , Fraiture et al, 2022 ). After obtaining a positive signal for the screening step, an identification step is conducted to demonstrate the presence of specific GMM strains by targeting unnatural associations of sequences specific to known GMM strains, as performed for any genetically modified organisms (GMO) ( ENGL, 2015 ).…”