Anticipating xenogenic pollution at the source: Impact of sterilizations on DNA release from microbial cultures

Abstract

18The dissemination of DNA and xenogenic elements across waterways is under scientific and public 19 spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits. Over decades, 20 prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and 21 pathogens from transgenic systems has focused on microbial inactivation. However, sterilization 22 methods have not been assessed for DNA release and integrity. Here, we investigated the fate of 23 intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces24 cerevisiae) cells, commonly used as microbial hosts for genetic modifications, such as in white 25 biotechnology. DNA release was tracked during exposure of these cultures to conventional 26 sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to 27 inactivate broths, healthcare equipment, and GMOs produced at kitchen table. The results show that 28 current sterilization methods are effective on microorganism inactivation but do not safeguard an 29 aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the 30 most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk 31 assessment on the emission of DNA fragments in urban systems and nature. 32 33 34 35 3 Graphical abstract 36 37 (created with BioRender) 38 39 45 (ARGs) or pathogenicity, and their transport across urban waterways through wastewater treatment 46 plants into nature 2,3 . Novel CRISPR-Cas technologies propel the engineering of microorganisms out of 47 industry boundary with do-it-yourself (DIY) kits available at kitchen table. Uncontrolled, diffuse 48 emission of GMO materials via domestic waste streams could be a threat.49 Current sterilization methods are proven to efficiently inactivate microorganisms. However, a key 50 knowledge gap remains on their impact on DNA and its potential release into industrial, clinical, and 51 domestic sewage. Common methods used to treat industrial broths, healthcare equipment and 52 surfaces, and domestic waste primarily involve autoclaving, glutaraldehyde, and microwaving, 53 respectively. 54Several studies have shown how DNA present in food products react with different sterilization 55 procedures 4-6 . Treatments such as irradiation and autoclaving affect DNA in meat products or edible 56 seeds by decreasing the total DNA content as well as causing DNA fragmentation, degradation and 57 denaturation 7,8 . However, the impact highly depends on the cell type, the sterilization method, and 58 the process conditions. 59Temperature, pressure, pH, and sterilization times significantly exert effects on DNA quality. For 60 instance, temperatures over 100 °C have resulted in significant DNA strand clipping and irreversible 61 loss of secondary structure 4 . Normal autoclaving (121°C between 5 to 20 min) of food and crops did 62 not impeded it to be available for PCR amplification 4,5,9 63Microwaving is co...