Anticipating Xenogenic Pollution at the Source: Impact of Sterilizations on DNA Release From Microbial Cultures

Abstract

The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121 • C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.

Facing the 2020 pandemic: What does cyberbiosecurity want us to know to safeguard the future?

“…With increased automation, dangers arise, for example, in the context of sterilization methods used in the healthcare and laboratory setting. For some methods, a very recent study [ 62 ] demonstrates that “integrity of released DNA is not completely compromised,” which is leading to the “danger of dissemination of DNA and xenogenic elements across waterways.” These findings were linked to temperature and time (e.g., short microwave exposure times or short exposure time to glutaraldehyde treatment were least effective). Parameters like these are both highly malleable and susceptible to manipulation, which will become an even bigger concern with “smart labs” of the future [ 21 ].…”

The Potential Benefits of N2 Gas Flushing Technology for Various Dairy Products: A Sustainable Approach That Proved to Be Multiadvantageous for Preserving the Quality and Safety of Raw Milk During Its Storage

“…On the other side, the fate of residual genomic DNA from dead cells following heat treatments remains questionable, taking into account that three studies showed that autoclave sterilization (121 • C/15 min), which is chemically more stringent than UHT sterilization, did not totally degrade DNA (Masters et al, 1998;Yap et al, 2013;Calderón-Franco et al, 2020). By considering a 10 5 cfu/ml raw milk sample, a rough estimation lead to around 24,500 AMR genes per ml of raw milk (Munsch- .…”

Antibiotic resistance response of activated sludge to sulfamethoxazole: insights from the intracellular and extracellular DNA fractions

“…Extracellular DNA (exDNA) is commonly released from the intracellular DNA (iDNA) during cell lysis (passive release) or excreted from live cells (active release). 20,21 Such exDNA can be present as a structural component in microbial aggregates, as part of the matrix of extracellular polymeric substances (EPS) 22 or floating freely in the water body. 23 Recent advances in methods for the extraction of exDNA allow for a deeper analysis of the composition and distribution of both DNA fractions in WWTPs and natural environments.…”