Developing a Freeze-Thaw Assay to Quantify Antibiotic Resistance Gene Transfer in Bacterial Co-Cultures

The hastening thaw of glaciers and permafrost due to climate change threatens to release archaic genetic material, including antibiotic resistance genes (ARGs), into aquatic environments. While the biological risks associated with this release have been recognized, it is vital to understand the effects that freeze-killed cells and their genetic material may have on living microbial communities.

This study aims to investigate whether DNA from freeze-killed cells can be taken up by living cells, specifically via transformation. Kanamycin-resistant MC1061 Escherichia coli, harboring the P3 plasmid, encoding the resistance gene aph(3’)-Ia, were subjected to five freeze-thaw cycles (-20°C/37°C) to simulate glacial thawing and were then co-cultured with kanamycin-sensitive DH5α E. coli. Colony forming units on kanamycin-selective and non-selective LB agar were used to measure kanamycin resistance frequency (KRF) across experimental and control groups. Co-cultures containing freeze-thawed donors cells exhibited a substantially elevated KRF compared to co-cultures with donor cells that had not undergone freeze-thaw cycles and control group estimated, considering no interaction due to HGT, demonstrating that HGT of ARGs occurred at a significant rate despite the majority of donor cells being dead. These findings are consistent with gene transfer occurring via transformation, likely facilitated by cell lysis of frozen donor cells, though this was not explicitly confirmed and alternative explanations, such as resistance arising from surviving donor cells, cannot be excluded. This work highlights a way in which melting environments may contribute to the global spread of antibiotic resistance.