Antibiotic Resistance Evolution of Bacterial Biofilm Via Natural Transformation Under Antibiotic and Heavy Metal Pressures

Bacterial biofilms can enhance tolerance to antibiotics, however, antibiotic-resistance evolution in biofilms under environmental stressors via horizontal gene transfer remains unclear. This study investigated the natural transformation mediated by extracellular DNA (eDNA) at single-cell level under the stresses of tetracycline, sulfamethoxazole, and Zn²⁺ to explore the antibiotic-resistance evolution in biofilms. The stressors at the sub-minimal inhibitory concentrations (MICs) enhanced antibiotic resistance genes (ARGs) transformation by 1.01-fold to 4.62-fold in B. subtilis biofilms and 1.01-fold to 6.94-fold in A. baylyi biofilms, respectively, compared to unstressed conditions; meanwhile, biofilm growth increased by 1.29-fold and 1.48-fold, respectively, with natural transformation. These results demonstrated that ARGs transformation enhanced bacterial adaptability to stresses. The stressors stimulated polysaccharide production by 1.06–3.15 times, offering protection against stressors penetration. They also induced reactive oxygen species production and membrane permeability by 1.02–2.83 and 1.02–2.04 times, respectively, promoting ARGs transformation. Additionally, the exogenous SOS response-related genes introduced by eDNA were upregulated by 1.03–7.06 times in recipient cells, facilitating biofilm tolerance to stresses via functional gene regulation. These findings reveal the critical role of natural transformation in biofilm resistance evolution under sub-MIC stressors, providing a theoretical basis for controlling antibiotic-resistance spreads.