Natural transformation of antibiotic resistance genes and the enhanced adaptability in bacterial biofilm under antibiotic and heavy metal stresses

Bacterial biofilms are hotspots for the natural transformation of antibiotic resistance genes (ARGs). Antibiotics and heavy metals at the sub-minimal inhibitory concentrations (sub-MICs) are ubiquitous in water environments, but their impact on the ARG dissemination via natural transformation in biofilms and the biofilm development remains poorly understood. This study found that the individual stressors including tetracycline, sulfamethoxazole, and Zn at the sub-MIC levels, significantly enhanced ARG transformation. Notably, Zn exhibited the most obvious effect, increasing transformation frequencies by up to 4.62-fold in B. subtilis and 6.42-fold in A. baylyi biofilms. Their combined stressors increased the higher ARG transformation compared to the individual. These stressors significantly elevated ARG transformation by stimulating reactive oxygen species generation, increasing membrane permeability, and enhancing polysaccharide production. Meanwhile, the bacterial adaptability in biofilm to stressors was achieved via ARG transformation, and the biofilm growth was increased by 25.4 % in B. subtilis and 49.6 % in A. baylyi , respectively, compared to biofilms without natural transformation. Except for ARG uptake via transformation, the enhanced bacterial adaptability in biofilms to stressors can also be attributed to the expression of the plasmid-borne SOS response-related genes. These findings broaden the understanding of the influence of sub-MIC stressors in ARG dissemination in biofilm.