Subminimal inhibitory concentrations of ampicillin and mechanical stimuli cooperatively promote cell-to-cell plasmid transformation in .

Horizontal gene transfer (HGT) is a bacterial evolution tool for improved survival. Although several environmental stimuli induce or promote HGT, the diversity and complexity of the environmental factors have not been sufficiently elucidated. In this study, we showed that the biofilm culture of at the air-solid interface in the presence of a subminimal inhibitory concentration (sub-MIC) of ampicillin (∼0.5-4 µg/mL) and subsequent mechanical stimulation (rolling small glass balls,  = 5-8 mm) cooperatively promoted horizontal plasmid transfer without the usual competence-inducing conditions. Either of the two treatments promoted plasmid transfer at a lower frequency than when the treatments were combined. The effect of several parameters on the two treatments was tested and then optimized, achieving a high frequency of plasmid transfer (up to 10 per cell) under optimal conditions. Plasmid transfer was DNase-sensitive for both treatments, demonstrating its mechanism of transformation. Plasmid transfer occurred using various strains, plasmids, ball materials, shaking conditions, and even the mechanical stimulation of brushing the biofilm with a toothbrush, indicating the conditional flexibility of this phenomenon. This is the first demonstration of the promoting effect of the combination of a sub-MIC antibiotic and mechanical stimulation on horizontal plasmid transfer between cells via transformation. Regarding environmental bacterial physiology, the aggregations or biofilms of bacterial cells may encounter both sub-MIC antibiotics and mechanical stimuli in some specific environments, therefore, this type of HGT could also occur naturally.