Various evolutionary trajectories lead to loss of activity of tobramycin-potentiating compounds in Burkholderia cenocepacia biofilms

Antibiotic adjuvants are commonly described as an alternative approach to overcome bacterial resistance towards conventional antibiotics. In this experiment, we investigated this statement for tobramycin (TOB) in combination with three adjuvants; baicalin hydrate (BH), a quorum sensing inhibitor, econazole (ECO) and miconazole (MICO), two antifungal agents that are repurposed as antibiotic adjuvants. We repeatedly exposed mature (24 hour old) Burkholderia cenocepacia J2315 biofilm cells to TOB alone (768ug/ml), or a combination of TOB with either BH (250uM), ECO (1uM) or MICO (1uM). We also included an untreated control. After a treatment, the remaining cells were quantified and the cells were allowed to regrow for another 48 hours. This process is one cycle. At the end of the experiment, biofilm cells were exposed to 15 cycles. DNA extraction was performed on the evolved cells and of cells from the start population. DNA sequencing was performed on these samples and single nucleotide polymorphisms compared to the start population were evaluated.