Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.