A coevolution experiment reveals parallel mutations in the AcrA-AcrB-TolC efflux pump that contributes to bacterial antibiotic resistance

One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic and/or acquired mechanisms. Here, we performed a coevolution experiment to understand long-term consequences of antibiotic production and antibiotic resistance for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We first observed that the F. johnsoniae ancestor could tolerate the B. thailandensis-produced antibiotic through efflux mechanisms. We then sequenced the genomes of clonal isolates from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications to the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of antibiotic resistance that were not observed in the evolved monoculture lines. Two mutations were found in tolC: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Placing the tolC 33 bp deletion back into the F. johnsoniae ancestor conferred some antibiotic resistance, but not to the degree of resistance observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis-produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis-produced antibiotics were involved in the inhibition of F. johnsoniae. This study reveals how long-term interspecies chemical interactions can result in a novel mutation in efflux that contribute to antibiotic resistance.