Evolution of antibiotic resistance in biofilm and planktonic P. aeruginosa populations exposed to sub-inhibitory levels of ciprofloxacin [PublishAheadOfPrint]

The opportunistic Gram-negative pathogen Pseudomonas aeruginosa, known for its intrinsic and acquired antibiotic resistance, has a notorious ability to form biofilms, which often facilitate chronic infections. The evolutionary paths to antibiotic resistance have mainly been investigated in planktonic cultures and are less studied in biofilms. We experimentally evolved P. aeruginosa PAO1 colony-biofilms and stationary-phase planktonic cultures for seven passages in the presence of sub-inhibitory levels (0.1 mg/L) of ciprofloxacin (CIP) and performed a genotypic (whole bacterial population sequencing) and phenotypic assessment of the populations. We observed a higher proportion of CIP resistance in the CIP-evolved biofilm populations compared to planktonic populations exposed to the same drug concentrations. However, the minimal inhibitory concentrations (MICs) of ciprofloxacin were lower in CIP-resistant isolates selected from biofilm population compared to the MICs of CIP-resistant isolates from the planktonic cultures. We found common evolutionary trajectories between the different lineages, with mutations in known CIP resistance determinants as well as growth condition-dependent adaptations. A general trend towards a reduction in type IV-pili dependent motility (twitching) in CIP-evolved populations, and towards loss of virulence associated traits in the populations evolved in the absence of antibiotic, was observed. In conclusion, our data indicate that biofilms facilitate the development of low-level mutational resistance, probably due to the lower effective drug exposure compared to planktonic cultures. These results provide a framework for the selection process of resistant variants and the evolutionary mechanisms in the two different growth conditions.

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