Lack of the Major Multifunctional Catalase KatA in Pseudomonas aeruginosa Accelerates Evolution of Antibiotic Resistance in Ciprofloxacin-Treated Biofilms

During chronic biofilm infections, Pseudomonas aeruginosa bacteria are exposed to increased oxidative stress as a result of the inflammatory response. As reactive oxygen species (ROS) are mutagenic, the evolution of resistance to ciprofloxacin (CIP) in biofilms under oxidative stress conditions was investigated. We experimentally evolved six replicate populations of P. aeruginosa lacking the major catalase Delta KatA in colony biofilms and stationary-phase cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of CIP or without CIP (eight replicate lineages for controls) under aerobic conditions. In CIP-evolved biofilms, a larger CIP-resistant subpopulation was isolated in the Delta katA strain than in the wild-type (WT) PAO1 population, suggesting oxidative stress as a promoter of the development of antibiotic resistance. A higher number of mutations identified by population sequencing were observed in evolved Delta katA biofilm populations (OP and control) than in WT PAO1 populations evolved under the same conditions. Genes involved in iron assimilation were found to be exclusively mutated in CIP-evolved AkatA biofilm populations, probably as a defense mechanism against ROS formation resulting from Fenton reactions. Furthermore, a hypermutable lineage due to mutL inactivation developed in one CIPevolved Delta katA biofilm lineage. In CIP-evolved biofilms of both the Delta katA strain and WT PAO1, mutations in nfxB, the negative regulator of the MexCD-OprJ efflux pump, were observed while in CIP-evolved planktonic cultures of both the Delta katA strain and WT PAO1, mutations in mexR and naID, regulators of the MexAB-OprM efflux pump, were repeatedly found. In conclusion, these results emphasize the role of oxidative stress as an environmental factor that might increase the development of antibiotic resistance in in vivo biofilms.