Lack of the major multifunctional catalase KatA in P.aeruginosa accelerates evolution of antibiotic resistance in ciprofloxacin-treated biofilms

During chronic biofilm infections, Pseudomonas aeruginosa 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 KatA in colony biofilms and stationary-phase cultures for seven passages in the presence of sub-inhibitory levels (0.1 mg/L) 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 ΔkatA compared to WT PAO1 population suggesting oxidative stress as a promoter of antibiotic resistance development.A higher number of mutations identified by population sequencing were observed in evolved ΔkatA biofilm populations (CIP and control) compared to WT PAO1 evolved under same conditions.

Genes involved in iron assimilation were found to be exclusively mutated in CIP-evolved ΔkatA 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 CIP-evolved ΔkatA biofilm lineage.In CIP-evolved biofilms of both ΔkatA 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 ΔkatA and WT PAO1, mutations in mexR and nalD, regulators of 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.