TY - JOUR

T1 - Lack of the Major Multifunctional Catalase KatA in Pseudomonas aeruginosa Accelerates Evolution of Antibiotic Resistance in Ciprofloxacin-Treated Biofilms

AU - Ahmed, Marwa N.

AU - Porse, Andreas

AU - Abdelsamad, Ahmed

AU - Sommer, Morten

AU - Høiby, Niels

AU - Ciofu, Oana

PY - 2019

Y1 - 2019

N2 - 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 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 ∆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 ∆katA biofilm populations (CIP 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 ∆katA 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 ∆katA biofilm lineage. In CIP-evolved biofilms of both the ∆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 ∆katA strain and WT PAO1, mutations in mexR and nalD, 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.

AB - 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 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 ∆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 ∆katA biofilm populations (CIP 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 ∆katA 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 ∆katA biofilm lineage. In CIP-evolved biofilms of both the ∆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 ∆katA strain and WT PAO1, mutations in mexR and nalD, 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.

KW - Antibiotic resistance

KW - Biofilm

KW - Catalase mutant

KW - Oxidative stress

KW - Pseudomonas aeruginosa

U2 - 10.1128/AAC.00766-19

DO - 10.1128/AAC.00766-19

M3 - Journal article

C2 - 31307984

AN - SCOPUS:85072627375

VL - 63

JO - Antimicrobial Agents and Chemotherapy

JF - Antimicrobial Agents and Chemotherapy

SN - 0066-4804

IS - 10

M1 - e00766-19

ER -