Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Ahmed MN

PROVIDER: S-EPMC6761533 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Ahmed Marwa N MN Porse Andreas A Abdelsamad Ahmed A Sommer Morten M Høiby Niels N Ciofu Oana O

Antimicrobial agents and chemotherapy 20190923 10

During chronic biofilm infections, <i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> lacking the major catalase KatA in colony biofilms and stationary-phase cultures for seven passages in ...[more]

PMID: 31307984

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Project description:Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.

Project description:OUTLINE:In chronic lung infections by Pseudomonas aeruginosa (PA) the bacteria thrive in biofilm structures protected from the immune system of the host and from antibiotic treatment. Increasing evidence suggests that the susceptibility of the bacteria to antibiotic treatment can be significantly enhanced by hyperbaric oxygen treatment. The aim of this study is to simulate the effect of ciprofloxacin treatment in a PAO1 biofilm model with aggregates in agarose when combined with hyperbaric oxygen treatment. This is achieved in a reaction-diffusion model that describes the combined effect of ciprofloxacin diffusion, oxygen diffusion and depletion, bacterial growth and killing, and adaptation of the bacteria to ciprofloxacin. In the model, the oxygen diffusion and depletion use a set of parameters derived from experimental results presented in this work. The part of the model describing ciprofloxacin killing uses parameter values from the literature in combination with our estimates (Jacobs, et al., 2016; Grillon, et al., 2016). Micro-respirometry experiments were conducted to determine the oxygen consumption in the P. aeruginosa strain PAO1. The parameters were validated against existing data from an HBOT experiment by Kolpen et al. (2017). The complete oxygen model comprises a reaction-diffusion equation describing the oxygen consumption by using a Michaelis-Menten reaction term. The oxygen model performed well in predicting oxygen concentrations in both time and depth into the biofilm. At 2.8 bar pure oxygen pressure, HBOT increases the penetration depth of oxygen into the biofilm almost by a of factor 4 in agreement with the scaling that follows from the stationary balance between the consumption term and diffusion term. CONCLUSION:In the full reaction-diffusion model we see that hyperbaric oxygen treatment significantly increases the killing by ciprofloxacin in a PAO1 biofilm in alignment with the experimental results from Kolpen et al. (Kolpen, et al., 2017; Kolpen, et al. 2016). The enhanced killing, in turn, lowers the oxygen consumption in the outer layers of the biofilm, and leads to even deeper penetration of oxygen into the biofilm.

Dataset Information

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

Publications

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

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