Diclofenac Degradation—Enzymes, Genetic Background and Cellular Alterations Triggered in Diclofenac-Metabolizing Strain Pseudomonas moorei KB4

Abstract

Diclofenac (DCF) constitutes one of the most significant ecopollutants detected in various environmental matrices. Biological clean-up technologies that rely on xenobiotics-degrading microorganisms are considered as a valuable alternative for chemical oxidation methods. Up to now, the knowledge about DCF multi-level influence on bacterial cells is fragmentary. In this study, we evaluate the degradation potential and impact of DCF on Pseudomonas moorei KB4 strain. In mono-substrate culture KB4 metabolized 0.5 mg L−1 of DCF, but supplementation with glucose (Glc) and sodium acetate (SA) increased degraded doses up to 1 mg L−1 within 12 days. For all established conditions, 4′-OH-DCF and DCF-lactam were identified. Gene expression analysis revealed the up-regulation of selected genes encoding biotransformation enzymes in the presence of DCF, in both mono-substrate and co-metabolic conditions. The multifactorial analysis of KB4 cell exposure to DCF showed a decrease in the zeta-potential with a simultaneous increase in the cell wall hydrophobicity. Magnified membrane permeability was coupled with the significant increase in the branched (19:0 anteiso) and cyclopropane (17:0 cyclo) fatty acid accompanied with reduced amounts of unsaturated ones. DCF injures the cells which is expressed by raised activities of acid and alkaline phosphatases as well as formation of lipids peroxidation products (LPX). The elevated activity of superoxide dismutase (SOD) and catalase (CAT) testified that DCF induced oxidative stress.

Application of Immobilized Biocatalysts in the Biotransformation of Non-Steroidal Anti-Inflammatory Drugs

“…The bacteria in which the complete decomposition of paracetamol has been described include the Pseudomonas moorei KB4 strain isolated from the activated sludge from the wastewater treatment plant Klimzowiec (Chorzów, Poland), which is also characterized by the ability to degrade diclofenac [24,37]. In turn, the Bacillus thuringiensis B1(2015b) strain, isolated from the soil of the chemical factory Organika-Azot in Jaworzno (Poland), belongs to the bacteria that break down ibuprofen and naproxen [38,39].…”

Evaluation of the Effects of Ag, Cu, ZnO and TiO2 Nanoparticles on the Expression Level of Oxidative Stress-Related Genes and the Activity of Antioxidant Enzymes in Escherichia coli, Bacillus cereus and Staphylococcus epidermidis

“…For Gram-positive bacteria, an additional incubation with lysosome at 37 • C for 1 h was performed in order to disintegrate the double-layer of peptidoglycan of the cell wall. The extracted total RNA was subjected to additional purification with RNase-free DNase (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) to digest the residual genomic DNA present in the samples [55]. The concentration and purity of collected RNA were assessed using an ND-1000 NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) through the measurement of the absorbance of acquired samples at 230, 260 and 280 nm and the calculating of 260/280 and 260/230 optical density (OD) ratios [36,41,55].…”

“…The extracted total RNA was subjected to additional purification with RNase-free DNase (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) to digest the residual genomic DNA present in the samples [55]. The concentration and purity of collected RNA were assessed using an ND-1000 NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) through the measurement of the absorbance of acquired samples at 230, 260 and 280 nm and the calculating of 260/280 and 260/230 optical density (OD) ratios [36,41,55]. Moreover, the quality and integrity of the obtained RNA samples were examined through agarose gel electrophoresis [55].…”

“…K1621, ThermoFisher Scientific, Waltham, MA, USA). For this purpose, 1 µg of total RNA from each sample was used [55]. Aliquots of cDNA were stored at −21 • C for further experiments.…”

Draft Genomic Sequences of Pseudomonas moorei Strains Isolated from Wild Cranberry Bogs in Massachusetts

“… Pseudomonas moorei , first isolated from Elbe river sediments in Germany, is notable for its ability to detoxify pharmaceutical industry environmental contaminants such as 4- and 5-chlorosalicylates, acetaminophen, and diclofenac ( 1 – 3 ), but nothing is known about the activities of these bacteria in pristine ecosystems. Two P. moorei strains were isolated from dune swale bog soils as part of a culture-dependent survey of bacteria within the Cape Cod National Seashore (42.070624 N, 70.210548 W and 42.064742 N, 70.117562 W) in early July 2012.…”