Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells.

Diclofenac (DCF) is one of the most commonly utilized non-steroidal anti-inflammatory drugs (NSAIDs), which is known to pose an ecotoxicological threat. In this study, from activated sludge and contaminated soil, we isolated four new bacterial strains able to degrade DCF under mono-substrate and co-metabolic conditions with glucose supplementation. We found that the effectiveness of DCF removal is strictly strain-specific and the addition of the primary substrate is not always beneficial. To assess the multidirectional influence of DCF on bacterial cells we evaluated the alterations of increasing concentrations of this drug on membrane structure. A significant increase was observed in the content of 17:0 cyclo fatty acid, which is responsible for reduced fluidity and profound changes in membrane rigidity. The cell injury and oxidative stress were assessed with biomarkers used as endpoints of toxicity, i.e. catalase (CAT), superoxide dismutase (SOD), lipids peroxidation (LPX), and both intra- and extracellular alkaline and acid phosphatase activity. Results indicated that DCF induced oxidative stress, frequently intensified by the addition of glucose. However, the response of the microbial cells to the presence of DCF should not be generalized, since the overall picture of the particular alterations greatly varied for each of the examined strains.

Genome Mining and Evaluation of the Biocontrol Potential of Pseudomonas fluorescens BRZ63, a New Endophyte of Oilseed Rape (Brassica napus L.) against Fungal Pathogens.

Endophytic bacteria hold tremendous potential for use as biocontrol agents. Our study aimed to investigate the biocontrol activity of Pseudomonas fluorescens BRZ63, a new endophyte of oilseed rape (Brassica napus L.) against Rhizoctonia solani W70, Colletotrichum dematium K, Sclerotinia sclerotiorum K2291, and Fusarium avenaceum. In addition, features crucial for biocontrol, plant growth promotion, and colonization were assessed and linked with the genome sequences. The in vitro tests showed that BRZ63 significantly inhibited the mycelium growth of all tested pathogens and stimulated germination and growth of oilseed rape seedlings treated with fungal pathogens. The BRZ63 strain can benefit plants by producing biosurfactants, siderophores, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and ammonia as well as phosphate solubilization. The abilities of exopolysaccharide production, autoaggregation, and biofilm formation additionally underline its potential to plant colonization and hence biocontrol. The effective colonization properties of the BRZ63 strain were confirmed by microscopy observations of EGFP-expressing cells colonizing the root surface and epidermal cells of Arabidopsis thaliana Col-0. Genome mining identified many genes related to the biocontrol process, such as transporters, siderophores, and other secondary metabolites. All analyses revealed that the BRZ63 strain is an excellent endophytic candidate for biocontrol of various plant pathogens and plant growth promotion.

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

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.

Comparative Genomics of Stenotrophomonas maltophilia and Stenotrophomonas rhizophila Revealed Characteristic Features of Both Species.

Although Stenotrophomonas maltophilia strains are efficient biocontrol agents, their field applications have raised concerns due to their possible threat to human health. The non-pathogenic Stenotrophomonas rhizophila species, which is closely related to S. maltophilia, has been proposed as an alternative. However, knowledge regarding the genetics of S. rhizophila is limited. Thus, the aim of the study was to define any genetic differences between the species and to characterise their ability to promote the growth of plant hosts as well as to enhance phytoremediation efficiency. We compared 37 strains that belong to both species using the tools of comparative genomics and identified 96 genetic features that are unique to S. maltophilia (e.g., chitin-binding protein, mechanosensitive channels of small conductance and KGG repeat-containing stress-induced protein) and 59 that are unique to S. rhizophila (e.g., glucosylglycerol-phosphate synthase, cold shock protein with the DUF1294 domain, and pteridine-dependent dioxygenase-like protein). The strains from both species have a high potential for biocontrol, which is mainly related to the production of keratinases (KerSMD and KerSMF), proteinases and chitinases. Plant growth promotion traits are attributed to the biosynthesis of siderophores, spermidine, osmoprotectants such as trehalose and glucosylglycerol, which is unique to S. rhizophila. In eight out of 37 analysed strains, the genes that are required to degrade protocatechuate were present. While our results show genetic differences between the two species, they had a similar growth promotion potential. Considering the information above, S. rhizophila constitutes a promising alternative for S. maltophilia for use in agricultural biotechnology.

Organic micropollutants paracetamol and ibuprofen-toxicity, biodegradation, and genetic background of their utilization by bacteria.

Currently, analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are classified as one of the most emerging group of xenobiotics and have been detected in various natural matrices. Among them, monocyclic paracetamol and ibuprofen, widely used to treat mild and moderate pain are the most popular. Since long-term adverse effects of these xenobiotics and their biological and pharmacokinetic activity especially at environmentally relevant concentrations are better understood, degradation of such contaminants has become a major concern. Moreover, to date, conventional wastewater treatment plants (WWTPs) are not fully adapted to remove that kind of micropollutants. Bioremediation processes, which utilize bacterial strains with increased degradation abilities, seem to be a promising alternative to the chemical methods used so far. Nevertheless, despite the wide prevalence of paracetamol and ibuprofen in the environment, toxicity and mechanism of their microbial degradation as well as genetic background of these processes remain not fully characterized. In this review, we described the current state of knowledge about toxicity and biodegradation mechanisms of paracetamol and ibuprofen and provided bioinformatics analysis concerning the genetic bases of these xenobiotics decomposition.

Paracetamol - toxicity and microbial utilization. Pseudomonas moorei KB4 as a case study for exploring degradation pathway.

Paracetamol, a widely used analgesic and antipyretic drug, is currently one of the most emerging pollutants worldwide. Besides its wide prevalence in the literature only several bacterial strains able to degrade this compound have been described. In this study, we isolated six new bacterial strains able to remove paracetamol. The isolated strains were identified as the members of Pseudomonas, Bacillus, Acinetobacter and Sphingomonas genera and characterized phenotypically and biochemically using standard methods. From the isolated strains, Pseudomonas moorei KB4 was able to utilize 50 mg L-1 of paracetamol. As the main degradation products, p-aminophenol and hydroquinone were identified. Based on the measurements of specific activity of acyl amidohydrolase, deaminase and hydroquinone 1,2-dioxygenase and the results of liquid chromatography analyses, we proposed a mechanism of paracetamol degradation by KB4 strain under co-metabolic conditions with glucose. Additionally, toxicity bioassays and the influence of various environmental factors, including pH, temperature, heavy metals at no-observed-effective-concentrations, and the presence of aromatic compounds on the efficiency and mechanism of paracetamol degradation by KB4 strain were determined. This comprehensive study about paracetamol biodegradation will be helpful in designing a treatment systems of wastewaters contaminated with paracetamol.

Properties of Antibiotic-Resistant Bacteria Isolated from Onsite Wastewater Treatment Plant in Relation to Biofilm Formation.

The aim of the present study was to determine some properties of antibiotic-resistant bacterial strains isolated from onsite wastewater technology in relation to biofilm formation, e.g., autoaggregation and motility. Additionally, biosurfactant production by the isolates was also evaluated. The ability of selected strains to develop a biofilm was assessed by using the crystal violet method, which allows to indirectly quantify the attached bacterial biomass (live, dead cells, and polysaccharides as well). Obtained results showed that 19 of the analyzed strains were able to produce biofilm after 72 h of incubation. The low values of surface tension in the range between 28 and 36 mN/m were observed in the bacteria, which are not able to produce biofilm or be classified as weak biofilm producers. Among biofilm-forming strains the highest autoaggregation index was observed for Mycobacterium brumae and Bacillus alcalophilus. Noteworthy, that some strains capable of biofilm formation showed no aggregation abilities or were characterized by low autoaggregative properties. The results of visual autoaggregation assay showed no visible flocs after given time of incubation. The results from motility test demonstrated that most of the analyzed strains were motile. Noteworthy, that up to now literature data about physiology, biofilm formation, and autoaggregative capabilities of bacteria isolated from onsite wastewater technology are very limited and this paper gives the information on the antibiotic-resistant bacteria with ability to form biofilm. Thus, the present study points to develop novel bioinocula in antibiotic degradation and to reach novel biofilm-dispersing agents produced by various bacteria that can be used as disinfectants or surface-coating agents to prevent microbial surface colonization and biofilm development.

Metabolic Responses of Bacterial Cells to Immobilization.

In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability.

Yersinia enterocolitica-Specific Infection by Bacteriophages TG1 and ϕR1-RT Is Dependent on Temperature-Regulated Expression of the Phage Host Receptor OmpF.

UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.