Modeling of Effectiveness of N3-Substituted Amidrazone Derivatives as Potential Agents against Gram-Positive Bacteria.

Prediction of the antibacterial activity of new chemical compounds is an important task, due to the growing problem of bacterial drug resistance. Generalized linear models (GLMs) were created using 85 amidrazone derivatives based on the results of antimicrobial activity tests, determined as the minimum inhibitory concentration (MIC) against Gram-positive bacteria: Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Nocardia corallina, and Mycobacterium smegmatis. For the analysis of compounds characterized by experimentally measured MIC values, we included physicochemical properties (e.g., molecular weight, number of hydrogen donors and acceptors, topological polar surface area, compound percentages of carbon, nitrogen, and oxygen, melting points, and lipophilicity) as potential predictors. The presence of R1 and R2 substituents, as well as interactions between melting temperature and R1 or R2 substituents, were also considered. The set of potential predictors also included possible biological effects (e.g., antibacterial, antituberculotic) of tested compounds calculated with the PASS (Prediction of Activity Spectra for Substances) program. Using GLMs with least absolute shrinkage and selection (LASSO), least-angle regression, and stepwise selection, statistically significant models with the optimal value of the adjusted determination coefficient and of seven fit criteria were chosen, e.g., Akaike's information criterion. The most often selected variables were as follows: molecular weight, PASS_antieczematic, PASS_anti-inflam, squared melting temperature, PASS_antitumor, and experimental lipophilicity. Additionally, relevant to the bacterial strain, the interactions between melting temperature and R1 or R2 substituents were selected, indicating that the relationship between MIC and melting temperature depends on the type of R1 or R2 substituent.

Evaluation of Biological Activity of New 1,2,4-Triazole Derivatives Containing Propionic Acid Moiety.

To this day, the quest to find new drugs is still a challenge due to the growing demands of patients suffering from chronic inflammatory diseases and the need for the individualization of therapy. The aim of this research was to synthesize new 1,2,4-triazole derivatives containing propanoic acid moiety and to investigate their anti-inflammatory, antibacterial and anthelmintic activity. Compounds 3a-3g were obtained in reactions of amidrazones 1a-1g with succinic anhydride. Several analyses of proton and carbon nuclear magnetic resonance (1H NMR, 13C NMR, respectively), as well as high-resolution mass spectra (HRMS), confirmed the structures of 1,2,4-triazole derivatives 3a-3g. Toxicity, antiproliferative activity and influence on cytokine release (TNF-α: Tumor Necrosis Factor-α, IL-6: Interleukin-6, IFN-γ: Interferon-γ, and IL-10: Interleukin-10) of the compounds 3a-3g were evaluated in peripheral blood mononuclear cells culture. Moreover, mitogen-stimulated cell culture was used for biological activity tests. The antimicrobial and anthelmintic activity of derivatives 3a-3g were studied against Gram-positive and Gram-negative bacterial strains and Rhabditis sp. culture. Despite the lack of toxicity, compounds 3a-3g significantly reduced the level of TNF-α. Derivatives 3a, 3c and 3e also decreased the release of IFN-γ. Taking all of the results into consideration, compounds 3a, 3c and 3e show the most beneficial anti-inflammatory effects.

Synthesis and Structural Study of Amidrazone Derived Pyrrole-2,5-Dione Derivatives: Potential Anti-Inflammatory Agents.

1H-pyrrole-2,5-dione derivatives are known for their wide range of pharmacological properties, including anti-inflammatory and antimicrobial activities. This study aimed to synthesize new 3,4-dimethyl-1H-pyrrole-2,5-dione derivatives 2a-2f in the reaction of N3-substituted amidrazones with 2,3-dimethylmaleic anhydride and evaluate their structural and biological properties. Compounds 2a-2f were studied by the 1H-13C NMR two-dimensional techniques (HMQC, HMBC) and single-crystal X-ray diffraction (derivatives 2a and 2d). The anti-inflammatory activity of compounds 2a-2f was examined by both an anti-proliferative study and a production study on the inhibition of pro-inflammatory cytokines (IL-6 and TNF-α) in anti-CD3 antibody- or lipopolysaccharide-stimulated human peripheral blood mononuclear cell (PBMC) cultures. The antibacterial activity of compounds 2a-2f against Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Esherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Mycobacterium smegmatis and Nocardia corralina strains was determined using the broth microdilution method. Structural studies of 2a-2f revealed the presence of distinct Z and E stereoisomers in the solid state and the solution. All compounds significantly inhibited the proliferation of PBMCs in anti-CD3-stimulated cultures. The strongest effect was observed for derivatives 2a-2d. The strongest inhibition of pro-inflammatory cytokine production was observed for the most promising anti-inflammatory compound 2a.

Methicillin-resistant Staphylococcus aureus and glycopeptide-resistant enterococci in fecal samples of birds from South-Eastern Poland.

BACKGROUND: The incidence of human infection and colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) has increased in the recent years. Environmental sources, including bird droppings, might play an important role as resistance reservoirs. RESULTS: Fresh fecal samples were collected from rooks and wild-living birds during the autumn-winter period of 2016/2017, and tested for the presence of bacteria associated with human diseases. Besides bacteria representing the genera Enterococcus, Campylobacter, Escherichia, and Staphylococcus, Enterobacter, Citrobacter, Proteus, Hafnia, and Pseudomonas were also identified. The susceptibility of S. aureus and Enterococcus spp. isolates to methicillin, and vancomycin and teicoplanin, respectively, was analyzed to assess the avian wildlife as a reservoir of MRSA and VRE strains. Twenty-two percent of all S. aureus isolates were methicillin-resistant. These strains were screened by polymerase chain reaction (PCR), using the most widely used primer sets specific for the mecA gene. Twenty percent of all Enterococcus strains were phenotypically vancomycin-resistant. The presence of van resistance genes in these strains was investigated by PCR using vanA and vanB gene-specific primers. A good correlation between mecA gene detection and disc diffusion data was observed, while some discrepancy was noted between the PCR data and the vancomycin/teicoplanin phenotypic resistance pattern. The incidence of strains resistant to methicillin and glycopeptide antibiotics in wild-living birds was twice that in rooks. CONCLUSIONS: The study suggests that rooks from urban areas and passerine birds from the natural habitat carry antibiotic-resistant Enterococcus spp. and S. aureus strains, probably reflecting the presence of such isolates in the environmental food sources.

Electrophoretic profiles of lipopolysaccharides from Rhizobium strains nodulating Pisum sativum do not reflect phylogenetic relationships between these strains.

Rhizobia that nodulate peas comprise a heterogeneous group of bacteria. The aim of this study was to investigate the relationship between phylogeny and electrophoretic and hydroxy fatty acid lipopolysaccharide (LPS) profiles of pea microsymbionts. Based on amplified fragment length polymorphism (AFLP) fingerprinting data, the pea microsymbionts were grouped into two clusters distinguished at 58% similarity level. Based on the concatenated 16S rRNA, recA, and atpD housekeeping gene data, the microsymbionts appeared to be most closely related to Rhizobium leguminosarum biovars viciae and trifolii. Applying cluster analysis to their LPS electrophoretic profiles, the strains were assigned to two major groups with different banding patterns. All hydroxy fatty acids common to R. leguminosarum and R. etli were detected in each examined strain. Differences in the proportions of 3- to ω-1 hydroxy fatty acids allowed us to distinguish two groups of strains. This classification did not overlap with one based on LPS electrophoretic profiles. No clear correlation was apparent between the genetic traits and LPS profiles of the pea nodule isolates.

ANTIBACTERIAL AND CENTRAL NERVOUS SYSTEM ACTIVITY OF (4,5-DIARYL-4H-1,2,4-TRIAZOL-3-YL)METHACRYLIC ACID DERIVATIVES.

The series of 1,2,4-triazole derivatives containing methacrylic acid moiety were synthesized in reaction of N3-substituted amidrazones with itaconic anhydride. Preliminary calculated bioavailability parameters of obtained compounds suggested good penetration via cell membranes and their good absorption after oral intake. Antimicrobial evaluation in vitio showed diverse activity of obtained triazoles mainly on Gram-positive bacterial strains. One derivative was also examined to determine the effect on the central nervous system of mice.

NEW HETEROCYCLIC OXIME ETHERS OF 1-(BENZOFURAN-2-YL)ETHAN- 1-ONE AND THEIR ANTIMICROBIAL ACTIVITY.

In this study, some O-benzyl (benzofuran-2-yl)ethan-1-one ether oximes were synthesized starting from 2-acetylbenzofuran. The structure elucidation of the compounds was performed by IR, 1H-NMR and 13C-NMR spectra. Antimicrobial activities of the compounds were examined and notable activity was observed.

Reactions of N(3) -substituted amidrazones with cis-1,2-cyclohexanedicarboxylic anhydride and biological activities of the products.

A series of novel compounds were synthesized in reactions of N(3) -substituted amidrazones with cis-1,2-cyclohexanedicarboxylic anhydride: linear, isoindole, and triazole derivatives. All new structures were confirmed by H(1) NMR and IR spectrometry as well as elemental analysis. Potential biological effects of new compounds were predicted with the Prediction of Activity Spectra for Substances (PASS) program. Antiviral, antibacterial, analgesic, and anti-inflammatory activities were experimentally verified.

Ozone-Based Eye Drops Activity on Ocular Epithelial Cells and Potential Pathogens Infecting the Front of the Eye.

Confirmation of the biological effectiveness of new ophthalmic preparations introduced in the market is an important element in maintaining the safety of using this type of medications. This study aimed to investigate the activity of Ozodrop® on human corneal and conjunctival epithelial cells, as well as its antibacterial and antifungal activity. Cytotoxicity analyses of ocular surface epithelial cells were performed in vitro by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Neutral Red uptake assays. The level of nitric oxide released by the cells was assessed by the Griess method. The reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical by the tested formulation was analyzed. Microbiological tests were also performed. It was found that the Ozodrop® preparation exhibited biological activity, but was less active than the reference antibiotics and the anti-yeast agent. The cytotoxic activity of the Ozodrop® formulation was dependent on the time of cell exposure to it. No toxic effect was observed in the short-term, for up to 3 h. It appeared after 24 h of exposure of the cells to the preparation. The drops showed antioxidant activity in the specified concentration range. They also stimulated the release of nitric oxide, mainly by corneal epithelial cells. The Ozodrop® formulation exhibits biological activity that can be considered useful in the treatment of infections in the front part of the eye.

Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment.

BACKGROUND: Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized. RESULTS: In this work, we extend our earlier studies to the characterization of rosR mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in rosR also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The rosR mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the rosR mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of rosR mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional rosR is important for competitive nodulation. CONCLUSIONS: This report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and R. leguminosarum bv. trifolii 24.2.

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi.

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC50 (10.04 ± 0.75 µg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 µg/ml). The EC50value reached 12.6 ± 1.51 µg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.

Anti-Candida albicans effect of the protein-carbohydrate fraction obtained from the coelomic fluid of earthworm Dendrobaena veneta.

An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

Synthesis and evaluation of new amidrazone-derived hydrazides as a potential anti-inflammatory agents.

ABSTRACT: The series of new hydrazide derivatives were synthesized in reactions of N3-substituted amidrazones with cyclic anhydrides as potential anti-inflammatory and antibacterial agents. The compounds were characterized by 1H-13C two-dimensional NMR techniques, which revealed the presence of two tautomeric forms in DMSO-d6 solutions, while the molecular structure of one species was confirmed by single-crystal X-ray diffraction. The anti-inflammatory effects of hydrazides on peripheral blood mononuclear cells were experimentally evaluated. Three compounds showed antiproliferative activity comparable to ibuprofen. One derivative demonstrated strong reduction of lymphocyte proliferation stimulated by anti-CD3 antibody (by 90%) and PHA, as well as low cell toxicity. The obtained compounds exhibited relatively weak antibacterial activity; they were more effective against Gram-positive bacterial strains.

PssP2 is a polysaccharide co-polymerase involved in exopolysaccharide chain-length determination in Rhizobium leguminosarum.

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.

Synthesis, crystal structure and biological activities of a novel amidrazone derivative and its copper(II) complex--a potential antitumor drug.

A new linear amidrazone derivative, 6-acetyl-cyclohex-3-enecarboxylic acid [1-pyridin-2-yl-1-(pyridyn-2-yloamin)meth-(Z)-ylidene] hydrazide, H(2)L (2) and its Cu(II) complex, [Cu(2)L(2)]·4H(2)O (3) were synthesized and characterized by elemental analysis, IR and (1)H NMR spectroscopy and cyclic voltammetry. Compound 2 was synthesized in the equimolar reaction of N(3)-substituted amidrazone with cis-1,2,3,6-tetrahydrophthalic anhydride. The Cu complex of 2 was obtained in the reaction with copper(II) acetate. The molecular structures of 2 and 3 were determined by X-ray crystallography. The parent ligand exists in its amide-hydrazone form in the solid state. The central amidrazone moiety has a Z configuration with respect to the double C=N bond. Coordination to the metal center promotes Z/E isomerization of the hydrazone group of the ligand. Compound 3 is a dinuclear four-coordinated Cu(II) complex with the amidrazone ligand behaving as a tetradentate double deprotonated chelating one. Several biological activities of 2 and 3 were examined in vitro; they were: antimicrobial properties against selected bacterial and fungal strains, suppression of phytohemagglutinin A (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMC) and their effects on tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) production. The cytotoxic activity of Cu(II) complex was determined with respect to the four carcinoma cell lines (SW 984, CX-1, L-1210, A-431). The studied complex exhibited significant cytotoxic effects (particularly against CX-1 colon carcinoma), comparable to those reported for cisplatin. Both compounds have shown a relatively low antibacterial activity and were devoid of antifungal properties.

The incomplete substitution of lipopolysaccharide with O-chain prevents the establishment of effective symbiosis between Mesorhizobium loti NZP2213.1 and Lotus corniculatus.

Mesorhizobium loti NZP2213.1 mutant obtained after random Tn5 mutagenesis of M. loti NZP2213 was inefficient in nitrogen fixation on Lotus corniculatus. The transposon insertion was located within an ORF with a sequence similarity to a putative glycosyl transferase from Caulobacter crescentus. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the mutant produced LPS of the same O-chain length but only half of the entire smooth LPS, compared to that of the parental strain. A greater diversity of the anomeric region as determined by NMR spectroscopy, reflected structural differences in the mutant repeating units represented by 6-deoxytalose, 2-OAc-6-deoxytalose, and 2-OMe-6-deoxytalose. In contrast to the completely O-acetylated 6-deoxytalose in wild-type OPS only partial O-acetylation was found in the mutant. The decrease of the LPS species with O-chains seems to be correlated with 6-deoxytalose deficiency. Microscopic examination of the nodules induced by the mutant revealed disturbances in infection thread development and premature senescence of symbiosomes. The impairment of mutant-induced symbiosomes to sustain latter stages of symbiosis could be a consequence of the decreased ratio of the hydrophobic to the hydrophilic LPSs.

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides.

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopies, including 2D (1)H/(1)H COSY, TOCSY, ROESY, and H-detected (1)H/(13)C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure: [Formula: see text] where 2-acetamido-2-deoxy-4-O-methyl-D-glucose (D-GlcNAc4Me) and methyl group on 2-substituted D-rhamnose (Me) shown in italics are present in approximately 80% and approximately 40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-D-rhamnose ( approximately 70%).

Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants.

Rhizobium leguminosarum biovar trifolii strain TA1 polysaccharide synthesis (pss) mutants in the pssD, pssP, pssT and pssO genes and altered in exopolysaccharide (EPS) synthesis were investigated. EPS-deficient mutants were also changed in lipopolysaccharide structure. All mutants exhibited varied sensitivities to detergents, ethanol and antibiotics, thus indicating changes in bacterial membrane integrity. Using pss mutants marked with the gusA gene, EPS-deficient mutants were found to have abnormalities in nodule development and to provoke severe plant defence reactions. The pss mutants that produced altered quantities of EPS with a changed degree of polymerisation generally occupied the younger developmental zones of the nodules and elicited moderate plant defence reactions.