Sensitive fluorescent imaging of <Emphasis Type="Italic">Salmonella enteritidis</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> using a polyvalent directed peptide polymer

The authors describe three fluorescein-conjugated peptides generated by cell-phage display for use as a diagnostic probes for fluorescent detection and imaging of Salmonella enteritidis and Salmonella typhimurium. The authors also designed a polyvalent-directed peptide polymer synthesized with poly-D-lysine and bifunctional succinimidyl 3-(2-pyridyldithio)propionate with an affinity and sensitivity that is higher by more than an order of magnitude compared to single peptides due to multiple binding site interactions. In order to establish a diagnostic system for food poisoning, imaging analysis was performed using fluorescence microscopy. The limit of detection of the diagnostic system based on polyvalent directed peptide interaction is 10² colony-forming units per mL for Salmonella.

Open image in new window

Graphical abstract Schematic of a fluorescent method for detection and imaging of Salmonella enteritidis and Salmonella typhimurium by using a fluorescein labeled polyvalent-directed peptide polymer (PDPP) with a high affinity and sensitivity as a diagnostic probe. The system uses a microplate reader and was applied to the detection of food poisoning.

     

Bischelated complexes of a dithiocarbazate <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">S</Emphasis> Schiff base ligand: synthesis,characterization and antimicrobial activities

The N,S bidentate proligand S-hexyl-β-N-(4-methoxybenzylidene)dithiocarbazate (HL), obtained by condensation of S-hexyldithiocarbazate with 4-methoxybenzaldehyde, has been used to synthesize six metal complexes, namely NiL₂, CuL₂, ZnL₂, CdL₂, PdL₂ and PbL₂, which have been characterized by physicochemical techniques and spectroscopic methods. Single crystal structural analyses for NiL₂, CuL₂ and PdL₂ show that these are square-planar complexes with each metal bischelated by the Schiff base in its deprotonated monoanionic form. In all three cases, the ligands show a trans configuration, although they crystallize in different space groups. All the metal complexes with the exception of the nickel derivative show a significant decrease in fluorescence intensity with respect to the free proligand HL. Free HL and all six complexes were tested for antibacterial activity against three pathogenic gram-negative organisms. The metal complexes show moderate although diverse activities; however, free HL as well as the copper(II) complex did not reveal any antibacterial activity against the tested organisms.     

Photochromism of an <Emphasis Type="Italic">N</Emphasis>-aminofulgimide-derived Schiff base

A Schiff base was synthesized from 5-methoxysalicylaldehyde and N-aminofulgimide, viz., 1-amino-3-[1-(2,5-dimethylthiophen-3-yl)ethylidene]-4-isopropylidenepyrrolidine-2,5-dione. The compound synthesized undergoes photochromic transformations typical of those of N-aminofulgimide studied earlier. A bathochromic shift of the absorption bands of the open and cyclic forms of the obtained Schiff base compared to the starting N-aminofulgimide was observed.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Analysis of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 Biofilm Protein Profile After Exposure to <Emphasis Type="Italic">n</Emphasis>-Butanolic <Emphasis Type="Italic">Cyclamen coum</Emphasis> Extract Alone and in Combination with Ciprofloxacin

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.     

Microcalorimetric study on the growth and metabolism of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Microcalorimetry is an experimental technique which allows us to precisely measure the energy released as a consequence of any transformation process. All organisms produce heat as a consequence of metabolism. The rate of heat production is an adequate measurement of metabolic activity of organisms and their constituent parts, cells and sub-cellular levels. Microorganisms produce small amounts of heat, in the order of 1–3 pW per cell. Despite the low quantity of heat produced by bacteria, their exponential replication in culture medium allows their detection using microcalorimetry. This study is a microcalorimetric study of the growth and metabolism of the bacterium Pseudomonas aeruginosa, using the heat liberated as a consequence of bacterial metabolism. With this aim, we used a Calvet microcalorimeter, inside which two Teflon screw-capped stainless steel cells were located (sample and reference). Experiments were carried out at final concentrations of 10⁶, 10⁵, 10³ and 10 CFU/mL, and a constant temperature of 309.65 K was maintained within the microcalorimeter. Recording the difference in calorific potential over time we obtained P. aeruginosa’s growth curves. The shape of these curves is characteristic and has a single phase. Thus, the heat flow curves were mathematically studied to calculate the growth constant and generation time of this bacterium.     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Grown in Residual Soybean Oil

Pseudomonas aeruginosa PACL strain, isolated from oil-contaminated soil taken from a lagoon, was used to investigate the efficiency and magnitude of biosurfactant production, using different waste frying soybean oils, by submerged fermentation in stirred tank reactors of 6 and 10 l capacities. A complete factorial experimental design was used, with the goal of optimizing the aeration rate (0.5, 1.0, and 1.5 vvm) and agitation speed (300, 550, and 800 rpm). Aeration was identified as the primary variable affecting the process, with a maximum rhamnose concentration occurring at an aeration rate of 0.5 vvm. At optimum levels, a maximum rhamnose concentration of 3.3 g/l, an emulsification index of 100%, and a minimum surface tension of 26.0 dynes/cm were achieved. Under these conditions, the biosurfactant production derived from using a mixture of waste frying soybean oil (WFSO) as a carbon source was compared to production when non-used soybean oil (NUSO), or waste soybean oils used to fry specific foods, were used. NUSO produced the highest level of rhamnolipids, although the waste soybean oils also resulted in biosurfactant production of 75–90% of the maximum value. Under ideal conditions, the kinetic behavior and the modeling of the rhamnose production, nutrient consumption, and cellular growth were established. The resulting model predicted data points that corresponded well to the empirical information.     

Characterization of Rhamnolipid Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolate Bs20

Rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20 is viscous sticky oily yellowish brown liquid with a fruity odor. It showed solubility at aqueous pH > 4 with optimum solubility at pH 7–7.5 and freely soluble in ethyl acetate. This biosurfactant has a very high surface activity as it could lower the surface tension of water to 30 mN/m at about 13.4 mg/L, and it exhibited excellent stabilities at high temperatures (heating at 100°C for 1 h and autoclaving at 121°C for 10 min), salinities (up to 6% NaCl), and pH values (up to pH 13). The produced biosurfactant can be used in the crude form either as cell-free or cell-containing culture broth of the grown bacteria, since both preparations showed high emulsification indices ranged between 59% and 66% against kerosene, diesel, and motor oil. These characters make the test rhamnolipid a potential candidate for use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. High-performance thin-layer chromatography densitometry revealed that the extracted rhamnolipid contained the two most active rhamnolipid homologues dirhamno dilipidic rhamnolipid and monorhamno dilipidic rhamnolipid at 44% and 56%, respectively, as compared to 51% and 29.5%, respectively, in a standard rhamnolipid preparation. The nature and ratio of these two rhamnolipid homologues showed to be strain dependent rather than medium-component dependent.     

Substrate specificity and function of acetylpolyamine amidohydrolases from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background

Pseudomonas aeruginosa, a Gram-negative, aerobic coccobacillus bacterium is an opportunistic human pathogen and worldwide the fourth most common cause of hospital-acquired infections which are often high mortality such as ventilator-associated pneumoniae. The polyamine metabolism of P. aeruginosa and particularly the deacetylation of acetylpolyamines has been little studied up to now. Results with other bacterial pathogens e.g., Y. pestis suggest that polyamines may be involved in the formation of biofilms or confer resistance against certain antibiotics.

Results

To elucidate the role of acetylpolyamines and their enzymatic deacetylation in more detail, all three putative acetylpolyamine amidohydrolases (APAHs) from P. aeruginosa have been expressed in enzymatic active form. The APAHs PA0321 and PA1409 are shown to be true polyamine deacetylases, whereas PA3774 is not able to deacetylate acetylated polyamines. Every APAH can hydrolyze trifluoroacetylated lysine-derivatives, but only PA1409 and much more efficiently PA3774 can also process the plain acetylated lysine substrate. P. aeruginosa is able to utilize acetylcadaverine and acetylputrescine as a carbon source under glucose starvation. If either the PA0321 or the PA1409 but not the PA3774 gene is disrupted, the growth of P. aeruginosa is reduced and delayed. In addition, we were able to show that the APAH inhibitors SAHA and SATFMK induce biofilm formation in both PA14 and PAO1 wildtype strains.

Conclusions

P. aeruginosa has two functional APAHs, PA0321 and PA1409 which enable the utilization of acetylpolyamines for the metabolism of P. aeruginosa. In contrast, the physiological role of the predicted APAH, PA3774, remains to be elucidated. Its ability to deacetylate synthetic acetylated lysine substrates points to a protein deacetylation functionality with yet unknown substrates.

     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

New tin(<Emphasis Type="SmallCaps">iv</Emphasis>) <Emphasis Type="Italic">o</Emphasis>-iminosemiquinone complexes

New tin(iv) mono- and bis-o-iminosemiquinone complexes were obtained by the exchange reaction of radical anion lithium salt of 4,6-di-tert-butyl-N-(2,6-diisopropylphenyl)-o-imino-benzoquinone with tin(iv) organochlorides. The compounds synthesized were characterized by EPR spectroscopy and X-ray diffraction analysis. Substituents on the tin atom were found to affect stability of paramagnetic metal derivatives formed.     

Synthesis of 6-methyl-8<Emphasis Type="Italic">H</Emphasis>-dibenzo[<Emphasis Type="Italic">a</Emphasis>,<Emphasis Type="Italic">g</Emphasis>]quinolizin-8-imines <Emphasis Type="Italic">via Reissert</Emphasis> compounds

Alkylation of Reissert compounds derived from 3-methylisoquinolines with several 2-cyanobenzylbromides followed by hydrolytic cleavage provided the corresponding 1-benzyl-3-methylisoquinolines. Treatment of the latter with methylmagnesiumiodide caused cyclization to the title compounds rather than formation of 2-acetylbenzylisoquinolines.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> nicotinate mononucleotide adenylyltransferase mutants <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD⁺ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD⁺, especially for one of the alleles, nadD72.     

Conformations and properties of <Emphasis Type="Italic">O</Emphasis>-Alkyl-<Emphasis Type="Italic">S</Emphasis>-(2-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dialkylamino)-ethylmethylthiophosphonates

Conformers of the biologically active compounds CH₃P(O)(OR)(SCH₂CH₂NR ₂ ^′ ), where (I) R = i-C₄H₉, R′ = C₂H₅ and (II) R = C₂H₅, R′ = i-C₃H₇, are calculated within the AM1 level of theory. The elongated and twisted forms with maximum and minimum distances between a nitrogen atom and those of a phosphorus tetrahedron, respectively, and bearing a syn and anti oriented alkoxy group relative to a phosphoryl oxygen, are studied. It is found that the differences between the energy, electronic, and geometric parameters of these forms are apparent in differences between their properties, e.g., the ability to participate in complexation and protonation, reactions that to some extent simulate the interaction between a substance and a biological object.     

<Emphasis Type="Italic">E. coli</Emphasis>, <Emphasis Type="Italic">P. aeruginosa</Emphasis>, and <Emphasis Type="Italic">B. cereus</Emphasis> Bacteria Sterilization Using Afterglow of Non-Thermal Plasma at Atmospheric Pressure

We developed and employed a new geometrical structure of dielectric barrier discharge in atmospheric pressure for bacterial broad spectrum sterilization. We utilized a plasma source having an AC power supply at 50 HZ and 5,400 V (rms value). We prepared suspensions of the Gram-negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) and a Gram-positive of Bacillus cereus with Luria–Bertani broth media up to OD_600 nm = 0.25 of McFarland standard. Afterglow of non-thermal atmospheric pressure plasma treated these suspensions. The influence of the atmospheric plasma afterglow on the species was assayed in different time durations 5, 10, and 15 min. The spectroscopic results of this investigation indicated that the survival reduction of the species can reach to 100% for P. aeruginosa in an exposure time of 10 min, E. coli and B. cereus in an exposure time of 15 min.