Effect of meso-substituents and medium properties on the photo- and pH-stability, penetration efficiency into bacterial and microscopic fungi cells of terpene-BODIPY conjugates.

In order to expand the arsenal of tools and areas for practical use of BODIPY dyes as bifunctional fluorescent theranostics, we studied the effect of the meso-substituents nature and medium properties on photo- and pH-stability, efficiency of singlet oxygen generation, and affinity to biostructures of terpene-BODIPY conjugates. The BODIPYs fused with myrtenol or thiotherpenoid via carboxylic acid residues exhibit high stability over a wide pH range and the presence of a bulky substituent at the meso-position of BODIPY conjugates increases their photostability two-fold compared to structurally related meso-unsubstituted analogues. Furthermore, the photodegradation rate of the conjugates directly depends on their ability to generate singlet oxygen and the course probability of the corresponding red-ox reactions involving reactive oxygen species. The conjugate of BODIPY with a thiotherpenoid demonstrated high ability to penetrate the membranes of filamentous and yeast-like fungi and bind to membrane of organelles in the fungal cell. At the same time, this compound also had a high ability to penetrate into biofilms of Staphylococcus aureus and Klebsiella pneumoniae and into bacterial cells within the matrix, which makes this compound promising for staining intracellular structures of eukaryotic cells and bacteria embedded into biofilms.

Alterations in Antibiotic Susceptibility of Staphylococcus aureus and Klebsiella pneumoniae in Dual Species Biofilms.

In the last decades, it has been shown that biofilm-associated infections in most cases are caused by rather two or even more pathogens than by single microorganisms. Due to intermicrobial interactions in mixed communities, bacteria change their gene expression profile, in turn leading to alterations in the biofilm structure and properties, as well as susceptibility to antimicrobials. Here, we report the alterations of antimicrobials efficiency in mixed biofilms of Staphylococcus aureus-Klebsiella pneumoniae in comparison with mono-species biofilms of each counterpart and discuss possible mechanisms of these alterations. In cell clumps detached from dual-species biofilms, S. aureus became insensitive to vancomycin, ampicillin, and ceftazidime compared to solely S. aureus cell clumps. In turn, the increased efficiency of amikacin and ciprofloxacin against both bacteria could be observed, compared to mono-species biofilms of each counterpart. Scanning electron microscopy and confocal microscopy indicate the porous structure of the dual-species biofilm, and differential fluorescent staining revealed an increased number of polysaccharides in the matrix, in turn leading to more loose structure and thus apparently providing increased permeability of the dual-species biofilm to antimicrobials. The qRT-PCR showed that ica operon in S. aureus became repressed in mixed communities, and polysaccharides are produced mainly by K. pneumoniae. While the molecular trigger of these changes remains undiscovered, detailed knowledge of the alterations in antibiotic susceptibility to given drugs opens doors for treatment correction options for S. aureus-K. pneumoniae biofilm-associated infections.

The Novel Chiral 2(5H)-Furanone Sulfones Possessing Terpene Moiety: Synthesis and Biological Activity.

Over the past decades, 2(5H)-furanone derivatives have been extensively studied because of their promising ability to prevent the biofilm formation by various pathogenic bacteria. Here, we report the synthesis of a series of optically active sulfur-containing 2(5H)-furanone derivatives and characterize their biological activity. Novel thioethers were obtained by an interaction of stereochemically pure 5-(l)-menthyloxy- or 5-(l)-bornyloxy-2(5H)-furanones with aromatic thiols under basic conditions. Subsequent thioethers oxidation by an excess of hydrogen peroxide in acetic acid resulted in the formation of the corresponding chiral 2(5H)-furanone sulfones. The structure of synthesized compounds was confirmed by IR and NMR spectroscopy, HRMS, and single crystal X-ray diffraction. The leading compound, 26, possessing the sulfonyl group and l-borneol moiety, exhibited the prominent activity against Staphylococcus aureus and Bacillus subtilis with MICs of 8 µg/mL. Furthermore, at concentrations of 0.4-0.5 µg/mL, the sulfone 26 increased two-fold the efficacy of aminoglycosides gentamicin and amikacin against S. aureus. The treatment of the model-infected skin wound in the rat with a combination of gentamicin and sulfone 26 speeded up the bacterial decontamination and improved the healing of the wound. The presented results provide valuable new insights into the chemistry of 2(5H)-furanone derivatives and associated biological activities.

Brightfield vs Fluorescent Staining Dataset-A Test Bed Image Set for Machine Learning based Virtual Staining.

Differential fluorescent staining is an effective tool widely adopted for the visualization, segmentation and quantification of cells and cellular substructures as a part of standard microscopic imaging protocols. Incompatibility of staining agents with viable cells represents major and often inevitable limitations to its applicability in live experiments, requiring extraction of samples at different stages of experiment increasing laboratory costs. Accordingly, development of computerized image analysis methodology capable of segmentation and quantification of cells and cellular substructures from plain monochromatic images obtained by light microscopy without help of any physical markup techniques is of considerable interest. The enclosed set contains human colon adenocarcinoma Caco-2 cells microscopic images obtained under various imaging conditions with different viable vs non-viable cells fractions. Each field of view is provided in a three-fold representation, including phase-contrast microscopy and two differential fluorescent microscopy images with specific markup of viable and non-viable cells, respectively, produced using two different staining schemes, representing a prominent test bed for the validation of image analysis methods.

Increasing the Efficacy of Treatment of Staphylococcus aureus-Candida albicans Mixed Infections with Myrtenol.

Infectious diseases caused by various nosocomial microorganisms affect worldwide both immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (-)-myrtenol and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and benzalkonium chloride on 64-81% of the clinical isolates of S. aureus and C. albicans, including MRSA and fluconazole-resistant fungi, while (-)-myrtenol increased the properties of amikacin and fluconazole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myrtenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an S. aureus-C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both (-)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by membrane damage since the treatment with both terpenes led to a significant drop in membrane potential similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it seems to be a promising agent to increase the efficiency of the treatment of infections caused by bacteria and be fungi of the genus Candida as well as mixed fungal-bacterial infections, including resistant strains.

Design, Spectral Characteristics, and Possibilities for Practical Application of BODIPY FL-Labeled Monoterpenoid.

This article describes the design and biological properties of a BODIPY FL-labeled monoterpenoid BF2-meso-(4-((1″R)-6″,6″-dimethylbicyclo[3.1.1]hept-2″-ene-2″)yl-methoxycarbonylpropyl)-3,3',5,5'-tetramethyl-2,2'-dipyrromethene conjugate (BODIPYmyrt). The fluorophore was characterized using X-ray, NMR, MS, and UV/vis spectroscopy. The conjugate exhibits a high quantum yield (to ∼100%) in the region 515-518 nm. BODIPYmyrt effectively penetrates the membranes of the bacterial and fungal cells and therefore can be used to examine the features of a broad spectrum of Gram-positive and Gram-negative bacteria and pathogenic fungi as well. Moreover, BODIPYmyrt exhibits a moderate tropism to the subcellular structures in mammalian cells (e.g., mitochondria), thereby providing an attractive scaffold for fluorophores to examine these particular organelles.

Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus-Pseudomonas aeruginosa dual-species biofilm.

In mixed infections, the bacterial susceptibility differs significantly compared to monocultures of bacteria, and generally the concentrations of antibiotics required for the treatment increases drastically. For S. aureus and P. aeruginosa dual species biofilms, it has been numerously reported that P. aeruginosa decreases S. aureus susceptibility to a broad range of antibiotics, including beta-lactams, glycopeptides, aminoglycosides, macrolides, while sensitizes to quinolones via secretion of various metabolites. Here we show that S. aureus also modulates the susceptibility of P. aeruginosa to antibiotics in mixed cultures. Thus, S. aureus-P. aeruginosa consortium was characterized by tenfold increase in susceptibility to ciprofloxacin and aminoglycosides compared to monocultures. The same effect could be also achieved by the addition of cell-free culture of S. aureus to P. aeruginosa biofilm. Moreover, similar increase in antibiotics efficacy could be observed following addition of S. aureus suspension to the P. aeruginosa mature biofilm, compared to P. aeruginosa monoculture, and vice versa. These findings open promising perspectives to increase the antimicrobial treatment efficacy of the wounds infected with nosocomial pathogens by the transplantation of the skin residential microflora.

Targeting Bacillus cereus cells: increasing efficiency of antimicrobials by the bornylpossessing 2(5Н)-furanone derivative.

Among a variety of antimicrobial compounds, the derivatives of 2(5H)-furanone exhibit different effects on Firmicutes and Proteobacteria. While inhibiting quorum-dependent biofilm formation and virulence factor expression by Gram-negative bacteria through specific interference with the AI-2 signaling pathways, these compounds demonstrate bactericidal effects against Gram-positive bacteria. Here we report that 3,4-dichloro-5(S)-[(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy]-2(5H)-furanone designed as F123 inhibits growth and biofilm formation by the food-poisoning bacterium Bacillus cereus at 8 µg/ ml and kills bacteria at 16 µg/ml. While the growth of Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis were also inhibited at 8-16 µg/ml of F123, no bactericidal effect on these strains was observed at concentrations up to 128 µg/ml, suggesting pronounced specificity of F123 for B. cereus. In a checker-board assay F123 increased the efficacy of amikacin, gentamicin and benzalkonium chloride against B. cereus with medians of fractional inhibitory concentration index of 0.38, 0.56 and 0.56, respectively. Moreover, the number of viable B. cereus cells in biofilm was reduced by more than 3 orders of magnitude at 64 µg/ml of F123, suggesting its chemotype as a promising enhancer for specific treatment of B. cereus-associated topical infections, including biofilm-embedded bacteria.

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus.

The gram-positive opportunistic bacterium Staphylococcus aureus is one of the most common causatives of a variety of diseases including skin and skin structure infection or nosocomial catheter-associated infections. The biofilm formation that is an important virulence factor of this microorganism renders the antibiotic therapy ineffective, because biofilm-embedded bacteria exhibit strongly increased tolerance to antimicrobials. Here, we describe a novel 3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone (F105), possessing a sulfonyl group and l-menthol moiety. Minimal inhibitory and bactericidal concentration values (MIC and MBC) of F105 were 10 and 40 mg/L, respectively, suggesting F105 biocidal properties. F105 exhibits pronounced activity against biofilm-embedded S. aureus and increases the efficacy of aminoglycosides (amikacin, gentamicin, and kanamycin) and benzalkonium chloride with fractional inhibitory concentration index values of 0.33-0.44 and 0.29, respectively, suggesting an alternative external treatment option, e.g., for wound infections. Moreover, low concentrations (0.5-1.3 mg/L) of F105 reduced the MICs of these antimicrobials twofold. By using confocal laser scanning microscopy and CFU counting, we show explicitly that F105 also restores the antimicrobial activity of gentamicin and ampicillin against S. aureus biofilms by several orders of magnitude. Biofilm structures were not destroyed but sterilized, with embedded cells being almost completely killed at twofold MBC. While F105 is quite toxic (CC50/MBC ratio 0.2), our data suggest that the F105 chemotype might be a promising starting point for the development of complex topical agents for combined anti-staphylococcal biofilm-therapies restoring the efficacy of some antibiotics against difficult to treat S. aureus biofilm.

Targeting microbial biofilms using Ficin, a nonspecific plant protease.

Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 µg/ml and six-fold at 1000 µg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 µg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

Inhibition of biofilm formation in Bacillus subtilis by new halogenated furanones.

Gram-positive bacteria can cause various infections including hospital-acquired infections. While in the biofilm, the resistance of bacteria to both antibiotics and the human immune system is increased causing difficulties in the treatment. Bacillus subtilis, a non-pathogenic Gram-positive bacterium, is widely used as a model organism for studying biofilm formation. Here we investigated the effect of novel synthesized chloro- and bromo-containing 2(5H)-furanones on biofilm formation by B. subtilis. Mucobromic acid (3,4-dibromo-5-hydroxy-2(5H)-furanone) and the two derivatives of mucochloric acid (3,4-dichloro-5-hydroxy-2(5H)-furanone)-F8 and F12-were found to inhibit the growth and to efficiently prevent biofilm formation by B. subtilis. Along with the low production of polysaccharide matrix and repression of the eps operon, strong repression of biofilm-related yqxM also occurred in the presence of furanones. Therefore, our data confirm that furanones affect significantly the regulatory pathway(s) leading to biofilm formation. We propose that the global regulator, Spo0A, is one of the potential putative cellular targets for these compounds.