Rapid detection of lytic antimicrobial activity against yeast and filamentous fungi.

A rapid method for assessing the lytic activity of antimicrobial agents against yeast and fungi has been developed. The assay is based on the release of the intracellular enzyme, maltase (alpha-glucosidase). The released maltase activity was measured colorimetrically by the production of p-nitrophenol from p-nitrophenyl-alpha-D-glucopyranoside (PNPG). The lytic activity of different antimicrobial compounds was measured against yeast cells or germinating spores of filamentous fungi. Lytic anti-yeast activity could be detected within 20 min incubation at 30 degrees C against Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans. Lytic anti-fungal activity appeared after 2 h of incubation at 30 degrees C against germinating spores of Aspergillus niger and Botrytis cinerea. Whole cells of either yeast or fungi did not hydrolyze sufficient PNPG within 3 h at 30 degrees C to yield a detectable color change. Lytic activity of enzymes (e.g., Lyticase), antibiotics (e.g., Amphotericin B), and an antibiotic-producing strain of bacteria were detected using the assay. The anti-yeast assay has been adapted to a 96-well microtiter format. Both assays provided a rapid, sensitive, and reproducible detection of lytic anti-yeast and anti-fungal activity.     

Rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation

Considering the increased incidence of bacterial infections and the emergence of multidrug resistant bacteria at the global level, we designed superparamagnetic iron oxide nanoparticles as nanosensors for the assessment of antimicrobial susceptibility through magnetic relaxation. In this report, we demonstrate that iron oxide nanosensors, either dextran-coated supplemented with Con A or silica-coated conjugated directly to Con A, can be used for the fast (1) quantification of polysaccharides, (2) assessment of metabolic activity and (3) determination of antimicrobial susceptibility in blood. The use of these polysaccharide nanosensors in the determination of antimicrobial susceptibility in the clinic or the field, and the utilization of these nanoprobes in pharmaceutical R&D are anticipated.     

Biocatalytic preparation of acylated derivatives of flavonoid glycosides enhances their antioxidant and antimicrobial activity

Enzymatic synthesis of acylated derivatives of a monosaccharidic flavonoid chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside as well as of a disaccharidic flavonoid chrysoeriol-7-[6'-O-acetyl-beta-D-allosyl-(1-->2)-beta-D-glucopyranoside], isolated from Greek endemic plants, was performed using an immobilized Candida antarctica lipase in non-toxic organic solvents. The influence of the reaction parameters such as the molar ratio of acyl donor to flavonoid, as well as the nature of the acyl donor, on the performance of the biocatalytic process was pointed out using the acylation of naringin as a model reaction. With vinyl laurate as acyl donor, the highest conversion was observed at relatively high molar ratio (>or=10), using acetone as solvent. Lipase exhibits specificity towards primary alcohol of the glucose moiety of both flavonoid glycosides. The introduction of an acyl group into glucosylated flavonoids significantly improved their antioxidant activity towards both LDL and serum model in vitro. Furthermore, the acylated derivative of disaccharidic flavonoid increased its antimicrobial activity against two Gram-positive bacteria.     

Polymer antimicrobial coatings with embedded fine Cu and Cu salt particles

Many diseases spread due to the bacterial infections, which cause significant economic and personal losses. Contact with infected surfaces is likely to catch infections. Hence, antimicrobial surfaces play an important role in public sectors that can prevent the spreading of disease and infection. Coatings on contact surfaces have been used to provide antimicrobial function. Copper (Cu), as one of the commonly used metals, has long been known to possess germ-killing properties. However, metallic Cu or Cu coatings have not been widely used for the purposes of antimicrobial due to the heavy weight, relatively high cost, limited corrosion resistance and low compatibility of the metal with substrates of non-metallic materials. We have recently developed a polymer-based coating system containing mixtures of fine particles of Cu and Cu salt, which provides excellent antimicrobial properties. The results indicate that the coating with embedded fine Cu salt showed higher antimicrobial property than the coating with only Cu due to the release of more Cu ions. The elimination of 10⁶ bacteria by contacting the polymer-Cu coatings needs 8 h, while contacting with the polymer-CuCl₂ coatings took only 20 min to kill the same amount of bacteria. We have also used transmission electron microscopy and synchrotron infrared microscopy technique to study the degradation of bacterial cell membrane to understand the mechanism of the antimicrobial function of Cu coating.     

An antimicrobial peptide with antimicrobial activity against Helicobacter pylori

An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had similarity to antimicrobial peptide odorranain-W1 but it has a different GLLR N-terminus. The cDNA encoding odorranain-HP was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms. Interestingly, odorranain-HP could exert antimicrobial capability against Helicobacter pylori, along with its antimicrobial activities similar to odorranain-W1. This is the first report of naturally occurring peptide with anti-H. pylori activity from amphibian skins.     

Development of the biological preparation enatin with broad-range antimicrobial action

Physiological and biochemical traits of epiphytic spore forming bacteria Bacillus pumilis BIM V-263 were examined. The nutrient medium and conditions for submerged cultivation of the strain were selected. The growth dynamics and antagonistic activity during cultivation in a laboratory fermenter ANKUM-2M were studied. The results provide grounds for development of the biological preparation Enatin with broad-range antimicrobial effect. The plant-protective and growth-stimulating effect of Enatin was examined in laboratory and field experiments. The preparation holds promise as means for biological control of crop pathogens.     

Novel gratisin derivatives with high antimicrobial activity and low hemolytic activity

The substitution of each constituent amino acid residue of gratisin (GR) with Ala residue indicated that each side chain structure of the constituent amino acid residues affect largely the antibiotic and hemolytic activities of GR. Among them, the substitution of Pro residues at positions 5 and 5′ with a cationic amino acid residues (Lys and Arg) results the high antibiotic activity and the low toxicity against human blood cells. Thus, we have found a novel position on the scaffold of GR at Pro^5,5′ residues whose modification will significantly lower the unwanted hemolytic activity and enhance the desired antibiotic activity.     

Aminoglycoside antibiotic derivatives: preparation and evaluation of toxicity on cochlea and vestibular tissues and antimicrobial activity

Aminoglycoside antibiotic derivatives such as neamine, methyl neobiosaminide B, 2-deoxystreptamine, tetra-azidoneamine, tetra-N-acetylneamine, tetra-N-carboxy-benzylneamine, tetra-N-carboxy-methylneamine and tetra-p-methoxy-benzyliminoneamine were prepared and evaluated as to their cochlear and vestibular toxicity. Methyl neobiosaminide B, the most promising derivative in the series showed selective, cochlea-dissociated vestibulotoxic activity and was considered to be a potential lead compound for the treatment of Ménière's disease. Antimicrobial properties of the compounds, qualitatively evaluated against a group of pathogenic bacteria, indicated that neomycin B sulfate, neamine as a free base and methyl-neobiosaminide B dihydrochloride show a broader range of activity when compared to the other derivatives.     

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH₂) showed the greatest antimicrobial activity (MIC₉₀ of 16 μM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Terpenes with antimicrobial activity from Cretan propolis

Five terpenes, the diterpenes: 14,15-dinor-13-oxo-8(17)-labden-19-oic acid and a mixture of labda-8(17),13E-dien-19-carboxy-15-yl oleate and palmitate as well as the triterpenes, 3,4-seco-cycloart-12-hydroxy-4(28),24-dien-3-oic acid and cycloart-3,7-dihydroxy-24-en-28-oic acid were isolated from Cretan propolis. Moreover, 18 known compounds were also isolated, seven of them for the first time as propolis components. All structures were established on the basis of spectroscopic analysis and chemical evidence. All isolated compounds were tested for antimicrobial activity against some Gram-positive and Gram-negative bacteria as well as against some human pathogenic fungi showing a broad spectrum of antimicrobial activity.     

Synthetic magainin analogues with improved antimicrobial activity

Based on modifications to enhance the alpha-helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.     

A novel chimeric peptide with antimicrobial activity

Beta‐lactamase‐mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co‐administration of beta‐lactam type antibiotics and beta‐lactamase inhibitors. Antimicrobial peptides are promising broad‐spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46–Tyr51 beta‐hairpin loop of beta‐lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta‐lactamase. Here, our goal was to modify this peptide for improved beta‐lactamase inhibition and cellular uptake. Motivated by the cell‐penetrating pVEC sequence, which includes a hydrophobic stretch at its N‐terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N‐terminus to facilitate uptake. Activity measurements of the peptide based on the 45–53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta‐lactamase inhibitor with a K_i value of 58 μM. Incubation of beta‐lactam‐resistant cells with peptide decreased the number of viable cells, while it had no effect on beta‐lactamase‐free cells, indicating that this peptide had antimicrobial activity via beta‐lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N‐terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta‐lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity

Currently, novel antibiotics are urgently required to combat the emergence of drug‐resistant bacteria. Antimicrobial peptides with membrane‐lytic mechanism of action have attracted considerable interest. Anoplin, a natural α‐helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin‐4 composed of d ‐amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin‐4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin‐4 treatment relative to anoplin. In conclusion, anoplin‐4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

New chitosan derivatives with potential antimicrobial activity

A series of four water-soluble chitosan derivatives differing in molecular mass, hydrophobicity, and charge was synthesized and tested for the intensity of their effects on Gram-negative and Gram-positive bacteria. It was shown that the tested compounds allowed the penetration of ethidium bromide into the bacteria, which showed increased permeability of their cell walls under the effect of chitosans. The tolerance to various chitosan derivatives differed in Gram-negative and Gram-positive bacteria. The Gram-negative bacteria were the most responsive to high-molecular chitosan and the Gram-positive ones, to N-,O-carboxypropylchitosan, whereas high-molecular chitosan had little effect. Research on the correlation between the structure and activity of the studied compounds revealed that depolymerization of chitosan reduced, and introduction of hydrophobic substantives in chitosan molecule significantly enhanced its permeability effect on bacterial cell walls. The obtained results provide a basis for the construction of new chitosan derivatives with antimicrobial activities.     

Compounds from Allium species with cytotoxic and antimicrobial activity

Garlic (Allium sativum L.) is a bulb-shaped plant belonging to the Allium genus which also includes onions (Allium cepa L.), leek (Allium ampeloprasum L. var. porrum Gay), shallot (Allium ascalonicum L), scallion (A. fistulosum L.) and chives (Allium schoenoprasum L.). The biological activity of garlic has been known since ancient times. Babylonians, Egyptians, Phoenicians, Greeks and Romans used garlic as a remedy for intestinal disorders, respiratory infections, skin diseases, bacterial infections, worms, wounds and tumors. In particular, before the discovery of antibiotics, garlic has been used against amoebic dysentery and epidemic diseases such as typhus, cholera, diphtheria, and tuberculosis. To date, more than 3,000 publications scientifically supported the use of garlic in the ethno-medicine. But what makes garlic and Allium species effective against cancer? The effect of garlic may arise from its antibacterial properties or from its ability to block formation on cancer-causing substances, half the activation of cancer causing substances, enhance DNA repair, reduce cell proliferation or induce cell death. Epidemiological studies have found that an increase of consumptions of Allium spp. reduce the risk of prostate and gastric cancers and this has been mainly related to two main classes of compounds: the apolar sulphur compounds and the polar saponins. These latter compounds, compared to the more studied thiosulphinates, have the advantages of not being pungent and more stable during cooking. Recently, there has been increasing scientific attention given to such compounds. In this paper, the literature about the major volatile and non-volatile organic compounds of garlic and other Allium plants has been reviewed. Particular attention is given to the compounds possessing antibacterial and cytotoxic activity in garlic and in the other Allium species and their mechanism of action.     

Secondary metabolites from Centaurea deusta with antimicrobial activity

The aerial parts of Centaurea deusta Ten. afforded in addition to several known compounds, mainly sesquiterpene lactones, one new eudesmanolide and one new elemane derivative. Structures of the new compounds were elucidated by spectroscopic methods. The in vitro antifungal and antibacterial activities of the isolated compounds was tested, using the microdilution method. All compounds tested showed high antifungal activity.     

Synthetic flavonoids with antimicrobial activity: a review

The emergence of drug-resistant microbes left us with a great need for new antimicrobial agents. Flavonoids, with their wide range of biological activities, are good candidates in this respect. Although naturally occurring flavonoids are the most studied ones, semi-synthetic or synthetic flavonoids have proven to have great potential, inhibiting and even killing microbes at concentrations below 1 μg ml⁻¹. The substitution pattern of these flavonoids often includes hydroxy groups, halogens or other heteroatomic rings, such as pyridine, piperidine or 1,3-dithiolium cations. However, the great variety in substituents makes it difficult to draw any definitive conclusion regarding their structure–activity relationship.     

Biological activity of 4-hydroxyisophthalic acid derivatives. Hydrazones with antimicrobial activity

The following hydrazono derivatives (I-XIX) of type (A) (sequence in text) where Rn = (sequence in text ) (I-XVII); (sequence in text) (XVIII); -CCl3 (XIX); and Xn = H (I); 2-Cl (II); 3-Cl (III); 4-Cl (IV); 2-NO2 (V); 3-NO2 (VI); 4-NO2 (VII); 2-OH (VIII); 3-OH (IX); 4-OH (X); 4-F (XI); 3,4-OCH3,OH (XII); 3,4,5-OCH3,OH,J (XIII); 3,4-OCH3,OCH3 (XIV); 2,4-Cl2 (XV); 3,4-Cl2 (XVI); 2,6-Cl2 (XVII); were prepared and characterized in an attempt to make available for testing a representative selection of hitherto unreported 4-hydroxyisophthalic acid derivatives. The new compounds in question were obtained in satisfactory yield by condensation of 4-hydroxyisophthalic acid hydrazide with the appropriate aldehydes. The prepared compounds were tested for their possible activity against Gram-positive (S. epidermidis, B. subtilis, B. anthracis) and Gram-negative bacteria (P. aeruginosa, B. melitensis, S. typhi O, S. typhi H, S. infantis, S. paratyphi B, E. coli Bb, E. coli 7075), and fungi (C. albicans, A. niger, S. cerevisiae). The "in vitro" antimicrobial assays were carried out using the paper disk technique (Kirby-Bauer modified). The influence of certain structural modifications on the antimicrobial activity was evaluated.