Investigation of Colloidal Structure and Biopharmaceutical Properties of New Antibacterial Composition of Gramicidin S

Introduction. Gramicidin S has been conventionally manufactured as buccal tablets. However, in the past decade, the interest in the development of spray formulations has been growing. Those formulations contain excipients that enhance the solubility of the antibiotic in water solutions. However, the real structure of gramicidin S containing sprays remains unrevealed.Aim. Investigation of colloidal structure and biopharmaceutical properties of new gramicidin S antibacterial composition.Materials and methods. The composition sample was obtained using gramicidin S dihydrochloride, propylene glycol, polysorbate-80, ethanol and purified water. Raman spectroscopy has been performed to determine the composition of the phases. Dynamic light scattering analysis was performed to characterize the composition particles. Release of gramicidin S was performed by dialysis method and the concentration was determined by HPLC. The antimicrobial properties were investigated in accordance with the requirements of the XIV edition of the Russian pharmacopoeia.Results and discussion. Dynamic light scattering analysis results show gramicidin S formulation particles having an average size in solution 5–50 nm and ζ-potential (–1.1: +7.9 mV). Based on the obtained data on the composition properties and formulation parameters it was classified as colloidal solution. The kinetic stability evaluation was performed. We compared the solubility in water and release parameters of the active pharmaceutical ingredient in the native state and in the micelles. The enhancement of the antimicrobial activity of the peptide in the colloidal solution was confirmed and ascribed to the synergic effect gramicidin S – surfactant.Conclusion. We reported the colloidal type of the composition, that aggregate gramicidin S at a concentration of 8 mg/mL. We found that gramicidin S inclusion into the colloidal solution led to significant efficiency increase, which reveals the potential to reduce the drug dose and side effects level.

The Antimicrobial Peptide Gramicidin S Enhances Membrane Adsorption and Ion Pore Formation Potency of Chemotherapy Drugs in Lipid Bilayers

We recently published two novel findings where we found the chemotherapy drugs (CDs) thiocolchicoside (TCC) and taxol to induce toroidal type ion pores and the antimicrobial peptide gramicidin S (GS) to induce transient defects in model membranes. Both CD pores and GS defects were induced under the influence of an applied transmembrane potential (≈100 mV), which was inspected using the electrophysiology record of membrane currents (ERMCs). In this article, I address the regulation of the membrane adsorption and pore formation of CDs due to GS-induced possible alterations of lipid bilayer physical properties. In ERMCs, low micromolar (≥1 μM) GS concentrations in the aqueous phase were found to cause an induction of defects in lipid bilayers, but nanomolar (nM) concentration GS did nothing. For the binary presence of CDs and GS in the membrane-bathing aqueous phase, the TCC pore formation potency is found to increase considerably due to nM concentration GS in buffer. This novel result resembles our recently reported finding that due to the binary aqueous presence of two AMPs (gramicidin A or alamethicin and GS), the pore or defect-forming potency of either AMP increases considerably. To reveal the underlying molecular mechanisms, the influence of GS (0–400 nM) on the quantitative liposome (membrane) adsorption of CD molecules, colchicine and TCC, was tested. I used the recently patented direct detection method, which helps detect the membrane active agents directly at the membrane in the mole fraction relative to its concentrations in aqueous phase. We find that GS, at concentrations known to do nothing to the lipid bilayer electrical barrier properties in ERMCs, increases the membrane adsorption (membrane uptake) of CDs considerably. This phenomenological finding along with the GS effects on CD-induced membrane conductance increase helps predict an important conclusion. The binary presence of AMPs alongside CDs in the lipid membrane vicinity may work toward enhancing the physical adsorption and pore formation potency of CDs in lipid bilayers. This may help understand why CDs cause considerable cytotoxicity.

First Draft Genome Sequence of Thermophilic Laceyella tengchongensis BKK01, Isolated from Municipal Solid Waste in Thailand

ABSTRACT Laceyella tengchongensis BKK01 is a thermophilic bacterium isolated from municipal solid waste. The genome of L. tengchongensis BKK01 includes a gene putatively encoding gramicidin S synthase. Gramicidin S has antibiotic activity against some bacteria and fungi. The newly sequenced 3.44-Mb draft genome of L. tengchongensis BKK01 will shed some light on the biosynthesis of gramicidin S.

Supreme activity of gramicidin S against resistant, persistent and biofilm cells of staphylococci and enterococci

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.

[Leu2]Gramicidin S preserves the structural properties of its parent peptide and forms helically aligned β-sheets

For crystallographic analysis, Leu was substituted for Orn in Gramicidin S (LGS) to suppress interactions with hydrophilic solvent molecules, which increased the flexibility of the Orn side chains, leading to disorder within the crystals. The asymmetric unit (C62H94N10O10·1.296C3H8O·1.403H2O) contains three LGS molecules (A, B and C) forming β-turn and intramolecular β-sheet structures. With the exception of one motif in molecule C, D-Phe-Pro turn motifs (Phe is phenylalanine and Pro is proline) were classed as type II′ β-turns. The peptide backbones twist slightly to the right along the long axis of the molecule. The puckering of Pro is in a Cγ-endo or twisted Cγ-endo–Cβ-exo form. Flanking molecules are arranged such that the angles (A...B = 104°, B...C = 139° and C...A = 117°) form helical β-sheets. Solvent molecules interact with the peptide backbones supporting the β-sheets. The forms of the replacement Leu side chains are consistent with the e-form of the Orn side chain in GS analogues. No hydrophilic region composed of solvent molecules, such as that observed in Gramicidin S hydrochloride (GS·HCl) crystals, was found. The perturbation of αH chemical shifts and coupling constants of CONH showed that the structural properties of GS·HCl and LGS are similar to each other in solution. CD spectra also supported the structural similarity. With the sequence cyclo(–Val–Leu–Leu–D-Phe–Pro–)2 (Val is valine and Leu is leucine), LGS lacks the amphiphilicity and antimicrobial activity of parental Gramicidin S (GS). However, the structure of LGS reflects the structural characteristics of GS and no disordering inconvenient for structural analysis was found. Thus, LGS could be a novel scaffold useful for studying β-turn and sheet structures.