Efficacy and mechanism of carvacrol with octanoic acid against mastitis causing multi-drug-resistant pathogens.

In the present investigation, we determined the in vitro antimicrobial activity of eight essential oils (EOs) and three medium-chain fatty acids (MCFAs) alone and in combination against Staphylococcus aureus ATCC 700698, Klebsiella pneumoniae ATCC 700603, and E. coli FcW5. The interactions between EOs and MCFAs were determined by fractional inhibitory concentration indices. Moreover, mode of action of selected bioactive components was studied by changes in bacterial surface charge, morphology, and membrane integrity assays. Among EOs, carvacrol (CAR), trans-cinnamaldehyde (TC), and thymol (TM) showed strong antimicrobial activity. In combination study, CAR+OA (octanoic acid), CAR+DA (decanoic acid), and TM+OA were observed as the most significant (P≤0.05) which were also confirmed through time-kill plots. Based on these results, CAR+OA were found to be most efficacious in terms of killing time (P≤0.05). Changes in the surface charge, morphology, and membrane integrity upon the combined treatment of CAR+OA were also observed, which ultimately leads to cell death. Results suggest that CAR+OA when used in combination offer a significant (P≤0.05) additive antimicrobial activity against the selected pathogenic bacteria. Therefore, these natural bioactive molecules could be interesting alternatives to conventional therapy for the control of mastitis caused by multi-drug-resistant pathogens in bovine animals to ensure the milk safety.

Epimers l- and d-Phenylseptin: How the relative stereochemistry affects the peptide-membrane interactions.

In recent decades, several epimers of peptides containing d-amino acids have been identified in antimicrobial sequences, a feature which has been associated with post-translational modification. Generally, d-isomers present similar or inferior antimicrobial activity, only surpassing their epimers in resistance to peptidases. The naturally occurring l-Phenylseptin (l-Phes) and d-Phenylseptin (d-Phes) peptides (FFFDTLKNLAGKVIGALT-nh2) were reported with d-epimer showing higher activity against Staphylococcus aureus and Xanthomonas axonopodis in comparison with the l-epimer. In this study, we combine structural (CD, solution NMR), orientational (solid-state NMR) and biophysical (ITC, DSC and DLS) studies to understand the role of the d-phenylalanine in the increase of the antimicrobial activity. Although both peptides are structurally similar in the helical region ranging from D4 to the C-terminus, significant structural differences were observed near the peptides' N-termini (which encompasses the FFF motif). Specific aromatic interactions involving the phenylalanine side chains of d-Phes is responsible to maintaining the F1-F3 residues on the hydrophobic face of the peptide, increasing its amphipathicity when compared to the l-epimer. The higher capability of d-Phes to exert an efficient anchoring in the hydrophobic core of the phospholipid bilayer indicates a pivotal role of the N-terminus in enhancing the interaction between the d-peptide and the membrane interface in relation to its epimer.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems.

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1-3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Antimicrobial metal-based nanoparticles: a review on their synthesis, types and antimicrobial action.

The investigation of novel nanoparticles with antimicrobial activity has grown in recent years due to the increased incidence of nosocomial infections occurring during hospitalization and food poisoning derived from foodborne pathogens. Antimicrobial agents are necessary in various fields in which biological contamination occurs. For example, in food packaging they are used to control food contamination by microbes, in the medical field the microbial agents are important for reducing the risk of contamination in invasive and routine interventions, and in the textile industry, they can limit the growth of microorganisms due to sweat. The combination of nanotechnology with materials that have an intrinsic antimicrobial activity can result in the development of novel antimicrobial substances. Specifically, metal-based nanoparticles have attracted much interest due to their broad effectiveness against pathogenic microorganisms due to their high surface area and high reactivity. The aim of this review was to explore the state-of-the-art in metal-based nanoparticles, focusing on their synthesis methods, types, and their antimicrobial action. Different techniques used to synthesize metal-based nanoparticles were discussed, including chemical and physical methods and "green synthesis" methods that are free of chemical agents. Although the most studied nanoparticles with antimicrobial properties are metallic or metal-oxide nanoparticles, other types of nanoparticles, such as superparamagnetic iron-oxide nanoparticles and silica-releasing systems also exhibit antimicrobial properties. Finally, since the quantification and understanding of the antimicrobial action of metal-based nanoparticles are key topics, several methods for evaluating in vitro antimicrobial activity and the most common antimicrobial mechanisms (e.g., cell damage and changes in the expression of metabolic genes) were discussed in this review.