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Tryptocidine C (TpcC), a Trp-rich cyclodecapeptide is a minor constituent in the antibiotic tyrothricin complex from Brevibacillus parabrevis. TpcC possesses a high tendency to oligomerise in aqueous solutions and dried TpcC forms distinct self-assembled nanoparticles. High-resolution scanning electron microscopy revealed the influence of different ethanol:water solvent systems on TpcC self-assembly, with the TpcC, dried from a high concentration in 15% ethanol, primarily assembling into small nanospheres with 24.3 nm diameter and 0.05 polydispersity. TpcC at 16 µM, near its CMC, formed a variety of structures such as small nanospheres, large dense nanospheroids and facetted 3-D-crystals, as well as sheets and coarse carpet-like structures which depended on ethanol concentration. Drying 16 µM TpcC from 75% ethanol resulted in highly facetted 3-D crystals, as well as small nanospheres, while those in 10% ethanol preparation had less defined facets. Drying from 20 to 50% ethanol led to polymorphic architectures with a few defined nanospheroids and various small nanoparticles, imbedded in carpet- and sheet-like structures. These polymorphic surface morphologies correlated with maintenance of fluorescence properties and the surface-derived antibacterial activity against Staphylococcus aureus over time, while there was a significant change in fluorescence and loss in activity in the 10% and 75% preparations where 3-D crystals were observed. This indicated that TpcC oligomerisation in solutions with 20-50% ethanol leads to metastable structures with a high propensity for release of antimicrobial moieties, while those leading to crystallisation limit active moieties release. TpcC nano-assemblies can find application in antimicrobial coatings, surface disinfectants, food packaging and wound healing materials.

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The widespread resistance of clinically relevant bacteria against established antibiotics emphasizes the urgent need for novel therapeutics. In this context, wound infections constitute a specific challenge, as most systemically applied antibiotics are insufficiently available at the site of infection. Therefore, the local treatment of infected wounds poses a particular challenge regarding the appropriate release kinetics of actives and their residence time in the wound bed. Consequently, design and development of novel, drug-loaded wound dressings constitute a major research focus for the effective treatment of wound infections. In this study, we employed electrospinning to design drug-loaded wound dressings, incorporating the therapeutically promising antimicrobial peptide tyrothricin. By parallel electrospinning, we combined different ratios of water-soluble polyvinylpyrrolidone and water-insoluble methacrylate copolymer (EudragitE), in order to take advantage of their specific mechanical stability and dissolution properties. We fabricated fiber mats constituting mechanically stable wound dressings with a controlled drug release profile, combining an initial burst release above the minimal inhibitory concentration of known wound pathogens and a subsequent prolonged antimicrobial effect of the active ingredient. Antimicrobial activity against Staphylococcusaureus and Staphylococcusepidermidis was successfully proven, thereby introducing our tyrothricin-loaded fiber mats as a promising prospective therapy against typical wound-associated pathogens.

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Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by Brevibacillus parabrevis, showed potent activity against chloroquine sensitive (CQS) Plasmodium falciparum. The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe1-Pro2-(Phe3/Trp3)-D-Phe4/D-Trp4)-Asn5-Gln6-(Tyr7/Phe7/Trp7)-Val8-(Orn9/Lys9)-Leu10 was proposed to have an important role in activity. CQS and resistant (CQR) P. falciparum strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for P. falciparum strains, other than for the CQS 3D7 strain. The Tyr7 in tyrocidine A (TrcA) with Phe3-D-Phe4 seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe7 in phenycidine A, the second peptide containing Phe3-D-Phe4, also showed CQ antagonism. Conversely, Trp7 in tryptocidine C (TpcC) with Trp3-D-Trp4 showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR P. falciparum strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated.

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The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5-L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-(Orn9/Lys9)-L-Leu10], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe3-D-Phe4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp3-D-Trp4. For the other variable aromatic residue position, Tyr7 was preferentially incorporated above Trp7, with a minor incorporation of Phe7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn9 to Lys9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.

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AIMS: The aims of this study were to explore the development of bacterial resistance and cross-resistance in four common human pathogens following realistic exposure to antibiotics found in over-the-counter (OTC) sore throat medicines: gramicidin, neomycin, bacitracin and tyrothricin. METHODS AND RESULTS: Bacterial exposure to in-use (concentration in the product before use) and diluted concentration (i.e. during use) of antibiotic where conducted in broth for 24 h or until growth was visible. The changes in bacterial susceptibility profile before and after exposure was determined using standardized ISO microdilution broth. Antibiotic testing was performed according to EUCAST guidelines. We demonstrated that test bacteria were able to survive exposure to the in-use concentrations of some antibiotics used in OTC medicines. Exposure to during use concentrations of bacitracin resulted in stable increase in minimal inhibitory concentration (MIC) (>8-fold) in Staphylococcus aureus and Acinetobacter baumannii. Exposure to tyrothricin resulted in a stable increase in MIC (2·4-fold) in Klebsiella pneumoniae, and exposure to neomycin resulted in a stable increase MIC (5000-fold higher than the baseline) in Streptococcus pyogenes. Clinical cross-resistance to other antibiotics (ciprofloxacin, fusidic acid, gentamicin, cefpodoxime, amoxicillin/clavulanic acid and cefotaxime) was also demonstrated following exposure to bacitracin or tyrothricin. Bacitracin exposure lead to a stable bacterial resistance after 10 passages. CONCLUSIONS: Our results indicate that OTC antibiotic medicines have the potential to drive resistance and cross-resistance in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Tackling antibiotic resistance is a high worldwide priority. It is widely accepted that the overuse and misuse of antibiotics increase the risk of the development and spread of antibiotic resistance within communities. A number of OTC sore throat products, widely available across the world for topical use in respiratory indications, contain locally delivered antibiotics. Our findings showed that these antibiotics in OTC medicines present a risk for emerging cross-resistance in a number of bacterial respiratory pathogens.

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Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by ß-sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H-bonding, possibly ß-sheet structures. The amides involved in intramolecular H-bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1 H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA-glucose interactions characterized by a dissociation constant around 200 µM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible.

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Chitosan-based film-forming gel is regarded as a promising vehicle for topical delivery of antimicrobial agents to skin wounds, since it protects from microbial infection and the cationic polymer itself possesses antibacterial activity. In this study, possible synergistic interaction against common skin pathogens between the cationic polymer and tyrothricin (TRC), a cyclic polypeptide antibiotic, was investigated, by determining the concentration to inhibit 90% of bacterial isolates (MIC). The addition of the polysaccharide to TRC dramatically reduced the MIC values of TRC by 1/33 and 1/4 against both methicillin-resistant and methicillinsusceptible Staphylococcus aureus, respectively. The synergism of TRC and chitosan combination against both strains was demonstrated by the checkerboard method, with a fractional inhibitory concentration index below 0.5. Moreover, co-treatment of TRC and chitosan exhibited antibacterial activity against Pseudomonas aeruginosa, due to the antibacterial activity of chitosan, whereas TRC itself did not inhibit the gram-negative bacterial growth. These findings suggested that the use of chitosan-based film for topical delivery of TRC could be an alternative to improve TRC antimicrobial activity against strains that are abundant in skin wounds.

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Objetivo: Avaliar a atividade antimicrobiana in vitro dos componentes (tirotricina, quinosol e tintura de malva) de um enxaguatório bucal contendo malva (Malvatricin®) sobre Streptococcus mutans, Lactobacillus spp. e um pool de micro-organismos da cavidade bucal. Método: Para a verificação do potencial antimicrobiano do enxaguatório bucal contendo malva (Malvatricin®) e de seus diferentes componentes, a metodologia utilizada foi a da difusão em ágar-cilindro em placas. Utilizaram-se dez placas com ágar Brain Heart Infusion para cada micro-organismo e foram testadas as seguintes soluções: Malvatricin®, tirotricina, quinosol e tintura de malva. Como controle positivo foi utilizada uma solução de clorexidina 0,12%. As placas foram incubadas por 24 horas a 37°C em anaerobiose. Após 24 horas, mensuraram-se os diâmetros das zonas de inibição. Utilizou-se o teste de Kruskal-Wallis, seguido pelo teste de comparações múltiplas, quando indicado, para comparar o efeito de cada substância sobre os micro-organismos testados. O nível de significância adotado foi de 5%. Resultados: Sobre Streptococcus mutans, Lactobacillus e um pool de micro-organismos da cavidade bucal, Malvatricin® apresentou valores de medianas 23,47; 8,61 e 11,23, respectivamente. O quinosol apresentou resultado semelhante ao de Malvatricin®, mostrando-se efetivo para inibir o crescimento microbiano de todos os micro-organismos (27,17; 9,33; 12,53). A tirotricina (0; 5,66; 0) e a tintura de malva (0; 0; 4,29) apresentaram pouca ou nenhuma atividade antimicrobiana. A clorexidina apresentou as maiores zonas de inibição frente a todos os micro-organismos testados (30,06; 15,54; 20,89). Conclusão: Os resultados do presente estudo sugerem que, dentre os componentes presentes na composição do produto comercial Malvatricin®, a substância quinosol apresenta a maior atividade antimicrobiana frente aos micro-organismos testados. E que a atividade antimicrobiana verificada para o produto comercial provavelmente deva-se à ação da substância quinosol, presente em sua composição.

Objective: To evaluate the in vitro antimicrobial activity of the components (tyrothricin, hydroxyquinoline and Malva sylvestris tincture) of a mouthwash containing Malva sylvestris (Malvatricin®) against Streptococcus mutans, Lactobacillus spp. and a pool of oral microorganisms. Method: In order to verify the antimicrobial potential of the Malva sylvestris-containing mouthwash (Malvatricin®) and its different components, was used the cylinder-agar diffusion plate methodology. Ten plates with Brain Heart Infusion agar were used for each microorganism and the following solutions were tested: Malvatricin®, tyrothricin, hydroxyquinoline and Malva sylvestris tincture. The positive control was a 0.12% chlorhexidine solution. The plates were incubated for 24 hours at 37°C in anaerobiosis. After 24 hours, the diameters of bacterial growth inhibition zones were measured. The Kruskal-Wallis test and the multiple-comparisons test (when required) were used to compare the effect of each substance against the test microorganisms. The significance level was set at 5%. Results: Malvatricin® presented medians of 23.47, 8.61 and 11.23, respectively, against Streptococcus mutans, Lactobacillus spp. and a pool of oral microorganisms. Hydroxyquinoline showed similar result to Malvatricin®, being effective to inhibit the growth of all microorganisms (27.17, 9.33 and 12.53, respectively). Tyrothricin (0, 5.66, 0, respectively) and Malva sylvestris tincture (0, 0, 4.29, respectively) showed little or no antimicrobial activity. Chlorhexidine produced the largest inhibition zones against the tested microorganisms (30.06, 15.54, 20.89, respectively). Conclusion: The results from this study suggest that among all components of Malvatricin®, hydroxyquinoline presented the greatest antimicrobial activity against the tested microorganisms, and that the antimicrobial activity of the commercial product is likely due to the action of the hydroxyquinoline present in its composition

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