Electrospun fibers loaded with antimicrobial peptides for treatment of wound infections.

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.

Tyrothricin--An underrated agent for the treatment of bacterial skin infections and superficial wounds?

The antimicrobial agent tyrothricin is a representative of the group of antimicrobial peptides (AMP). It is produced by Bacillus brevis and consists of tyrocidines and gramicidins. The compound mixture shows activity against bacteria, fungi and some viruses. A very interesting feature of AMPs is the fact, that even in vitro it is almost impossible to induce resistances. Therefore, this class of molecules is discussed as one group that could serve as next generation antibiotics and overcome the increasing problem of bacterial resistances. In daily practice, the application of tyrothricin containing formulations is relatively limited: It is used in sore throat medications and in agents for the healing of infected superficial and small-area wounds. However, due to the broad spectrum antimicrobial activity and the low risk of resistance development it is worth to consider further fields of application.

Trends in the susceptibility of methicillin-resistant Staphylococcus aureus to nine antimicrobial agents, including ceftobiprole, nemonoxacin, and tyrothricin: results from the Tigecycline In Vitro Surveillance in Taiwan (TIST) study, 2006-2010.

This study investigated the in vitro susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) to nine antimicrobial agents in Taiwan. A total of 1,725 isolates were obtained from 20 hospitals throughout Taiwan from 2006 to 2010. The minimum inhibitory concentrations (MICs) of the nine agents were determined by the agar dilution method. The MICs of mupirocin and tyrothricin were determined for 223 MRSA isolates collected from 2009 to 2010. For vancomycin, 99.7 % were susceptible; however, 30.0 % (n = 517) exhibited MICs of 2 µg/ml and 0.3 % (n = 6) demonstrated intermediate susceptibility (MICs of 4 µg/ml). Nearly all isolates (≥ 99.9 %) were susceptible to teicoplanin, linezolid, and daptomycin. The MIC90 values were 2 µg/ml for ceftobiprole and 1 µg/ml for nemonoxacin. The MIC90 values of mupirocin and tyrothricin were 0.12 and 4 µg/ml, respectively. MIC creep was noted for daptomycin during this period, but not for vancomycin, teicoplanin, linezolid, or tigecycline. For isolates with vancomycin MICs of 2 µg/ml, the MIC90 values were 2 µg/ml for teicoplanin, 0.5 µg/ml for daptomycin, and 0.5 µg/ml for tigecycline. Those values were four- to eight-fold higher than those among isolates with vancomycin MICs of 0.5 µg/ml (2, 0.06, and 0.12 µg/ml, respectively). Of the nine MRSA isolates exhibiting non-susceptibility to vancomycin (n = 6), teicoplanin (n = 1), daptomycin (n = 2), or tigecycline (n = 1), all had different pulsotypes, indicating the absence of intra-hospital or inter-hospital spread. The presence of a high proportion of MRSA isolates with elevated MICs (2 µg/ml) and MIC creep of daptomycin might alert clinicians on the therapy for serious MRSA infections in Taiwan.

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus.

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.

Repurposing FDA-approved drugs to combat drug-resistant Acinetobacter baumannii.

OBJECTIVE: The rising occurrence of drug-resistant pathogens accentuates the need to identify novel antibiotics. We wanted to identify new scaffolds for drug discovery by repurposing FDA-approved drugs against Acinetobacter baumannii, an emerging Gram-negative nosocomial drug-resistant pathogen. MATERIALS AND METHODS: In this study, we screened 1040 FDA-approved drugs against drug-susceptible A. baumannii ATCC 17978 and drug-resistant A. baumannii BAA-1605. RESULTS AND DISCUSSION: Twenty compounds exhibited significant antimicrobial activity (MIC ≤8 mg/L) against ATCC 17978 while only five compounds showed such activity against BAA-1605. Among the most notable results, tyrothricin, a bactericidal antibiotic typically active only against Gram-positive bacteria, exhibited equipotent activity against both strains. CONCLUSION: The paucity of identified compounds active against drug-resistant A. baumannii exemplifies its ability to resist antimicrobials as well as the resilience of drug-resistant Gram-negative pathogens. Repurposing of approved drugs is a viable alternative to de novo drug discovery and development.

Differential assay for high-throughput screening of antibacterial compounds.

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.

Identification of cultivable microorganisms from root canals with apical periodontitis following two-visit endodontic treatment with antibiotics/steroid or calcium hydroxide dressings.

The study was aimed at comparing the efficacy of disinfection of root canals with periapical radiolucencies when treated with either antibiotics/steroid medicaments (Ledermix or Septomixine) or a calcium hydroxide paste (Calasept). Microbiological samples were taken before and after two-visit endodontic treatment from 88 canals with apical periodontitis. All of the canals but one (87 of 88) had cultivable growth before treatment. After dressing with Ledermix, Septomixine, or Calasept, the percentages of canals remained with positive growth were 48% (13 of 27), 31% (8 of 26), and 31% (11 of 35), respectively. The chi(2) tests showed there were no significant differences in the number of canals with positive growth or mean colony forming units counts after instrumentation, irrigation and dressing. In the Ledermix group, 38 strains of bacteria were recovered. The Septomixine group had 25 strains, and the Calasept group had 25 strains. Gram-positive facultative anaerobic cocci (including staphylococci and streptococci) were more prevalent than the Gram-negative obligate anaerobic rods after treatment in all three groups. Similarities in the reduced number of canals with residual growth, and the prevalence of Gram-positive facultative anaerobic cocci suggest that the use of different inter-appointment dressings produced similar microbiological outcomes. However, factors other than the antimicrobial effectiveness of intracanal medicaments may also be responsible for the results observed.

Fungicidal effect of tyrothricin on Candida albicans.

Tyrothricin, a polypeptide antibiotic, is active against yeast cells. Tyrothricin was rapidly fungicidal towards Candida albicans. Concentration of four times the minimum inhibitory (25 mg l-1) reduced the yeast numbers by more than 3 log10 within 1 h. Similar results were obtained in a flow cytometric antifungal activity assay using the new two-colour probe for yeast viability, FUN-1, which measures impairment of metabolic activity. The respiratory activity of Candida albicans, measured in a XTT kinetic assay, was significantly reduced in comparison with controls by 3.12 mg l-1 of the substance. Because fungicidal concentrations of tyrothricin are locally achievable in patients, an evaluation of the local effect of tyrothricin in patients suffering from mucosal infections with Candida species should be considered.

Decontamination of the oral cavity. Effect of six local anti-microbial preparations in comparison to water and parafilm as controls.

Six preparations (four liquid and two solid) were tested in a double-blind crossover design for their anti-bacterial effect on aerobic and anaerobic bacteria in saliva of 12 volunteers. The four liquid preparations contained either tyrothricin, hexetidine, hydrogen peroxide or ethanol and were tested against a rinse with water as control. The two solid preparations, in the form of lozenges, contained tyrothricin in doses of 4 or 10 mg and were tested against parafilm. A single rinse with either tyrothricin or hexetidine resulted in a significant reduction of aerobic and anaerobic bacteria in saliva which lasted for the whole 1 h observation period and was considerably more effective than hydrogen peroxide or ethanol. Similar results were seen with the solid preparations. The lozenge containing 10 mg tyrothricin was the most effective and was the only formulation capable of reducing oral aerobic bacterial counts by a factor of approximately 100.

Tyrocidine-induced modulation of the DNA conformation in Bacillus brevis.

Using the [3H]trimethylpsoralen photobinding method [Sinden, R.R., Carlson, J.O. & Pettijohn, D.E. (1980) Cell 21, 773-783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension-relaxing and tension-inducing activities. Addition of tyrocidine to vegetative cells reduced by one-half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross-links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA-tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin.

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.

In vitro antibacterial activity of gramicidin and tyrothricin.

815 recent clinical isolates of different species representing pathogenic or saprophytic constituents of human microbial flora were submitted to agar dilution tests. In concentrations above 64 micrograms/ml tyrothricin is in general equally as gramicidin or slightly more effective whereas below 8 micrograms/ml gramicidin is mostly more effective than tyrothricin. Of 401 streptococci all were inhibited by tyrothricin and gramicidin in concentrations up to 96 micrograms/ml; staphylococci were more resistant. The pathogenic species of 130 strains of gram-negative cocci were more susceptible than the saprophytic species. Haemophilus strains were mostly resistant to gramicidin and to a lesser extent to tyrothricin. Enterobacteriaceae were totally resistant to both antibiotics.

Induction of sporulation in Bacillus brevis. 1. Biochemical events and modulation of RNA synthesis during induction by tyrocidine.

Under conditions of severe nitrogen starvation, brought about by nutritional shift-down, Bacillus brevis ATCC 8185 was unable to sporulate unless supplemented with the peptide antibiotic tyrocidine. The induction of sporulation was highly specific for tyrocidine and required only very low concentrations of the peptide (5 microM). Tyrocidine-induced sporulation was accompanied by the typical sporulation-specific events (e.g. extracellular protease production and dipicolinate synthesis) as well as the formation of linear gramicidin. The addition of tyrocidine produced acute inhibition of RNA synthesis that was followed by a limited activation of transcription near the time of onset of linear gramicidin synthesis, when the first sporulation-specific changes were observed. These results provide direct evidence for a role of tyrocidine in sporulation of B. brevis and suggest that the action of the peptide antibiotic may involve the control of transcription. Such a notion is supported by earlier studies on the effects of tyrocidine and linear gramicidin on purified RNA polymerase.

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin.

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.

Antibacterial action of gramicidin S and tyrocidines in relation to active transport, in vitro transcription, and spore outgrowth.

The cyclopeptide antibiotic gramicidin S or tyrocidine in concentrations of 2 to 4 mumol/mg of membrane protein inhibited the active transport of [3H]alanine and [3H]uridine in membrane vesicles isolated from Bacillus brevis and Bacillus subtilis. We used one analog of gramicidin S and two of tyrocidine A to study the relationship between peptide structure and antibacterial action as seen in inhibiting active transport and in vitro transcription and in delaying spore outgrowth. The data showed that [Ser2,2']-gramicidin S, in which the two ornithine residues were replaced by two serines, was at least 50 times less active antibacterially and gave a low response in transport inhibition and delay of spore outgrowth compared with the natural peptide. The antibacterial activity of [Val6]-tyrocidine A was twice lower than that of tyrocidine A, and it also showed a considerable reduction in transport and transcription inhibition. [Orn7]-tyrocidine A containing two ornithine residues in positions corresponding to those in gramicidin S was almost inactive in all functions tested. The correlation between peptide structure and activity is discussed.

Functions of the peptide antibiotics tyrocidine and gramicidin. Induction of conformational and structural changes of superhelical DNA.

The peptide antibiotic tyrocidine which is produced by Bacillus brevis and is probably involved in sporogenesis, unwinds superhelical plasmids in vitro at low peptide: DNA ratios, as found by gel electrophoresis. At higher peptide concentrations, the DNA is packed tightly leading to apparent nuclease stability of the complex and inhibition of RNA synthesis. The addition of the linear gramicidin, another peptide antibiotic synthesized by the same bacterial strain, partially restores transcription by breaking down the tightly packed DNA X peptide complex. The complexed DNA, after nuclease digestion, is retained on a nitrocellulose filter, but loses its affinity for the filter in the presence of gramicidin. The results are discussed with respect to possible functions of the two peptides within in the cell.

[The antibacterial activity of a gel for burns and wounds containing tyrothricin, fomocaine, diphenhydramine and 8-hydroxyquinoline (author's transl)]. / Uber die antibakterielle Wirkung eines Brand- und Wundgels mit den Komponenten Tyrothricin, Fomocain, Diphenhydramine und 8-Hydroxychinolin.

The antibacterial activity of a new jelly for burns and wounds with the components tyrothricin, 8-hydroxyquinoline, fomocaine and diphenhydramine (Herit) is described. The biological availability of tyrothricin and 8-hydroxyquinoline from the gel is in accordance with minimum inhibitor concentrations (MIC). Against strains of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans the jelly was most effective.