Composition for Inactivating Spores by Means of Antimicrobial Peptides

Abstract

A composition, which comprises at least one natural, recombinant, or synthetic human antimicrobial peptide, is selected from a human defensin or cathelicidin or functional fragments or combinations thereof, for inactivating bacterial spores.

Claims

1. The use of a composition, comprising: selecting at least one natural, recombinant, or synthetic human antimicrobial peptide from a human defensin or cathelicidin, or functional fragments or combinations thereof, for inactivating bacterial spores.

2. The use as claimed in claim 1, wherein the spores are of aerobic or anaerobic bacteria.

3. The use as claimed in claim 1, wherein the bacterial spores are derived from bacteria selected from at least one of the following: Clostridium sporogenes, Clostridium difficile, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Clostridium novyi, Clostridium histolyticum, Clostridium sordellii, Clostridium ramosum, Clostridium innocuum, Clostridium septicum or Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Bacillus licheniformis, Bacillus circulans, and Bacillus coagulans.

4. The use as claimed in claim 1, further comprising: using the composition for an inhibition of a germination of the bacterial spores to form vegetative cells.

5. The use as claimed in claim 1, further comprising: selecting the human defensin or cathelicidin from a group comprising human beta-defensin-3, cathelicidin LL-37, or fragments or combinations of one or more thereof.

6. The use as claimed in claim 1, further comprising: using the composition as a disinfectant.

7. The use as claimed in claim 1, further comprising: using the composition as a disinfectant of material surfaces.

8. The use as claimed in claim 1, further comprising: using the composition for disinfection in mammals including humans.

9. The use as claimed in claim 8, further comprising: using the composition on human hands, a mucous membrane, or a wound.

10. The use as claimed in claim 1, further comprising: using the composition as medicament for preventing a recurrence in a case of infections initiated by the bacterial spores.

11. A composition comprising: at least one natural, recombinant, or synthetic human antimicrobial peptide selected from a human defensin or cathelicidin, or functional fragments or combinations thereof, wherein the composition is used in an inactivation of bacterial spores.

12. The composition as claimed in claim 11, wherein the composition is used as disinfectant or medicament.

13. The composition as claimed in claim 11, wherein the composition is used in therapy or prophylaxis of an infection initiated by the bacterial spores.

Description

[0056] The figures are described below.

[0057] FIG. 1 shows the results of a treatment of a suspension of vegetative cells and spores after a heat-treatment to kill the vegetative cells with the exemplary antimicrobial peptides LL37 and hBD3 as a function of the concentration of the antimicrobial peptide; and

[0058] FIG. 2 shows an electron microscopy of C. difficile spores which had been incubated with gold-labeled HBD3 peptides. HBD3 was able to penetrate into the spores (black particles); and

[0059] FIG. 3 shows an electron microscopy of C. difficile spores. In this C. difficile strain, the spores were destroyed structurally by HBD3.

EXAMPLES

Embodiments of the Invention

[0060] Effect of Antimicrobial Peptides on C. difficile Spores

[0061] The effects of antimicrobial peptides on C. difficile spores were investigated by first enriching a bacterial suspension with spores so that at least 70% spores were present. The spore suspension was incubated for 60 minutes with various defensins (HBD1, HBD2, HBD3) and the cathelicidin LL37 (recombinant defensins: Peptanova Sandhausen, HBD1 order No. 4337; HBD2; 4338, HBD3 4382/recombinant LL37: Innovagen Lund Sweden, order No. SP-LL-37-1). Thereafter, aliquots of the samples were plated out on Columbia blood agar and incubated anaerobically for at least 4 days. The germinated spores in an untreated control and the preparations containing antimicrobial peptides were counted.

[0062] It became apparent that, in the case of the samples which were incubated with HBD3 or LL37, significantly fewer germinated spores were present in comparison with the controls.

[0063] In order to able to establish with certainty whether either germinating bacteria are rapidly killed by the presence of antimicrobial peptides in the medium or the spores are in fact prevented from germinating, the experiment was repeated, but at the end of the incubation time, the peptides were washed out and the medium was free of peptides.

[0064] Despite the wash-out, the inhibitory effect of HBD3 and LL37 was nevertheless surprisingly maintained, meaning that it was also possible to demonstrate in these experiments that both defensins and cathelicidins are effective against spores.

[0065] In further experiments, the inventers wanted to make the unambiguous assignment that resultant bacterial colonies can be attributed with certainty to the germinated spores and not to the steady growth of residual vegetative cells. To this end, incubation and wash-out were followed by killing the residual vegetative cells using heat (61° C., 30 min). This heat treatment kills only the vegetative cells, but not the spores.

[0066] These experiments revealed a concentration-dependent decrease in germinating spores for the two effective antimicrobial peptides, with HBD3 already showing in lower concentrations a somewhat higher efficacy. The results of said experiments are summarized in FIG. 1. There, the different effects of LL37 in comparison with HBD3 on germinating spores are reproduced in a graph.

[0067] It is clear that just a low concentration (2.5 μg/mL) of HBD3 is sufficient for greatly lowering the number of germinating spores. Whereas there is still a distinct difference between the effect of LL37 and HBD3 at low concentrations, said difference is hardly noticeable at larger concentrations of antimicrobial peptides (10 μg/mL).

[0068] In further experiments with gold-labeled HBD3 peptides, it was possible to demonstrate that HBD3 penetrates into the exosporium, the outer wall of the spores, and destroys parts of the spore structure. These results are depicted strikingly in FIGS. 2 and 3.

[0069] To date, it was only known that antimicrobial peptides, especially the defensin HBD3 and the cathelicidin LL37, kill vegetative cells effectively and that the effect can moreover be intensified in conjunction with antibiotics. What is completely new by contrast is the finding that spores as well can be destroyed by the treatment with antimicrobial peptides.

[0070] Furthermore, electron micrographs confirm the penetration of HBD3 directly into the spores. According to the current prior art, there is no known antibiotic or peptide which would be able to achieve this.

[0071] The best known mechanism of action of antimicrobial peptides is pore formation in the bacterial membrane. What is completely new and surprising is that said mechanism of action can also be applied to the spore wall. Since the cell wall structure of bacteria greatly differs compared to the cell wall structure of bacterial spores, for example antibiotics which have one mode of action within the cell membrane are completely ineffective against spores, it was not possible to assume that human antimicrobial peptides are necessarily active against bacterial spores.