Abstract:
Antimicrobial peptides set forth in SEQ ID Nos: 1-5 are described. The peptides have activity against Gram positive bacteria and against Gram negative bacteria. A bactericidal composition is also provided, which may comprise an amount between 0.5 μg/mL and 1024 μg/mL of the peptides and excipients.

Description:
The present invention relates to antimicrobial peptides such as for example, the peptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. These peptides show activity against Gram positive bacteria and against Gram negative bacteria. It is also provided a bactericidal composition comprising an amount between 0.5 μg/ml and 1024 μg/ml of the peptides: and excipients. 
     BACKGROUND OF THE INVENTION 
     The treatment of chronic wounds and ulcers is complex and difficult and represents a challenge for the health system as a whole, having a strong impact on economy in general. In particular, the problem of recurrent infections with bacterial strains resistant to antimicrobial treatments. 
     Even though nowadays effective treatments are available for the majority of the infections, antibiotics abuse, carried out over many decades, has led to the generation of strains resistant to common-use antimicrobials by means of horizontal transfer of genes between pathogenic microorganisms. 
     This progressive decrease in the efficacy of first-choice antibiotics and the limited therapeutic approaches available for the wound treatment and healing, emphasize the need for new classes of drugs and their means of application. 
     Presently, some bactericidal peptides exist. Each peptide has different characteristics in terms of the minimum inhibitory concentrations that are to be used for achieving the desired bactericidal activity, in addition, they show differences in solubility, cytotoxicity, specificity and target against different pathogens. 
     Patent document U.S. Pat. No. 6,696,238 discloses polypeptides and defensins used as antimicrobials in the preparation of culture media. 
     BRIEF DESCRIPTION OF THE INVENTION 
     Antimicrobial peptides are provided, in a preferred embodiment the peptides may be those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID No:5. The peptides have activity against Gram positive bacteria and against Gram negative bacteria. 
     A bactericidal composition is provided, comprising an amount between 0.5 μg/mL and 1024 μg/mL of the peptides mentioned in the paragraph above; and excipients. 
     The use of the peptides 1 is provided for preparing a medicament for bacterial infections. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows circular dichroism (CD) spectra of the peptides of the invention in an aqueous solution; wherein it is shown that the peptides are not structured in aqueous buffer, and with a characteristic minimum at approximately 200 nm. Omi: Omiganan peptide, p1: peptide 1; p2: peptide 2, p3: peptide 3, p4: peptide 4, p5: peptide 5. 
         FIG. 2  shows circular dichroism (CD) spectra of the peptides of the invention in a solution with SDS, wherein conformational changes are observed in the peptides 1, 2 and 5, which are consistent with the formation of alpha-helix structures. Omi: Omiganan peptide, p1: peptide 1; p2: peptide 2, p3: peptide 3, p4: peptide 4, p5: peptide 5. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The fundamental characteristic of the peptides of the invention is their sequence and the amino acids assembly within the peptide chain which has been designed with the aim of enhancing selectivity of action over the bacterial strains, decreasing its potential cytotoxic action, which is very common in these types of peptides. 
     The peptides disclosed herein are active against Gram positive and Gram negative bacteria, including several types of  Staphilococcus  and  Pseudomonas . The MIC values (minimum inhibitory concentration) are especially interesting in some strains. New synthetic peptides are disclosed in the present invention which are amidated or not amidated at the C-terminal end, and which have bactericidal activity in strains relevant for clinical issues and are resistant to traditional antibiotics. These peptides may be incorporated in a suitable vehicle for application, for instance, topically, which would provide a correct solution to the problem of superficial wounds with a high risk of infection. Additionally, they are easy to apply products and have a wide bactericidal spectrum, which solves the increasing problem of multiresistant bacterial strains. 
     Five peptides were synthesized having antibacterial activity, in a preferred embodiment the peptides were the following: 
     
       
         
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                   
                 peptide 1: 
               
             
          
           
               
                 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                 WPKWWKWKRRWGRKKAKKRRG 
               
               
                   
               
               
                   
                 peptide 2: 
               
             
          
           
               
                 (SEQ ID NO: 2) 
               
             
          
           
               
                   
                 GLLKKWLKKWKEFKRIVGY 
               
               
                   
               
               
                   
                 peptide 3: 
               
             
          
           
               
                 (SEQ ID NO: 3) 
               
             
          
           
               
                   
                 FGKEKKAWWRRRKWLK 
               
               
                   
               
               
                   
                 peptide 4: 
               
             
          
           
               
                 (SEQ ID NO: 4) 
               
             
          
           
               
                   
                 TTCDLLSGVGLPNVPQPLHCVFRGNRKIKW 
               
               
                   
               
               
                   
                 peptide 5: 
               
             
          
           
               
                 (SEQ ID NO: 5) 
               
             
          
           
               
                   
                 RIVQRIKKWLLKWKKLGY 
               
               
                   
               
               
                   
                 known peptide (Omiganan): 
               
             
          
           
               
                 (SEQ ID NO: 6) 
               
             
          
           
               
                   
                 ILRWPWWPWRRK. 
               
             
          
         
       
     
     The 5 designed and synthesized peptides of the invention, plus omiganan (peptide of the state of the art) were evaluated for their antimicrobial capacity in microdilution assays for determining the minimum inhibitory concentration (MIC). A first panel of 8 representative strains was analyzed in these assays, encompassing Gram positive and Gram negative bacteria. The results of the obtained MIC are outlined in Table 1, expressed as μg/ml of each peptide. The lowest MIC values indicate a higher antimicrobial potency. 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 pep- 
                 pep- 
                 pep- 
                 pep- 
                 pep- 
               
               
                   
                   
                 tide 
                 tide 
                 tide 
                 tide 
                 tide 
               
               
                 Organism 
                 OMI 
                 1 
                 2 
                 3 
                 4 
                 5 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Staphylococcus warneri 
                 
                 4 
                 4 
                 8 
                 8 
                 &gt;256 
                 4 
               
               
                 M6823 
               
               
                 
                   Staphylococcus cohnii 
                 
                 8 
                 8 
                 8 
                 4 
                 &gt;256 
                 4 
               
               
                 M6767 
               
               
                 
                   Staphylococcus aureus 
                 
                 16 
                 16 
                 64 
                 128 
                 &gt;256 
                 16 
               
               
                 ATCC29213 
               
               
                 
                   Escherichia coli 
                 
                 32 
                 32 
                 16 
                 64 
                 &gt;256 
                 4 
               
               
                 ATCC25922 
               
               
                 
                   Staphylococcus aureus 
                 
                 16 
                 8 
                 32 
                 128 
                 &gt;256 
                 4 
               
               
                 M6794 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 32 
                 16 
                 16 
                 128 
                 &gt;256 
                 8 
               
               
                 ATCC27853 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 64 
                 16 
                 32 
                 128 
                 &gt;256 
                 8 
               
               
                 M13513 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 64 
                 &gt;128 
                 16 
                 128 
                 &gt;256 
                 8 
               
               
                 M13540 
               
               
                   
               
             
          
         
       
     
     Subsequently, the capacity of the peptides of the invention for lysing human erythrocytes was evaluated, as a measure of their cytotoxicity (Table 2). The assays were carried out according to the Examples and are expressed as a percentage of total lysis obtained with either water or the surfactant Tween 20. In this assays, the lower hemolysis percentage, the lower expected cytotoxic activity. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 Hemolysis % 
                 Hemolysis % 
               
               
                 Sample 
                 Concentration 
                 OD 540 nm 
                 (1% Tween20) 
                 (water) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 peptide 1 
                 128 ug/ml 
                 0.067 
                 4 
                 3.4 
               
               
                 peptide 2 
                 128 ug/ml 
                 0.641 
                 46.3 
                 39.8 
               
               
                 peptide 3 
                 128 ug/ml 
                 0.036 
                 1.7 
                 1.5 
               
               
                 peptide 4 
                 128 ug/ml 
                 0.04 
                 2 
                 1.72 
               
               
                 peptide 5 
                 128 ug/ml 
                 0.163 
                 11.1 
                 9.5 
               
               
                 Omiganan 
                 128 ug/ml 
                 0.171 
                 11.6 
                 10 
               
               
                 Tween20 
                 1% 
                 1.37 
                 100 
                 86 
               
               
                 PBS 
                 — 
                 0.013 
                 0 
                 0 
               
               
                 water 
                 — 
                 1.59 
                 116.2 
                 100 
               
               
                   
               
             
          
         
       
     
     As can be seen, peptides 1, 2 and 5 have higher antimicrobial potency values. The hemolysis values are lower or similar to those of the Omiganan peptide. Even though peptide 2 has high hemolysis values, it was decided to go on with its analysis since in an eventual topical use these values would not be an impediment. In a preferred embodiment, peptides 1 and 5 are selected, which have the highest therapeutic index values, understood as the value indicating the antimicrobial potency over the cytotoxicity percentage. 
     Subsequently, an analysis of a panel of clinical isolates including strains with different resistance mechanisms against conventional antibiotics was carried out, all of which are of clinical relevance. In Tables 3 and 4 the results obtained when analyzing MIC of the selected peptides 1, 2 and 5, are shown, plus the control peptide Omiganan. In Table 3 the results for a panel of Gram negative bacteria are shown, and in Table 4 for a panel of Gram negative bacteria. In these tables the detected gene/s responsible for the resistance against antibiotics for each strain, are included. 
     In this studies, it may be seen that peptide 5 is up to eight times more active than the known peptide Omiganan in  Pseudomona aeuruginosa . A similar thing is observed with  Klebsiella pneumoniae , where great improvements in potency of the peptides 2 and 5 are observed, while peptide 1 has a lower activity than the Omiganan peptide for that strain. 
     
       
         
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLES 3 and 4 
               
               
                   
                   
               
             
             
               
                   
                 MIC (ug/ml) 
               
             
          
           
               
                 Strain 
                 ID No. 
                 Resistance Gene 
                 Omiganan 
                 peptide 1 
                 peptide 2 
                 peptide 5 
               
               
                   
               
             
          
           
               
                   Pseudomonas aeruginosa  (n = 12) 
               
             
          
           
               
                 
                   P. aeruginosa 
                 
                 M13513 
                 kpc 
                 256 
                 32 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M11005 
                 kpc 
                 512 
                 64 
                 64 
                 128 
               
               
                 
                   P. aeruginosa 
                 
                 M7723 
                 kpc 
                 256 
                 64 
                 64 
                 32 
               
               
                 
                   P. aeruginosa 
                 
                 M7728 
                 imp 
                 512 
                 128 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M5109 
                 vim 
                 256 
                 128 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M5200 
                 vim + ges 
                 512 
                 64 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M7525 
                 spm 
                 512 
                 64 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M7712 
                 spm 
                 512 
                 64 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M5470 
                 ND 1   
                 512 
                 128 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 M7907 
                 per 
                 128 
                 128 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 COS 12p 
                 ND. 
                 512 
                 64 
                 64 
                 64 
               
               
                 
                   P. aeruginosa 
                 
                 ATCC 27853 
                 WT 
                 256 
                   
                 64 
                 64 
               
             
          
           
               
                   Acinetobacter  sp. (n = 10) 
               
             
          
           
               
                 
                   A. baumanii 
                 
                 M13523 
                 oxa-51 
                 64 
                 64 
                 32 
                 4 
               
               
                 
                   A. baumanii 
                 
                 FAV-1 
                 per + oxa-51 + oxa-58 
                 64 
                 64 
                 16 
                 16 
               
               
                 
                   A. baumanii 
                 
                 M5179 
                 oxa-51 
                 64 
                 64 
                 32 
                 32 
               
               
                 
                   A. baumanii 
                 
                 M7978 
                 imp 
                 64 
                 64 
                 16 
                 8 
               
               
                 
                   A. junii 
                 
                 M9013 
                 oxa-51 + imp 
                 64 
                 32 
                 32 
                 8 
               
               
                 
                   A. baumanii 
                 
                 M5277 
                 per 
                 32 
                 64 
                 32 
                 8 
               
               
                 
                   A. baumanii 
                 
                 M5949 
                 oxa-23 + oxa-GVI 
                 128 
                 256 
                 32 
                 16 
               
               
                 
                   A. baumanii 
                 
                 M7489 
                 oxa-51 + tem 
                 32 
                 64 
                 8 
                 16 
               
               
                 
                   A. baumanii 
                 
                 M9665 
                 oxa-51 
                 64 
                 128 
                 4 
                 32 
               
               
                 
                   A. baumanii 
                 
                 M5282 
                 oxa-51 
                 16 
                 64 
                 8 
                 8 
               
             
          
           
               
                   Klebsiella pneumoniae  (n = 12) 
               
             
          
           
               
                 
                   K. pneumoniae 
                 
                 M5825 
                 ges-3 + ctx-m-2 
                 64 
                 &gt;1024 
                 8 
                 32 
               
               
                 
                   K. pneumoniae 
                 
                 M7647 
                 vim-like + ctx-m-2 + 
                 1024 
                 &gt;1024 
                 32 
                 32 
               
               
                   
                   
                 tem-1 + shv-1 
               
               
                 
                   K. pneumoniae 
                 
                 M13540 
                 kpc 
                 256 
                 &gt;1024 
                 16 
                 64 
               
               
                 
                   K. Pneumoniae 
                 
                 M9885 
                 kpc 
                 256 
                 &gt;1024 
                 64 
                 32 
               
               
                 
                   K. Pneumoniae 
                 
                 M11245 
                 kpc + per-2 
                 32 
                 1024 
                 16 
                 8 
               
               
                 
                   K. pneumoniae 
                 
                 M1803 
                 ctx-m-2 + per-2 + 
                 1024 
                 &gt;1024 
                 32 
                 64 
               
               
                   
                   
                 tem-1 + shv + oxa-9 
               
               
                 
                   K. pneumoniae 
                 
                 M9140 
                 cit 
                 64 
                 1024 
                 32 
                 32 
               
               
                 
                   K. pneumoniae 
                 
                 M9310 
                 ctx-m-2 + shv-1 + 
                 64 
                 &gt;1024 
                 16 
                 32 
               
               
                   
                   
                 tem-1 
               
               
                 
                   K. pneumoniae 
                 
                 M9375 
                 ctx-m-2 + shv-1 + 
                 32 
                 1024 
                 64 
                 16 
               
               
                   
                   
                 tem-1 
               
               
                 
                   K. pneumoniae 
                 
                 M9170 
                 oxa-GIII 
                 128 
                 &gt;1024 
                 32 
                 64 
               
               
                 
                   K. pneumoniae 
                 
                 M9491 
                 mox 
                 128 
                 1024 
                 64 
                 64 
               
               
                 
                   K. pneumoniae 
                 
                 FAV3 
                 WT 
                 128 
                 1024 
                 128 
                 128 
               
             
          
           
               
                   Escherichia coli  (n = 9) 
               
             
          
           
               
                 
                   E. coli 
                 
                 Cos15 
                 per + tem 
                 64 
                 128 
                 64 
                 64 
               
               
                 
                   E. coli 
                 
                 M9884 
                 WT 
                 64 
                 128 
                 32 
                 32 
               
               
                 
                   E. coli 
                 
                 M9209 
                 kpc 
                 64 
                 128 
                 64 
                 128 
               
               
                 
                   E. coli 
                 
                 M7859 
                 cit 
                 64 
                 128 
                 32 
                 16 
               
               
                 
                   E. coli 
                 
                 NEU23 
                 oxa-GIII + tem 
                 32 
                 128 
                 32 
                 32 
               
               
                 
                   E. coli 
                 
                 ABC11 
                 ctx-m-2 
                 64 
                 128 
                 32 
                 32 
               
               
                 
                   E. coli 
                 
                 LCA1 
                 ctx-m-2 + tem-1 
                 64 
                 128 
                 64 
                 64 
               
               
                 
                   E. coli 
                 
                 M5306 
                 ctx-m-2 + tem-1 + 
                 64 
                 128 
                 4 
                 64 
               
               
                   
                   
                 per-2 
               
               
                 
                   E. coli 
                 
                 ATCC 25922 
                 WT 
                 64 
                 256 
                 32 
                 32 
               
               
                   
               
             
          
           
               
                   
                 MIC (ug/ml) 
                   
               
             
          
           
               
                 Strain 
                 ID No. 
                 Resistance gene 
                 Omiganan 
                 peptide 1 
                 peptide 2 
                 peptide 5 
               
               
                   
               
             
          
           
               
                   Staphylococcus aureus  (n = 11) 
                   
               
             
          
           
               
                 
                   S. aureus 
                 
                 ATCC29213 
                 WT 
                 32 
                 32 
                 64 
                 32 
               
               
                 
                   S. aureus 
                 
                 M6794 
                 mecA 
                 64 
                 32 
                 64 
                 64 
               
               
                 
                   S. aureus 
                 
                 204 
                 ermA 
                 64 
                 64 
                 64 
                 32 
               
               
                 
                   S. aureus 
                 
                 28 
                 ermA 
                 32 
                 32 
                 64 
                 64 
               
               
                 
                   S. aureus 
                 
                 33 
                 msrA 
                 32 
                 32 
                 128 
                 64 
               
               
                 
                   S. aureus 
                 
                 239 
                 ermC 
                 32 
                 32 
                 64 
                 64 
               
               
                 
                   S. aureus 
                 
                 M6276 
                 ermA + lnuA 
                 32 
                 16 
                 128 
                 64 
               
               
                 
                   S. aureus 
                 
                 M2832 
                 mecA 
                 64 
                 32 
                 128 
                 64 
               
               
                 
                   S. aureus 
                 
                 M4046 
                 mecA 
                 32 
                 32 
                 128 
                 32 
               
               
                 
                   S. aureus 
                 
                 M6820 
                 mecA 
                 128 
                 64 
                 128 
                 64 
               
               
                 
                   S. aureus 
                 
                 M6784 
                 mecA 
                 32 
                 32 
                 32 
                 32 
               
             
          
           
               
                 Coagulase Negative  Staphylococcus  (CNS) (n = 12) 
                   
               
             
          
           
               
                 
                   S. epidermidis 
                 
                 M2919 
                 mecA 
                 8 
                 16 
                 16 
                 8 
               
               
                 
                   S. epidermidis 
                 
                 M2921 
                 mecA 
                 8 
                 8 
                 8 
                 8 
               
               
                 
                   S. saprophyticus 
                 
                 M4070 
                 mecA 
                 8 
                 16 
                 32 
                 8 
               
               
                 
                   S. saprophyticus 
                 
                 M2981 
                 mecA 
                 8 
                 16 
                 8 
                 8 
               
               
                 
                   S. epidermidis 
                 
                 M2923 
                 WT 
                 8 
                 16 
                 16 
                 16 
               
               
                 
                   S. epidermidis 
                 
                 M2931 
                 WT 
                 16 
                 16 
                 16 
                 8 
               
               
                 
                   S. haemolyticus 
                 
                 M2976 
                 mecA 
                 4 
                 16 
                 8 
                 8 
               
               
                 
                   S. haemolyticus 
                 
                 M3014 
                 WT 
                 4 
                 16 
                 8 
                 8 
               
               
                 
                   S. hominis 
                 
                 M2973 
                 mecA 
                 4 
                 4 
                 4 
                 4 
               
               
                 
                   S. hominis 
                 
                 M2967 
                 mecA 
                 4 
                 8 
                 8 
                 8 
               
               
                 
                   S. warnerii 
                 
                 M6823 
                 mecA 
                 8 
                 8 
                 8 
                 8 
               
               
                 
                   S. cohnii 
                 
                 M6767 
                 mecA 
                 4 
                 16 
                 16 
                 8 
               
             
          
           
               
                   Enterococcus  spp. (n = 17) 
                   
               
             
          
           
               
                 
                   E. faecium 
                 
                 M2304 
                 vanA 
                 4 
                 16 
                 16 
                 8 
               
               
                 
                   E. faecium 
                 
                 M2664 
                 vanA 
                 8 
                 16 
                 8 
                 8 
               
               
                 
                   E. faecium 
                 
                 M2619 
                 vanB 
                 16 
                 32 
                 16 
                 16 
               
               
                 
                   E. faecium 
                 
                 M2481 
                 vanB 
                 16 
                 32 
                 16 
                 16 
               
               
                 
                   E. faecium 
                 
                 ZAP95 
                 WT 
                 16 
                 32 
                 16 
                 16 
               
               
                 
                   E. faecium 
                 
                 M6261 
                 N.D 
                 16 
                 32 
                 16 
                 16 
               
               
                 
                   E. faecalis 
                 
                 M4899 
                 vanB 
                 256 
                 256 
                 128 
                 256 
               
               
                 
                   E. faecalis 
                 
                 M6534 
                 vanB 
                 256 
                 128 
                 256 
                 256 
               
               
                 
                   E. faecalis 
                 
                 M4992 
                 vanA 
                 128 
                 128 
                 128 
                 128 
               
               
                 
                   E. faecalis 
                 
                 M6383 
                 vanA 
                 128 
                 128 
                 128 
                 128 
               
               
                 
                   E. faecalis 
                 
                 M4449 
                 vanA 
                 128 
                 128 
                 128 
                 128 
               
               
                 
                   E. faecalis 
                 
                 ATCC 51299 
                 vanB 
                 256 
                 256 
                 256 
                 256 
               
               
                 
                   E. faecalis 
                 
                 ATCC 29212 
                 WT 
                 128 
                 64 
                 128 
                 128 
               
               
                 
                   E. faecalis 
                 
                 M6983 
                 vanA 
                 128 
                 64 
                 128 
                 128 
               
               
                 
                   E. gallinarum 
                 
                 M2723 
                 vanC1 + vanA 
                 16 
                 32 
                 32 
                 16 
               
               
                 
                   E. gallinarum 
                 
                 M2685 
                 vanC1 + vanA 
                 16 
                 16 
                 16 
                 16 
               
               
                 
                   E. raffinosus 
                 
                 M6187 
                 vanA 
                 4 
                 ND 
                 8 
                 8 
               
               
                   
               
             
          
         
       
     
     In order to evaluate the structure that the peptides acquire in aqueous solution and in the presence of a surfactant such as SDS, a circular dichroism (CD) assay was performed. This way, the differences between the structured acquired by the peptides and their biological activity could be correlated. 
     The CD spectra of the peptides in aqueous solution show that said peptides are not structured in aqueous buffer, with a characteristic minimum at approximately 200 nm. With the addition of SDS micelles, conformational changes are generated in peptides 2 and 5, which are consistent with the formation of alpha-helix structures, with two characteristic minimums, close to 208 and 222 nm. Peptide 1 is also subjected to a similar transition, however the structure level acquired is much lower than that observed in peptides 2 and 5. The CD spectrum of peptide 3 is almost invariable with the addition of SDS micelles, indicating the persistence of a disorganized conformation. In the case of Omiganan, the spectrum is significantly modified in the presence of SDS, with the band at 200 nm diminishing and a new band appearing near 230 nm, which may be a result of the interaction between side chains in tryptophan ( FIGS. 1 and 2 ). These experimental results match the theoretical parameters obtained by computational analysis. 
     Physical-chemical parameters of the peptides theoretically analyzed for each sequence are summarized in Table 5. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 pep- 
                 pep- 
                 pep- 
                 pep- 
                 pep- 
               
               
                   
                 tide 1 
                 tide 2 
                 tide 3 
                 tide 4 
                 tide 5 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Isoelectric point 
                 13.10 
                 10.89 
                 12.25 
                 9.85 
                 11.75 
               
               
                 Net charge 
                 +12 
                 +6 
                 +7 
                 +3 
                 +7 
               
               
                 Hydrophobicity * 
                 +29.04 
                 +20.51 
                 +23.38 
                 +19.05 
                 +16.10 
               
               
                 Alpha-helix content ** 
                 1.03 
                 5.35 
                 1.54 
                 0.11 
                 7.07 
               
               
                   
               
               
                 * Wimley-White Scale Kcal mol −1   
               
               
                 ** According to AGADIR algorithm 
               
             
          
         
       
     
     When the non-amidated peptides of the invention were assessed the results shown in Table 6 were obtained. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 NON-AMIDATED PEPTIDES, MIC (μg/ml) IN AGAR DILUTION 
               
             
          
           
               
                 Species 
                 ID No. 
                 Resistance Gene 
                 Omiganan 
                 peptide 1 
                 peptide 2 
                 peptide 3 
                 peptide 4 
                 peptide 5 
               
               
                   
               
             
          
           
               
                 
                   S. warnerii 
                 
                 M6823 
                 Oxa-R 
                 16 
                 16 
                 8 
                 32 
                 &gt;128 
                 8 
               
               
                 
                   S. cohnii 
                 
                 M6767 
                 mecA 
                 16 
                 16 
                 16 
                 32 
                 &gt;128 
                 8 
               
               
                 
                   S. aureus 
                 
                 M6794 
                 MRSA 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
               
               
                 
                   E. coli 
                 
                 ATCC 25922 
                 WT 
                 &gt;128 
                 &gt;128 
                 128 
                 &gt;128 
                 &gt;128 
                 64 
               
               
                 
                   S. aureus 
                 
                 ATCC29213 
                 MSSA 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
               
               
                 
                   P. aeruginosa 
                 
                 ATCC 27853 
                 WT 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
               
               
                 
                   P. aeruginosa 
                 
                 M13513 
                 KPC 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 &gt;128 
               
               
                 
                   K. pneumoniae 
                 
                 M13540 
                 KPC 
                 &gt;128 
                 &gt;128 
                 64 
                 &gt;128 
                 &gt;128 
                 &gt;128 
               
               
                   
               
             
          
         
       
     
     For the concentrations studied: peptide 1 has activity against  S. warnerii  and  S. cohnii ; peptide 2 has activity against  S. warnerii, S. cohnii, E. coli  and  K. pneumoniae , peptide 3 has activity against  S. warnerii  and  S. cohnii , peptide 4 does not have activity against any of the tested strains and peptide 5 has activity against  S. warnerii, S. cohnii  and  E. coli.    
     The present invention is better illustrated with the following examples, which are not to be construed as a limitation to the scope thereof. On the contrary, it must be clearly understood that other embodiments, modifications and equivalents thereof may be apparent for a person skilled in the art, after reading the present description without departing from the spirit of the present invention and/or scope of the annexed claims. 
     EXAMPLES 
     The design of the peptides was aimed at generating amphipathic, cationic sequences, of the alpha-helix type. To this end, multiple alignment informatics tools, simulators of physical-chemical properties such as hydrophobicity or alpha-helix content, were used. All of said informatics software (BLAST, CLUSTAL, AGADIR, ExPASY) are freeware and available through the Internet. 
     In this analysis, the sequences and amino acids were set forth in specific consensus positions, when available, or semi-conserved motifs in some known peptides. Taking into consideration these diverse parameters, five in silico peptides were designed, having between 18 and 30 amino acids (SEQ ID NOs: 1-5). 
     Synthesis and Purification 
     The peptide synthesis was carried out by means of automated SPPS (solid phase peptide synthesis) synthesis and subsequently analyzed by mass spectrometry and purified by reverse phase HPLC. 
     Also, each peptide was amidated in the C-terminal end. 
     Circular Dichroism Analysis 
     Far-UV Circular Dichroism 
     Secondary structure content was studied by far-UV Circular dichroism spectroscopy, using a JASCO J 810 equipment (Jasco Corp., Tokio, Japan) calibrated with (+)10-camphorsulfonic acid. Measurements were carried out under a 8 l/h-nitrogen gas flux, at a temperature of 20° C., controlled by a Peltier-type system (JASCO). 
     Spectra between 185 and 320 nm were recorded, using a 0.1 cm-optical path cell. Concentration of the peptides were 40 μM, dissolved in 10 mM sodium phosphate buffer pH 7.0, or in the same buffer with 10 mM sodium dodecyl sulfate (SDS). Sensitivity was 100 millidegrees. Scan speed was 50 nm/min, a response time of 1 s and a bandwidth of 1 nm. An average of five spectra was performed for each sample. The average was corrected by buffer absorption and then baselined to zero using the average of readings at 290 and 320 nm. Finally, the data were smoothed using a Golay polynomial Savizky fourth grade, with a window of ten points. The spectra were converted to mean molar ellipticity per residue by using the equation: [θ]=θ/(10× c×n×d ) wherein [θ] is the molar ellipticity (in degrees×cm 2 ×dmol −1 ), θ is the ellipticity in millidegrees, n is the number of residues in the peptide and c its molar concentration, d the length of the cell in centimeters. 
     In vitro sensitivity assay of aerobically growing bacteria, using the disc diffusion method was performed according to the standards in the M2 document of the CLSI ( Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Disk Susceptibility Tests .) using the non-amidated peptides 
     In brief, intermediate solutions (10×) of the antimicrobial non-amidated peptides were prepared by serial dilutions 1:2, 1:4 and 1:8 by two-fold dilutions. Then, one part of the solution of the antimicrobial peptide 10× was added to nine parts of melted bacteriological agar. The antibiotic solution was added for each dilution, in the melted agar and cooled to 45-50° C. in a water bath. Then, the agar-antibiotic mixture was placed in a Petri dish until a 3-4 mm depth was reached. The inoculum is prepared with a turbidity of 0.5 in the McFarland scale (approximately 1-2 10 8  CFU/ml). The final inoculum required for the agar dilution test is 10 4  colony forming units (CFU) per “spot” having 5-8 mm in diameter. The inoculated plates must be incubated upside down at 35° C. for a period of 16-20 hs. MIC was recorded as the value of the lower dilution completely inhibiting bacterial development, without considering the development of a single colony or a slight film caused by the inoculum deposit. The final point in these cases will correspond to the concentration at which there is more than 80% reduction in growth as compared with the control. 
     Analysis of Antimicrobial Activity 
     Antimicrobial activity was analyzed by means of the determination of the MIC (minimum inhibitory concentration) by the microdilution technique. The technique was carried out in the standard fashion according to ( CLSI M 07- A 9 Vol. 32 No 2. January 2012). The peptide concentration range used was from 0.5 μg/mL to 512 μg/mL in two-fold serial dilutions. The used medium was MH Difco broth supplemented with cations, at a final concentration of Ca ++ 20-25 mg/L and Mg ++ 10-12.5 mg/L. The working inoculum used corresponds to a 1/100 dilution of a bacterial suspension equivalent to 0.5 in the Mc Farland scale. 
     Analysis of Hemolytic Activity 
     Evaluation of cytotoxic activity was performed by means of an erythrocyte hemolysis assay (according to  Pure Appl. Chem ., Vol. 79, No. 4, pp. 717-728, 2007). In brief, from a volume of heparinized human whole blood, 3 times that volume of PBS is added and centrifuged 10 minutes at 1500 rpm, repeating the washing two more times. 10% blood solutions in PBS were prepared with that pellet. Subsequently, each group was added the corresponding peptide at the indicated concentration and incubated at 37° C. for 30 minutes. After incubation time, each tube was centrifuged at 10000 rpm for 5 minutes and the supernatant was read in a spectrophotometer at 600 nm. 
     Bacterial Strains 
     78 isolates were studied, previously characterized in the Reference National Laboratory INEI-ANLIS of the Instituto Carlos Malbrán, with different antibiotic resistance mechanisms. 
     40 Gram positive strains, 37 INEI-ANLIS isolates were analyzed plus three ATCC reference strains: 
     
         
         
           
               Staphylococcus aureus  (11 isolates) 
               Staphylococcus  Coagulase Negative (12 isolates: 4  S. epidermidis,  2  S. saprophyticus,  2  S. haemolyticus,  2  S. hominis,  1  S. warnerii , 1  S. cohnii ) 
               Enterococcus  spp. (17 isolates: 6  E. faecium,  8  E. faecalis,  2  E. gallinarum , 1  E. raffinosus )
 
The resistance mechanisms for this group, as may be seen in Table 4, were as follows: WT, vanA, vanB, vanC, mecA, ermA, ermC, msrA, InuA.
 
43 Gram negative strains, 41 INEI-ANLIS isolates, plus three reference ATCC strains were analyzed:
 
               Pseudomonas aeruginosa  (12 isolates) 
               Acinetobacter  sp. (10 isolates: 9  A. baumanii,  1  A. junii ) 
               Klebsiella pneumoniae  (12 isolates) 
               Escherichia coli  (9 isolates) 
           
         
       
    
     The resistance mechanisms for this group, as may be seen in Table 4, were as follows: (tem-1, cmy, cit, shv-1, ctx-m-2, per-2, ges-1/3, veb-1, oxa-9, oxa-23, oxa-58, vim-11, imp-1/13/16, spm-1, kpc-2).