Emergence of antibiotic resistant strains due to widespread use of antibiotics and dearth of new antibiotics has resulted in looking for new antimicrobial agents with new targets and unique mechanism of action. AMPs are gaining importance due to their superior and dynamic mechanism of action compared to antibiotics 
. AMPs have existed for millions of years; however resistance to antimicrobial peptides has not been reported. Computer-assisted peptide design, combinatorial libraries and structure based designs are some of the methods used for designing novel AMPs 
. Another way to develop novel antimicrobial peptides is by using recombinant bacteriophages engineered to display short random peptide coding sequences in their genome. Combinatorial phage-display is a powerful tool for the selection of short peptides binding to any target, biological or non-biological 
. Cell surface recognition is considered to be a crucial event in biological events. Finding new scaffolds that recognize cell surface can be useful in diagnostics and therapeutics 
. Since cell surfaces are composed of complex molecular compositions they can provide unique binding sites. One approach to identify ligands that binds to cell surfaces is by using phage-display. Phage-display has been used successfully in number of applications, including vaccine development, protein drug discovery, and to generate diagnostic and therapeutic peptides 
. Several peptides with antimicrobial property have been selected by phage-display indicating that phage-display libraries play an important role in drug development 
. Phage-display peptide as inhibitors against essential bacterial enzymes is another approach that scientists are using to develop novel antimicrobial peptides. A recent study used phage-display to select peptides against the enzyme I component of the E. coli
phosphotransferase system (PTS) thereby inhibiting the cell growth 
. This enzyme is present only in bacteria and is absent in eukaryotes thereby making it highly selective and specific. In this study we used a whole-cell phage-display approach to identify peptides binding to the cell surface of E. coli
. By using this approach we observed that a specific sequence, represented by EC5 (RLLFRKIRRLKR) repeated multiple times (5 out of 10 clones). Interestingly the aligned sequences contained conserved Arginine (R) and Lysine (L) residues. These Arginine and Lysine residues have been shown to be major components of antimicrobial peptides 
. Majority of native antimicrobial peptides have net charge ranging from +2 to +8 and hydrophobic value ranging from 41% to 50% 
. EC5 showed features common to antimicrobial peptides: net positive charge of +7 and hydrophobic value of 41%. Sequence analysis of EC5 suggested that it was a cationic α-helical peptide.
EC5 showed antimicrobial properties deemed bactericidal by structure analysis of the peptide and hence we investigated the bactericidal activity of the peptide in vitro
. The best bactericidal AMP kills bacteria in vitro
, including certain antibiotic-resistant pathogens, with MICs ranging from 1 to 8 µg/ml 
. EC5 is a narrow spectrum antibacterial agent and was most effective against gram-negative bacteria tested, with an MIC and MBC of 8 µg/ml for E. coli
strain. P. aeruginosa
on the other hand demonstrated an MIC of 8 µg/ml for ATCC strain 27853 and 16–32 µg/ml for P. aeruginosa
ATCC 12121. The EC5 peptide showed no activity against Gram-positive strains and appeared to be more active against Gram-negative strains with a MIC of 4–128 µg/ml. Since AMPs are known to often show reduced MIC values when full MHB medium is used for testing, we also tested the MIC values of EC5 using cation-supplemented MHB. However we found no difference in the MIC values of EC5 when tested by both the methods suggesting that the presence of salts in MHB does not interfere with the activity of EC5.
While AMPs are effective in vitro, they may lose their activity in vivo, when given intravenous since human blood may have factors such as proteins or small nucleic acids that can adsorb AMPs and hinder their activity. In this study we developed an ex vivo assay using human plasma and platelets as the test medium to evaluate the extent and duration of EC5 efficacy. The EC5 peptide was incubated along with the test organisms into the medium and incubated for 2 h. In this experimental setup, EC5 exhibited potent bactericidal activity in homologous plasma and inhibited E. coli and P. aeruginosa at concentration of 50 µg/ml. However, at lower concentration it did not retain the similar effect as in conventional media. EC5 in the presence of platelets suspended in plasma showed complete inhibition of E. coli at 25 and 50 µg/ml. However at 12.5 µg/ml it showed only 4.5 log10 reduction in CFU/ml; Also, the activity of EC5 was lower against P. aeruginosa in the presence of platelets suspended in plasma compared to what is observed in conventional media. These observations suggest that some plasma factors may be interfering with, or masking the effect of EC5 at lower concentrations.
Safety analysis such as the non-hemolysis of chRBCs (even at concentration of 500 µg/ml) and non-cytotoxicity of the peptide suggests a potential for EC5 to be an effective drug candidate. Since the peptide MIC and peptide concentrations inducing hemolysis differ by more than an order of magnitude, the data indicates that EC5 therapeutic index for the treatment of bacterial infections could be favorable. Many AMPs kill bacterial cells by disrupting their membrane integrity. We investigated the interaction of peptides with membranes using membrane permeabilization assays. EC5 caused rapid increase in outer membrane permeabilization at lower concentration, below MBC, which was followed by cytoplasmic depolarization. The changes correlated well with cell killing and cytoplasmic depolarization at the same time. However, polymyxin B at 3.125 µg/ml resulted in complete inhibition of CFU within 5 min of exposure of the peptide, but only minimal release of diSC3
5 from the cells after 5 min of exposure. Polymyxin B causes cell death prior to cytoplasmic depolarization whereas for EC5 both the events appear to occur at the same time. Our observations suggest that EC5 may disrupt the cytoplasmic membrane, causing rapid depolarization, and inhibition of macromolecular synthesis as seen by ATP inhibition and rapid cell death. In order to confirm our hypothesis we also investigated the peptide-membrane interaction using molecular dynamic simulations. EC5 was simulated with POPE
POPG membrane bilayer model using the Hex docking server (http://hexserver.loria.fr/
) and Cluspro protein-protein docking server (Version 2.0). Docking results suggested that EC5 may lie parallel to the membrane and translocate across the cytoplasmic membrane. These results suggest that EC5 penetrates bacterial-mimicking membranes as a result of electrostatic interactions which are essential for peptides to interact with membrane surface. The peptide then integrates into the cell membrane, causing depolarization and cell death 
. Currently Gram-negative bacteria such as E. coli
and P. aeruginosa
are causing concern due to the rapid spread of extremely resistant strains to traditional antibiotics.
The use of polymyxin B was abandoned previously since the antibiotic showed high toxicity, especially nephrotoxicity 
. EC5 showed potent in vitro
and low cytotoxicity, suggesting their use as drug template for the development of new antibacterial drugs. Since EC5 shows membrane-permeabilizing properties it can also be used in combination with conventional antibiotics to facilitate the entry of drugs into the cells. Combination therapy with antibiotics can potentially be used to broaden the antimicrobial spectrum to treat multiple-drug resistant strains. 
. Our future studies would include developing improved analogs of EC5 with improved antimicrobial activity against other pathogens using different approaches such as substitution of amino acids, inclusion of D-amino acids or beta-amino acids and cyclization of peptides and peptoid mimics to name a few. Drug-resistant strains of E. coli
are a serious health problem and hence AMPs such as EC5 may have potential therapeutic applications. In conclusion, these studies demonstrate that peptides with antimicrobial activity can be selected from random phage libraries and may prove useful in the development of novel bactericidal agents.