Cationic peptides with broad spectrum activity

Abstract

The present invention relates generally to peptides, and more specifically to peptides with anti-biofilm and / or anti-microbial activity.

Description

CATIONIC PEPTIDES WITH BROAD SPECTRUM ACTIVITY FIELD

[0001] The present invention relates generally to peptides, and more specifically to peptides with anti-biofilm and / or anti-microbial activity. BACKGROUND

[0002] The current therapy for infectious diseases is facing twin threats [Boucher, H. W., et al. (2009). Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clinical Infectious Diseases, 48(1), 1–12; Coates, A. R. M., & Hu, Y. (2007). Novel approaches to developing new antibiotics for bacterial infections. British Journal of Pharmacology, 152(8), 1147–1154]. On the one hand, antibiotic resistance is rising rapidly [Zaman, S. B., et al. (2017). A Review on Antibiotic Resistance: Alarm Bells are Ringing. Cureus, 9(6), e1403] while, on the other, there are a limited number of effective novel compounds or strategies under development or entering the clinic. Adding to the problem is the issue of microbial biofilms, which are surface-associated bacterial communities that grow in a protective polymeric matrix [Koo, H., et al. (2017). Targeting microbial biofilms: Current and prospective therapeutic strategies. Nature Reviews. Microbiology, 15(12), 740–755]. Bacterial growth as biofilms is a major lifestyle adaptation for bacteria in nature as well as in industrial and clinical settings and some consider biofilm to represent the natural growth state of bacteria [Penesyan, A., et al. (2021). Three faces of biofilms: A microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. npj Biofilms and Microbiomes, 7(1), Article 1]. Indeed, biofilms have been associated with 65% or more of all clinical infections and 80% of chronic infections [Assefa, M., & Amare, A. (2022). Biofilm-Associated Multi-Drug Resistance in Hospital-Acquired Infections: A Review. Infection and Drug Resistance, 15, 5061–5068] and conventional antibiotics have proven relatively ineffective when prescribed as treatments [Khan, J., et al. (2021). Challenges of antibiotic resistance biofilms and potential combating strategies: A review.3 Biotech, 11(4), 169]. With regards to antibiotic resistance, bacterial cells growing within a biofilm can result in as much as 1000-fold decrease in susceptibility to antimicrobial agents, due to gene expression changes of bacteria within the biofilm, poor antibiotic penetration into the biofilm, and the stationary phase growth of bacteria underlying the surface layer [Hancock, R. E. W., Alford, M. A., & Haney, E.F. (2021). Antibiofilm activity of host defence peptides: Complexity provides opportunities. Nature Reviews Microbiology, 19(12), 786-797; Antunes, L. C. M., & Ferreira, R. B. (2011). Biofilms and bacterial virulence. Reviews and Research in Medical Microbiology, 22(1), 12; Høiby, N., et al. (2010). Antibiotic resistance of bacterial biofilms. International Journal of Antimicrobial Agents, 35(4), 322–332]. Biofilm-associated infections are widespread and contribute to various tissue-related infections including: sinusitis, otitis media, endocarditis, dental plaque, lung infections (particularly in cystic fibrosis patients), urinary tract infections and chronic wounds [Lebeaux, D., Ghigo, J.-M., & Beloin, C. (2014). Biofilm-related infections: Bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiology and Molecular Biology Reviews : MMBR, 78(3), 510–543]. In addition, biofilms growing on the surface of medical devices are also a major source of antibiotic resistant infections and these commonly occur on the surface of catheters, breast implants, contact lenses, prosthetic joints, and pacemakers [Lebeaux, D., Ghigo, J.-M., & Beloin, C. (2014). Biofilm-related infections: Bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiology and Molecular Biology Reviews : MMBR, 78(3), 510–543].

[0003] Cationic antimicrobial host defense peptides (HDPs) [Mookherjee, N., et al. (2020). Antimicrobial host defence peptides: Functions and clinical potential. Nature Reviews. Drug Discovery, 19(5), 311–332; Haney, E. F., Straus, S. K., & Hancock, R. E. W. (2019). Reassessing the host defense peptide landscape. Frontiers in Chemistry, 7, 43] have been implicated as having potential to address biofilm-associated infections [Hancock, R. E. W., Alford, M. A., & Haney, E. F. (2021) Antibiofilm activity of host defence peptides: Complexity provides opportunities. Nature Microbiology Reviews, 19:786-797; Overhage, J., et al. (2008). Human host defense peptide LL-37 prevents bacterial biofilm formation. Infection and Immunity, 76(9), 4176–4182; Haney, E. F., et al. (2019). Influence of non-natural cationic amino acids on the biological activity profile of innate defense regulator peptides. Journal of Medicinal Chemistry, 62(22), 10294–10304; Haney, E. F., et al. (2018). Computer-aided discovery of peptides that specifically attack bacterial biofilms. Scientific Reports, 8, 1871; de la Fuente- Núñez, C., et al. (2015). D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections. Chemistry & Biology, 22(2), 196–205; de la Fuente-Núñez, C., et al. (2014). Broad-spectrum anti-biofilm peptide thattargets a cellular stress response. PLoS Pathogens, 10(5), e1004152; PCT / CA2007 / 001453, filed 21 August 2007, published under No. WO 2008 / 022444 on 28 February 2008; PCT / US2014 / 052993, filed 27 August 2014, published under WO 2015 / 038339 on 19 March 2015; PCT / IB2019 / 050643, filed 25 January 2019, published under WO 2019 / 145913 on 1 August 2019.] HDPs are produced by virtually all complex organisms and represent a major component of the organism’s innate defenses against infection. HDPs have been implicated as potential alternatives to antibiotics [Rima, M., et al. (2021). Antimicrobial Peptides: A Potent Alternative to Antibiotics. Antibiotics, 10(9), 1095; Fjell, C. D., et al. (2012). Designing antimicrobial peptides: Form follows function. Nature Reviews Drug Discovery, 11(1), 37–51; Baltzer, S. A., & Brown, M. H. (2011). Antimicrobial Peptides – Promising Alternatives to Conventional Antibiotics. Microbial Physiology, 20(4), 228–235].

[0004] Toxicity and / or aggregation tendencies are however obstacles to developing these molecules as novel therapeutics [Haney, E. F., et al. (2017). Aggregation and its influence on the immunomodulatory activity of synthetic innate defense regulator peptides. Cell Chemical Biology, 24(8), 969-980.e4]. SUMMARY

[0005] In one aspect, disclosed herein is an isolated peptide. In an alternative aspect, the disclosure includes an isolated polynucleotide encoding the peptide as described herein.

[0006] In some embodiments, the isolated peptide includes the amino acid sequence of Peptide 1: AA1– AA2– AA3– AA4– AA5– AA6– AA7– AA8– AA9– AA10– AA11– AA12– NH2Peptide 1 where AA1is Arg, Lys, Trp, Ala, or Leu, AA2is Val or Trp, AA3is Arg, Ile, Leu, or Val, AA4is Val, Trp, or Leu, AA5 is Ile or Trp, AA6 is Arg or Trp, AA7 is Val or Ile, AA8 is Gln, Lys, Val, Ile, Trp, or Arg,AA9 is Trp, Leu, Val, Lys, AA10is Arg, AA11is Ala, Ile, Arg, Trp, Val, Leu, or Lys, and AA12is Ile or Arg.

[0007] In some embodiments, in the isolated peptide including the amino acid sequence of Peptide 1: AA1is Arg or Lys, AA2is Val, AA3 is Arg, or Ile, AA4is Val or Trp, AA5is Ile or Trp, AA6 is Arg, AA7 is Val, AA8is Gln or Lys, AA9 is Trp or Leu, AA10 is Arg, AA11is Ala, Ile or Arg and AA12is Ile.

[0008] In some embodiments, the isolated peptide includes an amino acid sequence as set forth in one or more of SEQ ID NOs: 3, 9-12, 15, 18, 42, 43 or a functional variant thereof.

[0009] In some embodiments, the isolated peptide includes an amino acid sequence as set forth in one or more of SEQ ID NOs: 1-104, or a functional variant thereof.

[0010] In some embodiments, the isolated peptide as described herein includes a non-natural amino acid equivalent, such as L-2-amino-3-guanidinopropionic acid, L-2-Amino-4- guanidinobutyric acid, L-Homoarginine, L-2,3-diaminopropionic acid or L-Ornithine.

[0011] In some embodiments, the isolated peptide includes an isolated polypeptide X1-A- X2, wherein A includes an isolated peptide as described herein; and where each X1 and X2 independently includes an amino acid sequence of n amino acids, where n is 0 to 50.

[0012] In some embodiments, there is provided an isolated polynucleotide encoding an isolated peptide as described herein.

[0013] In some embodiments, there is provided a pharmaceutical composition including an isolated peptide as described herein, in combination with an antibiotic, such as an aminoglycoside, a beta-lactam, a penicillin, a cephalosporin, a quinolone, a fluoroquinolone, a carbapenem, a tetracycline, a polymyxin, a glycopeptide or a macrolide. In some embodiments, the antibiotic may be amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethyl- succinate / gluceptate / lactobionate / stearate, such as penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, imipenem, meropenem, panipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, polymyxin B, colistin, benzalkonium-Cl, benzalkonium-Cl, chlorhexidine, H2O2, polyvinylpyrrolidone–iodine, levofloxacin, linezolid, synercid, colimycin, methotrexate, daptomycin, phosphonomycin, cotrimoxazole, EDTA or AgNO3.

[0014] In some aspects, there is provided a method of inhibiting the growth of a bacterium, or of a bacterial biofilm, by contacting the bacterium or bacterial biofilm with an inhibition effective amount of a peptide or pharmaceutical composition as described herein. The bacterium may be, or the bacterial biofilm may include, a gram negative bacterium or a gram positive bacterium. The bacterium may be, or the bacterial biofilm may include, Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA), such as MRSA USA300, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, such as Enterococcus faecium #2-1, Enterobacter sp. including Enterobacter cloacae, such as Enterobacter cloacae 218R1, Pseudomonas aeruginosa, such as P. aeruginosa PAO1, Klebsiella pneumonia, such as Klebsiella pneumonia KPLN649, S. epidermidis, Escherichia coli, such as E. coli O157:H7, Salmonella enteritidis ssp Typhimurium, Campylobacter, Burkholderia cepacia complex or Acinetobacter baumannii, such as Acinetobacter baumannii Ab5075. The bacterium may be, or the bacterial biofilm may include, MRSA and the peptide may include the amino acid sequenceset forth in SEQ ID NO:2. The bacterium may be, or the bacterial biofilm may include, P. aeruginosa and the peptide may include the amino acid sequence set forth in SEQ ID NOs: 3, 9- 12, 15, 18, 42, or 43.

[0015] The bacterium or bacterial biofilm may be associated with an infection, such as a skin infection, sinusitis, septic shock or abscess. The bacterial biofilm may be an oral multispecies biofilm or an oral plaque biofilm.

[0016] In some aspects, there is provided a method of ameliorating resistance to an antibiotic, or of enhancing the efficacy of an antibiotic, by contacting a cell with an effective amount of a peptide as described herein in combination with the antibiotic, such as a aminoglycoside, a beta-lactam, a penicillin, a cephalosporin, a quinolone, a fluoroquinolone, a carbapenem, a tetracycline, a polymyxin, a glycopeptide or a macrolide. In some embodiments, the antibiotic may be amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethyl- succinate / gluceptate / lactobionate / stearate, such as penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, imipenem, meropenem, panipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, polymyxin B, colistin, benzalkonium-Cl, benzalkonium-Cl, chlorhexidine, H2O2, polyvinylpyrrolidone–iodine, levofloxacin, linezolid, synercid, colimycin, methotrexate, daptomycin, phosphonomycin, cotrimoxazole, EDTA or AgNO3.

[0017] In some aspects, there is provided the use of an isolated peptide as described herein for inhibiting the growth of a bacterium or a bacterial biofilm, for ameliorating resistance to an antibiotic, or for enhancing the efficacy of an antibiotic.

[0018] In some aspects, there is provided an article of manufacture, such as a collagen dressing, a commercial sterile bandage, an oral rinse, a medical device, a catheter, or a prosthetic device, including an isolated peptide as described herein.

[0019] In alternative embodiments of the various aspects, the peptide may be bound to a solid support or surface.

[0020] This summary does not necessarily describe all features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows biofilm inhibition of MRSA biofilms by SPOT-synthesized peptides comprised of modular components of peptides DJK5, RI-1018 and RI-1002. Peptides were evaluated at three different concentrations corresponding to treatment with 10 µl of the peptide stock solution (relative concentration = 1) as well as ½ and ¼ dilutions of this concentration. The percent biofilm inhibition of the peptides was calculated at each peptide concentration tested and this was compared to the percent inhibition observed for three independent replicates of DJK5 (shown as black dashed lines). Data points corresponding to peptides that prevented biofilm formation better than DJK5 at the two highest concentrations are highlighted in black while those peptide with comparable or worse activities relative to DJK5 are shown in gray. Three peptides: AB101-D (RI1002N8-DJK5C4, diamonds), AB102-D (DJK5C-RI1002N, squares), and AB103- D (DJK5C-DJK5N, triangles) proved to be more active than DJK5 alone.

[0022] Figures 2A-B show biofilm inhibition activity of >95% pure peptides against MRSA (A) and P. aeruginosa PAO1 (B) biofilms. Peptides were evaluated in duplicate for MRSA and triplicate for PAO1 and error bards represent standard deviation.

[0023] Figures 3A-B show antibiofilm activity of D-amino acid peptides DJK-5, AB101-D, AB102-D and AB103-D against luminescent strains of MRSA (A) and PAO1 (B) biofilms grown on skin organoids. Biofilms were established on the surface of the organoid skin for 24 hrs and then treated with 0.1% solution of peptide dissolved in water for 4 hrs. The reduction in CFUs recovered from the skin surface upon peptide treatment is reflected by the amount of light emitted from the luminescent bacteria present within the biofilm on the skin surface (bottom panel).

[0024] Figures 4A-D show antibiofilm activity of D-amino acid peptides in an organoid model of biofilm skin infection. MRSA (A and B) or P. aeruginosa (C and D) biofilms were established on the surface of N / TERT epithelial skin grown in on a transwell filter support for 24-hours. DJK- 5, AB101-D, AB102-D or AB103-D peptides were added (either alone on in combination with ciprofloxacin) to collagen dressings and then placed on the skin surface to mimic treatment of a biofilm-associated skin infection. Biofilms were treated for 24-hours and then the bacteriarecovered from the skin surface (A and C), or from the collagen dressing (B and D) were enumerated.

[0025] Figures 5A-D show efficacy of AB103-D loaded Medvance adhesive foam dressings in an organoid model of biofilm-associated skin infections. Briefly, 160 µl of aqueous peptide and / or antibiotic solution was used to hydrate an ~1 cm piece of Medvance foam dressing and this was applied to 24-hr old biofilms formed by luminescent strains of MRSA (A), S. epidermidis (B), P. aeruginosa LESB58 (C) and A. baumanii (D) grown on the surface of differentiated Ker-CT epidermal cells. Shown are the CFUs recovered after 24-hr treatment from the skin surface (left panels) and from the applied dressing (right panels).

[0026] Figures 6A-B show in vivo activity of D-amino acid antibiofilm peptides in a porcine model of biofilm wound infections. Peptide (PEP1 = DJK5; PEP3 = AB103-D) solutions were used to hydrate Puracol® resorbable dressings made of collagen either alone or in the presence of antibiotic (ABX1 = ciprofloxacin). Briefly, partial thickness wounds (10mm x 7mmx 1mm deep) were created on the backs of the animals then infected with log-phase cultures of bacteria and allowed biofilms to form for 24-hrs. Dressings were applied to infected wounds of either MRSA S. aureus USA300 (A) or P. aeruginosa 27312 (B) as either a single treatment (day 1) or twice treated (Day 1 and 4) and all wounds were excised on day 7 for CFU renumeration. The amount of peptide applied in all treatments was 10 mg while 1mg of ciprofloxacin was used in and treatment containing antibiotic. Mupirocin and silver sulfadiazine (SSD) were used as positive controls.

[0027] Figures 7A-B show resistance development to antibiotic, peptide or a combination of the two in a serial passage experiment. Resistance was developed against MRSA (A) or PAO1 (B) by serially passaging bacteria for 20-days in microtitre plates at the concentration of antimicrobial closest to the MIC where bacterial growth was observed. Data is plotted as the fold change in MIC relative to the MIC recorded during the first passage. The experiment was carried out three separate times and individual resistance trajectories are shown as solid, dashed or dotted lines for each treatment condition.

[0028] Figures 8A-C show cytotoxicity of D-amino acids peptides AB101-D, AB102-D and AB103-D determined in vitro. Cytotoxicity was assessed against PBMCs (A), Human bronchiolar epithelial cells (HBEs, B) and N / TERT keratinocytes (C) using the LDH release assay. In all cases, the peptides exhibited low toxicity towards cells within the concentration range evaluated.

[0029] Figure 9 shows biofilm inhibition of MRSA biofilms by SPOT-synthesized DJK5 derivatives. Three independent DJK5 peptide SPOT samples (dashed lines) were assessed for their biofilm inhibition activity against MRSA biofilms grown in 10% tryptic soy broth supplemented with 0.1% glucose. All SPOT samples of DJK5 derivatives with more potent antibiofilm activity have been coloured black while all samples with roughly equivalent or poorer antibiofilm activity compared to the parent peptide DJK5 are indicated in gray. Peptide names in the legend correspond to the DJK5 derivative evaluated and specifically refer to the amino acid residue that has been substituted within the parent sequence of DJK5 (for example, the sequence of “R3” is vqrrairvrvir- NH2compared to the DJK5 sequence of vqwrairvrvir-NH2).

[0030] Figure 10 shows killing of microbes in 3-day-old oral plaque biofilms exposed to the peptides for 1 and 3 days. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, respectively (appearing as light gray and dark gray regions). The scale bar represents 200 μm. The proportion of dead biofilm bacterial cell volume after exposure to the peptides or water for 1 or 3 days was quantified based on the amount of green and red staining present within the sample. Groups labeled with same letters denote no statistical difference (p > 0.05). Peptide names are the same as those in Table 1 with the leading “AB” dropped from the peptide names.

[0031] Figure 11 shows impact of brief exposure to peptides after one minute on 3-day-old plaque biofilms. The proportion of dead biofilm bacterial cell volume after different peptide treatments for 1 minute was quantified based on the amount of green and red staining within the biomass (B). Groups labeled with same letters denote no statistical difference (p > 0.05).

[0032] Figures 12A-B show toxicity of DJK-5 Derived Peptides. Hemolysis was evaluated against red blood cells in PBS (A) while cytotoxicity was evaluated against peripheral blood mononuclear cells (PBMCs) isolated from healthy donors using the LDH cytotoxicity assay (B).

[0033] Figures 13A-B show toxicity of AB103-D Derived Peptides. Hemolysis was evaluated against red blood cells in PBS (A) while cytotoxicity was evaluated against peripheral blood mononuclear cells (PBMCs) isolated from healthy donors using the LDH cytotoxicity assay (B).

[0034] Figures 14A-B show murine subdermal toxicity assessment of AB103-D derived peptides. Clinical scores were recorded for each peptide treated mouse one hour after peptide administration (A). In general, a higher clinical score corresponds to more toxic effects of the peptides and the maximum possible clinical score according to the grading scheme used was 40.The presence of a cyst or lesion was recorded 16 hours after peptide administration and the size of the cyst was measured using calipers (B). Data shown are the mean of two independent replicates at a peptide dose of 4 mg / kg.

[0035] Figures 15A-C show peptides AB101-D, AB102-D and AB103-D compared to DJK5 in a murine sinusitis model of bacterial infections. MRSA USA300, nasal cavity (A) P. aeruginosa LESB58 (B and C, nasal cavity); MRSA USA300, lung tissue (D) P. aeruginosa LESB58 (E and F, lung tissue); and MRSA USA300, weight change (G) P. aeruginosa LESB58 (H and I, weight change).

[0036] Figures 16A-C show peptides AB101-D, AB102-D and AB103-D compared to DJK5 in 3-day-old oral multispecies biofilms. Daily peptide treatment for 3 days (A), two days (B) or one day (C).

[0037] Figures 17A-B show peptide AB103-D compared to peptide DJK5 following brief exposure in oral multispecies biofilms. DJK5 treatment (A) and AB103-D treatment (B).

[0038] Figures 18 shows dead bacterial cell volume upon treatment with peptide AB103-D compared to DJK5 in oral multispecies biofilms.

[0039] Figures 19A-B show antibiofilm effects of peptide AB103-D on oral plaque biofilms compared to oral rinse solutions. Representative single plaque donor sample (A) and representative donor biofilm (B).

[0040] Figures 20A-D show antibiofilm and antibacterial activity of AB103-D derivatives compared to DJK5. MRSA USA300, planktonic inhibition (A) and P. aeruginosa PAO1, planktonic inhibition (B); MRSA USA300, biofilm inhibition (C) and P. aeruginosa PAO1, biofilminhibition (D). DETAILED DESCRIPTION

[0041] The present disclosure provides, in part, isolated peptides that have inhibitory activity against bacteria, including bacterial biofilms, planktonic bacteria and / or abscesses.

[0042] In some embodiments, the present disclosure provides isolated “broad spectrum peptides”. A “broad spectrum peptide,” as used herein, refers to a peptide that exhibits antimicrobial activity against both Gram positive and Gram negative bacteria. In some embodiments, a broad spectrum peptide in accordance with the present disclosure exhibits activityagainst the biofilm growth state of a bacterium. In alternate embodiments, a broad spectrum peptide in accordance with the present disclosure exhibits activity against the planktonic growth state of a bacterium. In some embodiments, a broad spectrum peptide in accordance with the present disclosure also exhibits low toxicity and low aggregation.

[0043] In some embodiments, a peptide according to the present disclosure can be an isolated peptide including the amino acid sequence of Peptide 1: AA1 – AA2 – AA3 – AA4 – AA5 – AA6 – AA7 – AA8 – AA9 – AA10 – AA11 – AA12 – NH2 Peptide 1 where AA1 can be Arg, Lys, Trp, Ala, or Leu, AA2can be Val or Trp, AA3can be Arg, Ile, Leu, or Val, AA5can be Ile or Trp, AA6 can be Arg or Trp, AA7 can be Val or Ile, AA8can be Gln, Lys, Val, Ile, Trp, or Arg, AA9can be Trp, Leu, Val, Lys, AA10 can be Arg, AA11 can be Ala, Ile, Arg, Trp, Val, Leu, or Lys, and AA12 can be Ile or Arg.

[0044] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg or Lys, AA2is Val, AA3 is Arg or Ile, AA4 is Val or Trp, AA5is Ile or Trp, AA6is Arg, AA7 is Val, AA8is Gln or Lys, AA9is Trp or Leu, AA10 is Arg, AA11 is Ala, Ile or Arg andAA12 is Ile.

[0045] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg, AA2is Val, AA3is Arg, AA4 is Val, AA5 is Ile, AA6is Arg, AA7 is Val, AA8 is Gln, AA9is Trp, AA10is Arg, AA11 is Ala, Ile, or Arg, and AA12is Ile.

[0046] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2is Val, AA3 is Arg, AA4 is Val, AA5is Ile, AA6 is Arg, AA7 is Val, AA8is Gln, AA9is Trp or Leu, AA10 is Arg, AA11is Ala, and AA12is Ile.

[0047] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where:AA1 is Arg, AA2is Val, AA3is Arg, AA4 is Val, AA5is Ile, AA6is Arg, AA7 is Val, AA8 is Gln or Lys, AA9is Trp, AA10 is Arg, AA11 is Ala, and AA12is Ile.

[0048] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2is Val, AA3 is Arg or Ile, AA4is Val, AA5is Ile, AA6 is Arg, AA7 is Val, AA8is Gln, AA9 is Trp, AA10 is Arg, AA11is Ala, and AA12is Ile.

[0049] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2 is Val, AA3 is Arg,AA4 is Val, AA5is Ile or Trp, AA6is Arg, AA7 is Val, AA8is Gln, AA9is Trp, AA10 is Arg, AA11 is Ala, and AA12is Ile.

[0050] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2is Val, AA3 is Arg, AA4is Val or Trp, AA5is Ile, AA6 is Arg, AA7is Val, AA8is Gln, AA9 is Trp, AA10 is Arg, AA11is Ala, and AA12 is Ile.

[0051] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg or Lys, AA2 is Val, AA3is Arg, AA4is Val, AA5 is Ile, AA6 is Arg,AA7 is Val, AA8is Gln, AA9is Trp, AA10 is Arg, AA11is Ala, and AA12is Ile.

[0052] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2 is Val, AA3 is Arg, AA4is Val, AA5is Ile, AA6 is Arg, AA7is Val, AA8is Gln, AA9 is Trp, AA10is Arg, AA11is Ala, and AA12 is Ile.

[0053] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Lys, AA2 is Val, AA3is Arg, AA4is Val, AA5 is Ile, AA6is Arg, AA7is Val, AA8 is Gln, AA9 is Trp,AA10 is Arg, AA11is Ala, and AA12is Ile.

[0054] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2 is Val, AA3 is Arg, AA4is Trp, AA5 is Ile, AA6 is Arg, AA7is Val, AA8is Gln, AA9 is Trp, AA10is Arg, AA11is Ala, and AA12 is Ile.

[0055] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg, AA2 is Val, AA3is Arg, AA4 is Val, AA5 is Trp, AA6is Arg, AA7is Val, AA8 is Gln, AA9is Trp, AA10is Arg, AA11 is Ala, and AA12 is Ile.

[0056] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2 is Val, AA3is Arg, AA4is Val, AA5 is Ile, AA6 is Arg, AA7is Val, AA8 is Gln, AA9 is Trp, AA10is Arg, AA11is Ile, and AA12 is Ile.

[0057] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg, AA2is Val, AA3is Arg, AA4 is Val, AA5 is Ile, AA6is Arg, AA7 is Val, AA8 is Lys, AA9is Trp, AA10is Arg, AA11 is Ala, and AA12is Ile.

[0058] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg,AA2 is Val, AA3is Arg, AA4is Val, AA5 is Ile, AA6is Arg, AA7is Val, AA8 is Gln, AA9 is Leu, AA10is Arg, AA11 is Ala, and AA12 is Ile.

[0059] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1 is Arg, AA2is Val, AA3is Ile, AA4 is Val, AA5is Ile, AA6is Arg, AA7 is Val, AA8 is Gln, AA9is Trp, AA10 is Arg, AA11 is Ala, and AA12is Ile.

[0060] In some embodiments, a peptide according to Peptide 1 includes an amino acid sequence where: AA1is Arg, AA2is Val, AA3 is Arg, AA4 is Val,AA5 is Ile, AA6is Arg, AA7is Val, AA8 is Gln, AA9is Trp, AA10is Arg, AA11 is Arg, and AA12 is Ile.

[0061] In some embodiments, a peptide according to Peptide 1 specifically excludes the amino acids Met, Phe, Pro, His, Asp, Glu, Ser, Thr, Cys, Tyr, and Asn.

[0062] In some embodiments, a peptide according to Peptide 1 specifically excludes the amino acids Asp, Glu, Ser and Thr.

[0063] In some embodiments, a peptide according to Peptide 1 specifically excludes the amino acids: Ile or Gln in the AA1position, Ala, Leu, Gly or Lys in the AA2position, Ala, Gln, or Gly in the AA3 position, Gln, Ala, Ile, or Gly in the AA4position, Leu, Val, Ala, Gln or Gly in the AA5position, Lys, Leu, Val, Gln, Ala, Ile or Gly in the AA6 position, Gln, Arg, Ala, Trp or Gly in the AA7 position, Leu, Ala or Gly in the AA8position, Gln, Ala or Gly in the AA9 position, Trp, Leu, Val, Lys, Ile, Gln, Ala or Gly in the AA10 position, Gly or Gln in the AA11position, and / or Trp, Val, Ala or Gly in the AA12position.

[0064] In some embodiments, a peptide according to the present disclosure may be an isolated peptide including an amino acid sequence as set forth in one or more of SEQ ID NOs: 1-104, or a functional variant thereof. In some embodiments, a peptide according to the present disclosure may be an isolated peptide including an amino acid sequence as set forth in one or more of SEQ ID NOs: 1-12 and 14-104, or a functional variant thereof.

[0065] In some embodiments, an isolated peptide according to the present disclosure may be a peptide including an amino acid sequence as set forth in one or more of SEQ ID NOs: 1-3, 9-12, 15, 18, 42, 43, or a functional variant thereof.

[0066] In some embodiments, an isolated peptide according to the present disclosure may be a peptide including an amino acid sequence as set forth in one or more of SEQ ID NOs: 3, 9-12, 15, 18, 42, 43, or a functional variant thereof.

[0067] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Lys, Trp, Ala, or Leu at the first position.

[0068] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Trp at the second position.

[0069] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Ile, Leu, or Val at the third position.

[0070] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Trp or Leu at the fourth position.

[0071] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Trp at the fifth position.

[0072] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Trp at the sixth position.

[0073] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Ile at the seventh position.

[0074] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Lys, Val, Ile, Trp, or Arg at the eighth position.

[0075] In some embodiments, an isolated peptide according to the present disclosure includesa peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Leu, Val, or Lys at the ninth position.

[0076] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Ile, Arg, Trp, Val, Leu, or Lys at the eleventh position.

[0077] In some embodiments, an isolated peptide according to the present disclosure includes a peptide including a single amino acid change from the amino acid sequence of SEQ ID NO: 3, where the single amino acid change is Arg at the twelfth position.

[0078] “Isolated” when used in reference to a peptide, refers to a peptide substantially free of proteins, lipids, nucleic acids, for example, with which it might be naturally associated. For example, in some embodiments, the present disclosure includes a peptide with the amino acid sequence set forth in Peptide 1 or any one or more of SEQ ID NOs: 1-104, or a functional variant thereof, as long as the bioactivity (e.g., anti-biofilm or antibacterial) of the peptide remains. In some embodiments, a peptide according to the present disclosure includes one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve D amino acids. In some embodiments, a peptide according to the present disclosure consists of all D amino acids.

[0079] A “functional variant” includes peptides containing one or more L-amino acids, non- natural amino acids, amidated amino acids, unamidated amino acids, enantiomers, retro-inverso derivatives, analogs, etc. In some embodiments, a functional variant may be a C-terminal derivative, such as a C-terminal amidate, C-terminal acylates, a C-terminal methyl ester or a C- terminal acetyl ester. In some embodiments, a peptide according to the present disclosure is amidated at the C-terminus.

[0080] In some embodiments, a peptide according to the present disclosure may exhibit broad spectrum activity, for example, any one or more of the peptides including an amino acid sequence as set forth in one or more of Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof.

[0081] In some embodiments, a peptide according to the present disclosure may exhibit activity against Gram positive bacteria, for example, any one or more of the peptides including an amino acid sequence as set forth in SEQ ID NOs: 16, 19, 22, 29, 32, 34, 35, 60-64, or a functional variant thereof.

[0082] In some embodiments, a peptide according to the present disclosure may exhibit activity against Gram negative bacteria, for example, any one or more of the peptides including anamino acid sequence as set forth in SEQ ID NOs: 17, 21, 23, 44, 48, 53, 65-69, or a functional variant thereof.

[0083] In some embodiments, a peptide according to the present disclosure may exhibit anti- biofilm activity, for example, any one or more of the peptides including an amino acid sequence as set forth in one or more of Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof.

[0084] In some embodiments, a peptide according to the present disclosure that exhibits broad spectrum anti-biofilm activity may include for example, any one or more of Peptide 1 and / or SEQ ID NOs: 3, 9-40, or for example, one or more of SEQ ID NOs: 3, 9-12, or 14-40, or a functional variant thereof.

[0085] In some embodiments, a peptide according to the present disclosure that exhibits anti- biofilm activity against Gram negative bacteria may include, for example, any one or more of the peptides including an amino acid sequence as set forth in one or more of SEQ ID NOs: 41-54, or a functional variant thereof.

[0086] In some embodiments, a peptide according to the present disclosure that exhibits broad spectrum antibacterial activity may include for example, any one or more of Peptide 1 and / or SEQ ID NOs: 3, 9-15, 18, 20, 27, 28, 33, 36, 37, 39- 43, 45-47, 49, 50-52, 54-59, or a functional variant thereof.

[0087] In some embodiments, a peptide according to the present disclosure may exhibit enhanced activity when compared to a reference peptide, such as one or more of D-amino acid peptides DJK5 (VQWRAIRVRVIR, SEQ ID NO: 105), D-1018 (VRLIVAVRIWRR, SEQ ID NO: 106) and / or D-1002 (VQRWLIVWRIRK, SEQ ID NO: 107). Accordingly, in some embodiments, a peptide according to the present disclosure that exhibits enhanced anti-biofilm activities, when compared to a reference peptide, such as peptide DJK5, D-1018 and / or D-1002, may include for example, any one or more of Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof.

[0088] By “enhance,” “enhanced” or “enhancing” means an increase in activity by any value between about 10% and about 90%, or of any value between about 30% and about 60%, or over about 100%, or an increase by about 1-fold, 2-fold, 5-fold, 8-fold, 10-fold or more, in comparison to a reference sample or molecule, such as a peptide, or a control. In some embodiments, the enhanced activity may be at least 15%. In some embodiments, the enhanced activity may be at least 2-fold. In some embodiments, the enhanced activity may be at least 5-fold. In someembodiments, the enhanced activity may be at least 8-fold. In some embodiments, the enhanced activity may be determined using relative IC50 values to determine which peptides exhibit enhanced activity compared to a reference peptide, where a peptide with a relative IC50 less than 1 would be considered as having enhanced activity relative to the reference peptide. The enhanced activity may be antimicrobial activity, antibacterial activity or antibiofilm activity against Gram negative and / or Gram positive bacteria.

[0089] “Anti-biofilm” relates to the ability to destroy, inhibit the growth of, or encourage the dispersal of, biofilms of living organisms, such as microorganisms. “Antimicrobial” as used herein means that a peptide according to the present disclosure can inhibit, prevent, or destroy the growth or proliferation of planktonic (free swimming) microbes such as bacteria, fungi, viruses, parasites or the like. “Antibacterial” as used herein means that a peptide according to the present disclosure can inhibit, prevent, or destroy the growth or proliferation of planktonic (free swimming) bacteria.

[0090] In some embodiments, a peptide according to the present disclosure may exhibit low toxicity. By “low toxicity” or “reduction in toxicity” is meant a decrease in peptide-induced cytotoxicity by any value between about 10% and about 90%, or of any value between about 30% and about 60%, or over about 100%, or a decrease by about 1-fold, 2-fold, 5-fold, 8-fold, 10-fold or more, in comparison to a reference sample or molecule, such as a reference peptide, such as one or more of peptide DJK5, D-1018 and / or D-1002, or a control. In some embodiments, a peptide that does not exhibit suitably low toxicity, for example, peptide AB109-D, is specifically excluded.

[0091] In some embodiments, a peptide according to the present disclosure may exhibit low aggregation. By “low aggregation” or “reduction in aggregation” is meant a decrease in peptide- induced aggregation i.e., the tendency of a peptide to self-assemble, for example through the interactions of their hydrophobic region(s) by any value between about 10% and about 90%, or of any value between about 30% and about 60%, or over about 100%, or a decrease by about 1-fold, 2-fold, 5-fold, 8-fold, 10-fold or more, in comparison to a reference sample or molecule, such as a reference peptide, such as one or more of peptide DJK5, D-1018 and / or D-1002, or a control. In some embodiments, a peptide according to the present disclosure that exhibits low aggregation, when compared to a reference peptide, such as one or more of peptide DJK5, D-1018 and / or D- 1002, or a control, may include for example, any one or more of Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof.

[0092] In some embodiments, a peptide according to the present disclosure may reducebacterial abscess formation when compared to a reference peptide, such as one or more of peptide DJK5, D-1018 and / or D-1002, or a control. In some embodiments, a peptide according to the present disclosure that reduces bacterial abscess formation, when compared to a reference peptide, such as one or more of peptide DJK5, D-1018 and / or D-1002, or a control, may include for example, any one or more of Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof. By “reduces bacterial abscess formation” or “reduction in bacterial abscess formation” is meant a decrease in abscess size by any value between about 10% and about 90%, or of any value between about 30% and about 60%, or over about 100%, or a decrease by about 1-fold, 2-fold, 5-fold, 8- fold, 10-fold or more, in comparison to the reference sample or molecule, such as a peptide, or a control.

[0093] In some embodiments, a peptide according to the present disclosure, in combination with another compound, such as a conventional antibiotic or other peptide, may exhibit enhancement of activity, as determined by fractional inhibitory concentration (FIC) index values, where an FIC index between 0.5 and 1 indicates enhancement of activity.

[0094] In some aspects, the present disclosure provides a method of inhibiting the growth of or causing dispersal of a bacterium in a biofilm including contacting the bacteroid and / or biofilm with an inhibiting effective amount of at least one peptide of the disclosure alone, or in combination with at least one antibiotic, such as a conventional antibiotic as known in the art. In some embodiments of this aspect, the bacterium may be Gram positive. In some embodiments of this aspect, the bacterium may be Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis. In some embodiments of this aspect, the bacterium may be Gram negative. In some embodiments of this aspect, the bacterium may be Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis ssp Typhimurium, Acinetobacter baummanii, Klebsiella pneumoniae, Enterobacter sp., Campylobacter or Burkholderia cepacia complex.

[0095] In some embodiments of this aspect, the contacting includes a peptide in combination with at least one antibiotic, such as a conventional antibiotic as known in the art. Classes of conventional antibiotics that can be used in combination with the peptides of the disclosure include, but are not limited to, aminoglycosides, ^-lactams, fluoroquinolones, vancomycin, and macrolides. In some embodiments of this aspect, the antibiotic is selected from the group consisting of aminoglycosides, ^-lactams, quinolones, and glycopeptides. In some embodiments of this aspect, the antibiotic may be selected from the group consisting of amikacin, gentamicin,kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethyl-succinate / gluceptate / lactobionate / stearate, penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, imipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, vancomycin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, teicoplanin, polymyxin B, colistin, benzalkonium-Cl, benzalkonium-Cl, chlorhexidine, H2O2, polyvinylpyrrolidone–iodine, levofloxacin, meropenem, cotrimoxazole or EDTA.

[0096] In some embodiments, the combination of a peptide according to the present disclosure with an antibiotic may provide synergistic therapy.

[0097] By “synergy” or “synergistic therapy,” as used herein, is meant the interaction of two or more compounds, such as a peptide as disclosed herein in combination with one or more of a conventional antibiotic compound, a further peptide as disclosed herein, or a known antimicrobial peptide, when their combined effect is greater than the sum of the effects seen when each compound is administered alone. The existence of a synergistic effect between a peptide as disclosed herein and a conventional antibiotic may be determined using standard measures, such as determining fractional inhibitory concentration (FIC) index values, where an FIC index of ≤ 0.5 indicates synergy.

[0098] In some embodiments of this aspect, the peptide may be bound to a solid support. In some embodiments, the peptide may be bound covalently or noncovalently. In some embodiments of this aspect, the solid support may be a medical device.

[0099] In some embodiments, the present disclosure provides a polynucleotide that encodes one or more of a peptide of the disclosure.

[0100] In some embodiments, the present disclosure provides a method of identifying an antibacterial and / or anti-biofilm peptide having 7 to 14 amino acids. The method may include contacting, under conditions sufficient for anti-biofilm activity, a test peptide with a microbe that will form or has formed one or more surface-associated biofilm colonies, and detecting a reducedamount of biofilm as compared to amount of biofilm in the absence of the test peptide. In one embodiment, the peptide may be synthesized on, or attached to, a solid support. In some embodiments, the peptides may retain anti-biofilm activity when cleaved from the solid support or may retain activity when still associated with the solid support. The microbe can be a Gram negative bacterium, such as Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis ssp. Typhimurium, Acinetobacter baumanii, Burkholderia spp., Klebsiella pneumoniae, Enterobacter sp., or Campylobacter spp. In another embodiment, the microbe can be a Gram positive bacterium, such as Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis. The detection can include detecting residual bacteria by confocal microscopy of coverslips with adhered bacteria in flow cells, after specific staining, or by measuring residual bacteria adherent to the plastic surface of a microtiter plate by removing free swimming (planktonic) bacteria and staining residual bacteria with crystal violet.

[0101] In some embodiments, the present disclosure provides a method of protecting a medical device from colonization with pathogenic biofilm-forming bacteria by coating at least one peptide onto the medical device. PEPTIDES

[0102] Peptides can be synthesized in solid phase, or as an array of peptides made in parallel on cellulose sheets (Frank, R. 1992) or by solution phase chemistry. These methods have been used to create a large number of variants through sequence scrambling, truncations and systematic modifications of peptide sequence, and a luciferase-based screen to investigate their ability to kill Pseudomonas aeruginosa planktonic cells (Hilpert K, et al. 2005). In some embodiments, a peptide in accordance with the present disclosure may be 7 to 14 amino acids in length, or any value or range in between, such as 7, 8, 9, 10, 11, 12, 13 or 14 amino acids, or 7 to 12 amino acids, or 8 to 14 amino acids, etc.

[0103] The “amino acid” residues of the peptides identified herein may be in the natural L- configuration or isomeric D-configuration (“D-amino acids”). In keeping with standard polypeptide nomenclature (J. Biol. Chem., 243:3557-59, (1969), abbreviations and chemical names for side chains (affixed to the alpha carbon of the backbone) for natural amino acid residues are as shown in the following table.1-Letter 3-Letter Amino Acid Side chain chemical name Y Tyr L-tyrosine 1-methyl-4-hydroxybenzyl G Gly L-glycine hydrogen F Phe L-phenylalanine methylbenzyl M Met L-methionine ethylthiomethyl A Ala L-alanine methyl S Ser L-serine hydroxymethyl I Ile L-isoleucine 1-methylpropyl L Leu L-leucine 2-methylpropyl T Thr L-threonine 1-hydroxyethyl V Val L-valine isopropyl P Pro L-proline pyrrolidine K Lys L-lysine α-aminobutyl H His L-histidine methyl-1H-imidazol-4-yl Q Gln L-glutamine propyl-3-carboxamide E Glu L-glutamic acid propyl-3-carboxylate W Trp L-tryptohan methyl-1H-indol-3-yl R Arg L-arginine propyl-3-guanidine D Asp L-aspartic acid ethyl-2-carboxylate N Asn L-asparagine ethyl-2-carboxamide C Cys L-cysteine methylsulphydryl

[0104] It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Peptides can be modified at the carboxy-terminus to remove the negative charge, often through amidation, esterification, acylation or the like.

[0105] Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule that would also have utility. For example, amino or carboxy terminal amino acids that may not be required for biological activity of the particular peptide can be removed. Peptides in accordance with the present disclosure may includeany analog, homolog, mutant, isomer or derivative of the peptides disclosed herein, so long as bioactivity as described herein remains. In general, the peptides are synthesized using L or D form amino acids, however, mixed peptides containing both L- and D- form amino acids can be synthetically produced. In addition, C-terminal derivatives can be produced, such as C-terminal amidates, C-terminal acylates, and C-terminal methyl and acetyl esters, in order to increase the anti-biofilm or antibacterial activity of a peptide of the disclosure. The peptide can be synthesized such that the sequence is reversed whereby the last amino acid in the sequence becomes the first amino acid, and the penultimate amino acid becomes the second amino acid, and so on (a “retro- inverso” or “RI” derivative).

[0106] In certain embodiments, the peptides of the disclosure may include peptide analogs and peptide mimetics. Indeed, the peptides of the disclosure include peptides having any of a variety of different modifications, including those described herein.

[0107] Peptide analogs of the disclosure may be generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the following sequences disclosed in the tables. The present disclosure clearly establishes that these peptides in their entirety and derivatives created by modifying any side chains of the constituent amino acids have the ability to inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, parasites or the like. The present disclosure further encompasses polypeptides up to about 50 amino acids in length that include the amino acid sequences and functional variants or peptide mimetics of the sequences described herein.

[0108] In another embodiment, a peptide of the present disclosure may be a pseudopeptide. Pseudopeptides or amide bond surrogates refers to peptides containing chemical modifications of some (or all) of the peptide bonds. The introduction of amide bond surrogates not only decreases peptide degradation but also may significantly modify some of the biochemical properties of the peptides, particularly the conformational flexibility and hydrophobicity.

[0109] To improve or alter the characteristics of the peptides of the present disclosure, protein engineering can be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can show, e.g., increased / decreased biological activity or increased / decreased stability. In addition, they can bepurified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Further, the peptides of the present disclosure can be produced as multimers including dimers, trimers and tetramers. Multimerization can be facilitated by linkers, introduction of cysteines to permit creation of interchain disulphide bonds, or recombinantly though heterologous polypeptides such as Fc regions.

[0110] One or more amino acids can be deleted from the N-terminus or C-terminus without substantial loss of biological function (see, e.g., Ron, et al. 1993). Accordingly, polypeptides having one or more residues deleted from the amino terminus fall within the scope of the present disclosure. Similarly, many examples of biologically functional C-terminal deletion mutants are known (see, e.g., Dobeli, et al., 1988). Accordingly, the present disclosure provides polypeptides having one or more residues deleted from the carboxy terminus. The disclosure also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini as described herein.

[0111] Other mutants in addition to N- and C-terminal deletion forms of the protein discussed above are included in the present disclosure. Thus, the disclosure further includes variations of the polypeptides that show substantial anti-biofilm and / or antibacterial activity. Such mutants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity.

[0112] There are two main approaches for studying the tolerance of an amino acid sequence to change, see, Bowie, et al., 1994. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. The effects of such changes can easily be assessed by employing artificial neural networks and quantitative structure activity analyses (Cherkasov, A., et al.2009).

[0113] Typically seen as “conservative substitutions” are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, and replacements among the aromatic residues Phe, Tyr and Trp. Thus, the peptide of the present disclosure can be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue can or cannot be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a pro-protein sequence.

[0114] Thus, the peptides of the present disclosure can include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the peptide. The following groups of amino acids represent equivalent changes: (1) Gln, Asn; (2) Ser, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp.

[0115] Arginine and / or lysine can be substituted with other basic non-natural amino acids including ornithine, citrulline, homoarginine, Nδ-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)- ethyl-L-ornithine, Nε-methyltrityl-L-lysine, and diamino-butyrate although many other mimetic residues are available. Favourable subtstitutions utilized here include: L-2-amino-3- guanidinopropionic acid (GPro); L-2-Amino-4-guanidinobutyric acid (But), L-Homoarginine (Har), L -2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), and L-Ornithine (Orn). Tryptophan residues can be substituted for homo-tryptophan, bromotryptophan and fluorotryptophan. The term “conservative variation” or “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that the substituted polypeptide at least retains most of the activity of the unsubstituted parent peptide. Such conservative substitutions are within the definition of the classes of the peptides of the disclosure.

[0116] The present disclosure further includes peptide fragments. More specifically, the present disclosure embodies purified, isolated, and recombinant peptides comprising at least any one integer between 6 and 504 (or the length of the peptides amino acid residues minus 1 if the length is less than 1000) of consecutive amino acid residues. The fragments may be at least 6, preferably at least 7 to 11, more preferably 12 to 14 consecutive amino acids.POLYNUCLEOTIDES

[0117] The disclosure includes polynucleotides encoding the peptides described herein. Exemplary polynucleotides encode peptides including those set forth in Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof, where the peptides have antibiofilm and / or antibacterial activity.

[0118] “Isolated” when used in reference to a polynucleotide, refers to a polynucleotide substantially free of proteins, lipids, nucleic acids, for example, with which it is naturally associated. As used herein, “polynucleotide” refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct. DNA encoding a peptide of the disclosure can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit. Polynucleotide sequences of the disclosure include DNA, RNA and cDNA sequences. A polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. Polynucleotides of the disclosure include sequences which are degenerate as a result of the genetic code. Such polynucleotides are useful for the recombinant production of large quantities of a peptide of interest, such as those set forth Peptide 1 and / or SEQ ID NOs: 1-104, or a functional variant thereof.

[0119] In the present disclosure, the polynucleotides encoding the peptides of the disclosure may be inserted into a recombinant “expression vector”. The term “expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of genetic sequences. Such expression vectors are preferably plasmids that contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence in the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. For example, the expression of the peptides can be placed under control of E. coli chromosomal DNA comprising a lactose or lac operon which mediates lactose utilization by elaborating the enzyme beta- galactosidase. The lac control system can be induced by IPTG. A plasmid can be constructed to contain the lacIq repressor gene, permitting repression of the lac promoter until IPTG is added. Other promoter systems known in the art include beta lactamase, lambda promoters, the protein A promoter, and the tryptophan promoter systems. While these are the most commonly used, othermicrobial promoters, both inducible and constitutive, can be utilized as well. The vector contains a replicon site and control sequences which are derived from species compatible with the host cell. In addition, the vector may carry specific gene(s) which are capable of providing phenotypic selection in transformed cells. For example, the beta-lactamase gene confers ampicillin resistance to those transformed cells containing the vector with the beta-lactamase gene. An exemplary expression system for production of the peptides is described in U.S. Pat. No.5,707,855.

[0120] Transformation of a host cell with the polynucleotide may be carried out by conventional techniques known to those skilled in the art. For example, where the host is prokaryotic, such as E. coli, competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl2 method using procedures known in the art. Alternatively, MgCl2 or RbCl could be used.

[0121] In addition to conventional chemical methods of transformation, the plasmid vectors may be introduced into a host cell by physical means, such as by electroporation or microinjection. Electroporation allows transfer of the vector by high voltage electric impulse, which creates pores in the plasma membrane of the host and is performed according to methods known in the art. Additionally, cloned DNA can be introduced into host cells by protoplast fusion, using methods known in the art.

[0122] DNA sequences encoding the peptides can be expressed in vivo by DNA transfer into a suitable host cell. “Host cells” are those in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that not all progeny are identical to the parental cell, since there may be mutations that occur during replication. However, such progeny are included when the terms above are used. Exemplary host cells include E. coli, S. aureus and P. aeruginosa, although other Gram negative and Gram positive organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.

[0123] The polynucleotide sequence encoding a peptide as described herein can be isolated from an organism or synthesized in the laboratory. Specific DNA sequences encoding the peptide of interest can be obtained by: 1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the peptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement ofmRNA is eventually formed that is generally referred to as cDNA.

[0124] The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known. In the present disclosure, the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons that are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation. In addition, virtually any peptide can be synthesized, including those encoding natural peptides, variants of the same, or synthetic peptides.

[0125] When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries that are derived from reverse transcription of mRNA that is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the peptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA / DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay, et al., Nuc. Acid Res., 11:2325, 1983). METHODS OF USE

[0126] The disclosure also provides a method of inhibiting the growth of a microbe or bacterium, or a bacterial biofilm or abscess, including contacting the microbe or bacterium with an inhibition effective amount of a peptide of the disclosure, including a peptide having an amino acid sequence set forth in Peptide 1 and / or SEQ ID NOs: 1-104, and functional variants thereof, where the peptides have antibiofilm, antimicrobial and / or antibacterial activity.

[0127] The term “contacting” refers to exposing the microbe or bacterium to the peptide so that the peptide can effectively inhibit, kill, or cause dispersal of microbes or bacteria growing in the biofilm state, in the planktonic state, or in an abscess. Contacting may be in vitro, for example by adding the peptide to a bacterial culture to test for susceptibility of the microbe or bacterium to the peptide or acting against biofilms that grow on abiotic surfaces or in an abscess. Contacting may be in vivo, for example administering the peptide to a subject with a microbial or bacterialdisorder, such as septic shock, abscess or infection. Contacting may further involve coating an object (e.g., medical device) such as a catheter or prosthetic device to inhibit growth of the bacterium or production of biofilms by the microbe or bacterium with which it comes into contact, thus preventing it from becoming colonized with the microbe or bacterium. “Inhibiting” or “inhibiting effective amount” refers to the amount of peptide that is required to cause an anti- biofilm bacteriostatic or bactericidal effect. Examples of bacteria that may be inhibited include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella enteritidis subspecies Typhimurium, Campylobacter sp., Burkholderia complex bacteria, Acinetobacter baumanii, Staphylococcus aureus, Enterococcus facaelis, Listeria monocytogenes, and oral pathogens. Other potential targets are well known to the skilled microbiologist.

[0128] The method of inhibiting the growth of biofilm bacteria may further include the addition of antibiotics for combination or synergistic therapy. Antibiotics can work by either assisting the peptide in killing bacteria in biofilms or by inhibiting bacteria released from the biofilm due to accelerated dispersal by a peptide of the disclosure. Those antibiotics most suitable for combination therapy can be easily tested by utilizing modified checkerboard titration assays that use the determination of Fractional Inhibitory Concentrations to assess synergy as further described below. The appropriate antibiotic administered will typically depend on the susceptibility of the biofilms, including whether the bacteria is Gram negative or Gram positive, and will be discernible by one of skill in the art. Examples of particular classes of antibiotics useful for synergistic therapy with the peptides of the disclosure include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., ciprofloxacin), carbapenems (e.g., imipenem), tetracyclines, vancomycin, polymyxins and macrolides (e.g., erythromycin and clarithromycin). The method of inhibiting the growth of bacteria may further include the addition of antibiotics for combination or synergistic therapy. The appropriate antibiotic administered will typically depend on the susceptibility of the bacteria such as whether the bacteria is Gram negative or Gram positive, or whether synergy can be demonstrated in vitro, and will be easily discernable by one of skill in the art. Further to the antibiotics listed above, typical antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin), macrolides (azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethylsuccinate / gluceptate / lactobionate / stearate), beta- lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin and piperacillin), or cephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, and cefsulodin) or carbapenems (e.g., imipenem, meropenem, panipenem), or monobactams (e.g., aztreonam). Other classes of antibiotics include quinolones (e.g., fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin), tetracyclines (e.g., doxycycline, minocycline, tetracycline), and glycopeptides (e.g., vancomycin, teicoplanin), for example. Other antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, linezolid, synercid, polymyxin B, colistin, colimycin, methotrexate, daptomycin, phosphonomycin and mupirocin.

[0129] The peptides or functional variants thereof may be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only the growth of a bacterium, but also a microbe, virus, parasite or fungus. These peptides are useful as antibiofilm agents, and / or anti-infective agents, including anti-bacterial agents, antiviral agents, antimicrobial or antifungal agents.

[0130] The disclosure further provides a method of protecting objects from bacterial colonization. Bacteria grow on many surfaces as biofilms. The peptides of the disclosure are active in inhibiting bacteria on surfaces. Thus, the peptides may be used for protecting objects such as medical devices from biofilm colonization with pathogenic bacteria by, coating or chemically conjugating, or by any other means, at least one peptide of the disclosure to the surface of the medical device. Such medical devices include indwelling catheters, prosthetic devices, and the like. Removal of bacterial biofilms from medical equipment, plumbing in hospital wards and other areas where susceptible individuals congregate and the like is also a use for peptides of the disclosure. TREATMENT REGIMES

[0131] The disclosure provides pharmaceutical compositions comprising one or a combination of a peptide in accordance with the present disclosure, for example, formulated together with a pharmaceutically acceptable carrier. Some compositions include a combination of multiple (e.g., two or more) peptides of the disclosure.

[0132] As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, detergents, emulsions, lipids, liposomes and nanoparticles, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular or topical administration. In another embodiment, the carrier is suitable for oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is compatible with the active compound, use thereof in the pharmaceutical compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0133] A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (See, e.g., Berge, et al., J. Pharm. Sci., 66: 1-19, 1977). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N’-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0134] In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., as a result of bacteria, fungi, viruses, parasites or the like) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease or condition in an amount sufficient to cure, or at least partially arrest, the symptomsof the disease or condition (e.g., biochemical and / or histologic), including its complications and intermediate pathological phenotypes in development of the disease or condition. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.

[0135] The pharmaceutical composition of the present disclosure should be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0136] When the active compound is suitably protected, as described above, the compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.

[0137] Pharmaceutical compositions of the disclosure also can be administered in combination therapy, i.e., combined with other agents. For example, in treatment of bacteria, the combination therapy can include a composition of the present disclosure with at least one agent or other conventional therapy. ROUTES OF ADMINISTRATION

[0138] A composition of the present disclosure can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. The phrases “parenteral administration” and “administered parenterally” mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraabscess, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection and infusion. The peptide of the disclosure can be administered parenterally by injection or by gradual infusion over time. The peptide can also be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems Further methods for delivery of the peptide include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation., or directly injected into abscesses.

[0139] The peptides may also be delivered via transdermal or topical application. Transdermal and topical dosage forms of the disclosure include, but are not limited to, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[0140] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal and topical dosage forms encompassed by this disclosure are well known to those skilled in the pharmaceutical arts, and will depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. For example, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1,3-diol, isopropyl myristate, isopropyl palmitate, lipids, nanoparticles, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., See, e.g., Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).

[0141] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with peptides as described herein. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0142] To administer a peptide of the disclosure by certain routes of administration, it can be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. The method of the disclosure also includes delivery systems such as microencapsulation of peptides into liposomes or a diluent. Microencapsulation also allows co- entrapment of antimicrobial molecules along with the antigens, so that these molecules, such as antibiotics, may be delivered to a site in need of such treatment in conjunction with the peptides of the disclosure. Liposomes in the blood stream are generally taken up by the liver and spleen. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan, et al., J. Neuroimmunol., 7: 27, 1984). Thus, the method of the disclosure is particularly useful for delivering antimicrobial peptides to such organs. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, Ed., 1978, Marcel Dekker, Inc., New York. Other methods of administration will be known to those skilled in the art.

[0143] Preparations for parenteral administration of a peptide of the disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0144] Therapeutic compositions typically must be sterile, substantially isotonic, and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0145] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Therapeutic compositions can also be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful in the present disclosure include: U.S. Patent No.4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No.4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No.4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0146] When the peptides of the present disclosure are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (or 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier. EFFECTIVE DOSAGES

[0147] “Therapeutically effective amount” as used herein for treatment of antimicrobial related diseases and conditions refers to the amount of peptide used that is of sufficient quantity to decrease the numbers of bacteria, viruses, fungi, and parasites in the body of a subject. The dosage ranges for the administration of peptides are those large enough to produce the desired effect. The amount of peptide adequate to accomplish this is defined as a “therapeutically effective dose.” The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient’s health, the patient’s physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition’s rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington’s (Remington’s Pharmaceutical Science, Mack Publishing Company, Easton, PA); Egleton, Peptides 18: 1431-1439, 1997; Langer Science 249: 1527-1533, 1990. The dosage regimen can be adjusted by the individual physician in the event of any contraindications.

[0148] Dosage regimens of the pharmaceutical compositions of the present disclosure are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0149] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

[0150] A physician or veterinarian can start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the disclosure is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present disclosure to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0151] An effective dose of each of the peptides disclosed herein as potential therapeutics for use in treating microbial diseases and conditions is from about 1 μg / kg to 500 mg / kg body weight, per single administration, which can readily be determined by one skilled in the art. As discussed above, the dosage depends upon the age, sex, health, and weight of the recipient, kind of concurrent therapy, if any, and frequency of treatment. Other effective dosage range upper limits are 50 mg / kg body weight, 20 mg / kg body weight, 8 mg / kg body weight, and 2 mg / kg body weight.

[0152] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0153] Some compounds of the disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, See, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (See, e.g., Ranade, J. Clin. Pharmacol., 29: 685, 1989). Exemplary targeting moieties include folate or biotin (See, e.g., U.S. Patent 5,416,016 to Low, et al.); mannosides (Umezawa, et al., Biochem. Biophys. Res. Commun., 153: 1038, 1988); antibodies (Bloeman, et al., FEBS Lett., 357: 140, 1995; Owais, et al., Antimicrob. Agents Chemother., 39: 180, 1995); surfactant protein A receptor (Briscoe, et al., Am. J. Physiol., 1233: 134, 1995), different species of which can comprise the formulations of the disclosure, as well as components of the invented molecules; p120 (Schreier, et al., J. Biol. Chem., 269: 9090, 1994); See also Keinanen, et al., FEBS Lett., 346: 123, 1994; Killion, et al., Immunomethods, 4: 273, 1994. In some methods, the therapeutic compounds of the disclosure are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In some methods, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the infection. The composition should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0154] “Anti-biofilm amount” as used herein refers to an amount sufficient to achieve a biofilm-inhibiting blood concentration in the subject receiving the treatment.

[0155] “Anti- bacterial amount” as used herein refers to an amount sufficient to achieve a bacterium-inhibiting blood concentration in the subject receiving the treatment. The “anti-bacterial amount” is generally recognized as safe for administration to a human is well known in the art, and as is known in the art, varies with the specific antibiotic and the type of bacterial infection being treated.

[0156] Because of the broad spectrum anti-biofilm properties of the peptides, they may also be used as preservatives or to prevent formation of biofilms on materials susceptible to microbial biofilm contamination. The peptides of the disclosure can be utilized as broad spectrum anti- biofilm agents directed toward various specific applications. Such applications include use of the peptides as preservatives for processed foods (organisms including Salmonella, Yersinia, Shigella, Pseudomonas and Listeria), either alone or in combination with antibacterial food additives such as lysozymes; as a topical agent (Pseudomonas, Streptococcus, Staphylococcus) and to kill odor producing microbes (Micrococci). The relative effectiveness of the peptides of the disclosure for the applications described can be readily determined by one of skill in the art by determining the sensitivity of biofilms formed by any organism to one of the peptides. FORMULATION

[0157] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this disclosure can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0158] Additional formulations suitable for other modes of administration include oral, intranasal, topical and pulmonary formulations, suppositories, and transdermal applications.

[0159] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, detergents like Tween or Brij, PEGylated lipids, cellulose, magnesium carbonate, methyl cellulose 25 cP, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hyluronic acid and hyperbranched polyglycerols. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0160] Topical application can result in transdermal or intradermal delivery, or enable activity against local biofilm infections. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.

[0161] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes (Paul et al., Eur. J. Immunol.25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998).

[0162] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0163] The disclosure provides a number of methods, reagents, and compounds that can be used for inhibiting microbial infections, and biofilm growth. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended embodiments, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a peptide” includes a combination of two or more peptides, and the like.

[0164] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0165] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In describing and claiming the present invention, the following terminology will be used.

[0166] From the foregoing description, various modifications and changes in the compositions and methods will occur to those skilled in the art. All such modifications coming within the scope of the appended embodiments are intended to be included therein. Each recited range includes all combinations and sub-combinations of ranges, as well as specific numerals contained therein.EXEMPLARY EMBODIMENTS EXAMPLE 1: MATERIALS AND METHODS

[0167] Peptide Synthesis: All peptides studied in larger amounts were synthesized to >95% purity and made commercially by GenScript (Piscataway, N.J., USA), or another suitable company. Highly pure synthetic peptides were made using standard solid phase Fmoc chemistry and then purified to >95% using reverse phase HPLC. The peptide identity was then confirmed by mass spectrometry. Synthetic peptide arrays synthesized on cellulose membranes by SPOT synthesis were prepared by Kinexus Bioinformatics Corp (Vancouver, BC, Canada) according to methods described previously [Hilpert, K., Winkler, D. F., & Hancock, R. E. W. (2007). Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nature Protocols, 2(6), 1333–1349; Frank, R. (2002). The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports– principles and applications. J Immunol Methods, 267(1), 13–26; Kramer, A., et al. (1997). Molecular Basis for the Binding Promiscuity of an Anti-p24 (HIV-1) Monoclonal Antibody. Cell, 91(6), 799–809; Kramer, A., et al. (1994). Combinatorial Cellulose-Bound Peptide Libraries: Screening Tools for the Identification of Peptides That Bind Ligands with Predefined Specificity. Methods, 6(4), 388–395]. SPOT peptide syntheses on cellulose were performed using a pipetting robot (Abimed, Langenfeld, Germany) and Whatman 50 cellulose membranes (Whatman, Maidstone, United Kingdom). Peptides were obtained associated with their cellulose membrane support but having been treated with ammonia gas to release the peptide from the membrane surface. Peptide stock solutions were prepared in endotoxin free water according to the mass of peptide (for 95% pure samples) or by adding a defined volume of water (200 µl) to the SPOT sample to solubilize the free peptide and prepare a stock peptide solution for subsequent screening assays [Etayash, H., Haney, E. F., & Hancock, R. E. W. (2021). Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology. Nature Protocols, 16(4), 1850–1870]. The concentration of the stock solutions of the SPOT peptide samples were determined by measuring the sample absorbance at 280 nm (due to the presence of Trp residues) on a Nanodrop instrument (ThermoFisher Scientific, Waltham, MA) and then applying Beer’s law [A (absorbance) = ε (extinction coefficient) * c (molar concentration) * l (pathlength in cm)] using the molar extinction coefficient for Trp residues of 5500 M-1cm-1.

[0168] In vitro Antibiofilm activity: Biofilm inhibition assays in microtitre plates were carried out according to protocols described previously [Haney, E. F., Trimble, M. J., & Hancock, R. E. W. (2021). Microtiter plate assays to assess antibiofilm activity against bacteria. Nature Protocols, 16(5), 2615–2632]. Briefly, 10 µl of a serially diluted peptide solution was mixed with 90 µl of a freshly prepared bacterial suspension (OD600= 0.01) in a 96-well polypropylene round bottom plate (Corning Inc. Corning, NY). Media used to grow biofilms for methicillin resistant Staphylococcus aureus (USA300 LAC strain, MRSA) was 10% tryptic soy broth (TSB) supplemented with 0.1% glucose while Pseudomonas aeruginosa PAO1 was grown in BM2 minimal media (62 mM potassium phosphate buffer, pH 7, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 μM FeSO4 and 0.4% (wt / vol) glucose). The plates were incubated overnight at 37°C and the following day, the media containing unbound bacteria was discarded, the adhered biofilm was rinsed three times with distilled water, and then stained with 0.1% crystal violet (CV) for ~30 minutes at room temperature. The unbound CV dye was subsequently rinsed away with distilled water (three times) and the biofilm-bound CV dye was released from the adhered biomass by adding 70% (v / v) ethanol to the well and gently mixing. The absorbance of the resuspended CV dye was quantified by recording the absorbance of each well at 595 nm on a BioTek Epoch Microplate Spectrophotometer. The percent biofilm inhibition of the peptide treated biofilms was calculated by comparison to the amount of CV stain present in the untreated controls (defined as 100%) and the sterility control wells (defined as 0%) found within the same polypropylene microtitre plate.

[0169] Synergy with Antibiotics: Biofilm synergy was assessed using the in vitro antibiofilm activity method described above and incubating the peptides with different concentrations of peptides and antibiotic in a standard checkboard assay layout [de la Fuente- Núñez, C., et al. (2015). D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections. Chemistry & Biology, 22(2), 196–205]. Briefly, 5 µl of a two-fold peptide gradient was set up horizontally in the wells of a 96-well polypropylene round bottom plate. A vertical two-fold antibiotic gradient was set up and 5 µl of each antibiotic solution was added to the corresponding vertical rows to establish the checkboard gradient for the synergy assay. Separate wells containing peptide or antibiotic gradients alone were prepared in parallel to allow for fractional inhibitory concentration (FIC) calculations (see below). Each well then received 90 µl of an OD600 = 0.01 bacterial suspension(either MRSA or PAO1 using the same growth media described above) and the plates were incubated overnight at 37°C. The following day, the microtitre plates were processed as above and stained with CV to quantify the amount of biofilm adhered to the plate. The percent biofilm inhibition of the peptide treated biofilms was calculated by comparison to the amount of CV stain present in the untreated controls (defined as 100%) and the sterility control wells (defined as 0%) found within the same polypropylene microtitre plate. The fractional inhibitory concentration (FIC) index was calculated as follows: FIC = [A] / MBICA + [B] / MBICB, where MBICA and MBICB are the MBICs of peptides A and B alone and [A] and [B] are the MBICs of A and B when in combination.

[0170] Minimal inhibitory concentration (MIC) against planktonic bacteria: The MIC of the synthetic peptides was determined in Mueller-Hinton broth (MHB) using the broth microdilution method described previously [Wiegand, I., Hilpert, K., & Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163–175]. Briefly, 10 µl of a 10x concentrated peptide solution was placed in the bottom of a well in a 96-well polypropylene round bottom plate (Corning Inc.). A bacterial suspension of ~5 x 105CFU / ml was prepared from an overnight culture and 90 µl of this was added to each well. The plates were incubated overnight at 37°C and the following day, the MIC was visually determined as the lowest peptide concentration that resulted in no visible growth in the well. Bacteria tested included P. aeruginosa PAO1, MRSA USA300 LAC, Staphylococcus haemolyticus, Staphylococcus epidermidis, Klebsiella pneumoniae and Streptococcus salivarius.

[0171] Oral Biofilm Model: Sterile HA disks were used as the plaque biofilm substrate. To allow for the formation of the salivary pellicle, saliva coated HA (sHA) disks were prepared by incubating with 400 μl infiltrated saliva for 4 hours. The supragingival plaque was collected from upper molars of healthy volunteers and mixed in the same batch of BHI. Bacterial suspension was adjusted to an OD405of 0.1, corresponding to ~3.0×107CFU / ml. The sHA disks were placed in the wells, each containing 1.8 ml of BHI, of 24-well plates. Each well was inoculated with 0.2 ml of dispersed plaque suspension. All disks were incubated in the BHI-plaque suspension under anaerobic conditions at 37°C for 3 days. To evaluate the short-term antibiofilm effect of peptides on 3-day-old preformed plaque biofilms, the sHA disks were rinsed in PBS for 1 minute then immersed in 1 ml of 10 μg / ml of peptide for one minute. Disks treated by sterile water were setas the control group. To evaluate the long-term effect of peptides on 3-day-old biofilms, the culture medium was replaced with 1.98 ml of fresh BHI then subjected to peptide treatments at a concentration of 10 μg / ml. The first two sHA disks from each peptide group were treated for 24 hours under anaerobic incubation at 37°C (1-day treatment). Another two sHA disks were treated a second and third time with the same peptide solution and cultured for another 24 and 48 hours (2-days and 3-days treatment total). The control disks with no peptide were included for each time period (1 and 3 days). Biofilms were stained with the fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, to characterize the effect of peptide and to quantify the proportion of dead bacterial volume by confocal microscopy.

[0172] In vitro skin organoid biofilm infection model: Peptide treatment of biofilms established on the surface of N-TERT or Ker-CT keratinocyte derived epithelial skin were performed as described previously [Wu, B., et al. (2021). Human organoid biofilm model for assessing antibiofilm activity of novel agents. npj Biofilms and Microbiomes, 7(1), 8]. In brief, keratinocyte cells were grown in Keratinocyte-SFM medium (ThermoFisher Scientific) supplemented with 25 μg / ml Bovine Pituitary Extract, 0.2 ng / ml human recombinant Epidermal Growth Factor 1-53 and 0.3 mM CaCl2at 37°C and 7.3% CO2(5% CO2for Ker-CT cells). The cell culture medium was refreshed every 2-3 days until ready for passage to the next flask at ≤40% confluency or to the Transwell inserts at 50-60% confluency. Epidermal skin was created by seeding 3 x 105cells on filter inserts (ThinCert™ Cell culture insert, Greiner bio-one) in a 12- well ThinCert™ Plate (Greiner bio-one) while submerged in DermaLife K Keratinocyte Complete Medium supplemented with LifeFactors (Lifeline Cell Technology). After forming a confluent monolayer on the inserts (~3–4 days), the culture medium was changed to a differentiation medium (DMEM / Ham’s F-12 / CnT-Prime 3D Barrier Media in a 3:1:4 ratio supplemented with 0.1 μg / mL hydrocortisone, 0.125 μg / mL isoproterenol, 0.25 μg / mL bovine insulin, 26.5 pM selenious acid, 5 mM L-serine, 5 μM L-carnitine, 1.6 mg / mL BSA, 25 μM palmitic acid, 15 μM linoleic acid, and 7 μM arachidonic acid). The apical side of the skin was air-exposed the next day to induce stratification. After 2–3 days, the concentration of linoleic acid in the differentiation medium was increased to 30 μM. The air-exposed skin was cultured at 37°C and 7.3% CO2for 10 days at which point one million luminescent bacterial cells suspended in PBS (5 μl of 2 × 108CFU / ml) were inoculated in the center of the epidermal skin growing on the Transwell insert. Luminescent strains of bacteria used to establish biofilms on the skinsurface include MRSA-lux, PAO1-lux, P. aeruginosa LESB58-lux, S. epidermidis-lux and A. baumannii-lux. The specimens were incubated at 37°C and 7.3% CO2to allow the establishment of a bacterial biofilm on the skin surface. One day after inoculation, 30 µl of treatment containing peptides and / or antibiotic was added to the skin surface for 4 hrs. To visualize biofilms on the skin surface, the luminescent bacteria on the skin surface were imaged using the ChemiDoc imaging System (Bio-Rad). To quantify bacterial counts recovered from the skin surface, skin samples, together with the filter inserts, were excised using a disposable scalpel, sonicated in 1.5 ml PBS, vortexed, serially diluted, and plated on LB agar plates.

[0173] Resistance Development: Resistance development was assessed by performing a serial passage experiment of bacteria grown in the presence of peptide and / or antibiotic. Briefly, a bacterial suspension (either MRSA USA300 or PAO1) was prepared to ~5 x 105CFU / ml in Mueller Hinton Broth (MHB) media from an overnight culture grown in LB at 37°C. Bacteria (90 µl of suspension) were mixed with peptide and / or antibiotic (10 µl) across a 2-fold dilution series and the plates were incubated overnight at 37°C. The following day, the minimal inhibitory concentration (MIC) was assessed as the lowest concentration of peptide and / or antibiotic that resulted in no visible growth. A fresh suspension of bacteria was then prepared to ~5 x 105CFU / ml using the bacteria obtained from the well with the lowest concentration at which growth was observed (i.e. at the MIC cutoff). This suspension was used to inoculate a fresh plate containing peptide and / or antibiotic across the same concentration gradient used previously and the plate was again incubated overnight at 37°C for subsequent MIC determination the following day. This serial passage was repeated for 20 days in three independent replicates to assess the fold change in MIC compared to passage one.

[0174] Hemolysis: Peptide induced hemolysis was evaluated according to methods described previously [Hilchie, A. L., et al. (2016). Mastoparan is a membranolytic anti-cancer peptide that works synergistically with gemcitabine in a mouse model of mammary carcinoma. Biochimica Et Biophysica Acta, 1858(12), 3195–3204]. Briefly, red blood cells were obtained, with human ethics approval, from the blood of healthy volunteers collected in a sodium heparin tube and then rinsed three times in PBS, centrifuging between washes, to obtain the hematocrit (100% RBC). A 2.5% RBCs suspension was exposed to peptide at varying concentrations and incubated at 37C in a 5% CO2 humidified atmosphere for 4 hours. Supernatants were collected following centrifugation (1100 rpm) and transferred to a flat bottom microtitre plate. The absorbance(490nm) of each supernatant was measured on a BioTek Epoch Microplate Spectrophotometer. Percent hemolysis was calculated by comparing the absorbance arising from peptide treated cells to RBCs treated with water (0% hemolysis) or lysed with 1% Triton X-100 (100% hemolysis).

[0175] Cytotoxicity Assays: Peptide cytotoxicity was assessed against peripheral blood mononuclear cells (PBMCs), a human bronchial epithelial cell line or a human keratinocyte cell line using the Cytotoxicity Detection Kit (Roche Diagnostics) which measures the enzyme activity of lactate dehydrogenase (LDH) released from damaged cells [Haney, E. F., et al. (2017). Aggregation and its influence on the immunomodulatory activity of synthetic innate defense regulator peptides. Cell Chemical Biology, 24(8), 969-980.e4]. Briefly, PBMCs were isolated from blood collected from healthy volunteers in sodium heparin tubes and then resuspended in RPMI media supplemented with 10% fetal bovine serum. All cell lines were grown under standard growth conditions optimized for each cell type to enable growth and proliferation under tissue culture conditions [Wu, B., et al. (2021). Human organoid biofilm model for assessing antibiofilm activity of novel agents. npj Biofilms and Microbiomes, 7(1), 8; Haney, E. F., et al. (2017). Aggregation and its influence on the immunomodulatory activity of synthetic innate defense regulator peptides. Cell Chemical Biology, 24(8), 969-980.e4]. Cells and peptide were incubated overnight and the following day, supernatants were collected and subjected to the LDH assay. Supernatants of cells treated with vehicle (water) or lysed with 2% Triton-X100 (added 1-hr prior to collection of sample supernatant) were used as the negative (0% toxicity) and positive (100% toxicity) controls, respectively.

[0176] Peptide Aggregation: Peptide aggregation was assessed qualitatively by examining samples of peptides mixed with PBMCs following overnight incubation at 37°C and 5% CO2. All samples were prepared in RPMI media containing 10% FBS the same manner as the cytotoxicity assays described above. Visualization of aggregates was done using a Nikon Eclipse TS100 Inverted Microscope at 10X magnification.

[0177] In vivo porcine dermal biofilm model: Peptide loaded dressings were evaluated using a porcine dermal biofilm infection model as described previously [Davis, S. C., et al. (2018). The wound‐healing effects of a next‐generation anti‐biofilm silver Hydrofiber wound dressing on deep partial‐thickness wounds using a porcine model. International Wound Journal, 15(5), 834–839]. Briefly, partial thickness wounds (10mm x 7mmx 1mm deep) were created on the backs of the animals then infected with log-phase cultures of bacteria and allowed biofilms toform for 24-hrs. Dressings were applied to infected wounds of either S. aureus USA300 (MRSA) or P. aeruginosa 27312 as either a single treatment (day 1) or twice treated (Day 1 and 4) and all wounds were excised on day 7 for CFU renumeration. The amount of peptide applied in all treatments was 10 mg while 1mg of ciprofloxacin was used in and treatment containing antibiotic. Mupirocin and silver sulfadiazine (SSD) were used as positive controls.

[0178] Subdermal murine toxicity model: A subdermal toxicity mouse model was used to evaluate whether peptides induced localized toxicity of treated cutaneous tissue. Mice were anesthetized with 2.5-3% isoflurane and their fur was removed by shaving followed by application of a chemical depilatory. The bare skin of each mouse was disinfected with ethanol prior to subcutaneous (SQ) injection of peptide (4-8 mg / kg) into the dorsum. Mice were visually assessed and received clinical scores for a battery of signs or symptoms (activity / behaviour change, appearance / grooming, hydration, pain, cyst formation), for which a score of zero was given to animals that appeared normal. Animals were weighed and clinical scores were totalled. Following an overnight (16 h) incubation, mice were scored and weighed then euthanized with CO2 followed by cervical dislocation. Mice were imaged and cysts (swollen, inflamed tissue) were measured using a caliper. Animal experiments were performed in accordance with the Canadian Council on Animal Care (CCAC) guidelines and were approved by the University of British Columbia Animal Care Committee. Female CD-1 mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All mice were 8 weeks of age and weighed 25 ± 5 g at the time of the experiment. Animals were group housed in cohorts of three to four littermates. Standard animal husbandry practices were employed. EXAMPLE 2: PEPTIDES

[0179] 12-mer peptide sequences harboring 4-mer, 6-mers and 8-mer blocks from at least two parent peptides of DJK5 (VQWRAIRVRVIR, SEQ ID NO: 105), D-1018 (VRLIVAVRIWRR, SEQ ID NO: 106) or D-1002 (VQRWLIVWRIRK, SEQ ID NO: 107) were synthesized on SPOT-peptide arrays and their antibiofilm activity at three different concentrations was assessed against MRSA biofilms in vitro.

[0180] Among the 15 novel peptide sequences screened in this manner, three exhibited consistently better biofilm inhibitory activity compared to the DJK5 samples at the two highest peptide concentrations evaluated for antibiofilm activity in a microtitre plate assay (Figure 1).These peptides were RI1002N8-DJK5C4 (renamed AB101-D), DJK5C-RI1002N (AB102-D), and DJK5C-DJK5N (AB103-D), and all had consistently better MRSA activities compared to DJK5 at 2 / 3 tested concentrations.

[0181] These peptides were synthesized to >95% purity to evaluate their activity in more sophisticated models of biofilm growth and biofilm-associated infections. The peptides were evaluated for their biofilm inhibition activity against MRSA biofilms (Figure 2A, Table 1). When tested against P. aeruginosa, two of the peptides (AB101-D and AB103-D) exhibited 4- fold or greater biofilm inhibition activity when compared to DJK5 (Figure 2B, Table 1).

[0182] These antibiofilm effects were further assessed against pre-formed biofilms and biofilm eradication was assessed using a metabolic dye, TTC, and calculating the 50% inhibitory concentration (IC50) required to inhibit biofilm metabolism (a function of cellular viability in the biofilms) based on dose-response curves resulting from 24-hr old (i.e. preformed) biofilms treated with peptide (Table 1). Table 1: Sequences and activities of peptides. All peptides consist exclusively of D-amino acids and are amidated at their C-terminus. Biofilm inhibition IC50values (lower value = improved activity) were calculated after 24 hours by crystal violet staining of biofilms grown in the presence of peptide. Biofilm eradication IC50values were calculated based on the absorbance of a metabolic dye, TTC, recorded from dose response curves of 24-hr preformed biofilms treated overnight with peptide. Biofilm Inhibition IC50Biofilm Eradication IC50SEQ ID Peptide (µM) (µM) NO: Name Sequence MRSA PAO1 MRSA PAO1 105 DJK-5 vqwrairvrvir- 0.35 7.4 1.6 3.8 NH21 AB101-D krirwvilrvir- 0.27 1.1 1.4 1.9 NH22 AB102-D rvrvirkrirwv- 0.15 16.5 3.7 23 NH2 3 AB103-D rvrvirvqwrai- 0.34 1.9 2.2 1.7 NH2

[0183] For these pre-formed biofilms, similar trends in the antibiofilm activities were observed with AB101-D and AB103-D exhibiting ≥2-fold enhanced antibiofilm eradication activity against P. aeruginosa.

[0184] AB101-D, AB102-D and AB103-D were further evaluated in more sophisticated biofilm growth models and consistently resulted in enhanced antibiofilm activity when compared to DJK-5 treatment. For example, all 3 peptides exhibited excellent antibiofilm activity against MRSA and PAO1 biofilms grown on a human skin organoid model of biofilm-associated skin infections (Figures 3A-B). Solutions containing the peptides were also loaded into collagen dressings to treat biofilm associated skin infections in organoid models (Figures 4A-D) and AB103-D and AB101-D showed clearly superior activity to both DJK-5 and ciprofloxacin. Aqueous peptide solutions containing AB103-D could also be loaded into commercial sterile bandages that were able to effectively reduce the bacterial burden on the surface of organoid skin infected with luminescent strains of MRSA, S. epidermidis, P. aeruginosa LESB58 and A. baumanii (Figures 5A-D). In these tests, the antibiofilm activity of these peptides was enhanced in the presence of ciprofloxacin demonstrating the potential of applying synthetic antibiofilm peptides with conventional antibiotics to achieve a synergistic effect. Importantly, relatively few CFUs could be recovered from the applied dressings loaded with peptide suggesting that little bacterial colonization or contamination of the bandage itself occurred. Similarly, AB103-D proved synergistic with several other antimicrobials in in vitro synergy experiments conducted against biofilms grown by P. aeruginosa PAO1 and MRSA USA 300 (Table 2).

[0185] Table 2: Synergy of AB103-D with conventional antibiotics and other antimicrobial agents against bacterial biofilms. This was performed by checkerboard titration as described in the methods against biofilms of P. aeruginosa PAO1 grown in BM2 minimal media and MRSA USA 300 LAC grown in 10% Tryptic soy broth (TSB) supplemented with 0.1% glucose. The minimum biofilm inhibitory concentration (MBIC) corresponds to the concentration of peptide or antibiotic that resulted in at least 70% reduction of adhered biofilm biomass, based on crystal violet staining. The MBIC of AB103-D alone against PAO1 was 2 µg / ml and 1 µg / ml for MRSA. An FIC index of ≤ 0.5 is formal synergy (bold) against biofilms while FIC’s between 0.5 and 1 (italics) indicates important enhancement of activity. Also shown is the MIC of each antibiotic comparing the inhibitory effect on planktonic cell growth in theirrespective biofilm growth media compared to the MICs determined in the standard MIC growth media, Mueller-Hinton broth (MHB). Table 2A: P. aeruginosa PAO1 Fold Decrease in Antibiotic MIC against planktonic cells Antibiotic used FIC concentration of MBIC against (µg / mL) with AB103-D against biofilms biofilmsAB103-D Antibiotic(µg / mL)MIC in BM2MIC in MHB mediummedium Ceftazidime 0.5 4-fold 4-fold 0.5 2 1 Cefepime 0.56 2-fold 16-fold 0.125 0.5 1 Ciprofloxacin 0.53 2-fold 32-fold 0.125 0.125 0.25 Levofloxacin 0.63 2-fold 8-fold 0.125 0.125 0.25 Tobramycin 1 2-fold 2-fold 2 2 0.5 Gentamicin 0.53 2-fold 32-fold 4 4 1Amikacin 1.06 Nochange16-fold 4 4 0.5Polymyxin B 0.56 16-fold 2-fold 2 2 2 Colistin 0.63 8-fold 2-fold 4 4 1 Piperacillin 1 2-fold 2-fold 1 4 8Meropenem 1.06 16-fold Nochange0.06 0.06 0.25Azithromycin 0.75 2-fold 4-fold 128 128 128 Clindamycin 1 2-fold 2-fold > 256 > 256 > 256 Aztreonam 0.56 2-fold 16-fold 1 8 4Norfloxacin 1.13 Nochange8-fold 0.25 0.25 0.5Trimethoprim 1 2-fold 2-fold 128 128 256 Cotrimoxazole 1 2-fold 2-fold 64 64 128Mupirocin - NoNo change change> 128 > 128 > 256Vancomycin 0.56 2-fold 16-fold 256 256 > 256 Benzalkonium-Cl0.51 2-fold 128-fold 16 16 16Chlorhexidine 0.63 8-fold 2-fold 4 4 3.125 H2O20.5 4-fold 4-fold 0.08% 0.08% 0.01% Polyvinylpyrrolidone–Iodine0.52 2-fold 64-fold 313 313 2500EDTA 1.02 Nochange64-fold 128 > 256 > 256AgNO3 1 2-fold 2-fold 0.03 0.03 0.125 Table 2B: MRSA USA 300 LACAntibiotic used Fold Fold Biofilm MBIC MIC in decr MIC in MHBwith AB103-DFICease decrease in 10% TSB, 10%TSB, AB103-D Antibiotic 0.1% glucose 0.1% glucose medium Ceftazidime 0.5 4-fold 4-fold 64 64 32 Cefepime 0.38 8-fold 4-fold 4 8 8 Ciprofloxacin 0.5 4-fold 4-fold 2 4 2 Levofloxacin 0.75 4-fold 2-fold 4 8 4 Tobramycin 0.38 8-fold 4-fold 0.125 0.125 0.125Gentamicin 1.13 Nochange8-fold 0.03 0.06 0.06Amikacin 1 2-fold 2-fold 0.25 0.5 0.5 Polymyxin B 0.5 4-fold 4-fold 2 2 4 Colistin 0.63 2-fold 8-fold 2 4 8 Piperacillin 0.63 2-fold 8-fold 4 4 4 Meropenem 0.31 16-fold 4-fold 0.25 0.25 0.125 Azithromycin 0.38 8-fold 4-fold 4 4 1 Clindamycin 0.635 2-fold 8-fold 0.5 0.5 0.125 Aztreonam 0.75 4-fold 2-fold > 128 > 128 > 128 Norfloxacin 0.5 4-fold 4-fold 32 32 32 Trimethoprim 0.56 2-fold 16-fold 8 8 2 Cotrimoxazole 0.56 16-fold 2-fold 4 4 2 Mupirocin 0.75 2-fold 4-fold 0.25 0.25 0.125 Vancomycin 0.38 8-fold 4-fold 0.25 0.25 0.25 Benzalkonium-Cl0.5 4-fold 4-fold 1 1 0.25Chlorhexidine 0.56 2-fold 16-fold 1 1 0.4 H2O2 0.63 8-fold 2-fold 0.0025% 0.0025% 0.0018% Polyvinylpyrrolidone–Iodine0.63 2-fold 8-fold 156 156 2500EDTA 0.38 4-fold 8-fold 128 128 64AgNO3 1.13 Nochange8-fold 0.25 0.25 0.25

[0186] In porcine models of skin infections, the peptides, especially AB103-D, worked as well or better than ciprofloxacin, mupirocin (for MRSA) or silver sulfadiazine (for P. aeruginosa) (Figures 6A-B), especially for extremely recalcitrant Gram-negative infections. While the peptides worked well on their own, they worked even better in combination with the antibiotic ciprofloxacin (Figures 4A-D, 5A-D, and 6A-B). Additionally, when evaluated in a murine model of abscess infections, treatment with AB101-D and AB103-D both resulted in reduced abscess size formed by P. aeruginosa LESB58 with AB103-D (77% decrease in abscess formation) exhibiting superior activity to the peptide DJK5 (only 54% decrease; i.e. residualabscesses were twice as large). Notably, the presence of the peptide prevented the evolution of resistance to the antibiotic ciprofloxacin in serial passage experiments (Figures 7A-B).

[0187] The peptides also exhibited a very favorable cytotoxicity profile since none of them caused appreciable toxicity against human peripheral blood mononuclear cells, ex-vivo passaged human bronchiolar epithelial cells or N / TERT keratinocytes (Figures 8A-C).

[0188] In aggregation studies, peptides were added to PBMCs in RPMI media containing 10% FBS and incubated overnight at 37°C in 5% CO2. The following day, the cells and / or peptide aggregates were visualized using a light microscope. Representative images of PBMCs isolated from three different healthy donors in the presence of 50 µg / ml peptide showed peptide aggregates in wells containing the highly aggregation-prone peptide, 1018, while some small aggregates appeared in wells containing AB101-D and AB103-D, but not to the same extent as 1018. No appreciable aggregation was observed for AB102-D, similar to the aggregation tendency seen for DJK5. EXAMPLE 3: AMINO ACID SUBSTITUTIONS

[0189] DJK-5 is a peptide comprised exclusively of D-amino acids and having the sequence vqwrairvrvir-NH2 (SEQ ID NO: 105)[de la Fuente-Núñez, C., et al. (2015). D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections. Chemistry & Biology, 22(2), 196–205]. A series of singly, doubly and triply substituted DJK-5 derivatives were synthesized on SPOT-peptide arrays, and their antibiofilm activity was assessed against MRSA biofilms in vitro (Figure 9). Notably, four novel peptide sequences were identified that exhibited antibiofilm activities that were ~2-4 fold stronger than DJK5 based on the results of the SPOT-synthesized peptide array screen, with improved activities vs. MRSA in the order of strength of AB104-D>AB105-D≈AB106- D≈AB107-D>DJK5 (Table 3). Interestingly, all of these single amino acid derivatives consist of non-obvious non-conservative substitutions compared to the parent sequence of DJK5 with a hydrophobic residue replacing a positively charged residue, or vice versa. An additional derivative, AB108-D, with activity that was only slightly lower than the DJK5 sample, was also carried through for analysis in subsequent experiments performed on 95% pure peptide samples.

[0190] Table 3: Names and sequences of DJK-5 derivatives with enhanced antibiofilm activity compared to DJK5 against MRSA biofilms. Note all peptides consist exclusively of D- amino acids and are amidated at their C-terminus. SEQ ID DJK5 New Peptide Name Sequence SPOT Array IC50NO: Variant MRSA 105 DJK5 - vqwrairvrvir-NH20.37 4 DJK5-r7l AB104-D vqwrailvrvir-NH2 0.15 5 DJK5-w3r AB105-D vqrrairvrvir-NH2 0.22 6 DJK5-r7i AB106-D vqwraiivrvir-NH20.21 7 DJK5-r9i AB107-D vqwrairvivir-NH2 0.23 8 DJK5-r5v AB108-D vqwrvirvrvir-NH2 0.53

[0191] To further examine the antibiofilm potential of these DJK-5 derivatives, their activity against oral plaque biofilms grown on hydroxyapatite (HA) disks, mimicking dental infections / plaque, was evaluated. HA is a natural occurring mineral form of calcium that is the main component in dental enamel and dentin, as well as bones. To establish biofilms on the HA substrate, human saliva-coated HA (sHA) disks were prepared by incubating sterile HA disks with 400 μl of infiltrated saliva for 4 hours. Supragingival plaque was collected from the upper molars of healthy volunteers and mixed in the same batch of brain-heart infusion (BHI) media. The bacterial suspension was adjusted to an OD405nm of 0.1, corresponding to 3.0×107CFU / ml. The sHA disks were then placed in the wells of a 24-well plate containing 1.8 ml of BHI. Each well was then inoculated with 0.2 ml of the dispersed plaque suspension. All disks were incubated in the BHI-plaque suspension under anaerobic conditions at 37°C for 3 days.

[0192] After the establishment of the plaque biofilms, the impact of long-term exposure to the DJK-5 derived peptides was evaluated for 1- or 3-days (Figure 10). Confocal microscopy images of 3-day-old plaque biofilms on HA disks treated daily with 10 μg / mL of peptide for 1 day were obtained. Similar images were obtained for biofilms treated daily for 3 days. Under these conditions, AB107-D demonstrated significantly enhanced killing of the oral plaque biofilms, while peptide AB106-D displayed activity comparable to peptide DJK-5, and the remaining 3 peptides were reasonably similar in activity to DJK-5.

[0193] Treatment of 3-day-old oral plaque biofilms with these DJK-5 derivatives for one minute, which is more reflective of how these peptides would be used in an oral mouth rinse application, still resulted in strong antibiofilm activity and substantial killing of the cells withinthe biofilm biomass (Figure 11). Confocal microscopic images of 3-day-old plaque biofilms on HA disks treated with peptides (10 μg / mL) for 1 minute were obtained. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, respectively. As with the long-term experiment, the most active peptides were again AB107- D and AB106-D, all with antibiofilm activity that was comparable to or better than peptide DJK- 5.

[0194] Importantly, none of these peptides proved to be overly toxic towards red blood cells or peripheral blood mononuclear cells (Figures 12A-B). In particular, the most active peptide, AB107-D, proved to be non-toxic at all concentrations evaluated while AB106-D was only marginally toxic at the highest peptide concentration evaluated.

[0195] The peptides were also evaluated for their tendency to aggregate when added to RPMI tissue culture media containing 10% fetal bovine serum in the presence of PBMCs. Peptides were added to PBMCs in RPMI media containing 10% FBS and incubated overnight at 37°C in 5% CO2. The following day, the cells and / or peptide aggregates were visualized using a light microscope. Peptide aggregates were seen in wells containing the highly aggregation-prone peptide 1018 at 50 µg / ml while 200 µg / ml of 1018 resulted in the formation of large aggregated mats of peptide. Minimal peptide aggregation was observed for any of the DJK-5 derivatives and only at the highest concentration of peptide evaluated. Compared to a highly aggregation prone peptide, 1018 [Haney, E. F., et al. (2017). Aggregation and its influence on the immunomodulatory activity of synthetic innate defense regulator peptides. Cell Chemical Biology, 24(8), 969-980.e4], most DJK5-derived peptides exhibited no peptide aggregation at either 50 or 200 µg / ml based on visual inspection of the wells by microscopy. Only peptide AB106-D displayed marginal peptide aggregation at the highest concentration of peptide evaluated. EXAMPLE 4: FURTHER ENHANCEMENT OF AB103-D

[0196] A series of single amino acid substitution variants of the AB103-D peptide were generated on cellulose arrays using SPOT-synthesis technology [Hilpert, K., Winkler, D. F., & Hancock, R. E. W. (2007). Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nature Protocols, 2(6), 1333–1349; Frank, R. (2002). The SPOT-synthesis technique. Syntheticpeptide arrays on membrane supports–principles and applications. J Immunol Methods, 267(1), 13–26] to test for enhanced Gram-negative activity against P. aeruginosa and determine which residues contribute to the antibiofilm effects of this peptide.96 AB103-D variants were generated by replacing each residue in the parent peptide with one of D-Arg, D-Lys, D-Glu, Gly, D-Ala, D-Ile, D-Leu, D-Val and D-Trp. The biofilm inhibitory activity of all 96 AB103-D variants was subsequently assessed against MRSA USA300 LAC or P. aeruginosa PAO1 in a microtitre plate assay [Haney, E. F., Trimble, M. J., & Hancock, R. E. W. (2021). Microtiter plate assays to assess antibiofilm activity against bacteria. Nature Protocols, 16(5), 2615–2632]. The biofilm inhibition ability of the AB103-D derivatives against MRSA biofilms was retained with many of the peptides displaying comparable activity to the parent peptide and a moderate (~8x) spread in potency from the most active to the least active peptide. However, 32 peptides had enhanced activity vs. MRSA (relative IC50<1) and broad-spectrum activity, while AB117-D, AB114-D, and 3 other peptides had nearly 2-fold better activity vs. MRSA (Table 4).

[0197] Table 4: AB103-D single amino acid derivatives with superior antibiofilm activity against P. aeruginosa PAO1 compared to AB103-D or DJK5. Relative IC50cf. AB103-D represents the improved (lower) IC50 of the compared peptide, such that 0.5 means a 2-fold improved IC50. DJK-5 data are inferred based on relative IC50values cf. AB103-D from Table 2. Mutations are described as per convention (e.g. v4w indicates that the D-valine at position 4 of the AB103-D sequence was replaced with D-tryptophan). The amino acid sequences use the one letter code (in lower case to reflect that they are D-amino acids). All peptides were amidated at their carboxy termini. Bolded IC50s represent superior activity to comparator. Relative IC50of Derivative cf. Relative IC50of AB103-D Derivative cf. DJK5 SEQ ID Peptide Mutatio MRS Pseudomona NO: Name n Sequence A s MRSA Pseudomonas 4A - Peptides with equivalent or better broad spectrum antibiofilm activity 3 AB103-D - rvrvirvqwrai 1 1 1 0.26 9 AB117-D r1k kvrvirvqwrai 0.51 0.59 0.51 0.15 10 AB110-D v4w rvrwirvqwrai 0.67 0.51 0.67 0.13 11 AB111-D i5w rvrvwrvqwrai 0.71 0.52 0.71 0.13 12 AB112-D a11i rvrvirvqwrii 0.95 0.42 0.95 0.11 13 AB109-D r3w rvwvirvqwrai 0.85 0.56 0.85 0.14 14 r1w wvrvirvqwrai 0.49 1.00 0.49 0.2615 AB114-D q8k rvrvirvkwrai 0.49 1.00 0.49 0.26 16 v2w rwrvirvqwrai 0.50 1.00 0.50 0.26 17 v7i rvrviriqwrai 0.52 1.00 0.52 0.26 18 AB115-D w9l rvrvirvqlrai 0.71 0.99 0.71 0.25 19 r1a avrvirvqwrai 0.74 1.00 0.74 0.26 20 r1l lvrvirvqwrai 0.75 1.00 0.75 0.26 21 q8v rvrvirvvwrai 0.75 1.00 0.75 0.26 22 w9v rvrvirvqvrai 0.81 1.00 0.81 0.26 23 v4l rvrlirvqwrai 0.82 1.00 0.82 0.26 24 w9k rvrvirvqkrai 0.85 1.00 0.85 0.26 25 i12q rvrvirvqwraq 0.87 1.00 0.87 0.26 26 i5r rvrvrrvqwrai 0.87 1.00 0.87 0.26 27 i12k rvrvirvqwrak 0.96 0.94 0.96 0.24 28 w9i rvrvirvqirai 0.91 1.00 0.91 0.26 29 v4k rvrkirvqwrai 0.92 1.00 0.92 0.26 30 v7k rvrvirkqwrai 0.93 1.00 0.93 0.26 31 w9r rvrvirvqrrai 0.93 1.00 0.93 0.26 32 i5k rvrvkrvqwrai 0.95 1.00 0.95 0.26 33 v2i rirvirvqwrai 0.95 1.00 0.95 0.26 34 v4r rvrrirvqwrai 0.96 1.00 0.96 0.26 35 v2q rqrvirvqwrai 0.97 1.00 0.97 0.26 36 v7l rvrvirlqwrai 0.97 1.00 0.97 0.26 37 r3k rvkvirvqwrai 0.98 1.00 0.98 0.26 38 v2r rrrvirvqwrai 0.98 1.00 0.98 0.26 39 i12l rvrvirvqwral 0.99 1.00 0.99 0.26 40 r1v vvrvirvqwrai 1.00 1.00 1.00 0.26B - Peptides with selectively enhanced antibiofilm activity against Gram-negative bacteria 41 a11w rvrvirvqwrwi 2.77 0.46 2.77 0.12 42 AB116-D r3i rvivirvqwrai 2.19 0.49 2.19 0.13 43 AB113-D a11r rvrvirvqwrri 1.02 0.56 1.02 0.14 44 q8i rvrvirviwrai 1.60 0.58 1.60 0.15 45 a11v rvrvirvqwrvi 1.81 0.60 1.81 0.15 46 r3l rvlvirvqwrai 1.95 0.60 1.95 0.15 47 a11l rvrvirvqwrli 1.17 0.65 1.17 0.17 48 q8w rvrvirvwwrai 1.90 0.67 1.90 0.17 49 r6w rvrviwvqwrai 1.30 0.70 1.30 0.18 50 i12r rvrvirvqwrar 1.31 0.71 1.31 0.18 51 r3v rvvvirvqwrai 2.17 0.72 2.17 0.18 52 a11k rvrvirvqwrki 1.28 0.77 1.28 0.20 53 q8r rvrvirvrwrai 2.58 0.83 2.58 0.21 54 r1g gvrvirvqwrai 1.51 0.90 1.51 0.23C - Peptides with equivalent or weaker antibiofilm activity than AB103-D 67 i12w rvrvirvqwraw 1.01 1.00 1.01 0.26 68 q8l rvrvirvlwrai 1.01 1.00 1.01 0.26v2a rarvirvqwrai 1.02 1.00 1.02 0.26 i5l rvrvlrvqwrai 1.02 1.00 1.02 0.26 r1i ivrvirvqwrai 1.04 1.00 1.04 0.26 r1q qvrvirvqwrai 1.04 1.00 1.04 0.26 v4q rvrqirvqwrai 1.07 1.00 1.07 0.26 i5v rvrvvrvqwrai 1.10 1.00 1.10 0.26 v2l rlrvirvqwrai 1.11 1.00 1.11 0.26 r6k rvrvikvqwrai 1.14 1.00 1.14 0.26 i12v rvrvirvqwrav 1.16 1.00 1.16 0.26 r3a rvavirvqwrai 1.17 1.00 1.17 0.26 r10k rvrvirvqwkai 1.21 1.00 1.21 0.26 a11g rvrvirvqwrgi 1.25 1.00 1.25 0.26 q8a rvrvirvawrai 1.27 1.00 1.27 0.26 v4a rvrairvqwrai 1.27 1.00 1.27 0.26 q8g rvrvirvgwrai 1.29 1.00 1.29 0.26 i12a rvrvirvqwraa 1.30 1.00 1.30 0.26 v2g rgrvirvqwrai 1.32 1.00 1.32 0.26 i12g rvrvirvqwrag 1.36 1.00 1.36 0.26 v4i rvriirvqwrai 1.36 1.00 1.36 0.26 r10q rvrvirvqwqai 1.39 1.00 1.39 0.26 v4g rvrgirvqwrai 1.47 1.00 1.47 0.26 a11q rvrvirvqwrqi 1.47 1.00 1.47 0.26 r3q rvqvirvqwrai 1.47 1.00 1.47 0.26 i5a rvrvarvqwrai 1.56 1.00 1.56 0.26 v2k rkrvirvqwrai 1.56 1.00 1.56 0.26 i5q rvrvqrvqwrai 1.59 1.00 1.59 0.26 v7q rvrvirqqwrai 1.68 1.00 1.68 0.26 r3g rvgvirvqwrai 1.70 1.00 1.70 0.26 w9q rvrvirvqqrai 2.02 1.00 2.02 0.26 r6l rvrvilvqwrai 2.10 1.00 2.10 0.26 v7r rvrvirrqwrai 2.13 1.00 2.13 0.26 r10w rvrvirvqwwai 2.22 1.00 2.22 0.26 r6v rvrvivvqwrai 2.23 1.00 2.23 0.26 r10l rvrvirvqwlai 2.27 1.00 2.27 0.26 r6q rvrviqvqwrai 2.32 1.00 2.32 0.26 r10g rvrvirvqwgai 2.50 1.00 2.50 0.26 w9a rvrvirvqarai 2.52 1.00 2.52 0.26 v7a rvrviraqwrai 2.56 1.00 2.56 0.26 r6a rvrviavqwrai 2.62 1.00 2.62 0.26 v7w rvrvirwqwrai 2.74 1.00 2.74 0.26 r10a rvrvirvqwaai 2.90 1.00 2.90 0.26 r10v rvrvirvqwvai 3.00 1.00 3.00 0.26 r6i rvrviivqwrai 3.12 1.00 3.12 0.26 i5g rvrvgrvqwrai 3.24 1.00 3.24 0.26101 v7g rvrvirgqwrai 3.68 1.00 3.68 0.26 102 w9g rvrvirvqgrai 3.69 1.00 3.69 0.26 103 r10i rvrvirvqwiai 3.79 1.00 3.79 0.26 104 r6g rvrvigvqwrai 3.87 1.00 3.87 0.26

[0198] With regards to specific residues, many D-amino acid substitutions were well tolerated in AB103-D without having a large impact on the biofilm inhibition activity against MRSA. Notably, the positive charges at positions 6 and 10 appear to be somewhat more important for the overall activity of the peptide, as is the presence of a hydrophobic residue at position 7 (although D-Lys is also well tolerated at this position). The two residues at both the N- and C-termini, as well as residues 4, 8 and 9, are all relatively tolerant of different amino acid substitutions without dramatically altering the antibiofilm activity.

[0199] Furthermore, the AB103-D derivatives were generally able to inhibit planktonic MRSA in microtiter plate assays (Table 5), but somewhat different inhibition effects and amino acid preferences were observed for AB103-D derivatives against planktonic bacterial cells (Table 5) cf. biofilm cells (Table 4).

[0200] Against P. aeruginosa PAO1 biofilms, only a few peptides demonstrated activity superior to that of AB103-D, notably AB109-D, AB110-D, AB111-D, AB112-D, and AB117-D, as well as those 14 peptides in category 4B, including AB113-D and AB116-D (Table 4) the remainder had similar activities to AB103-D (which was itself superior to DJK-5; Table 1). Improved peptides vs. Gram negative P. aeruginosa included five substitutions in the D-Ala residue at position 11, which tolerated a change to a positively charged residue, four substitutions at D-Arg residue position 3 e.g. to a hydrophobic residue and three substitutions at D-Gln position 8 that was enhanced by substitutions for a D-Arg, D-Ile or D-Trp. Regarding D-Trp residues, it also appears that there is a preference for this residue to appear between positions 3 and 6, cf. the parent sequence.

[0201] The sequences of some of the most active AB103-D derivatives with better antibiofilm activity against both MRSA and PAO1 biofilms when compared to the parent peptide, AB103-D, are shown in Table 4. Similarly, the in vitro activity of the AB103-D derived peptides against planktonic cells of MRSA and PAO1 are summarized in Table 5. In both cases, the peptides have been ranked according to their measured activities and any peptide withsuperior activity to the parent peptide AB103-D or inferred superiority to DJK5 is highlighted in bold.

[0202] Table 5: Antibacterial activity against MRSA or P. aeruginosa PAO1 planktonic cells of excellent single amino acid substitution derivatives of AB103-D. Shown are the relative IC50values calculated for each peptide compared to the IC50values for AB103-D peptides alone against MRSA (1.96 µM) and Pseudomonas (16.02 µM). Peptides improved activity cf. the parent sequence (FC ≤ 1.5) against either MRSA or PAO1 are shown in bold. All peptides were amidated at their C-termini. Relative Planktonic IC50of Derivative SEQ ID Peptide cf. AB103-D NO: Name Mutation Sequence MRSA Pseudomonas 5A: Peptides with enhanced broad-spectrum antibacterial activity 3AB103-D rvrvirvqwrai 1 112 AB112-D a11i rvrvirvqwrii 0.52 0.22 13 AB109-D r3w rvwvirvqwrai 0.52 0.27 47 a11l rvrvirvqwrli 0.56 0.26 11 AB111-D i5w rvrvwrvqwrai 0.49 0.34 43 AB113-D a11r rvrvirvqwrri 0.53 0.32 10 AB110-D v4w rvrwirvqwrai 0.65 0.33 49 r6w rvrviwvqwrai 0.61 0.37 9 AB117-D r1k kvrvirvqwrai 0.64 0.34 45 a11v rvrvirvqwrvi 0.59 0.41 42 AB116-D r3i rvivirvqwrai 0.69 0.35 14 r1w wvrvirvqwrai 0.48 0.64 41 a11w rvrvirvqwrwi 0.86 0.32 36 v7l rvrvirlqwrai 0.64 0.53 50 i12r rvrvirvqwrar 0.49 0.70 51 r3v rvvvirvqwrai 0.52 0.71 27 i12k rvrvirvqwrak 0.44 0.82 18 AB115-D w9l rvrvirvqlrai 0.46 0.83 52 a11k rvrvirvqwrki 0.69 0.61 46 r3l rvlvirvqwrai 0.71 0.61 54 r1g gvrvirvqwrai 0.56 0.78 37 r3k rvkvirvqwrai 0.52 0.90 33 v2i rirvirvqwrai 0.66 0.81 55 v2k rkrvirvqwrai 0.63 0.85 20 r1l lvrvirvqwrai 0.68 0.83 39 i12l rvrvirvqwral 0.64 0.88 15 AB114-D q8k rvrvirvkwrai 0.72 0.81 40 r1v vvrvirvqwrai 0.74 0.82 56 v2l rlrvirvqwrai 0.74 0.857 v2g rgrvirvqwrai 0.70 0.978 v4i rvriirvqwrai 0.92 0.799 a11q rvrvirvqwrqi 0.87 0.908 w9i rvrvirvqirai 0.96 0.87B: Peptides with enhanced activity against Gram-positive bacteria 9 v4k rvrkirvqwrai 0.47 2.000 r1i ivrvirvqwrai 0.63 1.046 v2w rwrvirvqwrai 0.63 2.001 r1q qvrvirvqwrai 0.69 2.004 v4r rvrrirvqwrai 0.71 1.095 v2q rqrvirvqwrai 0.75 2.002 i12g rvrvirvqwrag 0.81 2.002 w9v rvrvirvqvrai 0.83 1.103 i12v rvrvirvqwrav 0.85 1.032 i5k rvrvkrvqwrai 0.86 2.004 v4q rvrqirvqwrai 0.92 2.009 r1a avrvirvqwrai 0.99 2.00C: Peptides with enhanced activity against Gram-negative bacteria 4 q8i rvrvirviwrai 1.49 0.397 v7i rvrviriqwrai 1.10 0.461 q8v rvrvirvvwrai 1.04 0.488 q8w rvrvirvwwrai 1.24 0.515 r6i rvrviivqwrai 2.18 0.566 r6l rvrvilvqwrai 1.26 0.773 q8r rvrvirvrwrai 2.40 0.797 i12w rvrvirvqwraw 1.50 0.803 v4l rvrlirvqwrai 1.03 0.838 q8l rvrvirvlwrai 1.10 0.849 v7a rvrviraqwrai 2.59 0.95D: Peptides with weaker antibacterial activity 6 a11g rvrvirvqwrgi 1.10 1.020 r10w rvrvirvqwwai 1.42 1.021 r6v rvrvivvqwrai 1.72 1.039 q8g rvrvirvgwrai 1.09 1.054 i5a rvrvarvqwrai 1.85 1.095 w9a rvrvirvqarai 4.13 1.104 w9k rvrvirvqkrai 1.70 1.106 i5r rvrvrrvqwrai 2.16 1.192 v4g rvrgirvqwrai 1.33 1.270 v7k rvrvirkqwrai 1.26 1.283 r3q rvqvirvqwrai 1.23 1.298 r10a rvrvirvqwaai 2.59 1.3404 r6g rvrvigvqwrai 3.62 1.517 v7w rvrvirwqwrai 2.08 1.7871 i5l rvrvlrvqwrai 1.02 2.00 87 r3g rvgvirvqwrai 1.07 2.00 73 r6k rvrvikvqwrai 1.11 2.00 77 q8a rvrvirvawrai 1.11 2.00 92 r10l rvrvirvqwlai 1.13 2.00 38 v2r rrrvirvqwrai 1.15 2.00 80 i12a rvrvirvqwraa 1.23 2.00 75 r10k rvrvirvqwkai 1.23 2.00 70 v2a rarvirvqwrai 1.27 2.00 74 r3a rvavirvqwrai 1.29 2.00 25 i12q rvrvirvqwraq 1.30 2.00 72 i5v rvrvvrvqwrai 1.38 2.00 78 v4a rvrairvqwrai 1.42 2.00 31 w9r rvrvirvqrrai 1.59 2.00 99 r10v rvrvirvqwvai 1.80 2.00 103 r10i rvrvirvqwiai 2.22 2.00 85 i5q rvrvqrvqwrai 2.33 2.00 101 v7g rvrvirgqwrai 2.74 2.00 93 r6q rvrviqvqwrai 2.76 2.00 81 r10q rvrvirvqwqai 3.03 2.00 86 v7q rvrvirqqwrai 3.37 2.00 96 r6a rvrviavqwrai 3.39 2.00 102 w9g rvrvirvqgrai 3.90 2.00 100 i5g rvrvgrvqwrai 3.94 2.00 88 w9q rvrvirvqqrai 4.31 2.00 89 v7r rvrvirrqwrai 4.78 2.00 94 r10g rvrvirvqwgai 4.79 2.00

[0203] Several AB103-D derivatives were synthesized to > 95% purity to validate the results of the SPOT-synthesized peptide array screen as well as evaluate their toxic effects in vitro and in vivo. The biofilm inhibition activity of all the AB103-D derivatives was evaluated against MRSA USA300 and P. aeruginosa PAO1 in a microtitre plate assay. Most of the AB103-D derived peptides displayed similar antibiofilm activity towards MRSA as the parent peptides DJK-5 or AB103-D, with observed minimum biofilm inhibitory concentrations that inhibited 90% of growth in the range of 0.5-1 µM (Table 6). Against PAO1 biofilms, almost all of the AB103-D derivatives (except AB116-D) exhibited superior antibiofilm activity compared to peptide DJK-5 with some (AB109-D, AB112-D and AB113-D) being up to 4-fold more active than AB103-D (Table 6).

[0204] Table 6: Biofilm inhibition activity of >95% pure AB103-D derived peptides against MRSA USA300 and P. aeruginosa PAO1 biofilms. The data are presented as the minimum inhibitory concentration of peptide that inhibited at least 90% of biofilm growth (MBIC90, in µM) based on the crystal violet stained biomass in a microtitre plate biofilm inhibition assay. Biofilms of MRSA were grown in 10% TSB media supplemented with 0.1% glucose while PAO1 biofilms were grown in BM2 minimal media. SEQ ID Peptide MRSA PAO1 NO: 105 DJK-5 0.5 16 3 AB103-D 1 8 13 AB109-D 1 2 10 AB110-D 1 4 11 AB111-D 0.5 4 12 AB112-D 1 2 43 AB113-D 0.5 2 15 AB114-D 0.5 8 18 AB115-D 1 8 42 AB116-D 1 >16 9 AB117-D 0.5 8

[0205] As with the DJK-5 derivatives above, the AB103-D derivatives were evaluated for their tendency to aggregate when added to RPMI tissue culture media containing 10% fetal bovine serum in the presence of PBMCs. Peptides were added to PBMCs in RPMI media containing 10% FBS and incubated overnight at 37°C in 5% CO2. The following day, the cells and / or peptide aggregates were visualized using a light microscope. Peptide aggregates were seen in wells containing the highly aggregation-prone peptide 1018 at 200 µg / ml resulting in the formation of large aggregated mats of peptide with similar mats observed for AB109-D and AB112-D. The same peptides, along with AB117-D exhibited peptide aggregation at 50 µg / ml. Minimal peptide aggregation was observed for all the other peptides at both 200 µg / ml and 50 µg / ml. At a peptide concentration of 200 µg / ml, both AB109-D and AB112-D strongly aggregated, similar to what was seen for the aggregation prone peptide, 1018. None of the other peptides exhibited as strong an aggregation tendency at this high concentration. At 50 µg / ml, peptide aggregation was again observed for AB109-D, AB112-D and slightly for AB117-D, similar to the aggregation observed for 1018 at the same concentration of peptide. None of theother peptides exhibited any visible tendency to aggregate under these conditions at this lower concentration, similar to the effects seen for peptide DJK-5 or in cells treated with water alone.

[0206] The AB103-D derivatives were also assessed for in vitro toxicity towards PBMCs as well as ability to lyse red blood cells. In general, most of the AB103-D peptides caused very little hemolysis (Figure 13A) and were relatively non-toxic towards PBMCs (Figure 13B) across the peptide concentration range evaluated. The only exception to this was AB109-D which caused substantial hemolysis at concentrations higher than 25 µg / ml. Notably, AB109-D was also the most toxic towards PBMCs at the highest peptide concentration evaluated of 200 µg / ml. This effect may be related to the tendency of AB109-D to aggregate under these conditions as similar effects were seen for the aggregation prone peptide 1018, although the effect was not as pronounced as for AB109-D.

[0207] To further assess the potential toxic effects of the AB103-D derived peptides, the non-aggregation prone peptides were evaluated in a murine model of subdermal toxicity. A comparison of the clinical scores among the mice treated with the various peptides revealed that most peptides only caused a slight increase in clinical score within 1 hour of peptide administration (Figure 14A). The average clinical scores for mice treated with AB103-D, AB111-D, AB114-D, AB115-D and AB117-D were all lower than the clinical scores obtained for mice treated with DJK-5 (clinical score = 2), indicating that these peptides were less toxic than DJK-5. An additional assessment of peptide-induced cytotoxicity in vivo was the development of a cyst or lesion on the back of the mice 16 hours after peptide administration. When administered at a relatively high dose of 4 mg / kg, most of the AB103-D derived peptides caused small cysts to form (Figure 14B), although it should be noted that this peptide concentration is higher than the effective dose of DJK-5 (3 mg / kg) that was previously used in an abscess model of high-density bacterial infection [Mansour, S. C., et al. (2016). Bacterial abscess formation is controlled by the stringent stress response and can be targeted therapeutically. EBioMedicine, 12, 219–226]. Notably, treatment with AB103-D, AB110-D, B111-D, AB114-D, AB115-D and AB116-D at this concentration all produced average cyst sizes that were smaller than those induced by DJK-5, again supporting reduced in vivo toxic effects of these peptides compared to DJK-5.

[0208] Finally, the AB103-D derivatives that were the least aggregation prone and which produced the lowest toxic effects in vivo were evaluated for their direct antibacterial activitytowards several pathogens of clinical relevance using a standard minimum inhibitory concentration (MIC) assay [Wiegand, I., Hilpert, K., & Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163–175]. Specifically, peptides AB110-D, AB111-D, AB114-D and AB115-D were tested against P. aeruginosa PAO1, MRSA USA 300, Staphylococcus haemolyticus, Staphylococcus epidermidis, Klebsiella pneumoniae and Streptococcus salivarius and compared against the antibacterial activity of DJK-5 and AB103-D (Table 7). Overall, the antibacterial potency of all of the AB103-D derivatives was stronger than that seen for DJK-5, particularly in the case of the Gram-negative pathogens P. aeruginosa and K. pneumoniae. All of the AB103-D derivatives exhibited comparable antibacterial potency to each other against the pathogens evaluated.

[0209] Table 7: Antibacterial activity of select AB103-D derivatives against clinically relevant bacterial pathogens. Values shows are the MIC (in µg / ml) of each peptide based on the mode of three biological replicates. ND = not determined. SEQ Peptide P. MRSA S. S. K. S. ID NO: aerugino USA30 haemolytic epidermidi pneumonia salivariu sa PAO1 0 us s e s 105 DJK-5 6 0.8 0.8 0.8 3 0.8 3 AB103- 2 0.8 0.8 0.8 0.8 0.8 D 10 AB110- 2 0.8 0.8 ND ND ND D 11 AB111- 2 0.8 0.8 0.8 0.8 0.8 D 15 AB114- 2 0.8 0.8 0.4 0.8 0.8 D 18 AB115- 2 0.8 0.8 0.8 0.8 0.8 D EXAMPLE 5: BROAD SPECTRUM ANTIBIOFILM ACTIVITY

[0210] Biofilm inhibition was measured according to the procedure outlined in [Haney, E. F., Trimble, M. J., & Hancock, R. E. W. (2021). Microtiter plate assays to assess antibiofilm activity against bacteria. Nature Protocols, 16(5), 2615–2632]. Bacterial strains tested include Enterococcus faecium #2-1 (clinical isolate), MRSA USA300, Klebsiella pneumonia KPLN649, Acinetobacter baumannii Ab5075, P. aeruginosa PAO1, Enterobacter cloacae 218R1 and E.coli O157:H7. All experiments were performed in BM2 minimal media except for MRSA USA300 (10% TSB + 0.1% glucose) and E. faecium (TSB + 1% glucose). Most biofilms were grown for 18-24 hours, except for K. pneumonia KPLN649 and E. faecium #2-1 which were grown for 48 hours. Biofilm growth was quantified based on crystal violet staining of the residual adhered biomass in the microtitre plate well and data were analyzed as a percentage relative to untreated biofilm growth (100%) and a sterility control (0%). Biofilm inhibition was defined as any concentration of peptide or antibiotic where at least 80% of biofilm growth was inhibited. Results shown are the average of three or four biological replicates (Table 8). Overall, AB103-D and AB107-D exhibited broad spectrum antibiofilm activity that was 2-4 fold more active than peptide DJK5. Accordingly, AB103-D and AB107-D exhibited enhanced broad spectrum antibiofilm activity of compared to DJK5, ciprofloxacin and vancomycin against ESKAPEE Pathogens.

[0211] Table 8:EXAMPLE 6: MURINE SINUSITIS MODEL

[0212] A murine sinusitis model of bacterial infections in the nasal cavity of mice [Alford et al. (2021). Murine model of sinusitis infection for screening antimicrobial and immunomodulatory therapies. Frontiers in Cellular and Infection Microbiology., 11, 621081] was used to determine the efficacy of D-amino acid peptides AB101-D, AB102-D and AB103-D compared to DJK5. Briefly, a bacterial suspension of MRSA USA300 (Figures 15A, D, G) or P. aeruginosa LESB58 (Figures 15 B, C, E, F, H, I) was instilled dropwise into the left naris of mice then peptide treatment (0.1 mg dose) was added one-hour post infection. Treatment was applied either as dropwise administration of a peptide solution (20 µl) to the infected naris of a mouse (Figures 15 A, B, D, E, G, H) or peptide was delivered using a Respimat® inhaler device (Figures 15 C, F, I). Mice were monitored and weighed once daily for up to three days then euthanized prior to collection of nasal lavage fluid and lung tissue for bacterial enumeration. The limit of detection is shown by the dotted line at log CFU = 2. Overall, AB102-D or AB103-D treatment reduced the amount of bacteria in the nasal cavity and lungs of mice much more than DJK5 treatment, regardless of treatment modality. Additionally, all non-DJK5 peptide treatments prevented weight loss experienced by infected control mice whereas DJK5 treatments did not prevent weight loss. Accordingly, the results indicated enhanced efficacy of D-amino acid peptides AB101-D, AB102-D and AB103-D compared to DJK5 in this murine model. EXAMPLE 7: ORAL MULTISPECIES BIOFILMS

[0213] Three-day-old plaque biofilms were grown on hydroxyapatite (HA) surfaces treated with different concentrations of peptide then visualized with confocal microscopy. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, respectively. The scale bar represents 200μm. Quantification of the proportion of dead biofilm cells within the total biovolume following daily peptide treatment for 3 days revealed that AB101-D, AB102-D and AB103-D killed more of the biofilm cells when treated with 1 or 5 µg / ml peptide solutions (Figure 16A). At 10 µg / ml, both AB101-D and AB103-D were superior to DJK5 at killing bacteria cells within the biofilm. AB103-D also killed more biofilm cells than DJK5 at all three peptide concentrations when 3-day-old plaque biofilms were treated for two days (Figure 16B) or one day (Figure 16C). For additional experimental details,see [Shen et al. (2009) Evaluation of the effect of two chlorhexidine preparations on biofilm bacteria in vitro: a three-dimensional quantitative analysis. Journal of Endodontics.35:981-985; and Shen, Stojicic & Haapasalo (2011) Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. Journal of Endodontics.37:657–661]. The results indicated enhanced activity of AB101-D, AB102-D and AB103-D compared to DJK5 in treating 3-day-old oral multispecies biofilms. EXAMPLE 8: ORAL MULTISPECIES BIOFILM DEVELOPMENT

[0214] Sterile HA disks were used as the plaque biofilm substrate. To allow for the formation of salivary pellicle, saliva coated HA (sHA) disks were prepared by incubating with 400μl infiltrated saliva from volunteers for 4 hours. The supragingival plaque was collected from upper molars and mixed in the same batch of BHI. Bacterial suspension was adjusted to an OD405nm of 0.1, corresponding to 3.0×107CFU / ml. The sHA disks were placed in the wells of a 24-well plate containing 1.8 ml of BHI. Each well was inoculated with 0.2 ml of dispersed plaque suspension. A 10 µg / ml solution of peptide AB103-D or DJK5 was added to the plaque suspension at the beginning of biofilm development, and maintained over the course of the experiment. All disks were incubated in the BHI-plaque suspension under anaerobic conditions at 37°C for 3 days and then analyzed by confocal microscopy. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, respectively. AB103-D treatment resulted in a significantly higher percentage of dead bacterial cell volume compared to DJK5 treatment (Figure 17A) and also led to reduced total biovolume of the peptide treated biofilms (Figure 17B). The results indicated enhanced effect of AB103-D on oral multispecies biofilm development compared to DJK5. EXAMPLE 9: BRIEF EXPOSURE TO PLAQUE BIOFILMS

[0215] After the formation of a 3-day-old biofilm, the HA disks were rinsed in PBS for 1 minute then immersed in 1 ml of a 10 μg / ml peptide solution for one minute once or three times. Disks treated by sterile water were set as the control group. Disks treated three times were immersed in PBS for 1 minute between each treatment. Confocal microscopy images of 3-day- old plaque biofilms on HA disks treated once or three times with peptide (10 μg / ml) for 1 minute were obtained. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide,which stain live cells green and dead cells red, respectively. The scale bar represents 200 μm. In all cases, AB103-D was significantly more effective than DJK5 at killing 3-day-old oral plaque biofilms based on quantification of the percentage of dead bacterial cell volume derived from the confocal microscopy imaging (Figure 18). The results indicated enhanced antibiofilm activity of AB103-D compared to DJK5 following brief exposure (1 min) of biofilms to peptides once or three times on 3-day-old plaque biofilms. EXAMPLE 10: PLAQUE BIOFILMS COMPARED ORAL RINSE SOLUTIONS

[0216] Briefly, after the formation of a 3-day-old biofilm, HA disks were rinsed in PBS for 1 minute and assigned to one of five treatment groups: (i) sterile water, (ii) Listerine Total Care Mouthwash (Johnson & Johnson Inc., Canada), (iii) GUM Paroex Mouthwash (Sunstar, USA), (iv) 0.12% CHX + 10 μg / mL of peptide DJK-5, and (v) 0.12% CHX + 10 μg / mL of peptide AB103-D. For each treatment, the disks were treated twice each day (i.e. at 8 am and 6 pm for the first and second time, respectively). Treatment consisted of a droplet of 40 μl of each solution spread on the surface of the biofilm for 1 minute, followed by rinsing with PBS for 1 minute and then placing the disk back into the 24-well plates under anaerobic conditions at 37 °C. On the 0, 1st, 3rd, 7th, 10th, and 14th day, three disks from each group were collected for confocal microscopy analysis. Biofilms were stained with fluorescent dyes SYTO-9 and propidium iodide, which stain live cells green and dead cells red, respectively. The scale bar represents 200 μm. A plot of the proportion of dead bacteria within the biofilm for a representative single plaque donor sample (Figure 19A) revealed that both peptide solutions killed the most bacteria within the plaque, with the AB103-D solution being more effective than DJK5. Analysis of the total biovolume recorded during the course of the experiment for a representative donor biofilm (Figure 19B) reveals that peptide treated samples had the lowest residual plaque biovolume, with AB103-D being superior than DJK5. The results indicated improved antibiofilm effects of AB103-D on 3-day-old oral plaque biofilms compared to treatment by various oral rinse solutions for 14 days. EXAMPLE 11: ANTIBIOFILM AND ANTIBACTERIAL ACTIVITY

[0217] The effect of peptide on bacterial and biofilm growth was assessed using the biofilm inhibition assay [Haney, E. F., Trimble, M. J., & Hancock, R. E. W. (2021) Figures 20A-D.Microtiter plate assays to assess antibiofilm activity against bacteria. Nature Protocols, 16(5), 2615–2632]. The effect of peptide on planktonic growth and biofilm growth of MRSA USA300 (A, C) and P. aeruginosa PAO1 (B, D) was assessed and compared to the activity seen for AB103-D (gray stars) and DJK5 (gray open circles). Data shown are the average of three biological replicates. Error bars have been omitted for clarity. In general, all of the peptides inhibited growth of MRSA USA300 biofilms to a similar extent while AB111-D, AB113-D and AB114-D were among the most active against planktonic cells. Against P. aeruginosa PAO1, all the peptides (except AB116-D) were more active than DJK5 at inhibiting planktonic and biofilm growth (4-8 fold more potent) with peptides AB109-D, AB112-D and AB113-D being among the most active. The results indicated enhanced antibiofilm and antibacterial activity of AB103- D derivatives compared to DJK5.

[0218] All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.

[0219] Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications are comprehended by the disclosure and can be practiced without undue experimentation within the scope of the embodiments, which are presented by way of illustration not limitation.

Claims

What is Claimed is:

1. An isolated peptide comprising the amino acid sequence of Peptide 1: AA1 – AA2 – AA3 – AA4 – AA5 – AA6 – AA7 – AA8 – AA9 – AA10 – AA11 – AA12 – NH2 Peptide 1 wherein AA1 is Arg, Lys, Trp, Ala, or Leu, AA2 is Val or Trp, AA3 is Arg, Ile, Leu, or Val, AA4is Val, Trp, or Leu, AA5is Ile or Trp, AA6is Arg or Trp, AA7is Val or Ile, AA8 is Gln, Lys, Val, Ile, Trp, or Arg, AA9 is Trp, Leu, Val, Lys, AA10 is Arg, AA11 is Ala, Ile, Arg, Trp, Val, Leu, or Lys, and AA12is Ile or Arg.

2. The isolated peptide of claim 1 wherein: AA1 is Arg or Lys, AA2 is Val, AA3is Arg, or Ile, AA4 is Val or Trp, AA5 is Ile or Trp, AA6is Arg, AA7is Val, AA8 is Gln or Lys, AA9is Trp or Leu, AA10is Arg, AA11 is Ala, Ile or Arg and AA12 is Ile.

3. The isolated peptide of claim 1 comprising an amino acid sequence as set forth in one or more of SEQ ID NOs: 3, 9-12, 15, 18, 42, 43 or a functional variant thereof.

4. An isolated peptide comprising an amino acid sequence as set forth in one or more of SEQ ID NOs: 1-104, or a functional variant thereof.

5. The isolated peptide of any one of claims 1 to 4 comprising a non-natural amino acid equivalent.

6. The isolated peptide of claim 5, wherein the non-natural amino acid equivalent is L-2- amino-3-guanidinopropionic acid, L-2-Amino-4-guanidinobutyric acid, L-Homoarginine, L-2,3-diaminopropionic acid or L-Ornithine.

7. An isolated polypeptide X1-A-X2, wherein A comprises a peptide of any one of claims 1 to 4; and wherein each X1 and X2 independently comprise an amino acid sequence of n amino acids, wherein n is 0 to 50.

8. An isolated polynucleotide encoding the peptide of any one of claims 1 to 7.

9. A pharmaceutical composition comprising the peptide of any one of claims 1 to 7 in combination with an antibiotic.

10. The pharmaceutical composition of claim 9 wherein the combination is synergistic.

11. The pharmaceutical composition of claim 9 or 10 wherein the antibiotic is a aminoglycoside, a beta-lactam, a penicillin, a cephalosporin, a quinolone, a fluoroquinolone, a carbapenem, a tetracycline, a polymyxin, a glycopeptide or a macrolide.

12. The pharmaceutical composition of any one of claims 9 to 11 wherein the conventional antibiotic is amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethyl- succinate / gluceptate / lactobionate / stearate, such as penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, imipenem, meropenem, panipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, , polymyxin B, colistin, benzalkonium-Cl, benzalkonium-Cl, chlorhexidine, H2O2, polyvinylpyrrolidone–iodine, levofloxacin, linezolid, synercid, colimycin,methotrexate, daptomycin, phosphonomycin, cotrimoxazole, EDTA or AgNO3.

13. A method of inhibiting the growth of a bacterium, or of a bacterial biofilm, comprising contacting the bacterium or bacterial biofilm with an inhibition effective amount of a peptide of any one of claims 1 to 8 or the pharmaceutical composition of any one of claims 9 to 12.

14. The method of claim 13 wherein the bacterium is, or the bacterial biofilm comprises, a gram negative bacterium or a gram positive bacterium.

15. The method of claim 13 or 14 wherein the bacterium is, or the bacterial biofilm comprises, Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Enterobacter sp. including Enterobacter cloacae, Pseudomonas aeruginosa, Klebsiella pneumonia, S. epidermidis, Escherichia coli, Salmonella enteritidis ssp Typhimurium, Campylobacter, Burkholderia cepacia complex or Acinetobacter baumannii.

16. The method of claim 15 wherein the MRSA is MRSA USA300, the Enterococcus faecium is Enterococcus faecium #2-1, the Enterobacter cloacae is Enterobacter cloacae 218R1, the P. aeruginosa is P. aeruginosa PAO1, the Klebsiella pneumonia is Klebsiella pneumonia KPLN649, S. epidermidis, the Escherichia coli is E. coli O157:H7 or the Acinetobacter baumannii is Acinetobacter baumannii Ab5075.

17. The method of claim 13 wherein the bacterium is, or the bacterial biofilm comprises, MRSA and the peptide comprises the amino acid sequence set forth in SEQ ID NO:

2.

18. The method of claim 13 wherein the bacterium is, or the bacterial biofilm comprises, P. aeruginosa and the peptide comprises the amino acid sequence set forth in SEQ ID NOs: 1-3, 9-15, 18, 42, 43.

19. The method of any one of claims 13 to 18 wherein the bacterium or bacterial biofilm is associated with an infection, sinusitis, septic shock or abscess.

20. The method of claim 19 wherein the infection is a skin infection.

21. The method of any one of claims 13 to 18 wherein the bacterial biofilm is an oral multispecies biofilm or an oral plaque biofilm.

22. A method of ameliorating resistance to an antibiotic, or of enhancing the efficacy of an antibiotic, comprising contacting a cell with an effective amount of a peptide of any one of claims 1 to 8 in combination with the antibiotic.

23. The method of claim 22 wherein the antibiotic is an aminoglycoside, a beta-lactam, a penicillin, a cephalosporin, a quinolone, a fluoroquinolone, a carbapenem, a tetracycline, a polymyxin, a glycopeptide or a macrolide.

24. The method of claim 22 wherein the antibiotic is amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethyl-succinate / gluceptate / lactobionate / stearate, such as penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, imipenem, meropenem, panipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, polymyxin B, colistin, benzalkonium-Cl, benzalkonium-Cl, chlorhexidine, H2O2, polyvinylpyrrolidone–iodine, levofloxacin, linezolid, synercid, colimycin, methotrexate, daptomycin, phosphonomycin, cotrimoxazole, EDTA or AgNO3.

25. The method of any one of claims 13 to 24 wherein the peptide is bound to a solid support or surface.

26. An article of manufacture comprising a peptide of any one of claims 1 to 8.

27. The article of manufacture of claim 26 wherein the article of manufacture is a collagen dressing, a commercial sterile bandage, an oral rinse, a medical device, a catheter, or a prosthetic device.

28. The article of manufacture of claim 26 or 27 wherein peptide is bound to the article of manufacture.

29. Use of the peptide of any one of claims 1 to 8 for inhibiting the growth of a bacterium or a bacterial biofilm, or for ameliorating resistance to an antibiotic, or for enhancing the efficacy of an antibiotic.

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