Novel antimicrobial peptides in tick attachment GLUE and tick immunomodulator proteins

Abstract

Embodiments of the disclosure include methods and compositions related to antimicrobial applications. In particular embodiments, the compositions comprise peptides for antimicrobial applications that are also found in tick attachment glue. In some embodiments, the disclosure concerns methods to treat bacterial, viral and fungal infections in an animal or insect and / or to clean an environment or matter of any kind.

Description

NOVEL ANTIMICROBIAL PEPTIDES IN TICK ATTACHMENT GLUE AND TICK IMMUNOMODULATOR PROTEINSBACKGROUND

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 553,319, filed February 14, 2024, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under All 19873, AI138129, AI081093, AI093858, and AI074789, awarded by the National Institute of Human Health. The government has certain rights in the invention.I. Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 13, 2025, is named TAMUP0006WO.xml and is 185 kilobytes in size.II. Technical Field

[0004] This disclosure relates at least to the fields of microbiology, molecular biology, bacteriology, virology, cell biology, acarology, medicine, antimicrobial medicine, preventing the spread of antibiotic resistant microbes from food animals, food safety, and environmental safety.III. Background

[0005] Ticks and tick-borne diseases are important to public and veterinary health. Globally, ticks transmit the highest diversity of disease agents than any arthropod vector and come second to mosquitoes in terms of human vector-borne disease impact [1], However, in the United States, human vector-borne diseases are primarily transmitted by ticks, as revealed by recent surveys that indicated that over a span of 12 years (2004-2016); 75% of reported human vector-borne diseases were tick-borne [2],

[0006] Although tick feeding can impact health and animal productivity because of stress, ticks are mostly known for their roles as vectors of tick-borne disease pathogens. They accomplish transmission of tick-borne disease pathogens during feeding. Tick salivary factors facilitatesuccessful feeding and transmission of at least of tick-borne pathogens by blacklegged (or Ixodes scapularis) and Lone Star (or Amblyomma americcmum) ticks. Hard ticks are described as pool feeders that feed by penetrating host skin with its barbed mouthpart. The barbed mouthpart disrupts host skin tissue to generate the wound in the skin (or feeding lesion or site) and then the ticks suck up blood that collects into the wound area. Tick attachment glue is secreted to secure tick mouthparts onto skin of the host (non-human animals and humans) so that ticks are not removed by the host during their long feeding period which may last for more than 14 days. The tick feeding style triggers tissue repair immune defenses to stop further blood loss. The tick responds by injecting immuno-modulatory proteins that block the immune defenses of the host [3], Proteins have been identified that the tick utilizes to successfully feed and transmit tick-borne pathogens. In particular, tick proteins have been identified that form tick attachment glue and tick immunomodulatory proteins that regulate the three-way interaction cascade between ticks and pathogens; ticks and the vertebrate host, and vertebrate host and pathogens [4-10], The tick feeding style of generating a wound in the skin of the host and then sucking up blood that bleeds into the wound (or feeding site) predisposes the host to secondary infections from microbes in the skin of the host

[0011] .

[0007] The present disclosure provides a long-felt need in the art of pathogen infection treatment and prevention.SUMMARY

[0008] Embodiments of the disclosure include methods and compositions related to antimicrobial compositions, which also may be referred to as antibiotic compositions. In particular embodiments, any composition encompassed herein may comprise one or more peptides that have antimicrobial activity. In specific embodiments, the peptides are identified from tick attachment glue, although in some embodiments the peptides originate from another source. In embodiments, the peptides have antimicrobial activity and may be used in any application thereof, such as a medical therapeutic and / or as a cleansing agent. One or some of the peptides may be used as an antimicrobial medical therapeutic, one or some of the peptides may be used as an antimicrobial medical preventative, and / or one or some of the peptides may be used as an antimicrobial cleansing agent, as examples only.

[0009] Microbial infections may be treated using antibiotic drugs. However, the overuse of antibiotics has selected for drug-resistant microbes that are difficult to treat. The antimicrobial peptides that are encompassed herein offer unique opportunities to treat both susceptible and drugresistant microbial infections. An advantage of antimicrobial peptides encompassed herein arises from the fact that they are novel and are not related to currently utilized antibiotics on the market. In a specific embodiment, it is less likely that circulating microbes have any resistance to one or more antimicrobial peptides encompassed herein.

[0010] In some embodiments, the compositions comprising one or more of the peptides herein are utilized for veterinary and / or human microbial infections. In a specific embodiment, the compositions of peptide(s) are utilized in livestock embodiments, including at least livestock production and environments where livestock are maintained. Examples of livestock include cattle, sheep, pigs, goats, horses, donkeys, mules, buffalo, oxen, llamas, camels, emus, chicken, turkeys, ducks, goose, deer, etc. The peptide compositions may be utilized in agriculture and / or aquaculture environments. Examples of aquaculture include at least fish, mollusks, shellfish, algae, etc. Specific examples include at least oysters, clams, mussels, shrimp, seaweeds, and fish such as salmon, black sea bass, sablefish, yellowtail, pompano, tilapia, etc.

[0011] Embodiments of the disclosure encompass novel antimicrobial peptides in tick attachment glue and tick immunomodulator proteins, and embodiments of the disclosure encompass their use for any purpose, including treating microbial infections in an animal of any kind.

[0012] In particular embodiments, some of the proteins in tick attachment glue and tick saliva immuno-modulatory proteins comprise antimicrobial peptides, at least some of which are active against clinical isolates from both human and / or veterinary patients. Significantly, antimicrobial peptides encompassed herein are active against extended spectrum beta lactamase-positive bacteria isolates that are known to be resistant against major classes of antibiotic drugs.

[0013] In certain embodiments, the antimicrobial peptides are utilized as new generation antibiotic therapeutics. In some embodiments, the analogs (or derivatives of antimicrobial peptides) are utilized as new generation antibiotic therapeutics. In some embodiments, antimicrobial peptides encompassed herein are used to decontaminate an environment, such as domestic animal fecal effluents, including before release into public waterways. In someembodiments, antibiotic therapeutics based on antimicrobial peptides, or their analogs are used in managing the emergence of antibiotic resistance.

[0014] In some embodiments, the source of a microbe infection treated by the peptide(s) disclosed herein is from a host at a site of a tick bite on the host, whereas in other embodiments the source of the microbe infection is from the tick itself.

[0015] In some embodiments, one or more of the peptides disclosed herein may be bacteriostatic, and in some embodiments, one or more of the peptides disclosed herein may be bactericidal. In some embodiments, one or more of the peptides disclosed herein may be bacteriolytic.

[0016] Embodiments of the disclosure include any peptides encompassed herein. Embodiments include any composition comprising one or more peptides comprising the sequence of any one of SEQ ID NO: 1-SEQ ID NO:210, or comprising a sequence that is at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto, respectively. In specific embodiments, the composition comprises peptides comprising the sequence of one or more of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Variants of any peptide disclosed herein may have 1, 2, 3, 4, 5, or more amino acid changes compared to any one of SEQ ID NO: 1-SEQ ID NO:210 peptides. Any composition may further comprise a pharmaceutically acceptable carrier. Any composition may be formulated as an antimicrobial composition. Any composition may be formulated as a therapeutic composition. Any composition may be formulated as an antimicrobial cleaning composition, including a surface cleaning composition and / or an environmental cleaning composition. In specific aspects, the surface is a floor, countertop, table, chair, couch, bathtub, shower, toilet, sink, door, appliance, or a combination thereof. The surface may be in an enclosed environment, such as an office building, a school, a university, a residence hall, a hospital, a library, a childcare center, a gym, a medical facility, a nursing home, a residence, a transportation entity, a theater, a hotel, a sporting venue, or a combination thereof. The surface may be the skin and / or hair of a mammal. In specific embodiments, the composition is formulated as a solid, or is on and / or in a solid substrate, such as a wipe, a cloth, a brush, a sponge, a mesh, or a combination thereof. In some embodiments, the solid substrate is a medical device or is used for medicinal purposes, and a device may be a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt,valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain; and / or wherein the solid substrate is a bandage. Any composition encompassed herein may be formulated as a liquid or spray. In some aspects, the composition further comprises an additional therapeutic agent, such as an antimicrobial agent. In some embodiments, the composition further comprises one or more metal cofactors, such as Mg2+, Ca2+, Fe2+, Cu2+, Zn2+, or a combination thereof. Embodiments include antibiotic compositions, comprising an effective amount of any one or more peptides encompassed herein. Embodiments include immunogenic compositions, comprising an effective amount of any one or more peptides encompassed herein. Embodiments include vaccine compositions, comprising an effective amount of any one or more peptides encompassed herein. Embodiments include any disinfectant solution comprising an effective amount of any one or more peptides encompassed herein.

[0017] In certain embodiments, there is a medical device, comprising an effective amount of any composition encompassed herein, including a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain. The composition may be on the surface and / or in the device. The composition may elute from the device. In some embodiments, there is a medical substrate, comprising an effective amount of any composition encompassed herein. In certain embodiments, the substrate is personal protective equipment, clothing, collection swab or collection device, a gel, a lotion, a bandage, mesh, tape, cloth, scaffold, or a combination thereof. The personal protective equipment or clothing may be a glove, gown, mask, face shield, or a combination thereof.

[0018] In certain embodiments, there are methods of inhibiting the growth of a microbe in and / or on an individual, surface, and / or substrate, comprising the step of providing an effective amount of one or more of any composition encompassed herein to the individual, surface, and / or substrate, respectively. In some embodiments, there are methods of reducing the risk of having a microbe infection, including reducing the risk of having one or more symptoms associated with a microbe infection. In specific embodiments, the microbe is pathogenic, a contaminant, or the microbe has one or more deleterious symptoms for an individual. The microbe may be a bacteria, virus, fungus, or protozoa, and in at least some cases the individual has one or more deleterioussymptoms from the microbe. The individual may have an internal infection of the microbe and / or topical infection of the microbe. The composition may be provided by injection, by inhalation, and / or topically to the individual.

[0019] Embodiments of the disclosure include methods of treating an individual for a microbial infection, comprising administering an effective amount of any composition encompassed herein. The individual may have an internal infection of the microbe and / or topical infection of the microbe. The composition may be provided by injection, by inhalation, and / or topically to the individual.

[0020] Embodiments of the disclosure include methods of disinfecting an environment and / or matter, comprising providing an effective amount of any composition encompassed herein to the environment. The environment may be terra firma or is a water source, such as ocean water, fresh water, brackish water, black water, or grey water, The water source may be an ocean, lake, river, stream, estuary, creek, pond, water well, water tank, irrigation, or a combination thereof. In some aspects, the terra firma is waste landfill, lagoon, Brownfields site, Superfund site, chemical storage facilities, industrial or municipal wastewater outfall, mining operation, source of agriculture pollution, source of aquaculture pollution, residential and / or commercial septic systems, stormwater runoff, underground injection well, or a combination thereof. In some aspects, the environment is a wastewater facility, food processing facility, agriculture farm, or aquaculture environment. In some embodiments, the matter is fecal matter.

[0021] Embodiments include kits comprising any composition encompassed herein. The kit may or may not further comprise an additional therapeutic agent, such as an antimicrobial.

[0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The subject matter of the disclosuremay be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] FIG. 1. The CM1-95 peptide has bactericidal activity. Various CMI-95 peptide concentrations were incubated with human isolates overnight at 37°C. Subsequently, 2pL of cultures were spotted on LB agar plates incubated overnight at 37°C. Lack of bacteria growth indicates bactericidal effects.

[0025] FIG. 2. The CMI-95 antimicrobial peptide acts by blocking the efflux pump, in certain embodiments. The efflux pumps of overnight cultured bacteria were first blocked using chemical inhibitors. Following blocking of the efflux pump, bacteria was allowed to uptake and accumulate the Nile Red substrate. Following uptake of the Nile Red substrate, the efflux pump was reactivated to allow bacteria to pump out the substrate. As shown, reactivating the efflux pump in presence of CMI-95 peptide interfered with bacteria’s ability to pump out the substrate (graph lines with triangles).

[0026] FIGS. 3A-3D. The CMI-95 antimicrobial peptide inhibited growth of bacteria in feces of cattle (3A and 3B) and mice (3C and 3D). Freshly collected feces of cattle (3 A) and mice (3C) were incubated with or without the CMI-95 antimicrobial peptide (125 pg / mL) at 37C for the indicated time periods. Aliquots (100 mL) were plated on LB agar plates incubated overnight at 37C. Subsequently, colony forming units were counted (3B and 3D). As shown in FIGS. 3B and 3D, the CMI-95 reduced colony forming units (CFU) by nearly more than 50% in feces of cattle and mice.

[0027] FIGS. 4A-4D. The CMI-95 antimicrobial peptide is safe as revealed cell culture assays. Incubating the CMI-95 antimicrobial peptide with purified cattle red blood cells did not cause hemolysis (4A). Likewise, the CMI-95 antimicrobial peptide had no cytolytic effects when incubated with Chinese hamster ovary (CHO) cells (4B), LnCAP cell derived from a metastatic lymph node lesion of human prostate cancer (4C), and Primary Prostate Epithelial Cells (4D).DETAILED DESCRIPTION

[0028] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0029] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0030] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0031] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0032] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0033] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0034] The term “subject,” as used herein, generally refers to an individual having that has or is suspected of having a microbial infection of any kind. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., have or be suspected of having a disease (that may be referred to as a medical condition), such as microbial infection. The subject may being undergoing or having undergone treatment. The subject may be asymptomatic. The subject may be healthy individuals but that are desirous of prevention of microbial infection. The term “individual” may be used interchangeably, in at least some cases. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individualmay comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. The embodiments of the disclosure apply to single individuals as well as pluralities of individuals.

[0035] Any of the embodiments encompassed herein concern peptides that are efficient to one or more resistant bacteria and / or are more efficient at withstanding deleterious effects of the acidity of the gut. Any of the embodiments encompassed herein concern peptides that are modified by the hand of man to be efficient to one or more resistant bacteria and / or to be more efficient at withstanding deleterious effects of the acidity of the gut, all including into comparison to a peptide that is not so modified.

[0036] In particular embodiments, an acid-resistant material encompasses the peptide(s) where the material lacks degradability at the neutral to acidic pH ranges of the gastro-intestinal tract but will degrade at the neutral to alkaline pH range expected for livestock feces (e.g. polylactic acid (PLA) and polyglycolic acid (PGA). This encapsulation prevents or reduces the release of the antimicrobial peptide into the gastro-intestinal tract (stomach, small intestine, and large intestine) of the individual. In certain embodiments, this material is designed to dissolve upon exposure to conditions specific to feces upon defecation. That is, upon degradation of the encapsulation material, the antimicrobial peptide will be active in the feces.I. General Embodiments

[0037] Embodiments of the present disclosure generally concern compositions having antimicrobial activity, and in specific embodiments the compositions comprise one or more isolated peptides. The compositions may comprise one or more of the peptides encompassed herein and / or one or more analogs of the peptides encompassed herein, such as a functionally active derivative of one or more peptides encompassed herein.

[0038] Microbial infections are a global threat affecting public and veterinary health worldwide. Microbial infections are treated using antibiotic drugs. The overuse of antibiotic drugs has resulted in selection of several microbes that are resistant at least to several known antibiotic drugs. This has resulted in serious problems of microbial infections that are difficult to treat. The antimicrobial peptides in this disclosure contribute to strategies to prevent the emergence ofmicrobes that are resistant to drugs. The antimicrobial peptides in this disclosure are novel and have not been previously used to treat the circulating microbes. Thus, there has not been the selection of microbes that are resistant to antimicrobial peptides in this disclosure.

[0039] In particular embodiments, drugs based on antimicrobial peptides described herein are for treatment of microbial infections in veterinary patients. In such aspects, there is no spill-over of resistant microbes from veterinary patients to the human population. In alternative embodiments, drugs based on antimicrobial peptides described herein are for treatment of microbial infections in humans and in veterinary patients.

[0040] In particular embodiments, antimicrobial peptides are used to mitigate the impact of domestic animal fecal effluents contaminating waterways. In some aspects, one or more of the antimicrobial peptides encompassed herein are effective against bacteria in feces of cattle and / or rodents. In certain aspects, these antimicrobial peptides and their derivates (or analogs) lead to new chemistries that are used to decontaminate domestic animals’ fecal effluents before releasing them into waterways, which is one of the major ways drug-resistant bacteria of animals could get into the human population.II. Specific Embodiments

[0041] The tick feeding style of disrupting host tissue and sucking up blood that bleeds into the feeding lesion (or site) predisposes microbial infections of the tick feeding site, which will compromise feeding and transmission of disease agents by ticks. To prevent bacteria colonization of the tick feeding site, ticks inject antimicrobial peptides (AMP) to suppress bacteria growth at the tick feeding site. Using in silico methods, the inventors have putative AMPs (n=290) in proteins that form the tick attachment glue (or tick cement) and tick immunomodulatory proteins of blacklegged and Lone star ticks. The inventors used the Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) analyses to identify 5 out of 21 synthetic AMPs with antibacterial activity against human and veterinary clinical isolates. One of the candidate peptides identified in this study, CM1-95, is active against Salmonella enterica enteritidis and E. coli clinical isolates including Extended- spectrum beta-lactamases (ESBL) positive and Kanamycin resistant E. coli. The ESBL enzymes confer resistance to multiple antibiotics, and therefore, in certain embodiments AMPs such as CM1-95 form the basis for developing newgeneration antibiotics and contribute to mitigating the impact of widespread antimicrobial resistance (AMR) estimated to kill millions of people.

[0042] Embodiments of the disclosure encompasses antimicrobial peptide sequences that are found in tick attachment glue (or tick cement) of the blacklegged tick (Ixodes scapularis) (n = 49, Table 1A) and the Lone Star tick (Amblyomma americanum) (n = 34, Table IB), tick saliva proteins of uninfected and Lyme disease agent infected blacklegged nymphs (n = 126, Table 1C), immunomodulatory proteins found in saliva of blacklegged ticks (n = 20, Table ID) and Lone Star ticks (n = 61, Table IE). These data are presented elsewhere herein.

[0043] Embodiments of the disclosure include antimicrobial peptides and derivatives (or analogs) based on amino acid sequences to be used in treatment of bacterial infections that are caused by both susceptible and / or antibiotic-resistant bacteria.

[0044] In specific embodiments, the CM1-95 antimicrobial peptide (LSRRWHQGIRVLLRAA; SEQ ID NO: 1) and its derivatives (or analogs) are used to treat several microbial infections of humans and veterinary patients. The CM1-95 peptide was found among proteins that form blacklegged tick attachment glue. As assessed by minimum inhibitory concentration (MIC is the lowest concentration in pg / mL of an antibiotic that inhibits the growth of a given strain of bacteria), the CM1-95 that was derived from blacklegged tick attachment glue protein has potent activity against 14 pathogenic bacteria strains (out of a 26 bacteria strain screen) that cause disease in humans and animals. The methods and results that established this claim are presented in Table 2A.

[0045] The 16 pathogenic bacteria strains that are susceptible to CM1 -95 antimicrobial peptide include the following:

[0046] BACTERIA STRAIN 1. Escherichia coli (isolate CFT073). MIC of 62.5 pg / mL of CM1-95 clears 70% of this strain. This strain was originally isolated from human clinical specimen (blood and urine) from a woman with acute pyelonephritis (bacterial infection causing inflammation of the kidneys). This strain is described as mesophilic, as it grows best in not too hot or too cold temperatures and its optimum growth temperature range is 20-45 degrees centigrade.

[0047] BACTERIA STRAIN 2. Acinetobacter baumanmi (isolate 17978). MIC of 15.625 pg / mL of CM1-95 clears 80% of this strain. This strain was originally isolated from a 4 month old infant with fatal meningitis in France in 1951.

[0048] BACTERIA STRAIN 3. Kanamycin-resistant Escherichia coli (isolate CFT073 Al). MIC of 125 pg / mL of CM1-95 clears more than 90% of this strain. This strain was originally isolated from blood and urine of a urosepsis (sepsis that originates from urinary tract infection) woman patient. This strain is resistant against killing by Kanamycin antibiotic.

[0049] BACTERIA STRAIN 4. Escherichia coli (0157 strain isolate EDL-933). MIC of 62.5 pg / mL of CM1-95 clears 99% of this strain. This strain was isolated from ground beef in 1982 during the enterohemorrhagic Escherichia coli outbreak in the United States.

[0050] BACTERIA STRAIN 5. Escherichia coli (0157 strain isolate EDL-931). MIC of 62.5 pg / mL of CM1-95 clears more than 88% of this strain. This strain was first isolated from feces of patients suffering from outbreak of gastrointestinal illness in Oregon in 1982.

[0051] BACTERIA STRAIN 6. Escherichia coli (isolate UTI89). MIC of 500 pg / mL of CM1- 95 clears more than 90% of this strain.

[0052] BACTERIA STRAIN 7. Escherichia coli (isolate ABU83972). MIC of 500 pg / mL of CM1-95 clears more than 90% of this strain.

[0053] BACTERIA STRAIN 8. Escherichia coli (veterinary isolate 23109007-1) extended spectrum beta-lactamase positive. This strain is resistant to any known antibiotics. MIC of 500 pg / mL of CM1-95 clears more than 90% of this strain.

[0054] BACTERIA STRAIN 9. Escherichia coli (veterinary isolate 23117003-1) extended spectrum beta-lactamase positive. This strain is resistant to any known antibiotics. MIC of 500 pg / mL of CM1-95 clears more than 90% of this strain.

[0055] BACTERIA STRAIN 10. Escherichia coli (veterinary isolate 23111007-1) extended spectrum beta-lactamase positive. This strain is resistant to any known antibiotics. MIC of 500 pg / mL of CM1-95 clears more than 90% of this strain.

[0056] BACTERIA STRAIN 11. Escherichia coli (veterinary isolate 23121002-1). MIC of 125 pg / mL of CM1-95 clears more than 85% of this strain.

[0057] BACTERIA STRAIN 12. Escherichia coli (veterinary isolate 23121004-1). MIC of 31.5 pg / mL of CM1-95 clears more than 85% of this strain.

[0058] BACTERIA STRAIN 13. Salmonella enterica (ATCC 13076). MIC of 125 pg / mL of CM1-95 clears more than 90% of this strain. This strain colonizes the intestinal tract of a variety of animals, especially humans and poultry and causes inflammation disease of the small intestine.

[0059] BACTERIA STRAIN 14. Pseudomonas aeruginosa (ATCC 27853). MIC of 250 pg / mL of CM1-95 clears more than 51%, while MIC of 500 pg / mL clears more than 70% of this strain. This strain colonizes the intestinal tract of a variety of animals, especially humans and poultry, and causes inflammation disease of the small intestine.

[0060] BACTERIA STRAIN 15. Pseudomonas aeruginosa (veterinary isolate 116-50). MIC of 250 pg / mL of CM1-95 clears more than 62.2%, while MIC of 500 pg / mL clears more than 70% of this strain. This strain colonizes the intestinal tract of a variety of animals, especially humans and poultry, and causes inflammation disease of the small intestine.

[0061] BACTERIA STRAIN 16. Pseudomonas aeruginosa (veterinary isolate 118-09). MIC of 250 pg / mL of CM1-95 clears more than 50.2%, while MIC of 500 pg / mL clears more than 78% of this strain. This strain colonizes the intestinal tract of a variety of animals, especially humans and poultry, and causes inflammation disease of the small intestine.

[0062] In embodiments, the CRT-I antimicrobial peptide (GTKKVHVIFNYKGKNLLINKEIR; SEQ ID NO:2) and its derivatives (or analogs) are used to treat medically important microbial pathogens. As assessed by minimum inhibitory concentration (MIC is the lowest concentration in pg / mL of an antibiotic that inhibits the growth of a given strain of bacteria), the antimicrobial peptide CRT-I that is contained within the blacklegged tick cement and saliva is effective against both gram-negative and gram-positive bacteria. The CRT-I antimicrobial peptide is active against medically important bacteria strains (in a screen of 3 pathogenic bacteria). The methods and results are presented in Table 2B.

[0063] The 2 pathogenic bacteria strains that are cleared by the CRT-I peptide include:

[0064] BACTERIA STRAIN 1. Acinetobacter baumannii (isolate la7978). MIC of 500 pg / mL of CRT-I clears 76.6% of this strain. This strain was originally isolated from human clinical specimen (blood and urine) from a woman with acute pyelonephritis (bacterial infection causing inflammation of the kidneys). This strain is described as mesophilic, because it grows best in not too hot or too cold temperatures and its optimum growth temperature range is 20-45 degrees centigrade.

[0065] BACTERIA STRAIN 2. Staphylococcus aureus (isolate SAI 16). MIC of 500 pg / mL of CRT-I clears 58.8% of this strain. This strain referred to as Methicillin-resistant Staphylococcus aureus is difficult to treat because it is resistant to many drugs.

[0066] In some embodiments, the CM4 antimicrobial peptide (LIRKLVGHIKGNRRNKHGHGGG; SEQ ID NO:3) and its derivatives (or analogs) are used to treat medically important microbial pathogens. As assessed by minimum inhibitory concentration (MIC is the lowest concentration in pg / mL of an antibiotic that inhibits the growth of a given strain of bacteria), the antimicrobial peptide CM4 that is contained within the blacklegged tick attachment glue is effective against both gram-negative and gram-positive bacteria. The CM4 peptide is active against medically important bacteria strains (screen of 3 pathogenic bacteria). The methods and results are presented in Table 2C.

[0067] The 2 pathogenic bacteria strains that are cleared by the CM4 peptide include:

[0068] BACTERIA STRAIN 1. Escherichia coli (CFT073). MIC of 250 pg / mL of CM4 peptide clears 43% of this strain. This strain was originally isolated from a 4-month-old infant with fatal meningitis in France in 1951.

[0069] BACTERIA STRAIN 2. Staphylococcus aureus (isolate SAI 16). MIC of 500 pg / mL of CRT-I clears 38% of this strain. This strain referred to as Methicillin-resistant Staphylococcus aureus is difficult to treat because it is resistant to many drugs.

[0070] Embodiments of the disclosure include the TCI-Asiaticum antimicrobial peptide (RRRNMVCWLVMSRRRKCVSTYRSQ; SEQ ID NO:4) and its derivatives (or analogs) that are used to treat medically important microbial pathogens. As assessed by minimum inhibitory concentration (MIC is the lowest concentration in pg / mL of an antibiotic that inhibits the growth of a given strain of bacteria), the antimicrobial peptide TCI-Asiaticum (TCI-asiat) peptide that is contained within the Hyalomma asiaticum tick is effective at least against gram-positive bacteria. The TCI-asiat antimicrobial peptide is active against medically important bacteria strains (screen of 3 pathogenic bacteria). The methods and results are presented in Table 2D.

[0071] The pathogenic bacteria strains that are cleared by the TCI-asiat peptide include:

[0072] BACTERIA STRAIN 1. Acinetobacter baumarmii (isolate 17978). MIC of 250 pg / mL of TCI-Asiaticum peptide clears 50% of this strain. This strain was originally isolated from a 4 month old infant with fatal meningitis in France in 1951.

[0073] BACTERIA STRAIN 2. Pathogenic Escherichia coli (isolate CFT073). MIC of 125 pg / mL of TCI-Asiaticum clears 60% of this strain. This strain was originally isolated from human clinical specimens (blood and urine) from a woman with acute pyelonephritis (bacterial infection causing inflammation of the kidneys). This strain is described as mesophilic, as it grows best innot too hot or too cold temperatures and its optimum growth temperature range is 20-45 degrees centigrade.

[0074] In particular embodiments, the CM1-95 antimicrobial peptide kills bacteria by lysing bacteria cells. The antimicrobial peptide activity could be expressed by stunting bacteria growth (bacteriostatic effect) or by breaking up bacteria cells (bacteriolytic). As assessed by Minimum Bactericidal Concentration, the CM1-95 antimicrobial peptide lyses the bacteria cells. This is based on the fact that there were no bacteria grown on LB agar plates following CM1-95 antimicrobial peptide treatment of liquid cultures. The methods and results that established this claim is presented in FIG. 3.

[0075] In particular embodiments, the CM 1-95 antimicrobial peptides are used to decontaminate domestic animal fecal effluents. The CM1-95 antimicrobial peptides and its derivatives (or analogs) may be used to reduce the pathogenic bacteria load in domestic animal fecal effluents before releasing into waterways. Co-culturing the CM1-95 peptide with cattle or mice fecal extracts reduced bacterial forming units by more than 50%. The methods and results that are presented in FIG. 2.

[0076] In particular embodiments, the CM1-95 peptide works by locking up the resistant nodulation cell division (RND) efflux pump of bacteria. In certain aspects, the consequence of this is that the bacteria will not be able to eliminate toxins and drugs. Treating pathogenic bacteria with the CM1-95 peptide blocked the bacteria's ability to eliminate drugs (blocks the RND efflux pump). This property may be used to kill resistant bacteria that have developed resistance mechanisms by pumping drugs out of the cell before the drugs have any affect. The methods and results that established this are presented in FIGS. 3A and 3B.

[0077] In particular embodiments, the CM1-95 antimicrobial peptide may cause poration of Acinetobacter baumannii bacteria. This was revealed by observations that blocking of the RND efflux pump in presence of the CM1-95 antimicrobial peptide did not stop the efflux of the Nile red fluorescent substrate. The methods and results that established this are presented in FIG. 4C.

[0078] In particular embodiments, the CM1-95 antimicrobial peptide is safe for use in animals of any kind. Cytotoxicity and hemolytic activity analysis of the CM1-95 antimicrobial peptide show that this peptide had no cytotoxicity against CHO (Chinese hamster ovary cells), LnCAP (lymph node carcinoma of the prostate cell line), and primary prostate epithelial cells. Likewise,the CM1 -95 antimicrobial peptide did not lyse red blood cells. The methods and results that established this are presented in FIG. 4.III. Examples of Peptides of the Disclosure

[0079] The antimicrobial peptides in this disclosure contribute to reducing the spread of antibiotic drug-resistant microbes, in particular aspects. The antimicrobial peptides presented here are novel and have not been previously used to treat microbes, including circulating microbes. Thus, in specific aspects microbes are not resistant to one or more of the antimicrobial peptides in this disclosure.

[0080] Embodiments of this disclosure include antimicrobial peptide amino acid sequences and information on some of the antimicrobial peptides that are potent against both veterinary and human clinical isolates. Additionally, at least some of the antimicrobial peptides are effective against bacteria that are in feces of cattle, for example. These antimicrobial peptides and their derivates (or analogs) allow for new chemistries that may be used in decontaminating domestic animal fecal effluents before releasing into waterways, in specific embodiments. The release of domestic animal fecal effluents is one of the major ways by which drug resistant bacteria from animals reach into the human population.

[0081] Examples of particular embodiments of antimicrobial peptides are provided below:

[0082] Some of the peptides in Tables 1A-1F have the same sequences and the same antimicrobial peptide number but have different protein sources (ID of protein sources are different), and this is reflected by having different names / peptide number.

[0083] Table 1 A: Antimicrobial peptides found in proteins that form Jxodes scapularis (or blacklegged) tick cement (or attachment glue).

[0084] Table IB: Antimicrobial peptides found in proteins that form Amblyomma americanum (or Lone star) tick cement (or attachment glue).

[0085] Table 1C: Antimicrobial peptides found in proteins that are found in saliva of uninfected and Borrelia burgdorferi infected Ixodes scapularis (or Black legged) nymphs.

[0088] Table IF: Antimicrobial peptides found in proteins that are found in ticks other that Ixodes scapularis and Amblyomma americanum (or Lone star).

[0089] As used herein, a “peptide” refers to a molecule comprising at least four amino acid residues. As used herein, the term “wild type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to have antimicrobial activity. A “modified peptide” or a “variant” or a “analog” or a “derivative” refers to a peptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type peptide. In some embodiments, a modified / variant peptide has at least one modified amino acid, such as compared to its wild-type sequence. It is specifically contemplated that a modified / variant peptide may be altered with respect to one or more amino acids yet still retain antimicrobial activity.

[0090] Where a peptide is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) peptide. The peptide may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode the peptide. The term “recombinant” may be used in conjunction with a peptide, and this generally refers to a peptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0091] In certain embodiments the size of a peptide (wild-type or modified) may comprise, but is not limited to, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid residues, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that peptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced antimicrobial activity, for purification purposes, etc ).

[0092] The peptides of the disclosure may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, (or any derivable range therein) or more variant amino acids or contiguous amino acids, or any range derivable therein, of SEQ ID NOs: 1-210.

[0093] In some embodiments, the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, (or any derivable range therein) of SEQ ID NOs: 1-210.

[0094] In some aspects there is a peptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) contiguous amino acids of any of SEQ ID NOS: 1-210.

[0095] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total peptide per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).1. Variant Polypeptides or Peptides

[0096] The following is a discussion of changing the amino acid subunits of a peptide to generate an equivalent, or even improved, second-generation variant peptide yet that still retains antimicrobial activity. For example, certain one or more amino acids may be substituted for other amino acids in a peptide sequence with or without appreciable loss of antimicrobial activity. Since in some embodiments it is the interactive capacity and nature of a peptide that defines the functional activity of the peptide, certain one or more amino acid substitutions can be made in a peptide sequence and in its corresponding DNA coding sequence, and nevertheless produce a peptide with similar or desirable properties. It is thus contemplated herein that various changes may be made in the peptide sequences without appreciable loss of their biological utility or activity.

[0097] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0098] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants, as examples only. A variation in a peptide of the disclosure may affect 1, 2, 3, 4, 5, or more non-contiguous or contiguous amino acids of the peptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto, respectively, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 1, 2, 3, 4, 5, or more substitute amino acids. In some embodiments, an N-terminal amino acid and / or a C-terminal amino acid are substituted. When there are two or more amino acids that are changed in the peptide, they may or may not be contiguous with another.

[0099] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where peptide expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0100] Deletion variants typically lack one or more residues of the native or wild type peptide. Individual residues can be deleted or a number of contiguous amino acids can be deleted.

[0101] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the peptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins that are multimers or concatemers of one or more peptides referenced herein.

[0102] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the peptide, and may be designed to modulate one or more properties of the peptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine;glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0103] Alternatively, substitutions may be “non-conservative”, in some cases such that a function or activity of the peptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class. In particular aspects, the non-conservative change still allows for the peptide to have antimicrobial activity.2. Considerations for Substitutions

[0104] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar peptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0105] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein or peptide is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8);tryptophan (-0.9); tyrosine (—1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the present disclosure, those that are within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included.

[0106] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (—1.0); methionine (-1.3); valine (—1.5); leucine (—1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0107] Additionally, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues important for activity or structure in similar peptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0108] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar peptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a peptide with respect to its three- dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be in an important location of the peptide, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or antimicrobial activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein_structure.

[0109] In some embodiments of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, (5) confer or modify other physicochemical or functional properties on such peptides, (6) retain or improve antimicrobial activity. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0110] In some embodiments, 1, 2, 3, 4, or 5 amino acids of LSRRWHQGIRVLLRAA (SEQ ID NO: 1) are changed and the resultant variant peptide retains antimicrobial activity. The change may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and / or 16 of SEQ ID NO: 1. The change(s) may or may not be conservative. The change(s) may comprise substitution of one amino acid for another at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and / or 16 of SEQ ID NO: 1.[0U1] In some embodiments, 1, 2, 3, 4, or 5 amino acids ofGTKKVHVIFNYKGKNLLINKEIR (SEQ ID NO:2) are changed and the resultant variant peptide retains antimicrobial activity. The change may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and / or 23 of SEQ ID NO:2. The change(s) may or may not be conservative. The change(s) may comprise substitution of one amino acid for another at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and / or 23 of SEQ ID NO:2.

[0112] In some embodiments, 1, 2, 3, 4, or 5 amino acids ofLIRKLVGHIKGNRRNKHGHGGG (SEQ ID NO:3) are changed and the resultant variant peptide retains antimicrobial activity. The change may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 of SEQ ID NO:3. The change(s) may or may not be conservative. The change(s) may comprise substitution of one amino acid for another at positionI, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , and / or 22 of SEQ ID NO:3.

[0113] In some embodiments, 1, 2, 3, 4, or 5 amino acids ofRRRNMVCWLVMSRRRKCVSTYRSQ (SEQ ID NO:4) are changed and the resultant variant peptide retains antimicrobial activity. The change may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24 of SEQ ID NO: 4. The change(s) may or may not be conservative. The change(s) may comprise substitution of one amino acid for another at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24 of SEQ ID NO:4.3. Specific Examples of Modifications

[0114] In embodiments, at least one peptide is modified by the hand of man in at least one manner to improve at least one characteristic of the peptide compared to a peptide that lacks that modification(s). In some embodiments, one or more modifications to a peptide improves the solubility, potency, and / or stability of the peptide. Any peptide encompassed herein may bemodified in any manner and at any position. In specific embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of a peptide may be modified. Any peptide encompassed herein may be isolated and / or synthetic.

[0115] Strategies for enhancing solubility may include increasing hydrophilicity, optimizing charge distribution, and / or utilizing PEGylation and / or glycosylation. Increasing hydrophilicity may include introducing one or more polar amino acids (e.g, Ser, Thr, Asp, Glu) at any area of the peptide, including at the termini or within non-essential hydrophobic regions, as examples only. One may also increase hydrophilicity by reducing excessive hydrophobic clustering while maintaining amphipathicity. In specific embodiments, excessive hydrophobic clustering encompasses when a peptide is completely insoluble or precipitates out in an aqueous solution or environment. For optimizing charge distribution, one can maintain or increase overall positive charge (Lys, Arg) to enhance interaction with bacterial membranes. In addition, one can avoid excessive clustering of positively charged residues to prevent aggregation. In some embodiments, excessive clustering of positively charged residues occurs when a peptide has many basic amino acid residues that causes the peptide to self aggregate at higher concentrations. To prevent peptide aggregation, replacing basic amino acid residues (arginine, lysine, and histidine), which contribute to aggregation through electrostatic interactions and hydrogen bonding, with neutral or negatively charged residues (e.g., glutamine, asparagine, glutamate, or aspartate) can help reduce intermolecular interactions. Additionally, incorporating proline can disrupt secondary structure formation, further minimizing aggregation. In some examples, the number of excessive basic amino acids that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more basic amino acids. In some examples, the number of basic amino acids that is excessive is 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more basic amino acids for the peptide. In embodiments wherein the peptide is modified by PEGylation or glycosylation, the peptide may have polyethylene glycol (PEG) and / or small sugar moieties to increase water solubility. A "small sugar moiety" may refer to a single, simple sugar molecule, such as glucose, fructose, or galactose, and adding one or more of these increases solubility but not the complexity of the peptide.

[0116] .Strategies for increasing potency of a peptide, such as increasing antimicrobial potency of a peptide, one can enhance membrane interaction, incorporate unnatural amino acids, optimize length and / or flexibility, and / or multimerize and / or cyclization of the peptide. To enhance membrane interaction, one can increase a-helical content and amphipathicity by modifyinghydrophobic / hydrophilic ratios, and / or one can replace weakly interacting residues with Trp, Phe, or He to improve membrane penetration. To incorporate unnatural amino acids include using D- amino acids, non-natural residues, or both to improve selectivity and resist degradation. To optimize length and flexibility, one can include truncating non-essential regions to reduce steric hindrance while maintaining antimicrobial function and / or introducing hinge regions (Gly or Pro) if increased flexibility is beneficial. For multimerization or cyclization, one can design dimeric or cyclic structures to enhance target binding affinity and reduce degradation.

[0117] Strategies for improving stability include increasing resistance to proteolysis, improving structural integrity, and evaluating thermal and pH stability. One can increase resistance to proteolysis by substituting L-amino acids with D-amino acids, such as to prevent enzymatic degradation; introduce N-terminal acetylation or C-terminal amidation to protect from exopeptidases; and / or to modify the peptide for cyclization (head-to-tail or disulfide bonds), such as to shield from proteases. One can improve structural integrity by strengthening secondary structures using disulfide bonds, lactam bridges, or stapling and / or to replace labile amino acids (e.g., Met, Cys) with more stable analogs. One can evaluate thermal and pH stability by adjusting amino acid composition for optimal stability at physiological temperatures and pH.IV. Pharmaceutical Compositions

[0118] In some embodiments, pharmaceutical compositions comprising one or more peptides encompassed herein are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject. In some embodiments, one or more of the peptides may be administered to the subject to protect against or treat a microbial infection. Alternatively, an expression vector encoding one or more such peptides may be given to a subject as a preventative agent or as a treatment. Additionally, such compositions can be administered in combination with an additional therapeutic agent (e.g., an additional antimicrobial composition.). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium, in at least some aspects.

[0119] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0120] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

[0121] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0122] The peptide compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0123] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. 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. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0124] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the 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 techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0125] Administration of the compositions may typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0126] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations may easily be administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0127] Pharmaceutical compositions of the present disclosure comprise an effective amount of one or more peptides dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises at least one peptide will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21stEd. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0128] The compositions may comprise any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0129] The peptide(s) may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present peptide(s) can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0130] The peptide(s) may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, oraerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

[0131] Further in accordance with the present disclosure, the composition of the present peptide(s) suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent, or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fdlers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0132] In accordance with the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

[0133] In a specific embodiment of the present disclosure, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can also be added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0134] In further embodiments, the present disclosure may concern the use of a pharmaceutical lipid vehicle compositions that include peptide(s), one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0135] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the peptide(s) may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0136] The actual dosage amount of a composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0137] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the number of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0138] In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.A. Alimentary Compositions and Formulations

[0139] In preferred embodiments of the present invention, the peptide(s) are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0140] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0141] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

[0142] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments,suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.B. Parenteral Compositions and Formulations

[0143] In further embodiments, peptide(s) may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[0144] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may 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. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0145] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0146] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the 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 techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.C. Miscellaneous Pharmaceutical Compositions and Formulations

[0147] In some embodiments of the disclosure, the active compound peptide(s) may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and / or inhalation.

[0148] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion baseonly. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

[0149] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and / or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

[0150] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject’s age, weight and the severity and response of the symptoms.V. Compositions of the Disclosure

[0151] Embodiments of the disclosure include compositions comprising one or more peptides comprising the sequence of any one of SEQ ID NO: 1-SEQ ID NO:210. In some aspects, the peptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ IDN0:4. When the composition is a liquid (including fluid or spray), it may be soluble in an acceptable excipient, such as an aqueous solution. When the composition is a solid, it may be of any type of solid and any shape. In some embodiments, the solid is a pill, table, film, capsule, etc. The composition may be formulated as an antimicrobial composition, a therapeutic composition, an immunogenic composition, a vaccine composition, a cleaning composition, and so forth. An antimicrobial cleaning composition may be a surface cleaning composition or an environmental cleaning composition. Such a cleaning composition may be applied to a floor, countertop, table, chair, couch, bathtub, shower, toilet, sink, door, appliance, a combination thereof, and so forth. The surface may or may not be in a medical facility, a nursing home, a residence, a transportation entity (car, train, boat, bus, airplane, etc.) a theater, a hotel, a sporting venue, or a combination thereof. In some embodiments, the surface being cleaned by compositions of the disclosure is the skin and / or hair / fur of a mammal.

[0152] In some embodiments, the composition is formulated as a solid, or is on and / or in a solid substrate, such as a wipe, a cloth, a brush, a sponge, a mesh, or a combination thereof. The solid substrate is a medical device or is used for medicinal purposes, such as a bandage, mesh, tape, cloth, scaffold, or a combination thereof.

[0153] Examples of devices that may comprise the composition in and / or on the surface include at least a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain.

[0154] In certain embodiments, a composition comprising one or more peptides of the disclosure also includes another therapeutic agent, such as another antimicrobial, such as amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole and trimethoprim, amoxicillin and clavulanate, levofloxacin, or a combination thereof.

[0155] In particular embodiments, the peptides of the disclosure are effective against bacteria, viruses, molds, protozoa, protists, fungi, and so forth. In particular embodiments, the compositions are effective against Gram positive bacteria, Gram negative bacteria, or both.

[0156] Embodiments of the disclosure include antibiotic compositions, immunogenic compositions, vaccine compositions, disinfectant solutions, and so forth comprising an effectiveamount of any composition encompassed herein comprising one or more peptides of the disclosure.VI. Examples of Methods of the Disclosure

[0157] Embodiments of the disclosure encompass methods of using compositions comprising any one or more peptides comprising the sequence of SEQ ID NO: 1-SEQ ID NO:210, and use may be for any purpose, including for antimicrobial activity. In specific embodiments, there is a method of inhibiting the growth of a microbe in and / or on an individual, surface, and / or substrate, comprising the step of providing an effective amount of any composition encompassed herein to the individual, surface, and / or substrate, respectively. The inhibiting may be to slow growth of the microbe, to pause growth of the microbe, or to kill the microbe. In some embodiments, there are methods of treating an individual for a microbial infection comprising administering an effective amount of a peptide composition encompassed herein.

[0158] The microbe is pathogenic, in particular embodiments. The microbe may be a bacteria, virus, fungus, protist, or protozoa. In specific aspects, the individual receiving the compositions has one or more deleterious symptoms from the microbe. The individual may have a microbe infection but may be asymptomatic, in some cases. An individual being administered an effective amount of the peptide composition may have an internal infection of the microbe and / or a topical infection of the microbe. In specific embodiments, the composition is provided by injection, by inhalation, and / or topically to the individual.

[0159] In particular embodiments, when an individual is administered the peptide composition, the composition may be administered once or more than once. In cases wherein the administration is more than once, the administration may be 1, 2, 3, or 4 times a day; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 times a week, once a month, twice a month, and so forth. When there are multiple administrations, they may or may not be by the same administration route. An individual may receive different peptides for the same microbial infection or may receive only one peptide for the same microbial infection. Some peptides may have a specific use, such as a specific type of infection (e.g., bacterial vs. viral). Within a type of infection, such as bacterial, one peptide may be used for one type of bacterial infection and another peptide may be used for another bacterial infection. Some peptides may be used for certain microbe genera.

[0160] In some embodiments, there are methods of disinfecting an environment, comprising providing an effective amount of a peptide composition encompassed herein to the environment. The environment may be terra firma or may be a water source, such as an ocean, lake, river, stream, estuary, creek, pond, water well, water tank, irrigation, or a combination thereof. In particular embodiments there are methods of disinfecting domestic animal fecal effluents, including prior to entering a water source. In some embodiments, there are methods of disinfecting a wastewater facility or a food production facility. The environment may or may not be a commercial agricultural farm.

[0161] Examples of terra firma that may be treated with one or more compositions of the present disclosure include any soil, land, ground, earth, dirt, dust, continent, island, zone, peninsula, mainland, landmass, isthmus, etc.

[0162] Examples of environments or areas of any kind that may be considered to be contaminated by one or more microbes and may be treated with one or more compositions of the disclosure include any facility or activity that stores, uses, or produces contaminants of concern that could find their way into a source of drinking water is a source of contamination, such as a source water protection area. Examples of contaminant sources include waste landfills, lagoons, Brownfields sites, Superfund sites, chemical storage facilities (such as oil and gas storage facilities or underground storage tanks), industrial and municipal wastewater outfalls (such as those addressed by the National Pollutant Discharge Elimination System (NPDES) permit program), mining operations, agriculture and other types of nonpoint sources of pollution, residential and / or commercial septic systems, stormwater runoff, such as from streets and lawns (and other nonpoint sources in urban areas), industrial facilities that use or store chemicals (such as tanneries, automotive body shops, dry cleaners, and others, such as those reported to the Toxic Release Inventory (TRI) program), underground injection wells, roads and hazardous materials transportation routes, and so forth.VII. Examples of Kits

[0163] Any of the antimicrobial compositions described herein may be comprised in a kit. In a non-limiting example, one or more peptides may be comprised in a kit. The kits may comprise suitably ali quoted one or more peptides. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits may generally include atleast one vial, test tube, flask, bottle, syringe or other container means, into which one or more peptides may be placed, and at least in some cases, suitably aliquoted. Where there are more than one component in the kit, the kit also may generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also may typically include a means for containing the one or more peptides and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vessels are retained.

[0164] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution may be an aqueous solution, including a sterile aqueous solution. The compositions may also be formulated into a syringeable composition. In some cases, the container means may itself be a syringe, pipette, and / or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and / or even applied to and / or mixed with the other components of the kit.

[0165] However, the components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0166] In some embodiments, the kit comprises an object that comprises one or more peptides of the disclosure. In some embodiments, the kit comprises an object intended for one or more peptides to be placed on and / or in, and in such cases the kit may also comprise composition(s) that comprise one or more of the peptides. The composition(s) may be applied to and / or in the object prior to use.

[0167] In some embodiments, the kit comprises a medical device, comprising an effective amount of a composition comprising one or more peptides, or the composition may be applied to and / or in the device later, prior to use. The device may be a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain.

[0168] In some embodiments, the kit comprises a medical substrate, comprising an effective amount of any composition encompassed herein. The kit may comprise the medical substratealready comprising the composition, or the composition may be separate from the medical substrate and applied later, prior to use. In some embodiments, the substrate is a bandage, mesh, tape, cloth, scaffold, or a combination thereof.VIII. Examples

[0169] The following examples are included to demonstrate certain aspects or embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects or embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the subject matter of the disclosure.EXAMPLE 1NOVEL ANTIMICROBIAL PEPTIDES IN TICK ATTACHMENT GLUE AND TICK IMMUNOMODULATOR PROTEINS

[0170] MATERIALS AND METHODS

[0171] In silico identification and synthesis of tick antimicrobial peptides.

[0172] In silico identification of candidate AMPs was done using three publicly available servers for selecting candidate AMPS. Briefly, Antimicrobial Peptide Scanner Vrl, a deep-neural network algorithm-based server (https: / / www.dveltri.com / ascan / vl / about.html) was used to scan and identify putative AMP segments in native peptides that are likely effective against Gramnegative (E. coll) or Gram-positive (5. aureus) bacteria.

[0024] The predicted segments were then compared with three other servers: 1) Antimicrobial Peptide Scanner Vr2 server that scans peptides to Anti-microbial peptide database (APD3)

[0025] , 2) AxPEP - a sequence-based machine learning algorithm (https: / / cbbio.online / AxPEP / )

[0026] and 3)AI4AMP - an Artificial intelligence (Al) based AMP prediction tool (https: / / axp.iis.sinica.edu.tw / AI4AMP / ) to further validate antimicrobial activity and safety (hemolytic activity) of all predicted AMP candidates

[0027] , Theinventors chose candidate sequences if they had a positive match with all three AMP prediction servers.

[0173] Defining the Minimal Inhibitory Concentration of synthetic antimicrobial peptides.

[0174] The inventors initially defined the Minimal inhibitory Concentration (MIC) of each of the synthetic AMPs (Tables 1A-1E) using the broth microdilution test according to CLSI recommendations

[0028] , The MIC assay is the gold standard assay that defines in vitro levels of susceptibility of bacteria to antibiotics and is used to make treatment decisions

[0013] , Briefly, overnight culture of bacteria was diluted in normal saline (0.9% sodium chloride) to make 5xl03cfu / mL to make the bacteria cell suspension. The antibiotic plate was prepared by adding 195 pl of media and 5 pl of peptide (from 40 mg / ml stock) to the first well of 96 well plate. Then, the peptides were serially diluted till 10th well (from 500 to 0.9 pg / ml) and rest of the 2 wells were left for blank media and cells only control (considered as 100% growth) respectively. 100 pl of prepared cell suspension was added to each well (accept blank). Ampicillin and kanamycin were included as an antimicrobial positive control. Plate was sealed with the parafilm to prevent it from drying and incubated for 24 h at 37°C. The MIC was determined by determining the optical density at A600nm on a microplate reader (Biotek, USA), and the percent inhibition was calculated by using formula: % Inhibition = (Average of OD600 of peptide treated well / Average of OD600 of cells only well) x 100.

[0175] All tests were performed multiple times in triplicates to confirm the authenticity of the data. The MIC of each of the 21 antimicrobial peptides was initially assayed against the laboratory Escherichia coli strain (DH5 alpha) and Staphylococcus aureus (SAI 16) followed by human and veterinary clinical isolates listed below.

[0176] Human and veterinary clinical isolates that were screened.1. Escherichia coli CFT073: mesophilic human pathogen that was isolated from Human clinical specimen - blood and urine from a woman with acute pyelonephritis. Causes urinary tract infections.2. Staphylococcus aureus (isolate SA-116) is a Methillin-resistant Staphylococcus aureus is difficult to treat because it is resistant to many drugs.3. Acinetobacter baumannii (isolate 17978). This strain was originally isolated from a 4 month old infant with fatal meningitis in France in 1951.Kanamycin resistant Escherichia coli (isolate CFT073 Al). This strain was orinally isolated from blood and urine of a urosepsis (sepsis that originates from urinary tract infection) woman patient. This strain is resistant against killing by Kanamycin antibiotic. Escherichia coli (0157 strain isolate EDL-933). This strain was isolated from ground beef in 1982 during the enterohemorrhagic Escherichia coli outbreak in the United States. Escherichia coli (0157 strain isolate EDL-931). This strain was first isolated from feces of patients suffering from outbreak of gastrointestinal illness in Oregon in 1982. Escherichia coli (isolate UTI89) is , a strain of uropathogenic E. coli recovered from a human patient with cystitis. Escherichia coli (isolate ABU83972) is asymptomatic bacteriuria (ABU) strain of E. coli, originally isolated from the girl having it from 3 years without symptoms. Escherichia coli 042 is a multidrug resistant bacteria that was isolated from a child with diarrhea. Escherichia coli (veterinary isolate 23109007-1) is an extended spectrum beta-lactamase (ESBL) positive bacteria that is resistant against most known antibiotic drugs. Escherichia coli (veterinary isolate 23117003-1) is an extended spectrum beta-lactamase (ESBL) positive bacteria that is resistant against most known antibiotic drugs. Escherichia coli (veterinary isolate 23111007-1) is an extended spectrum beta-lactamase positive. This strain is resistant to any known antibiotics. Escherichia coli (veterinary isolate 23121002-1) is an extended spectrum beta-lactamase negative bacteria that is resistant against most known antibiotic drugs. Escherichia coli (veterinary isolate 23121004-1) is an extended spectrum beta-lactamase negative bacteria that is resistant against most known antibiotic drugs. Salmonella enterica (ATCC 14028). This strain was isolated from pooled heartand liver tissue of four weeks old chicken and is commonly associated with enteric disease Salmonella enterica (ATCC 13312). This strain is a serotype of Choleraesuis strain NCTC 5735. It is a whole genome sequenced strain used for control in enteric disease research, infectious disease research and zoonotic diseases research. Salmonella enterica (ATCC 15480). This strain causes enteric diseases and used in vaccine production.18. Salmonella enterica (ATCC 13076). It is whole genome sequenced strain used as control in enteric disease research, infectious disease research, zoonotic disease research and media testing.19. Staphylococcus aureus (ATCC29213) was isolated from a wound.20. Staphylococcus aureus (ATCC25953). It is awhole geome sequenced clinincal isolate and is used as control species for infectious disease testings.21. Staphylococcus aureus (ATCC BAA-977). This strain was clinically isolated from wound infection in 2003 and is used as a positive control for clyndamycin resistant tests.22. Staphylococcus aureus (ATCC BAA-976). This strain was clinically isolated from tracial aspirate in 2003 and is used as a negative control for clyndamycin resistant tests.23. Pseudomonas aeruginosa (ATCC27853). This is a whole genome sequenced strain isolated from blood culture, it is used as a reference strain in susceptibility testing and infectious diseases research.24. Pseudomonas aeruginosa (116-50). This strain is a clinical isolate abtained from the dog wound.25. Pseudomonas aeruginosa (118-09). Phenotypically different isolate from the ear infection of the same dog from which 116-50 strain was obtained.26. Pseudomonas aeruginosa (118-10). Phenotypically different isolate from the ear infection of the same dog from which 116-50 strain was obtained.

[0177] Evaluating if CM1-95 antimicrobial peptide has bactericidal effects.

[0178] To confirm the MIC readouts, the inventors also determined the Minimum Bactericidal Concentration (MBC) as described

[0029] , The MBC test was to confirm if CM1-95 has static (stunted growth) or lytic (kill cells) effects against bacteria cells. In the MBC assay, aliquots (2 pL) were spotted of the MIC test samples on the agar plates. The expectation was that if the CM1- 95 had bacteriostatic effects one can visualize the revival of bacterial growth and if CM1-95 has bactericidal activity, one would not see bacteria growth. This assay was conducted for 7 strains and its ongoing.

[0179] Mechanisms by which CM1-95 antimicrobial peptide kills bacteria.

[0180] Drug efflux is one of the principal mechanism microbes exploit to acquire multidrug resistance (MDR) properties. The efflux pump proteins are the dominant cause of drug efflux thatare overexpressed in the resistant bacteria and disconnect the drug to its target by throwing it out of the bacterial system. Bacterial efflux pumps can be classified into major 5 groups that are important because of the clinically relevant with respect to antibiotic resistance. The important efflux pumps family are Major facilitator superfamily (MFS), small MDR (SMR) family, multidrug and toxic compound extrusion (MATE) family, ATP binding cassette (ABC), and the resistant nodulation cell division (RND)

[0030] ,

[0181] To gauge insights into mechanisms of the CM1-95 antimicrobial activity, the effects of the CM1-95 antimicrobial peptide were assessed on the RND pump using the Nile red substrate as described [31, 32], In this method, the RND efflux pump of an overnight culture was blocked using 2,4 Dinitrophenol (2,4DNP; disruption of ATP), carbonyl cyanide 3 -chlorophenylhydrazone (CCCP, an ATP disruptor), and 2-deoxy-d-glucose (competitive inhibitor for glucose). These cells were then loaded with Nile red, which in absence of the RND pump accumulated in the cells. Subsequently, the RND efflux pump of cells treated with or without 125 micrograms of CM1-95 was turned on by adding glucose. Subsequently, the fluorescence emission of the Nile red substrate that was retained in cells was measured for 200 sec with excitation at 552 nm, and emission at 636 nm on plate reader. The expectation was that if the RND efflux pump is active, the inventors expected low to no fluorescence emission, as much of the substrate will be pumped out, and vice versa if the RND pump was blocked (protocol outlined elsewhere herein).

[0182] Effect of CM1-95 antimicrobial peptide on bacteria in feces of mice and cattle. Prompted by observations that the CM 1-95 antimicrobial peptide was effective against Enterobacteriaceae bacteria that colonize the gastrointestinal tract

[0033] , we sought to investigate the effect of this peptide on bacteria colonies in cattle and mice feces. Freshly collected feces of cattle or mice were suspended in sterile phosphate buffered saline. Subsequently, 100 microliters of the fecal extract were inoculated into 1 mL Lauria broth media with and without 125 micrograms of the CM1-95 antimicrobial peptide. The culture was then incubated at 37°C with shaking for 4 h. Samples (100 microliters) were plated onto plate Lauria Broth agar plates, incubated overnight and colony forming units were manually using a counter.

[0183] Safety evaluation of the CM1-95 antimicrobial peptide.

[0184] a) Hemolytic activity.

[0185] To gauge insights into safety of the CM1-95 antimicrobial peptide, hemolytic effects were assayed against purified cattle red blood cells (RBC). Cattle blood was collected inVACUETTE® TUBE 9 ml K3E K3EDTA (Greiner Bio-One North America Tnc., USA) to avoid coagulation and was kept at 4°C and was used within 12h to avoid excessive cell lysis. The noncoagulated blood was centrifuged at 1500xg for 5 min and the level of plasma fraction was marked on the tube. Subsequently, plasma was discarded and an equal volume of normal saline (0.9% NaCl) was added till the marked line and mixed by inversion. Same steps were used 5 times to wash the blood cells and after the last washing equal amount of Phosphate buffer saline (PBS) was added and the resulting solution was diluted 1 :50 with PBS, this blood PBS mixture was used for the assay. The assay was performed in 96 well plate (Corning Incorporated, USA) where 10 pL of CM1-95 was added in triplicate to make final concentration (in 200pl) of 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25pg / ml, 15.625pg / ml and 7.8pg / ml. For positive control, 2% triton X was used and PBS was used as negative control. Diluted RBCs (190 pL) was added to the wells and the plates were incubated for 1 h at room temperature. The plate was centrifuged at 1500xg for 5 min and the supernatant was carefully transferred in the clear well. To evaluate RBC lysis, optical density of the supernatant was measured at wavelength of 450 nm. Percent (%) hemolytic activity was calculated by using formula as follows. Percent (%) hemolysis = (absorbance of test sample - absorbance of diluent) / (absorbance of positive control -absorbance of diluent) xlOO.

[0186] b) Cytotoxicity assays.

[0187] Cytotoxicity of the CM1-95 antimicrobial peptide was tested against 3 immortalized cell lines including, Chinese hamster ovary (CHO) cell line, LnCAP cell line derived from a metastatic lymph node lesion of human prostate cancer, and Primary Prostate Epithelial Cells; Normal, Human (HPrEC). The cells were trypsin-EDTA harvested and suspended in the respective media. LNCap was harvested in RPMI media, CHO was harvested in DMEM and HPrEC was harvested in prostate epithelial cell growth media (PrECGM). Then 100 pL of cells were seeded on 96 well tissue culture plate (Genesee Scientific, USA) at the cell density of IxlO5cells / mL. Cells were allowed to adhere and grow for 24 h at 37°C with 4% CO2 in humidified incubator. After 24 h, the culture media was replaced by fresh media and the CM1-95 antimicrobial peptide was added to the medium at different concentration (500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25pg / ml, 15.625pg / ml and 7.8pg / ml) and the cells were allowed to grow for 24 h. Next day, the media was removed and replaced with media without fetal bovine serum (FBS) with added 1 mg / mL tetrazolium salt 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT). The plate was incubated for 3 h at 37°C and then 200 pL of DMSO was added to eachwell to stop the reaction. To solubilize MTT formazan product (purple blue color) the plate was shaken for 10 min and the OD intensity was measured at 450 nm. The study was repeated multiple times in triplicate to measure the consistency of the data.

[0188] In silica scanners identify antimicrobial peptides (AMP) in tick proteins.

[0189] The inventors successfully utilized web AMP scanners to identify putative AMP sequences in tick attachment glue and immuno-modulatory proteins. The AMP sequences were identified in proteins that form attachment glue of blacklegged tick (Ixodes scapularis, n = 49, Table 1A) and Lone Star ticks (Amblyomma americanum, n = 34, Table IB). Likewise, the inventors identified putative AMP sequences in immuno-modulatory proteins of uninfected and Lyme disease agent infected blacklegged ticks nymphs (n = 126, Table 1C), Uninfected adult blacklegged (n = 20, Table ID), and uninfected adult Lone Star ticks (n = 61, Table IE). These comprised AMPs that were identified positively by all the servers and were a final list of AMP candidates for in vitro testing for activity against veterinary and human clinical isolates. In this study, the antibiotic activity of 21 synthetic peptides that were predicted with 95-100% confidence was synthesized and screened.

[0190] Five of the 21 synthetic peptides have antimicrobial activity as revealed by MIC.

[0191] The inventors successfully utilized the broth microdilution test to screen MIC and detect the antimicrobial activity of 21 synthetic peptides. This analysis revealed that at least 5 (CM1-95, CRT-I, CRT-A, CM4, and TCI-Asiaticum) of the 21 peptides were active and showed antimicrobial activity (Table 2). Of these 5 antimicrobial peptides, in particular embodiments the CM1-95 (LSRRWHQGIRVLLRAA; SEQ ID NO: 1) is the most potent (Table 2A). The CM1-95 antimicrobial peptide was effective against 16 different bacteria strains. At MIC of 15.2 micrograms / mL, the CM1-95 antimicrobial peptide cleared Acinetobacter baumannii (strain 17978; 80% clearance). Similarly, at MIC of 62.5 micrograms / mL, the CM1-95 antimicrobial peptide cleared the non-pathogenic laboratory E. coli strain DH5 alpha (more than 90%) and human clinical isolates: pathogenic A. coli (strain CFT073; more than 70%), and the 0157 E. coli (strain EDL931 more than 99%). Likewise, at MIC of 125 micrograms / mL, the CM1-95 peptide cleared more than 90% of Kanamycin-resistant E. coli, the 0157 E. coli (EDL933) by more than 99%, E. coli (veterinary isolate 121002-1) by more than 85%, and Salmonella enterica (ATCC13076) by more than 90%. Likewise, at MIC of 500 micrograms / mL, the CM1-95 antimicrobial peptides cleared the extended spectrum beta-lactamase (ESBL) positive E. coliveterinary isolates 1 17003-1 and 231109007-1 by more than 90%, E.coli human isolates UTI89 and ABU83972 by more than 90% and Pseudomonas aeruginosa, ATCC27853, ATCC116-50, and ATCC 11809 by 70-78% (Table 2A).

[0192] The other 4 effective peptides included CRT-I (GTKKVHVIFNYKGKNLLINKEIR; SEQ ID NO:2), which at MIC of 125 micrograms / mL cleared more than 91% of the laboratory E. coli strain as well as more than 58 and 77% of Staphylococcus aureus (SAI 16) and Acinetobactor baumannii (17978) at MIC of 500 micrograms / mL (Table 2B). Similarly, the CM4 (LIRKLVGHIKGNRRNKHGHGGG; SEQ ID NO:3) respectively cleared more than 43% and 38% of pathogenic E. coli (strain CFT073) and Staphylococcus aureus (SAI 16) at MIC of 250 and 500 micrograms / mL respectively (Table 2C). The fourth active antimicrobial peptide, TCI- asiat (RRRNMVCWLVMSRRRKCVSTYRSQ; SEQ ID NO:4) cleared 60% of pathogenic E. coli (strain CFT073) and Acinetobactor baumannii at MIC of 125 and 250 micrograms / mL respectively (Table 2D). Lastly, at MIC of 125 micrograms / mL, the CRT-A antimicrobial peptide cleared more than 56% of the laboratory E. coli strain (not yet tested against other strains).

[0193] TABLES 2A-2D: ANTIMICROBIAL ACTIVITY OF EXAMPLES OF SYNTHETIC PEPTIDES

[0194] Table 2A: Antimicrobial activity of CM1-95 peptide.Table 2B: Antimicrobial activity of CRT-I peptide.Table 2C : Antimicrobial activity of CM4 peptide.Table 2D: Antimicrobial activity of TCI-Asiaticum peptides.The CM1-95 antimicrobial peptide has bactericidal activity.

[0195] The data (FIG. 1) show that the CM1-95 antimicrobial peptide has bactericidal (or causes lysis of bacteria) activity against five of the 16 strains that are susceptible to CM1-95 (Table 2A). As shown (FIG. 1), bacteria did not grow when an aliquot (2 pL) of an overnight bacteriaculture treated with the CM 1 -95 antimicrobial peptide was spotted onto LB agar plates. The CM 1 - 95 antimicrobial peptide bacteriolytic activity was observed at MIC of 15.2 pg / mL for Acinetobactor baumannii, at MIC of 62.5 pg / mL against pathogenic E. coli (strain CFT073) and 0157 E. coli (strain EDL931), at MIC of 125 pg / mL against 0157 E. coli (strain EDL933), and at MIC of 250 pg / mL against Kanamycin resistant E. coli. As expected, no bactericidal activity was observed against CMl-95-resistant bacteria; Staphylococcus aureus and E. coli strain 042 (FIG.1)The CM1-95 antimicrobial peptide acts by blocking the efflux pump.

[0196] The data (FIG. 2) show that the CM1-95 antimicrobial peptide may exert its function by disrupting (or blocking) the bacteria’s mechanism to expel toxicants (or xenobiotics). In the RND efflux (or pump) test, it was expected that turning on of the RND pump would have resulted in depleted fluorescence in cells. However, as shown (FIGS. 2A and 2B), the CM1-95 peptide disrupted the ability of Kanamycin-resistant pathogenic E. coli (CFT073 Al) and non-resistant pathogenic E. coli (CFT073) to pump out the Nile red substrate as revealed by higher fluorescence intensity in bacteria with activated efflux pumps that was treated (Green triangle line graph in FIGS. 2A and 2B) with the CM1-95 antimicrobial peptide (125 pg / mL) compared to bacteria with activated efflux pumps but were not treated (blue circle line graph in FIGS. 2A and 2B) with the CM1-95 peptide. Data in FIG. 2C suggest that the CM1-95 antimicrobial peptide might have caused pores in the cell wall of Acinetobacter baumannii . It was observed that fluorescence intensity was relatively similar in bacteria with the activated efflux pump in presence or absence of the CM1-95 antimicrobial peptide. This observation suggested that the substrate was diffusing out of the Acinetobacter baumannii which suggests the high possibility of poration and leaking of Nile red substrate from the cells, in certain aspects (FIG. 2C).The CM1-95 antimicrobial peptide kills bacterial colonies in cattle and mice feces.

[0197] The data revealed that colony forming units in feces of cattle or mice were reduced by more than 50% when incubated with the CM1-95 antimicrobial peptide for 4 hours at 37°C (FIGS. 3A, 3B, 3C, and 3D). Food animal fecal contamination of the food chain is the major source of food borne infections such as 0157 E. coli and Salmonella. Thus, in particular aspects the CM1- 95 peptide is used to decontaminate domestic animal fecal waste before disposal.The CM 1-95 antimicrobial peptide is safe.

[0198] The results show that at MIC of 500 pg / mL, the CM1-95 peptide did not show hemolytic activity. More than 90% of viable RBCs were observed in the presence of 500 pg / mL of the CM1-95 peptide compared to 2% tritonX treatment (positive control). Similarly, the CM1- 95 peptide did not show cytotoxicity against the three cell lines. There was 100% survival of HPrEC, 95% of LnCAP, and 80% survival of CHO cell lines (FIG. 4). These data show that the CM 1-95 peptide is not toxic under in-vitro and ex-vivo conditions.

[0199] This study has successfully utilized in silico methods to identify antimicrobial peptides in proteins that form the tick attachment glue and tick immuno-modulatory proteins (n=290). These antimicrobial peptides represent a useful resource for discovery of novel therapeutics of drug resistant microbial infection of humans and veterinary patients, in specific aspects.EXAMPLE 2PEPTIDES AS DECONTAMINATION AGENTS FOR ANIMAL WASTE

[0200] In specific embodiments, any one or more of the antimicrobial peptides encompassed herein may be used as a pass-through decontaminating drug or chemical agent to reduce diseasecausing bacterial load in animal waste, such as in cattle fecal droppings. Any of the peptides encompassed herein may be encapsulated in any suitable material to protect the active ingredient during digestion in the stomach and intestines of the animal. In specific embodiments, the peptide(s) is encapsulated in an acid-resistant material (pH sensitive material) that will not degrade at the neutral to acidic pH ranges of the gastro-intestinal tract but will degrade at the neutral to alkaline pH range expected for livestock feces (e.g. polylactic acid (PLA) and polyglycolic acid (PGA). The encapsulation will prevent the release of the antimicrobial peptide into the gastrointestinal tract (stomach, small intestine, and large intestine). In specific embodiments, this encapsulating material is designed to dissolve upon exposure to conditions specific to feces upon defecation. Upon degradation of the encapsulation material, the antimicrobial peptide will be active in the feces. When released, the peptide will kill the microbes in the animal feces. This embodiment mitigates the environmental and public health risks associated with bacterial contamination from animal waste by targeting and neutralizing pathogens effectively in situ.

[0201] In specific embodiments, the CM1-95 antimicrobial peptide (as one example) is used as a pass-through decontaminating drug or chemical agent to reduce disease-causing bacterial loadin animal fecal droppings, such as in cattle fecal droppings. The CM1-95 is encapsulated in an acid-resistant material designed to protect the active ingredient during digestion in the stomach and intestines. This encapsulating material will be designed to degrade upon being shed by the cattle, releasing the CM1-95 antimicrobial peptide directly into the feces in the environment. When released, the CM1-95 peptide at an effective dosage, such as ranging from 125-500 micrograms per milliliter, has been shown to kill more than 50% of colony-forming bacterial units (CFUs) in cattle feces. The range of concentration of any peptide may be 125-500, 125-400 125-300, 125- 200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 micrograms per milliliter.

[0202] This approach mitigates the environmental and public health risks associated with bacterial contamination from cattle waste by targeting and neutralizing pathogens effectively in situ.EXAMPLE 3PEPTIDES FOR INSECT PATHOGENS

[0203] In some embodiments, any one or more of the antimicrobial peptides encompassed herein are utilized as an additive to edible insecticides, such as for targeting insects (cockroaches and / or filth flies, such as the common house fly) in environments where animals (including humans) are present. As one example, the antimicrobial peptide(s) are utilized as an additive to edible insecticides targeting household insects such as cockroaches and houseflies. Upon ingestion by the insects, the antimicrobial peptide(s) are released in the insect midguts, where it will effectively kill pathogens such as bacteria present in their digestive systems. By eliminating these bacteria, the peptide prevents insects from shedding and spreading pathogenic microorganisms into the environment. This reduces the insect spreading of disease-causing bacteria to humans and animals through contaminated surfaces, food, and / or water.

[0204] In specific embodiments, the CM1-95 antimicrobial peptide (as one example) is utilized as an additive to edible insecticides, such as for targeting insects in environments where animals (including humans) are present. As one example, the CM1-95 antimicrobial peptide is utilized as an additive to edible insecticides targeting household insects such as cockroaches and houseflies, targeting household insects such as cockroaches and houseflies. Upon ingestion, the CM1-95 antimicrobial peptide is released in the insect midguts, where it will effectively killbacteria present in their digestive systems. By eliminating these bacteria, the peptide will prevent insects from shedding and spreading pathogenic microorganisms into the environment. This application holds significant potential to reduce the insect spreading of disease-causing bacteria to humans and animals through contaminated surfaces, food, and water.* * *

[0205] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. De La Fuente J, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: Ticks as vectors of pathogens that cause disease in humans and animals. Frontiers in Bioscience. 2008. doi: 10.2741 / 32002. Rosenberg R, Lindsey NP, Fischer M, Gregory CJ, Hinckley AF, Mead PS, et al. Vital Signs: Trends in Reported Vectorborne Disease Cases - United States and Territories, 2004- 2016. MMWR Morb Mortal Wkly Rep. 2018. doi: 10.15585 / mmwr.mm6717el3. Cupp EW. Biology of ticks. The Veterinary clinics of North America. Small animal practice. 1991. doi: 10.1016 / S0195-5616(91)50001-24. Tirloni L, Kim TK, Pinto AFM, Yates JR, da Silva Vaz I, Mulenga A. Tick-host range adaptation: Changes in protein profiles in unfed adult Ixodes scapularis and Amblyomma americanum saliva stimulated to feed on different hosts. Front Cell Infect Microbiol. 2017. doi: 10.3389 / fcimb.2017.005175. Lewis LA, Radulovic ZM, Kim TK, Porter LM, Mulenga A. Identification of 24h Ixodes scapularis immunogenic tick saliva proteins. Ticks Tick Borne Dis. 2015. doi : 10.1016 / j .ttbdis.2015.03.0126. Kim TK, Tirloni L, Bencosme-Cuevas E, Kim TH, Diedrich JK, Yates JR, et al. Borrelia burgdorferi infection modifies protein content in saliva of Ixodes scapularis nymphs. BMC Genomics. 2021. doi: 10.1186 / sl2864-021-07429-07. Kim TK, Tirloni L, Pinto AFM, Moresco J, Yates JR, da Silva Vaz I, et al. Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding. PLoS Negl Trop Dis. 2016. doi:10.1371 / journal.pntd.00043238. Hollmann T, Kim TK, Tirloni L, Radulovic ZM, Pinto AFM, Diedrich JK, et al. Identification and characterization of proteins in the Amblyomma americanum tick cement cone. Int J ParasitoL 2018. doi : 10.1016 / j .ijpara.2017.08.0189. Radulovic ZM, Kim TK, Porter LM, Sze SH, Lewis L, Mulenga A. A 24-48 h fed Amblyomma americanum tick saliva immuno-proteome. BMC Genomics. 2014. doi: 10.1186 / 1471-2164-15-51810. Kim TK, Tirloni L, Pinto AFM, Diedrich JK, Moresco JJ, Yates JR, et al. Time-resolved proteomic profile of Amblyomma americanum tick saliva during feeding. PLoS Negl Trop Dis. 2020. doi: 10.1371 / journal.pntd.000775811. Moorhouse DE. The attachment of some ixodid ticks to their natural hosts. Proc 2nd Int Congr Acarol. 1967; 319-327.12. Skames RC, Watson DW. Antimicrobial Factors of Normal Tissues and Fluids. Bacteriol Rev. 1957;21: 273-294. doi: 10.1128 / br.21.4.273-294.195713. Steiner H, Hultmark D, Engstrom A, Benni ch H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981. doi: 10.1038 / 292246a014. Ganz T, Selsted ME, Lehrer RI. Defensins. European Journal of Haematology. 1990. doi : 10.11 11 / j .1600-0609.1990.tb00339.x15. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci U S A. 1987. doi: 10.1073 / pnas.84.15.544916. Brogden KA. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology. 2005. doi: 10.1038 / nrmicro 109817. Lai Y, Gallo RL. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends in Immunology. 2009. doi: 10.1016 / j .it.2008.12.00318. Uttenweiler-Joseph S, Moniatte M, Lagueux M, Van Dorsselaer A, Hoffmann JA, Bulet P. 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Claims

WHAT IS CLAIMED IS:

1. A composition comprising one or more peptides comprising the sequence of any one of SEQ ID NO:1-SEQ ID NO:210, or comprising a sequence that is at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto, respectively.

2. The composition of claim 1, wherein the peptide comprises the sequence of SEQ ID NO:1.

3. The composition of claim 1, wherein the peptide comprises the sequence of SEQ ID NO:2.

4. The composition of claim 1, wherein the peptide comprises the sequence of SEQ ID NO:3.

5. The composition of claim 1, wherein the peptide comprises the sequence of SEQ ID NO:4.

6. The composition of any one of claims 1-5, wherein the composition comprises peptides comprising the sequence of one or more of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

7. The composition of any one of claims 1-6, wherein the composition further comprises a pharmaceutically acceptable carrier.

8. The composition of any one of claims 1-6, wherein the composition is formulated as an antimicrobial composition.

9. The composition of claim 8, wherein the composition is formulated as a therapeutic composition.

10. The composition of claim 8, wherein the composition is formulated as an antimicrobial cleaning composition.

11. The composition of claim 8, wherein the composition is formulated as an edible composition.

12. The composition of claim 11, wherein the antimicrobial cleaning composition is a surface cleaning composition or an environmental cleaning composition.

13. The composition of claim 12, wherein the surface is a floor, countertop, table, chair, couch, bathtub, shower, toilet, sink, door, appliance, or a combination thereof.

14. The composition of claim 12 or 13, wherein the surface is in an enclosed environment.

15. The composition of claim 14, wherein the enclosed environment is an office building, a school, a university, a residence hall, a hospital, a library, a childcare center, a gym, a medical facility, a nursing home, a residence, a transportation entity, a theater, a hotel, a sporting venue, or a combination thereof.

16. The composition of claim 12, wherein the surface is the skin and / or hair of a mammal.

17. The composition of any one of the preceding claims, wherein the composition is formulated as a solid, or is on and / or in a solid substrate.

18. The composition of claim 17, wherein the solid substrate is a wipe, a cloth, a brush, a sponge, a mesh, or a combination thereof19. The composition of claim 17 or 18, wherein the solid substrate is a medical device or is used for medicinal purposes.

20. The composition of claim 19, wherein the device is a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain; and / or wherein the solid substrate is a bandage.

21. The composition of any one of claims 1-16, wherein the composition is formulated as a liquid or spray.

22. The composition of any one of the preceding claims, further comprising an additional therapeutic agent.

23. The composition of claim 22, wherein the additional therapeutic agent is antimicrobial.

24. The composition of any one of the preceding claims, further comprising one or more metal cofactors.

25. The composition of claim 24, wherein the metal cofactor is Mg2+, Ca2+, Fe2+, Cu2+, Zn2+, or a combination thereof.

26. The composition of any one of claims 1-25, wherein the peptide has one or more modifications by the hand of man.

27. The composition of claim 26, wherein the one or more modifications enhance solubility, increase antimicrobial potency, and / or improve stability.

28. An antibiotic composition, comprising an effective amount of the composition of any one of claims 1-27.

29. An immunogenic composition, comprising an effective amount of the composition of any one of claims 1-28.

30. A vaccine composition, comprising an effective amount of the composition of any one of claims 1-29.

31. A composition comprising disinfectant solution, comprising an effective amount of the composition of any one of claims 1-28.

32. A medical device, comprising an effective amount of the composition of any one of claims 1- 27.

33. The device of claim 32, wherein the device is a catheter, surgical mesh, drive line, syringe, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain.

34. The device of claim 32 or 33, wherein the composition is on the surface and / or in the device.

35. The device of any one of claims 32-34, wherein the composition may elute from the device.

36. A medical substrate, comprising an effective amount of the composition of any one of claims 1-31.

37. The medical substrate of claim 34, wherein the substrate is personal protective equipment, clothing, collection swab or collection device, a gel, a lotion, a bandage, mesh, tape, cloth, scaffold, or a combination thereof.

38. The medical substrate of claim 35, wherein the personal protective equipment or clothing is a glove, gown, mask, face shield, or a combination thereof.

39. A method of inhibiting the growth of a microbe in and / or on an individual, surface, and / or substrate, comprising the step of providing an effective amount of the composition of any one of claims 1-31 to the individual, surface, and / or substrate, respectively.

40. The method of claim 39, wherein the microbe is pathogenic, a contaminant, or the microbe has one or more deleterious symptoms for an individual.

41. The method of claim 39 or 40, wherein the microbe is a bacteria, virus, fungus, or protozoa.

42. The method of any one of claims 39-41, wherein the individual has one or more deleterious symptoms from the microbe.

43. The method of any one of claims 39-42, wherein the microbe is a bacterium.

44. The method of any one of claims 39-43, wherein the individual has an internal infection of the microbe.

45. The method of any one of claims 39-44, wherein the individual has a topical infection of the microbe.

46. The method of any one of claims 39-45, wherein the composition is provided by injection, by inhalation, and / or topically to the individual.

47. The method of any one of claims 39-46, wherein the individual is an animal or insect.

48. The method of claim 47, wherein the animal is a human, dog, cat, cow, sheep, pig, goat, horse, donkey, mule, buffalo, ox, llama, camel, emu, chicken, turkey, duck, goose, snail, or deer.

49. The method of claim 47, wherein the insect is a cockroach, fly, or mosquito.

50. The method of claim 49, wherein the fly is a filth fly.

51. The method of claim 50, wherein the filth fly is a house fly, blow fly, flesh fly, stable fly, cluster fly, fruit fly, phorid fly, drain fly, or is a fungus gnat.

52. The method of any one of claims 39-51, wherein the microbe comprises one or more of the following:

53. A method of treating an individual for a microbial infection, comprising administering an effective amount of the composition of any one of claims 1-31.

54. The method of claim 53, wherein the individual has an internal infection of the microbe.

55. The method of any one of claims 53-54, wherein the individual has a topical infection of the microbe.

56. The method of any one of claims 53-55, wherein the composition is administered by injection, by inhalation, and / or topically to the individual.

57. The method of any one of claims 53-56, wherein the individual is an animal or insect.

58. The method of claim 53, wherein the individual is an animal or insect.

59. The method of claim 58, wherein the animal is a human, dog, cat, cow, sheep, pig, goat, horse, donkey, mule, buffalo, ox, llama, camel, emu, chicken, turkey, duck, goose, snail, or deer..

60. The method of claim 58, wherein the insect is a cockroach, fly, or mosquito63. The method of claim 60, wherein the fly is a filth fly.

64. The method of claim 63, wherein the filth fly is a house fly, blow fly, flesh fly, stable fly, cluster fly, fruit fly, phorid fly, drain fly, or is a fungus gnat.

65. The method of any one of claims 53-64, wherein the microbe comprises one or more of the following:

66. A method of disinfecting an environment or matter, comprising providing an effective amount of the composition of any one of claims 1-31 to the environment.

67. The method of claim 66, wherein the environment is terra firma or is a water source.

68. The method of claim 67, wherein the water source is ocean water, fresh water, brackish water, black water, or grey water.

69. The method of claim 67 or 68, wherein the water source is an ocean, lake, river, stream, estuary, creek, pond, water well, water tank, irrigation, industrial wastewater outfall, agricultural wastewater outfall, municipal wastewater outfall, or a combination thereof.

70. The method of claim 67, wherein the terra firma is waste landfill, lagoon, Brownfields site, Superfund site, chemical storage facilities, mining operation, source of agriculture pollution, source of aquaculture pollution, residential and / or commercial septic systems, stormwater runoff, underground injection well, or a combination thereof.

71. The method of claim 66, wherein the environment is a wastewater facility, food processing facility, agriculture farm, or aquaculture environment.

72. The method of any one of claims 66-71, wherein the matter comprises fecal matter.

73. A kit comprising the composition of any one of claims 1-31.

74. The kit of claim 73, further comprising an additional therapeutic agent.

75. The kit of claim 74, wherein the additional therapeutic agent is antimicrobial.

76. A method of reducing a pathogenic load of the intestine of an individual, comprising the step of the individual consuming a composition comprising any one or more of the peptides comprising the sequence of any one of SEQ ID NO: 1-SEQ ID NO:210.

77. The method of claim 76, wherein the individual is an animal or insect.

78. The method of claim 77, wherein the animal is a human, dog, cat, cow, sheep, pig, goat, horse, donkey, mule, buffalo, ox, llama, camel, emu, chicken, turkey, duck, goose, snail, or deer..

79. The method of claim 62, wherein the insect is a cockroach, fly, or mosquito.

80. The method of claim 61, wherein the consuming is by oral consumption by the individual.

81. The method of claim 61, wherein the consuming is by an insect consuming insect spray.

82. The method of claim 79, wherein the fly is a filth fly.

83. The method of claim 82, wherein the filth fly is a house fly, blow fly, flesh fly, stable fly, cluster fly, fruit fly, phorid fly, drain fly, or is a fungus gnat.

84. The method of any one of claims 76-83, wherein the pathogenic load comprises one or more of the following:

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