Peptide, set of peptides, composition, uses of at least one peptide, method for preventing and / or treating an infection and kit

Abstract

The present invention refers to the field of peptides, particularly with antimicrobial agent activity. Particularly, the present invention refers to peptides comprising the amino acid sequences as defined in the document, useful for treating and / or preventing an infection, preferably for treating and / or preventing animal mastitis, and / or as a preservative and / or as an immunomodulator. The present invention also refers to a set of peptides, to a composition, to uses of at least one peptide or the set of peptides, to a method for treating and / or preventing an infection and a kit.

Description

PEPTIDE, SET OF PEPTIDES, COMPOSITION, USES OF AT LEAST ONE PEPTIDE, METHOD FOR PREVENTING AND / OR TREATING AN INFECTION AND KIT

[0001] The present invention refers to the field of peptides, particularly with antimicrobial agent activity. Particularly, the present invention refers to peptides comprising the amino acid sequences as defined in the document, useful for treating and / or preventing an infection, preferably for treating and / or preventing animal mastitis, and / or as a preservative and / or as an immunomodulator. The present invention also refers to a set of peptides, to a composition, to uses of at least one peptide or the set of peptides, to a method for treating and / or preventing an infection and a kit.

[0002] Mastitis has been characterized as an inflammation of the mammary gland resulting from the invasion and colonization by microorganisms (REYES-JARA et al, 2016), which may affect several animal species, including bovines (KURJOGI and KALIWAL, 2014). In bovines, the disease starts in the papillary duct, leading the microorganisms to the mammary gland, causing the migration of blood serum proteins and leukocytes to the inflammation site (THOMPSON-CRISPI et al, 2014; REYES-JARA et al, 2016). This migration of host defensive cells may have different magnitude and duration, according to the virulence and concentration of the microorganisms involved, as well as the stage of the disease (BHARATHAN and MULLARKY, 2011).

[0003] Initially, the innate immunologic response is triggered, recruiting neutrophils to phagocytize the bacteria present in the mammary gland, together with the production of oxygen reactive species and the migration of antimicrobial agent peptides (AMPs), such as lactoferrin, β-lactoglobulin and defensins (WELLNITZ and BRUCKMAIER, 2012). Furthermore, cytokines are produced, such as interleukin-8, as well as other factors with antimicrobial agent activities, triggered by the early response by mammary epithelial cells (BRENAUT et al, 2014). The adaptive immune response may also be triggered, mediated by T and B cells, in order to eliminate the infection (THOMPSON-CRISPI et al, 2014).

[0004] Bovine mastitis consists of a disease with multifactor etiology and its cause may be related to a wide spectrum of pathogenic agents, more frequently bacteria (ASLI et al, 2017).

[0005] Therefore, the disease may be divided into two categories: contagious and environmental. Within the contagious category, there are bacteria which lodge in the cow udder, such as Staphylococcus sp., Streptococcus sp., Mycoplasma sp. and Corynebacterium sp. In the environmental category, bacteria present in the cow’s environment are involved, such as the coliform bacteria Escherichia coli and Klebsiella sp., as well as Streptococcus sp. (ABEBE et al, 2016). These bacteria are linked to soil and feces contamination, since they normally live in the cow’s intestine and soil, invading the udder when the animal makes direct contact with the contaminated site (IRAGUHA et al, 2015).

[0006] Particularly, the S. aureus bacterium deserves attention. This is a commensal and opportunistic pathogen microorganism for several organisms, including dairy cattle. This bacterium has been considered one of the main etiological agents for bovine mastitis (clinical and subclinical) and, possibly, one of the most studied microorganisms in this context, since S. aureus is one of the hardest pathogens to control (BARBOZA-CORONA et al, 2009; CASTRO et al, 2018; GOMES et al, 2016; TARTAGLIA et al, 2018; HOLMES and ZADOKS et al, 2011). The internalization, survival and propagation capacity within several host cell types, such as neutrophils, macrophages, mammary epithelial cells and peripheral blood lymphocytes, is one of the strategies by which S. aureus evades the immune response and the antibiotics action, besides allowing the dissemination of the infection (AITKEN et al, 2011; FRAUNHOLZ and SINHA, 2012; GÜNTHER et al, 2017; HOQUE et al, 2018; KAMARUZZAMAN et al, 2017; TARTAGLIA et al, 2018).

[0007] Another classification of the disease consists in the division between clinical and subclinical mastitis, which varies according to the intensity of the inflammatory process. The former is characterized by visible alterations, in which the gland is affected, causing milk abnormalities and reducing its volume, as well as a temperature increase and swelling in the udder (REYES-JARA et al, 2016). On the other hand, subclinical mastitis does not show any visible symptoms, but presents important effects to milk composition, such as a higher number of somatic cells (CCS – desquamation cells of the mammary epithelium, milk secretor cells and defense cells), only detectable by laboratory tests. The presence of too many CCS (> 200,000 cells mL-1) means the presence of pathogenic microorganisms which must be controlled, reducing the milk value (KURJOGI and KALIWAL, 2014; REYES-JARA et al, 2016; ASLI et al, 2017). Furthermore, subclinical mastitis may contaminate other animal species and disseminate bacteria among farms, since their transmission goes on unnoticed (REYES-JARA et al, 2016). Both clinical and subclinical mastitis present extrinsic factors which facilitate the entry of infectious microorganisms in the mammary gland, such as hygiene errors, mammary lesions and inadequate handling, which are important predispositions for the emergence of the disease (ABDALHAMED et al, 2018).

[0008] Methods for preventing and treating bovine mastitis:

[0009] Several studies point out, as the most frequently adopted prevention practices by milk producers, teat disinfection before and after milking, together with the use of gloves during milking, as well as vaccines against mastitis. The treatment methods, in turn, include the use of antibiotics during lactation and dry cow therapy (OLDE RIEKERINK et al, 2010; AGHAMOHAMMADI et al, 2018).

[0010] When dealing with disease prevention, teat disinfection is the simpler and less expensive method available for use by producers. However, the usually adopted antiseptic substances, such as iodine or chlorhexidine, are not sufficiently effective to prevent mastitis or even reduce the disease, due to bacterial resistance (REYES-JARA et al, 2016). As a consequence, other prevention methods were also developed, such as vaccines. Hoelzer and contributors (2018) reported that the currently used vaccines for animal production are attenuated live vaccines and inactivated vaccines, which may be used to prevent or control a given infection. Some vaccines against mastitis are already on the market, while others are in experimental stages, with varied effectiveness rates.

[0011] However, based on the current vaccines, which are not ideally effective, despite being easy or safe for use (HOELZER et al, 2018), it is still necessary to make use of further procedures, such as antibiotic intervention, to guarantee infection control (ISMAIL, 2017).

[0012] In relation to dry cow therapy (or drying period), it may be considered a prevention as well as a treatment method, since it may avoid and / or eliminate intramammary infections by means of antibiotics (DERAKHSHANI et al, 2018). The recurrent intramammary infections during the drying period may be due to existing infections (originating from the last lactation) or caused by new infections (occurring during the drying period and the birth) (GREEN et al, 2007). Furthermore, these infections during the drying period are, in most cases, due to the high exposition to environmental pathogens (GREEN et al, 2007).

[0013] The dry cow therapy consists in the application of intramammary antibiotics with prolonged action, which brings the consumption of 11 tons of antibiotics per year (BONSAGLIA et al, 2017). Nevertheless, trying to reduce bovine mastitis incidence rate would contribute to improving the well-being of the affected animals, since they suffer with discomfort and severe pain (THOMPSON-CRISPI et al, 2014). However, the use of antibiotics brings several problems, such as the residues eliminated in the milk (PEREIRA et al, 2016). Also, the indiscriminate use of this type of drug contributes to the evolution of antimicrobial agent resistance and, in animal production, may result in the dissemination of resistant strains among animals and humans (BONSAGLIA et al, 2017). Furthermore, the excessive use of antibiotics may affect the mammary gland microbiome and other corporal systems, where beneficial bacteria may also become resistant and have negative influence over the health of the animal (BONSAGLIA et al, 2017).

[0014] Considering the prevention methods as previously mentioned, we can observe that the most effective method to prevent and treat bovine mastitis is antibiotic therapy. Since bovine mastitis is considered a dangerous disease, it may also be harmful to human beings who consume the dairy products derived from those sick animals (THOMPSON-CRISPI et al, 2014). Therefore, the use of antibiotics becomes essential and still is the best way to eliminate the bacterial strains involved in an intramammary infection. Therefore, in cases where the disease is very severe, we need to select specific antibiotics for the strains that were isolated, avoiding the ones with wide spectrum, so to reduce the indiscriminate and inappropriate use of these drugs (REYES-JARA et al, 2016).

[0015] Antimicrobial agent residues in milk:

[0016] Antimicrobial agents are widely used to prevent and treat animal diseases, mainly in livestock raising, where the animals are a source of food, such as bovines, poultry and swines (ADESOKAN et al, 2015; VAN BOECKEL et al, 2015). In the United States, for example, high rates of antimicrobial agents are annually used for the production of animals intended for food production, corresponding to about 80% of the total use of antimicrobial agents (VAN BOECKEL et al, 2015).

[0017] Antimicrobial agent residues cause wide losses in the dairy industry. This is because the tolerable concentration ends up higher than allowed. This concentration is defined by several bodies regulating international food standards, such as the Food and Drug Administration (FDA), in the United States, Codex Alimentarius jointly by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) (PEREIRA, et al, 2016; ONDIEKI et al, 2017; MOUDGIL et al, 2019). These regulations establish that the residues present in any food may not cause harmful effects to humans. However, these regulations are not always followed and monitored (PEREIRA et al, 2016; ONDIEKI et al, 2017).

[0018] The use of antibiotics is a big issue, not only for the treatment of bovine mastitis, but of other diseases as well. Concerning mastitis, the main issue is the generation of undesired residues in milk, which is a consequence of the routine and inappropriate use of antimicrobial agents, aside from not always following the abstinence period (PEREIRA et al, 2016). These residues are eliminated through milk in lactating mammals and, in other animals, by way of urine and feces. In relation to residue elimination through milk, humans who consume said milk are exposed to risks, such as: resistance to drugs, hyper-sensitivity, mutagenicity, carcinogenicity, teratogenicity and disturbance of the normal intestinal microbiota (REYES-JARA et al, 2016; PEREIRA et al, 2016; ONDIEKI et al, 2017). Some of the resistance mechanisms to antibiotics more commonly reported in gram-negative bacterial strains are presented by Cardoso (2019), including the action of b-lactamases to hydrolyze the b-lactam ring of antibiotics, super expression of efflux bombs, reduction in porin expression, target changes through mutations in specific genes, as well as changes to the physical and chemical properties of the cellular surface. Concerning the elimination of antimicrobial agent residues though feces and urine, this problem entails microbiome imbalance, and the contamination of both the soil and water (PEREIRA et al, 2016).

[0019] Furthermore, bacteria and toxins present in the milk from cows with mastitis may give zoonotic potential to the disease, since every dairy product and derivatives become vehicles for transmission of the bacterial infection (ABEBE et al, 2016). Particularly, in this context, the bacterium S. aureus draws attention once again, for being considered a common cause of acute diseases transmitted by food (WHO, 2014). This is because of the highly thermostable staphylococcal enterotoxins produced by S. aureus, representing a frequent cause of staphylococcal food poisoning around the world (CASTRO et al, 2018; GRUNERT et al, 2018; JOHLER et al, 2015). In this sense, contaminated milk may act as a pathogen transmission route (DUARTE et al, 2018). Furthermore, many drugs used in animals are identical or related to drugs for human use, which may enable microbial resistance, depending on the exposition of animals or humans to these drugs (ONDIEKI et al, 2017).

[0020] The dangerousness of these residues varies according to the pharmacokinetic characteristics and properties of the drug, in addition to the biological, chemical and physical processes occurring in the organism of the animal. Therefore, it is very important to screen and monitor antimicrobial agent residue levels, so to reduce the adverse effects on human health, and allow for food of animal origin to be of high quality and provided with safety (MAJDINASAB et al, 2017).

[0021] It is important to point out the economic and animal health losses. In this regard, mastitis is one of the most dispersed diseases in the world, with high prevalence and burden in the dairy industry (THOMPSON-CRISPI et al, 2014). It results in high expenses (MONISTERO et al, 2018) attributed to veterinary services, antibiotic treatment, discarded milk and significant reduction of milk production and sales (THOMPSON-CRISPI et al, 2014; ABEBE et al, 2016; MONISTERO et al, 2018). Furthermore, a healthy herd may be affected, causing the death of the animals and / or early slaughter (THOMPSON-CRISPI et al, 2014; ABEBE et al, 2016).

[0022] Considering all the global problems generated by bovine mastitis, producers, researchers and different government bodies are searching for solutions to reduce the prevalence of the disease, aiming to also reduce the use of antibiotics (CHENG et al, 2014; REYES-JARA et al, 2016). These alternatives include more effective vaccines, bacteriophages or natural compounds, which may bring an effective approach to the handling of bovine mastitis (GOMES and HENRIQUES, 2016). Therefore, the group of natural compounds offers innovative options, such as the use of antimicrobial agent peptides.

[0023] Antimicrobial agent peptides as promising therapeutic alternatives:

[0024] Antimicrobial agent peptides (AMPs) are protein molecules originated from gene expression, in a constitutive form or in response to lesions or infections. They are also present in all life forms (PETERS et al, 2010; BROGDEN and BROGDEN, 2011). It has been suggested that, from the ancestral metazoans to the current mammals, immune system mechanisms have been used to eliminate pathogenic micro-organisms, and AMPs represent a part of the immune system.

[0025] A significant part of AMPs may also be called defense peptides (or “Host Defense Peptides” – HDPs). They received said terminology due to their multifunctionality (HANEY et al, 2019). In the innate immune system, they represent a large fraction of the more than 2000 AMPs which have already been experimentally characterized (LEE et al, 2018). In the processes related to the defense system of organisms, AMPs act as immune response modulators, as well as being capable of neutralizing endotoxins and acting indirectly in the pathogenic micro-organism depuration (MYLONAKIS et al, 2016).

[0026] In prokaryote organisms, AMPs are produced and used in the competition for an ecological niche, being produced by one bacterium to eliminate another one, in the defense of micro-organisms (HASSAN et al, 2012). In eukaryotes, these AMPs act in the defense against infections, due to their wide antimicrobial agent activity spectrum against a range of pathogens (CARMONA-RIBEIRO and DE MELO CARRASCO, 2014).

[0027] AMPs also have other variations in their biological activity, not restricted to fighting only bacteria. They may also present biological activity against tumor cells, viruses, protozoa or fungi, being able to act in synergy, besides presenting anti-inflammatory effect (LAVERTY et al, 2011). This range of biological activities gives the AMPs high therapeutic potential, which may be related to the fact of showing different molecular structures (MAHLAPUU et al, 2016).

[0028] Among the secondary structures present in AMPs, we highlight: α-helix, β-sheet and random-coil (TAKAHASHI et al, 2010; NGUYEN et al, 2011; MAHLAPUU et al, 2016). The distribution of AMPs filed with the Antimicrobial agent Peptides Database (APD) (http: / aps.unmc.edu / AP / main.php), according to their phi and psi twist angles, solved by nuclear magnetic resonance, may be found, e. g. in Fjell et al (2012). AMPs with α-helix structures acquire this conformation, primarily, when they come into contact with a biological membrane. Examples of peptides in this group, as already researched, include: human cathelicidin LL-37 and human lactoferrin (MAHLAPUU et al, 2016). AMPs with β-sheet structures, on the other hand, suffer less conformational changes, presenting themselves more structurally ordered in aqueous solution. This behavior is due to the rigid structure present in the β-sheets, stabilized by disulfide bonds. This class is represented, e.g., by bovine lactoferrin and gomesins (MAHLAPUU et al, 2016). Finally, random-coil does not have a defined secondary structure, but some of them may bend themselves when interacting with membranes, forming an amphipathic structure. This AMP class presents the higher portion of its amino acid residues as arginine, tryptophan, proline and histidine, and they are represented by indolicidins (MAHLAPUU et al, 2016).

[0029] AMPs are also different in other aspects, such as their amino acid residue composition, molecular masses and loads, besides having varied tridimensional structures (BROGDEN and BROGDEN, 2011). They have between 12 and 50 amino acid residues, they are generally cationic, with positive net load varying between +2 and +9, and practically 50% of their residues are hydrophobic (CZYZEWSKI et al, 2016).

[0030] Two of these main characteristics stand out among AMPs: cationicity and hydrophobicity. Cationicity (depending on the liquid load) makes the AMPs become selective to negative membranes (bacterial or of tumor cells) and not to zwitterionic membranes (mammal cells). Hydrophobicity, on the other hand, promotes better interaction between AMPs and fatty acid chains, enhancing the link to the lipopolysaccharides (LPS) present in bacterial membranes (NGUYEN et al, 2011). The interactions with bacterial membranes are enhanced due to these physical-chemical properties, wherein the wide spectrum activity may be of a membrane or intracellular (CZYZEWSKI et al, 2016).

[0031] AMPs’ routes of action are diverse and complex, and, in some cases, not fully elucidated, or causing controversies in literature. Generally speaking, the membrane severance mechanisms mainly occur, in summary, due to the amphipathic properties of the AMPs, wherein the interaction with membranes may or may not be mediated by receptors (KUMAR et al, 2018; MISAWA et al, 2019). In addition to membrane targets, AMPs may interact with intracellular targets, interrupting processes such as enzymatic activity, cellular wall, protein or DNA synthesis (NGUYEN et al, 2011; MAHLAPUU et al, 2016), or act as signaling molecules in sub-lethal concentrations (SANI and SEPAROVIC, 2016). The routes of action for AMPs differ from those associated to conventional antibiotics: while antibiotics act upon a specific target, AMPs may interact with several targets, being able to act in different ways, thus making the development of microbial resistance become more difficult (SINGH and ABRAHAM, 2014; MISHRA et al, 2018). AMPs cause bacterial death due to multiple and complementary actions, which may vary depending on some factors (MAHLAPUU et al, 2016).

[0032] Thus, although AMPs present a great alternative to the use of antibiotics, they can also present challenges, such as low bioavailability or short half-life time, low solubility, instability and susceptibility to proteolytic degradation (CZYZEWSKI et al, 2016; LAU and DUNN, 2018), added to bacterial resistance in rare cases (MARIA-NETO et al, 2012; CARDOSO et al, 2017). Therefore, optimized and effective AMPs continue to be a desirable alternative for infection treatment.

[0033] In this sense, the present invention is revealed, disclosing useful peptides for the treatment and / or prevention of infections, preferably for the treatment and / or prevention of animal mastitis, and / or as a preservative and / or as an immunomodulator. The present invention also refers to a set of peptides, a composition, uses of at least one peptide or the set of peptides, a method for preventing and / or treating an infection and a kit. The peptides of the present invention are effective and safe, representing a desirable alternative to the use of antibiotics for the treatment and / or prevention of mastitis, particularly bovine mastitis, bypassing said adverse effects, inherent to the use of antibiotics in this context. These and other advantages of the present invention will become more evident below.

[0034] An object of the present invention is, therefore, a peptide comprising the amino acid sequence as defined by SEQ ID NO. 1 (KRWKKFFRKVIKVF), SEQ ID NO. 2 (KRWKKFFRKVLKFF) or SEQ ID NO. 3 (KRWKKYRKVIKFF).

[0035] In a preferred embodiment of the peptide of the present invention, the peptide consists of the amino acid sequence as defined by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0036] In a further preferred embodiment of the peptide of the present invention, the peptide has, on its amino terminal end, an amide e / or is cyclized.

[0037] In another further preferred embodiment of the peptide of the present invention, the peptide is used as an antimicrobial agent, and / or as a preservative, and / or as an immunomodulator.

[0038] In another further preferred embodiment of the peptide of the present invention, the peptide is intended for treatment and / or prevention of an infection.

[0039] In another further preferred embodiment of the peptide of the present invention, the peptide is intended for the treatment and / or prevention of animal mastitis, preferably from the Bovidae family, more preferably a bovine.

[0040] A second object of the present invention refers to a set of peptides comprising two or more peptides, as defined by the present invention.

[0041] A third object of the present invention refers to a composition comprising at least one peptide, as defined by the present invention, or the set of peptides, as defined by the present invention.

[0042] In a preferred embodiment of the composition of the present invention, the composition further comprises one or more diluents and / or one or more pharmaceutically acceptable excipients and / or one or more biologically active compounds and / or one or more carriers and / or one or more adjuvants.

[0043] In a further preferred embodiment of the composition of the present invention, the composition is presented in the form of a solution, mixture, powder, granules, aerosol or freeze-dried.

[0044] In another further preferred embodiment of the composition of the present invention, the composition is antimicrobial agent, preferably for the treatment and / or prevention of an infection, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.

[0045] In another further preferred embodiment of the composition of the present invention, the at least one peptide is used in the composition as a preservative; and / or as an immunomodulator.

[0046] A fourth object of the present invention refers to the use of at least one peptide, as defined by the present invention, or the set of peptides, as defined by the present invention, for the production of a composition.

[0047] In a preferred embodiment of the use of the at least one peptide, or the set of peptides, the composition is an antimicrobial agent, preferably for the treatment and / or prevention of an infection, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.

[0048] In a further preferred embodiment of the use of the at least one peptide, or the set of peptides, the at least one peptide is used in the composition as a preservative and / or as an immunomodulator.

[0049] A fifth object of the present invention refers to the use of at least one peptide, as defined by the present invention, as an antimicrobial agent, and / or as a preservative, and / or as an immunomodulator.

[0050] In a preferred embodiment, the at least one peptide is used as an antimicrobial agent, preferably for the treatment and / or prevention of an infection, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.

[0051] A sixth object of the present invention refers to a method to prevent and / or treat an infection, by the administration of a therapeutically effective quantity of at least one peptide, as defined by the present invention, or the set of peptides, as defined by the present invention, or the composition, as defined by the present invention, to a human being or animal.

[0052] A preferred embodiment of the present invention refers to a method for preventing and / or treating animal mastitis, more preferably from the Bovidae family, preferably a bovine.

[0053] A seventh object of the present invention refers to a kit, comprising at least one peptide, as defined by the present invention, or the set of peptides, as defined by the present invention, or the composition, as defined by the present invention, and methods for its dilution and / or application.

[0054] The present invention may also be understood based on the details presented by the Figures.Fig.1A

[0055] shows the experimental result of the treatment of mice infected with S. aureus (1×108 CFU ml-1) and with mastitis, 72 h after the infection. There was a statistical difference between the untreated control group and the group treated with 64 mM of the peptide of the present invention. One-way ANOVA tests were performed. **** p < 0.0001 (Example 20).Fig.1B

[0056] shows the experimental result of the treatment of mice infected with S. aureus (1×108 CFU ml-1) and with mastitis, 96 h after the infection. There was a statistical difference between the untreated control group and the group treated with 64 mM of the peptide of the present invention. One-way ANOVA tests were performed. **** p < 0.0001 (Example 20).Fig.2

[0057] shows the experimental result of the treatment of bovines with subclinical mastitis treated with a carrier or with the peptide of the present invention. The selected animals presented positive bacterial culture for the species S. aureus on the first day and received respective treatments on days 2, 3 and 4. On the fifth day, the CFU content in the milk of the treated animals was followed. The results are expressed in log10 CFU / ml milk (Example 21).Fig.3

[0058] shows representative results of chromatographic profiles for the residue evaluation of the peptide of the present invention in bovine milk. The lower panel shows the mixtures of milk and peptide at T0 (lower left chromatogram) and T1 (lower right chromatogram). The red arrow indicates the retention time of the peptide (Example 22).Description of the Sequences

[0059] SEQ ID NO. 1 refers to the sequence of the peptide of the present invention (KRWKKFFRKVIKVF), which may have one amide on its amino terminal end (KRWKKFFRKVIKVF-NH2 or (SEQ ID NO. 1-NH2)) and / or be cyclized.

[0060] SEQ ID NO. 2 refers to the sequence of the peptide of the present invention (KRWKKFFRKVLKFF), which may have one amide on its amino terminal end (KRWKKFFRKVLKFF-NH2 or (SEQ ID NO. 2-NH2)) and / or be cyclized.

[0061] SEQ ID NO. 3 refers to the sequence of the peptide of the present invention (KRWKKYRKVIKFF), which may have one amide on its amino terminal end (KRWKKYRKVIKFF-NH2 or (SEQ ID NO. 1-NH2)) and / or be cyclized.Detailed Description of the Invention

[0062] The present invention refers to peptides comprising the amino acid sequences as defined by the document, useful for the treatment and / or prevention of an infection, preferably for the treatment and / or prevention of animal mastitis, and / or as a preservative and / or an immunomodulator. The present invention also refers to a set of peptides, a composition, uses of at least one peptide or the set of peptides, to a method for preventing and / or treating an infection and to a kit.

[0063] Throughout the present document, unless indicated otherwise, the limits of a range of values are included in said range. Furthermore, each range of values includes all numbers and fractions covered within their respective ranges.

[0064] Throughout the present document, the singular forms “a”, “an” and “the” include both the singular and their corresponding plurals and vice-versa, unless the context clearly states otherwise.

[0065] Throughout the present document, the term “at least one” is equivalent to the term “one or more”, and means one or more members, or at least one member of a group of members. These terms include any one among ≥ 1, ≥ 2, ≥ 3, ≥ 4, ≥ 5, ≥ 6, ≥ 7 etc. of said members, up to all said members.

[0066] Throughout the present document, the terms “about” or “approximately”, when referring to a measurable value as a parameter, a quantity, a temporary duration and similar, mean variations of the specified value, such as + / -10% or lower variations, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, whenever reasonable, wherein these variations are appropriate for each suitable parameter of the present invention. It is to be understood that the value which the modifier “about” refers to is likewise specifically, and preferably, disclosed.

[0067] Throughout the present document, words and terms such as “preferably”, “particularly”, “for example”, “such as”, “more particularly”, “more preferably” and similar, as well as their variations, should be understood as entirely optional characteristics, preferred embodiments or possible non-exhaustive examples, without limiting their scope.

[0068] Throughout the present document, the word “comprises” and any of its variations, such as “comprise” or “comprising”, should be understood as “open terms”, which may imply the inclusion of additional elements or groups of elements, which were not explicitly disclosed, and without limitation. Likewise, the words “including” and “encompassing”, and any of their variations.

[0069] Throughout the present document, the word “consists” and any of its variations, such as “consist” or “consisting”, should be understood as “closed terms”, which may not imply the inclusion of additional elements or groups of elements, which were not explicitly disclosed, indicating a limitation.

[0070] When not explicitly indicated otherwise, every acronym, expression and / or technical term should be understood under its generally used and widely known meaning in the technical field of the present invention. In some cases, terms with commonly understood meanings are defined by the present document in order to bring clarity and / or as a quick reference, and the inclusion of these definitions in the present document should not be necessarily understood as representing a substantial difference over the general understanding in the state of the art.

[0071] When not explicitly indicated otherwise, the techniques and procedures disclosed or referred to by the present document are generally well understood and employed using conventional methodology, based on the available literature and the knowledge of those skilled in the art without undue experimentation. It is worth mentioning that the present invention, wherever appropriate, is not limited to the disclosed methodology, protocols, cell strains, animal genus or species, constructions and specific reagents, which may clearly vary.

[0072] Throughout the present document, every title and subtitle is used only for convenience and should not be understood as a limitation of the present invention.

[0073] Throughout the present document, all mentioned references, books, articles, patent documents and others should be understood as incorporated by reference.

[0074] Thus, the present invention refers to a peptide comprising an amino acid sequence as defined by SEQ ID NO. 1 (KRWKKFFRKVIKVF), SEQ ID NO. 2 (KRWKKFFRKVLKFF) or SEQ ID NO. 3 (KRWKKYRKVIKFF).

[0075] Throughout the present document, the word “peptide” should be understood as a chain of amino acid monomers linked by peptide bonds. Every peptide has an N-terminal residue and a C-terminal residue on the corresponding ends of the peptide. Furthermore, throughout the present document, the word “peptide” should be understood as comprising any number of consecutive residues. Additionally, the words “peptide”, “polypeptide” and “protein” should be understood as synonyms; they may be interchangeable, and should not be understood as having a given number of residues.

[0076] In a preferred embodiment of the present invention, the peptide consists of the amino acid sequence as defined by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0077] Furthermore, throughout the present document, the word “peptide” should be understood as having any form, whether linear, with native tridimensional conformation, or of particular interest. The peptides of the present invention may be synthetically synthesized (for example, by solid phase synthesis techniques), recombinantly originated (for example, by using an expression cassette under control of a specific promoter) or from any pertinent technology in the field of the invention as known by an expert in the art.

[0078] According to the present invention, structural modifications to improve the in vivo stability of the peptides, as well as to improve the molecular properties related to its mechanism of action, resulting in an improvement of the pharmacological (peptidomimetic) activity, if any, should be considered as being within the scope of the present invention.

[0079] Such modifications may be specifically limited to protect or substitute the labile bond (peptide bond), e. g. from the introduction of atypical portions / fragments (non-peptides), or even to change peptide conformation. Promising molecular modifications (non-peptides) are those attempting to mimic the molecular properties and structure of the peptide.

[0080] Possible molecular modification examples would be (GENTILUCCI et al, 2010): pseudopeptides, which present modifications in the main chain of at least one peptide bond with isosteric or isoelectronic groups, such as reduced amides, azopeptides, retroinversed peptides and peptoids; substitution by unnatural amino acids, such as the substitution of L-amino acids with respective D-enantiomers; use of N-alkyl amino acids, alpha-substituted alpha-amino acids, beta-substituted alpha-amino acids, proline analogues, gamma- and beta-amino acids; cyclization, such as macrolactones; ether, biaryl, disulfide or other bridges mimicking the previously mentioned ones; N and C terminal portion link for the main chain, or N or C terminal portion link to amino acid side chains. According to a preferred embodiment of the present invention, the peptide has an amide on its amino terminal end, which may comprise and, preferably, consist of a construction as follows: (SEQ ID NO. 1)-NH2, (SEQ ID NO. 2)-NH2 or (SEQ ID NO. 3)-NH2. In a further preferred embodiment of the present invention, the peptide, comprising and / or consisting of amidated or non-amidated SEQ ID NO. 1, SEQ ID NO. 2 and / or SEQ ID NO. 3, is cyclized. Both the amidation and cyclization may advantageously make the peptide become more stable. Its techniques are known and available in literature, and / or the peptide thus modified may also be commercially acquired.

[0081] Other possible changes are also within the scope of the present invention. An expert in the art will recognize that preserving substitutions resulting in the alteration of an amino acid by another chemically similar amino acid in a sequence are within the scope of the present invention. Said preserving substitutions providing functionally similar amino acids are known in the art.

[0082] The peptide of the present invention is preferably antimicrobial agent. Throughout the present document, the terms “antimicrobial agent” or “antimicrobial agent” and similar should be understood as a peptide which kills or inhibits the development or growth of micro-organisms, and the term “antimicrobial agent activity” and similar should also be understood as the action of killing or inhibiting the development or growth of micro-organisms.

[0083] Throughout the present document, the term “micro-organism(s)” should be widely understood, so to include one or more among bacteria (including e. g. super-resistant bacteria), fungi, yeasts, viruses and / or protozoa, particularly those ones relevant to public health, be it human or animal health. Therefore, it does not distinguish between the types of micro-organisms listed above.

[0084] Preferably, antimicrobial agent action is intended for the treatment and / or prevention of an infection, whether in human beings or animals. Therefore, this includes any infection caused by, contributed by, or which has the presence of one or more micro-organisms. In the context of the present invention, preferably, the peptide with antimicrobial agent action is intended for the treatment and / or prevention of an infection, such as mastitis, be it in human beings or animals. Preferably, mastitis is present in animals, preferably from the Bovidae family, more preferably a bovine.

[0085] The present invention also refers to a set of peptides comprising two or more peptides, as defined by the present invention. Throughout the present document, the term “set of peptides” should be understood as a grouping, collection or aggregate comprising at least two peptides, as defined by the present invention. In this context, the term “at least two” refers to the quantity of 2 to 30 peptides, as well as any range between said values. Preferably, the set of peptides, according to the present invention, comprises between 2 and 8 peptides, particularly between 3 and 7 peptides, more particularly between 4 and 6 peptides. Preferred examples of peptide sets of the present invention comprise any combination among the following peptides: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 1-NH2, SEQ ID NO. 2-NH2, SEQ ID NO. 3-NH2, cyclized SEQ ID NO. 1, cyclized SEQ ID NO. 2, cyclized SEQ ID NO. 3, cyclized SEQ ID NO. 1-NH2, cyclized SEQ ID NO. 2-NH2 and cyclized SEQ ID NO. 3-NH2. The sets of peptides of the present invention comprise e. g. the peptides of SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 2 and SEQ ID NO. 3; SEQ ID NO. 1 and SEQ ID NO. 3; SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; SEQ ID NO. 1-NH2 and SEQ ID NO. 2-NH2; SEQ ID NO. 2-NH2 and SEQ ID NO. 3-NH2; SEQ ID NO. 1-NH2 and SEQ ID NO. 3-NH2; SEQ ID NO. 1-NH2, SEQ ID NO. 2-NH2 and SEQ ID NO. 3-NH2; cyclized SEQ ID NO. 1 and cyclized SEQ ID NO. 2; cyclized SEQ ID NO. 2 and cyclized SEQ ID NO. 3; cyclized SEQ ID NO. 1 and cyclized SEQ ID NO. 3; cyclized SEQ ID NO. 1, cyclized SEQ ID NO. 2 and cyclized SEQ ID NO. 3; cyclized SEQ ID NO. 1-NH2 and cyclized SEQ ID NO. 2-NH2; cyclized SEQ ID NO. 2-NH2 and cyclized SEQ ID NO. 3-NH2; cyclized SEQ ID NO. 1-NH2 and cyclized SEQ ID NO. 3-NH2; cyclized SEQ ID NO. 1-NH2, cyclized SEQ ID NO. 2-NH2 and cyclized SEQ ID NO. 3-NH2.

[0086] The present invention also refers to a composition comprising at least one peptide or the set of peptides, as defined by the present invention. “Composition” means, according to the present document, that the at least one peptide or the set of peptides have one or more diluents, excipients, ingredients, compounds (biologically active or otherwise), carriers, adjuvants and / or additional solutions, such as cosmetically and / or pharmaceutically acceptable solutions, depending on the purpose of said composition, which may vary. Preferably, the composition comprises the at least one peptide or the set of peptides of the present invention and, additionally, one or more diluents and / or one or more pharmaceutically acceptable excipients and / or one or more biologically active compounds and / or one or more carriers and / or one or more adjuvants.

[0087] Throughout the present document, the word “diluent” should be understood as any substance intended to dilute, preserving the properties of the matter being diluted. Thus, it should be understood, according to the present invention, that any conventional diluent, in any form, usually employed for dilution, is within the scope of the present invention. Acceptable diluents are properly known by an expert in the art. Diluents include, but are not limited to: water, alcohol and sterile solutions.

[0088] Throughout the present document, the term “pharmaceutically acceptable excipients” should be understood as any vessel or substance which is not a pharmaceutically active ingredient. Thus, it should be understood, according to the present invention, that any conventional pharmaceutically acceptable excipient, in any form, usually employed to act as an excipient, is within the scope of the present invention. Pharmaceutically acceptable excipients are properly known by an expert in the art and may also be chosen from Remington: The Science and Practice of Pharmacy, Remington, 22nd edition, or Handbook of pharmaceutical excipients, Rowe, 8th edition, incorporated by the present document as reference. The pharmaceutically acceptable excipient includes but is not limited to: buffer or saline solution, stabilizer, surfactant, solubilizer, emulsifier and preservative.

[0089] Throughout the present document, the term “biologically active compounds” should be understood as any substance or compound exerting a biologically active effect. Thus, it should be understood, according to the present invention, that any additional conventional biologically active compound or substance, in any form, usually employed to act as such, is within the scope of the present invention. They may exert their function by the same route or by similar or different routes. They may have a plurality of related action routes and / or independent action mechanisms. Biologically active compounds include, but are not limited to: enhancers, agonists etc.

[0090] Throughout the present document, the word “carrier” should be understood as a transporter, a directing group or any structure which the peptide may be incorporated to or associated with, so to guide and / or facilitate and / or improve the triggering of an immune response by a human being or an animal. Therefore, it should be understood that, according to the present invention, any conventional carrier, in any form, usually employed to work as a carrier is within the scope of the present invention. Adequate carriers are properly known by an expert in the art. Carriers include, but are not limited to: colloidal gold particles, antibodies or their fragments, polymers, vesicles and nanovesicles. In a preferred embodiment of the present invention, the carrier is a nonionic polymer, such as a nonionic polymer derived from cellulose, such as hydroxyethyl cellulose (commercially known as Natrosol), among others.

[0091] Throughout the present document, the word “adjuvant” should be understood as any substance that modules or increases the action of the peptides of the present invention. Therefore, it should be understood, according to the present invention, that any conventional adjuvant, in any form, usually employed in compositions for this purpose, is within the scope of the present invention. Adjuvants include, but are not limited to: Freund’s complete adjuvant, Freund’s incomplete adjuvant, aluminum hydroxide, silica and saponin.

[0092] It is important to highlight that the composition of the present invention may be prepared by any conventional method disclosed in literature with this purpose, and / or known by an expert in the art, without any undue experimentation.

[0093] According to the present invention, the composition may be in any form, for application by any means. In a preferred embodiment of the present invention, the composition is in the form of a solution, mixture, powder, granules, aerosol or freeze-dried.

[0094] Likewise, similarly to the at least one peptide or set of peptides of the present invention, the composition is preferably antimicrobial agent, preferably for the treatment and / or prevention of an infection in both human beings and animals, more preferably for treatment and / or prevention of mastitis, preferably in an animal, even more preferably from the Bovidae family, preferably a bovine. Furthermore, the at least one peptide may also, or alternatively, work in the composition as a preservative and / or as an immunomodulator.

[0095] Throughout the present document, the term “preservative” refers to the capacity to preserve or conserve a solution, composition or similar, precisely by the antimicrobial agent action of the peptides disclosed by the present invention. Thus, in an embodiment of the present invention, the composition is a preservative composition.

[0096] Throughout the present document, the term “immunomodulator” refers to the modulation capacity of the immune response by the peptides disclosed by the present invention. This modulation may vary, depending on the context in question. Therefore, in an embodiment of the present invention, the composition is an immunomodulator composition. Known and desirable immune responses, depending on the context are e.g. from type Th1, Th2, Th17 etc.

[0097] The present invention also refers to the use of the at least one peptide or the set of peptides, as defined by the present invention, for the production of a composition. Preferably, the composition is as defined by the present document, which is also an object of the present invention. Therefore, also in the context of the present use, the composition is preferably antimicrobial agent, preferably for the treatment and / or prevention of an infection in both human beings and animals, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine. Likewise, also in the context of the present use, the at least one peptide of the composition is also, or alternatively, intended for use as a preservative and / or as an immunomodulator.

[0098] The present invention also refers to the use of at least one peptide, as defined by the present invention, as an antimicrobial agent and / or as a preservative and / or as an immunomodulator. Preferably, the use is as an antimicrobial agent, preferably for the treatment and / or prevention of an infection, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.

[0099] The present invention also refers to a method for preventing and / or treating an infection, comprising the administration of a therapeutically effective quantity of at least one peptide or the set of peptides, or of the composition, as defined by the present invention, to a human being or animal. Preferably, the method is intended to prevent and / or treat mastitis in both human beings and animals, preferably in an animal, more preferably from the Bovidae family, preferably a bovine.

[0100] Throughout the present document, the term “therapeutically effective quantity” refers to the quantity of peptide which is sufficient to result in a therapeutic effect on said human being or animal. The therapeutically effective quantity may be readily determined by an expert in the art following routine procedures, without undue experimentation, considering the form of the composition, the route of administration, the specific composition used, clinical factors, age and weight of the human being or animal, among others. In the case of bovines, particularly, the therapeutically effective quantity is between about 32 µM / ml and about 128 µM / ml, preferably between about 40 µM / ml and about 120 µM / ml, even more preferably between about 48 µM / ml and about 112 µM / ml, of at least one, or of each peptide, intramammarily (such as by way of an intramammary syringe), to each teat, for at least 1 day, more preferably for two days, even more preferably for three days. Additional doses may be required, such as for 4, 5 or 6 days of treatment.

[0101] Finally, the present invention refers to a kit, comprising at least one peptide, the set of peptides, or the composition, as defined by the present invention, and means for their dilution and / or application. Therefore, in a preferred example of the present invention, the kit comprises one or more peptides, in one or more adequate tubes or containers of its own, in their freeze-dried form, and a diluent solution, such as a buffer solution. It may additionally or alternatively comprise means for its application, such as a syringe, if applicable. It may also additionally comprise instructions for use.

[0102] The preferred characteristics and embodiments of the peptide, the set of peptides, the composition, the uses of the at least one peptide or the set of peptides, the method for preventing and / or treating an infection and the kit of the present invention, as disclosed above, may be equally predicted to all objects, even if said relations have not been explicitly indicated.

[0103] The present invention may be better understood based on the non-limitative examples disclosed below.Methodology

[0104] Example 1

[0105] Peptide Sequences of the Invention

[0106] Based on antimicrobial agent patterns and with the assistance from the genetic algorithm Joker, the present invention has disclosed the following peptide sequences, which were commercially synthesized: SEQ ID NO. 1 (KRWKKFFRKVIKVF), SEQ ID NO. 2 (KRWKKFFRKVLKFF) and SEQ ID NO. 3 (KRWKKYRKVIKFF), which may or may not have an amide (-NH2) on the amino terminal end, and / or be cyclized. The obtained results with the peptide of SEQ ID NO. 1, with an amide on the amino terminal end (KRWKKFFRKVIKVF-NH2 or (SEQ ID NO. 1)-NH2) are shown below. Peptides according to SEQ ID NO. 1 (not amidated), SEQ ID NO. 2 (amidated and not amidated) and SEQ ID NO. 3 (amidated and not amidated) showed equivalent results.

[0107] Example 2

[0108] Obtaining bacterial strains

[0109] Three S. aureus strains (02 / 18, 353 / 17 and Aurora isolated – strains denominated 111, 117 and 118 in the bacterial bank of S-Inova Biotech, from Universidade Católica Dom Bosco - UCDB), obtained from Girolando cows with mastitis, owned by farms located in the towns of Rochedo (19º57’11.5’’S 54º253’17.1’’W), Campo Grande (20º28’09.2’’S 54º37’08.7’’W) and Jaraguari (20º08’22.3’’S 54º24’01.0’’W) in the State of Mato Grosso do Sul, Brazil, were used for all biological trials performed in the present study.

[0110] Example 3

[0111] Characterization of bacterial growth

[0112] So to determine the exponential phase (log) and the colony forming units (CFU ml-1), S. aureus strains (strains 111, 117 and 118) were cultivated in Mueller-Hinton agar medium (MHA) overnight at 37 ºC. On the following day, three different colonies of each bacterium were put in 50 ml conic tubes, with Mueller-Hinton broth (MHB) overnight at 37 ºC with 200 rpm stirring. A 100 µl portion of the culture was subsequently added to 4,900 µl MHB and, from that, punctual readings were taken every 30 min, in a spectrophotometer (Eppendorf BioPhotometer) at a wavelength of 600 nm. After the determination of the log phase (exponential) for each strain, the values of colony forming units (CFU ml-1) were determined, based on O.D. corresponding to half the log phase (50% of exponential growth). Therefore, a 100 µl portion of the culture overnight was added to 4,900 µl MHB to allow each bacterium to reach the ideal O.D. Then, 100 µl of the bacterial suspension were added to a 900 µl 0.9% saline solution in 1 to 10 dilutions, wherein 50 µl of the last 5 dilutions were seeded in MHA and incubated in a greenhouse at 37 ºC, for later colony counting. Colonies were manually counted, and the total value (a plate containing between 100 and 500 colonies) was calculated by the following equation:

[0113] N=(C × 10^D) / [(5×10)] ^(-2)

[0114] wherein: N = bacterial concentration, expressed in CFU ml-1; C = average number of colonies per plate; D = dilution number; 5 X 10-2 ml = volume used for seeding (50 µl). All experiments were performed in biological triplicates, following the protocol proposed by Wiegand et al (2008).

[0115] Example 4

[0116] Peptide synthesis and mass determination

[0117] The peptides of the present invention were synthesized with 95% purity by means of solid phase, F-moc strategy (9-fluorenylmethoxycarbonyl), using RINK amide as a resin for coupling amino acid residues. The purified peptides were qualitatively analyzed by using a mass spectrometer MALDI-ToF / ToF UltraFlex III (Bruker Daltonics). For the analysis, freeze-dried peptides were dissolved in ultrapure water, mixed in a saturated solution of a matrix constituted by α-cyan-4-hydroxycinamic acid (1:3), placed on an Anchorchip type plate and let to crystallize at room temperature. The calibration was performed by using a Peptide calibration standard II (Bruker Daltonics) as molecular mass standards for positive reflected operation mode (700-3000 Da).

[0118] Example 5

[0119] Trials of growth inhibition for free-swimming S. aureus strains treated with antimicrobial agent peptides

[0120] The minimum inhibitory concentration (MIC) was determined by using the broth microdilution method, according to Wiegand et al (2008), on a flat bottom 96-well microplate, using MHB as a culture medium. Briefly, the peptides were prepared and stocked at a concentration of 1 mm and, when added to the plate, they were diluted in 50 µl MHB to a 32 µm concentration, followed by serial dilutions of 16, 8, 4, 2 and 1 µm. The plates were kept at room temperature until the addition of the bacterial suspension. Subsequently, 50 µl adjusted bacterial suspension (1 x 106 CFU ml-1) were applied to each well of the microplate containing the peptides, so to reach the final trial concentration of 5 x 105 CFU ml-1. Bacterial cultures in MHB and cyprofloxacin (under the same concentrations as the peptides) were used as negative and positive controls, respectively. The microplates were incubated at 37 ºC and the O.D. reading was performed at 600 nm with a microplate reader (Multiskan Go, Thermo Scientific), after 18 h of incubation without stirring. The experiments were developed with three technical and three biological replications.

[0121] Example 6

[0122] Minimum bactericidal concentration (MBC) trials

[0123] This procedure depends on MIC results, and therefore, only the wells which concentrations showed inhibition upon evaluation were seeded on Petri dishes. Thus, 10 µl of each one of the three biological replications, originated from the wells which inhibited bacterial growth, were placed on Petri dishes containing MHA, and incubated at 37 ºC for 24 h, so to evaluate a possible bactericide or bacteriostatic effect of the peptides against bacterial strains.

[0124] Example 7

[0125] Hemolytic trials

[0126] The trial to determine hemolytic activity was performed according to the method disclosed by the works of Park et al (2004) and Dassanayake et al (2018), with a few modifications. The blood used for the experiment was heterogenic Swiss mouse blood. Additionally, bovine blood was also used. For blood collection, syringes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant were used. 1 ml of mouse blood (anesthetized with ketamine and xylazine) was collected by cardiac puncture, and 2 ml bovine blood were collected by way of venipuncture by the jugular vein. The blood was centrifuged at 400 X g (centrifuge Z 326 K, HERMLE Labortechnik) for 5 min, and the supernatant was discarded. The precipitate (erythrocytes) was washed for three times with a saline solution and re-suspended in the same solution, with 400 X g centrifugations for 2 min each. 50 µl of the suspension were placed on a 96-well plate, adding 50 µl peptide in 100 µM (L¬Exp) concentration, with serial dilutions of 50, 25, 12.5, 6.25 and 3.1 µm. The samples were subsequently incubated at room temperature for 1 h. The plate was then centrifuged at 1,400 X g for 2 min and 50 µl of the supernatant were transferred to a new 96-well plate. The freed hemoglobin was monitored by the microplate reader (Multiskan Go, Thermo Scientific), with absorbance measurement at a wavelength of 415 nm. As positive control (100% lysis), Triton X-100 was used (2% v / v, final concentration, LTx100), and, as a negative control, erythrocytes were treated with a saline solution (L0). The hemolysis percentage was calculated by using the following equation:

[0127] % hemolysis=((L_Exp-L_0)) / ((L_Tx100-L_0))×100

[0128] Example 8

[0129] Experimental animals

[0130] 8 to 10 week-old female Swiss mice were used, weighing between 25 and 30 g. The animals were kept at a constant temperature (23 ºC) and at a light / dark cycle, with free access to food and water.

[0131] Example 9

[0132] Lactation induction in mice

[0133] Animal groups, groups 1 (G1), 2 (G2), 3 (G3) and 4 (G4) (n=10 per group), received different metoclopramide hydrochloride monohydrate (MHM) concentrations or saline, per gavage, for a 14-day period as follows: G1: Saline (v = 50 µl); G2: MHM (70 µg kg-1); G3: MHM (140 µg kg-1) and G4: MHM (280 µg kg-1). The weight increase was followed during the treatment period. On the fifteenth day, the animals were euthanized by deep sedation (150 mg kg-1 ketamine and 15 mg kg-1 xylazine) and the mammary glands were removed by a surgical process, for histological analysis.

[0134] Example 10

[0135] Preparation of S. aureus 118 strain for later infection into murine models

[0136] An isolated S. aureus (118) colony, originated from a clinical bovine mastitis isolate, was inoculated in MHB and incubated for 12 h at 37 ºC. The S. aureus cells were cultivated overnight, transferred to a fresh MHB medium and cultivated until the exponential phase (O.D. at 600 nm of ~1.2). The cellular culture was centrifuged and re-suspended in a saline solution buffered with sterile phosphate (PBS) and adjusted to a final quantity of 1 X 108 CFU ml-1 using the following equation:

[0137] UFC [(mL)]^(-1)= [(D.O.)] 600nm x 2.5 x 10^8

[0138] Example 11

[0139] Mastitis model in mice

[0140] Groups of animals (n = 10) received the drug CMM 140 µg kg-1 by gavage. After 14 days of lactation induction, the infection of the mammary gland with S. aureus 118 was performed on previously anesthetized animals (ketamine 150 mg kg-1 and xylazine 7.5 mg kg-1). The bacterial concentration of 1 X 108 CFU ml-1 was inoculated, only once, with the help of a 6 mm x 0.25 mm needle directly to the mammary gland of the animal, for a final volume of 100 µl (BROUILLETTE et al, 2004). The animals were photo documented daily and, after a defined period, they were euthanized by deep sedation (ketamine 150 mg kg-1 and xylazine 7.5 mg kg-1) and the mammary tissue was removed to recover the bacterial load.

[0141] Example 12

[0142] Recovering the bacterial load from the mammary glands

[0143] After euthanizing the animals, their mammary glands were surgically removed, weighed and macerated with 500 ml PBS, with the help of a tissue macerator, divided into two serially diluted portions, and seeded in MHA by microdrops (GODSHALL et al, 2002). After seeding, the plates were incubated for 24 h at 37 ºC and the number of CFU ml-1 was expressed as CFU Log per milliliter of sample.

[0144] Example 13

[0145] Treatment of female mice with mastitis using the peptides of the invention

[0146] After 14 days of lactation induction, as disclosed by the present document, the mammary gland was infected with S. aureus in the previously anesthetized animals (ketamine 150 mg kg-1 and xylazine 15 mg kg-1). A bacterial load of 1 X 108 CFU ml-1 was inoculated, only once, by using a 1 ml syringe and a gauge 33 needle with a blunt tip. The animals were classified in groups, as follows: saline; saline + Plasil (metoclopramide); S. aureus + Plasil; S. aureus + Plasil + cyprofloxacin (10 mg kg-1); S. aureus + Plasil + peptide (SEQ ID NO. 1)-NH2 (32 µm); and S. aureus + Plasil + peptide (SEQ ID NO. 1)-NH2 (64 µm). To receive the treatments with peptide and antibiotic (cyprofloxacin), the animals in each group were anesthetized daily with a lidocaine ointment on the abdomen, and the treatment administration was carried out. The administration of the treatments was performed with the help of a gauge 33G blunt tip needle (as previously disclosed by the present document) to the mammary gland of the animal, for a final volume of 100 µl. Each group of animals (n = 4 to 8 per group) was euthanized at the predetermined times: saline, saline + Plasil (metoclopramide) and S. aureus + Plasil groups were euthanized after 24 h; S. aureus + Plasil, S. aureus + Plasil + cyprofloxacin (10 mg kg-1), S. aureus + Plasil + peptide (SEQ ID NO. 1)-NH2 (32 µm) and S. aureus + Plasil + peptide (SEQ ID NO. 1)-NH2 (64 µm) groups were euthanized after 48, 72 and 96 h. The purpose was to follow treatment evolution. Euthanasia was carried out by deep sedation (ketamine 150 mg kg-1 and xylazine 15 mg kg-1) and the mammary tissue was removed. The mammary gland was cut off and sent to analysis, e. g. of the bacterial load recovery from the mammary gland.

[0147] Example 14

[0148] Bovine mastitis model

[0149] Bovines of the Girolando race were used, weighing about 350 kg. The animals were kept with free access to pasture, water and food after milking. The animals (n = 4) were monitored for three consecutive weeks for the microbiological analysis of the udders. The analysis comprised daily observations of: clinical signs, black bottom mug, somatic cell count (SCC), California mastitis test (CMT) and microbiological analysis. After three weeks, the animals were submitted to the experiment. The animals were classified in two groups: a group identified with subclinical mastitis treated with the vehicle (G1) (n = 2) and a group identified with subclinical mastitis and treated with the peptide of the present invention (G2) (n = 2) (MARTINS et al, 2007). For the intramammary treatment, a sterile 10 ml syringe was used with the Natrosol vehicle and / or peptide with a final volume of 10 ml (1.25 mg peptide with a final volume of 10 ml Natrosol – 64 µM / ml – per syringe). The animals were treated with one syringe per teat for 3 days (every 24 hours), under daily evaluation, at the moment of milking and milk discard, watching for clinical signs, black bottom mug, SCC, CMT and microbiological analysis. For the microbiological analysis, a portion of the milk was collected in a sterile tube. The samples were seeded in MHA and Baird Parker Agar (BP) by spreading. After seeding, the plates were incubated for 24 h at 37 ºC and the number of CFU ml-1 was expressed as CFU log per milliliter of sample.

[0150] Example 15

[0151] Residue evaluation of the peptides of the present invention in milk

[0152] To evaluate the stability and degradation of the peptides in milk after the treatment period in infected animals, they were tracked by high efficiency liquid chromatography. For this purpose, the milk of healthy cows was collected and mixed with the peptides under the concentration of 64 µm and analyzed right after mixing (T0) and 5 days after mixing (T1), simulating the treatment time. Samples T1 were incubated at 37 ºC and all of them were centrifuged at 24,000 X g for 10 min to remove milk fat. They were subsequently filtered with a 0.22 µm pore size filter and analyzed in a High Performance Liquid Chromatography (HPLC) equipment Shimadzu LC system (LC Solution software, LC-20AR pumps, SIL10AF automatic injector, SPDM40 photodiode array (PDA) detector and CBM-40 controller system, Kyoto, Japan). Chromatography was carried out with a Venusil ASB C18 column (250 x 4.6 mm, 5 µm, Bonna-Agela Technologies) with 25 µl injection volume. The mobile phase constituted of a mixture of water and acetonitrile and the runs were carried out using a 5-95% acetonitrile linear gradient, applying a constant flow of 1 ml min-1, and monitored with a detection wavelength of 216 nm. The control samples (milk and peptide) were analyzed, as well as the mixtures at T0 and T1. The runs were carried out in triplicate.

[0153] Results

[0154] Example 16

[0155] Bacterial growth characterization

[0156] The characterization of S. aureus bacterial strains (111, 117 and 118) was carried out in relation to their growth curve, by which the lag, log and stationary phases were determined. Thus, the working optical density (O.D.) was determined for 50% of the log phase, the bacteria concentration in each O.D. was established in different experiments, and the results were expressed in colony forming units per ml (CFU ml-1), as follows: 111 strain: O.D. = 1.1 and 3.56 X 1010 CFU ml-1; 117 strain: O.D. = 0.6 and 4.62 X 1010 CFU ml-1; and 118 strain: O.D. = 1.2 and 1.41 X 108 CFU ml-1.

[0157] Example 17

[0158] Antimicrobial agent activity spectrum

[0159] In order to evaluate the efficacy of peptides (in vitro antimicrobial agent activity) against S. aureus bacterial strains (111, 117 and 118), the minimum inhibitory concentration (MIC) trial was carried out with the peptides of the present invention. Bacterial cells were incubated with the peptides under the concentrations of 1-32 µm between 18 and 24 h. As a result, we could observe that, under the highest tested concentration of 32 µm, the peptide was capable of inhibiting by 100% the growth of target strains, with the same activity profile in different tested strains, wherein the MIC (minimum bactericide concentration – MBC) was 8 (8) µM in all three cases.

[0160] Example 18

[0161] Hemolytic trials

[0162] Peptide hemolytic activity (HC50) was evaluated against erythrocytes originated from healthy bovines and murines. We could observe that, under the highest tested concentration of 100 µm, the peptide did not result in hemolysis of the used cells (>100).

[0163] Example 19

[0164] Lactation induction in mice followed by bacterial inoculum determination

[0165] After confirming the antibacterial potential and the lack of hemolytic activity for the peptides, lactation was induced in female mice, followed by the bacterial inoculum determination to establish a mastitis profile. For that purpose, the animals were treated with Plasil for seven days, as previously disclosed by the present document. Then, three bacterial loads were tested (1 X 106, 1 X 107 and 1 X 108 CFU ml-1), aiming to establish an initial inoculum capable of inducing mastitis for up to 96 h after infection. As a result, we could observe that the 1 X 108 CFU ml-1 load showed itself as appropriate for a final inoculum for mastitis induction, followed by treatment with the candidate peptides with intramammary administrations.

[0166] Example 20

[0167] Treatment of female mice with mastitis using the peptides of the present invention

[0168] The treatment of mice with induced mastitis was, thus, carried out with the most commonly used antibiotic in the clinical practice (cyprofloxacin), as well as with the peptide of the present invention. The experiments were carried out as previously disclosed by the present document. After inducing lactation and establishing mastitis, the animals were treated with cyprofloxacin (64 µm) and the peptide (SEQ ID NO. 1) NH2 (32 and 64 µm) and evaluated at different times after the infection (48, 72 and 96 h). Statistically significant results, by means of one-way ANOVA, were observed for the animals treated with the peptide of the present invention, at 64 µM, at 72 h (, **** p < 0.0001) and 96 h (, **** p < 0.0001) after the infection. At those times, said peptide was capable of reducing the bacterial load between 10 and 1,000 times, as compared to the non-treated control.

[0169] Example 21

[0170] Treatment of bovines with subclinical mastitis with the peptides of the present invention

[0171] Subclinical mastitis was identified by means of the mug test; CMT and bacterial culture in milk. After identifying subclinical mastitis and the presence of the S. aureus bacterium, bovines were selected for the experiment. The animals were treated with vehicle or with the peptides of the present invention for three days every 24 hours, as previously indicated, with daily follow-up concerning clinical signs, mug test, CMT and milk sample for bacterial culture. As a result, important differences between the vehicle group and the group treated with the peptide of the present invention were observed on days 4 and 5, proving that the peptide of the present invention was capable of reducing the bacterial load in -1Log10, when compared to the first day of the same group. Surprisingly, after 5 days, there was no bacterial growth on selective culture media for S. aureus bacterium, thus reducing in -3Log10 the bacterial load in comparison with the first day, when the experiment started ().

[0172] Example 22

[0173] Residue evaluation of the peptides of the present invention in milk

[0174] shows the evaluation chromatograms for residues of the peptides of the present invention in milk at the times T0 and T1. The top chromatogram, to the left, shows the chromatographic profile of the peptide control with holding time of 20.25 min under the methodology applied to the trial. The milk control (top chromatogram, to the right) shows that there are no other compounds in milk detected with those same holding times, thus indicating that the methodology could be used to detect the peptide of the present invention after mixed to the milk. On the bottom chromatogram, to the left, the detection results of the peptide of the present invention at T0 show that the peptide signal appears within the expected holding time, but with lower intensity, which could be due to an immediate interaction between the peptide and different milk compounds. Finally, the bottom chromatogram, to the right, indicates that no residue of the peptide of the present invention was found after the standard treatment time of the experiment, thus suggesting that the milk is free from peptide residues, presenting the same chromatographic standard for the milk without the addition of the peptide of the present invention.Bibliography

[0175] ABDALHAMED, A. M.; ZEEDAN, G. S. G.; ZEINA, H. Isolation and identification of bacteria causing mastitis in small ruminants and their susceptibility to antibiotics, honey, essential oils, and plant extracts.Vet World,v. 11, n. 3, p. 355-362, 2018.

[0176] ABEBE, R. et al. Bovine mastitis: prevalence, risk factors and isolation ofStaphylococcus aureusin dairy herds at Hawassa milk shed, South Ethiopia.BMC Vet Res,v. 12, n. 1, p. 270, 2016.

[0177] ADESOKAN, H. K. et al. Pattern of antimicrobial usage in livestock animals in south-western Nigeria: The need for alternative plans.Onderstepoort J Vet Res,v. 82, n. 1, p. 816, 2015.

[0178] AGHAMOHAMMADI, M. et al. Herd-level mastitis-associated costs on canadian dairy farms.Front Vet Sci,v. 5, p. 100, 2018.

[0179] ASLI, A. et al. Antibiofilm and antibacterial effects of specific chitosan molecules onStaphylococcus aureusisolates associated with bovine mastitis.PLoS One,v. 12, n. 5, p. e0176988, 2017.

[0180] AITKEN, S. L.; CORL, C. M.; SORDILLO, L. M. Immunopathology of mastitis: insights into disease recognition and resolution. J Mammary Gland Biol Neoplasia, v. 16, p. 291-304, 2011.

[0181] BARBOZA-CORONA, J. E. et al. Activity of bacteriocins synthesized byBacillus thuringiensisagainstStaphylococcus aureusisolates associated to bovine mastitis. Vet Microbiol, v. 138, n. 1-2, p. 179-183, 2009.

[0182] BHARATHAN, M.; MULLARKY, I. K. Targeting mucosal immunity in the battle to develop a mastitis vaccine.J Mammary Gland Biol Neoplasia,v. 16, n. 4, p. 409-19, 2011.

[0183] BONSAGLIA, E. C. R. et al. Milk microbiome and bacterial load following dry cow therapy without antibiotics in dairy cows with healthy mammary gland.Sci Rep, v. 7, n. 1, p. 8067-8067, 2017.

[0184] BRENAUT, P. et al. Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection toStaphylococcus aureus.Vet Res,v. 45, p. 16, 2014.

[0185] BROGDEN, N. K.; BROGDEN, K. A. Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals?Int J Antimicrob Agents,v. 38, n. 3, p. 217-25, 2011.

[0186] BROUILLETTE, E. et al. Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms ofStaphylococcus aureus.Vet Microbiol, v. 101, n. 4, p. 253-262, 2004.

[0187] CARDOSO, M. H. et al. Comparative NanoUPLC-MS(E) analysis between magainin I-susceptible and -resistantEscherichia colistrains.Sci Rep, v. 7, n. 1, p. 4197-4197, 2017.

[0188] CARDOSO, M. H.Atividades antibacteriana e antibiofilme de dois peptídeos helicoidais:Pa-MAP 1.9 e EcDBS1R5. 2019. 119 (Tese de Doutorado). Universidade de Brasília, Brasília-DF.

[0189] CARMONA-RIBEIRO, A. M.; DE MELO CARRASCO, L. D. Novel formulations for antimicrobial peptides.Int J Mol Sci,v. 15, n. 10, p. 18040-83, 2014.

[0190] CASTRO, A.; SILVA, J.; TEIXEIRA, P.;Staphylococcus aureus, a food pathogen: Virulence factors and antibiotic resistance. In: Foodborne diseases. Academic Press, 2018. p. 213-238.

[0191] CHENG, G. et al. Antibiotic alternatives: the substitution of antibiotics in animal husbandry?Front Microbiol,v. 5, p. 217, 2014.

[0192] CZYZEWSKI, A. M. et al.In vivo,in vitro, andin silicocharacterization of peptoids as antimicrobial agents.PLoS One,v. 11, n. 2, p. e0135961-e0135961, 2016.

[0193] DASSANAYAKE, R. P. et al. Antimicrobial activity of bovine NK-lysin-derived peptides onMycoplasma bovis.PLoS One,v. 13, n. 5, p. e0197677, 2018.

[0194] DERAKHSHANI, H. et al. Composition of the teat canal and intramammary microbiota of dairy cows subjected to antimicrobial dry cow therapy and internal teat sealant.J Dairy Sci,v. 101, n. 11, p. 10191-10205, 2018.

[0195] DUARTE, F. C. et al. Bacteremia causada porStaphylococcus aureus: Uma análise de quinze anos da sensibilidade a antimicrobianos em um hospital terciário do Brasil. Rev Epidemiol Controle Infecç, v. 8, n. 3, p. 232-238, 2018.

[0196] FJELL, C. D. et al. Designing antimicrobial peptides: form follows function.Nat Rev Drug Discov,v. 11, n. 1, p. 37-51, 2012.

[0197] FRAUNHOLZ, M.; SINHA, B. IntracellularStaphylococcus aureus: Live-in and let die.Front Cell Infect Microbiol, v. 2, a. 43, p. 1-10, 2012.

[0198] GENTILUCCI, L.; DE MARCO, R.; CERISOLI, L. Chemical Modifications Designed to Improve Peptide Stability: Incorporation of Non-Natural Amino Acids, Pseudo-Peptide Bonds, and Cyclization.Curr Pharm Des, v. 16, n. 28, p. 3185–3203, 2010.

[0199] GODSHALL, C. J. et al. Genetic background determines susceptibility during murine septic peritonitis.J Surg Res, v. 102, n. 1, p. 45-49, 2002.

[0200] GOMES, F.; HENRIQUES, M. Control of bovine mastitis: old and recent therapeutic approaches.Curr Microbiol,v. 72, n. 4, p. 377-82, 2016.

[0201] GREEN, M. J. et al. Cow, farm, and management factors during the dry period that determine the rate of clinical mastitis after calving.J Dairy Sci,v. 90, n. 8, p. 3764-3776, 2007.

[0202] GRUNERT, T., STESSL, B., WOLF, F. et al. Distinct phenotypic traits of Staphylococcus aureus are associated with persistent, contagious bovine intramammary infections. Sci Rep, v. 8, a. 15968, 2018.

[0203] GÜNTHER, J. et al. DifferentiatingStaphylococcus aureusfromEscherichia colimastitis:S. aureustriggers unbalanced immune-dampening and host cell invasion immediately after udder infection. Sci Rep, v. 7, n. 1, p. 4811, 2017.

[0204] HANEY, E. F.; STRAUS, S. K.; HANCOCK, R. E. W. Reassessing the host defense peptide landscape.Front Chem,v. 7, p. 43, 2019.

[0205] HASSAN, M. et al. Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance.J Appl Microbiol,v. 113, n. 4, p. 723-36, 2012.

[0206] HOELZER, K. et al. Vaccines as alternatives to antibiotics for food producing animals. Part 1: challenges and needs.Vet Res,v. 49, n. 1, p. 64-64, 2018.

[0207] HOLMES, M. A.; ZADOKS, R. N. Methicillin resistant S. aureus in human and bovine mastitis. J Mammary Gland Biol Neoplasia, v. 16, p. 373-382, 2011.

[0208] HOQUE, M. N. et al. Molecular characterization ofStaphylococcus aureusstrains in bovine mastitis milk in Bangladesh. Int J Vet Sci Med, v. 6, n. 1, p. 53-60, 2018.

[0209] IRAGUHA, B.; HAMUDIKUWANDA, H.; MUSHONGA, B. Bovine mastitis prevalence and associated risk factors in dairy cows in Nyagatare District, Rwanda.J S Afr Vet Assoc,v. 86, n. 1, p. 1228-1228, 2015.

[0210] ISMAIL, Z. B. Mastitis vaccines in dairy cows: Recent developments and recommendations of application.Vet World,v. 10, n. 9, p. 1057-1062, 2017.

[0211] JOHLER, S. et al. Outbreak of staphylococcal food poisoning among children and staff at a Swiss boarding school due to soft cheese made from raw milk.J Dairy Sci, v. 98, n. 5, p. 2944-2948, 2015.

[0212] KAMARUZZAMAN, N. F. et al. Bactericidal and anti-biofilm effects of polyhexamethylene Biguanide in models of intracellular and biofilm ofStaphylococcus aureusisolated from bovine mastitis.Front Microbiol, v. 8, a. 1518, 2017.

[0213] KUMAR, P.; KIZHAKKEDATHU, J. N.; STRAUS, S. K. Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibilityin vivo.Biomolecules,v. 8, n. 1, 2018.

[0214] KURJOGI, M. M.; KALIWAL, B. B. Epidemiology of bovine mastitis in cows of Dharwad District.Int Sch Res Notices,v. 2014, p. 968076, 2014.

[0215] LAU, J. L.; DUNN, M. K. Therapeutic peptides: Historical perspectives, current development trends, and future directions.Bioorg Med Chem,v. 26, n. 10, p. 2700-2707, 2018.

[0216] LAVERTY, G.; GORMAN, S. P.; GILMORE, B. F. The potential of antimicrobial peptides as biocides.Int J Mol Sci,v. 12, n. 10, p. 6566-6596, 2011.

[0217] LEE, E. Y.; WONG, G. C. L.; FERGUSON, A. L. Machine learning-enabled discovery and design of membrane-active peptides.Bioorg Med Chem, v. 26, n. 10, p. 2708-2718, 2018.

[0218] MAHLAPUU, M. et al. Antimicrobial peptides: An emerging category of therapeutic agents.Front CellInfectMicrobiol,v. 6, p. 194, 2016.

[0219] MAJDINASAB, M. et al. An overview on recent progress in electrochemical biosensors for antimicrobial drug residues in animal-derived food.Sensors (Basel),v. 17, n. 9, 2017.

[0220] MARIA-NETO, S. et al. Deciphering the magainin resistance process ofEscherichia colistrains in light of the cytosolic proteome.Antimicrob Agents Chemother,v. 56, n. 4, p. 1714-1724, 2012.

[0221] MARTINS FILHO L., et al. Indução experimental de mastite subclínica em vacas da raça Holandesa mediante inoculação intramamária de Staphylococcus aureus (estirpe ATCC 25923).ARS VETERINARIA, v. 23, n. 2, p. 75-80, 2007.

[0222] MISAWA, T. et al. Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues.Med Chem Comm,v. 10, n. 6, p. 896-900, 2019.

[0223] MISHRA, A. K. et al. Tryptophan-rich and proline-rich antimicrobial peptides.Molecules,v. 23, n. 4, 2018.

[0224] MONISTERO, V. et al.Staphylococcus aureusisolates from bovine mastitis in eight countries: genotypes, detection of genes encoding different toxins and other virulence genes.Toxins (Basel),v. 10, n. 6, 2018.

[0225] MOUDGIL, P. et al. Validation of HPLC multi-residue method for determination of fluoroquinolones, tetracycline, sulphonamides and chloramphenicol residues in bovine milk.Food Anal Methods,v. 12, n. 2, p. 338-346, 2019.

[0226] MYLONAKIS, E. et al. Diversity, evolution and medical applications of insect antimicrobial peptides.Trans R Soc Lond, B, Biol Sci,v. 371, n. 1695, p. 20150290, 2016.

[0227] NGUYEN, L. T.; HANEY, E. F.; VOGEL, H. J. The expanding scope of antimicrobial peptide structures and their modes of action.Trends Biochem Sci,v. 29, n. 9, p. 464-472, 2011.

[0228] ONDIEKI, G. K. et al. Antimicrobial residues and compositional quality of informally marketed raw cow milk, Lamu West Sub-County, Kenya, 2015.Pan Afr Med J,v. 28, n. Suppl 1, p. 5, 2017.

[0229] PARK, Y. et al. Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12).Biotechnol Lett,v. 26, n. 6, p. 493-8, 2004.

[0230] PEREIRA, R. V. V. et al. Ingestion of milk containing very low concentration of antimicrobials: longitudinal effect on fecal microbiota composition in preweaned calves.PLoS One, v. 11, n. 1, p. e0147525, 2016.

[0231] PETERS, B. M.; SHIRTLIFF, M. E.; JABRA-RIZK, M. A. Antimicrobial peptides: primeval molecules or future drugs?PLoS Pathog,v. 6, n. 10, p. e1001067, 2010.

[0232] REMINGTON, J. P.; ALLEN, L. V.Remington: The Science and Practice of Pharmacy, 22ª edição, London: Pharmaceutical Press, 2013.

[0233] REYES-JARA, A. et al. antibacterial effect of copper on microorganisms isolated from bovine mastitis.Front Microbiol,v. 7, p. 626, 2016.

[0234] ROWE, R.Handbook of Pharmaceutical Excipients, 8ª edição, Pharmaceutical Press, 2017.

[0235] SANI, M.-A.; SEPAROVIC, F. How membrane-active peptides get into lipid membranes.Acc Chem Res,v. 49, n. 6, p. 1130-1138, 2016.

[0236] SINGH, N.; ABRAHAM, J. Ribosomally synthesized peptides from natural sources.J Antibiot (Tokyo),v. 67, n. 4, p. 277-89, 2014.

[0237] TAKAHASHI, D. et al. Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity.Biochimie,v. 92, n. 9, p. 1236-41, 2010.

[0238] TARTAGLIA, N. R. et al.Staphylococcus aureusextracellular vesicles elicit an immunostimulatory response in vivo on the murine mammary gland. Front Cell Infect Microbiol, v. 8, p. 277, 2018.

[0239] THOMPSON-CRISPI, K. et al. Bovine mastitis: frontiers in immunogenetics.Front Immunol,v. 5, p. 493, 2014.

[0240] VAN BOECKEL, T. P. et al. Global trends in antimicrobial use in food animals.P Natl Acad Sci USA, v. 112, n. 18, p. 5649-5654, 2015.

[0241] WELLNITZ, O.; BRUCKMAIER, R. M. The innate immune response of the bovine mammary gland to bacterial infection.Vet J,v. 192, n. 2, p. 148-52, 2012.

[0242] WORLD HEALTH ORGANIZATION (WHO). Antimicrobial resistance: global report on surveillance, 2014.

[0243] WIEGAND, I.; HILPERT, K.; HANCOCK, R. E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.Nat Protoc,v. 3, n. 2, p. 163-75, 2008

Claims

1. Peptide, characterized by comprising an amino acid sequence as defined by SEQ ID NO. 1 (KRWKKFFRKVIKVF), SEQ ID NO. 2 (KRWKKFFRKVLKFF) or SEQ ID NO. 3 (KRWKKYRKVIKFF).

2. Peptide of claim 1, characterized by consisting of the amino acid sequence as defined by SEQ ID NO. 1 (KRWKKFFRKVIKVF), SEQ ID NO. 2 (KRWKKFFRKVLKFF) or SEQ ID NO. 3 (KRWKKYRKVIKFF).3.Peptide of any of claims 1 or 2, characterized by having, on its amino terminal end, one amide and / or being cyclized.4.Peptide of any of claims 1 to 3, characterized by being used as an antimicrobial agent and / or a preservative and / or an immunomodulator.5.Peptide of claim 4, characterized by being intended for the treatment and / or prevention of an infection.6.Peptide of claim 5, characterized by being intended for the treatment and / or prevention of animal mastitis, preferably from the Bovidae family, more preferably a bovine.7.Set of peptides, characterized by comprising two or more peptides, as defined by any of claims 1 to 6.8.Composition, characterized by comprising at least one peptide, as defined by any of claims 1 to 6, or the set of peptides, as defined by claim 7.9.Composition of claim 8, characterized by further comprising one or more diluents and / or one or more pharmaceutically acceptable excipients and / or one or more biologically active compounds and / or one or more carriers and / or one or more adjuvants.10.Composition of any of claims 8 or 9, characterized by being presented in the form of a solution, mixture, powder, granules, aerosol or freeze-dried.11.Composition of any of claims 8 to 10, characterized by being an antimicrobial agent, preferably for the treatment and / or prevention of an infection, more preferably for treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.12.Composition of any of claims 8 to 11, characterized by the at least one peptide being used in the composition as a preservative and / or as an immunomodulator.13.Use of at least one peptide, as defined by any of claims 1 to 6, or the set of peptides, as defined by claim 7, characterized by being intended for the production of a composition.14.Use of claim 13, characterized by the composition being an antimicrobial agent, preferably for the treatment and / or prevention of an infection, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.15.Use of any of claims 13 to 14, characterized by the at least one peptide being used in the composition as a preservative and / or as an immunomodulator.16.Use of at least one peptide, as defined by any of claims 1 to 3, characterized by being as an antimicrobial agent and / or a preservative and / or an immunomodulator.17.Use of claim 16, characterized by being as an antimicrobial agent, preferably for the treatment and / or prevention of an infection, such as by bacteria, super-resistant bacteria and / or fungi, more preferably for the treatment and / or prevention of animal mastitis, even more preferably from the Bovidae family, preferably a bovine.18.Method for preventing and / or treating an infection, characterized by being performed by the administration of a therapeutically effective quantity of at least one peptide, as defined by any of claims 1 to 6, the set of peptides, as defined by claim 7, or the composition, as defined by any of claims 8 to 10, to a human being or animal.19.Method of claim 18, characterized by being intended to prevent and / or treat animal mastitis, more preferably from the Bovidae family, preferably a bovine.20.Kit, characterized by comprising at least one peptide, as defined by any of claims 1 to 6, or the set of peptides, as defined by claim 7, or the composition, as defined by any of claims 8 to 12, and means for their dilution and / or application.

Images