Antimicrobial therapy

Abstract

To provide a composition effective for treating microbial skin infections, atopic dermatitis and the like.SOLUTION: Methods and compositions comprising hogocidin peptides (SH-lantibiotics), derivatives and variants are provided. Also provided are methods and compositions, including probiotic compositions, that utilize strains of Staphylococcus hominis and Staphylococcus epidermidis that produce hogocidin, hogocidin-like peptides, or other inhibitors of skin pathogens. Methods of treating microbial skin infections and atopic dermatitis are also provided.SELECTED DRAWING: Figure 1A-B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application No. 62 / 157,248, filed May 5, 2015, and U.S. Provisional Application No. 62 / 300,274, filed February 26, 2016, the disclosures of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant Nos. R01AI083358, AR067547, and HHSN-272201000020C, all of which were awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing Reference This application has been submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named Sequence_ST25.txt, created on May 4, 2016, and is 45 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION The present disclosure relates to methods and compositions for treating infections and modulating the microbiota of the skin and mucous membranes to treat diseases or disorders associated with or exacerbated by dysbiosis. [Background technology]

[0005] Small cationic antimicrobial peptides (AMPs) are naturally occurring antibiotics of the innate immune system. AMPs are widely distributed in animals and plants and are among the most ancient host defense factors. Their spectrum of activity includes Gram-positive and Gram-negative bacteria, as well as fungi and certain infectious agents. As pathogenic microorganisms become increasingly resistant to traditional antibiotics, researchers are exploring these endogenous antibiotics as potential sources or novel treatments for various infectious diseases.

[0006] Patients with atopic dermatitis (AD) suffer from recurrent skin infections with Staphylococcus aureus (SA) and dysbiosis of their skin microbiota. Increased susceptibility to SA is associated with impaired innate immune defenses, including abnormal barrier function and reduced induction of antimicrobial peptides (AMPs), such as cathelicidin and β-defensin.

[0007] Symptoms of atopic dermatitis, also called eczema or atopic eczema, include: dry skin that forms a rash; scaly, swollen, red skin; a rash on the face or the insides of the knees, elbows, or wrists; weeping blisters; changes in skin color after repeated episodes; thickened, cracked, dry, scaly, or rough-looking skin in patches; and severe itching (pruritus), especially at night, accompanied by raw, sensitive, swollen skin from scratching. The signs and symptoms of atopic dermatitis (eczema) vary greatly from person to person and may include red to brownish-gray patches, particularly on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the crooks of the elbows and knees, and in young children, on the face, scalp, back of the head, ears, legs, feet, arms, hands, and buttocks; and small bumps that may leak fluid and form scabs when scratched. Atopic dermatitis most often begins before the age of 5 and can persist into adolescence and adulthood. For some people, it periodically flares up and then resolves for periods, even for several years. The skin changes caused by atopic dermatitis may promote the high susceptibility of these patients to colonization and infection by Staphylococcus aureus.

[0008] Dysbiosis involves an imbalance in the flora (bacterial flora) of the skin or mucous membranes, including the nose, mouth, eyes, urogenital tract, and intestinal flora, resulting in an excess of species such as Staphylococcus aureus and an under-representation of other species. Generally, in a healthy flora, non-pathogenic bacteria secrete inhibitors or are able to simply occupy all available niches, thereby directly inhibiting or indirectly eliminating pathogens that could otherwise establish an infectious state or promote the development of disease or disease-like conditions, such as atopic dermatitis. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure provides compositions and methods for treating disorders associated with skin dysbiosis. These disorders, associated with an imbalance in normal skin flora and the overgrowth of skin pathogens such as Staphylococcus aureus, result in skin infections, atopic dermatitis, and psoriasis, among other conditions. The present disclosure provides compositions and methods for treating these disorders by restoring healthy skin flora using antimicrobial peptides derived from bacteria common in healthy skin flora, or by directly administering probiotic compositions containing strains derived from healthy skin flora or rare viable flora cultured from the skin of patients diagnosed with flora dysbiosis, which can kill or inhibit the growth of pathogenic species on the skin or species associated with disease-like microbial imbalances.

[0010] Specifically, the present disclosure provides thickened topical compositions containing one or more probiotic bacterial strains, preferably strains of the genus Staphylococcus, more preferably the disclosed strains of Staphylococcus hominis and Staphylococcus epidermidis. These strains can be isolated from healthy skin flora by the methods disclosed herein, or isolated as viable flora cultured from the skin of patients diagnosed with flora dysbiosis, and identified by secreted peptide sequences, fatty acid methyl ester profiles, and / or the antimicrobial peptide codon organization disclosed herein. The probiotic strains of the present disclosure can be provided in a live, lyophilized, or reconstitutable form. Additionally, the present disclosure provides compositions containing the Staphylococcus epidermidis and Staphylococcus hominis strains described herein, which can be formulated for topical administration to the skin, scalp, or mucous membranes. The present disclosure further provides compositions wherein the probiotic bacterial strain comprises one or more of Staphylococcus epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5 and / or AMT5-G6, and / or Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1 and / or AMT4-D12.

[0011] Compositions of the present disclosure may also include conditioned culture medium or isolated antimicrobial compounds derived from the strains described herein, such as the peptide designated herein as hogocidin. The present disclosure contemplates the use of heterologously expressed or synthetic hogocidin, hogocidin derivatives, or hogocidin-like peptides. The present disclosure also provides compositions comprising hogocidin peptides, derivatives, or variants, and cathelicidin peptides, derivatives, or variants. Furthermore, the present disclosure provides a composition of any of the above embodiments, wherein the peptide comprises one or more D-amino acids, one or more unnatural amino acids, and / or one or more post-translational modifications. The present disclosure provides a composition of any of the above embodiments, wherein the peptide is substantially purified from other peptides. The present disclosure provides a composition of any of the above embodiments, wherein the peptide is partially purified from other peptides. The present disclosure provides a composition, wherein the peptide is present in a crude extract. The present disclosure provides a composition of any of the above embodiments, wherein the peptide is formulated for topical administration.

[0012] The present disclosure provides a composition of any of the above embodiments, wherein the formulation includes, but is not limited to, a lotion, ointment, or spray, or cream, or oily suspension.

[0013] The present disclosure provides a composition of any of the above embodiments, wherein the hogoshidin peptide, derivative or variant comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 2 or 4 comprising unnatural amino acids, SEQ ID NO: 2 or 4 comprising D-amino acids, or SEQ ID NO: 2 or 4 comprising a fusion construct.

[0014] The present disclosure also provides a method for inhibiting the spread of and / or reducing the risk of microbial infection, comprising contacting the microorganism with an effective amount of a composition of the present disclosure. In one embodiment, the contacting is in vivo. In another embodiment, the in vivo contacting is by topical administration. The present disclosure further provides a method for treating skin or mucosal infection, atopic dermatitis, psoriasis, acne, or other disorders associated with skin dysbiosis by applying to the skin or mucosa an effective amount of a composition disclosed herein to a subject in need thereof.

[0015] The present disclosure provides a method of treating atopic dermatitis, comprising contacting a subject having or suspected of having atopic dermatitis with an effective amount of a probiotic composition comprising one or more bacterial strains disclosed herein.

[0016] The present disclosure provides a method for treating atopic dermatitis, the method comprising contacting a subject having or suspected of having atopic dermatitis with an effective amount of a hogoshidin peptide, derivative or variant.

[0017] The present disclosure provides a method for treating atopic dermatitis or skin dysbiosis by contacting the affected area with a composition comprising a bacterial strain that secretes hogoshidin, firmocidin, an SH-lantibiotic peptide, an SH-antibacterial agent, an SE-lantibiotic peptide, or an SE-antibacterial agent, wherein the bacterial strain includes Staphylococcus hominis strain A9, Staphylococcus hominis strain B1, Staphylococcus hominis strain C2, Staphylococcus hominis strain D3, Staphylococcus hominis strain E4, Staphylococcus hominis strain F5, Staphylococcus hominis strain F6, Staphylococcus hominis strain F7, Staphylococcus hominis strain F8, Staphylococcus hominis strain F9, Staphylococcus hominis strain F10, Staphylococcus hominis strain F11, Staphylococcus hominis strain F12, Staphylococcus hominis strain F13, Staphylococcus hominis strain F14, Staphylococcus hominis strain F15, Staphylococcus hominis strain F16, Staphylococcus hominis strain F17, Staphylococcus hominis strain F18, Staphylococcus hominis strain F19, Staphylococcus hominis strain F20, Staphylococcus hominis strain F21, Staphylococcus hominis strain F22, Staphylococcus hominis strain F31, Staphylococcus hominis strain F32, Staphylococcus hominis strain F41, Staphylococcus hominis strain F52, Staphylococcus hominis strain F6, Staphylococcus hominis strain F7, Staphylococcus hominis strain F8, Staphylococcus hominis strain F9, Staphyl strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, and Staphylococcus epidermidis strain AMT5-G6, etc. The present disclosure provides the above methods and compositions, further comprising a cathelicidin peptide.

[0018] The present disclosure provides compositions comprising a topical thickened formulation of one or more probiotic bacterial strains, and optionally a probiotic compound, a protectant, a moisturizer, an emollient, an abrasive, salt, and / or a surfactant. The one or more probiotic bacterial strains comprise one or more bacterial strains of the genus Staphylococcus, and the compositions are formulated for the topical treatment of dysbiotic disorders of the skin, scalp, or mucous membranes. In one embodiment, the one or more probiotic bacterial strains comprise Staphylococcus epidermidis, Staphylococcus hominis, or a combination of Staphylococcus epidermidis and Staphylococcus hominis. In a further embodiment, the one or more probiotic bacterial strains comprise Staphylococcus epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5, and / or AMT5-G6. In another embodiment, the one or more probiotic bacterial strains comprise Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1, and / or AMT4-D12. In yet another embodiment, each probiotic bacterial strain exhibits a fatty acid methyl ester profile corresponding to one of those shown in any of Figures 11, 12, 13, 14, 15, 16, 17, 18, or 19. In another embodiment, the one or more probiotic bacterial strains produce peptides having a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 55, and any combination thereof, wherein such peptides are optionally post-translationally modified. In another embodiment, the one or more probiotic bacterial strains are provided in a viable form. In yet another embodiment, the one or more probiotic bacterial strains are provided in a lyophilized or freeze-dried form or in a spray-dried form. In a further embodiment, the probiotic bacteria can be reconstituted into a viable form.

[0019] The present disclosure also provides a method for treating skin or mucosal infections, atopic dermatitis, psoriasis, mastitis, acne, or other disorders associated with skin dysbiosis in humans or other mammals by applying to the skin or mucosa an effective amount of a composition described herein and in the preceding paragraphs. In one embodiment, the composition is applied topically. In a further embodiment, the composition is formulated as a cream, ointment, spray, powder, oil, concentrate, or poultice.

[0020] The present disclosure also provides a composition comprising one or more of a hogoshidin peptide, derivative or variant, an SH-lantibiotic peptide, an SH-antimicrobial agent, an SE-lantibiotic peptide, and / or an SE-antimicrobial agent; and further comprising one or more thickeners, solvents, emulsifiers, or pharmaceutically acceptable carriers or excipients. In one embodiment, the composition further comprises a cathelicidin peptide, derivative or variant. In still further or alternative embodiments, the hogoshidin peptide, derivative or variant, SH-lantibiotic peptide, and / or SE-lantibiotic peptide comprises one or more D-amino acids or unnatural amino acids. In still further embodiments, the hogoshidin peptide, SH-lantibiotic peptide, SH-antimicrobial agent, SE-lantibiotic peptide, or SE antimicrobial agent is produced in situ by one or more of Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, Staphylococcus epidermidis strain AMT5-G6, and Staphylococcus epidermidis strain MO34. In yet another embodiment of any of the above, the peptide is formulated for topical administration. In still further embodiments, the formulation comprises a lotion, ointment cream, powder, unguent, oil, or spray. In another embodiment of any of the above, the hogoshidin peptide, derivative, or variant comprises a sequence selected from SEQ ID NO: 2 or SEQ ID NO: 4, or an active fragment thereof (e.g., a mature form) that has antibacterial activity.In yet another embodiment, one or more of the hogoshidin peptides, derivatives or variants, SH-lantibiotic peptides, SH-antibacterial agents, SE-lantibiotic peptides, SE antibacterial agents, and cathelicidin peptides, derivatives or variants are provided as extracts or lysates of Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis AMT5-C5, Staphylococcus epidermidis strain AMT5-G6, and Staphylococcus epidermidis strain MO34.

[0021] The present disclosure also provides a method for treating a skin or mucosal infection or atopic dermatitis in a subject, comprising contacting the subject with an effective amount of a composition comprising one or more of a hogoshidin peptide, derivative or variant, an SH-lantibiotic peptide, an SH-antibacterial agent, an SE-lantibiotic peptide, and optionally a cathelicidin peptide, derivative or variant. In one embodiment, the contacting is by topical administration or, optionally, by contacting the subject with one or more of the SH-lantibiotic or bacteriocin-producing Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1, AMT4-D12, and Staphylococcus epidermidis strains AMT5-G6 and MO34.

[0022] The present disclosure also provides recombinant vectors comprising a polynucleotide encoding a polypeptide at least 95% identical to SEQ ID NO: 2 or 4, or a biologically active fragment thereof having antibacterial activity. In one embodiment, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO: 2 or 4. In yet another embodiment, the vector comprises a polynucleotide encoding a polypeptide from about amino acid 32 to about amino acid 61 of SEQ ID NO: 2. In a further embodiment, the vector comprises a polynucleotide encoding a polypeptide from about amino acid 29 to about amino acid 66 of SEQ ID NO: 4. In another embodiment, the vector comprises a polynucleotide at least 95% identical to SEQ ID NO: 1 or 3, and encoding the polypeptide of SEQ ID NO: 2 or 4, respectively. In yet another embodiment of the above embodiments, the vector comprises a fragment of SEQ ID NO: 1 or 3. In a further embodiment of any of the above, the vector is an expression vector.

[0023] The present disclosure also provides host cells genetically engineered to express the recombinant vectors of the present disclosure. In one embodiment, the host cell is a non-pathogenic attenuated host cell.

[0024] The present disclosure also provides recombinant polypeptides produced by the host cells of the present disclosure. In another embodiment, the recombinant polypeptides are purified from the host cell culture.

[0025] The present disclosure also provides compositions comprising the disclosed host cells.

[0026] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0027] [Figure 1A-B]Figure 1A-D. The ratio of cultivable staphylococci relative to staphylococcal DNA is higher in lesional skin of atopic dermatitis. Figure 1A: Total cultivable Staphylococcus species were counted on selective mannitol salt agar plates from 49 subjects with atopic dermatitis (AD) and 30 subjects without AD. Figure 1B: CFU results for growth of Staphylococcus aureus from 30 non-atopic subjects and 49 atopic dermatitis patients are shown. [Figure 1C-D] Figures 1A-D. The ratio of culturable staphylococci relative to staphylococcal DNA is higher in lesional skin of atopic dermatitis. Figure 1C: The abundance of total Staphylococcus species DNA was determined by quantitative PCR (qPCR) for DNA from 14 non-atopic and 37 atopic subjects. Relative CFU (rCFU) was determined by comparison with a standard of known CFU of Staphylococcus epidermidis (ATCC 12228). Figure 1D: The ratio of viable Staphylococcus species CFU to the relative abundance of staphylococci determined by DNA was calculated for each corresponding skin site. [Figure 2] Figures 2A-C. Atopic dermatitis is colonized by coagulase-negative staphylococci with low levels of antibacterial activity. Figure 2A: Coagulase-negative staphylococci (CoNS) with antibacterial activity against S. aureus were determined by high-throughput assay of individual culture isolates, and the percentage of total colonies that inhibited S. aureus growth was determined. Figure 2B: The percentage of CoNS with antibacterial activity was determined on days 1, 7, and 14 from the same subjects. Figure 2C: The ratio of viable Staphylococcus species to the abundance of staphylococcal DNA was determined from the same subjects as in Figure 2B. *P<0.05, ****P<0.0001. Eleven atopic subjects and eleven nonatopic subjects were randomly selected for analysis in panels B and C. [Figure 3]Figures 3A-B. Antibacterial coagulase-negative staphylococci correlate with the absence of S. aureus colonization. Figure 3A: The percentage of antibacterial CoNS in each sample is plotted against the abundance of S. aureus cultured from each subject. Quadrants are divided based on the frequency of antibacterial CoNS (>50% or <50%) and the detection of viable S. aureus (<1 CFU / cm2 or >1 CFU / cm2). The percentage of subjects in each quadrant relative to total subjects is shown. Figure 3B: The frequency of antibacterial CoNS in S. aureus culture-negative subjects (white) and S. aureus culture-positive subjects (black). Data are means ± SE for 29 nonatopic subjects and 41 non-lesional or 40 lesional sites from atopic subjects. [Figure 4] Figures 4A-B. Diverse bacterial species have antibacterial activity. Antibacterial and non-antibacterial CoNS species were identified by DNA sequencing of full-length 16S rRNA from randomly isolated colonies with or without antibacterial activity. Figure 4A: Percentage of identified CoNS species with antibacterial activity from five non-atopic subjects. Figure 4B: Percentage of identified CoNS species from antibacterial and non-antibacterial colonies isolated from subjects with atopic dermatitis. Up to 48 CoNS isolates were sequenced from each individual. Pie charts show the relative proportions of antibacterial CoNS (solid) and non-antibacterial CoNS (open) colonies from each AD subject. [Figure 5]Figure 5A-B. Identification of antimicrobial peptides from a coagulase-negative Staphylococcus strain (SH-A9) within the skin microbiota. Figure 5A: Amino acid sequences and predicted mono- and disulfide bonds from two antimicrobial peptides purified from Staphylococcus hominis isolated from nonatopic skin. The peptides are designated hogoshidin-α (SEQ ID NO: 2, aa 32-61) and hogoshidin-β (SEQ ID NO: 4, aa 29-66) (SH-lantibiotics α and β). The calculated molecular masses of the hypothetical mature forms of hogoshidin-α [3152.52 (M+H)] and hogoshidin-β [3548.04 (M+H)] are identical to the observed molecular masses [m / z 3152.22 and 3547.71 (M+H)], respectively. Figure 5B shows the dose-response curves of the antibacterial activity of hogoshidin-α and hogoshidin-β against Staphylococcus aureus. Co-incubation with an antimicrobial peptide produced by human skin (LL-37) demonstrates synergistic activity. Data represent the mean ± SE of triplicate assays. Arrows indicate the minimum inhibitory concentration (MIC) of each AMP, defined as a 3-log reduction in viable bacteria compared to the control. Dha: 2,3-didehydroalanine. Dhb: (Z)-2,3-didehydrobutyrin. [Figure 6] Abundance of Staphylococcus aureus DNA in non-atopic, non-lesional, and lesional areas of atopic skin. 14 and 37 DNA swabs were obtained from 30 recruited normal and 50 recruited atopic dermatitis patients, respectively. Abundance of Staphylococcus aureus DNA was determined by qPCR using species-specific primers targeting the S. aureus -specific femA gene. Relative CFU (rCFU) of S. aureus DNA was determined by comparison with known CFU of S. aureus (ATCC 35556). Density of viable bacteria or bacterial DNA was normalized across the swabbed area. AD: atopic dermatitis. [Figure 7A]Figure 7A-B. Purification and mass spectrometry analysis of AMP produced by Staphylococcus hominis (SH-A9) isolated from nonatopic skin. AMP was purified from the culture supernatant of a representative antibacterial isolate of Staphylococcus hominis by HPLC using a CapcelPac C8 column (Figure 7A). The final step of the five purification steps is shown. The inset panel shows the antibacterial activity of each fraction in a radial diffusion assay against Staphylococcus aureus. Fractions with antibacterial activity were characterized by MALDI-TOF-MS (Figure 7B). [Figure 7B] Figure 7A-B. Purification and mass spectrometry analysis of AMP produced by Staphylococcus hominis (SH-A9) isolated from nonatopic skin. AMP was purified from the culture supernatant of a representative antibacterial isolate of Staphylococcus hominis by HPLC using a CapcelPac C8 column (Figure 7A). The final step of the five purification steps is shown. The inset panel shows the antibacterial activity of each fraction in a radial diffusion assay against Staphylococcus aureus. Fractions with antibacterial activity were characterized by MALDI-TOF-MS (Figure 7B). [Figure 8-1] Representative example of amino acid loss in genome-guided MALDI-TOF / TOF analysis of hogosidin-β (SH-lantibiotic-β). The amino acid sequence of purified hogosidin-β was obtained from amino acid loss in the MS / MS fragmentation spectrum of the precursor mass 3547.7 m / z. Dha: 2,3-didehydroalanine. Dhb: (Z)-2,3-didehydrobutyrine. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9]Figures 9A-B. Organization of the gene cluster encoding the hogoshidin precursor and lantibiotic biosynthetic genes in a Staphylococcus hominis strain (SH-A9) isolated from nonatopic skin. Figure 9A shows the order of the lantibiotic precursors (A1 and A2; SH-lantibiotic-α and β) and biosynthetic genes (C, T, and M) on the Staphylococcus hominis SH-A9 genome. Figure 9B lists the hypothetical genes, loci, and putative functions. This Staphylococcus hominis strain contains multiple copies of the lantibiotic-related gene cluster. [Figure 10] 1 shows the high-throughput method used in the present disclosure. [Figure 11A] 11A-B. Chromatograms showing the results of FAME analysis of S. epidermidis strains MO-34 and MO-38 identified by the methods provided in the present disclosure. [Figure 11B] 11A-B. Chromatograms showing the results of FAME analysis of S. epidermidis strains MO-34 and MO-38 identified by the methods provided in the present disclosure. [Figure 12A] 12A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains A9 and C2 identified by the methods provided in this disclosure. [Figure 12B] 12A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains A9 and C2 identified by the methods provided in this disclosure. [Figure 13A] 13A-B. Chromatograms showing the results of FAME analysis of S. epidermidis strains A11 and AMT1-A9 identified by the methods provided in this disclosure. [Figure 13B] 13A-B. Chromatograms showing the results of FAME analysis of S. epidermidis strains A11 and AMT1-A9 identified by the methods provided in this disclosure. [Figure 14A]14A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT2-A11 and AMT3-A12 identified by the methods provided in this disclosure. [Figure 14B] 14A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT2-A11 and AMT3-A12 identified by the methods provided in this disclosure. [Figure 15A] 15A-B Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT4-C2 and AMT4-G1 identified by the methods provided in this disclosure. [Figure 15B] 15A-B Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT4-C2 and AMT4-G1 identified by the methods provided in this disclosure. [Figure 16A] 16A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT4-D12 and Staphylococcus epidermidis AMT5-C5 identified by the methods provided in this disclosure. [Figure 16B] 16A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains AMT4-D12 and Staphylococcus epidermidis AMT5-C5 identified by the methods provided in this disclosure. [Figure 17A] 17A-B. Chromatograms showing the results of FAME analysis of Staphylococcus epidermidis strain AMT5-G6, identified by the methods provided in this disclosure, and Staphylococcus hominis strain C4, which does not produce hogoshidin. [Figure 17B] 17A-B. Chromatograms showing the results of FAME analysis of Staphylococcus epidermidis strain AMT5-G6, identified by the methods provided in this disclosure, and Staphylococcus hominis strain C4, which does not produce hogoshidin. [Figure 18A] Figures 18A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains C5 and C6, which do not produce hogoshidin. [Figure 18B] Figures 18A-B. Chromatograms showing the results of FAME analysis of Staphylococcus hominis strains C5 and C6, which do not produce hogoshidin. [Figure 19] Figure 19A. Chromatogram showing the results of FAME analysis of S. epidermidis strain MO1, which does not produce SE lantibiotics or SE antibacterials. [Figure 20A-B] Figures 20A-E. Implantation of antimicrobial CoNS reduces the survival of S. aureus on skin. Figure 20A: Effect of Staphylococcus hominis on the survival of S. aureus on pig skin. Viable hogoshidin-producing S. hominis A9 (1 × 10 CFU / cm) was compared to UV-killed and washed A9 strain, viable S. hominis strains that do not produce AMP activity (C4, C5, and C6), or a control containing vehicle cream only using a pig skin assay. Data represent the mean ± sem of five independent assays. Figure 20B: Effect of bacterial implantation on the survival of S. aureus on mouse skin. S. aureus was applied at 1 × 10 CFU / cm onto the shaved dorsal skin of mice. Two hours later, control (vehicle only), active Staphylococcus hominis (A9), or inactive strains (C4, C5, and C6) were applied at equal concentrations (1 x 10 CFU / cm). Recovery of S. aureus 20 hours after application of CoNS or control is shown. Data represent the mean ± sem of six mice. The inactive strains had no effect. [Figure 20C-D] Figures 20A-E. Transplantation of antibacterial CoNS reduces the survival of S. aureus on the skin. Figure 20C: Workflow for human autologous microbiota transplantation (AMT) in S. aureus colonized AD subjects. Figure 20D: Characterization of CoNS clones used for AMT. The antibacterial class of each clone was identified by whole genome sequencing. [Figure 20E]Figures 20A-E. Implantation of antimicrobial CoNS reduces the survival of S. aureus on the skin. Figure 20E: Effect of implantation of antimicrobial CoNS on the survival of S. aureus on the skin of subjects with AD. S. aureus survival was measured by colony counts of swabs taken before implantation (baseline) and 24 hours after treatment. The difference in S. aureus between the vehicle and AMT arms is shown as S. aureus CFU Δ%. [Figure 21A] Figures 21A-C. Hypothetical antibacterial genes identified in the anti-S. aureus strains AMT1-A9 (Figure 21A), AMT2-A12 (Figure 21B), and AMT3-A12 (Figure 21C) used in autologous microbiota transplantation. Whole genome sequences of active CoNS clones were obtained by miSeq and analyzed on the RAST server (rast.nmpdr.org) to identify antibacterial classes. [Figure 21B-C] Figures 21A-C. Hypothetical antibacterial genes identified in the anti-S. aureus strains AMT1-A9 (Figure 21A), AMT2-A12 (Figure 21B), and AMT3-A12 (Figure 21C) used in autologous microbiota transplantation. Whole genome sequences of active CoNS clones were obtained by miSeq and analyzed on the RAST server (rast.nmpdr.org) to identify antibacterial classes. [Figure 22A] Figures 22A-B. Hypothetical antibacterial genes identified in the anti-S. aureus strains AMT4-C2 (Figure 22A) and AMT4-G1 (Figure 22B) used in autologous microbiota transplantation. Whole genome sequences of active CoNS clones were obtained by miSeq and analyzed on the RAST server (rast.nmpdr.org) to identify antibacterial classes. [Figure 22B] Figures 22A-B. Hypothetical antibacterial genes identified in the anti-S. aureus strains AMT4-C2 (Figure 22A) and AMT4-G1 (Figure 22B) used in autologous microbiota transplantation. Whole genome sequences of active CoNS clones were obtained by miSeq and analyzed on the RAST server (rast.nmpdr.org) to identify antibacterial classes. [Figure 23]Figures 23A-C. Hypothetical antibacterial genes identified in the anti-S. aureus strains AMT4-D12 (Figure 23A), AMT5-C5 (Figure 23B), and AMT5-G6 (Figure 23C) used in autologous microbiota transplantation. Whole-genome sequences of active CoNS clones were obtained by miSeq and analyzed on the RAST server (rast.nmpdr.org) to identify antibacterial classes. [Figure 24-1] Figure 24 shows dose-dependent killing curves of antimicrobial peptides purified from the culture supernatant of S. epidermidis strain A11 isolated from normal skin. The indicated bacterial species (1 x 10 CFU / mL) were incubated with various concentrations of purified S. epidermidis A11 antimicrobial peptide (including SEQ ID NO: 55) in 50% Mueller-Hinton broth / 50% PBS at 37°C for 24 hours. Propionibacterium acnes was incubated in 100% Reinforsed-Clostridial medium under anaerobic conditions. S. epiA11: S. epidermidis strain A11. S. epi12228: S. epidermidis strain ATCC12228. S. homA9: Staphylococcus hominis strain A9. S. aur113: S. aureus strain 113. P.ac: Propionibacterium acnes ATCC 6919 strain; E.coli: Escherichia coli ATCC 25922; P.aeruginosa: Pseudomonas aeruginosa ATCC 14213. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 24-3] This is a continuation of Figure 24-2. [Figure 25A] Figures 25A-B show data on the antimicrobial peptide purified from the culture supernatant of a clinical isolate of Staphylococcus epidermidis (strain A11) by HPLC (A). The active fraction (fractions 33-34) was analyzed by MALDO-TOF mass spectrometry to estimate the molecular weight of the active antimicrobial peptide (B). The observed molecular weight was 3484.90 (m / z). The N-terminal sequencing of the peptide is provided in SEQ ID NO:55. [Figure 25B]Figures 25A-B show data on the antimicrobial peptide purified from the culture supernatant of a clinical isolate of Staphylococcus epidermidis (strain A11) by HPLC (A). The active fraction (fractions 33-34) was analyzed by MALDO-TOF mass spectrometry to estimate the molecular weight of the active antimicrobial peptide (B). The observed molecular weight was 3484.90 (m / z). The N-terminal sequencing of the peptide is provided in SEQ ID NO:55. DETAILED DESCRIPTION OF THE INVENTION

[0028] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a probe" includes a plurality of such cells, reference to "a cell" includes a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0029] Additionally, the use of "or" means "and / or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0030] It is further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that, in some specific instances, an embodiment can alternatively be described using the words "consisting essentially of" or "consisting of."

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods and materials are now described.

[0032] All publications mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies described therein that may be used in connection with the teachings herein. With respect to any term appearing in one or more publications that is similar or identical to a term expressly defined in this disclosure, the definition of the term expressly provided in this disclosure shall control in all respects.

[0033] Atopic dermatitis is a common chronic skin disease characterized by epidermal barrier dysfunction and recurrent skin inflammation. The severity of the disease is associated with dysbiosis of the skin microbiota and the high susceptibility of these patients to colonization and infection by Staphylococcus aureus.

[0034] A unified model of atopic dermatitis pathogenesis has emerged with the realization that immunity is codependent on functions provided by the epithelium. For example, the production of antimicrobial peptides (AMPs) provides direct bactericidal activity against invading pathogens. In healthy skin, AMPs such as cathelicidin and β-defensin are increased after injury. However, the skin of patients with atopic dermatitis has a reduced ability to produce certain AMPs, which is associated with an increased rate of infection with Staphylococcus aureus, a pathogen that is supposed to be killed by these AMPs. Furthermore, S. aureus exacerbates the symptoms of atopic dermatitis, resulting in immune dysfunction such as TH2 lymphocyte skewing, decreased AMPs, exacerbated allergic responses, and disruption of the skin barrier.

[0035] Previous studies of patients with atopic dermatitis have shown that the bacterial flora present in these patients differs from that found on the skin of non-atopic subjects. The microbiota of patients with atopic dermatitis is less diverse and typically exhibits a higher abundance of Staphylococcus species. Without wishing to be bound by any particular theory, it has been hypothesized that dysbiosis of the skin microbiota may contribute to the pathophysiology of this disease. Specifically, the diverse community of microorganisms that normally comprise the microbiota has been suggested to contribute to skin homeostasis. For example, in mice, Staphylococcus epidermidis can control inflammation after injury, influence T cell development, and induce the expression of AMPs. Furthermore, the microbiota can produce its own AMPs, which can synergize with AMPs produced by host cells. Therefore, in addition to the deleterious effects of colonization by Staphylococcus aureus, dysbiosis of the microbiota in atopic dermatitis may contribute to the disease by causing a loss of their beneficial functions.

[0036] Existing antibiotic therapies nonspecifically kill bacteria, affecting the homeostasis of the resident microbiota. An imbalanced microbiota contributes to the pathogenesis of inflammatory skin diseases such as atopic dermatitis, rosacea, and acne vulgaris. The present disclosure provides compositions and formulations for disinfecting surfaces or treating infections without the safety risks of nonspecific antibiotics. Furthermore, the present disclosure provides a probiotic approach, in which a subject can be provided with live Staphylococcus hominis or Staphylococcus epidermidis strains that can produce necessary antibacterial compounds in situ while simultaneously restoring the characteristics of healthy skin flora.

[0037] Staphylococcus hominis (S. hominis) is a major component of the microbiota of healthy human skin. Recent studies have shown that S. epidermidis protects human skin by preventing pathogenic infections through the production of phenol-soluble modulins (PSMs) and small molecule antibiotics called "fermocidins," which function as additional antimicrobial compounds on normal human skin (see, e.g., U.S. Patent Publication No. 2013 / 0331384A1, the disclosure of which is incorporated herein by reference). Furthermore, lipoteichoic acid produced by S. epidermidis benefits human skin by suppressing skin inflammation during wound repair. The present disclosure provides the use of live S. epidermidis and / or S. hominis cells or cultures to restore or enhance normal skin flora to support wound healing, prevent infection, and restore skin barrier function.

[0038] In the disclosed method, in which microbial abundance is directly assessed in atopic and non-atopic subjects by both culture and DNA quantification techniques, the limitation of DNA sequencing is that it cannot distinguish between viable and dead organisms. Surprisingly, the relative ability to culture viable bacteria compared to DNA measurements was significantly different between non-atopic and atopic dermatitis patients. Approximately 10 times more bacterial DNA was detected compared to the CFU of cultured bacteria in non-atopic skin compared to atopic lesion skin. These findings indicated that bacterial survival rates were lower on non-atopic skin than on atopic lesion skin.

[0039] One explanation for the lower survival rate of bacteria on nonatopic skin is more effective surface antibacterial activity. AMPs such as LL-37 and hBD-2 and -3 are expressed at lower levels in the inflamed skin of atopic patients than in the inflamed skin of normal subjects, but expression of these AMPs is lower in noninflamed skin. Therefore, the high ability of noninflamed normal skin to kill bacteria is likely not due to the expression of these host AMPs. The high frequency of antibacterial CoNS observed on nonatopic skin indicates that these resident bacteria are important for resisting colonization by pathogens. Supporting this, Staphylococcus aureus colonization was detected only in subjects with a low frequency of CoNS strains with antibacterial activity. CoNS, which can inhibit biofilm formation, were also observed in the nasal mucosa and inhibited nasal colonization by S. aureus. The observed lack of direct antibacterial activity from the bacterial community present on the skin of patients with atopic dermatitis defines a previously unknown defect in the innate defense system of these individuals.

[0040] High-throughput screening for antibacterial activity in over 7,500 individual isolates of coagulase-negative staphylococci (CoNS) cultured from skin swabs of 50 lesional and non-lesional sites from 30 healthy control subjects and patients with atopic dermatitis (AD) identified several CoNS isolates with antibacterial activity. Healthy subjects had a high frequency of CoNS isolates with antibacterial activity against Staphylococcus aureus (75.26 ± 6.59%), whereas bacteria isolated from AD non-lesional and lesional skin had significantly lower activity [22.83 ± 5.10% and 15.76 ± 4.10%, respectively (p < 0.0001)]. Notably, subjects with a low frequency of antibacterial CoNS isolates were also colonized by SA. 16S rRNA sequencing demonstrated that antibacterial activity was detected in various strains of CoNS, including Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus warneri, and Staphylococcus capitis. Two prokaryotic AMPs with molecular weights of 3152.2 Da and 3550.7 Da were identified using HPLC, protein sequencing by MALDI-TOF-MS2, and genome sequencing. Furthermore, as shown in Nakatsuji, T. et al. (2016), Nature Medicine, paper number NMED-A78395A, submitted March 29, 2016, which is incorporated herein by reference in its entirety, application of functional CoNS isolates to ex vivo model systems, animal models, and autografts in human subjects demonstrated a reduction in S. aureus levels in infected skin. For example, application of this antimicrobial CoNS isolate to SA-colonized mouse skin was effective in reducing SA survival by over 90% compared with application of an AD CoNS strain. These findings indicate that the microbiota is a first line of defense against SA and that AD dysbiosis has a major functional link with SA colonization.

[0041] Several CoNS species have been identified that produce antibacterial activity against Staphylococcus aureus. Several laboratory strains of Staphylococcus epidermidis and Staphylococcus warneri have previously been described to produce lantibiotics capable of inhibiting the growth of other bacteria, but these were not detected in human populations. Furthermore, several CoNS species isolated based on their antibacterial activity were not previously suspected of possessing antibacterial functions. To better understand these, two previously unknown lantibiotics were identified that had potent activity against Staphylococcus aureus and exhibited high levels of synergy with the host AMP LL-37. The genes encoding these lantibiotics were common in nonatopic individuals. This discovery indicates the potential for further analysis of the host defense functions of healthy human skin microbiota and may provide a genetic approach to predicting microbiota activity. Metagenomic sequencing and correlation with functional screening of the microbiota could be of great benefit in the treatment of patients with atopic dermatitis and other skin diseases.

[0042] This disclosure provides evidence that the bacterial community present on normal human skin provides an important shield against Staphylococcus aureus. Again, without intending to be bound by any particular theory, dysfunction in this microbiota-mediated antibacterial defense system may allow S. aureus to colonize the skin and further exacerbate the disease in atopic dermatitis. This observation suggests that a cutaneous bacteriotherapy strategy may be useful as a way to suppress S. aureus without the use of pharmacologic antibiotics. Given the complex nature of this disease, an ideal therapeutic approach for atopic dermatitis should involve targeting both repair of the intrinsic epidermal barrier and optimization of the immune defenses provided by the microbiota.

[0043] This disclosure also describes novel antimicrobial peptides (AMPs) derived from the culture supernatant of a clinical isolate of Staphylococcus hominis. These AMPs are referred to herein as hogoshidin-α and hogoshidin-β. Hogoshidin exerts antibacterial and bactericidal activity against Staphylococcus aureus (S. aureus) but does not inhibit the growth of commensal bacteria on the skin, such as Staphylococcus epidermidis. Thus, this disclosure demonstrates the availability of antibiotics with potent yet selective activity against pathogens and the high safety profile typically found in the human skin microbiota, providing a probiotic approach to treating these conditions.

[0044] The term "antimicrobial," as used herein, means that the peptide destroys, inhibits, or prevents the growth or proliferation of microorganisms (e.g., bacteria, fungi, and / or viruses). Similarly, the term "antiviral," as used herein, means that the peptide destroys, inhibits, or prevents the growth or proliferation of viruses or virus-infected cells. The term "anti-tumor," as used herein, means that the peptide prevents, inhibits the proliferation of, or destroys tumor cells. Similarly, the term "antifungal," means that the peptide prevents, destroys, or inhibits the proliferation of fungi.

[0045] As used herein, "probiotic" refers to a process that provides live or attenuated microbial cultures, or lysates, lyophilizates, or extracts of such cultures, to supplement or replace elements of healthy skin or mucosal flora. Attenuated vectors for delivery to the skin can include viruses or bacteria that have been genetically modified to (a) render the vector nonpathogenic, (b) reduce pathogenicity, (c) be replication-defective, or (d) be nonantigenic. Other attenuation techniques are known in the art. Attenuation is typically achieved by knocking out genes or disrupting gene coding sequences or expression control elements so that considerations (a) through (c) or (d) are achieved. Such techniques are known in the art, and many such attenuated bacterial and viral vectors are known.

[0046] "Hogoshidin" is composed of two distinct domains: an N-terminal "pro-sequence" domain and a C-terminal domain of mature hogoshidin. Mature hogoshidin-α comprises a sequence from about amino acid 32 to about amino acid 61 of SEQ ID NO:2 (e.g., beginning at about amino acid 30, 31, 32, or 33 of SEQ ID NO:2 and extending to about amino acid 59, 60, or 61 of SEQ ID NO:2). It is readily apparent to one of skill in the art that the pre-pro form of hogoshidin-α is about 61 amino acids long, and post-translational processing provides the mature form. Depending on the expression system and organism, the mature form may be processed slightly differently depending on the proteases present. Furthermore, it is also readily apparent that the pre-pro form of hogoshidin-α may be used in the methods, compositions, and kits of the present disclosure, and the pre-pro form may be processed in vitro or in vivo before or after administration.

[0047] Similarly, the mature form of hogoshidin β comprises the sequence of about amino acid 29 to about amino acid 66 of SEQ ID NO:4 (e.g., beginning at about amino acid 27, 28, 29, or 30 of SEQ ID NO:4 and extending to about amino acid 64, 65, or 66 of SEQ ID NO:4). It is readily apparent to one of skill in the art that the pre-pro form of hogoshidin β is about 66 amino acids in length, and is post-translationally processed to provide the mature form. Depending on the expression system and organism, the mature form may be processed slightly differently depending on the proteases present. Furthermore, it is also readily apparent that the pre-pro form of hogoshidin β can be used in the methods, compositions, and kits of the present disclosure, and the pre-pro form may be processed in vitro or in vivo before or after administration.

[0048] Polypeptides comprising SEQ ID NO:2 are typically cleaved after amino acid number 31 of SEQ ID NO:2, although one of skill in the art will recognize that the cleavage site can vary by one to three amino acids in either direction of amino acid number 31 of SEQ ID NO:2, depending on the enzyme used, the expression system used, and / or the conditions under which proteolytic cleavage of the polypeptide occurs.

[0049] Polypeptides comprising SEQ ID NO:4 are typically cleaved after amino acid number 28 of SEQ ID NO:4; however, one of skill in the art will recognize that the cleavage site may vary by one to three amino acids in either direction toward amino acid number 31 of SEQ ID NO:4, depending on the enzyme used, the expression system used, and / or the conditions under which proteolytic cleavage of the polypeptide occurs.

[0050] The genetic code is well understood by those skilled in the art and is routine for creating polynucleotides encoding desired polypeptide sequences. The present disclosure also provides polynucleotides encoding the polypeptides of the present disclosure. For example, the present disclosure provides SEQ ID NOs: 1 and 3, which encode the polypeptides of SEQ ID NOs: 2 and 4.

[0051] As used herein, the term "hogoshidin peptide" refers to a peptide that is about 30 to about 50 amino acids in length and has the sequence set forth in SEQ ID NO: 2 or 4, or a post-translationally modified version thereof: aa32 to aa61 of SEQ ID NO:2 - KCSWWNASCHLGNNGKICTVSHECAAGCNL (SEQ ID NO:56) aa29 to aa66 of SEQ ID NO:4 -ATPTITTSSATCGGIIVAASAAQCPTLACSSRCGKRKK (SEQ ID NO:57) The term "matured form of hogoshidin" refers to a mature form of hogoshidin comprising an amino acid chain comprising:

[0052] In one embodiment, the method provides a hogoshidin derivative comprising: (a) a peptide at least 90% identical to the hogoshidin peptide of SEQ ID NO: 2 or 4 and having antibacterial activity; (b) a post-translationally processed mature form of (a); (c) a fragment of the hogoshidin peptide that is about 15-40 amino acids in length and has antibacterial activity; (d) a fusion protein comprising (a) to (c) above and having antibacterial activity; (e) a peptide comprising any of (a), (b), (c), or (d), in which one or more amino acids comprise a D-amino acid, and the peptide has antibacterial activity; and (f) any of the above, a retro-inverso peptide comprising the antibacterial peptide. In some further embodiments, the method provides a hogoshidin derivative comprising a lanthionine or methyllanthionine residue, or a hogoshidin derivative modified to contain a lanthionine or methyllanthionine residue. An analog, derivative, variant, or variant need not have the same activity as the hogoshidin peptide from which the analog, derivative, conservative variant, or variant is derived, so long as it has antibacterial activity. In another embodiment, the disclosure provides a hogoshidin polypeptide comprising at least one conservative amino acid difference compared to the polypeptide of SEQ ID NO: 2 or 4.

[0053] The present disclosure also provides a polypeptide comprising the sequence of SEQ ID NO: 55 at its N-terminus, wherein the polypeptide has antibacterial activity and an observed molecular weight of 3484.90 (m / z). In a further embodiment, the polypeptide is produced by Staphylococcus epidermidis A11.

[0054] The present disclosure also provides compositions comprising substantially pure hogoshidin peptides or derivatives, including a pharmaceutically acceptable excipient. The present disclosure also provides compositions comprising probiotic formulations including one or more hogoshidin- or fermocidin-producing bacterial strains.

[0055] The term "purified," as used herein, refers to a peptide that is substantially free of other proteins, lipids, and polynucleotides (e.g., cellular components with which an in vivo produced peptide is naturally associated). Typically, the peptide is at least 70%, 80%, or most commonly 90% pure by weight. As described in more detail below, the composition may further comprise a cathelicidin peptide or derivative thereof.

[0056] A "variant" is an antimicrobial peptide (e.g., a hogoshidin peptide of the present disclosure) that is an altered form of a reference antimicrobial peptide. For example, the term "variant" includes antimicrobial peptides produced by the methods disclosed herein in which at least one amino acid (e.g., about 1-10 amino acids) of the reference peptide is substituted with another amino acid. The term "reference" peptide refers to any of the antimicrobial peptides of the present disclosure (e.g., polypeptides consisting of SEQ ID NOS: 2 and 4 or their mature forms) from which a variant, derivative, analog, or conservative variation is derived. The term "derivative" includes hybrid peptides containing at least a portion of each of two antimicrobial hogoshidin peptides. Derivatives can be generated by adding one or several amino acids (e.g., 1-5) to an antimicrobial peptide without completely inhibiting the antimicrobial activity of the peptide. Additionally, C-terminal derivatives, such as C-terminal methyl esters, can be generated and are encompassed by the present disclosure.

[0057] The present disclosure also includes peptides that are conservative variants of the peptides exemplified herein. The term "conservative variant" as used herein refers to a polypeptide in which at least one amino acid is replaced by another biologically, chemically, or structurally similar residue. Examples of conservative variants include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine, for another, or the substitution of one polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine, and threonine. Structurally conservative variants include substitutions of alanine for serine (and vice versa), isoleucine for threonine (and vice versa), arginine for lysine (and vice versa), and tyrosine, phenylalanine, tryptophan, and histidine for any other member of the group. The term "conservative variant" also encompasses peptides with a substituted amino acid in place of an unsubstituted parent amino acid; typically, antibodies raised against a substituted polypeptide will also specifically bind the unsubstituted polypeptide.

[0058] As used herein, "SH-lantibiotic" means a compound that comprises a post-translationally modified peptide produced by Staphylococcus hominis, optionally containing one or more lanthionine or methyllanthionine moieties, and that exhibits antibacterial activity against one or more non-Staphylococcus hominis species.

[0059] As used herein, "SH-antibacterial agent" means a compound that includes a non-lantibiotic compound produced or secreted by Staphylococcus hominis, which may optionally include one non-lantibiotic peptide, and that exhibits antibacterial activity against one or more non-Staphylococcus hominis species.

[0060] As used herein, "SE-lantibiotic" refers to a compound that comprises a post-translationally modified peptide produced by Staphylococcus epidermidis, optionally containing one or more lanthionine or methyllanthionine moieties, and that exhibits antibacterial activity against one or more non-S. epidermidis species. In one embodiment, the peptide comprises the sequence of SEQ ID NO: 55.

[0061] As used herein, "SE-antibacterial agent" means a compound that includes a non-lantibiotic compound produced or secreted by Staphylococcus epidermidis, which may optionally include one or more non-lantibiotic peptides, and that exhibits antibacterial activity against one or more non-Staphylococcus epidermidis species.

[0062] Hogoshidin peptide variants of the present disclosure can be identified by screening large collections or libraries of random peptides or peptides of interest using one of a number of animal models, such as CRAMP knockout mice, which exhibit increased susceptibility to skin infections. Hogoshidin peptide variants can be, for example, a collection of peptides related in amino acid sequence by having various substitutions based on such sequences, for example, SEQ ID NOs: 2 and 4.

[0063] Peptide libraries include, for example, tagged chemical libraries containing peptides and peptidomimetic molecules. Peptide libraries also include those generated by phage display technology. Phage display technology includes the expression of peptide molecules on the surface of phage, as well as other methodologies that associate or can associate protein ligands with their encoding nucleic acids. Methods for generating phage display libraries, including vectors and methods for diversifying the population of expressed peptides, are known in the art (see, e.g., Smith and Scott, Methods Enzymol, 217:228-257 (1993); Scott and Smith, Science, 249:386-390 (1990); and Huse, WO 91 / 07141 and WO 91 / 07149). These or other known methods can be used to generate phage display libraries in which the displayed peptides can be cleaved and assayed for antibacterial activity. If desired, the peptide population can be assayed for activity, the active population can be sorted, and the assay repeated to isolate active peptides from the population. Other methods for generating peptides useful in the present disclosure include, for example, rational design and mutagenesis based on the amino acid sequence of the hogoshidin peptide set forth in SEQ ID NO: 2 or 4.

[0064] A hogoshidin peptide variant may be, for example, a peptidomimetic, a non-amino acid chemical structure that mimics the structure of the hogoshidin peptide of SEQ ID NO: 2 or 4 (or its mature form) but retains antimicrobial / antibacterial activity. Such mimetics are generally characterized as exhibiting similar physical characteristics, such as size, charge, or hydrophobicity, in the same spatial arrangement found in the hogoshidin peptide counterpart. A specific example of a peptidomimetic is a compound in which the amide bond between one or more amino acids is replaced by, for example, a carbon-carbon bond or other bond known in the art (see, e.g., Sawyer, Peptide Based Drug Design, ACS, Washington (1995)).

[0065] The amino acids of the hogoshidin peptides, variants, or peptidomimetics of the present disclosure are selected from the 20 naturally occurring amino acids, including L- and D-amino acids, unless otherwise specified. The use of D-amino acids is particularly useful for extending the lifespan of proteins or peptides. Polypeptides incorporating D-amino acids are resistant to proteolytic digestion. The term amino acid also refers to compounds, including amino acid analogs, chemically modified amino acids, including natural amino acids not normally incorporated into proteins (e.g., norleucine), and chemically synthesized compounds having properties known in the art to be characteristic of amino acids, so long as they can be substituted within a peptide to retain the peptide's biological activity. For example, glutamine can be an amino acid analog of asparagine, so long as they can be substituted within active fragments of hogoshidin peptides, variants, etc., to retain antimicrobial / antibacterial activity. Other examples of amino acids and amino acid analogs are listed in Gross and Meienhofer, *The Peptides: Analysis, Synthesis, Biology*, Academic Press, Inc., New York (1983). An amino acid may also be an amino acid mimetic, which is a structure having functional groups in substantially the same spatial arrangement as an amino acid, but not necessarily having both the "-amino" and "-carboxyl" groups characteristic of an amino acid.

[0066] Polypeptides and peptides of the present disclosure can be synthesized by commonly used methods, such as those involving t-BOC or FMOC protection of the alpha-amino group. Both methods involve stepwise synthesis, with a single amino acid added at each step, starting from the C-terminus of the polypeptide or peptide (see Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Polypeptides and peptides of the present disclosure can also be synthesized by well-known solid-phase peptide synthesis methods, such as those described by Merrifield, J. Am. Chem. Soc., 85:2149, 1962, and Stewart and Young, Solid Phase Peptides Synthesis, Freeman, San Francisco, 1969, pp. 27-62. If desired, peptides can be quantified by solid-phase Edman degradation.

[0067] Using a synthesizer, the hogoshidin peptide (ie, the mature form of SEQ ID NO: 2 or 4) can be produced without the need for post-translational processing.

[0068] The present disclosure also includes isolated polynucleotides (e.g., DNA, cDNA, or RNA) encoding the polypeptides and peptides of the present disclosure. Included are polynucleotides encoding analogs, mutants, conservative variations, and variants of the polypeptides and peptides described herein. The term "isolated," as used herein, refers to a polynucleotide that is substantially free of proteins, lipids, and other polynucleotides with which the in vivo produced polynucleotide is naturally associated. Typically, the polynucleotide is at least 70%, 80%, or 90% isolated from other materials, and conventional methods for synthesizing polynucleotides in vitro can be used in place of in vivo methods.

[0069] As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger genetic construct (e.g., by operably linking a promoter to a polynucleotide encoding a peptide of the present disclosure). Numerous genetic constructs (e.g., plasmids and other expression vectors) are known in the art and can be used to produce the peptides of the present disclosure in cell-free systems or in prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian) cells. Taking into account the degeneracy of the genetic code, one of skill in the art can readily synthesize polynucleotides encoding the peptides of the present disclosure. The polynucleotides of the present disclosure can readily be used in conventional molecular biology methods to produce the peptides of the present disclosure.

[0070] DNA encoding the hogoshidin peptides and derivatives thereof of the present disclosure can be inserted into an "expression vector." The term "expression vector" refers to a genetic construct, such as a plasmid, virus, or other vehicle known in the art, that can be genetically engineered to contain a polynucleotide encoding a polypeptide of the present disclosure. Such expression vectors are typically plasmids containing a promoter sequence that promotes transcription of the inserted gene sequence in a host cell. Expression vectors typically contain an origin of replication and a promoter, as well as a gene that allows phenotypic selection of transformed cells (e.g., an antibiotic resistance gene). A variety of promoters, including inducible and constitutive promoters, can be used in the present disclosure. Typically, expression vectors contain a replicon site and regulatory sequences derived from a species compatible with the host cell.

[0071] Transformation or transfection of host cells with the polynucleotides of the present disclosure can be carried out using conventional techniques well known to those skilled in the art. For example, when the host cell is E. coli, competent cells capable of DNA uptake can be prepared using CaCl2, MgCl2, or RbCl methods known in the art. Alternatively, physical means such as electroporation or microinjection can be used. Electroporation can transfer polynucleotides into cells by high-voltage electrical impulses. Furthermore, polynucleotides can be introduced into host cells by protoplast fusion using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, are also known.

[0072] A "host cell" encompassed by the present disclosure is any cell in which a polynucleotide of the present disclosure can be used to express a hogoshidin peptide, derivative, or variant of the present disclosure. This term also includes the progeny of the host cell. Useful host cells include bacterial cells, fungal cells (e.g., yeast cells), plant cells, and animal cells. For example, the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be carried out by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology (1986)). Representative examples of suitable hosts include fungal cells such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS, or Bowes melanoma; and plant cells. Selection of an appropriate host is deemed within the scope of one skilled in the art given the teachings herein. In one embodiment, the host cell can comprise a bacterial cell present in the normal bacterial flora of the skin that has been engineered to express or overexpress a hogoshidin peptide or other antimicrobial peptide of the present disclosure. These engineered bacterial cells can then be used as a probiotic such that they are applied to the skin.

[0073] The host cell may be a eukaryotic host cell (e.g., a mammalian cell). In one embodiment, the host cell is a mammalian production cell adapted to grow in cell culture. Examples of such cells commonly used in the industry are CHO, VERO, BHK, HeLa, CV1 (including Cos; Cos-7), MDCK, 293, 3T3, C127, myeloma cell lines (especially murine), PC12, and W138 cells. Chinese hamster ovary (CHO) cells have been widely used for the production of several complex recombinant proteins, such as cytokines, clotting factors, and antibodies (Brasel et al., Blood 88:2004-2012 (1996); Kaufman et al., J. Biol Chem 263:6352-6362 (1988); McKinnon et al., J. Mol Endocrinol 6:231-239 (1991); Wood et al., J. Immunol 145:3011-3016 (1990)). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., Proc Natl Acad Sci USA 77:4216-4220 (1980)) are commonly used CHO host cell lines because an efficient DHFR-selectable and amplifiable gene expression system allows high-level recombinant protein expression in these cells (Kaufman, Meth Enzymol 185:527-566 (1990)). In addition, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and recombinant proteins expressed in them have been extensively characterized and approved for use in clinical manufacturing by regulatory agencies.

[0074] Polynucleotides encoding the polypeptides and peptides of the present disclosure can be isolated from cells (e.g., cultured cells) or produced in vitro. A DNA sequence encoding a desired hogoshidin peptide can be obtained by: 1) isolating a double-stranded DNA sequence from genomic DNA; 2) chemically producing a polynucleotide encoding the desired hogoshidin peptide; or 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from donor cells (i.e., cDNA production). Standard procedures for isolating a desired cDNA sequence include the formation of plasmids or phages containing a cDNA library derived from reverse transcription of mRNA in donor cells with high levels of gene expression. When used in conjunction with polymerase chain reaction technology, even rare gene products can be cloned.

[0075] The peptides of the present disclosure can be isolated using any of a variety of art-known methods for protein purification. For example, preparative chromatographic separation and immunological separation (such as those using monoclonal or polyclonal antibodies) can be used. A carrier peptide can facilitate the isolation of a fusion protein containing a peptide of the present disclosure. A purification tag can be operably linked to the hogoshidin peptide of the present disclosure. For example, glutathione-S-transferase (GST) allows purification using a glutathione-agarose affinity column. When either protein A or the ZZ domain from Staphylococcus aureus is used as a tag, purification can be achieved in a single step using an IgG-Sepharose affinity column. The pOprF peptide, the N-terminal half of Pseudomonas aeruginosa outer membrane protein F, is a prominent protein species in outer membrane preparations and can therefore be easily purified. If desired, the fusion peptide can be purified using a reagent that specifically reacts with (e.g., specifically binds to) the hogoshidin peptide of the fusion peptide. For example, monoclonal or polyclonal antibodies that specifically bind to hogoshidin peptides can be used in conventional purification methods. Techniques for producing such antibodies are well known in the art.

[0076] Fusion constructs comprising a polypeptide linked to a hogoshidin peptide of the present disclosure may be linked at either the amino or carboxy terminus of the peptide. Typically, the polypeptide linked to the hogoshidin peptide is sufficiently anionic so that the hogoshidin peptide has a neutral or negative net charge. The anionic polypeptide can correspond to a naturally occurring protein or can be completely artificial in design. Functionally, the polypeptide linked to the hogoshidin peptide ("carrier polypeptide") may serve to stabilize the hogoshidin peptide and protect it from proteases, although the carrier polypeptide need not have been shown to serve such purposes. Similarly, the carrier polypeptide may facilitate transport of the fusion peptide. Examples of usable carrier polypeptides include anionic pre-propeptides and anionic outer membrane peptides. Examples of carrier polypeptides include glutathione S-transferase (GST), protein A from Staphylococcus aureus, two synthetic IgG-binding domains of protein A (ZZ), outer membrane protein F from Pseudomonas aeruginosa, and protein transduction domains. The present disclosure is not limited to the use of these polypeptides; other suitable carrier polypeptides are known to those of skill in the art. In another embodiment, a linker moiety comprising a protease cleavage site can be operably linked to a hogoshidin peptide or variant of the present disclosure. For example, the linker can be operable between domains of a fusion protein (e.g., a fusion protein comprising a hogoshidin peptide and a carrier polypeptide). Because protease cleavage recognition sequences are generally only a few amino acids in length, the linker moiety can include the recognition sequence within a flexible spacer amino acid sequence, such as GGGGS (SEQ ID NO: 6). For example, a linker moiety comprising a cleavage recognition sequence for adenoviral endopeptidase can have the sequence GGGGGGSMFGGAKKRSGGGGGG (SEQ ID NO: 7). If desired, the spacer DNA sequence can encode a protein recognition site for cleavage of the carrier polypeptide from the hogoshidin peptide.Examples of such spacer DNA sequences include, but are not limited to, protease cleavage sequences, such as those for factor Xa protease, methionine, tryptophan, and glutamic acid codon sequences, and pre-prodefensin sequences. Factor Xa is used for proteolytic cleavage at the factor Xa protease cleavage sequence, while chemical cleavage by cyanogen bromide treatment releases peptides at methionine or related residues. Furthermore, the fusion product can be cleaved by inserting a codon for tryptophan (cleavable by o-iodosobenzoic acid) or glutamic acid (cleavable by Staphylococcus proteases). Insertion of such spacer DNA sequences is not a requirement for producing a functional hogoshidin peptide, and such sequences can enhance the stability of the fusion peptide. Because the pre-prodefensin sequence is negatively charged, it is contemplated within the present disclosure that other DNA sequences encoding negatively charged peptides may also be used as spacer DNA sequences to stabilize the fusion peptide.

[0077] The present disclosure also provides a method for inhibiting bacterial growth by contacting the bacteria with an inhibitory-effective amount of a peptide of the present disclosure. The term "contacting" refers to exposing the bacteria to the peptide so that the peptide can inhibit, kill, or lyse the bacteria. The present disclosure also provides a method for inhibiting a skin disease or disorder and / or a bacterial infection, comprising placing a probiotic preparation containing a peptide or bacteria secreting an antimicrobial molecule on or within a subject to inhibit or prevent the growth of pathogens or undesirable microorganisms. Contacting an organism with a hogoshidin peptide of the present disclosure can be performed in vitro, for example, by adding the peptide to a bacterial culture to test the susceptibility of the bacteria to the peptide or by contacting a bacterially contaminated surface with the peptide. Alternatively, contacting can be performed in vivo, for example, by administering the peptide to a subject suffering from or susceptible to a bacterial infection. Furthermore, contacting can be performed by exposing the bacteria to a probiotic preparation containing a bacterial strain that produces a hogoshidin peptide or other peptide or non-peptide inhibitor of bacterial growth. In vivo contacting includes both parenteral and topical administration. "Inhibition" or "inhibitory effective amount" refers to an amount of peptide sufficient to cause, for example, a bacteriostatic or bactericidal effect. Bacteria that can be affected by the peptides of the present disclosure include both gram-negative and gram-positive bacteria.For example, bacteria that may be affected include Staphylococcus aureus, Streptococcus pyogenes (group A), Streptococcus sp. (viridans group), Streptococcus agalactiae (group B), Streptococcus bovis, Streptococcus sp. (anaerobic species), Streptococcus pneumoniae, and Enterococcus sp.; gram-negative cocci, e.g., Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis; gram-positive bacilli, e.g., Bacillus anthracis, Bacillus subtilis, Propionibacterium acnes (P. Acnes, Corynebacterium diphtheriae, and the diphtheroids (aerobic and anaerobic) Corynebacterium sp., Listeria monocytogenes, Clostridium tetani, Clostridium difficile, Escherichia coli, Enterobacter species, Proteus mirabilis and other genera and species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella, Serratia sp., and Campylobacter jejuni. Infection with one or more of these bacteria can result in diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne vulgaris, wound infections, congenital infections, fasciitis, bronchitis, and various abscesses, hospital-acquired infections, and opportunistic infections.Fungal organisms may also be affected by the hogoshidin peptides of the present disclosure, including dermatophytes (e.g., Microsporum canis and other Microsporum species; and Trichophyton species, such as T. rubrum and T. mentagrophytes), yeasts (e.g., Candida albicans, C. tropicalis, or other Candida species), Saccharomyces cerevisiae, Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur, and the like. furfur (Pityropsporon orbiculare, or P. ovale), Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus nidulans, and other Aspergillus species, Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomyces dermatitides, Histoplasma capsulatum, capsulatum, Coccidioides immitis, and Sporothrix schenckii. Methods of inhibiting bacterial growth can also include contacting the bacteria with the peptide in combination with one or more antibiotics.

[0078] The peptides of the present disclosure can be administered to any host, including humans or non-human animals, in an amount effective to inhibit the growth of bacteria, viruses, or fungi. Thus, the peptides are useful as antibacterial, antiviral, and / or antifungal agents. Bacterial strains producing the peptides are useful as probiotic agents.

[0079] Peptides can be administered to a subject using any of a variety of methods known in the art. For example, peptides of the present disclosure can be administered parenterally by injection or by gradual infusion over time. Peptides can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, topically, or transdermally. In another embodiment, hogoshidin peptides of the present disclosure can be formulated for topical administration (e.g., as lotions, creams, sprays, gels, oily suspensions, or ointments). Examples of commercially available formulations include topical lotions, creams, soaps, wipes, powders, and devices such as gauze pads for wound coverage. They may also be formulated in liposomes to reduce toxicity or increase bioavailability or stability. Other methods for peptide delivery include encapsulation of peptides in microspheres or proteinoids, aerosol delivery (e.g., to the lungs), or oral delivery involving transdermal delivery (e.g., via iontophoresis or transdermal electroporation). Other administration methods are known to those skilled in the art.

[0080] Preparations for parenteral administration of the peptides of the present disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, saline, and buffered media, alcoholic / aqueous solutions, and emulsions or suspensions. Examples of parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be included, such as other antibacterial agents, antioxidants, chelating agents, inert gases, and the like.

[0081] The present disclosure provides methods for inhibiting localized bacterial or fungal-associated disorders by contacting or administering a therapeutically effective amount of a peptide or skin probiotic of the present disclosure to a subject having or at risk of having such a disorder. The term "inhibition" refers to preventing or ameliorating a sign or symptom of the disorder (e.g., rash, pain, etc.). Examples of disease symptoms that may be ameliorated include elevated blood levels of TNF in the subject, fever, hypotension, neutropenia, leukopenia, thrombocytopenia, disseminated intravascular coagulation, adult respiratory distress syndrome, shock, and organ failure. Examples of subjects that can be treated in the present disclosure include human or animal subjects at risk for or suffering from toxemia, such as endotoxemia resulting from gram-negative bacterial infection, envenomation, or liver failure. Other examples include subjects with dermatitis and skin infections, such as mastitis and especially bovine mastitis, or injuries that expose the subject to infection by gram-positive or gram-negative bacteria or fungi. Examples of candidate patients include patients suffering from infections caused by Escherichia coli, Haemophilus influenzae B, Neisseria meningitidis, Staphylococcus (Staphylococcus), or Streptococcus pneumoniae. Other patients include those suffering from gunshot wounds, renal or liver failure, trauma, burns, immunodeficiency infections (e.g., HIV / SIV / FIV infection), hematopoietic neoplasms, multiple myeloma, Castleman's disease, or myocardial myxoma. Those skilled in the medical arts can readily employ conventional criteria to identify appropriate subjects for treatment in accordance with the present disclosure.

[0082] The term "therapeutically effective amount," as used herein with respect to treating a subject suffering from a disease or disorder, refers to an amount of hogoshidin peptide sufficient to ameliorate the signs or symptoms of the disease or disorder. For example, a therapeutically effective amount can be measured as an amount sufficient to reduce a subject's symptoms of dermatitis or rash by measuring the frequency and severity of skin inflammation. Typically, a subject is treated with an amount of hogoshidin peptide sufficient to reduce the symptoms of the disease or disorder by at least 50%, 90%, or 100%. Generally, the optimal dosage of the peptide depends on the disorder and factors such as the patient's weight, type of bacterial or fungal infection, weight, sex, and severity of symptoms. Nevertheless, an appropriate dosage can be readily determined by one of skill in the art. Typically, an appropriate dosage is 0.5-40 mg peptide / kg body weight, e.g., 1-8 mg peptide / kg body weight.

[0083] If desired, a suitable therapeutic regimen can combine administration of the peptide(s) or probiotic composition of the present disclosure with administration of one or more additional therapeutic agents (e.g., TNF inhibitors, antibiotics, etc.). The peptide(s), other therapeutic agents, and / or antibiotic(s) can be administered simultaneously or sequentially. Suitable antibiotics include aminoglycosides (e.g., gentamicin), beta-lactams (e.g., penicillins and cephalosporins), quinolones (e.g., ciprofloxacin), and novobiocin. Generally, antibiotics are administered in bactericidal amounts. However, the present peptides provide a method for increasing antibiotic activity. Typically, the hogoshidin peptide and antibiotic are administered within 48 hours of each other (e.g., 2-8 hours apart, or may be administered simultaneously). A "bactericidal amount" is an amount sufficient to achieve a bacterial-killing blood concentration in the subject being treated. According to its conventional definition, an "antibiotic," as used herein, is a chemical compound that inhibits the growth of or kills microorganisms in a dilute solution. The term also includes synthetic antibiotics (eg, analogs) known in the art.

[0084] The peptides of the present disclosure can be used, for example, as preservatives or sterilants for materials susceptible to microbial or viral contamination. For example, the peptides can be used as preservatives in processed foods (e.g., to inhibit organisms such as Salmonella, Yersinia, Listeria, and Shigella). If desired, the peptides can be used in combination with antibacterial food additives such as lysozyme. The peptides and / or probiotics of the present disclosure can also be used as topical agents, for example, to inhibit Pseudomonas or Streptococcus or to kill odor-producing microorganisms (e.g., Micrococcus). The optimal amount of the hogoshidin peptides of the present disclosure for any given application can be easily determined by one of skill in the art.

[0085] The hogoshidin and / or probiotic of the present disclosure are also useful for promoting wound repair and tissue regeneration. Matrix metalloproteinases (MMPS) are inflammatory enzymes that degrade proteins in various tissues. Recent scientific studies have shown elevated levels of proteases (e.g., MMPs) in chronic wound exudate, the fluid that bathes the wound bed. These excess proteases cause the degradation of important extracellular matrix proteins and the inactivation of important growth factors essential for the wound healing process. This may contribute to a suboptimal healing environment, which results in delayed wound healing.

[0086] The compositions provided herein can be used simultaneously with the following other antibacterial agents: sulfonamides, such as sulfamethizole, sulfisoxazole, sulfamonomethoxine, sulfamethizole, salazosulfapyridine, and silver sulfadiazine; quinolone antibacterials, such as nalidixic acid, pipemidic acid trihydrate, enoxacin, norfloxacin, ofloxacin, tosufloxacin tosylate, ciproxacin hydrochloride, lomefloxacin hydrochloride, sparfloxacin, and fleroxacin; and antituberculosis drugs, such as isoniazid and ethambutol. antimycobacterial agents, such as diaphenylsulfone and rifampicin; antiviral agents, such as idoxuridine, acyclovir, vidarabine, and ganciclovir; anti-HIV agents, such as zidovudine, didanosine, zalcitabine, indinavir sulfate ethanolate, and ritonavir; antispirochetal agents; antibiotics, such as For example, tetracycline hydrochloride, ampicillin, piperacillin, gentamicin, dibekacin, kanendomycin, lividomycin, tobramycin, amikacin, fradiomycin, sisomicin, tetracycline, oxytetracycline, rolitetracycline, doxycycline, ampicillin, piperacillin, ticarcillin, cephalothin, cephapirin, cephaloridine, cefaclor, cephalexin, cefroxadine, cefadroxil, cefamandole, cefotoam, cefuroxime, cefotiam, cefotiam hexetil, cefoxitin, cefotiam, cefotiam hexetil, cefotiam ... Examples include furoxime axetil, cefdinir, cefditoren pivoxil, ceftazidime, cefpiramide, cefsulodin, cefmenoxime, cefpodoxime proxetil, cefpirome, cefozopran, cefepime, cefsulodin, cefmenoxime, cefmetazole, cefminox, cefoxitin, cefbuperazone, latamoxef, flomoxef, cefazolin, cefotaxime, cefoperazone, ceftizoxime, moxalactam, thienamycin, sulfazecin, aztreonam or a salt thereof, griseofulvin, and lankacidin group.

[0087] In humans, there are several classes of known antimicrobial peptides (AMPs), including α-defensins, β-defensins, and cathelicidins. Cathelicidins are found in several mammalian species. Cathelicidin production is induced in response to epithelial wounds or infectious challenges, or suppressed by the pathogenic mechanisms of certain bacterial pathogens, such as Shigella dysenteriae. Cathelicidin expression is also differentially affected in certain chronic inflammatory disorders. In psoriasis, cathelicidin levels are elevated and secondary infections are rare, whereas in atopic dermatitis, cathelicidin expression is absent and bacterial or viral superinfections are common. The therapeutic benefits of cathelicidins have been experimentally demonstrated, including reduced bacterial colonization in skin wounds after topical administration and improved pulmonary bacterial clearance by cathelicidin overexpression via viral gene transfer. The hogoshidin peptides of the present disclosure exhibit synergistic effects with cathelicidins. Thus, in some embodiments, formulations, compositions, and methods include both hogoshidin and cathelicidin. In some embodiments, a topical formulation (e.g., a lotion, ointment, or aerosol spray) can include both cathelicidin and hogoshidin peptides (or derivatives thereof).

[0088] Cathelicidin proteins are composed of two distinct domains: an N-terminal "cathelin-like" or "pro-sequence" domain and a C-terminal domain of mature AMP. The C-terminal domain of cathelicidin is among the earliest mammalian AMPs and exhibits potent, rapid, and broad-spectrum killing activity. The term "cathelin-like" derives from the similarity of its N-terminal sequence to cathelin, a 12 kDa protein isolated from porcine neutrophils that shares similarity with the cystatin superfamily of cysteine ​​protease inhibitors.

[0089] Cathelicidin was the first AMP discovered in mammalian skin because it is expressed in neutrophils, myeloid myeloid cells, and most epithelial sources, and is present in wound fluid. In neutrophils, cathelicidin is synthesized as a full-length precursor and targeted to secondary granules where it is stored. Upon stimulation, the full-length cathelicidin protein is proteolytically processed, releasing the bactericidal activity of the C-terminal peptide from the cathelin-like domain.

[0090] The C-terminal 37 amino acids of human cathelicidin (LL-37) have been characterized. LL-37, originally called FALL39, was named for the first four N-terminal amino acids and the total number of residues (i.e., 39) in this domain. LL-37 is a peptide predicted to contain an amphipathic alpha helix and lacks cysteines, which distinguishes it from all other previously isolated human peptide antibiotics of the defensin family, each of which contains three disulfide bridges. Full-length human cathelicidin (sometimes referred to as full-length LL-37) includes the cathelin-like precursor protein and the C-terminal LL-37 peptide, and thus contains 170 amino acids (SEQ ID NO: 5).

[0091] A polypeptide comprising SEQ ID NO:5 has a number of different domains. For example, there is a single domain comprising the sequence set forth from about 1 to about 29-31 of SEQ ID NO:5. The single domain is typically cleaved after amino acid number 30 of SEQ ID NO:5; however, one of skill in the art will recognize that the cleavage site may vary by one to three amino acids in either direction from amino acid number 30 of SEQ ID NO:5, depending on the enzyme used, the expression system used, and / or the conditions under which proteolytic cleavage of the polypeptide occurs. Another domain comprises an N-terminal domain designated the cathelin-like domain. The cathelin-like domain comprises from about amino acid number 29 (e.g., 29-31) to about amino acid number 128 (e.g., 128-131) of SEQ ID NO:5. Yet another domain of SEQ ID NO:5 comprises a C-terminal domain designated LL-37. The LL-37 domain comprises from about amino acid number 128 (e.g., 128-134) to amino acid number 170 of SEQ ID NO:5. LL-37 comprises the amino acid sequence set forth in SEQ ID NO:5. MKTQRNGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEFKRIVQRIDDFLRNLVPRTES (SEQ ID NO: 5)

[0092] The mechanism by which cationic human antimicrobial peptides kill bacteria and fungi is generally via binding of the peptides to the microbial cell membrane, which subsequently disrupts the membrane's proton gradient and integrity.

[0093] Vitamin D3 (or its analogs) in combination with hogoshidin (and in some embodiments, cathelicidin) can be administered systemically to treat systemic infections, particularly pneumonia, sepsis, and TB (tuberculosis). It can also be applied topically to treat infectious skin disorders. It can be used in combination with antibiotics or to treat immunosuppressed patients, such as HIV-positive individuals. In combination with an immunostimulatory approach, it can therapeutically address cancer.

[0094] The compositions and methods of the present disclosure may also include treating skin dysbiosis disorders by administering antimicrobial compounds or organisms that secrete antimicrobial compounds, or by administering probiotic compositions containing organisms that support skin health. In some embodiments, the composition includes a second active agent (e.g., an antibiotic, vitamin D3, cathelicidin, etc.).

[0095] In some embodiments, the compositions described herein comprise a probiotic organism. In further embodiments, the probiotic organism is a bacterium. In further embodiments, the bacterium comprises a component of normal skin flora. In further embodiments, the bacterium comprises a strain of Staphylococcus hominis. In other embodiments, the bacterium comprises a strain of Staphylococcus epidermidis. In other embodiments, the probiotic organism comprises a mixture of strains. In some embodiments, the mixture of strains comprises multiple strains of Staphylococcus hominis. In other embodiments, the mixture of strains comprises multiple strains of Staphylococcus epidermidis. In other embodiments, the mixture of strains comprises one or more strains of Staphylococcus hominis and one or more strains of Staphylococcus epidermidis. In some embodiments, the composition comprises one or more strains in addition to Staphylococcus hominis and / or Staphylococcus epidermidis. In some further embodiments, the additional strain comprises one or more strains from the genera Staphylococcus, Lactobacillus, or Lactococcus. For example, specific formulations may include Staphylococcus hominis or Staphylococcus epidermidis, and in particular, Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, and / or Staphylococcus epidermidis strain AMT5-G6. Such formulations typically have a 10% or greater efficacy when applied to the skin of a subject. 3 ~10 6 CFU / cm 2 Such a preparation contains a sufficient amount of bacterial cells to provide a final density of approximately 10 4 ~about 10 7 CFU / g, or 10 to approximately 10 5 CFU / g, or approximately 10 5 ~about 10 9The formulation may comprise a concentration of 0.1 CFU / g. Such formulations may include multiple strains of Staphylococcus hominis and / or Staphylococcus epidermidis, and may further include Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such species or strains known in the art that form part of the normal, healthy flora of the skin or mucous membranes. In some embodiments, the Staphylococcus hominis strains described above comprise 100% of the bacterial cells in the formulation. In some further embodiments, Staphylococcus hominis comprises 90-100%, 85-95%, 70-80%, 75-85%, 60-70%, 65-75%, 50-60%, 55-65%, 40-50%, 45-55%, 30-40%, 35-45%, 20-30%, 25-35%, 10-20%, 15-20%, 1-10%, 5-15%, or less than 1% of the bacterial cells in a given formulation, with the remainder of the colony forming units being provided by Staphylococcus epidermidis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such strains as are known in the art that form part of the normal, healthy skin or mucosal flora. In some embodiments, the above-mentioned S. epidermidis strain comprises 100% of the bacterial cells in the formulation. In some further embodiments, Staphylococcus epidermidis comprises 90-100%, 85-95%, 70-80%, 75-85%, 60-70%, 65-75%, 50-60%, 55-65%, 40-50%, 45-55%, 30-40%, 35-45%, 20-30%, 25-35%, 10-20%, 15-20%, 1-10%, 5-15%, or less than 1% of the bacterial cells in a given formulation, with the remainder of the colony forming units being provided by Staphylococcus hominis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such strains as are known in the art that form part of the normal, healthy flora of the skin or mucous membranes.In some embodiments, bacteria other than Staphylococcus hominis or Staphylococcus epidermidis comprise about 50% or less of the bacterial cells in a formulation. In some embodiments, the bacteria comprise less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the bacterial cells in a given formulation. In some embodiments, bacteria other than Staphylococcus hominis or Staphylococcus epidermidis may include Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such strains known in the art that form part of the normal, healthy flora of skin or mucous membranes. In some embodiments, the formulation comprises about 60% Staphylococcus hominis of the strains listed above and about 40% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 50% Staphylococcus hominis of the strains listed above and about 50% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 40% Staphylococcus hominis of the strains listed above and about 60% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 70% Staphylococcus hominis of the strains listed above and about 30% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 30% Staphylococcus hominis of the strains listed above and about 70% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 80% Staphylococcus hominis of the strains listed above and about 20% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises about 20% Staphylococcus hominis of the strains listed above and about 80% Staphylococcus epidermidis of the strains listed above, hi some embodiments, the formulation comprises about 90% Staphylococcus hominis of the strains listed above and about 10% Staphylococcus epidermidis of the strains listed above.In some embodiments, the formulation comprises greater than about 90% Staphylococcus hominis of the strains listed above and less than about 10% Staphylococcus epidermidis of the strains listed above. In some embodiments, the formulation comprises less than about 10% Staphylococcus hominis of the strains listed above and greater than about 90% Staphylococcus epidermidis of the strains listed above.

[0096] As used herein, autologous transplantation refers to the transplantation of a bacterial strain from one site to another or to the same site in the same subject, regardless of whether the strain is cultured prior to administration. In some embodiments, a bacterial strain obtained from a subject is grown in culture and then transplanted back into the subject.

[0097] As used herein, allogeneic transplantation refers to the transplantation of a bacterial strain from one subject to another, or the administration to a subject of a composition comprising a bacterial strain that is not obtained from on or in the subject's own body.

[0098] Such recovery can be achieved by swabbing, scraping, wiping, cutting, and removing tissue on which one of the bacterial strains described herein is present; optionally by growing and isolating a single colony from an agar plate or otherwise using methods known in the art; optionally by growing a growth culture of the isolated bacteria or an untreated swab, wipe, scraping, tissue, or other isolate in liquid or solid culture according to methods known in the art; optionally by recovering the bacteria from the growth culture by centrifugation, filtration, gravity settling, scraping, or other means known in the art; by formulating the bacteria or crude isolate with a thickening agent, carrier, or excipient; and contacting the formulation with the subject in the area determined to require transplantation.

[0099] As used herein, prebiotic compounds include polysaccharides, hydrolysates, salts, herbal extracts, or any other compounds sufficient to promote the growth of the relevant probiotic strain when used in combination with the strain, e.g., yeast hydrolysate at a concentration of less than about 40% (w / w), microcrystalline cellulose at a concentration of less than about 10% (w / w), and / or sucrose at a concentration of less than about 10% (w / w). Other examples of prebiotics that can be adapted for use with skin bacteria include inulin, glucooligosaccharides, isomaltooligosaccharides, lactosucrose, polydextrose, soybean oligosaccharides, and xylooligosaccharides, as well as those disclosed in Gibson, GR and Roberfroid, M, (Eds.) Handbook of Prebiotics, CRC press (2008); Roberfroid, M., J. Nutr. 137(3):830S-837 (2007) and Slavin, J. Nutrients 5(4):1417-1435 (2013), each of which is incorporated herein by reference in its entirety.

[0100] In some embodiments, the method comprises contacting a subject with a probiotic and / or prebiotic composition described herein. In some embodiments, such contacting comprises autologous transplantation. In some embodiments, such contacting comprises allogeneic transplantation, in which elements of the skin or mucosal flora are transplanted from a second subject (donor) to a first subject in need thereof. For example, in some embodiments, a bacterial strain disclosed above is identified and isolated from the second subject, grown in an appropriate medium under conditions known in the art to promote bacterial growth, followed by harvesting the bacterial cells, mixing the harvested cells with a predetermined formulation at a predetermined concentration according to the present disclosure, and applying the mixture to the affected area of ​​the first subject. In some embodiments, such a composition comprises a standardized formulation, e.g., a formulation in which the concentrations of components are fixed and do not vary from subject to subject. In some embodiments, formulations are developed independently for each subject, based on criteria such as, but not limited to, the composition of the subject's own skin or mucosal flora; the subject's disease state and treatment history; the nature and severity of the subject's condition; the nature and severity of concurrent skin or mucosal infections; the presence of other antibacterial compounds, including systemic antibiotics, in the subject's body; and other criteria known or readily apparent to one of skill in the art.

[0101] In some embodiments, the composition comprises a cream, ointment, oily suspension, or unguent, wherein the probiotic bacteria are incorporated into a moisturizer or emulsion as described below or in Nakatsuji, T. et al. (2016), Nature Medicine Submitted Manuscript No. NMED-A78395A, submitted March 29, 2016. In some embodiments, the composition comprises a patch or compress, wherein the bacteria are combined with suitable excipients and incorporated into a fabric, gel matrix, or polymer sheet. Suitable excipients and carriers for topical administration are known in the art and include thickeners, emulsifiers, fatty acids, polysaccharides, polyols, and polymers and copolymers, including, but not limited to, alginate, microcrystalline cellulose, polylactic acid, polylactic-co-glycolic acid, petrolatum, and many others known in the art.

[0102] In some embodiments, the composition comprises bacterial culture medium, conditioned bacterial culture medium, and / or a bacterial culture. In some embodiments, the composition comprises a filtrate or supernatant of a bacterial culture medium. In some embodiments, the composition comprises a lyophilized culture medium. In some embodiments, the composition comprises a lyophilized conditioned culture medium made from a filtrate or supernatant of a bacterial culture medium.

[0103] In some embodiments, the methods described herein include supporting skin health in a subject. In further embodiments, the methods include providing treatment for skin dysbiosis and disorders resulting therefrom. In some embodiments, the methods include providing treatment for bacterial skin infections. In some embodiments, the treatment includes identifying a subject with skin dysbiosis, bacterial infection, mastitis, a burn or other wound, atopic dermatitis, psoriasis, or other chronic skin condition; and administering a probiotic composition disclosed herein to the site of the condition in need of treatment. Determining the appropriate mode of administration for a given formulation (ointment, gel, patch, etc.) can be performed by one skilled in the art of treating skin infections. In some further embodiments, the probiotic composition is reapplied at regular time intervals. In some embodiments, the probiotic composition is reapplied every three days. In some embodiments, the probiotic composition is reapplied every two days. In some embodiments, the probiotic composition is reapplied every two days. In some embodiments, the probiotic composition is reapplied daily. In some embodiments, the probiotic composition is reapplied two or more times daily. In some embodiments, the probiotic composition is reapplied weekly. In some embodiments, the probiotic composition is applied only once.

[0104] In some embodiments, the method includes providing treatment for a Staphylococcus aureus infection, including methicillin- or oxacillin-resistant Staphylococcus aureus. In some further embodiments, the method includes diagnosing a Staphylococcus aureus infection; and applying a probiotic and / or prebiotic composition disclosed herein to the site of infection, where such a composition can kill or inhibit the growth of Staphylococcus aureus through the production of antimicrobial compounds, through competition for resources within the skin or mucosal biota, or by other means. Determining the appropriate mode of administration for a given formulation (ointment, gel, patch, etc.) can be performed by one skilled in the art of treating skin infections. In some further embodiments, the probiotic composition is reapplied at regular time intervals (e.g., daily, every two days, every three days, weekly, etc.). It will be apparent to one of skill in the art that in other embodiments, similar or identical steps are applied to provide treatment for Pseudomonas aeruginosa infections, or infections from bacteria of the genera Pseudomonas, Staphylococcus, Propionibacterium, Streptococcus, or Vibrio, or uncharacterized pathogens. In some embodiments, the method includes providing treatment for infections with unknown or uncharacterized pathogens. In some embodiments, the method includes providing treatment for polymicrobial infections. In some embodiments, the method includes administering such treatment to a burn or wound. In some embodiments, the method includes providing treatment for a chronic skin condition. In some embodiments, such a condition is atopic dermatitis, psoriasis, or other chronic skin condition.

[0105] The following examples are intended to illustrate but not limit the disclosure, and while they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. [Example]

[0106] [Example 1] Isolation of CoNS strains and antimicrobial peptides. Adult patients with atopic dermatitis and age-matched non-atopic subjects were recruited. Demographic data are shown in Table 1. All experiments involving human subjects were conducted in accordance with institutionally approved IRB protocols.

[0107] [Table 1]

[0108] Measurement of bacterial abundance. Viable surface bacteria and bacterial DNA were recovered from pre-measured areas (approximately 3 × 10 cm) of lesional skin in the antecubital fossa and non-lesional skin on the upper arm at least 2 cm away from the lesion site. Similar recoveries were obtained from non-atopic subjects at the same skin site. Skin was swabbed with cotton swabs pre-moistened with tryptic soy broth (TSB) to recover viable bacteria or Tris-EDTA buffer to recover bacterial DNA. Viable bacterial samples were plated onto mannitol salt agar containing egg yolk to identify coagulase-negative staphylococci (CoNS). Total genomic DNA was extracted using a QIAamp DNA Micro Kit (Qiagen), and DNA abundance was determined by quantitative real-time PCR (qPCR) using species- or genus-specific primers. Of the 50 patients recruited, one subject with atopic dermatitis had no detectable viable staphylococci or staphylococcal DNA. Therefore, data are reported for 49 AD subjects.

[0109] Bacterial DNA quantification. To recover bacterial DNA, a cotton swab pre-moistened with Tris-EDTA buffer containing 0.1% Triton X-100 and 0.05% Tween-20 (w / v) was used to scrub a pre-measured area similar to that used for bacterial culture. Bacterial cells were lysed with proteinase K, followed by purified achromopeptidase (Wako Chemical) and Ready-Lyse® (Epicentre Inc.). Total genomic DNA was purified using a QIAamp DNA Micro Kit (Qiagen) and eluted with 50 μL of elution buffer. Bacterial DNA abundance was determined by quantitative real-time PCR (qPCR) using species- or genus-specific primers (Table 2). To determine the relative CFU (rCFU) of Staphylococcus spp. DNA, a standard curve was generated using genomic DNA extracted from a standard derived from a known CFU of Staphylococcus epidermidis (ATCC 12228). The specificity of all primer pairs was confirmed by melting curve analysis and comparison with standard curves.

[0110] [Table 2]

[0111] Screening for antibacterial activity. Up to 84 individual colonies of CoNS isolates from each skin site were randomly picked and transferred to 96-well cluster tubes containing TSB. Each plate also contained a non-antibacterial strain of Staphylococcus epidermidis (ATCC 1457) as a negative control, a known antibacterial strain of Staphylococcus hominis (see below) as a positive control, and a blank well containing no bacteria. CoNS were incubated overnight at 37°C with shaking. Growth was monitored at OD 600 The bacteria were removed by centrifugation and then sterilized by filtration through a 0.22 μm membrane. The antibacterial activity released from each colony was evaluated using 1 × 10 4 Antibacterial strains were assessed by mixing colony-forming units (CFU) with S. aureus. Antibacterial strains were defined as those that showed growth after 22 hours of less than 50% of the growth seen in the negative control (I50 ) were defined as strains suppressed by CoNS. Poor CoNS colonies were grown from a subset of recruited subjects. Thus, data are reported for 29 non-atopic subjects and 41 non-lesional and 40 lesional sites from atopic subjects. All CoNS isolates were cryopreserved for species identification. Full-length 16S rRNA genes were amplified from 48 representative colonies using universal 16S primers 27-F and 1525-R. Amplicons were sequenced from both ends by the Sanger method.

[0112] Purification of AMPs produced by Staphylococcus hominis. Sterile conditioned medium from a representative antibacterial Staphylococcus hominis strain isolated from a healthy subject was used to further identify molecules with antibacterial activity on normal skin, present in low amounts on atopic skin. The active material was precipitated with ammonium sulfate (70% saturation), dissolved in HO, and applied to a Sep-Pak cartridge (Waters Co.). The active fraction was eluted with 30% acetonitrile in HO and subjected to HiTrap® SP (GE Healthcare Life Sciences) separation, with the active material eluted with 125 mM NaCl. A third stage of HPLC purification was performed using a CapCel Pak C8 (5 μm, 300 Å, 4.6 × 250 mm) (Shiseido Co.) with a linear gradient of 5% to 50% acetonitrile in 0.1% (v / v) TFA at 0.8 mL / min.

[0113] Identification of AMPs produced by Staphylococcus hominis. Antibacterial activity was purified from sterile conditioned medium of a representative antibacterial Staphylococcus hominis strain isolated from a nonatopic subject. The secondary structure of the purified active molecule was determined by MALDI-TOF / TOF, Edman end-sequencing, and genome sequencing.

[0114] Mass spectrometry. Mass spectra of HPLC-purified AMP from Staphylococcus hominis were recorded using a MALDI-TOF / TOF Bruker Autoflex™ Speed ​​instrument (Bruker Daltonics) controlled by FlexControl software (Bruker Daltonics). Mass spectrometry was performed in positive ion reflectron mode using cyano-4-hydroxycinnamic acid (CHCA) 10 mg / mL (Sigma-Aldrich) as the matrix (CHCA) dissolved in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). Full-scan mass spectra were acquired in positive ion reflectron mode over the mass range of 1000–4000 m / z. Each mass spectrum was the result of 750 averaged laser shots with the laser intensity set to approximately 65% ​​of the full laser intensity and the detector gain enhanced to 8 × 4 GS / s (selected within the Bruker FlexControl software). MALDI-MS / MS spectra of the manually selected ion m / z 3547 were acquired using TOF / TOF collision-induced dissociation with a 5 Da window. Each MS / MS spectrum was the result of 1000 averaged laser shots, with the laser intensity set to approximately 60% as selected in the software and the detector gain enhanced to 10 × 4 GS / s. The resulting mass spectra were analyzed using Flex analysis software (Bruker Daltonics). Spectra were calibrated against PepMix internal standard solution.

[0115] N-terminal protein sequencing. The N-terminal amino acid sequence of purified hogoshidin-α (fraction 30, FIG. 7A) was analyzed by 15 cycles of Edman degradation on a Procise® 494HT protein sequence system (Applied Biosystems). The predicted mature form of hogoshidin-α is shown in FIG. 5A.

[0116] Protein sequencing by MALDI-TOF / TOF analysis. The N-terminal region of hogoshidin-β from Staphylococcus hominis (fraction 32, Figure 8) contains modified amino acids, so the sequence could not be obtained by Edman degradation. Therefore, the entire protein sequence of this AMP was obtained based on genome-guided MALDI-TOF / TOF analysis. Analysis of the Staphylococcus hominis nucleotide sequence was performed using the Antibiotic and Secondary Metabolite Analysis Shell-AntiSMASH platform to identify secondary metabolite biosynthetic gene clusters. AntiSMASH results provided one gene cluster for lanthipeptides with potential candidates at loci 2050–2250 (Figure 9A). NCBI BlastP analysis of genes involved in synthesis and modification showed high homology to class 2 lanthipeptides. A core peptide with the sequence ATPTITTSSATCGGIIVAASAAQCPTLACSSRCGKRKK (amino acids [aa] 29-66 of SEQ ID NO: 4) was cleaved from the leader peptide at a GG cleavage site common to type 2 lantibiotics. By combining genome mining and MS / MS fragmentation, the mature form of hogoshidin-β was predicted (Figure 5A).

[0117] Genome sequencing. Because the protein sequence of the AMP from Staphylococcus hominis did not match any molecules found in existing genome databases, whole-genome sequencing of an antibacterial strain of Staphylococcus hominis was performed. Genomic DNA was purified using the UltraClean® Microbial DNA Isolation Kit (MO Bio). A whole-genome DNA sequencing library was constructed using the Nextera-XT DNA Sample Preparation Kit (Illumina) according to the supplier's protocol. The final library was sequenced by paired-end sequencing (300 × 300) on an Illumina MiSeq™. Sequenced reads were de novo assembled using SPAdes 2.5.1 with k-mer lengths of 21, 33, 55, 77, and 127 and flagged for "careful" analysis. All generated scaffolds were subjected to a six-frame translation using translate Whole Genome Multi Chromosome.pl ([http: / / ]proteomics.ucsd.edu / Downloads / ), and this output was then queried for matches to peptide fragments identified by mass spectrometry.

[0118] Antibacterial assay. The purified fractions were tested for antibacterial activity in a radial diffusion assay using Staphylococcus aureus (ATCC 35556) strain. Briefly, molten TSB agar (10 mL) was inoculated with Staphylococcus aureus (1 × 10 6 The test sample was mixed with 1 × 10 CFU and poured into a 10 cm Petri dish. 2–4 μL of the test sample was applied to small wells punched into the agar plate. The plate was incubated overnight at 37°C to allow visible bacterial growth. Antibacterial activity was indicated by a clear zone (no bacterial growth) around the well. The antibacterial activity of hogoshidin was demonstrated against Staphylococcus aureus (1 × 10 5The number of viable bacteria (CFU / mL) was assessed by incubating with two-fold serial dilutions of purified hogoshidin in half-strength Muller-Hinton broth (MHB) in PBS for 24 hours at 37°C. After incubation, the number of viable bacteria was measured by counting CFU after spreading ten-fold serial dilutions of the bacteria on TSB agar plates. The MBC was determined as a 3-log reduction (99.9%) of viable bacteria after 24 hours of incubation.

[0119] Statistical analysis. Paired t-tests were used to compare lesional samples with nonlesional samples within atopic subjects, and independent t-tests were used to compare nonatopic samples with atopic samples. Longitudinal mixed models were also fitted for the frequency of antimicrobial CoNS and the ratio of viable staphylococci to staphylococcal DNA over time. Each model included lesion type, visit, and their interaction period as fixed effects, while a compound symmetry structure was used to account for correlations between samples obtained from the same subject at multiple time points. The frequency of antimicrobial CoNS used a multinomial distribution with a cumulative logit link and categorized percentages (≤20, 21–79, ≥80) to account for the bimodal distribution. Statistical analysis was performed using SAS (version 9.3) software.

[0120] Staphylococcal survival is increased in atopic dermatitis. To assess the relationship between the abundance of cultivable / viable Staphylococcus species and noncultivable / dead Staphylococcus species, bacterial density was measured by both manual colony counts and genus-specific 16S ribosomal DNA qPCR. Consistent with previous reports, total Staphylococcus species and Staphylococcus aureus from lesional skin on the forearms of patients with atopic dermatitis were culturable in greater numbers than those from nonlesional skin of these patients or nonatopic subjects (Figures 1A and 1B). DNA abundance measurements showed a similar trend (Figures 1C and 6). However, the results of these two independent techniques differed significantly between lesional and nonatopic skin of atopic subjects. In lesional skin of subjects with atopic dermatitis, culture- and DNA-based results were similar (Figure 1D). In contrast, in non-atopic skin, the ratio of cultured CFU to relative CFU based on DNA abundance was approximately 0.1, suggesting low survival of bacteria on the skin of non-atopic subjects.

[0121] Antibacterial activity of the skin microbiota. Viable CoNS from 30 non-atopic subjects (2029 colonies) and 50 atopic subjects (5695 colonies) were isolated and their effect on S. aureus survival was characterized. The majority of CoNS clones isolated from non-atopic subjects (75.26 ± 35.49%) were observed to inhibit S. aureus growth (Figure 2A). In contrast, only a small proportion of CoNS found in atopic skin possessed this activity [22.83 ± 32.64% (non-lesional) and 15.76 ± 25.92% (lesional)]. This difference in the antibacterial function of CoNS isolated from each population was stable and reproducible over time, as seen after 2 weeks of repeated swabbing (Figure 2B). The increase in the ratio of culturable staphylococci to total staphylococcal DNA also remained stable over 2 weeks in this cohort (Figure 2C). These data indicate that the skin of patients with atopic dermatitis supports the growth of CoNS bacteria but allows the survival of strains whose antimicrobial function differs from that found in nonatopic skin.

[0122] As shown in Figure 1 , only 3% of nonatopic subjects had positive S. aureus cultures (>1 CFU / cm 2 ), whereas 57% of the atopic subjects were culture-positive for S. aureus. To determine whether the antibacterial activity detected from CoNS was associated with S. aureus survival, we compared the frequency of antibacterial CoNS to measurements of viable S. aureus (Figure 3A). Surprisingly, all patients with viable S. aureus had a lower frequency of antibacterial CoNS. Furthermore, this frequency was significantly higher in the S. aureus culture-positive group (>1 CFU / cm) than in the S. aureus-negative group. 2 ) (Fig. 3B). These data indicate that antimicrobial CoNS are protective against S. aureus colonization.

[0123] Identification of antibacterial bacterial species on skin. To further identify CoNS species with antibacterial activity, a random subset of bacterial colonies was selected for full-length 16S rRNA gene sequencing. In nonatopic skin, the predominant species of antibacterial CoNS were Staphylococcus epidermidis or Staphylococcus hominis (Figure 4A). In atopic subjects, antibacterial CoNS members included Staphylococcus pasteuri, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus epidermidis, and Staphylococcus hominis. However, in most subjects, similar species were identified within groups found to have antibacterial and non-antibacterial functions (Figure 4B). These observations indicate that antibacterial activity cannot be predicted at the species level. Overgrowth of functionally inactive CoNS strains is thought to occur in patients with atopic dermatitis.

[0124] Antibacterial peptides produced by the microbiota. Genetic and biochemical approaches were used to determine the identity of the antibacterial activity detected in CoNS strains. Bacteriocins are a group of AMPs produced by several CoNS species. However, DNA encoding the known bacteriocins epiA, pepA, eciA, and elkA was not detected by PCR in nonatopic skin (n = 14) (Table 2, primer sequences). Therefore, experiments were conducted to purify and identify the source of activity from a representative colony of antibacterial Staphylococcus hominis isolated from a nonatopic subject. Reverse-phase chromatography revealed two independent peaks (3152.2 and 3547.7 Da) associated with antibacterial activity (Figure 7). N-terminal amino acid sequencing of the 3152.2 Da peptide revealed KCSWWNAA. The complete sequence of the 3547.7 Da peptide was obtained by genome-guided MALDI-TOF / TOF analysis (Figure 8). Alignment of the mass spectrometry and amino acid sequences to the genome sequence of this Staphylococcus hominis strain revealed that these novel AMPs were encoded within the gene cluster of lanM, lanC, and lanT homologs (Figure 9), consistent with their identity as lantibiotics. Because these AMPs were previously unknown, they were named hogoshidin-α (3152.2 Da) and -β (3547.7 Da), derived from the Japanese word "hogo," meaning "protection." These newly described AMPs were readily detectable by PCR in 50% of 14 nonatopic individuals. The predicted secondary structures of mature hogoshidin-α and hogoshidin-β are shown in Figure 5A. The minimum bactericidal concentrations (>99.9% killing) of purified hogoshidin-α and -β against S. aureus were 0.625 μM and 1.25 μM, respectively (Figure 5B), demonstrating potent activity compared with conventional AMPs produced on human skin. Importantly, co-incubation of each hogoshidin peptide with the human skin cathelicidin AMP LL-37 exhibited potent synergistic antibacterial activity against S. aureus (Figure 5B), indicating that microbiota-derived AMPs enhance the host's innate immune defenses to resist S. aureus.

[0125] [Example 2] FAME analysis of bacterial strains. Because the lipid composition of whole bacterial cells (mainly cell membranes), which can be represented by the relative abundance of fatty acid methyl esters present in saponified and methylated samples of bacterial cell extracts, is highly specific to each strain, the identified strains were subjected to fatty acid methyl ester (FAME) analysis. Bacterial strains were cultured and harvested according to standard techniques. Cells were subjected to saponification and methylation, and then extracted into the mobile phase solvent for gas chromatography. The samples were loaded and run according to the instrument manufacturer's instructions. The resulting chromatograms are shown in Figures 11-19.

[0126] [Example 3] Commensal bacteria protect skin from colonization by Staphylococcus aureus. After clarifying the relationship between AMP-producing commensal CoNS and colonization by S. aureus and identifying the active peptides produced by these strains, experiments were performed to test whether the presence of these bacteria reduces colonization by S. aureus. Clinical CoNS isolates were applied to the surface of disinfected porcine skin to which a predetermined amount of S. aureus had first been applied. A significant reduction in S. aureus survival was observed at an estimated bacterial density on normal human skin (1 × 10 5 CFU / cm 2 This was seen after a single application of Staphylococcus hominis A9 at a density consistent with that observed in the control group (Figure 20A). Application of S. hominis A9 that had been killed and washed prior to application, or the use of other S. hominis strains that did not demonstrate antibacterial activity in culture, did not affect S. aureus survival. Similarly, a single application of active S. hominis to the backs of mice to which a given amount of S. aureus had been applied reduced S. aureus survival on the skin (Figure 20B). In contrast, application of an inactive strain at a similar density did not inhibit S. aureus.

[0127] Finally, to directly test the ability of functionally screened and isolated commensal bacteria to inhibit S. aureus in humans, we conducted experiments examining the effects of applying these bacteria to the skin of subjects with AD. As previously shown, strains with antibacterial activity were rare in the overall CoNS community of these subjects, but could be identified if sufficient colonies were screened. Five AD patients with positive S. aureus cultures were recruited to participate in this study. CoNS clones with antibacterial activity were identified and expanded for planned reapplication to the subjects (autotransplantation). Each selected clone was sequenced, and clusters of lantibiotic-related genes were identified in five S. epidermidis or S. hominis strains, with colicin V genes found in two S. epidermidis strains and one S. hominis strain (Figures 21–23). In a double-blind study, vehicle cream alone or bacteria formulated in a cream was applied once to the skin of each arm, followed by measurement of S. aureus 24 hours later. The selected strains were 1 x 10 5 CFU / cm 2 These functionally defined, autologous CoNS strains were applied to a total final concentration of 0.01 mg / ml, a density similar to previous assessments of bacterial abundance on normal human skin. A single application of these functionally defined, autologous CoNS strain(s) significantly reduced S. aureus CFUs within 24 hours compared to baseline (Figure 20E).

[0128] [Example 4] Ex vivo transplantation of antimicrobial CoNS into pig skin and mice. Frozen pig skin sheets were obtained from Loretta Tomlin Animal Technologies (Livermore, CA) and disinfected with 3% chloroxylenol using a surgical brush. Skin sheets were cut into 2.5 cm x 2.5 cm pieces and rinsed >20 times with sterile PBS to remove chloroxylenol residue. At least 24 h before bacterial application, the skin on the backs of randomly selected C57BL6 female mice, 6 weeks old, was shaved, treated with depilatory cream, and rinsed with water. The shaved skin was washed twice with alcohol swabs to remove any originally colonizing bacteria. All experiments, including those involving live animals, were conducted in accordance with the approved guidelines for the care and use of laboratory animals.

[0129] Staphylococcus aureus (ATCC35556) (1 × 10 5 CFU / cm 2 ) was challenged percutaneously onto pig skin (2.5 × 2.5 cm) or onto the dorsal skin of mice (2 × 2 cm). Staphylococcus hominis strain A9 isolated from a non-AD subject, which produces the Sh-lantibiotic (hogoshidin), or Staphylococcus hominis strain isolated from the lesional skin of an AD subject, which did not produce antibacterial activity in culture, was used at 1 × 10 7 The test was formulated into a skin moisturizer that was confirmed to have no effect on bacterial viability at CFU / g. The test was then performed with either the anti-S. aureus strain A9, UV-sterilized Staphylococcus hominis A9, or the inactive strains C4, C5, and C6 (1 × 10 5CFU / 10 μL) or vehicle was then applied to the surface of pig skin or mouse dorsal skin for 20 hours (Figures 20A and 20B). Purified lantibiotics (0.5 nmol), Staphylococcus hominis A9 conditioned medium (50 μL) were applied to the surface of disinfected pig skin. The pig skin was incubated at 30°C in a 6-well plate. Viable bacteria were collected from the skin surface using a Catch-All Swab pre-wetted with TSB as described above. Bacteria were vigorously suspended in 1 mL of TSB by vortexing the swab head. Ten-fold serial dilutions of the bacterial suspension were spread on egg yolk tellurite-containing Baird-Parker agar for selective enumeration of Staphylococcus aureus. S. aureus (large black colonies with a halo) was differentiated from S. hominis (small gray colonies without a halo) on the selective agar plate.

[0130] [Example 5] Autologous Microbiota Transplantation. The approach of autologous microbiota transplantation (AMT) for AD patients has been formally approved by the US Food and Drug Administration (FDA), and this protocol has been submitted as an Investigational New Drug (IND) application (UCSD approval number 15786). At the screening visit, AD patients were screened for S. aureus colonization at the lesion sites of both antecubital fossae. Meanwhile, skin samples were obtained by swabbing non-lesional skin on the AD patient's upper arm and screened for CoNS strains that produced antibacterial activity against S. aureus. The species of antibacterial CoNS isolates were identified by Sanger sequencing of the full-length 16S rRNA gene. Glycerol stocks of CoNS isolates were stored at -80°C until the second visit, when the patients underwent transplantation therapy. Each CoNS strain was individually grown overnight in TSB. Each CoNS strain was cultured at 1 × 10 in a skin moisturizer confirmed not to affect bacterial viability. 7The formulations were based on CFU / g. Only a single strain of S. epidermidis or S. hominis with antibacterial activity was isolated from three patients. In these cases, a single strain of CoNS was formulated. Three and two antibacterial strains of S. hominis or S. epidermidis were isolated from two AD patients (Figure 20D). In these cases, equal CFU of each CoNS was formulated at 10 7 At the second visit, the relevant areas were measured and the baseline CFU of viable S. aureus on the lesions on both forearms was quantified as described above. One arm was dosed at 10 mg / cm 2 and treated with AMT formulation at 1 x 10 5 CFU / cm 2 The other arm was simply treated with the same amount of moisturizer. All treatments were performed in a double-blind manner, and all results were analyzed without blinding. Subjects avoided bathing, showering, exercise, or applying any topical products to their arms and wore clean long-sleeved shirts to avoid cross-contamination with CoNS applied to the other arm until the next visit. At the third visit, S. aureus CFU was measured in the involved area. The difference in S. aureus viability between the vehicle and AMT arms was calculated as [AMT (χ time) - vehicle (χ time)] / AMT (baseline), yielding S. aureus CFU Δ% (χ = 0 or 24 hours) (Figure 20E).

[0131] Statistical Analysis. For all experiments, at least three or more biological replicates were used, and these are indicated in the figure legends. For all mouse experiments, at least six mice were used per treatment group. Therefore, for reported differences, the sample sizes used provided sufficient power for reliability. Paired t-tests (two-tailed) were used to compare lesioned and non-lesioned samples within AD subjects, and independent t-tests (two-tailed) were used to compare non-AD and AD samples. For non-normally distributed variables, such as CoNS with Sh-antibiotic-α (%), non-parametric approaches were used, such as the Wilcoxon-Mann-Whitney test for AD versus non-AD samples and the Wilcoxon signed-rank test for lesioned versus non-lesioned samples within AD subjects. Longitudinal mixed models were also fitted for the frequency of antibacterial CoNS and the ratio of viable staphylococci to staphylococcal DNA over time. Each model included lesion type, visit, and their interaction period as fixed effects, while a compound symmetry structure was used to account for correlations between samples obtained from the same subject at multiple time points. The frequencies of antimicrobial CoNS were calculated using a multinomial distribution with a cumulative logit link and categorized percentages (≤20, 21–79, ≥80) to account for the bimodal distribution. Statistical analyses were performed using SAS (version 9.3) and R software (version 3.1.1).

[0132] [Example 6] Allogeneic transplantation. 10 of SH-A9, SH-C2, SE-A11, AMT1, AMT2, AMT3, AMT4-C2, AMT4-G1, AMT4-D12, AMT5-C5, AMT5-G6 and / or SE-MO34 strains. 5CFU / g are formulated into a skin moisturizer that has been confirmed not to affect bacterial viability. Subjects are identified based on the presence of atopic dermatitis and / or active Staphylococcus aureus infection. Subjects are instructed to avoid bathing, showering, exercise, or applying any topical products to the affected area for three days. Eligible patients will show a significant reduction in Staphylococcus aureus levels in the treated area after seven days (a 3-log or greater reduction in recoverable Staphylococcus aureus colony counts). Eligible patients will show a clinically observable reduction in Staphylococcus aureus infection and / or atopic dermatitis symptoms that continues for at least 14 days after initial treatment.

[0133] [Example 6] Purification of AMPs produced by Staphylococcus epidermidis. Sterile conditioned medium from a representative antibacterial S. epidermidis strain A11 was used to further identify molecules with antibacterial activity on normal skin, which was found to be less abundant in atopic skin. The active material was precipitated with ammonium sulfate (70% saturation), dissolved in HO, and applied to a Sep-Pak cartridge (Waters Co.). The active fraction was eluted with 40% acetonitrile in HO and subjected to HiTrap® SP (GE Healthcare Life Sciences) separation, with the active material eluted with 500 mM NaCl. The third stage of HPLC purification was performed using a CapCel Pak C8 (5 μm, 300 Å, 4.6 × 250 mm) (Shiseido Co.) with a linear gradient of 5% to 50% acetonitrile in 0.1% (v / v) TFA at 0.8 mL / min.

[0134] Identification of AMPs produced by S. epidermidis. Antibacterial activity was purified from sterile conditioned medium of a representative antibacterial S. epidermidis strain A11 isolated from a nonatopic subject. The purified active molecule was characterized by MALDI-TOF / TOF and Edman end-sequencing.

[0135] Mass spectrometry. Mass spectra of HPLC-purified AMP from S. epidermidis A11 were recorded using a MALDI-TOF / TOF Bruker Autoflex™ Speed ​​instrument (Bruker Daltonics) controlled by Flex Control software (Bruker Daltonics). Mass spectrometry was performed in positive ion reflectron mode using cyano-4-hydroxycinnamic acid as the matrix (CHCA) 10 mg / mL (Sigma-Aldrich) dissolved in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). Full-scan mass spectra were acquired in positive ion reflectron mode over the mass range of 1000–6000 m / z. Each mass spectrum was the result of 750 averaged laser shots with the laser intensity set to approximately 65% ​​of the full laser intensity and the detector gain enhanced at 8 × 4 GS / s (selected within the Bruker Flex Control software). The resulting mass spectra were analyzed using Flex analysis software (Bruker Daltonics). Spectra were calibrated against PepMix internal standard solution.

[0136] N-terminal protein sequencing. The N-terminal amino acid sequence of the purified product (fractions 33-34, Figure 25A) was analyzed by 15 cycles of Edman degradation on a Procise® 494HT protein sequence system (Applied Biosystems). The N-terminal sequence is given in SEQ ID NO: 55.

[0137] Although a number of embodiments of the present invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0138] The present disclosure includes the following sequence information: SEQUENCE LISTING <110> The Regents of the University of California <120> ANTIMICROBIAL THERAPY <130> PA25-442 <140> <141> 2016-05-05 <150> US 62 / 157,248 <151> 2015-05-05 <150> US 62 / 300,274 <151> 2016-02-26 <160> 57 <170> PatentIn version 3.5 <210> 1 <211> 186 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(186) <400> 1 atg agt aaa tta gaa cta ctt aat gaa tct aaa gca at tat ctt gaa 48 Met Ser Lys Leu Glu Leu Glu Asn Ser Lys Ala Asn Tyr Leu Glu 1 5 10 15 aaa ctt act gat gaaaa att GA ga acg gaa gca tac ggc ggt aaa 96 Lys Leu Thr Asp Glu Lys Ile Glu Glu Thr Glu Ala Tyr Gly Gly Lys 20 25 30 tgt tct tgg tgg aat gca tca tgt cat tta gga aat aat ggg aaa att 144 Cys Ser Trp Trp Asn Ala Ser Cys His Leu Gly Asn Asn Gly Lys Ile 35 40 45 tgt aca gtt tct cat gag tgt gca gca gga tgt aat tta taa 186 Cys Thr Val Ser His Glu Cys Ala Ala Gly Cys Asn Leu 50 55 60 <210> 2 <211> 61 <212> PRT <213> Staphylococcus hominis <400> 2 Met Ser Lys Leu Glu Leu Leu Asn Glu Ser Lys Ala Asn Tyr Leu Glu 1 5 10 15 Lys Leu Thr Asp Glu Lys Ile Glu Glu Thr Glu Ala Tyr Gly Gly Lys 20 25 30 Cys Ser Trp Trp Asn Ala Ser Cys His Leu Gly Asn Asn Gly Lys Ile 35 40 45 Cys Thr Val Ser His Glu Cys Ala Ala Gly Cys Asn Leu 50 55 60 <210> 3 <211> 201 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(201) <400> 3 atg ttt agt aaa aat ttc caa aga aat gaa aag atg gaa aat act ttg Met Phe Ser Lys Asn Phe Gln Arg Asn Glu Lys Met Glu Asn Thr Leu 1 5 10 15 aaa aag gta agt tca gct aat gat gtg aat gga gga gct aca ccg act Lys Lys Val Ser Ser Ala Asn Asp Val Asn Gly Gly Ala Thr Pro Thr 20 25 30 att act aca tca tca act tgt ggt ggt att gtt gcg gca agt 144 Thr Thr Ser Ser Only Thr Cys Gly Gly Ile Val Only Ser 35 40 45 gct gct cag tgt ccg aca tta gct tgc tct tct aga tgt gga aaa aga Ala Ala Gln Cys Pro Thr Leu Ala Cys Ser Ser Arg Cys Gly Lys Arg 50 55 60 aaaaaaa 201 Lys Lys 65 <210> 4 <211> 66 <212> PRT <213> Staphylococcus hominis <400> 4 Met Phe Ser Lys Asn Phe Gln Arg Asn Glu Lys Met Glu Asn Thr Leu 1 5 10 15 Lys Lys Val Ser Ser Ala Asn Asp Val Asn Gly Gly Ala Thr Pro Thr 20 25 30 Thr Thr Ser Ser Only Thr Cys Gly Gly Ile Val Only Ser 35 40 45 Ala Ala Gln Cys Pro Thr Leu Ala Cys Ser Ser Arg Cys Gly Lys Arg 50 55 60 Lys Lys 65 <210> 5 <211> 170 <212> PRT <213> Homo sapiens <400> 5 Met Lys Thr Gln Arg Asn Gly His Ser Leu Gly Arg Trp Ser Leu Val 1 5 10 15 Leu Leu Leu Leu Gly Leu Val Met Pro Leu Ala Ile Ile Ala Gln Val 20 25 30 Leu Ser Tyr Lys Glu Ala Val Leu Arg Ala Ile Asp Gly Ile Asn Gln 35 40 45 Arg Ser Ser Asp Ala Asn Leu Tyr Arg Leu Leu Asp Leu Asp Pro Arg 50 55 60 Pro Thr Met Asp Gly Asp Pro Asp Thr Pro Lys Pro Val Ser Phe Thr 65 70 75 80 Val Lys Glu Thr Val Cys Pro Arg Thr Thr Gln Gln Ser Pro Glu Asp 85 90 95 Cys Asp Phe Lys Lys Asp Gly Leu Val Lys Arg Cys Met Gly Thr Val 100 105 110 Thr Leu Asn Gln Ala Arg Gly Ser Phe Asp Ile Ser Cys Asp Lys Asp 115 120 125 Asn Lys Arg Phe Ala Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu 130 135 140 Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Asp Asp Phe 145 150 155 160 Leu Arg Asn Leu Val Pro Arg Thr Glu Ser 165 170 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic linker <400> 6 Gly Gly Gly Gly Ser 1 5 <210> 7 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic linker <400> 7 Gly Gly Gly Gly Gly Gly Ser Met Phe Gly Gly Ala Lys Lys Arg Ser 1 5 10 15 Gly Gly Gly Gly Gly Gly 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus aureus-specific PCR primer <400> 8 aactgttggc cactatgagt 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus aureus-specific PCR primer <400> 9 ccagcattac ctgtaatctc g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis-specific PCR primer <400> 10 tcagcagttg aaggacagat 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis-specific PCR primer <400> 11 ccagaacaat gaatggttaa gg 22 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus-genus specific 16S sequence PCR primer <400> 12 tttgggctac acacgtgcta caatggacaa 30 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus-genus specific 16S sequence PCR primer <400> 13 aacaacttta tgggatttgc wtga 24 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal sequence of bacterial 16S rRNA PCR primer <400> 14 agagtttgga tcmtggctca g 21 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Universal sequence of bacterial 16S rRNA PCR primer <400> 15 aaggaggtgw tccarcc 17 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epidermn PCR primer <400> 16 gattcaggag ctgaaccaag a 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epidermn PCR primer <400> 17 ttgaagccct gccaatctaa 20 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis pep5 PCR primer <400> 18 ctgatgaact tgaacctcaa actg 24 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis pep5 PCR primer <400> 19 gacactgtaa ataaacgcgt agc 23 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epicidin280 PCR primer <400> 20 gcaactagac aggtatgtcc taaa 24 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epicidin280 PCR primer <400> 21 catctaagat taaatgaggg tggtt 25 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epilancin K7 PCR primer <400> 22 taagtccgca atctgctagt g 21 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus epidermidis epilancin K7 PCR primer <400> 23 cagtaatatt gcaaccgcat gt 22 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus hominis Hogocidin-alpha PCR primer <400> 24 attributes of actacts <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Staphylococcus hominis Hogocidin-alpha PCR primer <400> 25 ttataatta catcctgctg cacac <210> 26 <211> 8 <212> PRT <213> Staphylococcus hominis <400> 26 Lys Cys Ser Asp Asp Asn White White 1 5 <210> 27 <211> 138 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(138) <400> 27 atg gat att ata aaa gta at aaa aca gaa aga atg at gat at aga Met Asp Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asp Asp Asn Arg 1 5 10 15 aaa att gta atg att ttt tct tta tac gat aca ttt ttt aac gct act Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20 25 30 aat aca cat aag cta aag agt atg aag ctt aat gcg aaa taa 138 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45 <210> 28 <211> 45 <212> PRT <213> Staphylococcus hominis <400> 28 Met Asp Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asp Asp Asn Arg 1 5 10 15 Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20 25 30 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45 <210> 29 <211> 168 <212> DNA <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(168) <400> 29 atg gta tgg atc cat ggt ggt ggt aac tta ggt ggt gct ggc tta gaa 48 Met Val Trp Ile His Gly Gly Gly Asn Leu Gly Gly Ala Gly Leu Glu 1 5 10 15 gat gct ttt gat ggt aat act tta gct aaa cat aca tca aaa att aaa 96 Asp Ala Phe Asp Gly Asn Thr Leu Ala Lys His Thr Ser Lys Ile Lys 20 25 30 tat gtt ttt gga aat ttg caa cct gaa aac cat tat gat gat att gat 144 Tyr Val Phe Gly Asn Leu Gln Pro Glu Asn His Tyr Asp Ile Asp 35 40 45 father father atc tca aaa father father The Lion's Gln 50 55 <210> 30 <211> 55 <212> PRT <213> Staphylococcus epidermidis <400> 30 Met Val Trp Ile His Gly Gly Gly Asn Leu Gly Gly Ala Gly Leu Glu 1 5 10 15 Asp Ala Phe Asp Gly Asn Thr Leu Ala Lys His Thr Ser Lys Ile Lys 20 25 30 Tyr Val Phe Gly Asn Leu Gln Pro Glu Asn His Tyr Asp Asp Ile Asp 35 40 45 Ile Ile Ile Ser Lys Gln Leu 50 55 <210> 31 <211> 207 <212> DNA <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(207) <400> 31 atg att tca gtg ata ctg ccg atg gaa gaa ata att att gca ata ata 48 Met Ile Ser Val Ile Leu Pro Met Glu Glu Ile Ile Ile Ala Ile Ile 1 5 10 15 aat aac gac tta ggc cat tta att ttt gag aat aaa aaa aat agt ggg 96 Asn Asn Asp Leu Gly His Leu Ile Phe Glu Asn Lys Lys Asn Ser Gly 20 25 30 ttt tct ttt ttc ata ata aaa cct ttc ata act aat att tat ttt cta 144 Phe Ser Phe Phe Ile Ile Lys Pro Phe Ile Thr Asn Ile Tyr Phe Leu 35 40 45 tca ggt att aaa aaa att tta caa aaa cag aga aaa tat tat acg atg 192 Ser Gly Ile Lys Lys Ile Leu Gln Lys Gln Arg Lys Tyr Tyr Thr Met 50 55 60 cta ata aaa gta taa 207 Leu Ile Lys Val 65 <210> 32 <211> 68 <212> PRT <213> Staphylococcus epidermidis <400> 32 Met Tool Ser Val Leu Tool Pro Met Glue Glue Toe Toe Toe Toe Toe 1 5 10 15 Asn Asn Asp Leu Gly His Leu Ile Phe Glu Asn Lys Lys Asn Ser Gly 20 25 30 Phe Ser Phe Phe Ile Lys Pro Phe Ile Thr Asn Ile Tyr Phe Leu 35 40 45 Ser Gly Ile Lys Ile Leu Gln Lys Gln Arg Lys Tyr Thr Met 50 55 60 Leu Ile Lys Valley 65 <210> 33 <211> 210 <212> DNA <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(210) <400> 33 atg aac ata tac tta aaa gta att tta act tct tta ttt ttt gct tta 48 Met Asn - I'll Be There For You 1 5 10 15 ata att ttt at gta act tat ata acg act aag caa tgg gga aca tcg 96 Ile Ile Phe Ile Val Thr Tyr Ile Thr Thr Lys Gln Trp Gly Thr Ser 20 25 30 tta ggt ttt tca tct tta tca ttt atc ggt aac ttt att tac gat tat 144 Leu Gly Phe Ser Ser Leu Ser Phe Ile Gly Asn Phe Ile Tyr Asp Tyr 35 40 45 tca acg aaa tta agt gat aaa aaa tat gaa aaa aga ata aat agc aac 192 Ser Thr Lys Leu Ser Asp Lys Lys Tyr Glu Lys Arg Ile Asn Ser Asn 50 55 60 aaa aaa gat aaa ctt tag 210 Lys Lys Asp Lys Leu 65 <210> 34 <211> 69 <212> PRT <213> Staphylococcus epidermidis <400> 34 Met Asn Ile Tyr Leu Lys Val Ile Leu Thr Ser Leu Phe Phe Ala Leu 1 5 10 15 Ile Ile Phe Ile Ile Val Thr Ile Ile Thr Thr Lys Gln Trp Gly Thr Ser 20 25 30 Leu Gly Phe Ser Ser Leu Ser Phe Ile Gly Asn Phe Ile Tyr Asp Tyr 35 40 45 Thr Lys Leu Serves Asp Lys Tyr Glu Lys Arg Ile Asn Serves Asn 50 55 60 Lys Lys Asp Lys Lion 65 <210> 35 <211> 183 <212> DNA <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(183) <400> 35 atg aaa at aac aaa at tta ttt gat tta gaa att aaa aaa ga aca 48 Met Lys Asn Asn Lys Asn Leu Phe Asp Leu Glu Ile Lys Lys Glu Thr 1 5 10 15 agt caa aac act gat gaa ctt gaa cct caa act gct cca cca gcg att 96 Ser Gln Asn Thr Asp Glu Leu Glu Pro Gln Thr Ala Gly Pro Ala Ile 20 25 30 aga gct tct gtg aaa caa tgt cag aaa act ttg aaa gct acg cgt tta 144 Arg Ala Ser Val Lys Gln Cys Gln Lys Thr Leu Lys Ala Thr Arg Leu 35 40 45 ttt aca gtg tct tgc aaa gga aaa aac gga tgt aaa tag 183 Phe Thr Val Ser Cys Lys Gly Lys Asn Gly Cys Lys 50 55 60 <210> 36 <211> 60 <212> PRT <213> Staphylococcus epidermidis <400> 36 Met Lys Asn Asn Lys Asn Leu Phe Asp Leu Glu Ile Lys Lys Glu Thr 1 5 10 15 Ser Gln Asn Thr Asp Glu Leu Glu Pro Gln Thr Ala Gly Pro Ala Ile 20 25 30 Arg Ala Ser Val Lys Gln Cys Gln Lys Thr Leu Lys Ala Thr Arg Leu 35 40 45 Phe Thr Will Be Cys Gly Lys and Gly Cys Lys 50 55 60 <210> 37 <211> 171 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(171) <400> 37 atg ga ac aac aaa aaa gat tta ttt gat tta ga atc ​​aaa aaa gat at at 48 Met Glu Asn Lys Lys Asp Leu Phe Asp Leu Glu Ile Lys Lys Asp Asn 1 5 10 15 atg gaa aat aat gaa tta gaa gct caa tct ctt ggt cct gca att 96 Met Glu Asn Asn Asn Glu Leu Glu Ala Gln Ser Leu Gly Pro Ala Ile 20 25 30 aag gca act aga cag gta tgt cct aaa gca aca cgt ttt gtt aca gtt 144 Lys Ala Thr Arg Gln Val Cys Pro Lys Ala Thr Arg Phe Val Thr Val 35 40 45 tct tgt aaa aaa agt gat tgt caa tag 171 Ser Cys Lys Ser Asp Cys Clean 50 55 <210> 38 <211> 56 <212> PRT <213> Staphylococcus hominis <400> 38 Met Glu Asn Lys Lys Asp Leu Phe Asp Leu Glu Ile Lys Lys Asp Asn 1 5 10 15 Met Glu Asn Asn Asn Glu Leu Glu Ala Gln Ser Leu Gly Pro Ala Ile 20 25 30 Lys Ala Thr Arg Gln Val Cys Pro Lys Ala Thr Arg Phe Val Thr Val 35 40 45 Ser Cys Lys Ser Asp Cys Clean 50 55 <210> 39 <211> 135 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(135) <400> 39 atg aaa gtt gtt aaa gaa ag aaaaa ctt tt gat ctt gac gtt aaa 48 Met Lys Val Val Lys Glu Lys Lys Glu Leu Phe Asp Leu Asp Val Lys 1 5 10 15 gta aat gcg aga gac atg aat aat tca gaa tca ggt cca cct aat aca Val Asn Ala Arg Asp Met Asn Asn Ser Glu Ser Gly Pro Pro Asn Thr 20 25 30 agt tta ata tgg tgt acg gat gga tgc gct aaa cgg taa 135 Ser Leu Ile Trp Cys Thr Asp Gly Cys Ala Lys Arg 35 40 <210> 40 <211> 44 <212> PRT <213> Staphylococcus hominis <400> 40 Met Lys Val Val Lys Glu Lys Lys Glu Leu Phe Asp Leu Asp Val Lys 1 5 10 15 Val Asn Ala Arg Asp Met Asn Asn Ser Glu Ser Gly Pro Pro Asn Thr 20 25 30 Ser Leu Ile Trp Cys Thr Asp Gly Cys Ala Lys Arg 35 40 <210> 41 <211> 138 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(138) <400> 41 atg gat att ata aaa gta at aaa aca gaa aga atg at gat at aga Met Asp Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asp Asp Asn Arg 1 5 10 15 aaa att gta atg att ttt tct tta tac gat aca ttt ttt aac gct act Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20 25 30 aat aca cat aag cta aag agt atg aag ctt aat gcg aaa taa 138 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45 <210> 42 <211> 45 <212> PRT <213> Staphylococcus hominis <400> 42 Met Asp Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asp Asp Asn Arg 1 5 10 15 Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20 25 30 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45 <210> 43 <211> 201 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(201) <400> 43 atg agt aat aaa gat tta gaa tta ttt aat aca gcc gggt gat tta ata 48 Met Ser Asn Lys Asp Leu Glu Leu Phe Asn Thr Ala Gly Asp Leu Ile 1 5 10 15 caa gaa tta aaa gat ggt gac cta aat atc cat tta tat ggt gaa tcg 96 Gln Glu Leu Lys Asp Gly Asp Leu Asn With His Leu Tyr Gly Glu Ser 20 25 30 144 144 144 Glu Ile Arg Lys Lys Ser Phe Ser Gln Lys Thr Gly Asn Asp Gly Lys 35 40 45 cat tgt aca att act tgg gaa tgt tct ata tgt cct act aaa act tgt 192 His Cys Thr Ile Thr Trp Glu Cys Ser Ile Cys Pro Thr Lys Thr Cys 50 55 60 tgg tgc taa 201 Trp Cys 65 <210> 44 <211> 66 <212> PRT <213> Staphylococcus hominis <400> 44 Met Ser Asn Lys Asp Leu Glu Leu Phe Asn Thr Ala Gly Asp Leu Ile 1 5 10 15 Gln Glu Leu Lys Asp Gly Asp Leu Asn Ile His Leu Tyr Gly Glu Ser 20 25 30 Glu Ile Arg Lys Lys Ser Phe Ser Gln Lys Thr Gly Asn Asp Gly Lys 35 40 45 His Cys Thr Ile Thr Trp Glu Cys Ser Ile Cys Pro Thr Lys Thr Cys 50 55 60 Trp Cys 65 <210> 45 <211> 117 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(117) <400> 45 atg gga act tca gag gta aga aaa gga aaa gga ggc ggt ttt agt acc 48 Met Gly Thr Ser Glu Val Arg Lys Gly Lys Gly Gly Gly Phe Ser Thr 1 5 10 15 gta acc gtt gta aca cca to gta ccg aca tcg aag tgt gcc tca to 96 Val Thr Val Val Thr Pro Ile Val Pro Thr Ser Lys Cys Ala Ser Ile 20 25 30 gta aaa cca tgt aac aaa taa 117 Val Lys Pro Cys Asn Lys 35 <210> 46 <211> 38 <212> PRT <213> Staphylococcus hominis <400> 46 Met Gly Thr Ser Glu Val Arg Lys Gly Lys Gly Gly Gly Phe Ser Thr 1 5 10 15 Val Thr Val Val Thr Pro Ile Val Pro Thr Ser Lys Cys Ala Ser Ile 20 25 30 Val Lys Pro Cys Asn Lys 35 <210> 47 <211> 165 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(165) <400> 47 atg act aaa ata act aaa gat gat ttg aaa aag att aca gaa aat cgt 48 Met Thr Lys With Thr Lys Asp Asp Leu Lys With Thr Glu Asn Arg 1 5 10 15 att gaa gca cgt aca cat cca acc gtt gtt cct gta agt gct gct gta 96 Ile Glu Ala Arg Thr His Pro Thr Val Val Pro Val Ser Ala Ala Val 20 25 30 tgc gga gtt gct act aaa tta gta cca aca tcg aaa tgt gct tca att 144 Cys Gly Val Ala Thr Lys Leu Val Pro Thr Ser Lys Cys Ala Ser Ile 35 40 45 gta aaa cca tgt at aaa taa 165 Val Lys Pro Cys Asn Lys 50 <210> 48 <211> 54 <212> PRT <213> Staphylococcus hominis <400> 48 Met Thr Lys With Thr Lys Asp Asp Leu Lys With Thr Glu Asn Arg 1 5 10 15 Ile Glu Ala Arg Thr His Pro Thr Val Val Pro Val Ser Ala Ala Val 20 25 30 Cys Gly Val Ala Thr Lys Leu Val Pro Thr Ser Lys Cys Ala Ser Ile 35 40 45 Val Lys Pro Cys Asn Lys 50 <210> 49 <211> 522 <212> DNA <213> Staphylococcus hominis <220> <221> CDS <222> (1)..(522) <400> 49 atg act ata gat tta gta atg ata ctt att ttg atg tat atg ata 48 Met Thr Ile Asp Leu Val Met Ile Leu Ile Ile Leu Met Tyr Met Ile 1 5 10 15 att ggt ttt aga aga ggc ctt tgg ctg aat agt ctt cat ttg tcg tct 96 Ile Gly Phe Arg Arg Gly Leu Trp Leu Asn Ser Leu His Leu Ser Ser 20 25 30 aca ctt gtc tca cta ttc to gcg cat cgt ttt tac caa tat ata tca 144 Thr Leu Val Ser Leu Phe Ile Ala His Arg Phe Tyr Gln Tyr Ile Ser 35 40 45 aaa caa atg att gtt ttt gtt cca ttt cct aaa aca gtt gct ttt gac 192 Lys Gln Met Ile Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55 60 acg cac ttc gca ttt caa tac cat gat gta caa caa cgt ttt gat act 240 Thr His Phe Ala Phe Gln Tyr His Asp Val Gln Gln Arg Phe Asp Thr 65 70 75 80 to gtg gca ttt tta tgt to gct ttt ata agt aag ttg ctt tta tat 288 Ile Val Ala Phe Leu Cys Ile Ala Phe Ile Ser Lys Leu Leu Leu Tyr 85 90 95 ctt att att gta act ttt gat aat ata gtg tca tat cat aat att cat 336 Leu Ile Ile Val Thr Phe Asp Asn Ile Val Ser Tyr His Asn Ile His 100 105 110 gtt aca agt cga ata ttg gga agc gta tta ggt agt att gca agt gtg 384 Val Thr Ser Arg Ile Leu Gly Ser Val Leu Gly Ser Ile Ala Ser Val 115 120 125 att gta ctg caa ctt gtt tta tat tta gta tct tta tat cct aat gaa 432 Ile Val Leu Gln Leu Val Leu Tyr Leu Val Ser Leu Tyr Pro Asn Glu 130 135 140 tgg att caa gaa agt tta aaa tac ggt tat tta agc cat att att cta 480 Trp Ile Gln Glu Ser Leu Lys Tyr Gly Tyr Ile Ile Ile Leu 145 150 155 160 ttt aag atg ccg ttt tta tca tct tat ata cta aat tta taa 522 Phe Lys Met Pro Phe Leu Ser Ser Tyr Ile Leu Asn Leu 165 170 <210> 50 <211> 173 <212> PRT <213> Staphylococcus hominis <400> 50 Met Thr Ile Asp Leu Val Met Ile Leu Ile Ile Met Tyr Met Ile 1 5 10 15 Ile Gly Phe Arg Arg Gly Leu Trp Leu Ser Leu His Leu Ser Ser 20 25 30 Thr Leu Val Ser Leu Phe Ile Ala His Arg Phe Tyr Gln Tyr Ile Ser 35 40 45 Lys Gln Met Ile Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55 60 Thr His Phe Ala Phe Gln Tyr His Asp Val Gln Gln Arg Phe Asp Thr 65 70 75 80 Ile Val Ala Phe Leu Cys Ile Ala Phe Ile Ser Lys Leu Leu Leu Tyr 85 90 95 Leu Ile Ile Val Thr Phe Asp Asn Ile Val Ser Tyr His Asn Ile His 100 105 110 Val Thr Ser Arg Ile Leu Gly Ser Val Leu Gly Ser Ile Ala Ser Val 115 120 125 Ile Val Leu Gln Leu Val Leu Tyr Leu Val Ser Leu Tyr Pro Asn Glu 130 135 140 Trp Ile Gln Glu Ser Leu Lys Tyr Gly Tyr Leu Ser His Ile Ile Leu 145 150 155 160 Phe Lys Met Pro Phe Leu Ser Ser Tyr Ile Leu Asn Leu 165 170 <210> 51 <211> 522 <212> Ms <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(522) <400> 51 atg ctc att gat ata gtt gtt ctt ctt att att tgt tac ttt ata gtg 48 Met Leu Ile Asp Ile Val Val Leu Leu Ile Ile Cys Tyr Phe Ile Val 1 5 10 15 ata ggg ttt cgt aga ggt att tgg tta tcg ata ttg cac ttt gct tct 96 Ile Gly Phe Arg Arg Gly Ile Trp Leu Ser Ile Leu His Phe Ala Ser 20 25 30 tca att gta tct tta tat att gcg tca caa cat tat caa tcg att gcg 144 Ser Ile Val Ser Leu Tyr Ile Ala Ser Gln His Tyr Gln Ser Ile Ala 35 40 45 caa cgt tta gtt gta ttt gtg cca ttt ccg aaa acg gtg gcg ttt gat 192 Gln Arg Leu Val Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55 60 atg gtc tat act ata cct tat gat cat ttg caa tac aga ttt gaa aaa 240 Met Val Tyr Thr Ile Pro Tyr Asp His Leu Gln Tyr Arg Phe Glu Lys 65 70 75 80 gtg ata gca ttt att ata ata ttt ggt atg tgt aag ctt att ttg tat 288 Val Ile Ala Phe Ile Ile Ile Phe Gly Met Cys Lys Leu Ile Leu Tyr 85 90 95 cta gtt gtt gtt aca ttt gat aat ata ata acg tat aaa aag ata cat 336 Leu Val Val Val Thr Phe Asp Asn Ile Ile Thr Tyr Lys Lys Ile His 100 105 110 tta gta agt cgg ata tcg agt gtc gtt ttg agt atc ata tcg gtt ttt 384 Leu Val Ser Arg Ile Ser Ser Val Val Leu Ser Ile Ile Ser Val Phe 115 120 125 ata tat tta caa att gga ctt tat tta tta tcg cta tat ccg cat tca 432 Ile Tyr Leu Gln Ile Gly Leu Tyr Leu Leu Ser Leu Tyr Pro His Ser 130 135 140 ttt ata cag tac caa tta tct caa tcg cta gta agt cga gtt gtg att 480 Phe Ile Gln Tyr Gln Leu Ser Gln Ser Leu Val Ser Arg Val Val Ile 145 150 155 160 go caa att cct tat tta tca caa ttt att tta aat tta taa 522 Glu Gln Ile Pro Tyr Leu Ser Gln Phe Ile Leu Asn Leu 165 170 <210> 52 <211> 173 <212> PRT <213> Staphylococcus epidermidis <400> 52 Met Leu Ile Asp Ile Val Val Leu Leu Ile Ile Cys Tyr Phe Ile Val 1 5 10 15 Ile Gly Phe Arg Arg Gly Ile Trp Leu Ser Ile Leu His Phe Ala Ser 20 25 30 Ser Ile Val Ser Leu Tyr Ile Ala Ser Gln His Tyr Gln Ser Ile Ala 35 40 45 Gln Arg Leu Val Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55 60 Met Val Tyr Thr Ile Pro Tyr Asp His Leu Gln Tyr Arg Phe Glu Lys 65 70 75 80 Val Had Already Had Not Had Gly Met Cys Lys Leu Had Leu Tyr 85 90 95 Leu Val Val Thr Phe Asp Asn Ile Thr Tyr Lys Lys Ile His 100 105 110 Leu Val Ser Arg Ile Ser Ser Val Val Leu Ser Ile Ser Val Phe 115 120 125 I'm Not Afraid Of Gln I'm Gly I'm Not Afraid 130 135 140 Phe Ile Gln Tyr Gln Leu Ser Gln Ser Leu Val Ser Arg Val Val Ile 145 150 155 160 Glu Gln with Pro Tyr and Gln Phe with Pro Tyr 165 170 <210> 53 <211> 1260 <212> DNA <213> Staphylococcus epidermidis <220> <221> CDS <222> (1)..(1260) <400> 53 atg att ggt aga aaa aaa gaa acc ctt tta aaa aac gaa gtt att tct 48 Met Ile Gly Arg Lys Lys Glu Thr Leu Leu Lys Asn Glu Val Ile Ser 1 5 10 15 gcg ttt act act ttt ttt acc tgc agt tat ata ata to gtt aat ggt 96 Ala Phe Thr Thr Phe Phe Thr Cys Ser Tyr Ile Ile Ile Val Asn Gly 20 25 30 ata ttg tta cat caa gca gga atg tct ttg tta tgg acg att ata gct 144 Ile Leu Leu His Gln Ala Gly Met Ser Leu Leu Trp Thr Ile Ile Ala 35 40 45 act act cta gtt tgt tgc att agt tgc atc ctt ctt ggt ata tat gct 192 Thr Thr Leu Val Cys Cys Ile Ser Cys Ile Leu Leu Gly Ile Tyr Ala 50 55 60 aat gtt cca cta att att ata cca gga atc ​​ggt gaa act att ttt ttt 240 Asn Val Pro Leu Ile Ile Ile Pro Gly Ile Gly Glu Thr Ile Phe Phe 65 70 75 80 act tat aca atc ​​att aaa agt cat tac tat aat tat cat gaa gcg cta 288 Thr Tyr Thr Ile Ile Lys Ser His Tyr Tyr Asn Tyr His Glu Ala Leu 85 90 95 gct att gtt ttg att tca ggt ttg att ttc act ttt at gca tac aca 336 Ala Ile Val Leu Ile Ser Gly Leu Ile Phe Thr Phe Ile Ala Tyr Thr 100 105 110 ccg ttt gct aga gtt cta gac aag tcc ata cca aag aat tta aaa gaa 384 Pro Phe Ala Arg Val Leu Asp Lys Ser Ile Pro Lys Asn Leu Lys Glu 115 120 125 gga ata act to ggt ata ggt ctg ttt atg gcg ttt gtt gga cta caa 432 Gly Ile Thr Ile Gly Ile Gly Leu Phe Met Ala Phe Val Gly Leu Gln 130 135 140 aac agc aaa ata att ata cca aac agg caa agt att gtt gag cta aac 480 Asn Ser Lys Ile Ile Ile Pro Asn Arg Gln Ser Ile Val Glu Leu Asn 145 150 155 160 cac ata aac att tat agt ggg tta gcg ata cta cta cta tta ttt gca 528 His Ile Asn Ile Tyr Ser Gly Leu Ala Ile Leu Leu Leu Leu Phe Ala 165 170 175 att gtt ata ttt act tta ggg acc aag ttg gct ttc ttt tat aca gta 576 Ile Val Ile Phe Thr Leu Gly Thr Lys Leu Ala Phe Phe Tyr Thr Val 180 185 190 att att ggt atc att ata tct ttt tta gct ggg att ata aag gtg aaa 624 Ile Ile Gly Ile Ile Ile Ser Phe Leu Ala Gly Ile Ile Lys Val Lys 195 200 205 tat cat ttt tat aat ttt agt ttg cga tca ata gta agc gag aat aat 672 Tyr His Phe Tyr Asn Phe Ser Leu Arg Ser Ile Val Ser Glu Asn Asn 210 215 220 att ttt agt tac agt ttt gat aaa ata ggt cat ttt tct ttt tgg tct 720 Ile Phe Ser Tyr Ser Phe Asp Lys Ile Gly His Phe Ser Phe Trp Ser 225 230 235 240 tta gtg ttc tca ctt act att ttg tta ctg ttt caa aat tta ggt aca 768 Leu Val Phe Ser Leu Thr Ile Leu Leu Leu Phe Gln Asn Leu Gly Thr 245 250 255 tta cat gga ttg aaa att aat gat aaa gta aaa ttg tca aga att ttt 816 Leu His Gly Leu Lys Ile Asn Asp Lys Val Lys Leu Ser Arg Ile Phe 260 265 270 aaa atg gtc ggt att act aac ata att tca agc tta ttt ggt gtg agt 864 Lys Met Val Gly Ile Thr Asn Ile Ile Ser Ser Leu Phe Gly Val Ser 275 280 285 tct aca gtt att gca gtc gaa agt tct act gca act cat tca gga gct 912 Ser Thr Val Ile Ala Val Glu Ser Ser Thr Ala Thr His Ser Gly Ala 290 295 300 aaa aca gga aaa gta tct att ttt gta ggt ata atg ttt ctt tta tct 960 Lys Thr Gly Lys Val Ser Ile Phe Val Gly Ile Met Phe Leu Leu Ser 305 310 315 320 ttg ttg ata atg ccc gtt to ata gca ata cct agt tta gtt gta tca 1008 Leu Leu Ile Met Pro Val Ile Ile Ala Ile Pro Ser Leu Val Val Ser 325 330 335 cct atc tta ata att gtt ggc ggt tta atg ttt act aat att aaa gaa 1056 Pro Ile Leu Ile Ile Val Gly Gly Leu Met Phe Thr Asn Ile Lys Glu 340 345 350 tta gat ttt aat gat atg act gaa ttt att cct tgt tat ata aca att 1104 Leu Asp Phe Asn Asp Met Thr Glu Phe Ile Pro Cys Tyr Ile Thr Ile 355 360 365 ata atg ata cca ctt act ttt gat att gca act gga atg gga ttt gga 1152 Ile Met Ile Pro Leu Thr Phe Asp Ile Ala Thr Gly Met Gly Phe Gly 370 375 380 ttt att tca tat gtt cta att aat ttt gta tgc aaa aaa acc gaa cgt 1200 Phe Ile Ser Tyr Val Leu Ile Asn Phe Val Cys Lys Lys Thr Glu Arg 385 390 395 400 tta aat cca att tta ata att att gct tta ctt ttt aca ata aat tta 1248 Leu Asn Pro Ile Leu Ile Ile Ile Ala Leu Leu Phe Thr Ile Asn Leu 405 410 415 go to tta caa taa 1260 Val Leu Gln <210> 54 <211> 419 <212> PRT <213> Staphylococcus epidermidis <400> 54 Met Ile Gly Arg Lys Lys Glu Thr Leu Leu Lys Asn Glu Val Ile Ser 1 5 10 15 Ala Phe Thr Thr Phe Phe Thr Cys Ser Tyr Ile Ile Ile Val Asn Gly 20 25 30 Ile Leu Leu His Gln Ala Gly Met Ser Leu Leu Trp Thr Ile Ile Ala 35 40 45 Thr Thr Leu Val Cys Cys Ile Ser Cys Ile Leu Leu Gly Ile Tyr Ala 50 55 60 Asn Val Pro Leu Ile Ile Ile Pro Gly Ile Gly Glu Thr Ile Phe Phe 65 70 75 80 Thr Tyr Thr Ile Ile Lys Ser His Tyr Tyr Asn Tyr His Glu Ala Leu 85 90 95 Ala Ile Val Leu Ile Ser Gly Leu Ile Phe Thr Phe Ile Ala Tyr Thr 100 105 110 Pro Phe Ala Arg Val Leu Asp Lys Ser Ile Pro Lys Asn Leu Lys Glu 115 120 125 Gly Ile Thr Ile Gly Ile Gly Leu Phe Met Ala Phe Val Gly Leu Gln 130 135 140 Asn Ser Lys Ile Ile Ile Pro Asn Arg Gln Ser Ile Val Glu Leu Asn 145 150 155 160 His Ile Asn Ile Tyr Ser Gly Leu Ala Ile Leu Leu Leu Leu Phe Ala 165 170 175 Ile Val Ile Phe Thr Leu Gly Thr Lys Leu Ala Phe Phe Tyr Thr Val 180 185 190 Ile Ile Gly Ile Ile Ile Ser Phe Leu Ala Gly Ile Ile Lys Val Lys 195 200 205 Tyr His Phe Tyr Asn Phe Ser Leu Arg Ser Ile Val Ser Glu Asn Asn 210 215 220 Ile Phe Ser Tyr Ser Phe Asp Lys Ile Gly His Phe Ser Phe Trp Ser 225 230 235 240 Leu Val Phe Ser Leu Thr Ile Leu Leu Leu Phe Gln Asn Leu Gly Thr 245 250 255 Leu His Gly Leu Lys Ile Asn Asp Lys Val Lys Leu Ser Arg Ile Phe 260 265 270 Lys Met Val Gly Ile Thr Asn Ile Ile Ser Ser Leu Phe Gly Val Ser 275 280 285 Ser Thr Val Ile Ala Val Glu Ser Ser Thr Ala Thr His Ser Gly Ala 290 295 300 Lys Thr Gly Lys Val Ser Ile Phe Val Gly Ile Met Phe Leu Leu Ser 305 310 315 320 Leo Leo Ile Met Pro Val Ile Ile Ala Ile Pro Ser Leo Val Val Ser 325 330 335 Pro Ile Leu Ile Ile Val Gly Gly Leu Met Phe Thr Asn Ile Lys Glu 340 345 350 Leu Asp Phe Asn Asp Met Thr Glu Phe Ile Pro Cys Tyr Ile Thr Ile 355 360 365 Ile Met Ile Pro Leu Thr Phe Asp Ile Ala Thr Gly Met Gly Phe Gly 370 375 380 Phe Ile Ser Tyr Val Leu Ile Asn Phe Val Cys Lys Lys Thr Glu Arg 385 390 395 400 Leu Asn Pro Ile Leu Ile Ile Ile Ala Leu Leu Phe Thr Ile Asn Leu 405 410 415 Val Leu Gln <210> 55 <211> 10 <212> PRT <213> Staphylococcus epidermidis <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa at 10 is V or L <400> 55 Lys Asn Gly Ala Tyr Lys Ala Gln Gly Xaa 1 5 10 <210> 56 <211> 30 <212> PRT <213> Staphylococcus hominis <400> 56 Lys Cys Ser Trp Trp Asn Ala Ser Cys His Leu Gly Asn Asn Gly Lys 1 5 10 15 Ile Cys Thr Val Ser His Glu Cys Ala Ala Gly Cys Asn Leu 20 25 30 <210> 57 <211> 38 <212> PRT <213> Staphylococcus hominis <400> 57 Ala Thr Pro Thr Ile Thr Thr Ser Ser Ala Thr Cys Gly Gly Ile Ile 1 5 10 15 Val Wing Wing Ser Wing Wing Gln Cys Pro Thr Leu Wing Cys Ser Ser Arg 20 25 30 How To Get A Lys Arg Lys Lys 35

Claims

1. 1. A composition comprising a topical concentrated formulation of one or more probiotic bacterial strains, and optionally a prebiotic compound, a protectant, a moisturizer, an emollient, an abrasive, a salt, and / or a surfactant, The one or more probiotic bacterial strains include one or more bacterial strains of the genus Staphylococcus; and The composition is formulated for the topical treatment of dysbiotic disorders of the skin, scalp, or mucous membranes.

2. 10. The composition of claim 1, wherein the one or more probiotic bacterial strains comprise Staphylococcus epidermidis, Staphylococcus hominis, or a combination of Staphylococcus epidermidis and Staphylococcus hominis.

3. 3. The composition of claim 2, wherein the one or more probiotic bacterial strains comprise Staphylococcus epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5 and / or AMT5-G6.

4. 3. The composition of claim 2, wherein the one or more probiotic bacterial strains comprise Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1 and / or AMT4-D12.

5. 3. The composition of claim 2, wherein each probiotic bacterial strain exhibits a fatty acid methyl ester profile corresponding to one of those shown in any of Figures 11, 12, 13, 14, 15, 16, 17, 18, or 19.

6. 3. The composition of claim 2, wherein the one or more probiotic bacterial strains produce peptides having sequences selected from the group consisting of SEQ ID NOs: 2, 4, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 55, and any combination thereof, wherein such peptides are optionally post-translationally modified.

7. The composition of claim 1 , wherein the one or more probiotic bacterial strains are provided in a viable form.

8. 10. The composition of claim 1, wherein the one or more probiotic bacterial strains are provided in a freeze-dried or spray-dried form.

9. 9. The composition of claim 8, wherein the probiotic bacteria can be reconstituted into a viable form.

10. 10. A method of treating skin or mucosal infections, atopic dermatitis, psoriasis, mastitis, acne, or other disorders associated with skin dysbiosis in humans or other mammals by applying an effective amount of the composition of any one of claims 1 to 9 to the skin or mucosa of a subject in need thereof.

11. The method of claim 10, wherein the composition is applied topically.

12. 11. The method of claim 10, wherein the composition is formulated as a cream, ointment, spray, powder, oil, concentrate, or poultice.

13. A composition comprising one or more of a hogoshidin peptide, derivative or variant, an SH-lantibiotic peptide, an SH-antibacterial agent, an SE-lantibiotic peptide, and / or an SE antibacterial agent; and further comprising one or more thickeners, solvents, emulsifiers, or pharmaceutically acceptable carriers or excipients.

14. 14. The composition of claim 13, further comprising a cathelicidin peptide, derivative or variant.

15. 15. The composition of claim 13 or 14, wherein the peptide comprises one or more D-amino acids or unnatural amino acids.

16. 14. The composition of claim 13, wherein the hogoshidin peptide, SH-lantibiotic peptide, SH-antibacterial agent, SE-lantibiotic peptide, or SE antibacterial agent is produced in situ by one or more of Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, Staphylococcus epidermidis strain AMT5-G6, and Staphylococcus epidermidis strain MO34.

17. 16. The composition of claim 13, 14 or 15, wherein the peptide is formulated for topical administration.

18. 18. The composition of claim 17, wherein the formulation comprises a lotion, ointment cream, powder, ointment, oil, or spray.

19. The composition of claim 13, wherein the hogoshidin peptide, derivative or variant comprises a sequence selected from SEQ ID NO: 2 or SEQ ID NO:

4.

20. 14. The composition of claim 13, wherein one or more of the hogoshidin peptide, derivative or variant, SH-lantibiotic peptide, SH-antibacterial agent, SE-lantibiotic peptide, SE antibacterial agent, and cathelicidin peptide, derivative or variant is provided as an extract or lysate of Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, Staphylococcus epidermidis strain AMT5-G6, and Staphylococcus epidermidis strain MO34.

21. A method for treating skin or mucosal infection or atopic dermatitis in a subject, comprising contacting the subject with an effective amount of a composition comprising one or more of a hogoshidin peptide, derivative or variant, an SH-lantibiotic peptide, an SH-antibacterial agent, an SE-lantibiotic peptide, and optionally a cathelicidin peptide, derivative or variant.

22. 22. The method of claim 21, wherein the contacting is by topical administration or, optionally, by contacting the subject with one or more of SH-lantibiotic or bacteriocin-producing Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1, AMT4-D12, and Staphylococcus epidermidis strains AMT5-G6 and MO34.

23. A recombinant vector comprising a polynucleotide encoding a polypeptide that is at least 95% identical to SEQ ID NO: 2 or 4, or a biologically active fragment thereof that has antibacterial activity.

24. 24. The recombinant vector of claim 23, wherein the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO: 2 or 4.

25. 24. The recombinant vector of claim 23, wherein the vector comprises a polynucleotide encoding a polypeptide from about amino acid 32 to about amino acid 61 of SEQ ID NO:

2.

26. 24. The recombinant vector of claim 23, wherein the vector comprises a polynucleotide encoding a polypeptide from about amino acid 29 to about amino acid 66 of SEQ ID NO:

4.

27. 24. The recombinant vector of claim 23, wherein the vector comprises a polynucleotide that is at least 95% identical to SEQ ID NO: 1 or 3 and encodes the polypeptide of SEQ ID NO: 2 or 4, respectively.

28. 27. The recombinant vector of claim 25 or 26, wherein the vector comprises a fragment of SEQ ID NO: 1 or 3.

29. A host cell genetically engineered to express the recombinant vector of any one of claims 23 to 28.

30. 30. The host cell of claim 29, wherein the host cell is a non-pathogenic attenuated host cell.

31. 30. A recombinant polypeptide produced by the host cell of claim 29.

32. 30. A composition comprising the host cell of claim 29.

33. 31. A composition comprising the host cell of claim 30.

34. 32. A composition comprising the polypeptide of claim 31.

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