Methods and compositions for treating skin diseases using recombinant microorganisms

Abstract

The present disclosure provides isolated plasmids, recombinant microorganisms, kits and methods for the treatment of microbial infections, particularly MRSA.

Description

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 442,855, filed February 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] background The frequency and severity of viral, bacterial, and fungal infections of the skin are increasing. For example, pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are rapidly increasing, while the availability of antibiotics to treat infections, especially those related to the skin, is limited. There is a significant need for new therapies for both the prevention and acute care of viral, bacterial, and fungal infections of the skin.

[0003] Overview The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or antibiotic-resistant genetic elements enables the modulation of therapeutic exposure and improved safety. The research described herein supports the potential safety and utility of live biotherapeutic bacterial strains whose growth can be controlled by D-alanine for the treatment of a variety of skin diseases and conditions. In particular, this disclosure is characterized in some embodiments by a recombinant D-alanine auxotrophic strain, artificially engineered to extracellularly secrete therapeutic proteins (e.g., bacterial and fungal cell wall hydrolases, any protective expression of host immune proteins) onto the skin surface and / or other suitable tissues for prophylactic, post-exposure prophylaxis, or therapeutic treatment of dysbiosis caused by the overgrowth of pathogenic bacteria or fungi attributable to primary or secondary superficial or severe skin diseases or conditions.

[0004] In a first aspect, this disclosure provides a recombinant microorganism comprising: a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive.

[0005] In another aspect, this disclosure provides a recombinant microorganism comprising: 1) a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; 2) a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive; and 3) a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive.

[0006] In some embodiments of the above aspects, one or more naturally occurring antibiotic resistance genes are selected from mupirocin resistance genes, ampicillin resistance genes, cefotaxime resistance genes, chloramphenicol resistance genes, ciprofloxacin resistance genes, co-trimoxazole resistance genes, nalidixic acid resistance genes, oxytetracycline resistance genes, streptomycin resistance genes, tetracycline resistance genes, and trimethoprim resistance genes.

[0007] In some embodiments of the above aspects and implementation schemes, one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

[0008] In some embodiments of the above aspects and implementation schemes, one or more naturally occurring lysogenic phage genes encode phage capsid proteins.

[0009] In some embodiments of the foregoing aspects and implementations, one or more D-alanine biosynthesis genes include the D-alanine aminotransferase gene (dat), the alanine racemase gene alar1, or alar2. In some embodiments of the foregoing aspects and implementations, deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes include deletions in dat, alar1, and alar2.

[0010] In some embodiments of the above aspects and implementation plans, the recombinant microorganism further comprises one or more genes encoding a heterologous gene.

[0011] In some embodiments of the foregoing aspects and implementation schemes, the heterologous gene is selected from genes encoding antimicrobial peptides or variants thereof, genes encoding antimicrobial biosynthetic enzymes or variants thereof, genes encoding enzymes or variants thereof, genes encoding enzyme inhibitors or variants thereof, genes encoding antigens or variants thereof, and genes encoding immunomodulatory peptides or variants thereof, or combinations thereof.

[0012] In some embodiments of the above aspects and implementation schemes, the heterologous gene encodes an antimicrobial peptide or a variant thereof.

[0013] In some embodiments of the above aspects and implementation schemes, one or more antimicrobial peptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

[0014] In some embodiments of the above aspects and implementation schemes, one or more microbial pathogens are selected from bacterial pathogens, fungal pathogens, or viral pathogens.

[0015] In some embodiments of the foregoing aspects and implementation schemes, one or more bacterial pathogens are selected from the genera Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, species of Cutibacterium spp., and Mycoplasma, or combinations thereof.

[0016] In some embodiments of the above aspects and implementation plans, the fungal infection is caused by fungi selected from the genera *Malassezia* spp., *Candida* spp., *Aspergillus*, *Cryptococcus*, and *Pneumocystis*.

[0017] In some implementation schemes of the above aspects and implementation schemes, the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

[0018] In some embodiments of the above aspects and implementation schemes, the antimicrobial peptide is selected from epidermoid lanthanide, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, chondroglycoprotein 1, chitosan trisaccharidase, mucin 9, chondroglycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucosylamylase, amylopectin, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectic acid lyase, lysostaphin, and zoocin. A. Millericin B. Muramidin Cpl-1, Lysozyme, Intralysin PlyC, Intralysin, PlyV12, Enterolysin A, Clostridium difficile autolysin (Acd), Autolysin (LytA), PL-1 amidase hydrolase, Nisin A, Nisin Z, Subtilisin, Epidermin, Gallidermin, Varitin Ba Ny266, Varitin 1140, Pep5, Epicidin 280, Epilancin K7, Nisin 481, Cytolysin, Nisin 3147, Staphylococcin C55, Salvaricin A, Lactocin S, Streptococcin A-FF2, Sublancin 168, Carnocin U149, Variacin 8. Cypemycin, cinnamycin, natriuretic peptide, angiotensin-converting enzyme inhibitory peptide, Mersacidin, and Actardine.

[0019] In some embodiments, one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains or variants thereof, and wherein the LEKTI protein, one or more LEKTI protein domains or variants thereof are secreted.

[0020] In some embodiments of the foregoing aspects and implementation schemes, a heterologous gene encodes an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and wherein 6-HAP is secreted.

[0021] In some embodiments of the above aspects and implementation schemes, the gene encoding the immune-modulating polypeptide or a variant thereof is a gene encoding lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

[0022] In some implementation schemes of the above aspects and implementation schemes, the recombinant microorganism is a bacterium, or a combination of bacteria.

[0023] In some embodiments of the above aspects and implementation plans, the recombinant microorganism is selected from Bifidobacterium, Brevibacterium, Corynebacterium, Dermatobacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or combinations thereof.

[0024] In some embodiments of the above aspects and implementation plans, the recombinant microorganism is Staphylococcus epidermidis (S. epidermidis).

[0025] In some embodiments of the above aspects and implementation schemes, recombinant microorganisms secrete one or more therapeutic peptides or variants thereof.

[0026] Therefore, in another aspect, this disclosure provides a pharmaceutical composition comprising a cell culture composition and a pharmaceutically acceptable carrier, said cell culture composition comprising one or more recombinant microorganisms described above or any other aspect of this disclosure herein.

[0027] In some embodiments of the above aspects and implementation plans, the cell culture composition is a live cell culture composition.

[0028] In some embodiments, the cell culture composition contains 0% to no more than 90% water.

[0029] In some implementations, the pharmaceutically acceptable carrier is selected from aqueous solutions, emulsions, creams, lotions, gels, or ointments.

[0030] On the other hand, this disclosure provides a method for treating a disease, symptom, or condition of a subject, the method comprising administering to the subject a recombinant microorganism comprising: a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive.

[0031] Therefore, in another aspect, this disclosure provides a method for treating a disease, symptom, or condition of a subject, the method comprising: administering to the subject a cell culture composition comprising a recombinant microorganism comprising: 1) a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; 2) a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive; and 3) a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive.

[0032] In some implementations, one or more naturally occurring antibiotic resistance genes are selected from mupirocin resistance genes, ampicillin resistance genes, cefotaxime resistance genes, chloramphenicol resistance genes, ciprofloxacin resistance genes, trimethoprim-sulfamethoxazole resistance genes, nalidixic acid resistance genes, oxytetracycline resistance genes, streptomycin resistance genes, tetracycline resistance genes, and trimethoprim resistance genes.

[0033] In some embodiments of the above aspects and implementation schemes, one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

[0034] In some embodiments of the above aspects and implementation schemes, one or more naturally occurring lysogenic phage genes encode phage capsid proteins.

[0035] In some embodiments of the foregoing aspects and implementations, one or more D-alanine biosynthesis genes include the D-alanine aminotransferase gene (dat), the alanine racemase gene alar1, or alar2. In some embodiments of the foregoing aspects and implementations, deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes include deletions in dat, alar1, and alar2.

[0036] In some embodiments of the above aspects and implementation plans, the recombinant microorganism further comprises one or more genes encoding a heterologous gene.

[0037] In some embodiments of the foregoing aspects and implementation schemes, the heterologous gene is selected from genes encoding antimicrobial peptides or variants thereof, genes encoding antimicrobial biosynthetic enzymes or variants thereof, genes encoding enzymes or variants thereof, genes encoding enzyme inhibitors or variants thereof, genes encoding antigens or variants thereof, and genes encoding immunomodulatory peptides or variants thereof, or combinations thereof.

[0038] In some embodiments of the above aspects and implementation schemes, the heterologous gene encodes an antimicrobial peptide or a variant thereof.

[0039] In some embodiments of the above aspects and implementation schemes, one or more antimicrobial peptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

[0040] In some embodiments of the above aspects and implementation schemes, one or more microbial pathogens are selected from bacterial pathogens, fungal pathogens, or viral pathogens.

[0041] In some embodiments of the above aspects and implementation schemes, one or more bacterial pathogens are selected from species of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Dermatobacterium, Propionibacterium, and Mycoplasma, or combinations thereof.

[0042] In some implementation schemes of the above aspects and implementation schemes, the fungal infection is caused by fungi selected from species of the genera *Malassezia*, *Candida*, *Aspergillus*, *Cryptococcus*, and *Pneumocystis*.

[0043] In some implementation schemes of the above aspects and implementation schemes, the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

[0044] In some embodiments of the above aspects and implementation schemes, the antimicrobial peptide is selected from epidermoid lanthanide, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, chondroglycoprotein 1, chitosan trisaccharidase, mucin 9, chondroglycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylan esterase, α-amylase, β-amylase, glucosylamylase, amylopectin, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectic acid lysin, lysostaphin, zoocin A, millericin B, and muramidase. Cpl-1, lysozyme, intracytolysin PlyC, intracytolysin, PlyV12, enterolysin A, autolysin (Acd) of Clostridium difficile (C. difficile), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilisin, epidermin, Gallidermin, varisin Ba Ny266, varisin 1140, Pep5, Epicidin 280, Epilancin K7, nisin 481, cytolysin, nisin 3147, staphylococcal C55, Salvaricin A, lactobacillus S, streptococcal A-FF2, Sublancin 168, Carnocin U149, Variacin 8. Cinnamomum cassia, cinnamomum cassia, angiotensin-converting enzyme inhibitory peptide, Mersacidin, and Actardine.

[0045] In some embodiments of the foregoing aspects and implementation schemes, one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains or variants thereof, and wherein the LEKTI protein, one or more LEKTI protein domains or variants thereof are secreted.

[0046] In some embodiments of the foregoing aspects and implementation schemes, a heterologous gene encodes an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and wherein 6-HAP is secreted.

[0047] In some embodiments of the above aspects and implementation schemes, the gene encoding the immune-modulating polypeptide or a variant thereof is a gene encoding lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

[0048] In some implementation schemes of the above aspects and implementation schemes, the recombinant microorganism is a bacterium, or a combination of bacteria.

[0049] In some implementation schemes of the above aspects and implementation schemes, the recombinant microorganism is selected from Bifidobacterium, Breobacterium, Corynebacterium, Dermatobacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Stomatococcus or combinations thereof.

[0050] In some embodiments of the foregoing aspects and implementation schemes, the recombinant microorganism is *Staphylococcus epidermidis*. In some embodiments of the foregoing aspects and implementation schemes, the recombinant microorganism secretes one or more therapeutic peptides or variants thereof.

[0051] In some embodiments of the foregoing aspects and implementations, the cell culture composition is a live cell culture composition. In some embodiments of the foregoing aspects and implementations, the cell culture composition contains 0% to no more than 90% water.

[0052] In some embodiments of the above-mentioned aspects and implementation plans, the subject is a mammal. In some embodiments of the above-mentioned aspects and implementation plans, the mammal is a human.

[0053] In the above-mentioned aspects and some implementation schemes, the disease, condition or symptom is a microbial infection, inflammatory disease or symptom, metabolic disease or symptom.

[0054] In some embodiments of the above aspects and implementation plans, microbial infections include one or more microbial pathogens selected from the genera *Staphylococcus*, *Streptococcus*, *Haemophilus*, *Moraxella*, *Escherichia*, *Enterobacter*, *Proteus*, *Klebsiella*, *Pseudomonas*, *Legionella*, *Chlamydia*, *Dermatobacterium*, *Propionibacterium*, *Mycoplasma*, *Malassezia*, *Candida*, *Aspergillus*, *Cryptococcus*, *Pneumocystis*, respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus, or combinations thereof.

[0055] In some embodiments of the above aspects and implementation plans, the skin disease or condition is an inflammatory skin disease or condition. In some embodiments of the above aspects and implementation plans, the skin disease or condition is skin dysbiosis. In some embodiments of the above aspects and implementation plans, the skin disease or condition is dermatitis. In some embodiments of the above aspects and implementation plans, the skin disease or condition is ichthyosis. In some embodiments of the above aspects and implementation plans, the skin disease or condition is psoriasis. In some embodiments of the above aspects and implementation plans, the skin disease or condition is dermatitis. In some embodiments of the above aspects and implementation plans, the skin disease or condition is an autoimmune vesicular disease. In some embodiments of the above aspects and implementation plans, the skin disease or condition is selected from: Natherton's syndrome, hidradenitis suppurativa, psoriasis, pustular psoriasis, plaque psoriasis and palmoplantar psoriasis, atopic dermatitis, acne, acneiform eruption, impetigo, folliculitis, acute suppurative paronychia, lymphangitis, necrotizing fasciitis and cellulitis.

[0056] On the other hand, this disclosure provides a recombinant D-alanine auxotrophic bacterial strain prepared by means of: selecting a bacterial strain; and performing the following steps in sequence: (1) deleting or substituting one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; (2) deleting or substituting one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and (3) deleting or substituting one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive, thereby preparing a recombinant D-alanine auxotrophic bacterial strain.

[0057] In a further embodiment, deletions or substitutions in one or more genes encoding the D-alanine biosynthesis gene include deletions in dat, alar1, and alar2. In some embodiments of the foregoing aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis. Brief description of the attached diagram Figure 1A Inhibition screening of Staphylococcus aureus (S. aureus) from the Micromyx-50 library of Staphylococcus epidermidis strains was described.

[0059] Figure 1B The inhibition of Staphylococcus aureus by strain SE25MM23 was described.

[0060] Figure 2The inhibition of Staphylococcus aureus SA25923, Staphylococcus aureus SA29213, Staphylococcus epidermidis NRRL, MRSA USA 300, Bacillus subtilis, and Gram-negative strains Pseudomonas aeruginosa and Escherichia coli (E. coli) by SE25 was described.

[0061] Figure 3 A graph depicts the growth of Staphylococcus aureus in conditioned cell-free SE25 or SE3 medium compared to TSB alone.

[0062] Figure 4 The antimicrobial activity of SE25 cell-free supernatant against coagulase-negative staphylococci from the skin of patients with ichthyosis was characterized. SE25 cell-free supernatant was tested against Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis NRRL B-4268, Staphylococcus epidermidis 1457, Staphylococcus epidermidis 25, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and CoNS isolates from ichthyosis, Staphylococcus lugdunensis, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus pettenkoferi, Staphylococcus saprophyticus, and Staphylococcus warneri.

[0063] Figure 5A A graph depicting the activity of SE25 cell-free supernatant against Staphylococcus aureus is shown, in which the growth of Staphylococcus aureus is reduced by approximately 7 log units (7-log).

[0064] Figure 5B A graph depicting the bactericidal activity of linezolid, levofloxacin, erythromycin and vancomycin against Staphylococcus aureus is presented.

[0065] Figure 6A Biofilm formation on plastics with crystal violet indicator is depicted. The figure shows that SE25 does not form a biofilm, while the positive control SE 1457 does.

[0066] Figure 6B The biofilm detection method using Congo red is described. SE25 forms red colonies, indicating that no biofilm has formed.

[0067] Figure 6C A diagram depicts the dispersion of a biofilm formed by Staphylococcus aureus in the cell-free supernatant of SE25 through successive dilutions.

[0068] Figure 7 The figure depicts the ability of SE25 to settle in reconstructed human epidermis (RHE) after 24 hours and 48 hours.

[0069] Figure 8 A graph depicting a 4-log reduction in Staphylococcus aureus ATCC 29213 caused by RHE colonization with SE25 is presented.

[0070] Figure 9 A diagram depicting SE25 colonization on RHE induces the expression of human β-defensin 2 from keratinocytes.

[0071] Figure 10 The epidermal biosynthesis gene cluster in SE25 was described.

[0072] Figure 11 The phiSpy gene cluster of the prophage in SE25 is described.

[0073] Figure 12 Auxotrophy testing was depicted on TSA plates with or without D-alanine (“DA100”) supplementation (plate A) or (plate B). SE480: SE25 wild-type, SE482: SE25 double-deletion auxotroph, SE484: SE25 triple-deletion auxotroph (both with ΔmupAΔ capsid), SE123: SEΔΔΔ triple-deletion auxotroph of NRRL B-4268.

[0074] Figure 13A The screening assays for strains Δalr1Δalr2Δdat SE25 were described.

[0075] Figure 13B The screening assay for strain Δalr1Δdat SE25 was described.

[0076] Figure 14A The anti-Staphylococcal activity of the Δalr1Δdat SE25 strain in an agar topcoat assay was depicted. The SE25 strain was grown on TSB at 37°C for 48 hours and topped with an agar topcoat containing Staphylococcus aureus ATCC 29213. Growth inhibition bands indicate anti-staphylococcal activity.

[0077] Figure 14BThe anti-Staphylococcal activity of the SE25 strain Δalr1Δalr2Δdat in an agar coating assay was depicted. The SE25 strain was grown on TSB at 37°C for 48 hours and covered with a top layer of agar containing Staphylococcus aureus ATCC 29213. Growth inhibition zones indicate anti-staphylococcal activity.

[0078] Figure 15A Screening assays for chloramphenicol (Cam-10)- and mupirocin (Mup-20)-sensitive colonies in wild-type SE25 were described. Circles indicate positive Cam-10 / Mup-20-sensitive colonies.

[0079] Figure 15B Screening assays for chloramphenicol (Cam-10)- and mupirocin (Mup-20)-susceptible colonies in DA-auxotrophic strains of SE25 were depicted. Circles indicate positive Cam-10 / Mup-20-susceptible colonies.

[0080] Figure 16A The anti-Staphylococcal activity of strain SE25 in an agar coating assay was depicted. Strain SE25 was grown on TSA at 37°C for 24 hours, followed by an additional 24 hours at 30°C, and covered with a top layer of agar containing Staphylococcus aureus ATCC 29213. Growth inhibition bands indicate anti-staphylococcal activity. SE25ΔmupAΔcapsid (SE480, SE481), SE25ΔmupAΔcapsidΔalr1Δdat (SE482, SE483), and SE25ΔmupAΔcapsidΔalr1Δalr2Δdat (SE484, SE485) were also described.

[0081] Figure 16B The anti-Staphylococcal activity of strain SE25 in an agar topcoat assay was depicted. Strain SE25 was grown on TSA at 37°C for 24 h, followed by an additional 24 h at 30°C, and covered with a top layer of agar containing Staphylococcus aureus ATCC 29213. Growth inhibition bands indicate anti-staphylococcal activity. Wild-type parent strain SE25 (SE398) was also used.

[0082] Figure 17 The results of agar coating assays of the SE25ΔepiA mutant strain (plates 1–4) using Staphylococcus aureus ATCC 29213, the negative wild-type control SE120 (NRRL B-4268; plate 5), and the positive control SE25 wild-type (plate 6) were described.

[0083] Figure 18A SE25 strains selected for epiA knockout screening were described.

[0084] Figure 18BPCR analysis depicted Cam-sensitive colonies (colonies 5, 8, 11, and 32) of the ΔepiA mutant strain.

[0085] Figure 19A The screening assay for candidate colonies of the D-alanine auxotrophic strain SE464, streaked on tryptone agar (TSA) in the absence of chloramphenicol and with 100 μg / mL D-alanine, is shown.

[0086] Figure 19B The screening assay shows the candidate colonies of the D-alanine auxotrophic strain SE464, which was streaked on TSA supplemented with 10 μg / mL chloramphenicol in the absence of D-alanine.

[0087] Figure 19C The identification of three clones (highlighted with red circles) grown solely on TSA supplemented with D-alanine is shown.

[0088] Figure 20A This is a chart depicting the growth kinetics of strains SE25 and SE484. Figure 20B The anti-Staphylococcus activity of strain SE25 in an agar coating assay was characterized. Strain SE25 was streaked onto a TSA plate, followed by the addition of 100 mg / mL D-alanine, and incubated at 37°C. After 48 hours of incubation, soft agar containing Staphylococcus aureus was poured onto plates and then incubated at 37°C for an additional 24 hours.

[0089] Figure 20C The anti-Staphylococcus activity of strain SE484 in an agar coating assay was characterized. Strain SE484 was streaked onto a TSA plate, followed by the addition of 100 mg / mL D-alanine, and incubated at 37°C. After 48 hours of incubation, soft agar containing Staphylococcus aureus was poured onto plates and then incubated at 37°C for an additional 24 hours.

[0090] Figure 20D The validation of SE484 for D-alanine auxotrophy and mupirocin sensitivity was described. SE484 was streaked onto plates containing TSA + / - 100 μg / ml D-alanine and TSA + / - 20 μg / ml mupirocin. The plates were incubated at 37°C for 48 hours, and data were recorded afterward.

[0091] Figure 21A The anti-Staphylococcus aureus activity of SE123 was described. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 4Prior to challenge with CFU-containing Staphylococcus aureus USA300 cells, reconstructed human epidermis (RHE) was inoculated with SE123 and incubated at 37°C and 5% CO2 in a tissue culture incubator for four hours. After an additional 24 hours of incubation, USA300 cell counts were determined by biopsy and dilution plate inoculation.

[0092] Figure 21B The anti-Staphylococcus aureus activity of SE484 was described. (The text abruptly shifts to a seemingly unrelated topic: "In use ~10...") 4 Prior to challenge with CFU-containing Staphylococcus aureus USA300 cells, reconstructed human epidermis (RHE) was inoculated with SE484 and incubated at 37°C and 5% CO2 in a tissue culture incubator for four hours. After an additional 24 hours of incubation, USA300 cell counts were determined by biopsy and dilution plate inoculation.

[0093] Detailed Explanation This disclosure relates to the use of engineered microorganisms designed for delivering therapeutic agents to the skin of a host, offering key benefits over the use of naturally occurring probiotic strains that secrete antimicrobial agents. For example, the engineered commensal organism can be modified to have desired characteristics that improve its colonization, lifespan, and antimicrobial properties compared to any inherent limitations on probiotics that may have limited or varied colonization success in the skin. This disclosure provides methods and compositions for producing engineered microorganisms that can secrete therapeutic agents (e.g., various human-derived or bacterial-derived antimicrobial agents).

[0094] According to some embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein said naturally occurring antibiotic resistance gene is inactive or absent. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein said naturally occurring lysogenic phage gene is inactive. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein said D-alanine biosynthesis gene is inactive.

[0095] According to some implementation schemes, the recombinant microorganism comprises: 1) a deletion or substitution in one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; 2) a deletion or substitution in one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive; and 3) a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive.

[0096] According to some embodiments, the one or more naturally occurring antibiotic resistance genes of the recombinant microorganism are selected from mupirocin resistance genes, ampicillin resistance genes, cefotaxime resistance genes, chloramphenicol resistance genes, ciprofloxacin resistance genes, trimethoprim-sulfamethoxazole resistance genes, nalidixic acid resistance genes, oxytetracycline resistance genes, streptomycin resistance genes, tetracycline resistance genes, and trimethoprim resistance genes. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is located on the chromosome or plasmid of the recombinant microorganism, or both. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is a mupirocin resistance gene.

[0097] In some embodiments of the above aspects and implementations, the naturally occurring antibiotic resistance gene is inactivated on the chromosome rather than on the plasmid. In some embodiments of the above aspects and implementations, the naturally occurring antibiotic resistance gene is inactivated on the plasmid rather than on the chromosome. In some embodiments of the above aspects and implementations, the naturally occurring antibiotic resistance gene is inactivated on both the plasmid and the chromosome.

[0098] In some embodiments of the foregoing aspects and embodiments, the naturally occurring antibiotic resistance peptide has 90% identity with the entire sequence of SEQ ID NO: 152. Therefore, in one embodiment, the naturally occurring antibiotic resistance peptide has at least about 95% identity with the entire sequence of SEQ ID NO: 152. Therefore, in one embodiment, the naturally occurring antibiotic resistance peptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 152. In another embodiment, the naturally occurring antibiotic resistance peptide comprises the sequence of SEQ ID NO: 152. In yet another embodiment, the naturally occurring antibiotic resistance peptide consists of the sequence of SEQ ID NO: 152.

[0099] In some embodiments of the foregoing aspects and embodiments, the naturally occurring antibiotic resistance peptide has 90% identity with the entire sequence of SEQ ID NO: 153. Therefore, in one embodiment, the naturally occurring antibiotic resistance peptide has at least about 95% identity with the entire sequence of SEQ ID NO: 153. Therefore, in one embodiment, the naturally occurring antibiotic resistance peptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 153. In another embodiment, the naturally occurring antibiotic resistance peptide comprises the sequence of SEQ ID NO: 153. In yet another embodiment, the naturally occurring antibiotic resistance peptide consists of the sequence of SEQ ID NO: 153.

[0100] According to some implementation schemes, naturally occurring lysogenic phage genes encode structural proteins essential for phage particle formation and cycling. In some embodiments of the above-mentioned aspects and implementation schemes, naturally occurring lysogenic phage genes encode phage structural capsid proteins.

[0101] In some embodiments of the above aspects and embodiments, the naturally occurring lysogenic phage polypeptide has 90% identity with the entire sequence of SEQ ID NO: 154. Therefore, in one embodiment, the naturally occurring lysogenic phage polypeptide has at least about 95% identity with the entire sequence of SEQ ID NO: 154. Therefore, in one embodiment, the naturally occurring lysogenic phage polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the naturally occurring lysogenic phage polypeptide comprises the sequence of SEQ ID NO: 154. In yet another embodiment, the naturally occurring lysogenic phage polypeptide consists of the sequence of SEQ ID NO: 154.

[0102] According to some embodiments, the recombinant microorganism is an auxotroph for one or more essential genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism is an auxotroph for D-alanine biosynthesis. In some embodiments, the D-alanine biosynthesis gene is deleted or mutated, resulting in inactivation. In some embodiments of the above aspects and embodiments, the recombinant microorganism contains a deletion or substitution in the D-alanine aminotransferase (dat) gene, the alanine racemic enzyme gene alar1, or alar2. In some embodiments of the above aspects and embodiments, the recombinant microorganism contains a deletion or substitution in the dat, alar1, and alar2 genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism contains a deletion or substitution in the dat and alar1 genes.

[0103] According to some embodiments, this disclosure provides a composition for treating bacterial infections in a subject, comprising a microorganism genetically engineered to express a therapeutic agent (e.g., an antimicrobial peptide) and supplied to the subject. According to some embodiments, the therapeutic agent is an antimicrobial peptide that is effective in treating bacterial infections and skin dysbiosis (e.g., atopic dermatitis, superficial fungal infections, such as dandruff, tinea, candidiasis, etc.). In some embodiments of the above aspects and embodiments, the therapeutic agent is a LEKTI protein, one or more LEKTI protein domains, or variants thereof. In some embodiments of the above aspects and embodiments, the therapeutic agent is 6-N-hydroxyaminopurine (6-HAP).

[0104] As used herein, the term "genetic modification" and its grammatical variations are used to describe microbial organisms (e.g., bacteria) that have been genetically modified or artificially engineered by introducing DNA prepared outside the microorganism. For example, introducing plasmid DNA containing new genes into bacteria will allow the bacteria to express those genes. Alternatively, DNA containing new genes can be introduced into bacteria and then integrated into the bacterial genome (chromosome and / or plasmid), where the bacteria will express those genes.

[0105] As used herein, the terms “treat,” “treating,” and “treatment,” and their grammatical variations, refer to providing a procedure, regimen, method, or treatment to a subject in which a physiological response or outcome is expected. In particular, the methods and compositions of this disclosure can be used to slow or delay the onset of pathogen infection, the development of infection symptoms, or to halt the progression of infection. However, because not every subject may respond to a particular treatment procedure, regimen, method, or treatment, treatment does not require the achievement of a desired physiological response or outcome in every subject or subject group, such as a patient population. Therefore, a given subject or subject group, such as a patient population, may fail to respond to treatment or respond inadequately.

[0106] According to some implementation schemes, recombinant microorganisms secrete antimicrobial peptides that effectively ameliorate the symptoms of bacterial pathogens. As used herein, the terms “ameliorate,” “ameliorating,” and their grammatical variations refer to reducing the severity of the subject’s infection symptoms.

[0107] As used herein, the term "prevention" refers to the complete or near-complete cessation of the occurrence of a disease or a disease-related condition or symptom, for example, when a patient or subject is susceptible to or at risk of contracting a respiratory disease. Prevention may also include suppression, for example, preventing the development of a respiratory disease, or post-exposure prophylaxis (e.g., preventing the development of a disease or disease-related symptoms after initial exposure to a pathogen associated with the disease).

[0108] As used in this article, the term “reduce the risk of…” refers to reducing the likelihood or probability of developing a respiratory disease, such as when a patient or subject is susceptible to or at risk of contracting a respiratory disease.

[0109] As used herein, the term "adjuvant" refers to an agent that enhances the pharmaceutical effect of another agent. As used herein, an antimicrobial agent expressed and secreted by an artificially engineered microorganism, compared to the efficacy level of the same type of microorganism that has not been artificially engineered to express and secrete an antimicrobial agent, is expressed and secreted by an artificially engineered microorganism by, for example, by enhancing the efficacy level (i.e., the ability to kill pathogenic bacteria and / or fungi) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 35%, at least 50%, at least 60%, at least 90%, and all amounts in between. Therefore, the antimicrobial agent expressed and secreted by the artificially engineered microorganism exerts its effect.

[0110] As used herein, the terms “antimicrobial agent,” “antimicrobial protein,” or “antimicrobial polypeptide” are used interchangeably and refer to any entity possessing antimicrobial activity, i.e., the ability to inhibit the growth and / or kill bacteria and / or fungi, such as Gram-positive and Gram-negative bacteria and fungi. An antimicrobial agent is any agent that, compared to the absence of an antimicrobial agent, causes the growth of bacteria and / or fungi to be inhibited or their viability to be reduced by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more than 70%, or any integer more between 30% and 70%. In other words, an antimicrobial agent is any agent that, compared to the absence of an antimicrobial agent, reduces the bacterial and / or fungal cell population by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more than 70%, or any integer more between 30% and 70%. In one embodiment, the antimicrobial agent is an agent that specifically targets bacterial cells. In another embodiment, the antimicrobial agent modifies (i.e., inhibits, activates, or increases) the pathway specifically expressed in bacterial cells. In some specific embodiments of the above aspects and implementations, the antimicrobial agent is a polypeptide, i.e., a polypeptide expressed and secreted by the artificially modified microorganism.

[0111] Antimicrobial agents may include chitinase, glucanase, or peptidoglycan hydrolase.

[0112] As used herein, the term "chitinase" refers to an enzyme capable of catalyzing the hydrolysis of β-1,4-linked N-acetylglucosamine polymers that form chitin chains, a major component of the fungal cell wall. Chitinases are expressed in plants in response to pathogens.

[0113] As used herein, the term "glucanase" refers to an enzyme capable of catalyzing the degradation or depolymerization of complex sugars. The glucanase in the composition is capable of degrading one or more of cellulosic sugars, lignin cellulose, cellulose, hemicellulose, and pectin. This enzymatic activity can be, but is not limited to, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetyxylan esterase, α-amylase, β-amylase, glucosylamylase, amylopectinase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, or pectic acid lyase activity. The glucanase in the composition may be capable of degrading one or more of β-glucan, cellulose, cellobiose, pNP-D-glucopyranoside, and xylan.

[0114] As used herein, the term "peptidoglycan hydrolase" refers to an enzyme that can degrade bacterial cell walls when exposed to external forces. Bacterial cell walls are composed of peptidoglycan chains cross-linked by flexible peptide side chains, providing strength and rigidity. The peptidoglycans of both Gram-positive and Gram-negative bacteria are characterized by repeating units of N-acetylglucosamine (NAG) and β-(1-4)-N-acetylmuramic acid (NAM) cross-linked by peptide stem chains attached to NAM residues. So-called peptidoglycan hydrolases (PGHs) are enzymes responsible for cleaving bonds within the peptidoglycan chains and side chain branches.

[0115] The terms “infection” or “microbial infection” used interchangeably in this document refer in their broadest sense to any infection caused by a microorganism, including bacterial, fungal, yeast, and protozoan infections.

[0116] In this disclosure, the subject can be a mammal. As used herein, "mammal" and its grammatical variations refer to any class of mammals. In this disclosure, mammals include, for example, humans, farm animals, livestock, laboratory animals, etc. Some examples of farm animals include cattle, pigs, horses, goats, etc. Some examples of livestock include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. Preferably, the mammal is a human.

[0117] As used herein, the term "effective amount" or "therapeutic effective amount" for a compound or composition disclosed herein is the amount of such compound or composition sufficient, when applied to a subject, to achieve the beneficial or desired results as described herein. Effective dosage forms, routes of administration, and doses may be determined empirically, and such determinations are within the capabilities of the art. Those skilled in the art will understand that doses will vary with route of administration, rate of excretion, duration of treatment, characteristics of any other drug administered, age, size, and species of the mammal (e.g., human patient), and similar factors well known in the medical and veterinary fields. Generally, a suitable dose of a composition according to this disclosure will be the amount of the composition at the lowest dose required to effectively produce the desired effect. An effective dose of a composition of this disclosure may be administered as two, three, four, five, six, or more sub-dose doses administered at appropriate intervals throughout the day.

[0118] As used herein, the term "application" means contact between a pharmaceutical composition, therapeutic composition, diagnostic agent, or composition and a recipient (preferred person). The therapeutic agents disclosed herein may be administered intranasally, topically, or into the nasal cavity or nasopharynx.

[0119] As used herein, the terms "peptide" or "protein" refer to a biomolecule or macromolecule composed of amino acid residues bonded together in a chain. The definition of peptide as used herein is intended to include proteins (typically of higher molecular weight) composed of long chains of one or more amino acid residues and small peptides (typically of lower molecular weight) consisting of a few amino acids. In other embodiments, a single amino acid, although technically not a peptide, is also considered to be within the scope of this disclosure.

[0120] The articles “a” and “an” used in this article should be understood as meaning “at least one”, unless explicitly stated otherwise.

[0121] This article uses the term “including” to mean the phrase “including but not limited to”, and it is used interchangeably with the phrase “including but not limited to”.

[0122] This document uses the term “or” to mean “and / or” and it may be used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0123] Microbial composition According to some embodiments, this disclosure provides microbial compositions comprising one or more of a wide range of bacteria applicable to mammalian skin. Examples include, but are not limited to, non-pathogenic and commensal bacteria. Bacteria suitable for use in this disclosure include, but are not limited to, *Bifidobacterium*, *Bryophyte*, *Corynebacterium*, *Dermatobacterium*, *Lactococcus*, *Streptococcus*, *Staphylococcus* (e.g., *Staphylococcus epidermidis* and / or *Staphylococcus hominis*), *Lactobacillus* (e.g., *Lactobacillus acidophilus*), *Pediococcus*, *Leuconostoc*, or *Staphylococcus*. According to some embodiments, the microbial composition comprises one or more of *Staphylococcus westermani*, *Streptococcus pyogenes*, *Streptococcus mitis*, *Cutibacterium acnes*, *Corynebacterium* species, *Acinetobacter johnsonii*, and *Pseudomonas aeruginosa*. According to some embodiments, other relevant or similar species found on the skin are used.

[0124] Some implementations involve the use of the bacteria Staphylococcus epidermidis. According to some implementations, the strain of Staphylococcus epidermidis to be used cannot produce a biofilm. Examples of this are Staphylococcus epidermidis strains ATCC 12228, NRRL B-4268, or SE25.

[0125] According to some embodiments, the recombinant microorganism is adapted to survive indefinitely or for a controlled duration on the surface of mammalian skin to provide a controlled or continuous supply of therapeutic peptides (e.g., antimicrobial agents). In some embodiments of the above aspects and embodiments, the antimicrobial agent is a small molecule or peptide. In some embodiments of the above aspects and embodiments, the recombinant microorganism coexists with naturally occurring commensal microorganisms on mammalian skin. In some embodiments of the above aspects and embodiments, the recombinant microorganism excludes the survival of naturally occurring commensal microorganisms on mammalian skin. According to some embodiments, the recombinant microorganism is adapted to reproduce on mammalian skin.

[0126] In other embodiments, the recombinant microorganism is no longer alive but contains an effective amount of antimicrobial peptides, such as epidermin-like lanthanide, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, chondroprotein 1, chitosan glycosylase, mucin 9, chondroprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylan esterase, α-amylase, β-amylase, glucosylamylase, amylopectinase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectic acid lysinase, lysostaphin, zoocin A, millericin B, and muramidase. Cpl-1, lysozyme, intracytolysin PlyC, intracytolysin, PlyVl2, enterolysin A, Clostridium difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilisin, epidermin, Gallidermin, variadin Ba Ny266, variadin 1140, Pep5, Epicidin 280, Epilancin K7, nisin 481, cytolysin, nisin 3147, staphylococcal C55, Salvaricin A, lactobacillusin S, streptococcal A-FF2, Sublancin 168, Carnocin U149, Variacin 8, spiculin, cinnamomumin, nisin, angiotensin-converting enzyme inhibitory peptide, Mersacidin, and Actagardine, or one or more of their therapeutically effective domains.

[0127] In some embodiments of the above-mentioned aspects and implementation plans, the recombinant microorganism is able to produce 6-N-hydroxyaminopurine (6-HAP).

[0128] In some embodiments of the above-mentioned aspects and implementation schemes, the antimicrobial agent is effective against bacterial pathogens, fungal pathogens, or viral pathogens. In some embodiments of the above-mentioned aspects and implementation schemes, the antimicrobial agent is effective against Staphylococcus spp., Streptococcus spp., Haemophilus spp., Moraxella spp., Escherichia spp., Enterobacter spp., Proteus spp., Klebsiella spp., Pseudomonas spp., Legionella spp., Chlamydia spp., Propionibacterium spp., Mycoplasma spp., Malassezia spp., Candida spp., Aspergillus spp., Cryptococcus spp., Pneumocystis spp., Respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus. In some embodiments of the above-mentioned aspects and implementation plans, the antimicrobial agent is effective against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, E. faecium, and Candida albicans.

[0129] According to some embodiments, the therapeutic protein comprises one or more LEKTI domains that are effective against serine proteases present in the skin, upper and lower respiratory tract tissues, other mammalian tissues, or in the upper and systemic circulation. According to some embodiments, the recombinant LEKTI domain compensates for naturally occurring defective endogenous LEKTI proteins or other defective serine protease inhibitors in the skin, upper and lower respiratory tract tissues, other mammalian tissues, or in the upper and systemic circulation. According to some embodiments, genetically modified bacteria are capable of self-replication while maintaining the ability to produce the recombinant protein, thereby providing a continuous supply of therapeutic agents.

[0130] According to some embodiments, the therapeutic protein comprises one or more proteins encoded by one or more SPINK genes. According to some embodiments, the one or more SPINK genes are selected from SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13, and SPINK14. According to some embodiments, the SPINK protein is SPINK5. SPINK genes can be obtained from any mammalian gene sequence, such as mouse, rat, rabbit, goat, sheep, horse, cattle, dog, primate, or human gene sequences. According to some embodiments, the SPINK gene sequence is a human gene sequence.

[0131] SPINK5 geneAccording to some embodiments, recombinant microorganisms are artificially modified to express the mammalian gene SPINK5, which encodes the LEKTI protein. The SPINK5 gene can be obtained from any mammal, such as a mouse, rat, rabbit, goat, sheep, horse, cattle, dog, primate, or human gene sequence. According to some embodiments, the SPINK5 gene sequence is a human gene sequence. According to some embodiments, recombinant microorganisms are artificially modified to contain a fragment of the SPINK5 gene.

[0132] According to some embodiments, the recombinant protein expressed by an artificially engineered microorganism comprises the peptide sequence according to SEQ ID NO: 109 (LEKTID6). According to some embodiments, the recombinant protein expressed by an artificially engineered microorganism comprises the peptide sequence according to SEQ ID NO: 109. According to some embodiments, the artificially engineered microorganism expresses one or more fragments of the peptide sequence according to SEQ ID NO: 103. In one embodiment, the fragment comprises one or more LEKTI domains. In a specific embodiment, the LEKTI domain is domain 6.

[0133] According to some embodiments, the recombinant microorganism comprises a sequence as disclosed herein that has at least about 75%, or 80%, or 85%, or 90%, or 95% identity with any one or more SEQ ID NOS listed herein. As used herein, the term “identity” and its grammatical form refer to the degree to which two nucleotide or amino acid sequences have the same residues at the same position in an alignment. Percentage (%) identity is calculated by multiplying the number of matches in the sequence alignment by 100 and dividing by the length of the aligned region (including internal gaps).

[0134] According to some embodiments, the recombinant protein expressed by artificially engineered microorganisms includes one or more protease repressive domains of the LEKTI protein. Some non-limiting examples include one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15. According to some embodiments, the recombinant protein expressed by artificially engineered microorganisms includes LEKTI repressive domain 6 or domains D8 through D11.

[0135] In some embodiments of the foregoing aspects and implementations, the LEKTI protein domain acts as a competitive or non-competitive inhibitor of one or more proteases present on or within mammalian skin. In some embodiments of the foregoing aspects and implementations, the LEKTI protein domain acts as a serine protease inhibitor. As used herein, the terms "protease" and "proteolytic enzyme" are used interchangeably, both referring to enzymes that perform proteolysis.

[0136] According to some embodiments, microorganisms are genetically modified by transfection / transformation with recombinant DNA plasmids encoding therapeutic peptides (e.g., LEKTI protein domains), small molecules, or metabolites. Other conventional or undiscovered methods for introducing DNA into microorganisms may also be used in this disclosure. According to some embodiments, the recombinant DNA plasmid comprises a sequence encoding a LEKTI protein domain and one or more secretory peptides and / or cell-penetrating peptides. According to some embodiments, the LEKTI domain is operatively linked to one or more recombinant protein domains, which are effective for enhancing secretion from microorganisms and / or penetration into mammalian tissues.

[0137] According to some embodiments, this disclosure provides a composition for the prevention or treatment of respiratory bacterial, viral, fungal, and other microbial infections, oropharyngeal cancer, respiratory cancers, neuronal degeneration in spinal cord injuries, multiple sclerosis, glioblastoma, and other oligodendrocyte pathologies, comprising a microorganism genetically modified to express and deliver through the mammalian nasal cavity one or more LEKTI protein domains or LEKTI proteins, wherein the LEKTI protein domains or LEKTI proteins are effective in inhibiting the serine protease activity of at least one serine protease in mammalian skin, upper and lower respiratory tract tissues, other mammalian tissues, or in the upper or systemic circulation. According to some embodiments, this disclosure provides a composition for the prevention or treatment of respiratory diseases (e.g., chronic rhinosinusitis). According to some embodiments, this disclosure provides a composition for the prevention or treatment of central nervous system disorders and diseases. As used herein, the term "recombinant" and its grammatical variations refer to or represent biological, protein, or genetic material formed from or using recombinant DNA comprising DNA fragments from different sources or different portions from the same source. For example, the term "recombinant DNA" refers to a DNA molecule formed by recombinant methods by splicing DNA fragments from different sources or different parts of the same source. In some embodiments, two or more different DNA sources are cut using restriction enzymes and ligated together using ligases. As another example, the terms "recombinant protein" or "recombinant domain" and their grammatical variations refer to a protein molecule formed by recombinant methods from DNA fragments spliced ​​from different sources or different parts of the same source. As yet another example, the terms "recombinant microorganism" or "recombinant bacteria" and their grammatical variations are interchangeable and refer to microorganisms containing one or more recombinant DNA / protein molecules.

[0138] According to some embodiments, the recombinant microorganism comprises a sequence as disclosed herein that has at least about 75%, or 80%, or 85%, or 90%, or 95% identity with any one or more SEQ ID NOs listed herein. As used herein, the term “identity” and its grammatical form refer to the degree to which two nucleotide or amino acid sequences have the same residues at the same position in an alignment. Percentage (%) identity is calculated by multiplying the number of matches in the sequence alignment by 100 and dividing by the length of the aligned region (including internal gaps).

[0139] According to some embodiments, microorganisms are genetically modified by transfection / transformation with one or more recombinant DNA plasmids encoding one or more therapeutic proteins (e.g., antimicrobial peptides, such as epiA, elastase inhibitor, hiracin, lysostaphin, and LL-37). Other conventional or undiscovered methods for introducing DNA into microorganisms may also be used in this disclosure. According to some embodiments, one or more recombinant DNA plasmids contain sequences encoding antimicrobial peptides.

[0140] In some embodiments of the above aspects and embodiments, the therapeutic protein has 90% identity with the entire sequence of SEQ ID NOs: 130-145. Therefore, in one embodiment, the therapeutic protein has at least about 95% identity with the entire sequence of SEQ ID NOs: 130-145. Therefore, in one embodiment, the therapeutic protein has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NOs: 130-145. In yet another embodiment, the therapeutic protein consists of the sequence of SEQ ID NOs: 130-145.

[0141] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment, such that the function of one nucleic acid sequence is regulated by or not impaired by the other. For example, when a promoter can regulate the expression of a coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter), the promoter and coding sequence are operably linked. The coding sequence can be operably linked to the regulatory sequence in a sense or antisense direction. In another example, two proteins can be operably linked so that the function of either protein is not impaired. Generally, operably linked means that the linked nucleic acid sequences are adjacent, and in the case of linking two protein coding regions, they are adjacent and in the same reading frame.

[0142] As used herein, the term "gene" can refer to a genomic gene that contains transcriptional and / or translational regulatory sequences and / or coding regions and / or untranslated sequences (e.g., introns, 5′- and 3′-untranslated sequences, and regulatory sequences). The coding region of a gene can be a nucleotide sequence encoding an amino acid sequence or a functional RNA (e.g., tRNA, rRNA, catalytic RNA, siRNA, miRNA, and antisense RNA). A gene can also be mRNA or cDNA corresponding to a coding region (e.g., exons and miRNA), optionally containing a 5′- or 3′-untranslated sequence linked thereto. A gene can also be an in vitro generated amplified nucleic acid molecule containing all or part of a coding region and / or a 5′- or 3′-untranslated sequence linked thereto.

[0143] As used herein, the term "one or more gene products" refers to RNA transcribed from a gene, or polypeptides encoded by a gene or translated from RNA.

[0144] As used herein, the terms "secretory peptide," "secretory sequence," "secretory tag," "signal peptide," or "output signal," and their grammatical variations, refer to any peptide sequence capable of targeting a synthetic protein to the cellular secretory pathway. In some embodiments of the foregoing aspects and embodiments, the secretory peptide may be located at the N-terminus of the recombinant protein and may co-translate or post-translationally target the tagged protein for secretion. In some embodiments of the foregoing aspects and embodiments, the secretory peptide is located at the C-terminus of the recombinant protein. In some embodiments, an antimicrobial peptide is fused to a secretory peptide. In some embodiments, an antimicrobial peptide is fused to both a secretory peptide and a propeptide.

[0145] Secretory peptides: According to some embodiments, one or more therapeutic recombinant peptides and / or one or more metabolites (e.g., LEKTI domains) are operatively linked to one or more secretion signals or output signals, which label proteins for transport via the secretory pathway. Any secretion signal that promotes the release of LEKTI proteins from bacterial cells can be used as a secretory peptide. Non-limiting examples of secretory peptide signals are illustrated in Table 1 below: Table 1: List of Secretory Peptides amino acid sequence SEQ ID NO: MKKLAFAITAASGAAAVLSHHDAEA 9 WLDNRAFSKKFVPVVMATSVALFFLNLAFA 10 MAKKFNYKLPSMVALTLFGTAFTAHQANA 11 MKKRFLSICTMTIAALATTTMVNTSYA 12 NLKKQSKLILIFICIFTFFIMIIQSQFLMG 13 MKIFKLTSLTLAALTLAFPFSHVAQA 14 MKKTVIASTLAVSLGIAGYGLSGHEAH 15 MKKNKFLVYLLSTALITPTFATQTAFA 16 MKTRQNKYSIRKFSVGASSILIAALLFMGGGSAQA 17 MKNNNETRRFSIRKYTVGVVSIITGITIFVSGQHAQA 18 MKKKLSYMITIMLAFTLSLALGLFFNSAHA 19 According to some implementation schemes, the therapeutic LEKTI domain is operatively linked to one or more signaling sequences of endogenous proteins derived from Staphylococcus epidermidis. Non-limiting examples of secretory signal peptides of endogenous proteins derived from Staphylococcus epidermidis are illustrated in Table 2 below: Table 2: List of signal peptides from Staphylococcus epidermidis According to some embodiments, a recombinant therapeutic polypeptide (e.g., a LEKTI domain) is operably linked to one or more secretion signal sequences derived from endogenous proteins of other bacteria. Non-limiting examples of secretion signal peptides derived from endogenous proteins of various bacteria are set forth in Tables 1 and 2.

[0146] According to some embodiments, a recombinant therapeutic polypeptide (e.g., a LEKTI domain) is operably linked to a cell-penetrating peptide sequence that enhances the ability of the recombinant therapeutic polypeptide to cross cell membranes. As used in the term describing the cell-penetrating peptide / recombinant therapeutic polypeptide, the term "enhances" means that the cell-penetrating sequence improves the ability of the recombinant therapeutic polypeptide to cross cell membranes relative to a recombinant therapeutic polypeptide lacking a cell-penetrating sequence.

[0147] Cell-penetrating peptides : According to some embodiments, one or more cell-penetrating peptides are used to mediate the delivery of a therapeutic protein in vivo without using cell surface receptors and without causing significant membrane damage. According to some embodiments, one or more cell-penetrating peptides are operably linked to a therapeutic protein to facilitate entry into skin cells (e.g., keratinocytes). Non-limiting examples are set forth in Table 3 below: Table 3: List of Cell-Penetrating Peptides According to some embodiments, the cell-penetrating peptide comprises a periodic amino acid sequence. Non-limiting examples of periodic cell-penetrating sequences include: polyarginine, R x n (where 4 < n < 17); polylysine, K x n (where 4 < n < 17); arginine repeats separated by 6-aminohexanoic acid residues (RAca), with 2 to 6 arginine repeats; arginine repeats separated by 4-aminobutyric acid (RAbu), with 2 to 6 arginine repeats; arginine repeats separated by methionine, with 2 to 6 arginine repeats; arginine repeats separated by threonine, with 2 to 6 arginine repeats; arginine repeats separated by serine, with 2 to 6 arginine repeats; and arginine repeats separated by alanine, with 2 to 6 arginine repeats.

[0148] According to some embodiments, one or more therapeutic recombinant polypeptides and / or one or more metabolites are operably linked to a cell-penetrating peptide, e.g., the RMR domain (SEQ ID NO: 4).

[0149] According to some implementation schemes, the LEKTI domain is operatively connected to the RMR domain (SEQ ID NO: 4).

[0150] According to some embodiments, the expression of the recombinant therapeutic peptide (e.g., the LEKTI domain) is controlled by an operator, and the amount of LEKTI available to mammalian skin is proportional to the availability of external factors. For example, in some embodiments, the recombinant LEKTI gene may be under the control of a xylose-inducible promoter (e.g., xylose repressor (xylR), xylose operator (xylO), xylose isomerase gene (xylA), including cis-acting catabolite response elements (CREs), and the amount of recombinant LEKTI protein available to mammalian skin is controlled by the amount of exogenous xylose available to the recombinant microorganism. According to some embodiments, the expression of the recombinant therapeutic peptide (e.g., the LEKTI domain) is controlled by a constitutively active promoter. According to some embodiments, the expression of the recombinant therapeutic peptide (e.g., the LEKTI domain) is controlled by a CmR promoter.

[0151] According to some embodiments, microorganisms are genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a recombinant therapeutic peptide (e.g., a LEKTI protein domain) and one or more antibiotic resistance genes. For example, some embodiments of the recombinant DNA plasmid contain a kanamycin resistance gene and / or a trimethoprim resistance gene; for example, dfrA. According to some embodiments, treating mammalian skin with an antibiotic (to which the recombinant microorganism is resistant) can be used to bias a population of symbiotic microorganisms toward microorganisms that produce a greater proportion of the recombinant therapeutic peptide. Other elements that may be present in the recombinant DNA plasmid include, but are not limited to, replication protein genes, such as members of the Rep superfamily of replication proteins. For example, in some embodiments, the recombinant DNA plasmid contains the repF gene.

[0152] This disclosure utilizes standard molecular biology techniques, such as those described by Sambrook et al., 2001. In some embodiments, the genetic construct used in the disclosure is based on plasmid pBT-2, which is an allele exchange shuttle vector between *Escherichia coli* and *Staphylococcus* species (Nakanishi, Oshida et al., 1986). In some embodiments, the gene for the recombinant therapeutic polypeptide is inserted into the plasmid. In some embodiments, the coding sequence is driven by a promoter, such as an inducible or constitutive one. Examples of inducible promoters include those activated by compounds (e.g., alcohols, sugars, metals, or tetracyclines) or physical factors (e.g., light or high temperature).

[0153] According to some embodiments, the recombinant DNA plasmid contains one or more sequences of the pUBTR vector. According to some embodiments, the recombinant LEKTI is operatively linked to an inducible promoter, ribosome binding site, output signal, and / or cell-penetrating peptide in the pUBTR vector. According to some embodiments, the recombinant LEKTI is operatively linked to an inducible promoter, ribosome binding site, output signal, and / or cell-penetrating peptide in the pUBTR vector. According to some embodiments, the pUBTR vector is pUBTR119.

[0154] According to some embodiments, the recombinant DNA plasmid contains the complete pKK30-LEKTI sequence. According to some embodiments, this disclosure provides a composition for treating skin diseases comprising microorganisms containing a complete pKK30-LEKTI plasmid construct.

[0155] According to some embodiments, the quantity or duration of availability of a recombinant therapeutic peptide (e.g., LEKTI protein) is controlled by the stability of the vector carrying the recombinant therapeutic peptide in microorganisms. For example, the persistence of the recombinant vector can be controlled by one or more elements of the plasmid, including those that provide beneficial genes to the host, plasmid stability mechanisms, and plasmid co-adaptation. For example, some plasmids can provide stable replication, active partitioning mechanisms, and mechanisms that ensure reliable inheritance of the plasmid to daughter cells during generations. (See, for example, JCBaxter, BEFunnell, Plasmid partition mechanisms, Microbiol. Spectr., 2(2014) PLAS-0023-2014 and Nils Hülter et al., Anevolutionary perspective on plasmid lifestyle modes, Current Opinion in Microbiology, Vol. 38, August 2017, pp. 74-80, each incorporated herein by reference in its entirety). According to some embodiments, this disclosure includes the use of all conventional selection and stabilization methods known to those skilled in the art.

[0156] Examples of proteins that can be applied according to this disclosure are mostly eukaryotic proteins. These may include, but are not limited to, single amino acids, small peptides, and large proteins. More specifically, genes encoding proteins useful as recombinant therapeutic proteins in this disclosure include, but are not limited to, the following genes: members of the interleukin gene family, including but not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15, and genes encoding their receptor antagonists; genes encoding hematopoietic growth factors, including but not limited to erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, leukemia inhibitory factor, and thrombopoietin, which are also contemplated in this disclosure; genes encoding neurotrophic factors, including but not limited to nerve growth factor, brain-derived neurotrophic factor, and ciliary neurotrophic factor; and genes encoding interferons, including but not limited to IFN-α, IFN-β, and IFN-γ. This disclosure further envisions genes encoding chemokines (e.g., the CC and CXC families of cytokines), genes encoding hormones (e.g., proinsulin and growth hormone), and genes encoding thrombolytic enzymes (including tissue plasminogen activator, streptokinase, urokinase, or other enzymes such as trypsin inhibitors). This disclosure further includes genes encoding tissue repair factors, growth and regulatory factors, such as, but not limited to, oncokinin M, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, bone morphogenetic protein, insulin-like growth factor, calcitonin, and transforming growth factor α and β. Further envisioned genes include those encoding structural proteins, such as fibrin, actin, collagen, myofibril, elastin, or scleroprotein. In some embodiments, the recombinant microorganism includes genes encoding antimicrobial peptides (e.g., avidin, bacterial or fungal cell wall hydrolases, elastase inhibitors, hiracin, lysostaphylin, LL-37, and epidermin, or variants thereof, or combinations thereof).

[0157] This disclosure also relates to allelic variants of LEKTI or portions thereof (one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15), and synthetic or mutant genes of SPINK (e.g., SPINK5) that have been modified to alter the expression or activity of, for example, recombinant proteins. It should also be noted that the degeneracy of a nucleic acid code can be considered as variation in nucleotide sequences encoding the same amino acid residues. Therefore, this disclosure includes nucleic acid residues capable of hybridization under moderately stringent conditions. Those skilled in the art can determine efficient combinations of salt and temperature to constitute moderately stringent hybridization conditions. Orthologs of LEKTI are also envisioned to exist in other species, such as dogs, sheep, rats, hamsters, chickens, and pigs. Therefore, another embodiment of this disclosure relates to a SPINK (e.g., SPINK5) nucleic acid encoding a polypeptide having at least about 70% to 80% identity, preferably 90% to 95% identity, and more preferably 98% to 99% identity with LEKTI or a portion thereof (one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15) as described in SEQ ID NO: 103. In a specific embodiment, the LEKTI domain is domain 6.

[0158] According to some implementation schemes, the LEKTI domain is selected from the following non-limiting examples, Tables 4 and 5.

[0159] Table 4: LEKTI amino acid sequence Table 5: SPINK5 nucleotide sequence As used herein, the term "auxotrophic" or "auxotrophic" refers to an organism that is unable to synthesize specific compounds required for its growth. An auxotroph is an organism that exhibits the aforementioned characteristics.

[0160] As used herein, the terms "alrA" and "alr" refer to the D-alanine racemase gene, including the normal allele of the alarA gene. In some embodiments, the alar gene (UniProtKB-Q8CNK7(ALR_STAES)) from Staphylococcus epidermidis encodes the D-alanine racemase protein (EC 5.1.1.1). In some embodiments, the locus identifiers SE1674 (alrl) and SE1079 (alr2) refer to specific Staphylococcus epidermidis D-alanine racemase genes.

[0161] As used herein, the term "dat" refers to the D-alanine aminotransferase gene, including the normal allele of the dat gene. In some embodiments, the dat gene from *Staphylococcus epidermidis* (UniProtKB-Q8CS41(DAAA_STAES)) encodes the D-alanine aminotransferase protein (EC: 2.6.1.21). In some embodiments, the locus identifier SE1423(dat) refers to a specific *Staphylococcus epidermidis* D-alanine aminotransferase gene. As used herein, the term "murI" refers to the glutamate racemase gene, including the normal allele of the murI gene. In some embodiments, the murI gene from *Staphylococcus epidermidis* (UniProtKB-Q8CPL0(MURl_STAES)) encodes the glutamate racemase protein (EC: 5.1.1.3). In some embodiments, the locus identifier SE0843(murl) refers to a specific *Staphylococcus epidermidis* glutamate racemase gene.

[0162] D-alanine auxotrophs of Staphylococcus aureus have been produced for the purpose of developing vaccines against MRSA. (Moscoso M et al., 27th ECCMID 22-25, April 2017, The Congress of ESCMID (P0473); Moscoso et al., Virulence (2018) Vol. 9(1): 604-620, the contents of each of which are incorporated herein by reference in their entirety). In this case, it was found necessary to knock out only the alanine racemic enzymes arl1 and arl2, as well as the dat gene.

[0163] According to some embodiments, recombinant microorganisms are artificially modified to express therapeutic peptides, small molecules, or metabolites to treat, prevent, or improve diseases, conditions, or infections. In some embodiments, the composition comprises artificially modified microorganisms. In some embodiments, the composition can treat, prevent, or improve bacterial and other microbial (i.e., viral) infections, oral cancer, CNS diseases or injuries, and non-infectious (i.e., chronic rhinosinusitis) or infectious nasal, oral, or respiratory diseases.

[0164] According to some implementation plans, non-infectious and infectious diseases may include, but are not limited to, non-allergic rhinitis, vasomotor rhinitis, non-allergic rhinitis with eosinophilic syndrome, drug-induced rhinitis, atrophic rhinitis, pemphigus, benign mucosal pemphigoid, linear IgA bullous dermatitis, herpetic dermatitis, acquired epidermolysis bullosa, erythema multiforme, lichen planus, asthma, allergic rhinitis, cough, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, acute bacterial rhinosinusitis, chronic rhinosinusitis, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, bronchitis, and pneumonia.

[0165] According to another aspect, this disclosure provides a kit for treating or improving the effects of bacterial and fungal infections (e.g., Staphylococcus aureus) in mammals in need, comprising: (1) a composition containing microorganisms genetically modified to express antimicrobial peptides; and (2) a reagent for applying said composition to mammalian skin. According to some embodiments, the microorganisms are adapted to survive on the surface of mammalian skin for a controlled duration and provide a controlled or continuous supply of antimicrobial peptides.

[0166] According to another aspect, this disclosure provides a kit for treating or improving the effects of a disease (e.g., RSV) in a mammal in need, comprising: (1) a composition containing a microorganism genetically modified to express a therapeutic agent (e.g., LEKTI protein or a LEKTI protein domain or a variant thereof); and (2) an agent for applying said composition to mammalian skin or nose. According to some embodiments, the microorganism is adapted to survive on the surface of mammalian skin for a controlled duration and to provide a controlled or continuous supply of antimicrobial peptides.

[0167] In some embodiments, artificially engineered microorganisms are applied to the skin of a subject. In some embodiments, artificially engineered microorganisms are applied to a subject suffering from a skin condition. In some embodiments, the skin condition is a skin disease. In some embodiments, the skin disease or condition is a skin dysbiosis. Dysbiosis in the skin and / or gut microbiome is associated with altered immune responses, thereby promoting the development of skin diseases. In some embodiments, the skin disease or condition is an inflammatory skin disease or condition. In some embodiments, the skin disease is attributable to the skin toxicity of one or more chemotherapeutic agents (including, but not limited to, epidermal growth factor receptor (EGFR) inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, RAS inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, and extracellular signal-regulated kinase (ERK) inhibitors); in some embodiments, the skin disease is ichthyosis. In some embodiments, the skin disease is Netherton's syndrome. In some embodiments, the skin disease is hidradenitis suppurativa. In some embodiments, the skin disease is a psoriatic disease (including, but not limited to, psoriasis, pustular psoriasis, plaque psoriasis, and palmoplantar psoriasis). In some embodiments, the skin disease is neutrophilic dermatitis. In some embodiments, the skin disease is atopic dermatitis. In some embodiments, the skin disease is an autoimmune vesicular disease. In some embodiments, the skin disease is acne and / or acneiform eruptions. In some embodiments, the skin disease is impetigo. In some embodiments, the skin disease is folliculitis. In some embodiments, the skin disease is acute suppurative paronychia. In some embodiments, the skin disease is lymphangitis. In some embodiments, the skin disease is cellulitis. In some embodiments, the skin disease is necrotizing fasciitis.

[0168] As used herein, the term “skin” (e.g., skin applied to a subject) refers to the outer layer of a subject’s (e.g., a mammal or a human) body.

[0169] As used in this article, the term "skin dysbiosis" refers to a condition where, for example, the skin's microbiome is imbalanced. As a result of skin dysbiosis, inflammatory skin diseases and conditions can develop in individuals, such as atopic dermatitis, eczema, seborrheic dermatitis, psoriasis, acne vulgaris, dandruff, and even skin cancer. Atopic dermatitis can refer to a chronic inflammatory skin condition.

[0170] Atopic dermatitis can be associated with scaly skin, pruritic rashes, and lesions. It can also be called atopic eczema or simply eczema. A hallmark of atopic dermatitis is itching, referring to the itchy condition that causes the rash. Any part of the skin can be affected by atopic dermatitis. Age plays a role in the localization of atopic dermatitis. For example, in infants, atopic dermatitis can affect the cheeks, scalp, trunk, and extremities; in young children, it can be confined to flexor areas; and in adolescents and adults, it can affect the hands and feet.

[0171] In addition to the components described above, the thematic kit will further include instructions for using the components and / or practicing the thematic methods. These instructions may be present in a variety of forms within the thematic kit, one or more of which may be present in the kit. One form of these instructions may be as printed information on a suitable medium or substrate, such as one or more sheets of paper on which information is printed, in the kit packaging, or in a packaging insert. Another form would be computer-readable media, such as a disk or CD on which the information has been recorded. Furthermore, another form of the instructions may be as a website address used via the Internet to access information on a remote website. Any convenient form may be present in the kit.

[0172] The kit components can be packaged in an aqueous medium or in lyophilized form. The kit will typically be packaged as comprising at least one vial, tube, bottle, syringe, or other container device in which the reagents can be placed, and preferably appropriately aliquoted. When additional components are provided, the kit will also typically contain a second, third, or other additional container for holding such components.

[0173] The kits disclosed herein will also generally include means for tightly containing reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers holding the desired vials.

[0174] preparation According to some embodiments, formulations used in accordance with this disclosure may contain any pharmaceutically effective amount of recombinant bacteria to produce a therapeutically effective amount of the desired antimicrobial peptide or one or more of its therapeutically effective domains, for example, at least by weight of about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, or about 4.0%. % to 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0% or more of recombinant bacteria, with an upper limit of about 90.0% by weight of recombinant bacteria.

[0175] According to some embodiments, the formulation used in accordance with this disclosure may contain, for example, at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5% or more of recombinant bacteria by weight.

[0176] According to some embodiments, the topical formulation can be any form suitable for application to the body surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, ointments, liniments, bioadhesives, suspensions, emulsions, etc., and / or can be prepared to contain liposomes, microclusters, and / or microspheres. Such formulations can be used in combination with an occlusive overlayer, thereby retaining moisture evaporated from the body surface during and after application. According to some embodiments, the formulation may include a live cell culture composition and may contain at least one artificially engineered bacterial strain that produces a therapeutically effective recombinant peptide or one or more of its therapeutically effective domains. The artificially engineered live cell culture composition can deliver the peptide directly to the skin for the treatment or prevention of abnormal skin conditions.

[0177] Topical formulations include those in which any one or more other active ingredients are dissolved or dispersed in dermatological mediators known in the art (e.g., aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Such mediators may include water, aqueous buffer solutions, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, tetraethylene glycol ether (glycofurol), or glycerin), and oils (e.g., mineral oils such as liquid paraffin, natural or synthetic triglycerides such as Miglyol™, or silicone oils such as polydimethylsiloxane). Depending on the characteristics of the formulation and its intended use and site of application, the dermatological mediators used may contain one or more components selected from the following list (e.g., components other than water when the formulation is a hydrogel): a solvent or soluble additive (e.g., β-cyclodextrin, such as hydroxypropyl β-cyclodextrin, or alcohols or polyols, such as ethanol, propylene glycol, or glycerin); a thickener (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, or carbomer); a gelling agent (e.g., polyoxyethylene-polyoxypropylene copolymer); a preservative (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorobutanol, benzoate, potassium sorbate, or EDTA or its salts); and one or more pH buffers (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate).

[0178] Pharmaceutically acceptable carriers may also be incorporated into formulations of this disclosure and may be any carrier conventionally used in the art. Examples include water, lower alcohols, higher alcohols, polyols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based and emulsion-based mixtures of such carriers. The terms “pharmaceutically acceptable” or “pharmaceuticalally acceptable carrier” are used herein to refer to compounds or compositions that may be incorporated into pharmaceutical formulations without causing undesirable biological effects or unwanted interactions with other components of the formulation. As used herein, “carrier” or “medium” refers to a carrier material suitable for incorporation into a composition for topical application. Useful carriers and mediators herein include any such materials known in the art that are nontoxic and do not interact harmfully with other components of the formulation comprising them. The term “aqueous” refers to a formulation containing water or becoming aqueous upon application to skin or mucous membrane tissue.

[0179] Film-forming agents form a protective film on the application site upon drying. This film inhibits the removal of the active ingredient and maintains its contact with the treated site. An example of a film-forming agent suitable for this disclosure is flexible collodion, US P. As described in Remington: The Science and Practice of Pharmacy, 19th edition (Easton, PA: Mack Publishing Co., 1995), page 1530, collodion is an ether / ethanol solution containing collodion (nitrocellulose), which evaporates to leave a collodion film. The film-forming agent may additionally act as a carrier. The solution dried to form a film is sometimes referred to as paint. Creams, as well as those well known in the pharmaceutical formulation field, are viscous liquids or semi-solid emulsions, and are oil-in-water or water-in-oil emulsions.

[0180] Cream bases are washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also known as the "internal" phase, typically contains petrolatum and aliphatic alcohols such as cetyl alcohol or octadecyl alcohol. The aqueous phase is usually (though not necessarily) larger in volume than the oil phase and usually contains a wetting agent. Emulsifiers in cream formulations are typically nonionic, anionic, cationic, or facultative surfactants.

[0181] Lotions are preparations applied to the skin surface without friction and are generally liquid or semi-liquid preparations, comprising particles of active agents present in an aqueous or alcoholic matrix. Lotions are typically solid suspensions and preferably contain oil-in-water emulsions. Lotions are preferred preparations for treating large body areas because they are easier to apply and offer greater fluidity. Uniform distribution of insoluble substances in the lotion is generally necessary.

[0182] Lotions typically contain suspending agents to produce better dispersion, as well as compounds to position and maintain the active agents in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, etc.

[0183] A solution is a homogeneous mixture prepared by dissolving one or more chemical substances (solutes) in a liquid, such that the molecules of the dissolved substance are dispersed among those molecules in the solvent. Solutions may contain other pharmaceutically or cosmetically acceptable chemical agents to buffer, stabilize, or protect the solute. Common examples of solvents used to prepare solutions are ethanol, water, propylene glycol, or any other acceptable medium. As is well known, gels are semi-solid, suspension-type systems. A single-phase gel contains organic macromolecules that are substantially uniformly distributed throughout a carrier liquid, which is generally aqueous but preferably also contains alcohols and optionally oils. Preferred "organic macromolecules," i.e., gelling agents, are cross-linked acrylic polymers, such as polymers from the "carbomer" family, for example, carboxypolyalkylenes commercially available under the trademark Carbopol. Also preferred are hydrophilic polymers, such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulose polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and methyl cellulose; gums, such as tragacanth gum and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersant, such as alcohol or glycerin, may be added, or the gelling agent may be dispersed by grinding, mechanical mixing, or stirring, or a combination thereof. Ointments, as is well known in the art, are generally semi-solid formulations based on petrolatum or other petroleum derivatives. As those skilled in the art will understand, the specific ointment matrix to be used is a matrix that will provide a variety of desired characteristics (e.g., emolliency, etc.). Like other carriers or media, the ointment matrix should be inert, stable, non-irritating, and non-sensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th edition (Easton, PA: Mack Publishing Co., 1995), pp. 1399-1404, ointment bases can be classified into four categories: oil-based bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oil-based ointment bases include, for example, vegetable oils, fats derived from animals, and semi-solid hydrocarbons derived from petroleum.

[0184] Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum.

[0185] The ointment base is a water-in-oil (W / O) emulsion or an oil-in-water (O / W) emulsion, and includes, for example, acetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weights; for further information, see Remington: The Science and Practice of Pharmacy.

[0186] Pastes are semi-solid dosage forms in which active agents are suspended in a suitable matrix. Depending on the characteristics of the matrix, pastes are classified into fatty pastes and those prepared from single-phase hydrogels. The matrix in fatty pastes is typically petrolatum or hydrophilic petrolatum. Pastes prepared from single-phase hydrogels usually contain carboxymethyl cellulose or similar materials as a matrix.

[0187] Enhancers are lipophilic co-enhancers, generally referred to as "plasticizing" enhancers, i.e., enhancers having a molecular weight in the range of about 150 to 1000, and a water solubility of less than about 1% by weight, preferably less than about 0.5% by weight, and most preferably less than about 0.2% by weight. The Hildebrand solubility parameter δ of the plasticizing enhancer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are aliphatic esters, aliphatic alcohols, and aliphatic ethers. Specific and most preferred examples of fatty acid esters include methyl lauryl acid, ethyl oleate, propylene glycol monolaurate, propylene glycol dilaurate, glyceryl monolaurate, glyceryl monooleate, isopropyl n-decanoate, and octyl dodecyl myristate. Aliphatic alcohols include, for example, octadecyl alcohol and oleyl alcohol, while aliphatic ethers include compounds in which a diol or triol (preferably C2-C4 alkyldiol or triol) is substituted by one or two aliphatic ether substituents.

[0188] Additional penetration enhancers will be known to those of ordinary skill in the field of local drug delivery and / or described in relevant textbooks and literature. See, for example, Percutaneous Penetration Enhancers, edited by Smith et al. (CRC Press, 1995) (included in this article as a reference).

[0189] In addition to the additives identified above, the compositions of this disclosure may also include a variety of other additives. These include, but are not limited to, antioxidants, astringents, fragrances, preservatives, emollients, pigments, dyes, humectants, propellants, and sunscreens, as well as other classes of materials whose presence may be desired for pharmaceutical or other purposes. Typical examples of optional additives included in formulations disclosed herein are as follows: preservatives, such as sorbates; solvents, such as isopropanol and propylene glycol; astringents, such as menthol and ethanol; emollients, such as polyalkylene methyl glucoside; humectants, such as glycerin; emulsifiers, such as glyceryl stearate, PEG-100 stearate, polyglycerol-3-hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols, such as polyethylene glycol; sunscreens, such as octyl methoxycinnamate (available commercially as Parsol MCX) and butyl methoxybenzoylmethane (available under the trade name Parsol 1789); antioxidants, such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, 6-tocopherol, ε-tocopherol, ζ-tocopherol, Z ... Λ-Tocopherol, n-tocopherol, and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soybean oil, palm oil, liquid fractions of shea butter, sunflower oil), animal oils (e.g., fully hydrogenated squalene), synthetic oils, silicone oils or waxes (e.g., cyclomethyl silicone and dimethyl silicone), fluorinated oils (typically perfluoropolyethers), aliphatic alcohols (e.g., cetyl alcohol) and waxes (e.g., beeswax, carnauba wax, and paraffin); skin-feel modifiers; and thickeners and structurants, such as expanded clay and cross-linked carboxylated polyenes commercially available under the trademark Carbopol. Other additives include beneficial agents, such as those that condition the skin (particularly the upper layers of the stratum corneum) and keep the skin soft and / or protect it by slowing down the decrease in its moisture content. Such conditioning agents and humectants include, for example, pyrrolidine carboxylic acids and amino acids; organic antimicrobial agents such as 2,4,4′-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid; anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti-seborrheic agents such as retinoic acid; vasodilators such as niacin; melanin production inhibitors such as kojic acid; and mixtures thereof. Further additional active agents include, for example, alpha-hydroxy acids, alpha-keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts, and antioxidants such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, 6-tocopherol, ε-tocopherol, ζ1-tocopherol, ζ2-tocopherol, η-tocopherol, and retinol (vitamin A) and / or their pharmaceutically acceptable salts, esters, amides, or other derivatives. Preferred tocopherol compounds are α-tocopherol. Additional agents include those capable of improving oxygen supply to skin tissue, such as those described, for example, in WO 94 / 00098 and WO 94 / 00109 of Gross et al., both of which are assigned to Lancaster Group AG (incorporated herein by reference). Sunscreens and UV-absorbing compounds may also be included. Non-limiting examples of such sunscreens and UV-absorbing compounds include aminobenzoic acid (PABA), avobenzone, sinoxalate, dihydroxybenzone, homosalate, menthyl anthranilate, oxtocrylene, octyl methoxycinnamate, octyl salicylate, oxybenzone, pardimethicone O, phenylbenzimidazole sulfonic acid, sulphone, titanium dioxide, triethanolamine salicylate, zinc oxide, ensolazole, meladidone, osinolate, otelisyl ester, and octyl cyanadiazine acrylate. See Section 352 of Chapter 1, Subsection D, of the overall introduction to this article, “Sunscreen drug products for over-the-counter human use”.

[0190] Other embodiments may include a variety of non-carcinogenic, non-irritating healing materials that promote treatment using formulations based on this disclosure. Such healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, gland extracts, or animal extracts, or safe therapeutic agents that may be added to the formulation to promote the healing of skin conditions.

[0191] The amounts of these various additives are those commonly used in the cosmetics industry, and range, for example, from about 0.01% to about 20% of the total weight of the topical formulation.

[0192] The formulations disclosed herein may also include conventional additives, such as opacifiers, fragrances, colorants, stabilizers, surfactants, etc. In some embodiments, other agents, such as antimicrobial agents, may also be added to prevent spoilage during storage, i.e., to inhibit the growth of microorganisms such as yeasts and molds.

[0193] Suitable antimicrobial agents are generally selected from methyl and propylparabens (i.e., methylparaben and propylparaben), sodium benzoate, sorbic acid, imidoliquirylic urea, and combinations thereof. In other embodiments, other agents may be added, such as inhibitors and inducers, i.e., to inhibit (i.e., glucose) or induce (i.e., xylose) the production of the polypeptide of interest. Such additives can be used as long as they are compatible with the formulation and do not interfere with the function of the formulation.

[0194] The formulation may also contain irritant additives to minimize or eliminate the possibility of skin irritation or damage caused by the chemical entity to be applied or other components of the composition.

[0195] Suitable anti-irritant additives include, for example: α-tocopherol; monoamine oxidase inhibitors, particularly benzyl alcohols, such as 2-phenyl-1-ethanol; glycerol; salicylates; ascorbic acid salts; ionotropic agents, such as monensin; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. If present, the anti-irritant additive may be incorporated into the composition at a concentration that effectively reduces irritation or skin damage, generally not exceeding about 20% by weight of the formulation, and more generally not exceeding about 5% by weight.

[0196] Creams, lotions, gels, ointments, pastes, etc., can be applied to the affected surface and gently rubbed in. Solutions can be applied in the same way, but more generally, a dropper, cotton swab, etc., will be used, and the solution will be carefully applied to the affected area.

[0197] The application regimen will depend on many readily identifiable factors, such as the severity of the condition and its responsiveness to initial treatment, but will generally involve application once or more daily on a continued basis. A person skilled in the art can easily determine the optimal amount of the formulation to be applied, the method of administration, and the repetition rate. Generally, the formulations of this disclosure are expected to be used in the range of once or twice a week up to once or twice daily.

[0198] The pharmaceutical compositions of this disclosure comprise one or more active ingredients, such as therapeutic agents, mixed with one or more pharmaceutically acceptable diluents or carriers, and optionally one or more other compounds, pharmaceuticals, ingredients, and / or materials. Regardless of the chosen route of administration, the reagents / compounds of this disclosure are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0199] Pharmaceutically acceptable diluents or carriers are well known in the art (see, for example, Remington, The Science and Practice of Pharmacy (21st edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Journal of Pharmacy). Pharmaceutical Association (Washington, DC) and includes sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starch, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate, and dicalcium hydrogen phosphate), sodium citrate, ectoine, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's solution, glucose injection, glucose and sodium chloride injection, lactate Ringer's solution), alcohols (e.g., ethanol, propanol, oleyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolate, poly(orthoester) and poly(anhydride)), elastomer matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and peanut), cocoa butter, waxes (e.g., suppository waxes), paraffin, silicone, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in the pharmaceutical compositions of this disclosure must be "acceptable" in the sense that it is compatible with other components of the formulation and harmless to the subject. Diluents or carriers suitable for the selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for the selected dosage form and method of administration can be determined using ordinary techniques in the art.

[0200] The pharmaceutical compositions disclosed herein may optionally contain additional ingredients and / or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1) fillers or swelling agents, such as starch, lactose, sucrose, glucose, mannitol, and silica; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, and gum arabic; (3) humectants, such as glycerin; and (4) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain silicates, sodium glycolate starch, and croscarmellose sodium. (5) Solubility inhibitors, such as paraffin; (6) Absorption accelerators, such as quaternary ammonium compounds; (7) Wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) Absorbents, such as kaolin and bentonite; (9) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol and sodium dodecyl sulfate; (10) Suspension agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and sorbitan anhydride, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum; (11) Buffers; (12) Excipients, such as lactose, lactose (milk (13) Sugar), polyethylene glycol, animal and vegetable fats, oils, waxes, paraffin, cocoa butter, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, salicylates, zinc oxide, aluminum hydroxide, calcium silicate, and polyamide powders; (14) Inert diluents, such as water or other solvents; (15) Preservatives; (16) Surfactants; (17) Dispersants; (18) Controlled-release or absorption-delaying agents, such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (19) Opacifiers; (20) Excipients; (21) Wetting agents; (22) Emulsifiers and suspending agents; (23) Solvents and emulsifiers, such as ethyl acetate. Alcohols, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, wheat germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents that make the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickeners; (27) coating materials, such as lecithin; and (28) sweeteners, flavoring agents, coloring agents, fragrances, and preservatives. Each such ingredient or material must be “acceptable” in the sense that it is compatible with the other ingredients of the formulation and harmless to the host.The ingredients and materials suitable for the selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for the selected dosage form and route of administration can be determined using common techniques in the art.

[0201] Dosage forms for topical or transdermal application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, and inhalers. One or more active agents / compounds may be mixed under aseptic conditions with a suitable pharmaceutically acceptable diluent or carrier. Ointments, pastes, creams, and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0202] Pharmaceutical compositions of this disclosure suitable for parenteral administration may comprise one or more reagents / compounds in combination with one or more pharmaceutically acceptable sterile isotonic or non-aqueous aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions just before use, which may contain suitable antioxidants, buffers, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners. For example, proper flowability can be maintained by using coating materials, maintaining the desired particle size in the case of dispersions, and by using surfactants. These pharmaceutical compositions may also contain suitable excipients, such as wetting agents, emulsifiers, and dispersants. It may also be desirable to include isotonic agents. Furthermore, prolonged absorption of the injectable drug form can be achieved by including agents with delayed absorption.

[0203] The following examples are provided to further illustrate the methods of this disclosure. These examples are merely illustrative and are not intended to limit the scope of this disclosure in any way. Example The following examples are provided to further illustrate the methods of this disclosure. These examples are merely illustrative and are not intended to limit the scope of this disclosure in any way.

[0204] Example 1 Phenotypic characterization of the antimicrobial activity of SE25 secretion Analysis of the antimicrobial activity of SE25, a positive-selection Staphylococcus epidermidis strain from the Micromyx-50 library, particularly against Staphylococcus aureus ATCC 29213, revealed that the activity of SE25 (a positive-selection Staphylococcus epidermidis strain) was significant. Figure 1A and 1BA Micromyx library was obtained from samples taken from the forearm of a human subject. The samples were cultured on selective Staphylococcus epidermidis plates and grown in TSB for later studies. Inhibition bands indicated that SE25 inhibited the growth of Staphylococcus aureus when it was covered by SE25 strain streaked on TSA plates and grown for 48 hours. The general experimental procedure involved inoculating the Staphylococcus epidermidis library into tryptone broth (TSB) and growing it at 37°C for one day. On day 2, the strain was replicated onto TSA plates and grown overnight at 37°C, followed by overnight growth at room temperature. The Staphylococcus epidermidis library was covered with the indicator strain Staphylococcus aureus and grown at room temperature from day 4 to day 5.

[0205] Further analysis revealed that SE25 possesses antimicrobial activity against other Gram-positive but not Gram-negative bacteria. Figure 2 SE25 was covered by Gram-positive bacteria Staphylococcus aureus SA25923, Staphylococcus aureus SA29213, MRSA USA300, Staphylococcus epidermidis NRRL B-4268, Bacillus subtilis, and Gram-negative strains Pseudomonas aeruginosa and Escherichia coli. Figure 2 Table 6 below summarizes the antimicrobial activity of SE25 against various pathogenic bacteria.

[0206] Table 6: Antimicrobial activity of SE25 strain against bacteria, as shown. SE25 cell-free supernatant has bactericidal activity. It secreted SE25, exhibiting anti-Staphylococcal activity. Staphylococcus aureus ATCC 29213 was grown in conditioned growth medium derived from SE25 culture, conditioned growth medium derived from SE3 culture (negative control), and unconditioned medium. Figure 3 Conditioned medium from SE25 cultures inhibited the growth of *Staphylococcus aureus* ATCC 29213. Conversely, neither conditioned nor unconditioned medium from SE3 cultures inhibited the growth of *Staphylococcus aureus* ATCC 29213. Cell-free conditioned medium was prepared by growing *Staphylococcus epidermidis* cultures in tryptone broth (TSB) at 30°C with shaking at 250 RPM for 24 hours. The conditioned medium, also known as cell-free supernatant (CFS), was sterilized by filtration (PES, 0.22 μm membrane) and the pH was adjusted to 6.5–7.5. Anti-*Staphylococcus aureus* activity was determined by mixing 90 μL of CFS with approximately 10 μL of 5 x 10⁻⁶ microtiter in a 96-well microtiter plate. 5The indicator strains for CFU, such as Staphylococcus aureus, are used in a mixture. The indicator strains are prepared in fresh TSB. The negative control assay includes fresh TSB medium and CFS without added bacteria. The cultures are grown overnight at 37°C and the Staphylococcus aureus bacterial growth is counted by subsequent plate inoculation for CFU counting.

[0207] The activity spectrum of SE25 CFS was determined against Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis NRRL B-4268, Staphylococcus epidermidis 1457, Staphylococcus epidermidis SE25, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and CoNS isolates of ichthyosis, Staphylococcus ludens, Staphylococcus aureus, Staphylococcus capitulata, Staphylococcus hemolyticus, Staphylococcus hominis, S. pettenkoferi, Staphylococcus saprophyticus, and Staphylococcus warwickii. SE25 CFS exhibited inhibitory activity against all tested strains except Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa.

[0208] Bactericidal activity is defined as the 3-log of the CFU count of the indicator strain within 24 hours of growth. 10 The reduction in activity. The antimicrobial activity inhibited growth to + / - 1 log [value] of the initial CFUs within 24 hours. 10 Inside the CFU. Figure 5A SE25 was shown to have bactericidal activity against Staphylococcus aureus ATCC 29213. SE25 cell-free supernatant reduced the bacterial load of Staphylococcus aureus from the initial inoculation by approximately 7-log. 10 ( Figure 5A Similarly, known antimicrobial agents levofloxacin and vancomycin reduce the CFU of Staphylococcus aureus by approximately 6.75-log. 10 .

[0209] SE25 does not generate biofilm on plastics and disperses Staphylococcus aureus biofilm. Biofilm formation activity and Staphylococcus aureus biofilm dispersibility of the strains were tested. Strains SE25, Staphylococcus epidermidis NRRL B-4268, and Staphylococcus epidermidis 1457 were incubated in polystyrene plastic wells to allow adhesion (…). Figure 6A Unbound and loosely bound bacterial cells were removed and the wells were washed. Crystal violet solution was used to stain attached cells that had adhered to the polystyrene pores and formed biofilms. The positive control, *Staphylococcus epidermidis* 1457, showed high biofilm formation upon crystal violet staining. SE25 strain cells did not form biofilms, as evidenced by the lack of crystal violet staining. *Staphylococcus epidermidis* NRRL B-4268 strain was also negative for biofilm formation. Figure 6AThe results were confirmed using Congo Red assays, with strain SE25 showing growth as red colonies (negative for biofilm formation). Figure 6B ).

[0210] Strain SE25 showed activity in dispersing Staphylococcus aureus biofilms. Figure 6C First, Staphylococcus aureus cultures were allowed to adhere and form biofilms in the plastic wells. Then, SE25 strain cultures were added in serial dilutions. Untreated Staphylococcus aureus cells remained in the biofilm, while after treatment with the SE25 dilutions, the Staphylococcus aureus biofilm dispersed. Figure 6C ).

[0211] SE25 is sensitive to antibiotics against Staphylococcus aureus. The susceptibility of the SE25 and SE-27a strains was tested using different antibiotics to determine the MIC, as shown in Table 7 below.

[0212] Table 7: MICs for antibiotic resistance assays on SE25 and NRRL B-4268 derived strains *There is no clinical resistance inflection point for mupirocin, but it has been confirmed that the SE25 strain carries the resistance gene mupA on its plasmid, which can resist more than 500 μg / ml.

[0213] Reconstructed human skin The experimental procedure for reconstructed human epidermis (RHE) involves seeding SE onto reconstructed human epidermal tissue prepared from keratinocytes cultured on inert polycarbonate medium. Following seeding, the SE is incubated for 24 or 48 hours. Figure 7 ). SE with 10 3 10 5 and 10 7 The inoculation concentration settled at RHE.

[0214] Using in adding 10 3 Staphylococcus aureus cells before 10 5 SE cells were seeded onto RHE and incubated for 48 hours for RHE competition assay. Figure 8 The settlement of SE25 reduced SA29213 by 4 log units. Figure 8 After an additional 24 hours, the RHEs were rinsed to remove unattached bacteria, and Staphylococcus aureus was counted by inoculation with CFU plates.

[0215] SE25 induces the production of antimicrobial peptides (AMP) in RHE 72 hours after inoculation. Figure 9AMP is known to have Staphylococcus aureus activity. SE25 and NRRL induce similar host responses.

[0216] Example 2 Whole genome sequence (WGS) analysis of strain SE25 and annotation of naturally occurring plasmids in the strain. Genome sequence analysis of strain SE25 revealed the presence of four plasmids: p36.326 kb (“p36.3”, possibly with multiple variants), p43.056 kb (“p43.1”), p24.273 kb (“p24.3”), and p9.793 kb (“p9.8”), as well as a chromosome of 2.402255 Mb size. A determinant for high-level mupirocin resistance was found on plasmid p24.3, which contains the mupA gene encoding the MupA protein (ileS2, isoleucyl-tRNA synthetase, EC 6.1.1.5). The gene encoded by this plasmid can determine resistance up to more than 64 μg / ml.

[0217] The subsystem characteristics identified in the plasmids are as follows: cofactors, vitamins, prosthetic groups, pigments (97); cell wall and capsule (36); virulence, disease and defense (43); potassium metabolism (3); photosynthesis (0); miscellaneous (10); phages, prophages, transposable factors, plasmids (13); membrane transport (26); iron acquisition and metabolism (24); RNA metabolism (36); nucleosides and nucleotides (78); protein metabolism (157); cell division and cell... Cycle (5); Mobility and chemotaxis (0); Regulation and cell signaling (28); Secondary metabolism (5); DNA metabolism (54); Fat, lipids and isoprene (45); Nitrogen metabolism (20); Dormancy and spore formation (9); Respiration (19); Stress response (36); Aromatic compound metabolism (3); Amino acids and derivatives (224); Sulfur metabolism (5); Phosphorus metabolism (13); and Carbohydrates (189).

[0218] For a summary of ORF annotations in the SE25 plasmid, see Table 8-11.

[0219] Table 8. Characteristics of the SE25-p36.326kb plasmid: The epidermin BGC gene is shown in bold.

[0220] Table 9. Characteristics of the SE25-p43.056kb plasmid “p43.1” Table 10. Characteristics of the SE25-p24.273kb plasmid “p24.3”. The MupA gene is shown in bold.

[0221] Table 11. Characteristics of the SE25-p9.793kb plasmid “p19.4” name Location size direction type Rep replication protein 91..957 867 ＝> CDS Hypothetical protein 1063..1194 132 ＝> CDS Hypothetical protein 1571..2500 930 ＝> CDS Dissociation enzymes / Integrases 3529..3840 312 ＝> CDS Hypothetical protein 4246..4488 243 ＝> CDS Hypothetical protein 4862..5227 366 <＝ CDS Hypothetical protein 5357..5725 369 <＝ CDS Hypothetical protein 5729..6433 705 <＝ CDS Lipoproteins, presumed 6538..6897 360 ＝> CDS Hypothetical protein 6959..7366 408 <＝ CDS Hypothetical protein 7821..9029 1209 ＝> CDS Genome analysis of strain SE25 identified the mupirocin resistance gene and the biosynthetic gene cluster of lanthipeptide. Figure 10 ), siderophores, prophages ( Figure 11 And the ica operator does not exist.

[0222] Example 3 Artificial modification of Staphylococcus epidermidis SE25 strain Based on the overall positive results of the phenotypic test, and particularly due to its bactericidal activity against Staphylococcus aureus on agar, reconstructed human epidermis (RHE) and liquid culture medium, as well as its adaptability to genetic manipulation, strain SE25 was selected as the expected strain for one or more further modifications.

[0223] The SE25 strain was artificially modified to have two (Δalr1Δdat) or three gene deletions (Δalr1Δalr2Δdat) plus additional deletions of the mupirocin resistance gene (ΔmupA) and the prophage gene (Δcapsid) for D-alanine auxotrophy.

[0224] Artificial modification of D-alanine auxotrophic type.

[0225] To artificially engineer the D-alanine auxotrophic strain SE25, the chromosomal regions laterally attached to the alar1, alar2, and dat genes were analyzed for homology with those of *Staphylococcus epidermidis* NRRL B-4268. In the presence of a few single nucleotide polymorphisms, homology was found in *Staphylococcus epidermidis* SE25 strain used for artificially engineering the D-alanine auxotrophic strain. ΔΔΔ The same knockout plasmid construct is suitable for the artificial modification of SE25 auxotrophic cells. Primer sequences, their specific uses, and PCR product sizes are listed in Table 12 below.

[0226] Table 12. Primers (SE1423) used for knockout of D-alanine aminotransferase • Overlap PCR using primers 1423-5F / 1423-3R: 1.5Kb • PCR product from wild type was obtained using primers 1423-5F / 1423-3R: 2.3 kb • F: Forward primer • R: Reverse primer • Added restriction sites for cloning are shown in bold underlined letters. In a 2018 study by Moscoso et al. (Moscoso, García et al. 2018), which described the construction of D-alanine auxotrophic strains of Staphylococcus aureus, it was reported that "...double ΔdatΔalr1 and triple ΔdatΔalr1Δalr2 mutants require exogenous D-alanine for growth." Similar to the work done for Staphylococcus aureus, two distinct SE25 auxotrophic strains were constructed in parallel by sequentially deleting the alar1, alar2, and dat genes ("triple mutant") or the alar1 and dat genes ("double mutant"). The two knockout variants were artificially engineered and screened for their ability to grow on agar plates with and without D-alanine, and the tests were repeated in liquid media with and without D-alanine supplementation. Figure 12 Both variants require the same concentration (100 μg / mL) of D-alanine, as in Staphylococcus epidermidis SE. ΔΔΔ As required.

[0227] The pJB-1423KO plasmid isolated from the dam- / dcm-Escherichia coli (NEB) strain was transformed into competent SE25Δalr1Δalr2 cells using TSA+chloramphenicol (10 μg / mL) plates. The presence of the pJBv1423KO plasmid in the transformants was confirmed by detecting a 1.5 kb PCR product using primers 1423-5F (EcoRI) and 1423-3R (SalGI). A 1.5 kb PCR product was observed, while a 2.3 kb PCR product was observed in reactions containing cell lysates from wild-type SE host cells. Cells from both confirmed clones were streaked onto fresh plates containing TSA+Cm (10 μg / mL)+D-alanine (100 μg / mL). The plates were incubated at 43°C for 48 hours for plasmid integration via homologous recombination. The isolated colonies were streaked again for purification at 43°C and growth for 48 hours. Four isolated colonies were inoculated into 3 mL of TSB + D-alanine (100 μg / mL) in 15 mL tubes to loop the plasmid backbone via a second round of homologous recombination. The cultures were incubated overnight at 30°C with shaking. A 50 μL aliquot of the culture was transferred to a 15 mL tube containing 3 mL of fresh TSB and DA100 medium. Cell plates from the first and second 30°C cultures were inoculated onto TSA + anhydrous tetracycline (ATC 2 μg / mL) + D-alanine (DA, 100 μg / mL). After incubation at 30°C for 2 days, approximately 100-200 colonies formed on the inoculated plates.

[0228] SE25Δalr1Δalr2Δdat (6 colonies) and SE25Δalr1Δdat (14 colonies) were screened for Cm-sensitivity on Cm10 TSA plates and validated. Figure 13A and 13B SE25Δalr1Δdat( Figure 14A SE25Δalr1Δalr2Δdat( Figure 14B It retains its anti-Staphylococcus aureus activity.

[0229] Artificial modification of Mupirocin's sensitivity.

[0230] In three strain variants (wild-type strain and two auxotrophs), 3063 bp of the 3072-bp ORF of the mupA gene on plasmid p24.3, which conferred resistance to the topical dermal antibiotic mupirocin, was deleted to artificially modify the sensitivity to mupirocin in the following three strains: SE25ΔmupA, SE25Δalr1ΔdatΔmupA, and SE25Δalr1Δalr2ΔdatΔmupA.

[0231] To eliminate a portion of the mupA ORF, flanking regions of 968 bp and 966 bp were amplified by PCR and assembled into a shuttle pJB38 plasmid with a temperature-sensitive Gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5α maintainer strain. The PCR-confirmed plasmid DNA was purified and sequenced to verify correct assembly and the absence of mutations. The plasmid with the confirmed expected sequence was transformed into the methylation-deficient E. coli strain NEB(dam- / dcm-). The plasmid DNA was purified and re-transformed into the SE25 strain via electroporation. A standard allele exchange procedure on chloramphenicol-resistant colonies was followed by screening for positive ΔmupA mutants on TSA plates with or without mupirocin. Figure 15A and 15B ).

[0232] Deletion of prophage capsid gene A putative prophage of 41,881 bp in size was identified on the chromosome, encoding more than 50 structural, replication, and lysis genes, as well as a repetitive integration site at the 3′ end of the ORF for the iron-sulfur cluster assembly protein SufB. The putative prophage gene encoding the structural capsid protein was deleted in all three ΔmupA-derived strains as a precaution to disable the potential release of mature lysis phage particles. The putative prophage is structurally similar to Staphylococcus aureus phage phi11, Siphoviridae staphylococcus phage phiMR25, Siphoviridae staphylococcus phage phiMR11, Siphoviridae staphylococcus phage phiSauS-IPLA88, phage 92, phage 88, 29, phage 55, and Siphoviridae Staphylococcus aureus phage phiNM2.

[0233] To delete 1338 bp from the 1353-bp capsid ORF, flanking regions of 1004 bp and 958 bp were amplified by PCR and assembled into a shuttle pJB38 plasmid containing a temperature-sensitive Gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5α maintenance strain. PCR-confirmed plasmid DNAs were purified and sequenced to verify correct assembly and the absence of mutations. Plasmids with confirmed expected sequences were transformed into the methylation-deficient E. coli strain NEBα(dam- / dcm-), purified, and re-transformed into the SE25 strain via electroporation. A standard allele exchange procedure on chloramphenicol-resistant colonies was followed by PCR screening for positive Δ capsid mutants.

[0234] Confirmation of the anti-staphylococcal activity of artificially modified strains The retention of antimicrobial activity against Staphylococcus aureus by agar coating assays of strains SE25ΔmupAΔcapsid, SE25ΔmupAΔcapsidΔalr1Δdat and SE25ΔmupAΔcapsidΔalr1Δalr2Δdat was determined (Figure 15).

[0235] Identification of the biosynthetic gene cluster encoding the epidermin-lanthion antibiotic biosynthesis gene carried by the plasmid p36.3. Gene annotation of the plasmid revealed the existence of a biosynthetic gene cluster (BGC) related to the synthesis of the epidermin-lanthion antibiotic peptide. The gene cluster has 100% identity with the previously reported epidermin biosynthetic gene cluster. The approximately 15kb BGC is annotated with the following genes: epiA, B, C, D, E, F, G, H, P, Q, T, signal peptidase I (EC 3.4.21.89), and several hypothetical proteins ( Figure 10To disrupt lanolin production in SE25, the ORF of the epiA gene encoding the 52-amino acid precursor (MEAVKEKNDL FNLDVKVNAK ESNDSGAEPR IASKFICTPG CAKTGSFNS YCC*) was truncated to 6 amino acids (including MEA-YCC*) in wild-type SE25 strains to confirm gene function through loss of function deletion.

[0236] To eliminate a portion of the epiA ORF, flanking regions of 994 bp and 974 bp were amplified by PCR and assembled into a shuttle pJB38 plasmid containing a temperature-sensitive Gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5α maintainer strain. Transformations were analyzed by PCR, and confirmed plasmid DNAs purified from positive clones were sequenced to verify correct assembly and the absence of mutations. Plasmids with confirmed expected sequences were transformed into the methylation-deficient E. coli strain NEB(dam- / dcm-), purified, and re-transformed into the SE25 strain via electroporation. Standard allelic exchange procedures on chloramphenicol-resistant colonies were followed by PCR screening for ΔepiA mutant colonies and finally Staphylococcus aureus cap assay for positive ΔepiA mutants and wild-type strains. Figure 16A and 16B ).

[0237] The results showed that in the Staphylococcus aureus agar coating assay, the SE25 ΔepiA mutant strain exhibited a loss / significant reduction in the growth inhibition zone, thus confirming the role of epiA in conferring anti-Staphylococcus aureus activity. Figure 17 ).

[0238] The reduced anti-SA activity in the ΔepiA mutant strain was also confirmed by liquid culture assays (not shown).

[0239] Example 4 Table 13. Exemplary Sequences Example 5 Production of Staphylococcus epidermidis strain SE484 To construct the *Staphylococcus epidermidis* strain SE484, three D-alanine biosynthesis genes were first deleted: alanine racemase 1 (alr1), alanine racemase 2 (alr2), and D-alanine aminotransferase (dat). This resulted in strain SE464, a D-alanine auxotroph that does not grow in media without D-alanine supplementation. Subsequently, the muprocin resistance gene (mupA, also known as iles2) and the prophage capsid gene were derived from the deletion of SE464, leading to the SE484 strain.

[0240] Artificial modification of D-alanine auxotrophic type As described in Example 3, three D-alanine biosynthesis genes and their flanking DNA sequences were each cloned into temperature-sensitive knockout plasmids. These plasmids were then transformed into parental *Staphylococcus epidermidis* strain SE25. Colonies were analyzed by PCR to verify plasmid integration at the target loci. A second recombination was then performed to remove the plasmids from the genome, followed by screening for plasmid excision from the genome and subsequent loss during growth in the absence of chloramphenicol selection. Phenotypic screening of potential candidates was conducted by growth on tryptone agar (TSA) containing D-alanine and / or chloramphenicol. Figures 19A-19C First, streak candidate colonies onto TSA containing 100 μg / ml D-alanine in the absence of chloramphenicol. Figure 19A Then, the same colonies were streaked onto TSA supplemented with 10 μg / ml chloramphenicol in the absence of D-alanine. Figure 19B Select candidate colonies that require D-alanine supplementation for growth. Figure 19C To verify successful deletions, flanking regions of approximately two thousand bases surrounding the deletions of alar1, alar2, and dat were amplified by PCR and sequenced. All deletions were confirmed by sequencing, and the deletion of the three D-alanine biosynthesis genes resulted in strain SE464, a D-alanine auxotroph that does not grow in media without D-alanine supplementation.

[0241] Artificial modification to increase mupirocin sensitivity and deletion of prophage capsid genes To generate SE484 from SE464, two subsequent genes were deleted: mupA (also known as ilse2) and the gene encoding the major capsid protein of the putative prophage, identified by the PhiSPY algorithm during whole-genome sequencing (WGS). Deletion was performed using the same allelic exchange strategy used for deleting the alanine racemase gene. Transformants were screened for mupirocin-sensitive and D-alanine auxotrophic colonies.

[0242] Example 6 Characterization of Staphylococcus epidermidis strain SE484 Whole-genome sequencing of strain SE484 confirmed that, apart from the expected deletion, it was identical to its parent strain SE25.

[0243] Growth profile of strain SE484 The growth profile of strain SE484 in TSB at 30°C was observed to be similar to that of its parent strain SE25, thus confirming that the modifications compared to the parent strain did not affect the physiology of SE484. Figure 20A The growth kinetics of the two strains were compared in the figure shown. Then, inhibition zone plate assays were performed to compare the activity of the two strains against Staphylococcus aureus. Here, SE25 and SE484 were each streaked onto TSA supplemented with 100 μg / ml D-alanine and incubated at 37°C. After 48 hours of incubation, soft agar containing Staphylococcus aureus was poured onto both plates and then incubated at 37°C for an additional 24 hours. Both Staphylococcus epidermidis strains showed similar inhibition of Staphylococcus aureus growth. Figure 20B-20C These results indicate that modifications to the five target genes resulting in strain SE484 do not affect its anti-Staphylococcus aureus properties.

[0244] Phenotypic and genotypic stability of SE484 Phenotypic and genotypic stability of SE484 was evaluated over 5 days under daily subculturing. To validate phenotypic stability, daily subculturing of SE484 was assessed for D-alanine auxotrophicity, mupirocin sensitivity, growth rate, CFU count, and antimicrobial activity against Staphylococcus aureus. To validate genotypic stability, the alar1, alar2, dat, and epiA genes were amplified by PCR in samples obtained from the first and fifth subculturings of SE484 culture.

[0245] The phenotypic and genotypic characteristics of SE484 were evaluated over 5 passages and 179 generations. SE484 was streaked onto TSA, TSA containing 100 μg / ml D-alanine, or TSA containing D-alanine and 20 μg / ml mupirocin, and the plates were incubated at 37°C for 48 hours. Figure 20D As shown, from the first to the fifth passage, the growth profile and overall colony morphology remained unchanged, while the SE484 culture only grew when D-alanine was supplemented at both time points, thus verifying the maintenance of the D-alanine auxotrophic phenotype. Furthermore, mupirocin sensitivity was verified by the fifth passage, and it was found to be present in all passages.

[0246] To assess the stability of the anti-Staphylococcal properties of SE484 after successive passages, plates containing glycerol stock solution prepared and collected at each passage were inoculated and covered with Staphylococcus aureus. Results showed no loss of anti-Staphylococcal activity, as clearance of Staphylococcus aureus was observed near the restripe lines of SE484 in all five passages. Furthermore, the observed clearance banding pattern was consistent from the first to the fifth passage, indicating that the stated antimicrobial activity of SE484 is stable for at least five passages.

[0247] Example 7 The inhibitory activity of Staphylococcus epidermidis strain SE484 against Staphylococcus aureus was quantified and compared with that of strains SE123 (SEΔΔΔ). For the measurements, a prophylactic method was used, in which reconstructed human epidermis (RHE) was inoculated with either SE123 or SE484 and incubated at 37°C, 5% CO2 in a tissue culture incubator for 4 hours. Then, ~10 4 CFU of *Staphylococcus aureus* MRSA strain USA300 was challenged with RHEs. After an additional 24 hours of incubation, USA300 cell counts were determined by biopsy and dilution plate inoculation. Results revealed that SE123 did not show a significant ability to inhibit USA300 growth. Figure 21A SE484 inhibited the growth of USA300 by approximately 3 log units after 24 hours of incubation. Figure 21B ).

[0248] These results indicate that Staphylococcus epidermidis SE484 possesses anti-Staphylococcus aureus activity, while SE123 (SEΔΔΔ) does not.

[0249] Introduced as a reference The entire disclosure of every patent document, including patent applications, scientific papers, government reports, websites, and other references mentioned herein, is incorporated herein by reference in its entirety for all purposes. In the event of a conflict of terminology, this specification shall prevail. All lists of sequences or Seq.ID. numbers disclosed herein are incorporated herein in their entirety.

[0250] The following references are specifically incorporated herein by reference for providing exemplary procedures or other details that supplement those described herein.

[0251] Although illustrative embodiments of this disclosure have been described herein, it should be understood that this disclosure is not limited to those described, and various other changes or modifications may be made by those skilled in the art without departing from the scope or spirit of this disclosure.

Claims

1. Recombinant microorganisms, including: The deletion or substitution of one or more genes encoding the D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive. The deletion or substitution of one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and The deletion or substitution of one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive.

2. The recombinant microorganism according to claim 1, wherein one or more naturally occurring antibiotic resistance genes are selected from mupirocin resistance genes, ampicillin resistance genes, cefotaxime resistance genes, chloramphenicol resistance genes, ciprofloxacin resistance genes, trimethoprim-sulfamethoxazole resistance genes, nalidixic acid resistance genes, oxytetracycline resistance genes, streptomycin resistance genes, tetracycline resistance genes, and trimethoprim resistance genes.

3. The recombinant microorganism according to claim 1 or claim 2, wherein one or more naturally occurring antibiotic resistance genes are muprocin resistance genes (mupA).

4. The recombinant microorganism according to any one of claims 1-3, wherein one or more naturally occurring lysogenic phage genes encode phage capsid proteins.

5. The recombinant microorganism according to any one of claims 1-4, wherein one or more D-alanine biosynthesis genes include the D-alanine aminotransferase (dat) gene, the alanine racemic enzyme gene alrl or alr2.

6. The recombinant microorganism according to any one of claims 1-5, wherein the deletion or substitution in one or more genes encoding the D-alanine biosynthesis gene includes deletion in dat, arl1, and arl2.

7. The recombinant microorganism according to any one of claims 1-5, wherein the recombinant microorganism further comprises one or more genes encoding a heterologous gene.

8. The recombinant microorganism according to claim 7, wherein the heterologous gene is selected from genes encoding antimicrobial peptides or variants thereof, genes encoding antimicrobial biosynthetic enzymes or variants thereof, genes encoding enzymes or variants thereof, genes encoding enzyme inhibitors or variants thereof, genes encoding antigens or variants thereof, and genes encoding immunomodulatory peptides or variants thereof, or combinations thereof.

9. The recombinant microorganism according to any one of the preceding claims, wherein the one or more antimicrobial peptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

10. The recombinant microorganism according to claim 9, wherein the one or more microbial pathogens are selected from bacterial pathogens, fungal pathogens or viral pathogens.

11. The recombinant microorganism according to claim 10, wherein the one or more bacterial pathogens are selected from Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Cutibacterium spp., and Mycoplasma, or combinations thereof.

12. The recombinant microorganism according to claim 10, wherein the fungal infection is caused by fungi selected from the genera *Malassezia* spp., *Candida* spp., *Aspergillus*, *Cryptococcus*, and *Pneumocystis*.

13. The recombinant microorganism of claim 10, wherein the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

14. The recombinant microorganism according to any one of claims 8-13, wherein the antimicrobial polypeptide is selected from epidermoid lanthanide, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, chondroglycoprotein 1, chitosan trisaccharidase, mucin 9, chondroglycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosaccharidase, acetylesterase, acetylxylan esterase, α-amylase, β-amylase, glucosylamylase, amylopectin, β-glucanase, hemicellulase, arabinosaccharidase, mannanase, pectin hydrolase, pectic acid lysin, lysostaphin, zoocin A, millericin B, and muramidase. Cpl-1, lysozyme, intracytolysin PlyC, intracytolysin, PlyVl2, enterolysin A, autolysin (Acd) of Clostridium difficile, autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilisin, epidermin, Gallidermin, varisin Ba Ny266, varisin 1140, Pep5, Epicidin 280, Epilancin K7, nisin 481, cytolysin, nisin 3147, staphylococcal C55, Salvaricin A, lactobacillus S, streptococcal A-FF2, Sublancin 168, Carnocin U149, Variacin 8, spiculin, cinnamomumin, nisin, angiotensin-converting enzyme inhibitory peptide, Mersacidin, and Actardine.

15. The recombinant microorganism according to any one of claims 1-6, wherein the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains or variants thereof, and wherein the LEKTI protein, one or more LEKTI protein domains or variants thereof are secreted.

16. The recombinant microorganism of claim 7, wherein the heterologous gene is a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and wherein 6-HAP is secreted.

17. The recombinant microorganism of claim 8, wherein the gene encoding the immunomodulatory polypeptide or a variant thereof is a gene encoding lipoteichoic acid (LTA) biosynthetic enzyme, and wherein the recombinant microorganism is capable of producing and secreting LTA.

18. The recombinant microorganism according to any one of claims 1-17, wherein the recombinant microorganism is a bacterium, or a combination of bacteria.

19. The recombinant microorganism according to any one of the preceding claims, wherein the recombinant microorganism is selected from Bifidobacterium, Brevibacterium, Corynebacterium, Dermatobacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof.

20. The recombinant microorganism according to claim 19, wherein the recombinant microorganism is Staphylococcus epidermidis.

21. The recombinant microorganism according to any one of the preceding claims, wherein the recombinant microorganism secretes one or more therapeutic polypeptides or variants thereof.

22. A pharmaceutical composition comprising a cell culture composition and a pharmaceutically acceptable carrier, said cell culture composition comprising one or more recombinant microorganisms according to any one of the preceding claims.

23. The pharmaceutical composition of claim 22, wherein the cell culture composition is a live cell culture composition.

24. The pharmaceutical composition according to claim 22 or 23, wherein the cell culture composition comprises 0% to no more than 90% water.

25. The pharmaceutical composition according to any one of claims 22-24, wherein the pharmaceutically acceptable carrier is selected from aqueous solutions, emulsions, creams, lotions, gels, or ointments.

26. A method for treating a subject's disease, symptom, or condition, said method comprising: The recombinant microorganism is applied to the subject, the recombinant microorganism comprising: deletion or substitution of one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; deletion or substitution of one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and deletion or substitution of one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive.

27. The method of claim 26, wherein the recombinant microorganism is in a cell culture composition.

28. The method of claim 26 or claim 27, wherein the cell culture composition is a live cell culture composition.

29. The method according to any one of claims 26-28, wherein the one or more naturally occurring antibiotic resistance genes are selected from mupirocin resistance genes, ampicillin resistance genes, cefotaxime resistance genes, chloramphenicol resistance genes, ciprofloxacin resistance genes, trimethoprim-sulfamethoxazole resistance genes, nalidixic acid resistance genes, oxytetracycline resistance genes, streptomycin resistance genes, tetracycline resistance genes, and trimethoprim resistance genes.

30. The method according to any one of claims 26-29, wherein one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

31. The method according to any one of claims 26-30, wherein one or more naturally occurring lysogenic phage genes encode phage capsid proteins.

32. The method according to any one of claims 26-31, wherein the deletion or substitution in one or more genes encoding the D-alanine biosynthesis gene includes deletion in dat, alar1, and alar2.

33. The method according to any one of claims 26-32, wherein the recombinant microorganism further comprises one or more genes encoding a heterologous gene.

34. The method of claim 33, wherein the heterologous gene is selected from genes encoding antimicrobial peptides or variants thereof, genes encoding antimicrobial biosynthetic enzymes or variants thereof, genes encoding enzymes or variants thereof, genes encoding enzyme inhibitors or variants thereof, genes encoding antigens or variants thereof, and genes encoding immunomodulatory peptides or variants thereof, or combinations thereof.

35. The method according to any one of claims 26-34, wherein the one or more antimicrobial peptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

36. The method of claim 35, wherein the one or more microbial pathogens are selected from bacterial pathogens, fungal pathogens or viral pathogens.

37. The method of claim 36, wherein the one or more bacterial pathogens are selected from Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacterium, and Mycoplasma, or combinations thereof.

38. The method of claim 36, wherein the fungal infection is caused by fungi selected from species of the genera *Malassezia*, *Candida*, *Aspergillus*, *Cryptococcus*, and *Pneumocystis*.

39. The method of claim 36, wherein the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

40. The method according to any one of claims 34-39, wherein the antimicrobial polypeptide is selected from epidermoid lanthanide, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, chondroglycoprotein 1, chitosan trisaccharidase, mucin 9, chondroglycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosaccharidase, acetylesterase, acetylxylan esterase, α-amylase, β-amylase, glucosylamylase, amylopectin, β-glucanase, hemicellulase, arabinosaccharidase, mannanase, pectin hydrolase, pectic acid lysin, lysostaphin, zoocin A, millericin B, and muramidase. Cpl-1, lysozyme, intracytolysin PlyC, intracytolysin, PlyV12, enterolysin A, Clostridium difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilisin, epidermin, Gallidermin, variadin B-aNy266, variadin 1140, Pep5, Epicidin 280, Epilancin K7, nisin 481, cytolysin, nisin 3147, staphylococcal C55, Salvaricin A, lactobacillus S, streptococcal A-FF2, Sublancin 168, Carnocin U149, Variacin 8, spiculin, cinnamomumin, nisin, angiotensin-converting enzyme inhibitory peptide, Mersacidin, and Actardine.

41. The method according to any one of claims 26-33, wherein the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains or variants thereof, and wherein the LEKTI protein, one or more LEKTI protein domains or variants thereof are secreted.

42. The method of claim 33, wherein the heterologous gene is a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and wherein 6-HAP is secreted.

43. The method of claim 34, wherein the gene encoding the immunomodulatory polypeptide or a variant thereof is a gene encoding lipoteichoic acid (LTA) biosynthetic enzyme, and wherein the recombinant microorganism is capable of producing and secreting LTA.

44. The method according to any one of claims 26-43, wherein the recombinant microorganism is a bacterium, or a combination of bacteria.

45. The method according to any one of claims 26-44, wherein the recombinant microorganism is selected from Bifidobacterium, Brevibacterium, Corynebacterium, Dermatobacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Sterculia, or a combination thereof.

46. ​​The method of claim 45, wherein the recombinant microorganism is Staphylococcus epidermidis.

47. The method according to any one of claims 26-46, wherein the recombinant microorganism secretes one or more therapeutic polypeptides or variants thereof.

48. The method according to any one of claims 26-47, wherein the cell culture composition is a live cell culture composition.

49. The method according to claims 26-48, wherein the cell culture composition comprises 0% to no more than 90% water.

50. The method according to any one of claims 26-49, wherein the subject is a mammal.

51. The method of claim 50, wherein the mammal is a human.

52. The method according to any one of claims 26-51, wherein the disease, condition or condition is selected from microbial infection, skin disease or condition, inflammatory disease or condition and metabolic disease or condition.

53. The method of claim 51, wherein the microbial infection comprises one or more microbial pathogens selected from Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacterium, Mycoplasma, Malassezia, Candida, Aspergillus, Cryptococcus, Pneumocystis, Respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus, or combinations thereof.

54. The method according to any one of claims 25-51, wherein the skin disease or condition is an inflammatory skin disease or condition.

55. The method according to any one of claims 25-51, wherein the skin disease or condition is a skin ecological disorder.

56. The method according to any one of claims 25-51, wherein the skin disease or condition is skin toxicity.

57. The method according to any one of claims 25-51, wherein the skin disease or condition is ichthyosis.

58. The method according to any one of claims 25-51, wherein the skin disease or condition is a psoriatic disease or condition.

59. The method according to any one of claims 25-51, wherein the skin disease or condition is dermatitis.

60. The method according to any one of claims 25-51, wherein the skin disease or condition is an autoimmune vesicular disease.

61. The method according to any one of claims 25-51, wherein the skin disease or condition is selected from: Natherton syndrome, hidradenitis suppurativa, psoriasis, pustular psoriasis, plaque psoriasis and palmoplantar psoriasis, atopic dermatitis, acne, acneiform eruption, impetigo, folliculitis, acute suppurative paronychia, lymphangitis, necrotizing fasciitis and cellulitis.

62. A recombinant D-alanine auxotrophic bacterial strain, prepared by a method comprising the following steps: Select bacterial strains; The following steps are performed in the following order: (1) deletion or substitution of one or more genes encoding a D-alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; (2) deletion or substitution of one or more genes encoding a naturally occurring antibiotic resistance gene, wherein the naturally occurring antibiotic resistance gene is inactive; and (3) deletion or substitution of one or more genes encoding a naturally occurring lysogenic phage gene, wherein the naturally occurring lysogenic phage gene is inactive. Thus, recombinant D-alanine auxotrophic bacterial strains were prepared.

63. The recombinant D-alanine auxotrophic bacterial strain according to claim 62, wherein the deletion or substitution in one or more genes encoding the D-alanine biosynthesis gene includes deletions in dat, alar1, and alar2.

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