Methods and compositions for treating skin disease with recombinant microorganisms

Abstract

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

Description

METHODS AND COMPOSITIONS FOR TREATING SKIN DISEASE WITH RECOMBINANT MICROORGANISMSRELATED APPLICATIONS

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

[0002] Viral, bacterial and fungal infections of the skin are increasing in frequency and severity. For example, pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are rapidly increasing with limited antibiotics available to treat infections, especially those associated with skin. There is significant need for new therapeutics for both prevention as well as acute care for viral, bacterial and fungal infections of the skin.SUMMARY

[0003] The development of novel bacterial strains whose growth can be controlled without the use of antibiotics, or genetic elements conferring antibiotic resistance, enables modulation of therapeutic exposure and improves safety. The studies described herein support the potential safety and utility of a live biotherapeutic bacterial strain whose growth can be controlled by D-alanine for use in the treatment of various diseases and disorders of the skin. In particular, the present disclosure features, in some embodiments, a recombinant, D-alanine auxotrophic strain that was engineered to secrete therapeutic proteins (e.g., bacterial and fungal cell wall hydrolases, any protective expression-host immunity proteins) extracellularly onto the surface of skin and / or other appropriate tissues to be used as prophylactic, post-exposure prophylactic, or therapeutic treatments for dysbiosis due to pathogenic bacterial or fungal overgrowth either primary or secondary to superficial or serious skin disease or disorder.

[0004] In a first aspect, the 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage gene is inactive.

[0005] In another aspect, the present 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 bacteriophage gene,wherein the naturally occurring lysogenic bacteriophage 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, the one or more naturally occurring antibiotic resistance gene is selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a co-trimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

[0007] In some embodiments of the above aspects and embodiments, the one or more naturally occurring antibiotic resistance gene is the mupirocin resistance gene.

[0008] In some embodiments of the above aspects and embodiments, the one or more naturally occurring lysogenic bacteriophage gene encodes a bacteriophage capsid protein.

[0009] In some embodiments of the above aspects and embodiments, the one or more D-alanine biosynthesis genes comprises D-alanine aminotransferase gene (dat), alanine racemase genes alrl or alr2. In some embodiments of the above aspects and embodiments, the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2.

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

[0011] In some embodiments of the above aspects and embodiments, the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or variants thereof, a gene encoding an antimicrobial biosynthesis enzyme or variants thereof, a gene encoding an enzyme or variants thereof, a gene encoding an enzyme inhibitor or variants thereof, a gene encoding an antigen or variants thereof, and a gene encoding an immune modulating polypeptide or variants thereof, or a combination thereof.

[0012] In some embodiments of the above aspects and embodiments, the heterologous gene encodes the antimicrobial polypeptide or variants thereof.

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

[0014] In some embodiments of the above aspects and embodiments, the one or more microbial pathogen is selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

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

[0016] In some embodiments of the above aspects and embodiments, the fungal infection is caused by a fungus selected from the group consisting of Malassezia spp., Candida spp., Aspergillus, Cryptococcus, and Pneumocystis.

[0017] In some embodiments of the above aspects and embodiments, the viral infection is caused by a virus selected from the group consisting of respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses.

[0018] In some embodiments of the above aspects and embodiments, the antimicrobial polypeptide is selected from the group consisting of an epidermin-like lantibiotic, a YM-1, a YM-2, an acidic mammalian chitinase (AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilage glycoprotein-39, a chondrocyte protein 39, an endoglucanase, an exoglucanase, an P-glucosidase, a cellobiohydrolase, an endo-l,4-P-xylanase, a P-xylosidase, an a- glucuronidase, an a-L-arabinofuranosidase, an acetylesterase, an acetylxylanesterase, an a-amylase, a P-amylase, a glucoamylase, a pullulanase, a P-glucanase, a hemicellulase, an arabinosidase, a mannanase, a pectin hydrolase, a pectate lyase, a lysostaphin, zoocin A, a millericin B, a muraminidase Cpl-1, a lysozyme, an endolysin PlyC, an endolysin, a PlyV12, an enterolysin A, an autolysin of C. difficile (Acd), an autolysin (LytA), an PL-1 amidase hydrolase, a Nisin A, a Nisin Z, a Subtilin, an Epidermin, a Gallidermin, a Mutacin B-a Ny266, a Mutacin 1140, a Pep5, an Epicidin 280, an Epilancin K7, a Lacticin 481, a Cytolysin, a Lacticin 3147, a Staphylococcin C55, a Salvaricin A, a Lactocin S, a Streptococcin A-FF2, a Sublancin 168, a Carnocin U149, a Variacin 8, a Cypemycin, a Cinnamycin, a Duramycin, an Ancovenin, a Mersacidin, and an Actagardine.

[0019] In some embodiments, the one or more heterologous genes encodes 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 above aspects and embodiments, the heterologous gene encodes an antimicrobial biosynthesis enzyme or variants thereof, wherein the antimicrobial biosynthesis enzyme is capable of producing 6-N-hydroxy aminopurine (6-HAP), and wherein 6-HAP is secreted.

[0021] In some embodiments of the above aspects and embodiments, the gene encoding an immune modulating polypeptide or variants thereof is a gene encoding a lipoteichoic acid (LT A) biosynthesis enzyme, and wherein the recombinant microorganism is capable of producing LT A and secreting LT A.

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

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

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

[0025] In some embodiments of the above aspects and embodiments, the recombinant microorganism secretes the one or more therapeutic polypeptides or variants thereof.

[0026] Accordingly, in another aspect, the present disclosure provides a pharmaceutical composition comprising a cell culture composition comprising one or more recombinant microorganisms of the above aspects or any other aspect of the disclosure delineated herein and a pharmaceutically acceptable carrier.

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

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

[0029] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, or an ointment.

[0030] In another aspect, the disclosure provides a method of treating a disease, disorder, or condition in 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage gene is inactive.

[0031] Accordingly, in another aspect, the present disclosure provides a method of treating a disease, disorder, or condition in a subject, the method comprising: administering to the subject a cell culture composition comprising 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage 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 embodiments, the one or more naturally occurring antibiotic resistance gene is selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a co-trimoxazoleresistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

[0033] In some embodiments of the above aspects and embodiments, the one or more naturally occurring antibiotic resistance gene is the mupirocin resistance gene.

[0034] In some embodiments of the above aspects and embodiments, the one or more naturally occurring lysogenic bacteriophage gene encodes a bacteriophage capsid protein.

[0035] In some embodiments of the above aspects and embodiments, the one or more D-alanine biosynthesis genes comprises D-alanine aminotransferase gene (dat), alanine racemase genes alrl or alr2. In some embodiments of the above aspects and embodiments, the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2.

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

[0037] In some embodiments of the above aspects and embodiments, the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or variants thereof, a gene encoding an antimicrobial biosynthesis enzyme or variants thereof, a gene encoding an enzyme or variants thereof, a gene encoding an enzyme inhibitor or variants thereof, a gene encoding an antigen or variants thereof, and a gene encoding an immune modulating polypeptide or variants thereof, or a combination thereof.

[0038] In some embodiments of the above aspects and embodiments, the heterologous gene encodes the antimicrobial polypeptide or variants thereof.

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

[0040] In some embodiments of the above aspects and embodiments, the one or more microbial pathogen is selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

[0041] In some embodiments of the above aspects and embodiments, the one or more bacterial pathogens are selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, CutibacteriumPropionibacterium spp., and Mycoplasma, or combinations thereof.

[0042] In some embodiments of the above aspects and embodiments, the fungal infection is caused by a fungus selected from the group consisting of Malassezia spp., Candida spp., Aspergillus, Cryptococcus, and Pneumocystis.

[0043] In some embodiments of the above aspects and embodiments, the viral infection is caused by a virus selected from the group consisting of respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses.

[0044] In some embodiments of the above aspects and embodiments, the antimicrobial polypeptide is selected from the group consisting of an epidermin-like lantibiotic, a YM-1, a YM-2, an acidic mammalian chitinase (AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilage glycoprotein-39, a chondrocyte protein 39, an endoglucanase, an exoglucanase, an P-glucosidase, a cellobiohydrolase, an endo-l,4-P-xylanase, a P-xylosidase, an a- glucuronidase, an a-L-arabinofuranosidase, an acetylesterase, an acetylxylanesterase, an a-amylase, a P-amylase, a glucoamylase, a pullulanase, a P-glucanase, a hemicellulase, an arabinosidase, a mannanase, a pectin hydrolase, a pectate lyase, a lysostaphin, zoocin A, a millericin B, a muraminidase Cpl-1, a lysozyme, an endolysin PlyC, an endolysin, a PlyV12, an enterolysin A, an autolysin of Clostridium difficile (C. difficile) (Acd), an autolysin (LytA), an PL-1 amidase hydrolase, a Nisin A, a Nisin Z, a Subtilin, an Epidermin, a Gallidermin, a Mutacin B-a Ny266, a Mutacin 1140, a Pep5, an Epicidin 280, an Epilancin K7, a Lacticin 481, a Cytolysin, a Lacticin 3147, a Staphylococcin C55, a Salvaricin A, a Lactocin S, a Streptococcin A-FF2, a Sublancin 168, a Carnocin U149, a Variacin 8, a Cypemycin, a Cinnamycin, a Duramycin, an Ancovenin, a Mersacidin, and an Actagardine.

[0045] In some embodiments of the above aspects and embodiments, the one or more heterologous genes encodes 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 above aspects and embodiments, the heterologous gene encodes an antimicrobial biosynthesis enzyme or variants thereof, wherein the antimicrobial biosynthesis enzyme is capable of producing 6-N-hydroxy aminopurine (6-HAP), and wherein 6-HAP is secreted.

[0047] In some embodiments of the above aspects and embodiments, the gene encoding an immune modulating polypeptide or variants thereof is a gene encoding a lipoteichoic acid (LT A) biosynthesis enzyme, and wherein the recombinant microorganism is capable of producing and secreting LT A.

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

[0049] In some embodiments of the above aspects and embodiments, the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, CutibacteriumPropionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or combinations thereof.

[0050] In some embodiments of the above aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis (S. epidermis). In some embodiments of the above aspects and embodiments, the recombinant microorganism secretes the one or more therapeutic polypeptides or variants thereof.

[0051] In some embodiments of the above aspects and embodiments, the cell culture composition is a living cell culture composition. In some embodiments of the above aspects and embodiments, the cell culture composition comprises 0% water to no more than 90% water.

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

[0053] In some embodiments of the above aspects and embodiments, the disease, disorder, or condition is a microbial infection, inflammatory disease or disorder, metabolism disease or disorder.

[0054] In some embodiments of the above aspects and embodiments, the microbial infection comprises one or more microbial pathogens selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, CutibacteriumPropionibacterium spp., Mycoplasma, Malassezia spp., Candida spp., Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses or combinations thereof.

[0055] In some embodiments of the above aspects and embodiments, the skin disease or disorder is an inflammatory skin disease or disorder. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a skin dysbiosis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a dermal toxicity. In some embodiments of the above aspects and embodiments, the skin disease or disorder is an ichthyosis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a psoriatic disease or disorder. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a dermatitis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is an autoimmune blistering disease. In some embodiments of the above aspects and embodiments, the skin disease or disorder is selected from the group consisting of: Netherton 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] In another aspect, the disclosure provides a recombinant D-Alanine auxotrophic bacterial strain prepared by a process comprising selecting a bacterial strain; carrying out in the following order: (1) a deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene, wherein the D- alanine biosynthesis gene is inactive; (2) 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 (3) a deletion or substitution in one or more genes encoding a naturally occurring lysogenic bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage gene is inactive,

[0057] thereby preparing a recombinant D-Alanine auxotrophic bacterial strain. In further embodiments, the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2. In some embodiments of the above aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis (S. epidermis).BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1A depicts inhibition screen of Staphylococcus aureus (S. aureus) from Micromyx-50 library of .S', epidermidis strains.

[0059] FIG. IB depicts inhibition of .S'. aureus by strains SE25 MM23.

[0060] FIG. 2 depicts inhibition of .S'. aureus SA25923, .S'. aureus SA29213, .S'. epidermidis NRRL, MRSA USA 300, Bacillus subtilis, and Gram-negative strains Pseudomonas aeruginosa and Escherichia coli (E. coll) by SE25.

[0061] FIG. 3 depicts graph showing the growth of .S'. aureus grown in conditioned cell-free SE25 or SE3 media compared to TSB alone.

[0062] FIG. 4 depicts anti-microbial activity of SE25 cell-free supernatant against coagulase-negative Staphylococcus from the skin of ichthyosis patients. SE25 cell-free supernatant was tested against .S'. aureus ATCC 29213, .S’. aureus ATCC 25923, .S’. epidermidis NRRL B-4268, .S’. epidermidis 1457, .S’. epidermidis 25, B. subtilis, E. faecalis, E. coli, P. aeruginosa, and Ichthyosis CoNS isolates, .S’. lugdunensis, S. auricularis, S. capitis, S. haemolyticus, S. hominis, S. pettenkoferi, S. saprophyticus, and .S’. warneri.

[0063] FIG. 5A depicts graph showing SE25 cell-free supernatant has activity against 5. aureus, where 5. aureus growth is reduced approximately by 7-log.

[0064] FIG. 5B depicts graph showing bactericidal activity against 5. aureus by linezolid, levofloxacin, erythromycin, and vancomycin.

[0065] FIG. 6A depicts biofilm formation on plastic with crystal violet indicator. The graph shows that SE25 does not form a biofilm, while the positive control SE 1457 does.

[0066] FIG. 6B depicts biofilm detection using Congo Red assay. SE25 forms red colonies indicating no biofilms were formed.

[0067] FIG. 6C depicts a graph showing the serial dilution of SE25 cell-free supernatant disperses biofilms formed by .S', aureus.

[0068] FIG. 7 depicts a graph showing SE25 is capable of colonizing reconstituted human epidermis (RHE) after 24 hrs and 48 hrs.

[0069] FIG. 8 depicts a graph showing that RHE colonized with SE25 leads to a 4-log reduction of 5. aureus ATCC 29213.

[0070] FIG. 9 depicts a graph showing that SE25 colonized on RHE induces expression of human - defensin 2 from the keratinocytes.

[0071] FIG. 10 depicts epidermin biosynthetic gene cluster in SE25.

[0072] FIG. 11 depicts prophage phiSpy gene cluster in SE25.

[0073] FIG. 12 depicts auxotrophy test on TSA plates with (plate A) or without (plate B) D-alanine (“DA100”) supplementation. SE480: SE25 wild-type, SE482: SE25 double-deletion auxotroph, SE484:SE25 triple -deletion auxotroph (all with AmupAAcapsid), SE123: SEAAA triple -deletion auxotroph of NRRL B-4268.

[0074] FIG. 13A depicts screening assays for Aalrl Aalr2 Adat SE25 strains.

[0075] FIG. 13B depicts screening assays for Aalrl Adat SE25 strains.

[0076] FIG. 14A depicts anti -.S', aureus activity of Aalrl Adat SE25 strains in agar overlay assays. SE25 strains were grown on TSB for 48 hours at 37°C and overlaid with top agar containing .S'. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity.

[0077] FIG. 14B depicts anti -.S'. aureus activity of Aalrl Aalr2 Adat SE25 strains in agar overlay assays. SE25 strains were grown on TSB for 48 hours at 37°C and overlaid with top agar containing .S'. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity.

[0078] FIG. 15A depicts screening assays for chloramphenicol (Cam-10)- and mupirocin (Mup-20)- sensitive colonies for wild-type SE25. Circles indicate positive Cam-10 / Mup-20 sensitive colonies.

[0079] FIG. 15B depicts screening assays for chloramphenicol (Cam- 10)- and mupirocin (Mup-20)- sensitive colonies for DA-auxotrophic strains of SE25. Circles indicate positive Cam-10 / Mup-20 sensitive colonies.

[0080] FIG. 16A depicts anti -.S', aureus activity of SE25 strains in agar overlay assays. SE25 strains were grown on TSA for 24 hours at 37 °C, followed by additional 24 hr at 30 °C and overlaid with top agar containing .S', aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity. SE25 AmupAAcapsid (SE480, SE481), SE25 AmupAAcapsid Aalrl Adat (SE482, SE483), and SE25 AmupAAcapsid Aalrl Aalr2 Adat (SE484, SE485).

[0081] FIG. 16B depicts anti -.S', aureus activity of SE25 strains in agar overlay assays. SE25 strains were grown on TSA for 24 hours at 37 °C, followed by additional 24 hr at 30 °C and overlaid with top agar containing .S', aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity. Wild-type parental SE25 strain (SE398).

[0082] FIG. 17 depicts agar overlay assay of SE25 AepiA mutant strains (plates 1 - 4) with .S', aureus ATCC 29213, negative wild-type control SE120(NRRL B-4268; plate 5) and positive control SE25 wild-type (plate 6).

[0083] FIG. 18A depicts SE25 strains screened for epiA knockout.

[0084] FIG. 18B depicts PCR analysis of Cam-sensitive colonies for AepiA mutant strains (colonies 5, 8, 11, and 32).

[0085] FIG. 19A shows screening assays for candidate colonies of D-Alanine Auxotrophy Strain SE464 streaked onto tryptic soy agar. (TSA) in the presence of 100 pg / mL D-alanine without chloramphenicol.

[0086] FIG. 19B shows screening assays for candidate colonies of D- Alanine Auxotrophy Strain SE464 streaked onto TSA supplemented with 10 pg / mL chloramphenicol in the absence of D- alanine.

[0087] FIG. 19C shows the identification of 3 clones (highlighted in red circle) that only grew on TSA supplemented with D-alanine.

[0088] FIG. 20A is a plot depicting the growth kinetics of SE25 and SE484 strains

[0089] FIG. 20B depicts anti -.S', aureus activity of SE25 strains in agar overlay assays. SE25 strains were streaked onto TSA, supplemented with streaked 100 mg / mL D-alanine and incubated at 37°C. After a 48 hour incubation, soft agar containing .S'. aureus was poured over the plates which were then incubated at 37°C for an additional 24 hours.

[0090] FIG. 20C depicts anti -.S'. aureus activity of SE484 strains in agar overlay assays. SE484 strains were streaked onto TSA, supplemented with streaked 100 mg / mL D-alanine and incubated at 37°C. After a 48 hour incubation, soft agar containing .S', aureus was poured over the plates which were then incubated at 37°C for an additional 24 hours.

[0091] FIG. 20D depicts the verification of D-alanine auxotrophy and Mupirocin sensitivity of SE484. SE484 was streaked onto TSA + / - 100 pg / ml D-alanine and TSA with D-alanine + / - 20 pg / ml of Mupirocin. Plates were incubated at 37°C, and data were recorded after a 48 hour incubation.

[0092] FIG. 21 A depicts the anti -.S', aureus activity of SEI 23. Reconstituted Human Epidermis (RHE) was inoculated with SE123 and incubated for four hours in a tissue culture incubator at 37°C, 5% CO2, prior to challenge with ~104CFU of .S', aureus USA300. After an additional 24 hour incubation, USA300 cell count was determined by punch biopsy and dilution plating.

[0093] FIG. 21B depicts the anti -.S', aureus activity of SE484. Reconstituted Human Epidermis (RHE) was inoculated with SE484 and incubated for four hours in a tissue culture incubator at 37°C, 5% CO2, prior to challenge with ~104CFU of .S', aureus USA300. After an additional 24 hour incubation, USA300 cell count was determined by punch biopsy and dilution plating.DETAILED DESCRIPTION

[0094] The present disclosure relates to the use of an engineered microbe designed to deliver a therapeutic agent to a subject’s skin, which provides key benefits over using naturally occurring antimicrobial-secreting probiotic strains. For example, an engineered commensal can be modified to have desirable characteristics that improve its colonization, longevity, and antimicrobial properties versus any inherent limitations to the probiotic, which may have limited or differential colonization success in the skin. The present disclosure provides for methods and compositions of an engineered microorganism that produces therapeutic agents that can be secreted, e.g., different human- or bacteria-derived antimicrobials.

[0095] 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 the naturally occurring antibiotic resistance gene is inactive or deleted. In some embodiments of theabove aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding a naturally occurring lysogenic bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage 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 the D-alanine biosynthesis gene is inactive.

[0096] According to some embodiments, 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage 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.

[0097] According to some embodiments, the recombinant microorganism the one or more naturally occurring antibiotic resistance gene is selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a co-trimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is located on the chromosome of the recombinant microorganism or on a plasmid, or both. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is mupirocin resistance gene.

[0098] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on the chromosome and not the plasmid. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on the plasmid and not the chromosome. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on both the plasmid and the chromosome.

[0099] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance polypeptide has 90% identity with the entire sequence of SEQ ID NO: 152. Accordingly, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 95% identity with the entire sequence of SEQ ID NO: 152. Accordingly, in one embodiment, the naturally occurring antibiotic resistance 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: 152. In another embodiment, the naturally occurring antibiotic resistance polypeptide comprises the sequence of SEQ ID NO: 152. In yet another embodiment the naturally occurring antibiotic resistance polypeptide consists of the sequence of SEQ ID NO: 152.

[0100] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance polypeptide has 90% identity with the entire sequence of SEQ ID NO: 153. Accordingly, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 95% identity with the entire sequence of SEQ ID NO: 153. Accordingly, in one embodiment, the naturally occurring antibiotic resistance 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: 153. In another embodiment, the naturally occurring antibiotic resistance polypeptide comprises the sequence of SEQ ID NO: 153. In yet another embodiment the naturally occurring antibiotic resistance polypeptide consists of the sequence of SEQ ID NO: 153.

[0101] According to some embodiments, the naturally occurring lysogenic bacteriophage gene encodes a structural protein essential for phage particle formation and cycle. In some embodiments of the above aspects and embodiments, the naturally occurring lysogenic bacteriophage gene encodes a bacteriophage structural capsid protein.

[0102] In some embodiments of the above aspects and embodiments, the naturally occurring lysogenic bacteriophage polypeptide has 90% identity with the entire sequence of SEQ ID NO: 154. Accordingly, in one embodiment, the naturally occurring lysogenic bacteriophage polypeptide has at least about 95% identity with the entire sequence of SEQ ID NO: 154. Accordingly, in one embodiment, the naturally occurring lysogenic bacteriophage 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 bacteriophage polypeptide comprises the sequence of SEQ ID NO: 154. In yet another embodiment the naturally occurring lysogenic bacteriophage polypeptide consists of the sequence of SEQ ID NO: 154.

[0103] According to some embodiments, the recombinant microorganism is auxotrophic for an essential gene or genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism is auxotrophic for D-alanine biosynthesis. In some embodiments, D- alanine biosynthesis genes are deleted or mutated resulting in inactivation. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in the D-alanine aminotransferase (dat) gene, alanine racemase genes alrl or alr2. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in the dat, alrl and alr2 genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in the dat and alrl genes.

[0104] According to some embodiments, the disclosure provides a composition for the treatment of a bacterial infection on a subject comprising a microbe genetically engineered to express a therapeutic agent, e.g., antimicrobial polypeptide, and provide to the subject. According to some embodiments,the therapeutic agent is an antimicrobial polypeptide which is effective to treat the bacterial infection and skin dysbiosis e.g., atopic dermatitis, superficial fungal infections, e.g., dandruff, tinea, candidiasis, etc.). In some embodiments of the above aspects and embodiments, the therapeutic agent is 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-hydroxy aminopurine (6-HAP).

[0105] As used herein, the term “genetically modified” and grammatical variations thereof are used to describe a microbial organism (e.g. , bacteria) that has been genetically modified or engineered by the introduction of DNA prepared outside the microbe. For example, the introduction of plasmid DNA containing new genes into bacteria will allow the bacteria to express those genes. Alternatively, the DNA containing new genes can be introduced to the bacteria and then integrated into the bacteria's genome, either chromosome and / or plasmid, where the bacteria will express those genes.

[0106] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean providing to a subject a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of pathogen infection, infection symptoms or delay the onset of the infection, or halt the progression of infection development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population may fail to respond or respond inadequately to treatment.

[0107] According to some embodiments, the recombinant microbe secrets an antimicrobial polypeptide at levels effective to ameliorate the symptoms of a bacterial pathogen. As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of an infection in a subject.

[0108] As used herein, the term “preventing” refers to completely or almost completely stopping the disease or condition or symptoms associated with a disease from occurring, for example, when a patient or subject is predisposed to a respiratory disease or at risk of contracting a respiratory disease. Preventing can also include inhibiting, e.g., arresting the development of a respiratory disease or postexposure prophylaxis (e.g., preventing development of a disease or symptoms associated with a disease after initial exposure to a pathogen associated with the disease).

[0109] As used herein, the term “reducing the risk of’ refers to lowering the likelihood or probability of a respiratory disease from occurring, for example when a patient or subject is predisposed to a respiratory disease or at risk of contracting a respiratory disease.

[0110] As used herein, the term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of another agent. As used herein, an antimicrobial agent expressed and secretedby an engineered microorganism, for example by enhancing the level of efficacy (i.e. the killing ability of pathogenic bacteria and / or fungi) 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 as compared to the level of efficacy of the same type of microorganism which has not been engineered to express and secrete an antimicrobial agent. Accordingly, the antimicrobial agent expressed and secreted by an engineered microorganism function.

[0111] The term “antimicrobial agent”, “antimicrobial protein” or “antimicrobial polypeptide” as used herein can be used interchangeably and refers to any entity with antimicrobial activity, i.e., the ability to inhibit the growth and / or kill bacterium and / or fungus, e.g. , Gram positive and gram negative bacteria and fungus. An antimicrobial agent is any agent which results in inhibition of growth or reduction of viability of a bacteria and / or fungus 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 between 30% and 70% or more, as compared to in the absence of the antimicrobial agent. Stated another way, an antimicrobial agent is any agent which reduces a population of bacterial and / or fungal cells, 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 between 30% and 70% as compared to in the absence of the antimicrobial agent. In one embodiment, an antimicrobial agent is an agent which specifically targets a bacteria cell. In another embodiment, an antimicrobial agent modifies (i.e. inhibits or activates or increases) a pathway which is specifically expressed in bacterial cells. In some embodiments of the above aspects and embodiments, the antimicrobial agent is a polypeptide, i.e., a polypeptide expressed and secreted by the engineering microorganism.

[0112] Antimicrobial agents can include chitinase, a glucanase, or a peptidoglycan hydrolase.

[0113] As used herein, the term “chitinase” refers to an enzyme capable of catalyzes the hydrolysis of the P-1,4 linked N-acetylglucosamine polymers that form chitin chains, a major component of fungal cell walls. Chitinases are expressed in plants in response to pathogens.

[0114] As used herein, the term “glucanase” refers to an enzyme capable of catalyzing the degradation or depolymerization of complex carbohydrates. A glucanase in the composition may be capable of degrading one or more of cellooligosaccharide, lignocellulose, cellulose, hemicellulose, and pectin. Such enzymatic activity may be, but is not limited to, endoglucanase, exoglucanase, -glucosidase, cellobiohydrolase, endo-l,4- -xylanase, -xylosidase, a-glucuronidase, a-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, a-amylase, -amylase, glucoamylase, pullulanase, -glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, or pectate lyase activities. The glucanase of the composition may be capable of degrading one or more of beta-glucan, cellulose, cellobiose, pNP- D-glucopyranoside and xylan.

[0115] As used herein, the term “peptidoglycan hydrolase” refer to an enzyme that can degrade bacterial cell walls when exposed externally. The bacterial cell wall consists of glycan strands whichare cross-linked by flexible peptide side chains, providing strength and rigidity to the bacterial cell wall. The peptidoglycan of both the Gram-positive and Gram-negative bacteria features repeating units of N- acetylglucosamine (NAG) and P-(l-4)-N-acetylmuramic acid (NAM) cross-linked by peptide stem chains attached to NAM residues. The so-called peptidoglycan hydrolases (PGHs) are the enzymes responsible for cleaving the bonds within the peptidoglycan chains and side -chains branches.

[0116] The term “infection” or “microbial infection” which are used interchangeably herein refers to in its broadest sense, any infection caused by a microorganism and includes bacterial infections, fungal infections, yeast infections and protozomal infections.

[0117] In the present disclosure, the subject may be a mammal. As used herein, a “mammal” and grammatical variations thereof means any category of mammal. In the present disclosure, mammals include, for example, humans, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. Preferably, the mammal is a human.

[0118] As used herein, the term "effective amount" or a "therapeutically effective amount" of a compound or composition disclosed herein is an amount of such compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a composition according to the disclosure will be that amount of the composition, which is the lowest dose effective to produce the desired effect. The effective dose of a composition of the present disclosure may be administered as two, three, four, five, six or more subdoses, administered separately at appropriate intervals throughout the day.

[0119] The term “administration” as used herein is meant to refer to contact of a pharmaceutical composition, therapeutic composition, diagnostic agent or composition to a recipient, preferably a human. Therapeutic agents disclosed herein can be administered intranasally, topically, or into the nasal cavity or nasopharyngeal cavity.

[0120] As used herein, the terms “polypeptide” or “protein” refer to biological molecules, or macromolecules composed of amino-acid residues bonding together in a chain. The definition of polypeptides used herein is intended to encompass proteins (generally higher molecular weight) composed of one or more long chains of amino acids residues and small peptides (generally lower molecular weight) of a few amino acids. In other embodiments, a single amino acid, although not technically a polypeptide, is also considered within the scope of this disclosure.

[0121] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

[0122] The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0123] The term “or” is used herein to mean, and is used interchangeably with, the term “and / or,” unless context clearly indicates otherwise.

[0124] Microbial compositions: According to some embodiments, the disclosure provides microbial compositions comprising one or more of a wide range of bacteria suitable for use on a mammal’s skin. Examples include, but are not limited to, non-pathogenic and commensal bacteria. Bacteria suitable for use in the present disclosure include, but are not limited to, Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., S. epidermidis and / or .S’, hominis), Lactobacillus (e.g., L. acidophilus), Pediococcus, Leuconostoc, or Oenococcus. According to some embodiments, microbial compositions comprise one or more of Staphylococcus warned, Streptococcus pyogenes, Streptococcus mitis, Cutibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, Pseudomonas aeruginosa. According to some embodiments, other related or similar species found on the skin are used.

[0125] Certain embodiments involve the use of bacterium Staphylococcus epidermidis. According to some embodiments, the strain of .S'. epidermidis to be used is incapable of producing biofilms. An example of this is .S. epidermidis strain ATCC 12228, NRRL B-4268, or SE25.

[0126] According to some embodiments, the recombinant microbe is adapted to live indefinitely or for a controlled duration on the surface of the mammal’s skin to provide a controlled or continuous supply of an therapeutic polypeptide, e.g. antimicrobial. In some embodiments of the above aspects and embodiments, the antimicrobial is a small molecule or a polypeptide. In some embodiments of the above aspects and embodiments, the recombinant microbe lives alongside commensal microorganisms naturally occurring on the mammal’s skin. In some embodiments of the above aspects and embodiments, the recombinant microbe lives to the exclusion of commensal microorganisms that naturally occur on the mammal’s skin. According to some embodiments, the recombinant microbe is adapted to multiply on the skin of the mammal.

[0127] In other embodiments, the recombinant microbe is no longer alive, but contains effective amounts of an antimicrobial polypeptide, e.g., epidermin-like lantibiotic, a YM-1, a YM-2, an acidic mammalian chitinase (AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilage glycoprotein-39, a chondrocyte protein 39, an endoglucanase, an exoglucanase, an P-glucosidase, a cellobiohydrolase, an endo-l,4-P-xylanase, a P-xylosidase, an a- glucuronidase, an a-L-arabinofuranosidase, an acetylesterase, an acetylxylanesterase, an a-amylase, a P-amylase, a glucoamylase, a pullulanase, a P-glucanase, a hemicellulase, an arabinosidase, amannanase, a pectin hydrolase, a pectate lyase, a lysostaphin, zoocin A, a millericin B, a muraminidase Cpl-1, a lysozyme, an endolysin PlyC, an endolysin, a PlyV12, an enterolysin A, an autolysin of C. difficile (Acd), an autolysin (LytA), an PL-1 amidase hydrolase, a Nisin A, a Nisin Z, a Subtilin, an Epidermin, a Gallidermin, a Mutacin B-a Ny266, a Mutacin 1140, a Pep5, an Epicidin 280, an Epilancin K7, a Lacticin 481, a Cytolysin, a Lacticin 3147, a Staphylococcin C55, a Salvaricin A, a Lactocin S, a Streptococcin A-FF2, a Sublancin 168, a Carnocin U149, a Variacin 8, a Cypemycin, a Cinnamycin, a Duramycin, an Ancovenin, a Mersacidin, and an Actagardine or therapeutically effective domain(s) thereof.

[0128] In some embodiments of the above aspects and embodiments, the recombinant microbe is capable of producing 6-N-hydroxy aminopurine (6-HAP).

[0129] In some embodiments of the above aspects and embodiments, the antimicrobial is effective against a bacterial pathogen, a fungal pathogen, or a viral pathogen. In some embodiments of the above aspects and embodiments, the antimicrobial is effective against Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacteria spp., Mycoplasma, Malassezia spp., Candida spp., Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses. In some embodiments of the above aspects and embodiments, the antimicrobial is effective against E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Streptococcus (Str.) pneumoniae, Str. pyogenes, Enterococcus (E.) faecalis, E. faecium, and Candida albicans.

[0130] According to some embodiments, the therapeutic protein comprises one or more LEKTI domains that are effective to inhibit serine proteases present within or on the skin, upper and lower airway tissues, other mammalian tissues, and in systemic circulation. According to some embodiments, the recombinant LEKTI domains compensate for the defective endogenous LEKTI protein, or other defective serine protease inhibitors, naturally produced within or on the skin, upper and lower airway tissues, other mammalian tissues, and in systemic circulation. According to some embodiments, the genetically altered bacteria are able to self-replicate while retaining the ability to produce the recombinant protein, thereby providing a continuous supply of therapeutic agent.

[0131] 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 the group consisting of SPINKI, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINKI3, and SPINK14. According to some embodiments, the SPINK protein is SPINK5. The SPINK gene can be obtained from any mammal, such as mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequences. According to some embodiments, the SPINK gene sequence is a human gene sequence.

[0132] SPINK5 gene: According to some embodiments, the recombinant microbe is engineered to express a mammalian gene, SPINK5, encoding LEKTI protein. The SPINK5 gene can be obtained from any mammal, such as mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequences. According to some embodiments, the SPINK5 gene sequence is a human gene sequence. According to some embodiments, the recombinant microbe is engineered to comprise a fragment of the SPINK gene.

[0133] According to some embodiments, the recombinant protein expressed by the engineered microbe comprises the peptide sequence according to SEQ ID NO: 109 (LEKTI D6). According to some embodiments, the recombinant protein expressed by the engineered microbe comprises the peptide sequence according to SEQ ID NO: 109. According to some embodiments, one or more fragments of the peptide sequence according to SEQ ID NO: 103 are expressed by the engineered microbe. In one embodiment, the fragment comprises one or more LEKTI domains. In a specific embodiment, the LEKTI domain is Domain 6.

[0134] According to some embodiments, the recombinant microbe comprises a sequence as disclosed herein that has at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to any one or more of the SEQ ID NOS listed herein. As used herein, the term “identity” and grammatical versions thereof means the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0135] According to some embodiments, the recombinant protein expressed by the engineered microbe comprises one or more protease inhibitory domains of the LEKTI protein. Some nonlimiting examples include one or more of domains DI, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dl l, D12, D13, D14, and D15. According to some embodiments, the recombinant protein expressed by the engineered microbe comprises LEKTI inhibitory domain 6 or domains D8 to DI 1.

[0136] In some embodiments of the above aspects and embodiments, the LEKTI protein domains act as a competitive or non-competitive inhibitor of one or more proteases present on or in the skin of a mammal. In some embodiments of the above aspects and embodiments, the LEKTI protein domain acts as a serine protease inhibitor. As used herein, the terms “protease” and “proteinase” are used interchangeably, with both terms referring to an enzyme that performs proteolysis.

[0137] According to some embodiments, the microbe is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a therapeutic polypeptide (e.g. , the LEKTI protein domains), a small molecule, or a metabolite. Other conventional or to-be-discovered methods for introducing DNA into a microbe may also be used in the present disclosure. According to some embodiments, the recombinant DNA plasmid comprises sequences encoding the LEKTI protein domain and one or more secretory peptides and / or cell penetration peptides. According to someembodiments, the LEKTI domains are operably linked to one or more recombinant protein domains that are effective to enhance secretion from the microbe and / or penetration of mammalian tissue.

[0138] According to some embodiments, the disclosure provides a composition for the prevention or treatment of a respiratory bacterial, viral, fungal, and other microbial infections, oral / pharyngeal cancers, cancers of the respiratory tract, neuron degeneration in spinal injury, multiple sclerosis, glioblastoma, and other oligodendrogliopathies comprising a microbe genetically modified to express and provide one or more LEKTI protein domains or LEKTI proteins intranasally of a mammal, wherein the LEKTI protein domains or LEKTI proteins are effective to inhibit serine protease activity of at least one serine protease in or on the mammal’s skin, upper and lower airway tissues, other mammalian tissues, or in general systemic circulation. According to some embodiments, the disclosure provides a composition for the prevention or treatment of respiratory diseases (e.g., chronic rhinosinusitis). According to some embodiments, the disclosure provides a composition for the prevention or treatment of central nervous system disorders and diseases. As used herein, the term “recombinant” and grammatical variations thereof means relating to or denoting an organism, protein, or genetic material formed by or using recombined DNA comprising DNA pieces from different sources or from different parts of the same source. For example, the term “recombinant DNA” means a DNA molecule formed through recombination methods to splice fragments of DNA from a different source or from different parts of the same source. In some embodiments, two or more different sources of DNA are cleaved using restriction enzymes and joined together using ligases. As another example, the term “recombinant protein” or “recombinant domains” and grammatical variations thereof means a protein molecule formed through recombination methods originating from spliced fragments of DNA from a different source or from different parts of the same source. As another example, the term “recombinant microbe” or “recombinant bacteria” and grammatical variations thereof are interchangeable and mean a microbe that comprises one or more recombinant DNA / protein molecules.

[0139] According to some embodiments, the recombinant microbe comprises a sequence as disclosed herein that has at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to any one or more of the SEQ ID NOs listed herein. As used herein, the term “identity” and grammatical versions thereof means the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0140] According to some embodiments, the microbe is genetically modified by transfection / transformation with one or more recombinant DNA plasmids encode one or more therapeutic proteins (e.g., antimicrobial polypeptide, e.g., epiA, elafin, hiracin, lysostaphin, and LL- 37). Other conventional or to-be -discovered methods for introducing DNA into a microbe may also beused in the present disclosure. According to some embodiments, the one or more recombinant DNA plasmids comprise sequences encoding the antimicrobial polypeptide.

[0141] 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. Accordingly, in one embodiment, the therapeutic protein has at least about 95% identity with the entire sequence of SEQ ID NOs: 130-145. Accordingly, 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 another embodiment, the therapeutic protein comprises the sequence of SEQ ID NOs: 130-145. In yet another embodiment the therapeutic protein consists of the sequence of SEQ ID NOs: 130-145.

[0142] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other or is not hindered by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (z.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, two proteins can be operably linked, such that the function of either protein is not compromised. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0143] The term “gene” used herein can be a genomic gene comprising transcriptional and / or translational regulatory sequences and / or a coding region and / or non-translated sequences (e.g. , introns, 5'- and 3 '-untranslated sequences and regulatory sequences). The coding region of a gene can be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene can also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and miRNA) optionally comprising 5'- or 3' untranslated sequences linked thereto. A gene can also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and / or 5'- or 3 '-untranslated sequences linked thereto.

[0144] The term “gene product(s)” as used herein refers to include RNA transcribed from a gene, or a polypeptide encoded by a gene or translated from RNA.

[0145] As used herein the term “secretory peptides” or “secretory sequences” or “secretion tags” or “signal peptides” or “export signals” and grammatical variations thereof means any peptide sequence that is capable of targeting the synthesized protein to the secretory pathway of a cell. In some embodiments of the above aspects and embodiments, the secretory peptide may be positioned on the N-terminal end of a recombinant protein, and may co-translationally or post-translationally target the tagged protein for secretion. In some embodiments of the above aspects and embodiments, the secretorypeptide is positioned at the C-terminal end of a recombinant protein. In some embodiments, an antimicrobial polypeptide is fused to a secretory peptide. In some embodiments, an antimicrobial polypeptide is fused to a secretory peptide and propeptide.

[0146] Secretion peptides: According to some embodiments, the therapeutic recombinant polypeptide(s) and / or metabolite(s), e.g., LEKTI domain, is operably linked to one or more secretion signals or export signals that tag the protein for transport through the secretory pathway. Any secretion signal that facilitates exit of the LEKTI protein out of the bacterial cell may be used as a secretion peptide. Non-limiting examples of secretion peptides signals are set forth in Table 1, below:TABLE 1: List of Secretion Peptides

[0147] According to some embodiments, the therapeutic LEKTI domain is operably linked to one or more signal sequences derived from endogenous proteins of .S', epidermidis. Non-limiting examples of secretion signal peptides derived from endogenous proteins of Staphylococcus epidermidis are set forth in Table 2 below:Table 2 : List of Staphylococcus epidermidis signal peptides

[0148] According to some embodiments, the recombinant therapeutic polypeptide, e.g., 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.

[0149] According to some embodiments, the recombinant therapeutic polypeptide, e.g., LEKTI domain, is operably linked to a cell penetration peptide sequence that enhances the ability of the recombinant therapeutic polypeptide to pass through a cell membrane. The term “enhance”, as used to describe the cell penetration peptide / recombinant therapeutic polypeptide, means that the cell penetration sequence improves the passage of recombinant therapeutic polypeptide through a cell membrane relative to the recombinant therapeutic polypeptide lacking the cell penetration sequence.

[0150] Cell penetration peptides: According to some embodiments, one or more cell penetrating peptides are used to mediate delivery of therapeutic proteins 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 therapeutic proteins to facilitate entry into skin cells e.g., keratinocytes). Non-limiting examples are set forth in Table 3, below:Table 3: List of Cell-penetration Peptides

[0151] According to some embodiments, cell penetrating peptides comprise periodic amino acid sequences. Non-limiting examples of periodic cell penetrating sequences include: Poly arginines, R x n (wherein 4<n<17); Polylysines, K x n (wherein 4<n<17); arginine repeats interspaced with 6- aminocaprotic acid residues (RAca), wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with 4-aminobutyric acid (RAbu), wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with methionine, wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with threonine, wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with serine, wherein there are 2 to 6 arginine repeats; and arginine repeats interspaced with alanine, wherein there are 2 to 6 arginine repeats.

[0152] According to some embodiments, a therapeutic recombinant polypeptide(s) and / or metabolite(s) is operably linked to a cell penetrating peptide, e.g., RMR domain (SEQ ID NO: 4).

[0153] According to some embodiments, the LEKTI domain is operably linked to an RMR domain (SEQ ID NO: 4).

[0154] According to some embodiments, expression of the recombinant therapeutic polypeptide, e.g. , LEKTI domain, is controlled by an operon and the amount of LEKTI provided to the mammal’ s skin is proportional to the availability of an extrinsic factor. 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 the cis-acting catabolite-responsive element (CRE), and the amount of recombinant LEKTI protein made available to the skin of the mammal controlled by the amount of exogenous xylose available to the recombinant microbe. According to some embodiments, the expression of the recombinant therapeutic polypeptide, e.g., LEKTI domain, is controlled by a promoter that is constitutively active. According to some embodiments, the expression of the recombinant therapeutic polypeptide, e.g., LEKTI domain is controlled by a CmR promoter.

[0155] According to some embodiments, the microbe is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding the recombinant therapeutic polypeptide, e.g., LEKTI protein domains, and one or more antibiotic resistance genes. For example, some embodiments of the recombinant DNA plasmid comprise a kanamycin resistance gene and / or a trimethoprim resistance gene; e.g., dfrA. According to some embodiments, treatment of the skin of the mammal with an antibiotic (for which the recombinant microbe is resistant) may be used to bias the population of commensal microbes toward a larger proportion of recombinant therapeutic polypeptide producing microbes. Other elements that may be present in the recombinant DNA plasmid include, without limitation, a replication protein gene, such as a member of the Rep superfamily of replication proteins. For example, in some embodiments the recombinant DNA plasmid comprises the repF gene.

[0156] The disclosure utilizes standard molecular biology techniques, e.g., those described in Sambrook et al. 2001. In some embodiments, the genetic construct used for this disclosure is based on a plasmid pBT-2, an allelic exchange shuttle vector between E. coli and Staphylococcal species (Nakanishi, Oshida et al. 1986). In some embodiments, the gene of the recombinant therapeutic polypeptide is inserted in the plasmid. In some embodiments, the coding sequence is driven by a promoter, e.g., inducible or constitutive. Examples of inducible promoters include those that are activated by chemical compounds such as alcohols, sugars, metals, or tetracycline, or by physical factors such as light or high temperatures.

[0157] According to some embodiments, the recombinant DNA plasmid comprises one or more sequences of the pUBTR vector. According to some embodiments, the recombinant EEKTI is operably linked to an inducible promoter, ribosome binding site, export signal, and / or cell penetrating peptide in the pUBTR vector. According to some embodiments, the recombinant LEKTI is operably linked to an inducible promoter, ribosome binding site, export signal, and / or cell penetrating peptide in the pUBTR vector. According to some embodiments, the pUBTR vector is pUBTRl 19.

[0158] According to some embodiments, the recombinant DNA plasmid comprises the pKK30- LEKTI-complete sequence. According to some embodiments, the present disclosure provides a composition for the treatment of a skin disease comprising a microbe comprising the pKK30-LEKTI- complete plasmid construct.

[0159] According to some embodiments, the amount or durations of availability of recombinant therapeutic polypeptide, e.g., LEKTI protein, is controlled by the stability of the vector harboring the recombinant therapeutic polypeptide in a microbe. For example, the persistence of a recombinant vector may be controlled by one or more elements of a plasmid including those that provide host-beneficial genes, plasmid stability mechanisms, and plasmid co-adaptation. For example, some plasmid may provide for stable replication, active partitioning mechanisms, and mechanisms that insure reliable inheritance of plasmids to daughter cells over generations. (See, e.g. , J.C. Baxter, B.E. Funnell, Plasmid partition mechanisms, Microbiol. Spectr., 2 (2014) PLAS-0023-2014 and Nils Hiilter et al., Anevolutionary perspective on plasmid lifestyle modes, Current Opinion in Microbiology, Volume 38, August 2017, Pages 74-80, each of which are incorporated by herein by reference in its entirety). According to some embodiments, the present disclosure includes the use of all conventional selection and stability methods known to a person of skill in the art.

[0160] Examples of proteins that can be administered according to the disclosure are mostly eukaryotic proteins. These can include, but are not limited to, single amino acids, small peptides, and large proteins. More particularly, genes encoding proteins that are useful in the disclosure as recombinant therapeutic proteins include, but are not limited to, the following gene: members of the interleukin family of genes, 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 receptor antagonists thereof; genes which encode 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, are also contemplated in the disclosure; genes encoding neurotropic factors, including but limited to, nerve growth factor, brain derived neurotropic factor and ciliary neurotropic factor; genes which encode interferons, including but not limited to, IFN- alpha, IFN-beta, and IFN-gamma. Further contemplated in the disclosure are genes encoding chemokines such as the C-C family and the C-X-C family of cytokines, genes encoding hormones, such as proinsulin and growth hormone, and genes encoding thrombolytic enzymes, including tissue plasminogen activator, streptokinase, urokinase or other enzymes such as trypsin inhibitor. The disclosure further includes genes which encode tissue repair factors, growth and regulatory factors such as, but not limited, oncostatin M, platelet-derived growth factors, fibroblast growth factors, epidermal growth factor, hepatocyte growth factor, bone morphogenic proteins, insulin-like growth factors, calcitonin and transforming growth factor alpha and beta. Further contemplated genes include genes encoding structural proteins such as filaggrin, actin, collagen, fibrillin, elastin, or scleroprotein. In some embodiments, the recombinant microorganism included genes encoding antimicrobial polypeptides (e.g., defensins, bacterial or fungal cell-wall hydrolases, elafin, hiracin, lysostaphin, LL-37, and epidermin, or a variant thereof, or a combination thereof.

[0161] The present disclosure also relates to allelic variants of LEKTI, or portions thereof (one or more of domains DI, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dl l, D12, D13, D14, and D15), as well as synthetic or mutated genes of SPINK (e.g. , SPINK5 ) that have been modified to change, for example, the expression or activity of the recombinant protein. It is also noted that degeneracy of the nucleic acid code can be considered variations in the nucleotide sequences that encode the same amino acid residues. Accordingly, the disclosure includes nucleic acid residues that are able to hybridize under moderately stringent conditions. One skilled in the art can determine effective combinations of salt and temperature to constitute a moderately stringent hybridization condition. It is also envisioned that orthologs of LEKTI are present in other species, for example, dog, sheep, rat, hamster, chicken and pig. Thereforein another embodiment of the present disclosure relates to SPINK (e.g., SPINKS) nucleic acids that encode polypeptides having at least about 70% to 80% identity, preferably 90% to 95% identity, more preferably 98% to 99% identity to LEKTI set forth in SEQ ID NO: 103 or portions thereof (one or more of domains DI, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, and D15). In a specific embodiment, the LEKTI domain is Domain 6.

[0162] According to some embodiments, the LEKTI domains are selected from the non-limiting examples below, Tables 4 and 5.Table 4: LEKTI Amino Acid SequencesTable 5: SPINK5 Nucleotide Sequence

[0163] As used herein, the term “auxotrophic” or “auxotrophy” refers to inability of an organism to synthesize a particular compound required for its growth. An auxotroph is an organism that displays this characteristic.

[0164] As used herein, the term "air A" and "air" refer to the D-alanine racemase gene, including normal alleles of the alrA gene. In some embodiments, the air gene from S. epidermidis (UniProtKB - Q8CNK7 (ALR_STAES) encodes a D-alanine racemase protein (EC 5.1.1.1). In some embodiments, the locus identifiers SE1674 ( lrl) and SE1079 (alr2) refer to specific .S', epidermidis D-alanine racemase genes.

[0165] As used herein, the term "dat" refers to the D-alanine aminotransferase gene, including normal alleles of the dat gene. In some embodiments, the dat gene from .S'. epidermidis (UniProtKB - Q8CS41 (DAAA_STAES)) encodes a D-alanine aminotransferase protein (EC:2.6.1.21). In some embodiments, the locus identifier SEI 423 dat) refers to a specific .S'. epidermidis D-alanine aminotransferase gene. As used herein, the term "murF refers to the glutamate racemase gene, including normal alleles of the murl gene. In some embodiments, the murl gene from .S', epidermidis (UniProtKB - Q8CPL0 (MURI_STAES)) encodes a glutamate racemase protein (EC:5.1.1.3). In some embodiments, the locus identifier SE0843 (murl) refers to a specific .S', epidermidis glutamate racemase gene.

[0166] D-alanine auxotrophs of .S', aureus have been produced for the purpose of producing 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 being incorporated by reference in its entirety herein). In this case, it was found necessary to knockout only the alanine racemase alrl and alr2, and also a dat gene.

[0167] According to some embodiments, the recombinant microbe is engineered to express a therapeutic polypeptide, a small molecule, or metabolite to treat, prevent, or ameliorate a disease, condition, or infection. In some embodiments, a composition comprises the engineered microbe. In some embodiments, the composition can treat, prevent, or ameliorate bacterial and other microbial (i.e. , viral) infections, oral cancers, CNS disease or injury, and non-infectious i.e., chronic rhinosinusitis) or infectious nasal, oral, or respiratory diseases.

[0168] According to some embodiments, the non-infectious and infectious diseases can include, but are not limited to, non-allergic rhinitis, vasomotor rhinitis, non-allergic rhinitic eosinophilic syndrome, rhinitis medicamentosa, atrophic rhinitis, pemphigus, benign mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, epidermolysis bullosa acquisita, 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.

[0169] According to another aspect, the present disclosure provides a kit for the treatment or amelioration of the effects of a bacterial and fungal infection, e.g., S. aureus, of a mammal in need thereof comprising: (1) a composition comprising a microbe that is genetically modified to express an antimicrobial polypeptide; and (2) reagents for applying the composition to the skin of the mammal. According to some embodiments, the microbes are adapted to live for a controlled duration on the surface of the mammal’s skin and to provide a controlled or continuous supply of the antimicrobial polypeptide.

[0170] According to another aspect, the present disclosure provides a kit for the treatment or amelioration of the effects of a disease, e.g., RSV, of a mammal in need thereof comprising: (1) a composition comprising a microbe that is genetically modified to express therapeutic agent, e.g. , LEKTI protein, or LEKTI protein domains, or variants thereof; and (2) reagents for applying the composition to the skin or nose of the mammal. According to some embodiments, the microbes are adapted to live for a controlled duration on the surface of the mammal’s skin and to provide a controlled or continuous supply of the antimicrobial polypeptide.

[0171] In some embodiments, the engineered microorganism is administered to a subject’s skin. In some embodiments, the engineered microorganism is administered to a subject that has a skin disorder. In some embodiments, the skin disorder is a skin disease. In some embodiments, the skin disease or disorder is a skin dysbiosis. Dysbiosis in the skin and / or gut microbiome is associated with an altered immune response, promoting the development of skin diseases. In some embodiments, the skin disease or disorder is a skin inflammatory disease or disorder. In some embodiments, the skin disease is a dermal toxicity due to one or more chemotherapeutics (including, but not limited to, Epidermal Growth Factor Receptor (EGFR) inhibitors, Bruton’s Tyrosine Kinase (BTK) inhibitors, RAS inhibitors, Mitogen- Activated Protein Kinases (MAPK) inhibitors, Extracellular signal-Regulated Kinases (ERK) inhibitors), In some embodiments, the skin disease is an ichthyoses. In some embodiments, the skin disease is Netherton syndrome. In some embodiments, the skin disease is hidradenitis suppurativa. In some embodiments, the skin disease is a psoriatic diseases (including, but not limited to, psoriasis, pustular psoriasis, plaque psoriasis, and palmoplantar psoriasis). In some embodiments, the skin disease is a neutrophilic dermatosis. In some embodiments, the skin disease is atopic dermatitis. In some embodiments, the skin disease is an autoimmune blistering disease. In some embodiments, the skin disease is acne and / or an acneiform eruption. 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.

[0172] The term “skin” (e.g., administered to the skin of a subject) as used herein refers to the outer layer of the body of a subject e.g., mammal or human).

[0173] As used herein, the term “skin dysbiosis” refers to a condition when the microbiome, for example of the skin, become imbalanced. As a result of skin dysbiosis, skin inflammatory disease and disorders can develop in a subject, such as atopic dermatitis, eczema, seborrheic dermatitis, psoriasis, acne vulgaris, dandruff, and even skin cancer. Atopic dermatitis can refer to a chronic inflammatory skin disorder.

[0174] Atopic dermatitis can be associated with scaly skin, itchy rashes, and lesions. Atopic dermatitis can also be referred to as atopic eczema or eczema. The hallmark of atopic dermatitis can be pruritus, which refers to an itching condition that leads to rashes. Any part of the skin can be affected by atopic dermatitis. Age can play a role in the localization of atopic dermatitis. For example, in infants, atopic dermatitis can affect cheeks, scalp, trunk, and extremities; in early childhood, atopic dermatitis can localize to flexural areas; and in adolescents and adults, atopic dermatitis can affect hands and feet.

[0175] In addition to the above components, the subject kits will further include instructions for use of the components and / or practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as a piece or pieces of paper on which the information is printed, in the packaging of the kit, or in a package insert. Yet another means would be a computer readable medium, such as diskette, or CD, on which the information has been recorded. Further, another means by which the instructions may be present is a website address used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

[0176] The components of the kits may be packaged either in aqueous media or in lyophilized form. The kits will generally be packaged to include at least one vial, test tube, flask, bottle, syringe or other container means, into which the described reagents may be placed, and preferably, suitably aliquoted. Where additional components are provided, the kit will also generally contain a second, third or other additional container into which such component may be placed.

[0177] The kits of the present disclosure will also typically include a means for containing the reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.Formulations

[0178] According to some embodiments the formulation for use according to the present disclosure can comprise any pharmaceutically effective amount of the recombinant bacteria to produce a therapeutically effective amount of the desired antimicrobial polypeptide or therapeutically effective domain(s) thereof, for example, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about0.3%, 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%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% or more by weight of recombinant bacteria, the upper limit of which is about 90.0% by weight of recombinant bacteria.

[0179] According to some embodiments, the formulation for use according to the present disclosure can comprise, 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 by weight of recombinant bacteria.

[0180] According to some embodiments, the topical formulation can be in any form suitable for application to the body surface, such as a cream, lotion, sprays, solution, gel, ointment, paste, plaster, paint, bioadhesive, suspensions, emulsions, or the like, and / or can be prepared so as to contain liposomes, micelles, and / or microspheres. Such a formulation can be used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation upon application to the body surface and thereafter. According to some embodiments, the formulation can include a living cell culture composition and can comprise at least one engineered bacterial strain that produces a therapeutically effective recombinant polypeptide or therapeutically effective domain(s) thereof. This engineered living cell culture composition can deliver the polypeptide directly to the skin for treating or preventing abnormal skin conditions.

[0181] Topical formulations include those in which any other active ingredient(s) is (are) dissolved or dispersed in a dermatological vehicle known in the art (e.g. aqueous or nonaqueous gels, ointments, water-in-oil or oil-in-water emulsions). Constituents of such vehicles can comprise water, aqueous buffer solutions, non-aqueous solvents (such as ethanol, isopropanol, benzyl alcohol, 2-(2- ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol or glycerol), oils (e.g. a mineral oil such as a liquid paraffin, natural or synthetic triglycerides such as Miglyol™, or silicone oils such as dimethicone). Depending, inter alia, upon the nature of the formulation as well as its intended use and site of application, the dermatological vehicle employed can contain one or more components (for example, when the formulation is an aqueous gel, components in addition to water) selected from the following list: a solubilizing agent or solvent (e.g. a P-cyclodextrin, such as bydroxypropyl [>- cyclodextrin, or an alcohol or polyol such as ethanol, propylene glycol or glycerol); a thickening agent (e.g. hydroxyethylceliulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer); a gelling agent (e.g. a polyoxyethylene-polyoxypropylene copolymer); a preservative (e.g. benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, a benzoate, potassium sorbate orEDTA or salt thereof); and pH buffering agent(s) (such as a mixture of dihydrogen phosphate and hydrogen phosphate salts, or a mixture of citric acid and a hydrogen phosphate salt).

[0182] A pharmaceutically acceptable carrier can also be incorporated in the formulation of the present disclosure and can be any carrier conventionally used in the art. Examples thereof include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water- based mixtures and emulsion-based mixtures of such carriers. The term "pharmaceutically acceptable" or "pharmaceutically acceptable carrier" is used herein to refer to a compound or composition that can be incorporated into a pharmaceutical formulation without causing undesirable biological effects or unwanted, interaction with other components of the formulation, "Carriers" or "vehicles" as used herein refer to carrier materials suitable for incorporation in a topically applied composition. Carriers and vehicles useful herein include any such materials known in the art, which are non-toxic and do not interact with other components of the formulation in which it is contained in a deleterious manner. The term "aqueous" refers to a formulation that contains water or that becomes water-containing following application to the skin or mucosal tissue.

[0183] A film former, when it dries, forms a protective film over the site of application. The film inhibits removal of the active ingredient and keeps it in contact with the site being treated. An example of a film former that is suitable for use in this disclosure is Flexible Collodion, US P. As described in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), at page 1530, collodions are ethyl ether / ethanol solutions containing pyroxylin (a nitrocellulose) that evaporate to leave a film of pyroxylin. A film former can act additionally as a carrier. Solutions that dry to form a film are sometimes referred to as paints. Creams, as is well known in the arts of pharmaceutical formulation, are viscous liquids or semisolid emulsions, either oil -in -water or water-in-oil.

[0184] Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

[0185] Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably, comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be uniformly spread.

[0186] Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl -cellulose, or the like.

[0187] Solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution can contain other pharmaceutically or cosmetically acceptable chemicals to buffer, stabilize or preserve the solute. Common examples of solvents used in preparing solutions are ethanol, water, propylene glycol or any other acceptable vehicles. As is of course well known, gels are semisolid, suspension-type systems. Single -phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol, and, optionally, an oil. Preferred organic macromolecules," i.e., gelling agents, are crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes that can be obtained commercially under the Carbopol trademark. Also preferred are hydrophilic polymers such as polyethylene oxides, polyoxyethylene -polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxy-propyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxy-propyl methylcellulose phthaiate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin, In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof. Ointments, as also well known in the art, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for a number of desirable characteristics, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating, and non-sensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases can be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.

[0188] 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.

[0189] Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in- water (O / W) emulsions, and include, for example, acetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; see Remington: The Science and Practice of Pharmacy for further information.

[0190] Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from singlephase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or thelike. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0191] Enhancers are those lipophilic co-enhancers typically referred to as "plasticizing" enhancers, i.e., enhancers that have a molecular weight in the range of about 150 to 1000, an aqueous solubility of less than about 1 wt.%, preferably less than about 0.5 wt.%, and most preferably less than about 0.2 wt.%. The Hildebrand solubility parameter 5 of plasticizing enhancers 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 fatty esters, fatty alcohols, and fatty ethers. Examples of specific and most preferred fatty acid esters include methyl laurate, ethyl oleate, propylene glycol nionolaurace, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers include compounds wherein a diol or triol, preferably a C2-C4 alkane diol or triol, are substituted with one or two fatty ether substituents.

[0192] Additional permeation enhancers will be known to those of ordinary skill in the art of topical drug delivery, and / or are described in the pertinent texts and literature. See, e.g., Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995) (incorporated herein by reference).

[0193] Various other additives can be included in the compositions of the present disclosure in addition to those identified above. These include, but are not limited to, antioxidants, astringents, perfumes, preservatives, emollients, pigments, dyes, humectants, propeliants, and sunscreen agents, as well as other classes of materials whose presence can be pharmaceutically or otherwise desirable. Typical examples of optional additives for inclusion in the formulations of the disclosure are as follows: preservatives such as sorbate; solvents such as isopropanol and propylene glycol; astringents such as menthol and ethanol; emollients such as poly alkylene methyl glucosides; humectants such as glycerine; emulsifiers such as glycerol stearate, PEG- 100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols such as polyethylene glycol; sunscreen agents such as octyl methoxyl cinnamate (available commercially as Parsol MCX) and butyl methoxy benzoylmethane (available under the tradename Parsol 1789); antioxidants such as ascorbic acid (vitamin C), a-tocopherol (Vitamin E), P-tocopherol, y-tocopherol, 5-tocopherol, 8-tocopherol, y - tocopherol, ZA-tocopherol, r| -tocopherol, and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soya bean oil, palm oil, liquid fraction of shea butter, sunflower oil), animal oils e.g., perhydrosqualene), synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (generally perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners and structurants such as swelling clays and cross-linked carboxypolyalkylenes that can be obtained commercially under the Carbopol trademark. Other additives include beneficial agents such as those materials that condition the skin (particularly, the upper layers of the skin in the stratum corneum) and keep it soft by retarding the decrease of its water content and / or protect the skin. Suchconditioners and moisturizing agents include, by way of example, pyrrolidine carboxylic acid and amino acids; organic antimicrobial agents such as 2,4,4'-trichloro-2 -hydroxy diphenyl ether (triclosan) and benzoic acid; anti- inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti- seborrhoeic agents such as retinoic acid; vasodilators such as nicotinic acid; inhibitors of melanogenesis such as kojic acid; and mixtures thereof. Further additional active agents including, for example, alpha hydroxyacids, alpha ketoacids, polymeric hydroxyacids, moisturizers, collagen, marine extract, and antioxidants such as ascorbic acid (Vitamin C), a-tocopherol (Vitamin E), P-tocopherol, y-tocopherol, 6- tocopherol, 8-tocopherol, y -tocopherol, ^-tocopherol, i] -tocopherol, and retinol (Vitamin A), and / or pharmaceutically acceptable salts, esters, amides, or other derivatives thereof. A preferred tocopherol compound is a-tocopherol. Additional agents include those that are capable of improving oxygen supply in skin tissue, as described, for example, in Gross, et al, WO 94 / 00098 and Gross, et al, WO 94 / 00109, both assigned to Lancaster Group AG (incorporated herein by reference). Sunscreens and UV absorbing compounds can also be included. Non-limiting examples of such sunscreens and UV absorbing compounds include aminobenzoic acid (PABA), avobenzone, cinoxate, dioxybenzone, homosalate, menthyl anthranilate, oxtocrylene, octyl methoxycmnamate, octyl salicylate, oxybenzone, padirnate O, phenylbenzirmdazole sulfonic acid, sulisobenzone, titanium dioxide, trolamine salicylate, zinc oxide, ensulizole, meradiraate, octinoxate, octisalate, and octocrylene. See Title 21. Chapter 1. Subchapter D. Part 352. "Sunscreen drug products for over-the-counter human use" incorporated herein in its entirety.

[0194] Other embodiments can include a variety of non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of the disclosure. Such healing materials can include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, glandular or animal extracts, or safe therapeutic agents that can be added to the formulation to facilitate the healing of dermal disorders.

[0195] The amounts of these various additives are those conventionally used in the cosmetics field, and range, for example, from about 0.01 % to about 20% of the total weight of the topical formulation.

[0196] The formulations of the disclosure can also include conventional additives such as opacifiers, fragrance, colorant, stabilizers, surfactants, and the like. In certain embodiments, other agents can also be added, such as antimicrobial agents, to prevent spoilage upon storage, i.e., to inhibit growth of microbes such as yeasts and molds.

[0197] Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof. In other embodiments, other agents can also be added, such as repressors and inducers, i.e., to inhibit (i.e. glycose) or induce (i.e. xylose) the production of the polypeptide of interest. Such additives can be employed provided they are compatible with and do not interfere with the function of the formulations.

[0198] The formulations can also contain irritation -mitigating additives to minimize or eliminate the possibility of skin irritation or skin damage resulting from the chemical entity to be administered, or other components of the composition.

[0199] Suitable irritation-mitigating additives include, for example: a-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-l -ethanol; glycerin; salicylates; ascorbates; ionophores such as monensin; amphophilic amines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. The irritation-mitigating additive, if present, can be incorporated into the compositions at a concentration effective to mitigate irritation or skin damage, typically representing not more than about 20 wt.%, more typically not more than about 5 wt.%, of the formulation.

[0200] A cream, lotion, gel, ointment, paste or the like can be spread on the affected surface and gently rubbed in. A solution can be applied in the same way, but more typically will be applied with a dropper, swab, or the like, and carefully applied to the affected areas.

[0201] The application regimen will depend on a number of factors that can readily be determined, such as the severity of the condition and its responsiveness to initial treatment, but will normally involve one or more applications per day on an ongoing basis. One of ordinary skill can readily determine the optimum amount of the formulation to be administered, administration methodologies and repetition rates. In general, it is contemplated that the formulations of the disclosure will be applied in the range of once or twice weekly up to once or twice daily.

[0202] The pharmaceutical compositions of the disclosure comprise one or more active ingredients, e.g. therapeutic agents, in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and / or materials. Regardless of the route of administration selected, the agents / compounds of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0203] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, ectoine, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, oleyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and try glycerides), biodegradable polymers (e.g., polylactide-poly glycolide, poly(orthoesters), and poly( anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor,sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the disclosure must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0204] The pharmaceutical compositions of the disclosure 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 extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surfaceactive agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with theother ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

[0205] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s) / compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0206] The pharmaceutical compositions of the present disclosure suitable for parenteral administrations may comprise one or more agent(s) / compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0207] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.EXAMPLES

[0208] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.EXAMPLE 1Phenotypic characterization of the secreted antimicrobial activities of SE25SE25 was a Screening Positive from the “Micromyx-50 Library”

[0209] Analysis of a Micromyx library of .S', epidermidis strains for antimicrobial activity, specifically anti -.S'. aureus ATCC 29213 activity, revealed .S'. epidermidis strain SE25 (FIGs. 1A and IB).Micromyx library was acquired by obtaining samples from the forearms of human subjects. Samples were cultures on a selective plate for .S', epidermidis and grown in TSB for later study. Zone of inhibition indicated that SE25 was able to inhibit growth of .S'. aureus when .S'. aureus was overlaid on SE25 strain streaked and grown for 48 hours on TSA plate. General experimental procedure included, the library of .S. epidermidis strains were inoculated in tryptic soy broth (TSB) and grown at 37 °C for one day. On day 2, the strains were copied onto a TSA plate and grown overnight at 37 °C and then at room temperature overnight. The library of .S'. epidermidis strains were overlaid by an indicator strain, .S'. aureus and grown at room temperature from day 4 to day 5.

[0210] Further analysis revealed that SE25 has antimicrobial activity against other Gram-positive bacteria, but not Gram-negative bacteria (FIG. 2). SE25 was overlaid by Gram-positive bacteria .S'. aureus SA25923, -S’. aureus SA29213, MRSA USA300, -S’. epidermidis NRRL B-4268, B. subtilis, and Gram-negative strains P. aeruginosa and E. coli (FIG. 2). The below Table 6 summarizes SE25 antimicrobial activity against various pathogenic bacteria.Table 6: Antimicrobial Activates of SE25 Strains Against Bacteria, as IndicatedSE25 Cell-free Supernatant has bactericidal activity

[0211] Anti-5, aureus activity of SE25 is secreted. 5. aureus ATCC 29213 was grown in either conditioned growth media from SE25 culture, conditioned growth media from SE3 culture (negative control), and unconditioned culture media (FIG. 3). Conditioned media from SE25 culture inhibited 5. aureus ATCC 29213 growth. On the other hand, neither conditioned media from SE3 culture nor unconditioned media inhibited 5. aureus ATCC 29213 growth. Cell free conditioned media were prepared by growing 5. epidermidis cultures in tryptic soy broth (TSB) for 24hr at 30°C while shaking at 250 RPM. Conditioned media, also known as cell-free supernatant (CFS), were sterilized through filtration (PES, 0.22pm membrane) and pH was adjusted to 6.5-7.5. Anti 5. aureus activity assays were performed by mixing 90 pL CFS in 96 well microtiter plate with 10 uL of approximately 5 x 105CFU of indicator strain, e.g., S. aureus. The indicator strain was prepared in fresh TSB. Assay negative controls included fresh TSB media and CFS with no bacteria added. The cultures were grown overnight at 37 °C and 5. aureus bacterial growth was enumerated by following plating for CFU counting.

[0212] Spectrum of activity of SE25 CFS were assayed against 5. aureus ATCC 25923, 5. epidermidis NRRE B-4268, 5. epidermidis 1457, 5. epidermidis SE25, B. subtilis, E.faecalis, E. coli, P. aeruginosa, and Ichthyosis CoNS isolates, 5. lugdunensis, S. auricularis, S. capitis, S. haemolyticus , 5. hominis, S. pettenkoferi, S. saprophyticus, and 5. wameri. SE25 CFS had inhibitory activity against all strains tested except for E. faecalis, E. coli , and P. aeruginosa.

[0213] Bactericidal activity is defined as a decrease in CFU counts by 3-logio of an indicator strain within 24 hours growth.. . Bacteriostatic activity inhibits growth to within + / -1 logio CFU of starting CFUs within 24 hours. FIG. 5A shows that SE25 has bactericidal activity against 5. aureus ATCC 29213. SE25 cell-free supernatant reduces bacterial load of 5. aureus from the starting inoculation by approximately 7-logio (FIG. 5A). Similarly, known antimicrobial agents, levofloxacin and vancomycin, reduced CFU of 5. aureus approximately 6.75-logio-SE25 Does Not Produce Biofilm on Plastic and Disperses S. aureus Biofilms

[0214] Strains were tested for biofilm formation activity and 5. aureus biofilm dispersal activity. Strains SE25, 5. epidermidis NRRE B-4268, and 5. epidermidis 1457 were incubated in polystyrene plastic wells to allow adherence(FIG. 6A). Unbound and loosely bound bacterial cells were removed and the wells were washed. Crystal violet solution was used to stain attached cells that adhered to the polysterine plastic well and created a biofilm. 5. epidermidis 1457, the positive control, showed high biofilm formation by crystal violet staining. SE25 strain cells did not form biofilm, as evidenced by the lack of crystal violet staining. 5. epidermidis NRRL B-4268 strain was also negative for biofilm formation (FIG. 6A). This result was confirmed with Conga Red assay where SE25 strain grew as red colonies (negative for biofilm formation) (FIG. 6B).

[0215] SE25 strain showed activity to disperse 5. aureus biofilms (FIG. 6C). First, 5. aureus cultures were allowed to adhere and form biofilms in plastic wells. Then SE25 strain culture was added in aserial dilution. Untreated .S', aureus cells remained in a biofilm, while the .S'. aureus biofilms were dispersed after treatment with a dilution of SE25 (FIG. 6C).SE25 is Sensitive to Anti-staphylococcal Antibiotics

[0216] Sensitivity of SE25 and SE-27a strains were tested with different antibiotics to determine MIC as shown in Table 7, below.

[0217] Table 7: MIC of Antibiotic Resistance Assay on SE25 and NRRL B-4268 Derivative Strains* There is no clinical resistance breakpoint for mupirocin, but SE25 strain was confirmed to carry resistance gene mupA on plasmid that can be resistant to over 500 pg / ml.Reconstructed Human Epidermis

[0218] Reconstructed human epidermis (RHE) experimental procedure includes inoculating SE on reconstructed human epidermis tissue, prepared from cultured keratinocytes on an inert polycarbonate medium. After inoculation, SE was incubated for 24 hours or 48 hours (FIG. 7). SE colonized RHE at 103, 105, and 107inoculation concentration.

[0219] RHE competition assay was performed with inoculating 105SE cells on the RHE and incubating for 48 hours prior to the addition of 103-S', aureus cells (FIG. 8). Colonization of SE25reduced SA29213 by 4-log (FIG. 8). After an additional 24 hours, the RHEs are rinsed to remove unattached bacteria, and .S', aureus enumerated by CFU plating.

[0220] SE25 induced antimicrobial peptide production (AMP) in RHE after 72 hours inoculation(FIG. 9). AMP is known to have .S'. aureus activity. SE25 and NRRL induced similar a host response.EXAMPLE 2Whole genome sequence (WGS) analysis of SE25 strain, and annotation of naturally occurring plasmids in the strain

[0221] Analysis of genomic sequences of the SE25 strain revealed presence of four plasmids p36.326kb (“p36.3”, with multiple variants possible), p43.056kb (“p43.1”), p24.273kb (“p24.3”) and p 9.793kb (“p9.8”) and chromosome of 2.402255 Mb size. A determinant of high-level resistance to mupirocin was found on plasmid p24.3, which has mupA gene encoding MupA protein (ileS2, Isoleucyl- tRNA synthetase, EC 6.1.1.5). this plasmid encoded gene can determine resistance up to over 64 pg / ml.

[0222] Subsystem features identified in the plasmids are as follows: cofactors, vitamins, prosthetic groups, pigments (97); cell wall and capsule (36); virulence, disease, and defenses (43); potassium metabolism (3); photosynthesis (0); miscellaneous (10); phages, prophages, transposable elements, plasmids (13); membrane transport (26); iron acquisition and metabolism (24); RNA metabolism (36); nucleosides and nucleotide (78); protein metabolism (157); cell division and cell cycle (5); motility and chemotaxis (0); regulation and cell signaling (28); secondary metabolism (5); DNa metabolism (54); fatty, lipids, and isoprenoids (45); nitrogen metabolism (20); dormancy and sporulation (9); respiration (19); stress response (36); metabolism of aromatic compounds (3); amino acids and derivatives (224); sulfur metabolism (5); phosphorous metabolism (13); and carbohydrates (189).

[0223] See Tables 8-11 for summary of annotation of the ORFs in SE25 plasmids.Table 8 Features on SE25-p36.326kb plasmid: Epidermin BGC genes are in bold.Table 9. Features on SE25-p43.056kb plasmid "p43.1"Table 10. Features on SE25-p24.273kb plasmid "p24.3". MupA gene is in bold.Table 11. Features on SE25-p9.793kb plasmid "pl9.4"

[0224] Mupirocin-resistance gene, biosynthetic gene cluster for epidermin lanthipeptide (FIG. 10), siderophore, prophage (FIG. 11) and the absence of the ica operon were identified by genomic analysis of SE25 strain.EXAMPLE 3Engineering of S. epidermidis SE25 strain

[0225] SE25 strain was selected as a prospective strain for further modification(s) based on overall positive results of phenotypic testing, and particularly for its bactericidal activities against 5. aureus on agar, reconstructed human epidermis (RHE), and in liquid media, and for its amenability to genetic manipulation.

[0226] The SE25 strain was engineered to be auxotrophic for D-alanine with either two (AalrlAdaf) or three gene deletions (Aah'l Aa!r2Adat). plus additional deletions of a mupirocin resistance gene ( mupA) and a prophage gene (Acapsid).Engineering of D-alanine auxotrophy.

[0227] To engineer the D-alanine auxotroph of SE25 strain, the chromosomal regions flanking the alrl, alr2 and dat genes were analyzed for homology to the .S', epidermidis NRRL B-4268. With few single nucleotide polymorphisms, the same knockout plasmid constructs that were used for engineering the D- alanine auxotrophic strain .S'. epidermidis SEAAA were found to be suitable for SE25 auxotroph engineering. Primer sequences, their specific uses and PCR product sizes are listed in Table 12 as shown below.Table 12. Primers for knockout of D-alanine aminotransferase (SE1423)• Overlapping PCR using primers 1423-5F / 1423-3R: 1.5 Kb• PCR product from wild type using primers 1423-5F / 1423-3R: 2.3 Kb• F: forward primer• R: reverse primer• Added restriction sites for cloning are shown in underlined bold face letters

[0228] In a 2018 study from Moscoso et al. (Moscoso, Garcia et al. 2018) describing the construction of 5. aureus D-alanine auxotrophic strains, it was reported that “...the double AdatAalrl and triple AdatAalrlAalr2 mutants required exogenous D-alanine for growth.” Similar to what was done for .S'. aureus, two different SE25 auxotrophic strains were constructed in parallel by consecutive deletions of either the alrl, alr2 and dat genes (a “triple mutant”), or alrl and dat genes (a “double mutant”). Both knockout variants were engineered and screened for the ability to grow on agar plates with and without D-alanine, and retested in liquid media with and without D-alanine supplementation (FIG. 12). Both variants required D-alanine at the same concentration (100 pg / mL) as required by the .S'. epidermidis SEAAA-

[0229] pJB-1423KO plasmid isolated from dam- / dcm- E.coli strain (NEB) was transformed into competent cells of SE25AalrlAalr2 using plates of TSA + chloramphenicol (10 pg / mL). The presence of the pJB-1423KO plasmid in transformants was confirmed by detecting the PCR product of 1.5 Kb using primers 1423-5F (EcoRI) and 1423-3R (SalGI). PCR product of 1.5 Kb was observed, while a PCR product of 2.3 Kb was observed in a reaction containing cell lysate from the wild-type SE host cells. Cells of two confirmed clones were streaked on fresh plates of TSA + Cm (10 pg / mL) + D-alanine (100 pg / mL). Plates were incubated at 43 °C for 48 hr for plasmid integration via homologous recombination. Isolated colonies were streaked again for purification at 43 °C and 48 hr growth. Four isolated colonies were inoculated into 3 mL TSB + D-alanine (100 pg / mL) in a 15-mL tube in order to loop out the plasmid backbone via a second round of homologous recombination. The cultures were shaken at 30 °C for overnight. An aliquot of 50ul culture was transferred to 15 ml tube containing 3 mL fresh TSB, DA100 medium.. Cells from first and second 30C cultures were plated on TSA + Anhydrotetracyclne (ATC 2 pg / mL) + D-alanine (DA, 100 pg / mL). After 2 days of incubation at 30 °C, about 100-200 colonies were formed on plates plated.

[0230] S 25AalrlAalr2Adat (6 colonies) and SE25 A alrl A dat (14 colonies) were screened out for Cm- sensitivity on CmlO TSA plates and verified (FIGs. 13A and 13B). SE25AalrlAdat (FIG. 14A) S 25AalrlAalr2Adat (FIG. 14B) and retained anti -.S', aureus activity.Engineering of mupirocin-sensitivity.

[0231] The 3063 bp out of 3072-bp ORF of mupA gene on plasmid p24.3, conferring resistance to the topical skin antibiotic mupirocin, was deleted in three strain variants (the wild-type strain and bothauxotrophs), in order to engineer sensitivity to mupirocin in resulted three strains: SE25 AmupA, SE25 Aalrl Adat AmupA and SE25 Aalrl Aalr2 Adat AmupA.

[0232] To delete part of mupA ORF, flanking regions of 968 bp and 966 bp were PCR amplified and assembled into shuttle pJB38 plasmid that has a temperature-sensitive gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5a maintenance strain. PCR-confirmed plasmid DNA was purified and sequenced to verify correct assembly and absence of mutations. A plasmid with the confirmed expected sequence was transformed into methylation-deficient E. coli strain NEB (dam- / dcm-). Plasmid DNA was purified and retransformed in SE25 strains by electroporation. The standard allelic exchange procedure on chloramphicol-resistant colonies was followed with screening for positive AmupA mutants on TSA plates with or without mupirocin (FIGs. 15A and 15B).Deletion of prophage capsid gene

[0233] A putative prophage was identified on the chromosome with a size of 41 ,881 bp, encoding over 50 structural, replication and lysis genes and the duplicated sites of integration in 3’ end of ORF for Iron-sulfur cluster assembly protein SufB gene. The putative prophage gene coding for the structural capsid protein was deleted in all three AmupA derivative strains, as a preventative measure to inactivate the potential release of mature lytic phage particles. The putative prophage is structurally similar to 5. aureus phage phi 11, Siphoviridae Staphylococcus phage phiMR25, Siphoviridae Staphylococcus phage phiMRl l, Siphoviridae Staphylococcus phage phiSauS-IPLA88, Bacteriophage 92, Bacteriophage 88, 29, Bacteriophage 55, and Siphoviridae Staphylococcus aureus phage phiNM2.

[0234] To delete 1338 bp out of 1353-bp capsid ORF, flanking regions of 1004 bp and 958 bp were PCR amplified and assembled into shuttle pJB38 plasmid that has temperature -sensitive gram-positive origin of replication. Assembled recombinant plasmid was transformed into E. coli host NEB 5a maintenance strain. PCR-confirmed plasmid DNAs were purified and sequenced to verify correct assembly and absence of mutations. A plasmid with confirmed expected sequence was transformed in methylation-deficient E. coli strain NEBa (dam- / dcm-). purified and re-transformed into the SE25 strains by electroporation. The standard allelic exchange procedure on chloramphenicol-resistant colonies was followed with screening for positive Acapsid mutants by PCR.Confirmation of anti-staphylococcal activities of engineered strains

[0235] Resulting strains SE25 AmupAAcapsid, SE25 AmupAAcapsid AalrlAdat, and SE25 AmupAAcapsid Aalrl Aalr2Adat were assayed for the retention of antimicrobial activities against .S'. aureus in agar overlay assays (FIG. 15).Identification of a plasmid-borne biosynthetic gene cluster encoding biosynthetic genes for an epidermin lantibiotic

[0236] The gene annotation of the p36.3 plasmid revealed presence of the biosynthetic gene cluster (BGC) involved in synthesis of an epidermin lantibiotic peptide. The gene cluster has 100% identity to the reported epidermin biosynthetic gene cluster. The BGC of about 15 kb was 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 (FIG. 10). In order to disrupt the lantibiotic production in SE25, the ORF of the epiA gene encoding 52 amino acid prepropeptide (MEAVKEKNDL FNLDVKVNAK ESNDSGAEPR IASKFICTPG CAKTGSFNS YCC*) was truncated to 6 amino acids (including MEA-YCC*) in the wild-type SE25 strain to confirm the function of the gene by the lost-of-function deletion.

[0237] To delete part of epiA ORF, flanking regions of 994 bp and 974 bp were PCR-amplified and assembled into shuttle pJB38 plasmid that has temperature -sensitive gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E.coli host NEB 5a maintenance strain. Transformants were analyzed by PCR and -confirmed plasmid DNAs purified from positive clones were sequenced to verify correct assembly and absence of mutations. A plasmid with the confirmed expected sequence was transformed into methylation-deficient E. coli strain NEB (dam- / dcm-). purified and re -transformed into SE25 strains by electroporation. The standard allelic exchange procedure on chloramphenicol-resistant colonies was followed by PCR screening for AepiA mutant colonies and a final .S', aureus overlay assay on positive AepiA mutants and wild-type strains (FIGs. 16A and 16B).

[0238] Results showed that in a 5. aureus agar overlay assay, SE25 AepiA mutant strains exhibited a loss / significant reduction in zones of growth inhibition, confirming a role for epiA in conferring anti-5. aureus activity (FIG. 17).

[0239] The reduction of anti-SA activity in AepiA mutant strains were also confirmed by liquid culture assays (not shown).EXAMPLE 4Table 13. Exemplary sequencesEXAMPLE 5Creation of S. epidermidis SE484 strain

[0240] To construct the .S', epidermidis strain SE484 the 3 D-alanine biosynthesis genes, namely, alanine racemasel (alrl), alanine racenase2 (alr2) and D-alanine aminotransferase (dat) were first deleted to generate the strain SE464, a D-alanine auxotroph that does not grow in media without D- alanine supplementation. Subsequent deletions of the mupirocin resistance gene (mupA also known as iles2), and a prophage capsid gene from SE464 created the SE484 strain.Engineering of D-alanine auxotrophy

[0241] As described in Example 3, each of the 3 D-alanine biosynthesis genes and their flanking DNA sequences were cloned individually into a temperature-sensitive knockout plasmid. These plasmids were subsequently transformed into the parent .S', epidermidis strain SE25. The resulting colonies were analyzed by PCR to verify integration of the plasmid at the target locus. Next, a second recombination was performed to remove the plasmid from the genome, following which colonies were screened for excision of the plasmid from the genome, and its subsequent loss during growth in the absence of chloramphenicol selection. Phenotypes of potential candidates were screened by growth on tryptic soy agar (TSA) with D-alanine and / or chloramphenicol (FIG. 19A - 19C). Candidate colonies were first streaked onto TSA in the presence of 100 pg / ml D-alanine without chloramphenicol (FIG. 19A). Next, the same colonies were streaked onto TSA supplemented with 10 pg / ml chloramphenicol in the absence of D-alanine (FIG. 19B). Candidate colonies that required D- alanine supplementation for growth were selected (FIG. 19C). To verify successful deletions, flanking regions of about two kilobases around the alrl , alr2 and dat deletions were amplified by PCR and sequenced. All deletions were confirmed by sequencing, and deletion of the 3 D-alanine biosynthetic genes resulted in strain SE464, a D-alanine auxotroph that does not grow in media without D-alanine supplementation.Engineering of mupirocin-sensitivity, and deletion of prophage capsid gene

[0242] To create the SE484 strain from the SE464 strain, 2 subsequent genes were deleted, namely, mupA (also referred to as ilse2), and the gene encoding the major capsid protein of a putative prophage identified by the PhiSPY algorithm during whole genome sequencing (WGS). Deletions were performed using the same allelic exchange strategy used for the deletion of the alanine racemase genes. Transformants were screened for colonies that were mupirocin-sensitive, as well as D-alanine auxotrophs.EXAMPLE 6Characterization of S. epidermidis SE484 strain

[0243] Whole genome sequencing of the SE484 strain confirmed that it was identical to the parent strain SE25, except for the intended deletions.Growth profile of SE484 strain

[0244] The growth profile of the SE484 strain at 30°C in TSB was observed to be comparable to that of the parent strain SE25, thus confirming that the modifications compared to the parent did not affect the physiology of SE484. Growth kinetics of both strains are compared in the plot shown in FIG.20A. Next, a zone of inhibition plate assay was performed to compare the activity of both strains against .S', aureus. Here, SE25 and SE484 were each streaked onto TSA supplemented with 100 pg / ml D-alanine, and incubated at 37°C. After 48 hours of incubation, soft agar containing .S'. aureus was poured over both plates, which were subsequently incubated at 37°C for an additional 24 hours. Both .S. epidermidis strains showed comparable inhibition of .S'. aureus growth (FIG. 20B - 20C). These results indicated that the modifications of the 5 target genes that resulted in strain SE484 did not affect its anti-S. aureus properties.Phenotypic and genotypic stability of SE484

[0245] The phenotypic and genotypic stability of SE484 was evaluated over 5 days with daily subculturing. To verify the phenotypic stability, daily passages of SE484 were assessed for D-alanine auxotrophy verification, mupirocin sensitivity, growth rates, CFU counts, and antimicrobial activity against .S', aureus. To verify genotypic stability, amplification of the air / , alr2, dat, and epiA genes was performed by PCR on samples obtained from passage 1 and passage 5 of SE484 cultures.

[0246] Various phenotypic and genotypic characteristics of SE484 were evaluated over time across 5 passages and 179 generations. SE484 was streaked onto either TSA, TSA with 100 pg / ml D-alanine, or TSA with D- alanine and 20 pg / ml of mupirocin, and plates were incubated at 37 °C for 48 hours. As shown in FIG. 20D, the growth profile and gross colony morphology remained unchanged from passage 1 though passage 5, with SE484 cultures growing only when supplemented with D-alanine at both time points, thus verifying the maintenance of the D-alanine auxotrophy phenotype. In addition, Mupirocin sensitivity was also verified through passage 5 and found to be present in all passages.

[0247] To assess the stability of the anti -.S', aureus properties of SE484 after serial passaging, glycerol stocks prepared and collected at each passage were plated and overlayed with .S', aureus. The results showed no loss of anti- .S', aureus activity, since clearance of .S', aureus was observed near the heavy streak of SE484 at all 5 passages. Furthermore, the pattern of zone of clearance observed was consistent from passage 1 through to passage 5, indicating that this antimicrobial activity of SE484 is stable for at least 5 passages.EXAMPLE 7S. epidermidis SE484 strain inhibits S. aureus growth

[0248] The anti -.S', aureus activity of strains SE484 and SEI 23 (SEAAA) were quantified and compared. A prophylactic approach was adopted for the measurements, wherein reconstituted human epidermis (RHE) was inoculated with either SE123 or SE484 and incubated for 4 hours in a tissue culture incubator at 37°C, 5% CO2. Next, the RHEs were challenged with ~104CFU of .S'. aureus MRSA strain USA300. Following an additional 24 hour incubation, the USA300 cell count was determined by punch biopsy and dilution plating. The results revealed that SEI 23 showed no significant ability to inhibit USA300 growth (FIG. 21A), whereas SE484 inhibited USA300 growth by approximately 3 logs after the 24 hour incubation (FIG. 21B).

[0249] These results indicated that the .S'. epidermidis SE484 has anti .S', aureus activity whereas SE123 (SEAAA) does not.INCORPORATION BY REFERENCE

[0250] The entire disclosure of each of the patent documents, including patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls. All sequence listings, or Seq. ID. Numbers, disclosed herein are incorporated herein in their entirety.

[0251] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0252] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.

Claims

CLAIMSWhat is claimed is:

1. 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage gene is inactive.

2. The recombinant microorganism of claim 1 , wherein the one or more naturally occurring antibiotic resistance gene is selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a co-trimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

3. The recombinant microorganism of claim 1 or claim 2, wherein the one or more naturally occurring antibiotic resistance gene is the mupirocin resistance gene (mupA).

4. The recombinant microorganism of any one of claims 1-3, wherein the one or more naturally occurring lysogenic bacteriophage gene encodes a bacteriophage capsid protein.

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

6. The recombinant microorganism of any one of claim 1-5, wherein the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2.

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

8. The recombinant microorganism of claim 7, wherein the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or variants thereof, a gene encoding an antimicrobial biosynthesis enzyme or variants thereof, a gene encoding an enzyme or variants thereof, a gene encoding an enzyme inhibitor or variants thereof, a gene encoding an antigen or variants thereof, and a gene encoding an immune modulating polypeptide or variants thereof, or a combination thereof.

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

10. The recombinant microorganism of claim 9, wherein the one or more microbial pathogens are selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

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

12. The recombinant microorganism of claim 10, wherein the fungal infection is caused by a fungus selected from the group consisting of 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 the group consisting of respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses.

14. The recombinant microorganism of any one of claims 8-13, wherein the antimicrobial polypeptide is selected from the group consisting of an epidermin-like lantibiotic, a YM-1, a YM-2, an acidic mammalian chitinase (AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilage glycoprotein-39, a chondrocyte protein 39, an endoglucanase, an exoglucanase, an P-glucosidase, a cellobiohydrolase, an endo-l,4-P-xylanase, a P-xylosidase, an a- glucuronidase, an a-L-arabinofuranosidase, an acetylesterase, an acetylxylanesterase, an a-amylase, a P-amylase, a glucoamylase, a pullulanase, a P-glucanase, a hemicellulase, an arabinosidase, a mannanase, a pectin hydrolase, a pectate lyase, a lysostaphin, zoocin A, a millericin B, a muraminidase Cpl-1, a lysozyme, an endolysin PlyC, an endolysin, a PlyV12, an enterolysin A, anautolysin of C. difficile (Acd), an autolysin (LytA), an PL-1 amidase hydrolase, a Nisin A, a Nisin Z, a Subtilin, an Epidermin, a Gallidermin, a Mutacin B-a Ny266, a Mutacin 1140, a Pep5, an Epicidin 280, an Epilancin K7, a Lacticin 481, a Cytolysin, a Lacticin 3147, a Staphylococcin C55, a Salvaricin A, a Lactocin S, a Streptococcin A-FF2, a Sublancin 168, a Carnocin U149, a Variacin 8, a Cypemycin, a Cinnamycin, a Duramycin, an Ancovenin, a Mersacidin, and an Actagardine.

15. The recombinant microorganism of any one of claims 1-6, wherein the one or more heterologous genes encodes 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 biosynthesis enzyme or variants thereof, wherein the antimicrobial biosynthesis enzyme is capable of producing 6-N-hydroxy aminopurine (6-HAP), and wherein 6-HAP is secreted.

17. The recombinant microorganism of claim 8, wherein the gene encoding an immune modulating polypeptide or variants thereof is a gene encoding a lipoteichoic acid (LT A) biosynthesis enzyme, and wherein the recombinant microorganism is capable of producing LT A and secreting LTA.

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

19. The recombinant microorganism of any one of the preceding claims, wherein the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or combinations thereof.

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

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

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

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

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

25. The pharmaceutical composition of any one of claims 22-24, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, or an ointment.

26. A method of treating a disease, disorder, or condition in 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 bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage 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 living cell culture composition.

29. The method of any one of claims 26-28, wherein the one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a co-trimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

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

31. The method of any one of claims 26-30, wherein the one or more naturally occurring lysogenic bacteriophage genes encode a bacteriophage capsid protein.

32. The method of any one of claims 26-31 , wherein the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2.

33. The method of 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 the group consisting of a gene encoding an antimicrobial polypeptide or variants thereof, a gene encoding an antimicrobial biosynthesis enzyme or variants thereof, a gene encoding an enzyme or variants thereof, a gene encoding an enzyme inhibitor or variants thereof, a gene encoding an antigen or variants thereof, and a gene encoding an immune modulating polypeptide or variants thereof, or a combination thereof.

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

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

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

38. The method of claim 36, wherein the fungal infection is caused by a fungus selected from the group consisting of Malassezia spp., Candida spp., Aspergillus, Cryptococcus, and Pneumocystis.

39. The method of claim 36, wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses and rhinoviruses.

40. The method of any one of claims 34-39, wherein the antimicrobial polypeptide is selected from the group consisting of an epidermin-like lantibiotic, a YM-1, a YM-2, an acidic mammalianchitinase (AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilage glycoprotein-39, a chondrocyte protein 39, an endoglucanase, an exoglucanase, an - glucosidase, a cellobiohydrolase, an endo-l,4- -xylanase, a -xylosidase, an a-glucuronidase, an a-L- arabinofuranosidase, an acetylesterase, an acetylxylanesterase, an a-amylase, a -amylase, a glucoamylase, a pullulanase, a -glucanase, a hemicellulase, an arabinosidase, a mannanase, a pectin hydrolase, a pectate lyase, a lysostaphin, zoocin A, a millericin B, a muraminidase Cpl-1, a lysozyme, an endolysin PlyC, an endolysin, a PlyV12, an enterolysin A, an autolysin of C. difficile (Acd), an autolysin (LytA), an PL-1 amidase hydrolase, a Nisin A, a Nisin Z, a Subtilin, an Epidermin, a Gallidermin, a Mutacin B-a Ny266, a Mutacin 1140, a Pep5, an Epicidin 280, an Epilancin K7, a Lacticin 481, a Cytolysin, a Lacticin 3147, a Staphylococcin C55, a Salvaricin A, a Lactocin S, a Streptococcin A-FF2, a Sublancin 168, a Carnocin U149, a Variacin 8, a Cypemycin, a Cinnamycin, a Duramycin, an Ancovenin, a Mersacidin, and an Actagardine.

41. The method of 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 biosynthesis enzyme or variants thereof, wherein the antimicrobial biosynthesis enzyme is capable of producing 6-N-hydroxy aminopurine (6-HAP), and wherein 6-HAP is secreted.

43. The method of claim 34, wherein the gene encoding an immune modulating polypeptide or variants thereof is a gene encoding a lipoteichoic acid (LT A) biosynthesis enzyme, and wherein the recombinant microorganism is capable of producing LTA and secreting LTA.

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

45. The method of any one of claims 26-44, wherein the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or combinations thereof.

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

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

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

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

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

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

52. The method of any one of claims 26-51 , wherein the disease, disorder, or condition is selected from the group consisting of: a microbial infection, a skin disease or disorder, an inflammatory disease or disorder, and a metabolism disease or disorder.

53. The method of claim 51, wherein the microbial infection comprises one or more microbial pathogens selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacteria spp., Mycoplasma, Malassezia spp., Candida spp., Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial viruses (RSV), influenza viruses, parainfluenza viruses, adenoviruses, and rhinoviruses or combinations thereof.

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

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

56. The method of any one of claims 25-51, wherein the skin disease or disorder is a dermal toxicity.

57. The method of any one of claims 25-51, wherein the skin disease or disorder is an ichthyosis.

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

59. The method of any one of claims 25-51, wherein the skin disease or disorder is a dermatitis.

60. The method of any one of claims 25-51, wherein the skin disease or disorder is an autoimmune blistering disease.

61. The method of any one of claims 25-51, wherein the skin disease or disorder is selected from the group consisting of: Netherton 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 process comprising: selecting a bacterial strain; carrying out in the following order: (1) a deletion or substitution in one or more genes encoding a D- alanine biosynthesis gene, wherein the D-alanine biosynthesis gene is inactive; (2) 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 (3) a deletion or substitution in one or more genes encoding a naturally occurring lysogenic bacteriophage gene, wherein the naturally occurring lysogenic bacteriophage gene is inactive, thereby preparing a recombinant D- Alanine auxotrophic bacterial strain.

63. The recombinant D-Alanine auxotrophic bacterial strain of claim 62, wherein the deletion or substitution in one or more genes encoding a D-alanine biosynthesis gene comprises a deletion in dat, alrl and alr2.

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