Methods and compositions for the prevention of microbial infection

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is being filed on 04 December 2018 as a PCT International Patent application, and claims the benefit of priority to U.S. Provisional Application No. 62 / 594,943, filed 05 December 201SEQUENCE LISTING

[0002] The present application includes a Sequence Listing in electronic format as a txt file entitled "Sequence-Listing," which was created on 03 December 2018 and which has a size of 128,065 kilobytes (KB). The contents of txt file "Sequence-Listing" are incorporated by reference herein.BACKGROUND OF THE DISCLOSUREFIELD OF THE DISCLOSURE

[0003] Methods and compositions are provided for durably influencing microbiological ecosystems (microbiomes) in a subject in order to resist infection and reduce recurrence of infection by an undesirable microorganism by decolonizing and durably replacing with a synthetic microorganism. Synthetic microorganisms are provided that may durably replace an undesirable microorganism under dermal or mucosal conditions, and that contain molecular modifications designed to enhance safety, for example, by self-destructing when exposed to systemic conditions, by reducing the potential for acquisition of virulence or antibiotic resistance genes, and / or by producing a desirable product at the site of the ecosystem in a subject.DESCRIPTION OF THE RELATED ART

[0004] Health care or community associated infection often results from colonizing microorganisms that overcome patient defenses. Inappropriate use of antibiotics may lead to mismanagement of the microbiome. One critical unintended consequence of the mismanagement of the microbiome has been the emergence of antibiotic resistant microorganisms.

[0005] Each individual is host to a vast population of trillions of microorganisms, composed of perhaps 10,000 different species, types and strains. These "commensal" organisms are found both on external sites (e.g. dermal) and on internal sites (e.g. gastrointestinal), and are necessary for survival of the human species. "Colonization" happens automatically through ongoing interactions with the environment.

[0006] The menagerie of microorganisms constitutes the "biome", a dynamic, structured, living system that in many cases, and in many ways, is essential for our health and wellness. A biomic structure is created by a vast combinatorial web of relationships between the host, the environment, and the components of the biome. The human microbiome is an ecosystem. It has a dynamic but persistent structure - it is "resilient" and has a "healthy" normal base state.

[0007] Nonetheless, under some circumstances the microbiome can be invaded and occupied by pathogenic microorganisms. This type of "colonization" may become a precursor to "infection". This kind of disruption to the microbiome can cause serious and even life-threatening disease.

[0008] One unintended consequence of the mismanagement of our biome has been the emergence of "antibiotic resistance". This happens when antibiotics and antiseptics do not fully eliminate the target microorganisms. The few survivors that show resistance to these materials then preferentially grow back ("recolonize") into an open environment (or vacated "niche") already cleared of competing organisms. The survivor organisms then dominate the space, usually retaining that resistance for their descendants. If exposed to a new killing agent they will tend to develop resistance to that as well. Over only a few generations these microorganisms can develop resistance to many or all of our known antibiotics, becoming the now famous "super-bugs", and along the way creating an enormous new global health problem.

[0009] A phenomenon called "recurrence" is at the heart of the process that creates antibiotic resistance. While methods to treat pathogenic infection exist, methods to prevent recurrence are effectively nonexistent.

[0010] Bacterial infections are the home territory of the emerging "super bug" phenomenon. The overuse and misuse of antibiotics has caused many strains of pathogenic bacteria to evolve resistance to an increasing number of antibiotic therapies, creating a massive global public health problem. As each new variation of antibiotic is applied to treat these superbugs, the inevitable process of selecting for resistant strains begins anew, and resistant variants of the pathogen quickly develop. Unfortunately, today bacteria are becoming resistant faster than new antibiotics can be developed.

[0011] Beyond cultivating antibiotic resistance, and frequently causing adverse health effects in the recipients, antibiotic treatments also have the undesirable effect of disrupting the entire microbiome, including both good and bad bacteria. This often creates new problems such as opening the microbiome to colonization by adventitious pathogens after the treatment.

[0012] Bacteria however have less leeway to adapt to different resources, as these requirements are more basic on a molecular level and are intrinsically defined in the genome. This allows the microbiome ecology to behave as more of an "ideal" system, leading to full exclusion of one of the identical strain competitors from the niche.

[0013] The community of organisms colonizing the human body is referred to as the microbiome. The microbiome is often subdivided for analysis into sections of geography (i.e. the skin microbiome versus the gastrointestinal microbiome) or of phylogeny (i.e. bacterial microbiome versus the fungal or protist microbiome).

[0014] Antibiotics are life-saving medicines, but they can also change, unbalance, and disrupt the microbiome. The microbiome is a community of naturally-occurring germs in and on the body-on skin, gut, mouth or respiratory tract, and in the urinary tracts. A healthy microbiome helps protect from infection. Antibiotics disrupt the microbiome, eliminating both "good" and "bad" bacteria. Drug-resistant bacteria-like MRSA, CRE, and C. difficile-can take advantage of this disruption and multiply. With this overgrowth of resistant bacteria, the body is primed for infection. Once subjects are colonized with resistant bacteria, the resistant bacteria can easily be spread to others. See "Antibiotic Resistance (AR) Solutions Initiative: Microbiome, CDC Microbiome Fact Sheet 2016". www.cdc.gov / drugresistance / solutions-initiative / innovations-to-slow-AR.html.

[0015] According to the Center for Disease Control (CDC), the top drug-resistant threats to the United States include Neisseria gonorrhoeae, multi-drug resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, vancomycin-resistant Enterococcus (VRE), multi-drug resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae, drug-resistant Tuberculosis, vancomycin-resistant Staphylococcus aureus, erythromycin-resistant Group A Streptococcus, and clindamycin-resistant Group B Streptococcus. See "Antibiotic / Antimicrobial Resistance (AR / AMR)", https: / / www.cdc.gov / drugresistance / biggest_threats.html.

[0016] Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted disease that can result in discharge and inflammation at the urethra, cervix, pharynx, or rectum. There are about 820,000 gonorrhea infections per year. Of these, there are about 246,000 drug-resistant gonorrhea infections: 188,600 tetracycline resistant, 11,489 reduced susceptibility to cefixime, 3,280 reduced susceptibility to ceftriaxone, and about 2,460 exhibit reduced susceptibility to azithromycin.

[0017] Acinetobacter is a type of gram-negative bacteria that is a cause of pneumonia or bloodstream infections among critically ill patients. Many of these bacteria have become very resistant to antibiotics. There are about 12,000 Actinobacter infections per year, including about 7,300 multidrug-resistant Actinobacter infections and 500 deaths.

[0018] Candidiasis is a fungal infection caused by yeasts of the genus Candida. There are more than 20 species of Candida yeasts that can cause infection in humans, the most common of which is Candida albicans. Candida yeasts normally live on the skin and mucous membranes without causing infection. However, overgrowth or invasion of these microorganisms can cause symptoms to develop. Symptoms of candidiasis vary depending on the area of the body that is infected. Candida is the fourth most common cause of healthcare-associated bloodstream infections in the United States. In some hospitals it is the most common cause. These infections tend to occur in the sickest patients. There are about 46,000 Candida infections per year, including about 3,400 fluconazole-resistant Candida infections, and 220 deaths.

[0019] Staphylococcus aureus is a common type of bacteria that is found on the skin. During medical procedures when patients require catheters or ventilators or undergo surgical procedures, Staphylococcus aureus can enter the body and cause infections. Methicillin-resistant Staphylococcus aureus (MRSA) causes a range of illnesses, from skin and wound infections to pneumonia and bloodstream infections that can cause sepsis and death. Staph bacteria, including MRSA, are one of the most common causes of healthcare-associated infections. There are about 80,461 severe MRSA infections per year, and about 11,285 deaths from MRSA per year. When Staphylococcus aureus becomes resistant to vancomycin, there are few treatment options available because vancomycin-resistant Staphylococcus aureus bacteria identified to date were also resistant to methicillin and other classes of antibiotics. There have been at least 13 cases of vancomycin-resistant Staphylococcus aureus in 4 states since 2002.

[0020] Streptococcus pneumoniae (S. pneumoniae, or pneumococcus) is the leading cause of bacterial pneumonia and meningitis in the United States. It also is a major cause of bloodstream infections and ear and sinus infections. There are about 1,200,000 drug resistant infections per year, with about 19,000 excess hospitalizations, and 7,000 deaths.

[0021] Group A Streptococcus (GAS) causes many illnesses, including pharyngitis (strep throat), streptococcal toxic shock syndrome, necrotizing fasciitis ("flesh-eating" disease), scarlet fever, rheumatic fever, and skin infections such as impetigo. Group A Streptococcus is the leading cause of necrotizing fasciitis ("flesh-eating" disease). There are about 1-2.6 million Strep throat infections peer year, including about 1,300 drug-resistant Group A Strep infections per year, and about 160 deaths.

[0022] Group B Streptococcus (GBS) is a type of bacteria that can cause severe illness in people of all ages, ranging from bloodstream infections (sepsis) and pneumonia to meningitis and skin infections. Group B Strep is the leading cause of serious microorganism infections in newborns. There were about 27,000 severe cases of GBS in 2011, including about 7,600 drug-resistant Group B Strep infections, and about 440 deaths.

[0023] Prior art methods of preventing infection and transmission of drug-resistant microorganisms in colonized individuals include screening and isolation, decolonization of the drug-resistant microorganism, and / or recolonization with a drug-susceptible microorganism.

[0024] Prior art methods employing suppression (decolonization) alone -such as use of antibiotics and antimicrobial agents- often fail because they are subject to high rates of recurrence. Decolonization is often insufficient when used alone to effectively prevent recurrence and / or transmission of the drug-resistant microorganism.

[0025] Among pathogenic microorganisms causing health care related infection, methicillin-resistant Staphylococcus aureus (MRSA) has been given priority because of its virulence and disease spectrum, multidrug resistant profile and increasing prevalence in health care settings. MRSA is the most common cause of ventilator-associated pneumonia and surgical site infection and the second most common cause of central catheter associate bloodstream infection.

[0026] Strategies involving screening of new hospital patients for MRSA, and isolating those who carry it, with or without decolonization have been shown to be somewhat effective in reducing transmission. However, this type of therapy is rather expensive requiring extra accommodations, with special containment and hygiene procedures.

[0027] Decolonization alone has been used in hospital patients in an attempt to reduce transmission and prevent disease in Staphylococcus aureus carriers. Decolonization may involve a multi-day regimen of antibiotic and / or antiseptic agents- for example, intranasal mupirocin and chlorhexidine bathing. Universal decolonization is a method employed by some hospitals where all intensive care unit (ICU) hospital patients are washed daily with chlorhexidine and intranasal mupirocin, but since its widespread use, MRSA infection rates in the U.S. have not significantly changed. In addition, microorganisms may develop resistance to chlorhexidine and mupirocin upon repeated exposure.

[0028] Decolonization when used alone may not be durable because the vacated niche may become recolonized with pathogenic or drug-resistant microorganisms.

[0029] For example, Shinefield et al., 1963, Amer J Dis Child 105, June 1963, 146-154, observed that colonization of newborn infants with strains of Staphylococcus aureus of the 52 / 52a / 80 / 81 phage complex by contact with a carrier was often followed by disease in babies and their family contacts. Shinefield also observed that control measures using antiseptic or antimicrobial agents applied to the infant lead to colonization with abnormal flora, consisting primarily of highly resistant coagulase negative staphylocci and Gram-negative organisms such as Pseudomonas and Proteus. Shinefield attempted to solve the problem by artificially colonizing newborns with staphylococcal strain 502a by nasal and / or umbilical inoculation. 502a is a coagulase positive strain of Staphylococcus aureus of low virulence, susceptible to penicillin, and incapable of being induced to produce beta-lactamase. It was shown that presence of other staphylococci interfered with acquisition of 502a. Persistence of colonization was at best 35% after 6 months to one year.

[0030] Boris M. et al,. "Bacterial Interference: Protection Against Recurrent Intrafamilial Staphylococcal Disease." Amer J Dis Child 115 (1968): 521-29, deliberately colonized ~4000 infants in first few hours of life with Staphylococcus aureus 502a (nares & umbilical stump). Virtually complete protection of babies from 80 / 81 infection was observed (babies were monitored for 1-year post inoculation). Although 5-15% of babies developed tiny treatment emergent vesicles that self-resolved in first 3 days post-treatment. Prior decolonization improves persistence of 502a up to 5-fold compared to placebo (saline) n=63. Controlled studies in recurrent furunculosis showed that decolonization with systemic antibiotics + nasal antimicrobial followed by application of 502a curtailed disease in 80% of patients.

[0031] Recolonization with a drug-susceptible strain may not be safe because the drug-susceptible strain may still cause systemic infection.

[0032] Shinefield et al., 1973, Microbiol Immunol, vol. 1, 541-547, reported using bacterial replacement including decolonization in treating patients with recurrent furunculosis. Chronic staphylococcal carriers were treated with antibiotic therapy including systemic antibiotics and application of antimicrobial cream to nasal mucosa. In an initial study, 31 patients received antibiotic therapy alone and exhibited a 74% recurrence rate of original strain. 18 patients received antibiotic treatment followed by 502a inoculation and exhibited 27% recurrence of original strain. A larger study of 587 patients resulted in 21% recurrence of original strain after 12 months. However, a high relapse rate was noted in patients with diabetes, eczema or acne. Disease associated with 502a was noted in 11 patients.

[0033] Aly et al., 1974 J Infect Dis 129(6) pp. 720-724, studied bacterial interference in carriers of Staphylococcus aureus. The carriers were treated with antibiotics and antibacterial soaps and challenged with strain 502a. Specifically, decolonization method involved oral dicloxacillin 8 days; neosporin in nose for 8 days, and trichlorocabanilide. It was found that full decolonization was needed to get good take. Day 7 showed 100% take, but at day 23 the take was down to 60 to 80%. The persistence data was 73% at 23 weeks for well-decolonized subjects, and only 17% persistence for partially decolonized subjects. Co-colonization was found in 5 / 12 subjects at day 3, 2 / 12 subjects at day 10, and 1 / 12 subjects at day 35 and at day 70.

[0034] Decolonization, followed by recolonization with a microorganism of the same genus, but a different species, may not be durable because the vacated niche is not adequately filled by the different species.

[0035] WO2009117310 A2, George Liu, assigned to Cedars-Sinai Medical Center, discloses methods for treatment and prevention of methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus (MSSA) using a decolonization / recolonization method. In one example, mice are treated with antibiotics to eradicate existing flora, including MRSA, and newly cleared surface area is colonized with bacteria of the same genus, but of a different species, such as Staphylococcus epidermidis. No specific data regarding recurrence is provided.

[0036] Administration of probiotics in an attempt to treat infection by pathogenic microorganisms may not be effective and may not be durable because the probiotic may not permanently colonize the subject.

[0037] U.S. Pat. No. 6,660,262, Randy McKinney, assigned to Bovine Health Products, Inc., discloses broad spectrum antimicrobial compositions comprising certain minerals, vitamins, cobalt amino acids, kelp and a Lactobacillus species for use in treating microbial infection in animals. Field trials in cattle and horses were performed, but the infectious bacterial strain or other infectious agent was not identified.

[0038] U.S. Pat. No. 6,905,692, Sean Farmer, assigned to Ganeden Biotech, Inc., discloses topical compositions containing certain combinations of probiotic Bacillus bacteria, spores and extracellular products for application to skin or mucosa of a mammal for inhibiting growth of certain bacterium, yeast, fungi, and virus. Compositions comprising Bacillus coagulens spores, or Bacillus species. culture supernatants and Pseudomonas lindbergii culture supernatants in a vehicle such as emu oil are provided. The disclosure states since probiotics do not permanently colonize the host, they need to be ingested or applied regularly for any health-promoting properties to persist.

[0039] U.S. Pat. No. 6,461,607, Sean Farmer, assigned to Ganeden Biotech, Inc., discloses lactic acid-producing bacteria, preferably strains of Bacillus coagulans, for the control of gastrointestinal tract pathogens in a mammal. Methods for selective breeding and isolation of probiotic, lactic acid-producing bacterial strains which possess resistance to an antibiotic are disclosed. Methods for treating infections with a composition comprising an antibiotic-resistant lactic-acid producing bacteria and an antibiotic are disclosed.

[0040] U.S. Pat. No. 8,906,668, assigned to Seres Therapeutics, provide cytotoxic binary combinations of 2 or more bacteria of different operational taxonomic units (OTUs) to durably exclude a pathogenic bacterium. The OTUs are determined by comparing sequences between organisms, for example as sharing at least 95% sequence identity of 16S ribosomal RNA genes in at least in a hypervariable region. The international patent application WO02 / 15896 describes the potentiation of antibiotic action against tolerant microbes by expression of an inhibitor of a bacterial tolerance gene The international patent application WO2016 / 210373 describes a bacterial cell comprising a toxin-antitoxin system being under control of an oxygen-inducible promoter. This cell is used for biosafety applications. The international patent application WO2017 / 008018) describes a treatment of bleeding disorders by the use of a microorganism comprising an output molecule causing blood clotting. This output molecule is under control of a promoter which is regulated by a heme-responsive transcription factor, i.e., expression of the promoter is in response to blood The international patent application WO 2017 / 059245 discloses a so-called deadman kill switch circuit comprising an effector (e.g., toxin) linked to a first promoter, as well as a modulator modulating (repressing) the expression of the effector linked to a second promoter. The expression of the modulator requires the presence of a selected set of at least two input agents. In the absence of these agents, the modulator expression is altered, resulting in expression of the effector

[0041] Prior art methods employing replacement of the original pathogenic microorganism (recolonization) alone are subject to poor colonization rates with the new microorganism. The process may fail if the recolonization is done incorrectly. Effective recolonization is critical but not sufficient when used alone to prevent recurrence.

[0042] Prior art methods involving both suppression (decolonization) of the original pathogenic microorganism and replacement (recolonization) with a new microorganism may give variable recurrence of the pathogenic microorganism depending on the specific method. Rather than waging an un-winnable war against commensal pathogenic or drug-resistant microorganisms, a better approach may be to manage the microbiome: to actively promote "good bugs" and their supporting system dynamics, while selectively suppressing the recurrence of specific pathogenic organisms. Improved methods to safely and durably prevent and reduce recurrence of infection by undesirable microorganisms, such as virulent, pathogenic and / or drug-resistant microorganisms, are desirable.SUMMARY OF THE INVENTION

[0043] The present invention is related to a synthetic microorganism, comprising a recombinant nucleotide comprising at least one kill switch molecular modification comprising a first cell death gene operatively associated with a first regulatory region comprising an inducible first promoter, wherein the first inducible promoter exhibits conditionally high level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma of at least three fold increase of basal productivity, wherein the at least one molecular modification comprises a cell death gene nucleotide sequence that is integrated to a chromosome of the synthetic microorganism, and measurable average cell death of the synthetic microorganism occurs within at least a preset period of time following induction of the first promoter. Advantageously the synthetic microorganism of the invention presents one or more of the following characteristics: It further comprises at least a second molecular modification (expression clamp) comprising an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is operably associated with a second regulatory region comprising a second promoter which is active (constitutive) upon dermal or mucosal colonization or in a complete media, but is not induced, induced less than 1.5-fold, or is repressed after exposure to blood, serum or plasma for at least 30 minutes, and wherein the antitoxin gene encodes an antisense RNA sequence capable of hybridizing with at least a portion of the first cell death gene; It is derived from a target microorganism having the same genus and species as an undesirable microorganism Its first promoter is upregulated by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold within at least 30 minutes, 60 minutes, 90 miutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or at least 360 minutes following exposure to blood, serum, or plasma, preferably wherein the first promoter is not induced, induced less than 1.5 fold, or is repressed in the absence of blood, serum, heme, or plasma. its second regulatory region comprises a second promoter is active upon dermal or mucosal colonization or in TSB media, but is repressed at least 2 fold upon exposure to blood, serum or plasma after a period of time selected from the group consisting of the group consisting of at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and at least 360 minutes, its measurable average cell death occurs within at least a preset period of time selected from the group consisting of_within at least 1, 5, 15, 30, 60, 90, 120, 180, 240, 300, and 360 minutes following exposure to blood, serum, heme, or plasma, and wherein this measurable average cell death is at least a 50% cfu, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% cfu count reduction following the preset period of time, preferably wherein the kill switch molecular modification reduces or prevents infectious growth of the synthetic microorganism under systemic conditions in a subject, the target microorganism is selected from a bacterial, fungal, or protozoal target microorganism, optionally and wherein the target microorganism is preferably a bacterial species capable of colonizing a dermal and / or mucosal niche and is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Escherichia, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas, optionally wherein the target microorganism is selected from the group consisting of Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissimum, Cutibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Escherichia coli, Streptococcus pyogenes, Streptococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcal intermedius, Streptococcus agalactiae, Streptococcus mutans, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens, optionally wherein the target microorganism is susceptible to at least one antimicrobial agent, is derived from a Staphylococcus aureus strain, wherein the cell death gene is selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM, or lysostaphin toxin gene, optionally wherein the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or a substantially identical nucleotide sequence, its inducible first promoter comprises or is derived from a gene selected from the group consisting of isdA (iron-regulated surface determinant protein A), isdB (iron-regulated surface determinant protein B), isdG (heme-degrading monooxygenase), hlgA (gamma-hemolysin component A), hlgA1 (gamma-hemolysin), hlgA2 (gamma-hemolysin), hlgB (gamma-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosyntheis protein), sbnD, sbnI, sbnE (lucA / lucC family siderophore biosynthesis protein), isdI, lrgA (murein hydrolase regulator A), lrgB (murein hydrolase regulator B), ear (Ear protein), fhuA (ferrichrome transport ATP-binding protein fhuA), fhuB (ferrichrome transport permease), hlb (phospholipase C), heme ABC transporter 2 gene, heme ABC transporter gene, isd ORF3, sbnF, alanine dehydrogenase gene, diaminopimelate decarboxylase gene, iron ABC transporter gene, threonine dehydratase gene, siderophore ABC transporter gene, SAM dep Metrans gene, HarA, splF (serine protease SplF), splD (serine protease SplD), dps (general stress protein 20U), SAUSA300_2617 (putative cobalt ABC transporter, ATP-binding protein), SAUSA300_2268 (sodium / bile acid symporter family protein), SAUSA300_2616 (cobalt family transport protein), srtB (Sortase B), sbnA (probable siderophore biosynthesis protein sbnA), sbnB, sbnG, leuA (2-isopropylmalate synthase amino acid biosynthetic enzyme), sstA (iron transport membrane protein), sirA (iron ABC transporter substrate-binding protein), isdA (heme transporter), and spa (Staphyloccocal protein A), optionally wherein the first promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, and 163, or a substantially identical nucleotide sequence thereof, the antitoxin gene is selected from the group consisting of a sprA1 antitoxin gene, sprA2 antitoxin gene, sprG antitoxin gene or sprF, holin antitoxin gene, 187-lysK antitoxin gene, yefM antitoxin gene, lysostaphin antitoxin gene, or mazE antitoxin gene, kpn1 antitoxin gene, sma1 antitoxin gene, relF antitoxin gene, rsaE antitoxin gene, or yoeB antitoxin gene, respectively, and optionally wherein the antitoxin gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319, or 322, or a substantially identical nucleotide sequence, its second promoter comprises or is derived from a gene selected from the group consisting of clfB (Clumping factor B), sceD (autolysin, exoprotein D), walKR(virulence regulator), atlA (Major autolysin), oatA (O-acetyltransferase A); phosphoribosylglycinamide formyltransferase gene, phosphoribosylaminoimidazole synthetase gene, amidophosphoribosyltransferase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylaminoimidazole-succinocarboxamide gene, trehalose permease IIC gen, DeoR faimly transcriptional regulator gene, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkylhydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein disaggregation chaperon gene, alkylhydroperoxidase gene, phosphofructokinase gene, gyrB, sigB, and rho, optionally wherein the second promoter is a P clfB (clumping factor B) and comprises a nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a substantially identical nucleotide sequence thereof. It further comprises a molecular modification selected from the group consisting of a virulence block molecular modification, and nanofactory molecular modification, wherein the virulence block molecular modification prevents horizontal gene transfer of genetic material from the undesirable microorganism, and wherein the nanofactory molecular modification comprises an insertion of a gene that encodes, a knock-out of a gene that encodes, or a genetic modification of a gene that encodes a product selected from the group consisting of an enzyme, amino acid, metabolic intermediate, and a small molecule. The present invention is also related to a composition comprising an effective amount of the synthetic microorganism of the invention, and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetening agent, flavoring agent, wetting agent, preservative, buffer, or absorbent, or a combination thereof, optionally further comprising a nutrient, prebiotic, commensal, and / or probiotic bacterial species. The present invention concerns also a single dose unit comprising the composition of the invention, comprising at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 CFU, or at least 1011 of the synthetic strain and a pharmaceutically acceptable carrier, optionally formulated for topical administration. A further aspect of the invention is related to the synthetic microorganism, the composition or the single dose unit of the invention for use in a method for eliminating and preventing the recurrence of a undesirable microorganism in a subject hosting a microbiome, comprising: a. decolonizing the host microbiome; and b. durably replacing the undesirable microorganism with the synthetic microorganism comprising at least one element imparting a non-native attribute, wherein the synthetic microorganism is capable of durably integrating to the host microbiome, and occupying the same niche in the host microbiome as the undesirable microorganism, wherein the decolonizing is performed on at least one site in the subject to substantially reduce or eliminate the detectable presence of the undesirable microorganism from the at least one site, optionally wherein the niche is a dermal or mucosal environment that allows stable colonization of the undesirable microorganism at the at least one site, wherein the ability to durably integrate to the host microbiome is determined by detectable presence of the synthetic microorganism at the at least one site for a period of at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, wherein the ability to durably replace the undesirable microorganism is determined by the absence of detectable presence of the undesirable microorganism at the at least one site for a period of at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, and wherein the ability to occupy the same niche is determined by absence of co-colonization of the undesirable microorganism and the synthetic microorganism at the at least one site, optionally wherein the absence of co-colonization is determined at least one week, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, and preferably wherein the at least one element imparting the non-native attribute is durably incorporated to the synthetic microorganism A last aspect of the invention is related to a kit comprising in at least one container, the synthetic microorganism, the composition or the single dose unit of the invention , and optionally at least a second container comprising a decolonizing agent, a sheet of instructions, at least a third container comprising a promoting agent, and / or an applicator.

[0044] Methods and compositions are provided for safely and durably influencing microbiological ecosystems (microbiomes) in a subject to perform a variety of functions, for example, including reducing the risk of infection by an undesirable microorganism such as virulent, pathogenic and / or drug-resistant microorganism.

[0045] Methods are provided herein to prevent or reduce the risk of colonization, infection, recurrence of colonization, or recurrence of a pathogenic infection by an undesirable microorganism in a subject, comprising: decolonizing the undesirable microorganism on at least one site in the subject to reduce or eliminate the presence of the undesirable microorganism from the site; and durably replacing the undesirable microorganism by administering a synthetic microorganism to the at least one site in the subject, wherein the synthetic microorganism can durably integrate with a host microbiome by occupying the niche previously occupied by the undesirable microorganism; and optionally promoting colonization of the synthetic microorganism within the subject.

[0046] The disclosure provides a method for eliminating and preventing the recurrence of a undesirable microorganism in a subject hosting a microbiome, comprising (a) decolonizing the host microbiome; and (b) durably replacing the undesirable microorganism by administering to the subject a synthetic microorganism comprising at least one element imparting a non-native attribute, wherein the synthetic microorganism Staphylococcus aureus (MRSA) strain that contains a staphylococcal chromosome cassette (SCCmec types I-III), which encode one (SCCmec type I) or multiple antibiotic resistance genes (SCCmec type II and III), and / or produces a toxin. In some embodiments, the toxin is selected from the group consisting of a Panton-Valentine leucocidin (PVL) toxin, toxic shock syndrome toxin-1 (TSST-1), staphylococcal alpha-hemolysin toxin, staphylococcal beta-hemolysin toxin, staphylococcal gamma-hemolysin toxin, staphylococcal delta-hemolysin toxin, enterotoxin A, enterotoxin B, enterotoxin C, enterotoxin D, enterotoxin E, and a coagulase toxin.

[0047] In some embodiments, the subject treated with a method according to the disclosure does not exhibit recurrence or colonization of the undesirable microorganism as evidenced by swabbing the subject at the at least one site for at least two weeks, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 24 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks after the administering step.

[0048] The disclosure provides a synthetic microorganism for durably replacing an undesirable microorganism in a subject. The synthetic microorganism comprises a molecular modification designed to enhance safety by reducing the risk of systemic infection. In one embodiment, the molecular modification causes a significant reduction in growth or cell death of the synthetic microorganism in response to blood, serum, or plasma. The synthetic microorganism may be used in methods and compositions for preventing or reducing recurrence of dermal or mucosal colonization or recolonization of an undesirable microorganism in a subject.

[0049] The disclosure provides a synthetic microorganism for use in compositions and methods for treating or preventing, reducing the risk of, or reducing the likelihood of colonization, or recolonization, systemic infection, bacteremia, or endocarditis caused by an undesirable microorganism in a subject.

[0050] The disclosure provides a synthetic microorganism comprising a recombinant nucleotide comprising at least one kill switch molecular modification comprising a first cell death gene operatively associated with a first regulatory region comprising an inducible first promoter, wherein the first inducible promoter exhibits conditionally high level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma of at least three fold increase of basal productivity. In some embodiments, the inducible first promoter exhibits, comprises, is derived from, or is selected from a gene that exhibits upregulation of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold within at least 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, or at least 360 min following exposure to blood, serum, or plasma.

[0051] In some embodiments, the synthetic microorganism comprises a kill switch molecular modification comprising a first cell death gene operably linked to a first regulatory region comprising a inducible first promoter, wherein the first promoter is activated (induced) by a change in state in the microorganism environment in contradistinction to the normal physiological (niche) conditions at the at least one site in the subject.

[0052] In some embodiments, the synthetic microorganism further comprises an expression clamp molecular modification comprising an antitoxin gene specific for the first cell death gene or a product thereof, wherein the antitoxin gene is operably associated with a second regulatory region comprising a second promoter which is constitutive or active upon dermal or mucosal colonization or in a complete media, but is not induced, induced less than 1.5-fold, or is repressed after exposure to blood, serum or plasma for at least 30 minutes. In some embodiments, the second promoter is active upon dermal or mucosal colonization or in TSB media, but is repressed by at least 2 fold upon exposure to blood, serum or plasma after a period of time of at least 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, or at least 360 min.

[0053] In some embodiments, the synthetic microorganism exhibits measurable average cell death of at least 50% cfu reduction within at least 1, 5, 15, 30, 60, 90, 120, 180, 240, 300, or 360 minutes following exposure to blood, serum, or plasma. In some embodiments, the synthetic microorganism exhibits measurable average cell death of at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% cfu count reduction within at least 1, 5, 15, 30, 60, 90, 120, 180, 240, 300, or 360 minutes following exposure to blood, serum, or plasma.

[0054] In some embodiments, the synthetic microorganism comprises a kill switch molecular modification that reduces or prevents infectious growth of the synthetic microorganism under systemic conditions in a subject.

[0055] In some embodiments, the synthetic microorganism comprises at least one molecular modification that is integrated to a chromosome of the synthetic microorganism.

[0056] In some embodiments, the synthetic microorganism is derived from a target microorganism having the same genus and species as an undesirable microorganism. In some embodiments, the target microorganism is susceptible to at least one antimicrobial agent. In some embodiments, the target microorganism is selected from a bacterial, fungal, or protozoal target microorganism. In certain embodiments, the target microorganism is capable of colonizing a dermal and / or mucosal niche.

[0057] In some embodiments, the target microorganism has the ability to biomically integrate with the decolonized host microbiome. In some embodiments, the synthetic microorganism is derived from a target microorganism isolated from the host microbiome. In some embodiments, the target microorganism is selected from a bacterial, fungal, or protozoal target microorganism.

[0058] In some embodiments, the target microorganism that is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas.

[0059] In some embodiments, the target microorganism is a bacterial species capable of colonizing a dermal and / or mucosal niche and is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas. In some embodiments, the target microorganism is selected from the group consisting of Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissimum, Cutibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Streptococcus pyogenes, Streptococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcal intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens. In a particular embodiment, a synthetic microorganism is provided that is derived from a Staphylococcus aureus strain. In some embodiments, the target strain is a Staphylococcus aureus 502a strain or RN4220 strain.

[0060] In some embodiments, the synthetic microorganism comprises a kill switch molecular modification comprising a cell death gene selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM, or lysostaphin toxin gene. In some embodiments, the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or a substantially identical nucleotide sequence.

[0061] In some embodiments, the inducible first promoter is a blood, serum, and / or plasma responsive promoter. In some embodiments, the first promoter is upregulated by at least 1.5 fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold within a period of time selected from the group consisting of at least 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, and at least 360 min following exposure to human blood, serum or plasma. In some embodiments, the first promoter is not induced, induced less than 1.5 fold, or is repressed in the absence of the change of state. In some embodiments, the first promoter is induced at least 1.5, 2, 3, 4, 5 or at least 6 fold within a period of time in the presence of serum, blood or plasma. In some embodiments, the first promoter is not induced, induced less than 1.5 fold, or repressed under the normal physiological (niche) conditions at the at least one site.

[0062] In some embodiments, the inducible first promoter comprises or is derived from a gene selected from the group consisting of isdA (iron-regulated surface determinant protein A), isdB (iron-regulated surface determinant protein B), isdG (heme-degrading monooxygenase), hlgA (gamma-hemolysin component A), hlgA1 (gamma-hemolysin), hlgA2 (gamma-hemolysin), hlgB (gamma-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosyntheis protein), sbnD, sbnI, sbnE (lucA / lucC family siderophore biosynthesis protein), isdI, lrgA (murein hydrolase regulator A), lrgB (murein hydrolase regulator B), ear (Ear protein), fhuA (ferrichrome transport ATP-binding protein fhuA), fhuB (ferrichrome transport permease), hlb (phospholipase C), heme ABC transporter 2 gene, heme ABC transporter gene, isd ORF3, sbnF, alanine dehydrogenase gene, diaminopimelate decarboxylase gene, iron ABC transporter gene, threonine dehydratase gene, siderophore ABC transporter gene, SAM dep Metrans gene, HarA, splF (serine protease SplF), splD (serine protease SplD), dps (general stress protein 20U), SAUSA300_2617 (putative cobalt ABC transporter, ATP-binding protein), SAUSA300_2268 (sodium / bile acid symporter family protein), SAUSA300_2616 (cobalt family transport protein), srtB (Sortase B), sbnA (probable siderophore biosynthesis protein sbnA), sbnB, sbnG, leuA (2-isopropylmalate synthase amino acid biosynthetic enzyme), sstA (iron transport membrane protein), sirA (iron ABC transporter substrate-binding protein), isdA (heme transporter), and spa (Staphyloccocal protein A). In some embodiments, the inducible first promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, and 163, or a substantially identical nucleotide sequence thereof.

[0063] In some embodiments, the synthetic microorganism comprises an expression clamp molecular modification comprising a second promoter operatively associated with an antitoxin gene that encodes an antisense RNA sequence capable of hybridizing with at least a portion of the first cell death gene.In some embodiments, the antitoxin gene encodes an antisense RNA sequence capable of hybridizing with at least a portion of the first cell death gene. In some embodiments, the antitoxin gene is selected from the group consisting of a sprA1 antitoxin gene, sprA2 antitoxin gene, sprG antitoxin gene or sprF, holin antitoxin gene, 187-lysK antitoxin gene, yefM antitoxin gene, lysostaphin antitoxin gene, or mazE antitoxin gene, kpn1 antitoxin gene, sma1 antitoxin gene, relF antitoxin gene, rsaE antitoxin gene, or yoeB antitoxin gene, respectively. In some embodiments, the antitoxin gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319, or 322, or a substantially identical nucleotide sequence.

[0064] In some embodiments, the second promoter comprises or is derived from a gene selected from the group consisting of clfB (Clumping factor B), sceD (autolysin, exoprotein D), walKR(virulence regulator), atlA (Major autolysin), oatA (O-acetyltransferase A); phosphoribosylglycinamide formyltransferase gene, phosphoribosylaminoimidazole synthetase gene, amidophosphoribosyltransferase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylaminoimidazole-succinocarboxamide gene, trehalose permease IIC gen, DeoR faimly transcriptional regulator gene, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkylhydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein disaggregation chaperon gene, alkylhydroperoxidase gene, phosphofructokinase gene, gyrB, sigB, and rho. In some embodiments, the second promoter is a P clfB (clumping factor B) that comprises a nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a substantially identical nucleotide sequence thereof.

[0065] In some embodiments, the synthetic microorganism comprises a virulence block molecular modification, and / or a nanofactory molecular modification. In some embodiments, the virulence block molecular modification prevents horizontal gene transfer of genetic material from the undesirable microorganism.

[0066] In some embodiments, the nanofactory molecular modification comprises an insertion of a gene that encodes, a knock out of a gene that encodes, or a genetic modification of a gene that encodes a product selected from the group consisting of an enzyme, amino acid, metabolic intermediate, and a small molecule.

[0067] The disclosure provides a composition comprising an effective amount of a synthetic microorganism according to the disclosure and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetening agent, flavoring agent, wetting agent, preservative, buffer, or absorbent, or a combination thereof. In some embodiments, the composition further comprises a promoting agent. In some embodiments, the promoting agent is selected from a nutrient, prebiotic, commensal, and / or probiotic bacterial species.

[0068] The disclosure provides a single dose unit comprising a composition or synthetic microorganism of the disclosure. In some embodiments, the single dose unit comprises at least at least about 10 5< , at least 10 6< , at least 10 7< , at least 10 8< , at least 10 9< , at least 10 10< CFU, or at least 10 11< of the synthetic strain and a pharmaceutically acceptable carrier. In some embodiments, the single dose unit is formulated for topical administration. In some embodiments, the single dose unit is formulated for dermal or mucosal administration.

[0069] The disclosure provides a synthetic microorganism, composition according to the disclosure for use in the manufacture of a medicament for use in a method eliminating, preventing, or reducing the risk of the recurrence of a undesirable microorganism in a subject.

[0070] The disclosure provides a kit for preventing or reducing recurrence of dermal or mucosal colonization or recolonization of an undesirable microorganism in a subject, the kit comprising in at least one container, comprising a synthetic microorganism, composition, or single dose of the disclosure, and optionally one or more additional components selected from a second container comprising a decolonizing agent, a sheet of instructions, at least a third container comprising a promoting agent, and / or an applicator.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 shows a diagram of a representative molecular modification inserted to a Staphyloccoccus aureus, e.g., BioPlx-01, to create a synthetic microorganism BioPlx strain. A cassette comprising the molecular modification comprises a kill switch and an expression clamp, including expression clamp (e.g., ClfB) promoter cloned to drive expression of the SprA1 antisense (antitoxin) RNA wherein the cassette is incorporated into the same expression module from a kill switch comprising a serum-responsive promoter (e.g.,P hlgA ) operably associated with SprA1 toxin gene. In this strain, serum / blood exposure activates the toxin (e.g., up to 350-fold or more) but not the antitoxin, and growth in TSB or on the skin activates antitoxin but not toxin. FIG. 2 shows shuttle vector PCN51 used to clone genes into an E coli - Staphylococcus aureus pass-through strain (IMO8B) for transfection of the vector into BioPlx-01 for evaluation. FIGs. 3A-3C shows Table 4A with primer sequences for recombinant construction of synthetic Staphylococcus aureus from strain BioPlx-01. FIGs. 4A-4D shows Table 4Bwith primer sequences for CRISPR construction of synthetic Staphylococcus aureus from strain BioPlx-01. FIG. 5A shows a genetic map of a pKOR1 Integrative Plasmid depicting the repF(replication gene of pE194ts), secY570 (N-terminal 570 nucleotides of secY including ribosome binding site), cat (chloramphenicol acetyltransferase), attP (page lambda attachment site), ori(-) (ColE1 plasmid replication origin), and bla (b-lactamase). (+) or (-) indicates functions in gram positive (+) or gram negative (-) bacteria. The Pxyl / tetO promoter and the transcription direction of the promoter are indicated by an arrow. FIG. 5B shows a genetic map of a pIMAY Integrative Plasmid. (accession number JQ62198). FIG. 6 shows fold-induction of the HlgA (gamma hemolysin) promoter candidate in a methicillin-susceptible Staphylococcus aureus strain BioPlx-01 by incubation with human serum. Expression was normalized to a housekeeping gene (gyrB) and was compared with that in cells growing logarithmically in liquid TSB media. FIG. 7 shows fold-induction of the SstA (iron transport) promoter candidate in a methicillin-susceptible Staphyococcus aureus strain BioPlx-01 by incubation with human serum. Expression was normalized to a housekeeping gene (GyrB) and was compared with that in cells growing logarithmically in liquid TSB media. FIG. 8 shows CRISPR gRNA target site intergenic region identified between 1,102,100 and 1,102,700 bp in the Staphylococcus aureus 502a genome, GenBank: CP007454.1. FIG. 9 shows a representative screen shot of CRISPRScan used to find putative gRNAs for use in CRISPR methods. FIG. 10 shows cassette for integration via CRISPR and layout of the pCasSA vector. Cap1A is a constitutive promoter controlling gRNA transcription. Target seq is targeting sequence, for example, with 10 possible cutting targets (1.1, 1.2 etc.). sgRNA is single-strand guide RNA (provides structural component). Xba1 and Xho1 are two restriction sites used to add the HA's to the pCasSA vector. HAs are homologous arms to use as templates for homology directed repair (typically 200 - 1000 bp). P rpsL -mCherry is a constitutive promoter controlling the "optimized" mCherry. P rpsL -Cas9 is a constitutive promoter controlling Cas9 protein expression. FIG. 11 shows vectors for use in the present disclosure. A is a vector used for promoter screen with fluorescence using pCN51. B is a vector for promoter screen with cell death gene. C is a vector for chromosomal integration using CRISPR. D is a vector for chromosomal integration using homologous recombination. Left & Right (or upstream and downstream) HA: homology arms to genomic target locus, CRISPR targeting: RNA guide to genomic locus, mCherry: fluorescent reporter protein, Cas9 protein: CRISPR endonuclease, kanR: kanamycin resistance, oriT: origin of transfer (for integration), and sma1: representative kill gene (restriction endonuclease). FIG. 12A-12C shows nucleotide sequence (SEQ ID NO: 131) of pIMAY Integrative Plasmid. (accession number JQ62198). FIG. 13A shows activity of promoter candidates isdA, isdB, hlgA2, hrtAB, isdG, sbnE, lrgA, lrgB, fhuA, fhuB, ear, hlb, splF, splD, dps, and SAUSA300_2617 at 1 min, 15 min and 45 min in serum and fold changes in gene expression vs. media by qPCR. FIG. 13B shows activity of promoter candidates isdA, isdB, hlgA2, hrtAB, isdG, sbnE, lrgA, lrgB, fhuA, fhuB, ear, hlb, splF, splD, dps, and SAUSA300_2617 at 1 min, 15 min and 45 min in blood and fold changes in gene expression vs. media by qPCR. FIG. 14 shows inducible inhibition of cell growth of synthetic microorganism pTK1 cells comprising a cell death toxin gene (sprA1) behind a cadmium promoter on a pCN51 plasmid (pTK1) which had been transformed into Staphylococcus aureus RN4220 cells. OD (630 nm) read at 2 hrs post induction. Wild-type 4220 cells showed good cell growth both in the absence of cadmium and in the presence of 500 nM and 1 uM cadmium. pTK1-1 and pTK1-2 cells showed good growth in the absence of cadmium, but cell growth was significantly inhibited in presence of 500 nM and 1 uM cadmium at 2 hours post induction. FIG. 15A shows a plasmid map of p174 (pRAB11_Ptet-sprA1) zoomed view of the region of the plasmid containing the Ptet-sprA cassette. FIG. 15B shows the p174 (pRAB11_Ptet-sprA1) whole plasmid in its native circular form. FIG. 15C shows photographs of plate dilutions at 6 hours synthetic microorganism Staphylococcus aureus 502a p174 cells comprising a cell death toxin gene (sprA1) behind an anhydrotetracycline promoter on a pRAB11-2 plasmid (p174) which had been transformed into Staphylococcus aureus 502a cells. The p174 plasmid containing a deleted spra1 antisense (Das). Plate dilutions at 10e-5 are shown after 6 hours of induction for uninduced (left) and induced (right) 502a p174 (tet-spra1Das) cells on BHI chlor10. The plate on the left (Uninduced) was uncountable at 10e-5 but at 10e-6 counted ~ 720 colonies. The induced plate on the right at 10e-5 produced 16 colonies. The survival percentage of induced cells at 6 hours post induction was 0.22%. FIG. 16 shows cell growth pre- and post-induction of four synthetic strains derived from Staphylococcus aureus 502a having a plasmid based inducible expression system comprising four different cell death gene candidates sprA1, 187-lysK, Holin, and sprG. The candidate cell death genes had been cloned behind an tetracycline inducible promoter on pRAB11 plasmids and transformed into Staphylococcus aureus 502a cells. Calculated OD600 readings were taken at T=0, 30, 60, 120, and 240 min after induction of AtC induced (+) strains illustrated by dashed lines (- - - - - -) and uninduced (-) strains indicated by solid lines (-) for BP_068 (502a pRAB11-Ptet-sprA1), BP_069 (502a pRAB11-Ptet-187lysK), BP_070 (502a pRAB11-Ptet-holin), and BP_071 (502a pRAB11-Ptet-sprG1) and compared to BP_001 (502a wt) in BHI media. Each of the induced (+) strains BP_068 (sprA1), BP_069 (187lysK) and BP_070 (holin) exhibited both (i)good cell growth pre-induction and (ii)significant inhibition of cell growth post-induction. BP_068 (+) exhibited the best inhibition of cell growth at each time point T=30, T=60, T=60, T=120 and T=240 min post-induction, so the sprA1 gene was selected for initial further development of a kill switch in Staphylococcus aureus 502a. FIG. 17 shows a bar graph showing difference in the colony forming units(cfu) / mL between T=0 (gray) and 240 min(black) of un-induced (-) and anhydrotetracycline induced (+)strains BP_068 (502a pRAB11-Ptet-sprA1), BP_069 (502a pPRAB11-Ptet-187lysK), BP_070 (502a pRAB11-Ptet-holin), and BP_071 (502a pRAB11-Ptet-sprG1) compared to BP_001 (502a wt) in BHI media. Each of the induced (+) strains BP_068 (sprA1), BP_069 (187lysK) and BP_070 (holin) exhibited both (i)good cell growth pre-induction and (ii)significant inhibition of cell growth post-induction. BP_068 exhibited the best inhibition of cell growth 240 min post-induction, so the sprA1 gene was selected for initial further development of a kill switch in Staphylococcus aureus 502a. FIG. 18 shows GFP expression fold change of induced (+) and uninduced (-) subcultures of Staphylococcus aureus strains BP_001, BP_055 and BP_076. FIG. 19 shows a map of the genome for Strain BP_076 (SA 502a, ΔsprA1::Ptet-GFP). FIG. 20 shows a map of plasmid constructed for making genomic integration in Staphylococcus aureus. FIG. 21 shows a map of PsbnA-sprA1 kill switch in Staphylococcus aureus 502a genome. Serum and blood responsive promoter PisdB is operably linked to sprA1 toxin cell death gene. FIG. 22 shows a map of a kill switch construction using serum and blood responsive promoter PisdB operably linked to sprA1 toxin cell death gene and an expression clamp comprising a second promoter clfB operably linked to sprA AS to prevent leaky expression of the toxin in the absence of blood or serum. The kill switch is incorporated to the Staphylococcus aureus 502a genome. FIG. 23 shows a growth curve of three strains when exposed to human serum compared to TSB: 502a - Staphylococcus aureus wild type, Staphylococcus aureus BP_011 - 502a ΔsprA1-sprA1(AS), and Staphylococcus aureus BP_084 - 502a ΔPsprA::PsbnA in which the kill switch is integrated to the genome of Staphylococcus aureus 502a. The dashed lines represent the strains grown in serum, and the solid lines represent the strains grown in TSB. After 180 minutes, the strain BP_084 with the integrated kill switch shows a growth curve that is significantly reduced compared to the wild type in serum and the kill switch in complex media. After 3 hours of exposure to human serum, the Staphylococcus aureus BP_084 (502a ΔPsprA::PsbnA) cells exhibited 98.84% measurable average cell death compared to the same BP_084 cells in TSB. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure relies upon a principle known as "bacterial replacement", or "niche exclusion", where one microorganism replaces and excludes another. In the field of ecology, competitive exclusion, or Gause's Law, states that two species that compete for the exact same resources cannot stably coexist. This is due to the fact that one of the competitors will possess some slight advantage over the other leading to extinction of the lesser competitor in the long run. In higher order organisms, this often leads to the adaptation of the lesser competitor to a slightly different ecologic niche.

[0073] Methods and compositions for durably managing the microbiome of a subject are provided. In embodiments, the microbiome is a dermal and / or mucosal microbiome (Exobiome). While methods to treat infection by a pathogenic microorganism exist, methods to prevent recurrence are effectively nonexistent.Infectious Agent - Staphylococcus aureus (MSSA and MRSA)

[0074] Classified since the early twentieth century as among the deadliest of all disease-causing organisms, each year around 500,000 patients in hospitals of the United States contract a staphylococcal infection, chiefly by Staphylococcus aureus. Up to 50,000 deaths each year in the USA are linked with Staphylococcus aureus infections. Staphylococcus aureus exists on the skin or inside the nostrils of 40-44% of healthy people. Staphylococcus aureus is also sometimes found in the mouth, gastrointestinal, genitourinary, and upper respiratory tracts. Some studies indicate even higher colonization prevalence. For example, Eriksen et al maintain that there is a higher percentage of transient or intermittent carriers that increase the prevalence number; sometimes to greater than 75%.Staphylococcus aureus 502a WT BioPlx-01WT ®< and Other Replacement and Blocking Strains

[0075] A Staphylococcus aureus 502a WT strain called BioPlx-01WT ®< is employed in example 1 and is a natural "wild-type" organism known to be relatively non-infectious, and which has no known side effects. It has been shown in BioPlx clinical studies to be highly effective in this intended application (occupying and blocking the required microbiomic niche to prevent the recurrence of MRSA).

[0076] The present methods prevent infection by durably replacing the (typically virulent and antibiotic-resistant) colonizing undesirable Staphylococcus aureus strain with a "blocking" organism - in this study the BioPlx01-WT Staphylococcus aureus 502a WT strain. This phenomenon is expected to be applied in a similar manner for any other pathogen replacement organism developed by BioPlx.

[0077] Other replacement strains such as synthetic strains are provided herein that are fully able to colonize the properly prepared skin and mucosal surfaces, and to occupy the ecologic niche used by this bacterial species, thereby blocking other variants from recolonizing that niche.

[0078] There are a very large number of Staphylococcus aureus variants (10,000+ genomes as of 9 / 2017), as well as a wide range of genetic cassettes and virulence factors associated with this species.

[0079] Methicillin-resistant Staphylococcus aureus (MRSA) refers to a class of antibiotic resistant variants of this common human commensal and sometimes pathogenic bacteria. It varies from the wild-type strain (MSSA - Methicillin Sensitive Staphylococcus aureus) by its carriage of a mecA cassette that allows MRSA strains to produce an alternate penicillin binding protein (PBP2A) that renders them resistant to treatment with most beta lactam and many other first-line antibiotics.

[0080] Methicillin-Resistant Staphylococcus aureus (MRSA) and Virulent Methicillin-Susceptible Staphylococcus aureus (vMSSA) are virulent, invasive variants of Staphylococcus aureus that colonize many humans, and which can further cause both superficial soft tissue and severe systemic infections. Colonization with MRSA or vMSSA is usually a required precursor to active Staph infection. Infection is caused by the bacteria colony on the skin or mucosal membranes, penetrating the outer immunological barrier and invading tissue or the blood stream through a wound, an incision, a needle puncture, or other break in the skin. This can lead to bacteremia and other systemic infections that have high mortality rates.

[0081] The present disclosure uses a generally passive strain of Staphylococcus aureus to replace and exclude MRSA or vMSSA from its usual place in the dermal / mucosal microbiome. The wild type interfering Staphylococcus aureus used by BioPlx is known to be poor at causing systemic disease, however, regardless of the level of variance or invasiveness virtually any microorganism can become an "accidental pathogen" through natural or accidental inoculation. This is particularly true in the case of Staphylococcus aureus.

[0082] The decolonization and BioPlx01 strain application methods developed by BioPlx allows the strains provided herein a massive numerical and positional competitive advantage. The consequences of this method provide a much longer effect of MRSA decolonization than a simple antiseptic destruction of the virulent MRSA strain. Early studies show a greater than 6 month total exclusionary effect of the BioPlx01 MRSA decolonization / recolonization process with the BioPlx product as opposed to prior literature demonstrating 45% recurrence of Staphylococcus aureus nasal colonization at 4 weeks and 60% at 12 weeks with the standard decolonization method alone. Overview of Indication

[0083] Staphylococcus aureus infections are a severe problem in both hospitals and community health settings. Methicillin-resistant Staphylococcus aureus (MRSA) is genetically different from other strains of Staphylococcus aureus, with genetic elements conferring resistance to the antibiotic methicillin and other (usually beta-lactam) antibiotics typically used to treat Staphylococcus aureus infections. MRSA strains carry a mecA expression cassette that allows MRSA strains to produce an alternate penicillin binding protein (PBP2A), and it's this mutation that confers resistance. Due to this resistance, MRSA is difficult to treat, making it a life-threatening problem in many cases. MRSA is frequently contracted in hospitals or other types of healthcare settings (Hospital Associated [HA]). These infections typically occur at the time of an invasive procedure such as surgery, intravenous catheterization, intubation, or artificial joint placement. Community-associated (CA) MRSA is typically spread by skin-to-skin contact, and the first symptoms tend to be large boils on the skin.

[0084] The BioPlx method using BioPlx strains is not a treatment for invasive MRSA disease, and therefore is not intentionally applied to a patient during the invasive disease state. The benefits of the BioPlx method can be demonstrated in a patient group that: 1) is at high risk for invasive disease, 2) has high morbidity and mortality from this increased risk to show significant clinical benefit, and has no other effective options for the prevention of invasive Staphylococcus aureus disease. These characteristics define the group of patients that the Centers for Disease Control have been tracking regarding the MRSA subset since 2005 who have already experienced invasive MRSA disease - 72,444 according to ABC surveillance data in 2014.

[0085] The surface of the human skin and mucosal layer where Staphylococcus aureus resides in the colonization state has a very different level of required nutrients as well as different environmental qualities than that inside the human body. It has been widely recognized that in order for bacteria to be successfully invasive, they must be able to adjust their needs and responses between the colonization and invasive states. This is accomplished by the bacterium sensing the changes between these environments and switching on or off certain gene cassettes allowing for the production of proteins more adapted to the new invasive state.

[0086] The BioPlx method, and specifically BioPlx01 strains, take advantage of this requirement by rearranging molecular instructions leading to the death of the organism in the operons of one or more of these specific cassettes. This creates a "holding strain" of colonizing Staphylococcus aureus that is unable to cause disease in the patient to whom it is introduced, but also does not allow other circulating Staphylococcus aureus strains that may normally colonize the human population to colonize this patient. This occurs through the ecologic premise of competitive exclusion.

[0087] The current "Standard of Care" for patients colonized with MRSA is not uniform. There are no guidelines as to the management of staphylococcal colonization in patients that are at high risk of recurrent disease. The IDSA Clinical Practice Guidelines for the Treatment of MRSA Infections in Adults and Children in 2011 provide only C-III level (the lowest - no data, expert opinion) support for decolonization procedures in patients with recurrent community-acquired skin and soft tissue infections and make no mention of the role of decolonization in the prevention of invasive MRSA disease. Some hospitals have pursued a broad screening and isolation program for all admitted patients to their institution, but this has not been shown to be effective owing to (including) poor durability of effect and lower baseline risk of the average hospitalized patient (i.e. UC Irvine MRSA outbreak.) Other hospitals therefore have reduced their attention to patients admitted to the ICU and cardiothoracic surgery cases only. This strategy has been shown to reduce MRSA clinical isolates as well as bloodstream infection from any pathogen. However, these are short term situational strategies designed to reduce risk of MRSA infection over a near time frame.

[0088] MRSA disease and colonization is a complicated epidemiologic problem for both the United States and the rest of the world. The manifestations of MRSA are broad from asymptomatic colonization to invasive disease states conferring high mortality and cost to the system. It is clear that the MRSA patients that have experienced invasive disease is medically distinct. They have a higher mortality than any other MRSA subpopulation. They have a higher treatment failure rate. They have a much higher risk for another invasive MRSA incident than any other group of patients. This makes this group an appropriate orphan group toward which the BioPlx method should be directed, and which would benefit from its use.

[0089] It can be concluded that decolonization is largely ineffective in durably clearing MRSA colonization, and leads to a high rate of recurrence. We have found that only decolonization in conjunction with active recolonization provides long term conversion from one organism (variant) to another.Recurrent Invasive MRSA as a Clinically Distinct Disease

[0090] Another indication is "prevention of recurrent invasive MRSA." Patients who have already experienced an episode of invasive MRSA infection have a greatly increased susceptibility to a subsequent invasive MRSA infection. The BioPlx technology provided herein works by occupying the niche in the microbiome that would normally have the potential to be occupied by a virulent form of MRSA.Invasive MRSA-caused Systemic Infection:

[0091] SA, including the variant MRSA, can exist in harmless coexistence on the surface of the skin and mucous membranes of at least 40% of all humanity, so the bacterium itself is not descriptive of disease; rather, its clinical presentation is definitional.

[0092] The whole of national and international authorities that define and monitor this condition concur that invasive MRSA infection is a separate and distinct disease from other conditions caused by this bacterium.

[0093] Simple colonization with any type of Staphylococcus aureus should not be considered a disease state. In fact, those humans with nutritional and environmental characteristics of their skin and mucosal biomes that are hospitable to Staphylococcus aureus must have some such niche occupant as part of their microbial flora to achieve a stable balanced "resting state" of their biome. The goal of any method would be to durably replace a MRSA strain on an at-risk patient with the product strain - in this case an antibiotic sensitive Staphylococcus aureus modified to be unable to survive within the human body in the invasive state.

[0094] To create invasive infectious disease, MRSA must abandon its passive commensal status, and breach the dermal / mucosal barrier, entering into the subdermal interstitial (interstitial fluid) or circulatory (blood, serum, plasma) areas. This "state change" initiates a new disease state, with new organism behaviors and relationships to the host.

[0095] Staphylococcus aureus bacteremia (SAB) is an important instance of this type of infection with an incidence rate ranging from 20 to 50 cases / 100,000 population per year (ranging from 64,600 to 161,500 cases per year). Between 10% and 30% of these patients will die from SAB. Invasive systemic MRSA bacteremia has a mortality rate of around 20%. Comparatively, this accounts for a greater number of deaths than for AIDS, tuberculosis, and viral hepatitis combined.

[0096] The latest report for which there is a CDC-US national case estimate for invasive MRSA disease (2014) is 72,444 cases. The number of patients with this disease is less than 200,000 per annum, and it may permit an orphan drug designation. MRSA can impact patients at three distinct levels: 1) colonization , 2) superficial infection - skin and soft tissue, and 3) systemic invasive infection. 1) Colonization. Staphylococcus aureus is a normal commensal organism permanently colonizing around one third of the human population, with transient colonization occurring in about one additional third of the population. MRSA variants of this organism occupy organism the microbiome niche, and have colonized approximately 2% of the population in the US (with a high degree of variability depending on location and occupation). MRSA colonization creates a standing reservoir of potentially infectious organisms located directly on the outer layer of our immune / defense system, and this poses an ongoing risk to the patient. 2) Superficial infection - skin and soft tissue infection. Skin-associated MRSA or skin and soft tissue infection is the most common of the two major disease state categories. It typically starts as a swollen, pus or fluid filled, boil that can be painful and warm to the touch, and at times accompanied by a fever. If left untreated, these boils can turn into abscesses that require surgical intervention for draining. For MRSA that's confined to the skin, surgical draining of abscesses may be the only necessary treatment, and antibiotics are not indicated. Skin and soft tissue infections are treated by surgically draining the boil and only administering antibiotics when deemed absolutely necessary. 3) Systemic invasive infection. MRSA bacteremia (invasive MRSA) is a systemic MRSA infection that is defined as the presence of MRSA in typically sterile sites, including the bloodstream, cerebrospinal fluid, joint fluid, bone, lower respiratory tract, and other body fluids. MRSA bacteremia has a far worse prognosis compared to MRSA infections confined to the skin, with 20% of cases resulting in death. The difference in prognosis, location of the infection, and clinical symptoms of the condition make it clinically distinct from skin and soft tissue infection MRSA infections. MRSA bacteremia causes multiple complications not seen in skin and soft tissue infections, including infective endocarditis, septic arthritis, and osteomyelitis. For invasive MRSA, daptomycin and vancomycin are recommended treatments in the U.S. Vancomycin has a relatively slow onset and poorly penetrates some tissues. Daptomycin has been shown to be effective, but treatment-emergent nonsusceptibility is an issue, in addition to the issue of vancomycin encouraging daptomycin resistance in MRSA. The difference in clinical symptoms as well as treatment methods for invasive MRSA provides clear evidence for invasive MRSA as a clinically distinct condition from MRSA Skin and soft tissue infections.

[0097] The BioPlx technology works by preventing the recurrence of an invasive MRSA infection in those who have been colonized (including those that have already experienced an invasive MRSA infection) and who have undergone a decolonization procedure. As a decolonization / recolonization microbioic method, the BioPlx technology would not be administered to "treat" a patient while they had a systemic MRSA infection. It would be applied subsequent to the clearance of a systemic MRSA infection (and a full body decolonization).

[0098] It is an established principle of medical nomenclature that a disease or condition is not simply synonymous with the causative agent. In the present case, MRSA-mediated systemic bacteremia (or other designations of invasive systemic disease) is unambiguously distinct from the other superficial skin and mucosal conditions that may be caused by, or associated with, MRSA, or by other Staphylococcus aureus strains. Invasive systemic MRSA-mediated disease has a clearly distinct diagnosis, pathology, treatment, and prognosis profile.

[0099] It's important to note that, based on the mechanism of action of BioPlx01 strains, patients are prevented from subsequent systemic MRSA infection, as opposed to treatment of invasive MRSA infection per se. So, "prevention of recurrent systemic MRSA infection" would be the most accurate description of the indication for BioPlx01 strains.

[0100] The target population of patients that have had invasive MRSA Infection, have been successfully cleared of the organism (typically through standard antibiotic intervention (e.g. Vancomycin), and yet have a high risk (rate) of MRSA recolonization, recurrence and the associated elevated risk of MRSA systemic reinfection.International and US Recognition of the Disease Designation:

[0101] A clear definition of this disease is put forth by the Centers for Disease Control and Prevention (CDC) as it has been actively monitoring this condition in the United States since 2005. The agency performs this monitoring utilizing the Active Bacterial Core surveillance system via the Emerging Infections Program (EIP). A case in this context is defined by the isolation of MRSA from a normally sterile body site. Normally sterile sites included blood, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid, joint / synovial fluid, bone, internal body site (lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, or ovary), or other normally sterile sites.

[0102] The CDC also created the National Healthcare Safety Network (NHSN) as a tracking system for more than 16,000 US healthcare facilities to provide data to guide prevention efforts. The Center for Medicare Services (CMS) and other payers use this data to determine financial incentives to healthcare facilities for performance. The system tracks MRSA bloodstream infections as a marker for invasive disease for epidemiologic purposes.

[0103] The MRSA mediated invasive disease state is also codified in the ICD9 and ICD10 system by a grouping of conditions each with their own numeric code specific for the causative agent MRSA. For example, sepsis due to MRSA is coded A41.02, pneumonia due to MRSA is coded J15.212. This further exemplifies the differential characterization that invasive MRSA disease is given in juxtaposition to superficial skin and soft tissue disease due to the same agent - code L03.114 (left upper limb example) with the follow code of B95.6 MRSA as the cause of disease classified elsewhere, which is attached to a variety of other infection codes to indicate MRSA as the cause of the disease condition.

[0104] The European Center for Disease Control (ECDC), a branch of the EU also surveilles invasive Staphylococcus aureus isolates by similar definition to the NHSN and tracks methicillin-resistance percentages but the reporting requirements do not produce an EU estimate of total annual cases.

[0105] Differentially, unlike systemic conditions, simple MRSA colonization is not itself typically regarded as a disease. Colonization however is considered a precondition for most invasive disease, as evidenced (for example) by studies that show that nasal Staphylococcus aureus isolates are usually identical to strains later causing clinical infection. This persistent colonization state reflects the ecological stability of this bacteria on skin and mucosal surfaces.

[0106] This colonization state is recorded in the ICD10 system, Z22.322, under the Z subheading which is reserved for factors influencing health status and contact with health services but not an illness or injury itself.The target orphan disease population:

[0107] The orphan disease population targeted for the BioPlx non-recurrence method is the group of people previously invasively infected (systemic infection) with MRSA (a population known to be susceptible), and who continue to suffer ongoing recolonization with MRSA. CDC monitors all U.S. cases of invasive MRSA infection. Multiple researchers have described this medically distinct population - patients who have already suffered one defined episode of invasive MRSA infection. This group is at increased risk for life threatening invasive disease as a result of their demonstrated susceptibility and their continued colonization.

[0108] In some embodiments, a method is provided for preventing recolonization, or preventing recurrence of MRSA-caused systemic invasive bacteremia, comprising prevention of (or prevention of recurrence of) a prerequisite MRSA colonization by 1) decolonization of MRSA from mucosal and dermal microbiomes, and 2) recolonization of these microbiomes with a synthetic Staphylococcus aureus (e.g., a BioPlx01 strain). The method is effective, through the effect of bacterial interference, operating through niche dynamics within the target dermal / mucosal microbiome ecosystem, because the synthetic Staphylococcus aureus (e.g., a BioPlx01 strain) serves to occupy specific niches, and thus blocks / prevents MRSA recolonization (blocks recurrence). The efficacy of this method has been demonstrated clearly in proof of principle studies provided herein.

[0109] SA is present as part of the normal microbiome of more than 40% of the total human population. The MSSA colonization state is common. The MRSA variant is found on around 1-2% of the US population, but in certain areas or demographics this level can be considerably higher. It is thought that MRSA has the ability colonize anyone within the Staphylococcus aureus susceptible population. Staphylococcus aureus lives most commonly on the surface of the skin and in the anterior nasal vestibules, but can also be found in smaller amounts in the deep oropharynx and gastrointestinal tract and in normal vaginal flora in some individuals.

[0110] In colonized individuals Staphylococcus aureus usually remains a non-invasive commensal bacterium simply occupying an ecologic niche and not causing disease. In a portion of those colonized however, this bacteria can cause disease either opportunistically or as a result of the increased likelihood of invasion due to some particular variant characteristics.

[0111] Approximately 23% of persistent MRSA carriers developed a discrete MRSA infection within one year after identification as a carrier.

[0112] Many Staphylococcus aureus variants have acquired genetic cassettes coding for virulence protein products that allow such strains to more effectively invade through the epidermal or mucosal tissue layers, and subsequently initiating deep or systemic infection. In colonization or infection the presence of the mecA cassette limits the treatment options for these patients, and a number of studies have documented the increased mortality rate associated with MRSA when compared to MSSA in bacteremia, endovascular infection and pneumonia.

[0113] It is not possible to predetermine whether an individual who is colonized with MRSA will eventually progress to invasive disease or not, so it is particularly important to identify and treat the entire population of patients who have a well-documented increased risk for invasive MRSA disease.MRSA-mediated invasive disease statistics:

[0114] MRSA was identified by British scientists in 1961 and the first American clinical case was documented in 1968. For the next 25 years, MRSA was regarded largely as an endemic hospital-based problem that was increasing in incidence, however starting in the mid to late 1990s, an increase of incidence of community-associated MRSA was seen mostly manifesting in superficial skin and soft tissue infections. Of greatest concern to the medical community has been the increase in invasive infections caused by MRSA. The increasing trend in incidence of invasive MRSA disease was seen throughout the 1990s and peaked in 2005.

[0115] The CDC tracks the incidence of invasive MRSA disease through the NHSN and the Emerging Infections Program - Active Bacterial Core surveillance system also starting in 2005. As compared to 2005, 2015 data shows that the overall incidence for invasive MRSA disease has decreased almost 50% from an incidence rate of 37.56 to 18.8. Expensive and laborious infection control interventions enacted in hospitals in response to this public health crisis has been given much of the credit for the decreased incidence, as the majority of the gain was seen in health care associated cases as opposed to community associated ones. Despite the gains that have been made over the past decade, invasive MRSA infections continue to be a prioritized public health issue. These infections can be very difficult to treat and treatment failure has been shown in nearly 25% of patients on proper therapy. Predicting which health care experienced patients are at risk for invasive MRSA is a challenging problem. Risk factors such as MRSA colonization, the presence of chronic open wounds and the presence of invasive devices have been elucidated.

[0116] The presence of these characteristics alone do not predict which patient will ultimately display invasive disease. However, one of the most predictive risk factors for a patient getting an invasive MRSA infection is having had a previous invasive MRSA infection. In the 2004-2005 data from the Active Bacterial Core Surveillance (ABCs) it was noted that almost 13% of their invasive cases went on to develop a second invasive MRSA infection during the 18 months of retrospective data evaluation. Another look at the EIP-ABC data in the calendar year 2011 found that 8% of these patients had more than one invasive MRSA infection separated by at least 30 days. The longer term risk of recurrent invasive MRSA infection is surely greater still as these estimates will miss earlier infections in these patients prior to the study time period and later ones that occur after the end date. Since Huang and Platt (2003) showed that 29% of hospitalized patients with known MRSA colonization or infection went on to develop a second MRSA infection (often severe) within an 18 month follow up, targeting this group to prevent recurrence of the invasive disease state could prevent approximately 17,500 subsequent invasive MRSA infections (using the most recent CDC data).

[0117] Invasive MRSA and skin and soft tissue infection from MRSA are both caused by the same pathogen. However, orphan designations are awarded based on the dyad of drug and disease. MRSA is a pathogen, and not a disease state. However, it can cause infection, and it's these different types of infectious disease that are being treated. Invasive MRSA comes with a far more severe prognosis as well as different clinical manifestations from MRSA confined to the skin or simply being colonized with MRSA. About 40% of the U.S. population is colonized with Staphylococcus aureus, typically found in the nose or on the skin. Generally, there are no signs of infection that would be considered "a disease state." However, systemic MRSA infection will manifest as high grade fever, chills, dizziness, chest pain, swelling of the affected area, headache, rash, cough, and other systemic symptoms. These two conditions are treated differently, where skin and soft tissue infections are typically treated by incising and draining the boils commonly associated with skin and soft tissue infections. Antibiotics and decolonization are only employed if there are signs of systemic or severe disease that has spread to multiple sites.

[0118] Invasive MRSA has an incidence rate of 20 to 50 cases / 100,000 people per year. 6a< With a current U.S. population of 326,199,002 (accessed on November 2, 2017 from www.census.gov / popclock), this means there are 163,100 cases of invasive MRSA infection in the U.S. per year conservatively, falling below the 200,000 patient criteria for FDA orphan designation. We searched for other sources of reported prevalence to confirm that we had calculated the most conservative estimate of this patient population. Hassoun et. al reported an incidence of 72,444 cases of invasive MRSA in the U.S. in 2014, which had decreased from 111,261 in 2005. 7a< Based on this, and assuming that the population will continue to decrease, we can assume that a prevalence of 163,029 patients with invasive MRSA in the U.S. in 2017 is a very conservative estimate. According to the CDC, there were more than 80,000 invasive MRSA infections and 11,285 related deaths in 2011.

[0119] To address this problem the present inventors have developed BioPlx01 strains, molecularly-altered strains of Staphylococcus aureus that are unable to cause disease but can reside in the microbiome niche that MRSA could take hold in. The lack of invasiveness of BioPlx01 strains is made possible by operons that are turned on upon contact with blood or plasma, triggering the death of the organism. A patient who has tested positive for MRSA and is experiencing systemic symptoms will undergo a full body decolonization before the BioPlx01 strain is administered, allowing it to occupy the niche that MRSA would have previously occupied in that patient's microbiome. By preventing virulent strains of MRSA from occupying the niche, these virulent strains cannot colonize, and subsequently invade sterile tissue sites. BioPlx01 strain is able to prevent recurrent systemic MRSA infections.

[0120] In one embodiment, a method for treatment of Staphylococcus aureus lung infections in patients with cystic fibrosis is provided.

[0121] In one embodiment, a method for treatment of Invasive Bacteremia is provided. Using the criteria adopted by CDC (Centers for Disease Control and Prevention), Invasive Bacteremia is indicated by the isolation of bacteria from a normally sterile body site. These may include blood, CSF, joint fluid, bone samples, lower respiratory tract samples and other sterile body fluids. This condition is related to, but is clearly distinguished from, simple bacterial colonization and bacteria mediated skin and soft tissue infection. It is accepted that the colonization state is a prerequisite for invasive disease in the vast majority of cases.MRSA and v-MSSA Mediated Invasive (Systemic) Bacterial Infection

[0122] Mediated by Staphylococcus aureus, MRSA Invasive Bacterial Infection may also be referred to commonly or in the literature as: MRSA bacteremia or sepsis, Systemic MRSA infection, MRSA bloodstream infections, invasive MRSA infection. Specific MRSA induced systemic conditions range from osteomyelitis, septic arthritis, pneumonia, endocarditis, bacteremia, toxic shock syndrome, to septic shock. The development of a method to prevent or reduce the recurrence of invasive MRSA disease in high-risk populations, through the mechanism of durably interfering with colonization of undesirable strains, would be a significant advance in the prevention of conditions typically required for invasive MRSA infection, and would reduce the likelihood of these patients suffering a subsequent invasive MRSA infection.

[0123] One objective of the present disclosure is to evaluate the BioPlx-01 WT material's ability to prevent the recurrence of MRSA in active healthy adult medical workers. This population is particularly at-risk for MRSA infection and has amongst the highest rates of MRSA colonization of any demographic. Successfully demonstrating a protective effect for this group would validate BioPlx-01 WT's efficacy in being able to prevent MRSA recurrence amongst effectively all those who are at risk.

[0124] "Recurrence" simply means "the bug comes back". Recurrence is of central importance to both disease evolution and control. With recurrence, the pathogen comes back again and again, and each time it goes through a survival cycle it "learns" to be more and more resistant to the antibiotics it has seen. Without this recurrence, once the pathogen is gone, it would stay gone, and that would be that. If there were no recurrence, there would be no pressure to evolve toward antibiotic resistance.

[0125] In various embodiments, the subject may be colonized with one or more pathogenic microorganisms. In certain embodiments, the undesirable microorganism is a drug-resistant pathogenic microorganism. The drug-resistant pathogenic microorganism may be selected from a Neisseria gonorrhoeae, fluconazole-resistant Candida, MRSA, drug-resistant Streptococcus pneumoniae, drug-resistant Tuberculosis, vancomycin-resistant Staphylococcus aureus, erythromycin-resistant Group A Streptococcus, and clindamycin-resistant Group B Streptococcus. https: / / www.cdc.gov / drugresistance / biggest_threats.html.

[0126] In one embodiment, the undesirable microorganism may be a drug-resistant pathogenic Staphylococcus aureus.

[0127] Staphylococci are the most abundant skin-colonizing bacterial genus and the most important causes of nosocomial infections and community-associated skin infections. The species Staphylococcus aureus may cause fulminant infection, while infections by other staphylococcal species are mostly subacute. Colonization is usually a prerequisite for infection. Otto 2010, Expert Rev Dermatol 2010 Apr; 5(2):183-195. However, not all invasive Staphylococcus aureus infections are preceded by detected colonization with identical strain. The non-correlative fraction may be explained either by the "direct inoculation" or "direct wound seeding" theory such as an intraoperative event from a second carrier, or incomplete detection of all of these patient's Staphylococcus aureus strains in colonization or colonization with the invasive strain in the time since the initial colonization surveillance.

[0128] SA is a common human commensal organism that is present (colonizes), typically without symptoms, in 30 to 50% of the (US) population. The asymptomatic carriage of Staphylococcus aureus by humans is the primary natural reservoir, although domestic animals, livestock, and fomites may serve as adjunctive reservoirs.

[0129] There are many different strains of Staphylococcus aureus, many of which can also act as serious pathogens. Symptoms of Staphylococcus aureus infections can be diverse, ranging from none, to minor Skin and soft tissue infections, to invasive life-threatening systemic disease such as endovascular infections, pneumonia, septic arthritis, endocarditis, osteomyelitis, foreign-body infections, sepsis, toxic shock and endocarditis. The anterior nasal mucosa has traditionally been thought to be the most frequent site for the detection of colonization of healthy carriers with Staphylococcus aureus. Several sites may become asymptomatically colonized including the nares, throat, axilla, perineum, inguinal region, and rectum.

[0130] MRSA isolates were once confined largely to hospitals, other health care environments, and patients frequenting these facilities. Since the mid-1990s, however, there has been an explosion in the number of MRSA infections reported in populations lacking risk factors for exposure to the health care system. This increase in the incidence of MRSA infection has been associated with the recognition of new MRSA clones known as community-associated MRSA (CA-MRSA). CA-MRSA strains differ from the older, health care-associated MRSA strains; they infect a different group of patients, they cause different clinical syndromes, they differ in antimicrobial susceptibility patterns, they spread rapidly among healthy people in the community, and they frequently cause infections in health care environments as well. David, Michael et al., 2010, Clin Microbiol Rev 23(3): 616-687.

[0131] Why recurrent CA-MRSA Skin and soft tissue infections are common is not known. The mechanism by which recurrence occurs is unclear. Possibilities include reinfection from persistent asymptomatic CA-MRSA carriage or after acquisition from environmental MRSA or after new MRSA acquisition from close human or animal contact. Skin and soft tissue infections caused by MSSA also recur but less frequently than those caused by MRSA.

[0132] Under constant antibiotic pressure, many Staphylococcus aureus variants have developed antibiotic resistance. Today penicillin resistance in Staphylococcus aureus is virtually universal, and general beta-lactam and related multi-antibiotic (methicillin) resistance is now widespread, creating a significant new class of antibiotic-resistant "super-bugs".

[0133] The pathogenic Staphylococcus aureus may be a drug-resistant Staphylococcus aureus, such as MRSA, or a vancomycin-resistant strain, such as VISA or VRSA. Alternatively, the pathogenic Staphylococcus aureus may be a virulent methicillin-susceptible Staphylococcus aureus (v-MSSA). v-MSSA is a high-virulence cause of life-threatening invasive infections. MRSA and v-MSSA are epidemic, and have a high human cost.

[0134] MRSA has become a serious public health problem in hospitals, clinics, prisons, barracks, and even in gyms and health clubs around the world. MRSA is a common cause of hospital-acquired infections (500k US patients / year), and increasingly, of community acquired infections which can be serious. For systemically invasive disease - 20% of cases result in death. MRSA is one of the most significant of the new antibiotic-resistant "super-bugs". While methods to treat Staphylococcus aureus infection exist, methods to prevent recurrence are effectively nonexistent. Recurrence of MRSA skin infections is found in 31% to 45% of subjects.

[0135] One effort to prevent recurrence includes decolonization. The first (and currently only) widely practiced step for preventing recurrence is decolonization. Unfortunately, simple decolonization is poor at preventing recurrence. Doctors can initially treat the microbial colonization or infection-for example MRSA or v-MSSA colonization / infection- with topical chemicals (e.g. chlorhexidine) or antibiotics. In many cases treatment with antibiotics may "clinically" eliminate the disease. Antiseptics and astringents may be used for decolonization (i.e., suppression) including tea tree oil and chlorhexidine. Antibiotics used for suppression include topical antibiotics for nasal decolonization such as mupirocin. Systemic antibiotics most frequently used for MRSA include vancomycin, first generation antibiotics such as cefazolin, cepahalothin, or cephalexin; and new generation antibiotics such as linezolid or daptomycin. In less serious MRSA cases, clindamycin or lincomycin may be employed. Nonetheless, with this decolonization alone the MRSA and v-MSSA pathogens typically recur-or grow back -nearly 1 / 2 of the time. This level of performance has naturally led to skepticism as to the efficacy of simple decolonization in preventing recurrence.

[0136] Clinicians often prescribe topical, intranasal, or systemic antimicrobial agents to patients with recurrent skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in an effort to eradicate the staphylococcal carrier state. Some agents can temporarily interrupt staphylococcal carriage, but none has been proved effective for prevention of skin infections caused by MRSA. Creech et al. Infect Dis Clin North Am. 2015 September ; 29(3): 429-464 .

[0137] In both the literature and in the hands of the present inventors, it has been found that the quality of decolonization is correlated to the recurrence rate observed, but simple decolonization rarely resulted in a durable, successful, outcome.

[0138] The present disclosure provides methods and compositions focused on preventing recurrence through the effective and durable modification of microbiome populations.

[0139] Methods for preventing or decreasing recurrence of a pathogenic microbial infection have been developed comprising suppressing a microbial infection or colonization.

[0140] A method to decrease recurrence of a pathogenic infection or decrease colonization of a undesirable microorganism in a subject is provided, comprising decolonizing the undesirable microorganism on at least one site in the subject to significantly reduce or eliminate the presence of the undesirable microorganism from the site; and replacing the undesirable microorganism by administering to the subject a synthetic second microorganism having the same genus and species as the undesirable microorganism.

[0141] The methods and compositions to prevent recurrence include replacement of the pathogenic microorganism by filling the biome niche occupied by the pathogen with a specially designed synthetic microorganism - or "good bug". By occupying the same biome niche, the "good bug" crowds out the pathogen, preventing it from recolonizing, or moving into (or back into) its preferred ecological neighborhood. One way to ensure the same biome niche is filled is by designing a synthetic microorganism starting from the same genus and species as the pathogenic microorganism.

[0142] The methods and compositions to prevent recurrence include promoting or supporting the synthetic microorganism - the "good bug" - by re-establishing key nutritional, chemical, or commensal environments that further promote the preferred organism and inhibit recolonization by the pathogen. For example, a commensal cluster may provide further layered defense in preventing the pathogen from moving back into its old ecological niche - it may help prevent recurrence.

[0143] The BioPlx method is enabled by state of the art methods / technologies including microbiomics, systems & computational biology; environment interactions (clusters & signaling); proprietary organisms (selected & modified); and variant and strain substitution strategies.

[0144] Replacement microorganisms are provided herein including (1)"BioPlx01-WT ®< variant" - a Staphylococcus aureus 502a wild-type microorganism with an established history of non-virulence and passive colonization which has been isolated, verified, and prepared for field trials using this strain cluster as described in Example 1; (2) "BioPlx01-KO ®< engineered variant", a synthetic Staphylococcus aureus strain that enhances safety by knocking out specific virulence genes; and (3) "BioPlx01-KS ®< engineered variant", a synthetic Staphylococcus aureus strain that embeds a molecular programmed cell death trigger to prevent invasive virulence. In some embodiments, the synthetic microorganism acts purely as a substitution for the pathogenic strain, without "new" infection or colonization.

[0145] An extensive proprietary library of fully characterized Staphylococcus aureus cultures (strains and variants) has been developed which is used for replacement organism sourcing; used for durability and competition analysis; used for Genotype / Phenotype comparative analysis; used for virulence genome / transcriptome clustering modeling; and used for signaling genome / transcriptome clustering modeling.

[0146] A Library of controlled commensal organisms is being developed for potential variant cluster co-administration with the BioPlx01-KS ®< variant.

[0147] Methods for Computational Microbiology are also being developed including Machine Learning; Modeling of complex dynamic microbiomic systems; Genome / Transcriptome / Proteome (Phenotypic) relationships; Virulence factor genetics and promoters; Modeling resilience and changes over time / condition; n-dimensional niche-forming relationships; and High dimensional cluster relationships.

[0148] Central to the present model anti-recurrence method is the principle of "non-co-colonization", meaning that only one species, and one variant of that species, can occupy the relevant skin or mucosal biome ecological niche at any one time. Underlying this simple and testable phenomenon are a host of deeper generative principles that combine to shape the emerging science of Microbiomics. Although widely generalizable, discussion of non-co-colonization in this section refers specifically to Staphylococcus aureus colonizations.Non-co-colonization

[0149] The principle of non-co-colonization (also known as "bacterial replacement") states that only one variant / strain of one species can occupy any given niche within the biome at any given time.

[0150] The central empirical phenomenon of non-co-colonization represents an aggregate effect: the consequence of the interaction of a large number of forces that can be found operating in complex systems, and which are only today becoming well characterized and mathematized.

[0151] Bacterial niches within the human biome that are specific to the species level underlie the present technology. If there were no specificity to biologic niche occupation, then intentional strain exchange would not be achievable, as would the experimentally demonstrated phenomenon of bacterial replacement.

[0152] Expectations for non-co-colonization are important for durability of the present method for prevention of recurrence of pathogenic colonization or infection. Variant-to-variant non-co-colonization has been demonstrated experimentally in the literature with strain / variant substitution (e.g., the Staphylococcus aureus 80 / 81 to 502a conversions of Shinefield et al., 1963) and has been confirmed in present clinical studies, as shown in Example 1.

[0153] Sustained species-to-species niche occupation is suspect because careful reading of the literature indicates that durability is low, and in vivo evidence is rare. A transient occupation may occur, but is not considered to be an important outcome, as we are only interested in durable outcomes.

[0154] Failure of durability in species-to-species substitution serves as evidence that specific niche-filling requires a "close variant" substitution. This is significant as only durable biomes can display the functional characteristics (such as resilience) required for an effective non-recurrence technology / product.

[0155] In the case of variant-to-variant replacement, such as that seen in the present disclosure with respect to MRSA anti-recurrence materials, no direct evidence from the literature has been identified as to whether the replacement requires a "biome disruptive event" (such as accidental or intentional decolonization by antimicrobials, antibiotics, etc.) or whether it can occur via a "slow competitive replacement" (one organism out competing another for resources, growth, etc.). However, overwhelmingly in human dermal biomes, only one strain colonizes a person "in toto", indicating that slow competitive replacement occurs. Further, the 55% success rate of anti-MRSA decolonization methods show that "biome disruptive events" can also induce durable biome changes. Both of these phenomena are expressions of non-co-colonization.

[0156] Non-co-colonization occurs in nature, for example, in the vast majority of cases only one variant of Staphylococcus aureus is detected within a single biome (over 95% of cases, with the balance likely caused by "transient conditions").

[0157] In specifying and evaluating non-co-colonization durability (efficacy) it is necessary to recognize three distinct scales of outcomes: (1) short-term - immediately post recolonization, (2) early stable stage - after shedding excess organisms, and (3) long-term - after a stable "new" biome is established.

[0158] In the short-term - immediately post recolonization, the decolonized biome is dominated by organisms applied "in excess" during recolonization - generating a type of adventitious and transient binding (like spreading peanut butter). Testing within this period can only confirm that the biome application has occurred. Duration = a few days, with subsequent shedding of excess organisms.

[0159] In the early stable stage - after shedding excess organisms, the biome per se is reestablishing its equilibrium state, but ostensibly with the replacement organism rather than the pre-existing pathogen. Confidence in this outcome is primarily due to the overwhelmingly large ratio (probably millions to one) of new organisms to surviving post-decolonization pathogens. It is expected that this will become a stable colonization with a high level of durability. Testing at this period would confirm that MRSA or vMSSA has been eliminated, and replacement strain has been re-colonized. Duration = weeks to months.

[0160] In the long-term - after stable "new" biome established will demonstrate not only the organism's ability to occupy or "take" a niche, but its ability to "hold" that niche. In some embodiments, this stage is used to evaluate how competitive the replacement strain or synthetic microorganism is against the current generation of new biome invaders (such as USA300). This question refers to the "new" replacement organism's ability to compete over time against a slow competitive replacement as well as by external forces that could be biome disruptive over time such as antibiotic or antiseptic exposures or frequent re-exposure to the pathogen - especially if the strains are differentially sensitive to this disruptor.

[0161] It is important to characterize the phenomenon of microorganism variant non-co-colonization, variant-versus-variant niche occupation, and the empirical evidence already developed that this phenomenon exists and is a strong force in the dermal biome ecosystem.

[0162] The law of "competitive exclusion" refers to the situation where only one organism dominates one niche.

[0163] One historical error in understanding this phenomenon is assuming this is a binary system, conceptually driven by either one or two variants. In fact, a large number of different microorganisms, for example various Staphylococcus aureus strains may be environmentally present at any one time, and over time.

[0164] It may be concluded that without the phenomenon of non-co-colonization, virtually all "staph-capable" biomes would inherently be highly variable mixed heterologous "soups" of multiple variants. Various possibilities are shown in Table 1. Table 1. Staphylococcus aureus (SA) niche compatibilities and expected outcomescaseNiche compatibilityCompetitive exclusionExpected Outcome1)one Staphylococcus aureus niche+one variant dominates (except transitional)2)one Staphylococcus aureus niche-always large number of variants (soup)3)multi Staphylococcus aureus niches+any smaller # of variants = # of discrete niches4)multi Staphylococcus aureus niches-always large number of variants (soup)

[0165] In Table 1, cases 2 & 4 can be eliminated, because co-colonization occurs in under 5% (in literature), and even in these cases the vast majority of co-colonization instances observed involve only one other organism. Case 3 can be considered as possible in a low number of cases (less than 5%) potentially relating to incomplete or non-overlapping footprints of the niche vs replacement organism.

[0166] There is no direct evidence from the literature as to whether the observed replacement of one variant for another (e.g. acquisition of MRSA) is caused by a biome-disruptive event or from a slow competitive replacement. However, it is empirically clear that only one strain at a time tends to colonize any individual biome (in toto). Biogeographically distinct and distant sites within a given biome strongly tend to have the same variant, and this occurs without any observable total body decolonization and replacement process, indicating that a rule-driven competitive replacement process occurs. The observation of competitive replacement is another expression of the principle of non-co-colonization.

[0167] In hypothetical cases where the replacement variant does not fill the niche completely there may be a weak tendency to co-colonization. In these cases, a variant cluster may be used to "fill the slots" with alternatives so that the co-colonization favors a synthetic replacement microorganism rather than the original pathogen. While this may involve the use of a different replacement microorganism, this is not recurrence - this is further blocking of recurrence.Current evidence of non-co-colonization

[0168] One large study looked at the prevalence of co-colonization in 3,197 positive Staphylococcus aureus samples taken from healthy patients in Oxfordshire, England followed longitudinally for up to two years; the point prevalence of having multiple strains of Staphylococcus aureus in nares samples was 3.4 to 5.8%. Votintseva et al., 2014 J Clin Microbiol, 52 (4): 1192-1200. Of the Staphylococcus aureus carriers who submitted swabs nearly every two months for two years, 11% had transient co-carriage. The study used an effective spa typing protocol that allowed for a sensitive procedure for finding even low proportion co-colonization strains. The interpretation of this data set shows that Staphylococcus aureus colonization is a dynamic process with low prevalence of multiple Staphylococcus aureus strains vying for presence in the same niche over time. A simple calculation can establish that the observed results are not simply the independent occupation of a non-specific niche. In this instance, 1000 patients were screened and 360 were found to be Staphylococcus aureus positive. In a non-specific niche scenario, .36 x .36, or 13%, (130 persons), would be expected to display co-colonization; however only 3.9% of the 360 carriers, (14 persons) at that primary point were in fact co-colonized, demonstrating the strain specificity of the microbiome niche for Staphylococcus aureus.

[0169] A small percentage of Staphylococcus aureus carriers may be transiently colonized with two different strains of Staphylococcus aureus at any incident time point. As discussed above, Votintseva et al, looked at all variants within MSSA and MRSA and reported point incidences of this phenomenon to be in the range of 3.4 - 5.8%. The paper looking only at mixtures of MRSA and MSSA (would only find species that differ at the mecA site) is predictably lower at 2.3%. If co-colonization was a stable state, mixtures of Staphylococcus aureus species would be expected in virtually all samples. This is not observed.

[0170] Another study looked at 680 patients presenting for any type of hospital admission. It was practice of the National Health Service at that time to screen all patients being admitted for MRSA. Dall'Antonia, M. et al., 2005, J Hospital Infect 61, 62-67. During this evaluation the protocol was refined to discover MSSA, MRSA and co-colonized MRSA and MSSA patients. MSSA alone was found in 115 patients (16.9%), MRSA alone was found in 56 patients (8.2%) and co-colonization was discovered in 4 patients (0.58%), again supporting the view of a strain-exclusive niche in the microbiome for Staphylococcus aureus. It supports the concept that one Staphylococcus aureus strain can prevent the establishment of another. The results suggested a lower percentage of co-colonized carriers as would be predicted by the null hypothesis indicating that there is a significant protective effect against one Staphylococcus aureus strain colonization by a previous occupying resident Staphylococcus aureus strain. The statistical significance was p<0.01. The protective effect of MSSA colonization against MRSA colonization was calculated to be 78% (CI: 29-99%).

[0171] A further study looked at non-concordant Staphylococcus aureus isolates in a population composed of HIV infected IV drug users in a methadone clinic. There were 121 baseline positive Staphylococcus aureus samples and 4 of these showed clear discordance among 3 colonies evaluated by PFGE. However, re-evaluation of these 4 samples showed that 2 of the 4 were concordant at second evaluation. No discordance was found after re-evaluating 18 samples first found to be concordant. Therefore 1.7 - 3.3% of this population was found to have co-colonization at a singular time point. Cespedes C. et al., J Infect Dis 2005; 191: 444-52.

[0172] Historical Evidence of decolonization / recolonization studies also show evidence of Non-Co-Colonization. This principle has been previously partially demonstrated during the 1960s and 1970s in the well-known 80 / 81 to 502a "bacterial interference" studies and clinical applications. Absence of co-colonization is shown in the early bacterial interference papers in the 1960s and 1970s, these papers also clearly demonstrate "competitive exclusion" in regulating co-colonization. Mixed cultures of both 80 / 81 as the resident strain and 502A as the donor strain were not observed, experimentally demonstrating non-co-colonization as a stable situation for the microbiome. (Shinefield et al., 1963; Shinefield at al., 1966; Shinefield et al., 1973; Aly et al., 1974; Boris et al., 1964; Light et al., 1967; Fine et al., 1967).

[0173] Without "non-equivalence" and "competitive exclusion", microbiome niches would consistently be filled with multiple strains of the same species of bacteria. The isolation in nature of a pure strain culture of Staphylococcus aureus from the nares would be a rare event if ever seen. The population dynamic in such a state would create a heterogeneous "soup" of many varieties of Staphylococcus aureus, as dictated by adventitious or random exposure from the environment. Any strain that the host has ever come in to contact with would have equal opportunity to colonize that space without competition or interference with any other strain variant (polyclonal colonization). The absence of this empirical result demonstrates "competitive exclusion".

[0174] Yet, the exclusion principle is not so rigid that once a niche is occupied no other variant can usurp its position. These observations demonstrate an exclusion principle that is robust, but that allows external species to challenge an occupying species by briefly sharing that niche while the ultimate competition for dominance in that space is being enacted. On some occasions "new" strains overcome the previous resident strain and establish a new dominant resident strain. On other occasions, the interloper is rebuffed and the resident strain repels the attempt at replacement and reestablishes singular dominance. In both of these scenarios, the co-colonized state is transient and unstable; present at a low frequency.Microbiomic Systems

[0175] Methods and compositions are provided to durably and safely prevent recurrence of a pathogenic microbial infection in a subject, comprising suppression of a pathogenic microorganism, replacement with a synthetic microorganism capable of occupying the same niche to durably exclude the pathogenic microorganism, and promotion of the synthetic microorganism for durable residence within the niche. This method is termed the BioPlx ®< method, as discussed above. In some embodiments, the subject is found to be colonized with the pathogenic microorganism prior to the suppression step.

[0176] In order to successfully work within the microbiome to promote the colonization of a desired organism in such a way as to produce a durable protective outcome requires that we know the "rules" of microbiomes: as discussed in greater detail in the sections following.

[0177] A non-co-colonization model has been developed to provide context and establish target product characteristics. The rationale for the present technology rests on the Microbiomic paradigm (biome / ecosystem / niche), and on the Microbiome having certain persistent and verifiable characteristics. The key discoverable metric rests on co-colonization statistics in literature modified by specifics on decolonization, testing, and other relevant conditions, followed by direct observations from the clinical study of example 1.

[0178] The skin microbiome in the subject is an entity, a persistent identifiable thing. Over 10,000 different species of microorganisms make up the skin microbiome. The skin biome is an ecosystem which may be defined as a system, or group of interconnected elements, formed by the interaction of a community of organisms with their environment. The skin microbiome ecosystem has a "healthy", or "normal" base state. The biome can be "healthy" or "sick" (dysbiosis), and can be invaded by pathogenic organisms - in other words the Microbiome can be invaded by a "Bad Bug" - such as MRSA - it can also become infected or contaminated by undesirable organisms or variants (dysbiosis). Dysbiosis is a term for a microbial imbalance or maladaptation on or inside the body, such as an impaired microbiota.

[0179] The skin microbiome has a structure created by a vast combinatorial web of relationships between the host and all of the components of the biome. The microbiome, or biome, is a dynamically structured complex system and is an "elastically resilient" ecosystem. The skin microbiome has a dynamic but persistent structure - it is "resilient", for example, even under conditions of massive cell death (e.g. washing, using ethanol, hand sanitizer, etc.) the biome regenerates in a similar form.Resilience

[0180] The human microbiome has the quality of resilience meaning that mild perturbations tend to re-correct toward a previous established baseline of species mixture and concentration. However, members of each niche can be successfully challenged for their place in that stable mixture either as a result of an acute external disruptive event (i.e. an antimicrobial medication or an antiseptic application) or as a slow competitive replacement.

[0181] In ecology, resilience is the capacity of an ecosystem to respond to a perturbation or disturbance by resisting damage and recovering quickly. Resilience refers to ecosystem's stability and capability of tolerating disturbance and restoring itself.

[0182] In the literature, the main mathematical definitions of resilience are based on dynamical systems theory, and more specifically on attractors and attraction basins. The human microbiome operates in many ways like a multi-basin complex system. It changes states or basins, but then resilience stabilizes that state. Martin, S. et al., 2011, in: Deffuant G., Gilbert N. (eds) Viability and Resilience of Complex Systems. Understanding Complex Systems. Springer, Berlin, Heidelberg, pp. 15-36.

[0183] The microbiome operates in many ways like a multi-attractor complex system - it can changes its states or basins, but then the resilience associated with that attractor stabilizes that state.

[0184] Ecological resilience is defined as the capacity of a system to absorb disturbance and reorganize while undergoing change so as to still retain essentially the same function, structure, identity and feedbacks. Mitra, C., et al., 2015, An integrative quantifier of multistability in complex systems based on ecological resilience, Nature, Scient. Rep., 5, 1-12.

[0185] The "competitive exclusion principle" provides that complete competitors cannot exist. The "axiom of inequality" states that no two things or processes in a real world are precisely equal. Hardin, 1960, Science, vol. 131, 1292- 1297, p. 1292. Based on Hardin's 'Axiom of Inequality' and the Competitive Exclusion Principle, long-term durability should only be achieved by close variant substitution, but would not likely be available with respect to species substitution. For example, MRSA and MSSA can co-colonize briefly - just like any other variants of Staphylococcus aureus can co-colonize in transient fashion. See Dall' Antonia, M. et al., 2005, J Hospital Infect 61, 62-67, disclosing a study of 680 patients presenting for any type of hospital admission and screened all patients being admitted for MRSA. During this evaluation the protocol was refined to discover MSSA, MRSA and co-colonized MRSA and MSSA patients. MSSA alone was found in 115 patients (16.9%), MRSA alone was found in 56 patients (8.2%) and co-colonization was discovered in 4 patients (0.58%), again supporting the view of a strain-exclusive niche in the microbiome for Staphylococcus aureus. It supports the concept that one Staphylococcus aureus strain can prevent the establishment of another.

[0186] Resilience may create recurrence - an observed natural phenomenon - as the existing (MRSA contaminated) biome tries to preserve itself.

[0187] However, resilience can also prevent MRSA recurrence - as exhibited by methods and compositions provided herein. By suppressing a pathogenic microorganism such as MRSA ("bad bug") colonized in a subject, and replacing with a safe synthetic microorganism ("good bug") of the same species, it has been established that the "good bug" durably prevents recurrence of the "bad bug"(prevents MRSA re-invasion).

[0188] A historical example of resilience creating durable, persistent substitution is seen in Staphylococcus aureus carriers and replacement with strain 502a. Aly et al., 1974 J Infect Dis 129(6) pp. 720-724, studied bacterial interference in carriers of Staphylococcus aureus. The carriers were treated with antibiotics and antibacterial soaps and challenged with Staphylococcus aureus strain 502a. It was found that full decolonization was needed to get good colonization of 502a. Day 7 showed 100% take, but at day 23 the take was down to 60 to 80%. The persistence data was 73% at 23 weeks for well-decolonized subjects. Thus, long-term durability is only achieved by close variant substitution. Commensal microflora (normal microflora, indigenous microbiota) can help recolonization dynamics, but they do not fulfill close variant durability requirements. The inventors have designed a method for obtaining a "passive" version of an organism or pathogen (same species) that is to be "replaced" or "excluded".

[0189] A relative stability in the microbial ecosystem of adults in the absence of gross perturbation has been suggested, and that long-term stability of human communities is not maintained by inertia, but by the action of restoring forces within a dynamic system. Relman, D.A., 2012, Nutr Rev.,70(Suppl 1): S2-S9.

[0190] Functional resilience is an intrinsic property of microbial communities and it has been suggested that state changes in response to environmental variation may be a key mechanism driving functional resilience in microbial communities. Song et al., 2015, Frontiers in Microbiology, 6, 1298. Seeking an integrated concept applicable to all microbial communities, Song et al. compared engineering and ecological resilience and reconciled them by arguing that resilience is an intrinsic property of complex adaptive systems which, after perturbation, recover their system-level functions and interactions with the environment, rather than their endogenous state.

[0191] Thus, a biome ecosystem has a dynamic but "stable elastoplastic equilibrium". Once perturbed the biome "tries" to return to equilibrium. At any given moment the biome ecosystem has an equilibrium "base state". Even under conditions of stress or massive cell death (e.g. washing, using ethanol, hand sanitizer, etc.) the biome is observed to typically regenerate in a similar form.

[0192] Microbiome ecosystems have "niches" defined by structure and internal and external interactions. One "fact" or "principal" of any biome structure is that different organisms occupy different "niches" in the biome, as defined / allowed by the structure of relationships. An ecological "niche" is the role and position a species has in its environment; how it meets its needs for food and shelter, how it survives, and how it reproduces. A species' niche includes all of its interactions with the biotic and abiotic factors of its environment. A biome "niche" has specific environmental factors and conditions including, for example, pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, and electrolyte concentration.

[0193] Different organisms occupy different "niches" in the biome, as defined / allowed by the relationships structure. Niches as durable features of the biome ecosystem. Each niche has boundary conditions; a virtual shape or "footprint" reflecting the shape, which is discussed in the context of the "Hutchinsonian niche".

[0194] The Hutchinsonian niche is an n-dimensional hypervolume, where the dimensions are environmental conditions and resources, that define the requirements of an individual or a species to practice "its" way of life, more particularly, for its population to persist. The "hypervolume" defines the multi-dimensional space of resources (e.g., light, nutrients, structure, etc.) available to (and specifically used by) organisms, and "all species other than those under consideration are regarded as part of the coordinate system."

[0195] A niche is a very specific segment of ecospace occupied by a single species. On the presumption that no two species are identical in all respects (i.e., Hardin's 'axiom of inequality') and the competitive exclusion principle, some resource or adaptive dimension will provide a niche specific to each species.

[0196] Niches are exclusive. Each organism competes with similar organisms for that niche, and the successful organism fills that niche. Two organisms do not / cannot fill the same niche because one will out-compete the other over time. Therefore, the coexistence of two organisms in the same biome over extended time periods means they do not fill the same niche.

[0197] Once a niche is left vacant, other organisms can fill that position. This is because one species does not have the same footprint as another species, so one species cannot fill the same niche as another species. Successful replacement requires that the same organism (e.g., same species or close variant) should be used to fill or durably replace within a niche. It is recognized that partial competition exists in the form of transient colonization / infection and is an observable phenomenon.

[0198] Partial competition for a single niche can occur. One organism can "narrow" the "niche width" of another by partial competition. This might be the case with Staphylococcus epidermidis vs. Staphylococcus aureus. S. epidermidis is a commensal bacterium that secretes a serine protease capable of disassembling preformed Staphylococcus aureus biofilms, when used in high enough concentrations. Sugimoto et al., J Bacteriol, 195(8) 1645-1655. However, there is an important distinction between an organism as a carrier of a toxic phenotypic expression (being temporarily massively overloaded by application at a site), vs that organism as a durable inhabitant of a niche that narrows or outcompetes the pathogen.

[0199] Interspecies co-colonization is a different phenomenon than the ability to durably fill and block an ecological niche. For example, Shu et al., 2013 demonstrate that fermentation of glycerol to form short chain fatty acids (SCFA) with Cutibacterium acnes (C. acnes), a skin commensal bacterium that can inhibit growth of USA300, the most prevalent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Shu demonstrates that SCFAs produced by C. acnes under anaerobic conditions inhibits Staphylococcus aureus growth in high concentrations. Shu et al., 2013 PLoS ONE 8(2): e55380. However, these bacteria and this fermentation capability of C. acnes are already present in the normal human skin biome without there being effective eradication or diminution of Staphylococcus aureus pathogenicity. There is not any reason to believe that a hyper-physiologic application of these substrates would accomplish the goal of reduction of Staphylococcus aureus colonization or incidence of disease.Decolonization / Recolonization

[0200] A method is provided to prevent or decrease recurrence of a pathogenic infection of a undesirable microorganism in a subject, comprising the steps of (i) suppressing (decolonizing) the undesirable microorganism on at least one site in the subject to reduce or eliminate the presence of the undesirable microorganism from the site; and (ii) replacing the undesirable microorganism by administering to the subject at the at least one site a synthetic second microorganism having the same genus and species as the undesirable microorganism. Optionally, the method further comprises (iii) promoting colonization of the synthetic microorganism, for example, at the site of administration.

[0201] In some embodiments, the undesirable microorganism is a pathogenic microorganism and the term suppress (S) refers to a process of suppressing, reducing or eliminating the pathogenic microorganism at one or more, two or more, three or more, four or more sites in a subject. For example, the undesirable microorganism may be subject to nasal, mucosal, and / or dermal decolonization protocols.

[0202] The term replace (R) refers to replacing the pathogenic microorganism with a synthetic microorganism that is benign, drug-susceptible, and / or incapable of causing systemic or pathogenic infection in the subject. The replacement microorganism may be a molecularly modified synthetic microorganism of the same species as the pathogenic microorganism. The synthetic microorganism may be a molecularly modified microorganism of the same species, different strain, as the pathogenic microorganism, such that the synthetic microorganism is able to colonize the site on the subject, but is unable to cause systemic infection in the subject. By filling the vacated niche of the pathogenic microorganism, the synthetic microorganism is able to eliminate re-colonization by the pathogenic microorganism in the subject and thereby decrease or eliminate recurrence of pathogenic infection.

[0203] The term promote (P) refers to methods and compositions to promote replacement synthetic microorganism in the subject, for example, by employing prebiotics and biome management, for example, by employing a biome modulator in order to promote and support the new biome comprising the synthetic microorganism.

[0204] These methods broadly define a platform technology (SRP), with specifically designed protocols developed to address specific medical conditions (e.g. MRSA). If the processes of S, R, and P are selected properly - opening and then filling and sustaining a specific biome niche - a "durable" persistent biome is created that is capable of repelling pathogenic colonization.

[0205] A method is provided to decrease recurrence or chance of systemic infection of a pathogenic microorganism in a subject, the method comprising suppressing the pathogenic microorganism on the subject to significantly reduce or eliminate the detectable presence of the pathogenic microorganism; and replacing the pathogenic microorganism by administering a synthetic microorganism to the subject, wherein the synthetic microorganism is capable of occupying the same niche as the pathogenic microorganism as evidenced by (1) having the same genetic background, or genus and species, as the pathogenic microorganism, and / or by (2) exhibiting durable detectable presence on the subject for at least 60 days following replacement. The method may include promoting the colonization of the synthetic microorganism on at least one site in the subject. In some cases, the subject may have been found to be colonized by the pathogenic microorganism.

[0206] Frequently, systemic infection of a subject with a pathogenic microorganism is preceded by colonization of the pathogenic microorganism in the subject. For example, a substantial proportion of cases of Staphylococcus aureus bacteremia appear to be of endogenous origin since they may originate from colonies in the nasal mucosa. For example, in one multicenter study of Staphylococcus aureus bacteremia, the blood isolates were identical to those from the anterior nares in 180 of 219 patients (82.2%). In a second study, 14 of 1278 patients who had nasal colonization with Staphylococcus aureus subsequently had Staphylococcus aureus bacteremia. In 12 of these 14 patients (86%), the isolates obtained from the nares were clonally identical to the isolates obtained from blood 1 day to 14 months later. See von Eiff et al., 2001, NEJM, vol. 344, No. 1, 11-16. Another study showed the relative risk of Staphylococcus aureus bacteremia was increased multi-fold in nasal carriers when compared to non-carriers, reporting an 80% match between the invasive isolate and previously found colonizing strain. Wertheim et al., Lancet 2004; 364: 703-705.

[0207] In some embodiments, the subject is found to be colonized with the pathogenic strain of the microorganism prior to systemic infection. In other embodiments, the subject may have been colonized or infected by a nosocomial (hospital-acquired) strain or community-acquired strain of a pathogenic microorganism.

[0208] The pathogenic microorganism may be a wild-type microorganism, and / or a pathogenic microorganism that may be colonized or detectably present in at least one site in the subject. The site may be a dermal or mucosal site in the subject. The one or sites of colonization may include skin and soft tissue including, but are not limited to, nares, throat, perineum, inguinal region, vagina, nasal, groin, perirectal area, finger webs, forehead, pharynx, axillae, hands, chest, abdomen, head, and / or toe webs.

[0209] The pathogenic microorganism may be a drug resistant microorganism. The Centers for Disease Control (CDC) recently published a report outlining the top 18 drug-resistant threats to the United States, see www.cdc.gov / drugresistance / biggest_threats. In some embodiments, the undesirable microorganism is selected from Neisseria gonorrhoeae, fluconazole-resistant Candida, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus, drug-resistant Streptococcus pneumoniae and drug-resistant tuberculosis, erythromycin-resistant Group A Streptococcus, and clindamycin-resistant Group B Streptococcus.

[0210] In some embodiments, the pathogenic microorganism is a MRSA.

[0211] The synthetic microorganism (a) must be able to fill the ecological niche in the at least one site in the subject so as to durably exclude the undesirable microorganism following suppression; and (b) must have at least one molecular modification comprising a first cell death gene operably linked to a first regulatory region comprising a first promoter that is activated (induced) by a change in state in the environment compared to the normal physiological conditions in at least one site in the subject.

[0212] The synthetic microorganism may be of the same genus and species as the undesirable microorganism, in order to enhance the ability to fill the niche and durably exclude the undesirable microorganism in at least one site in the subject.

[0213] In some embodiments, the disclosure provides a synthetic microorganism that is not a pathogen and cannot become an accidental pathogen because it does not have the ability to infect the subject upon change in state, e.g., upon exposure to blood or serum. The synthetic microorganism comprises at least one molecular modification comprising a first cell death gene operably linked to a first regulatory region comprising a first promoter that is activated (induced) by a change in state in the environment compared to the normal physiological conditions in at least one site in the subject. For example, if the site in the subject is a dermal or mucosal site, then exposure to blood or serum is a change in state resulting in cell death of the synthetic microorganism. For example, average cell death of the synthetic microorganism may occur within 6 hours, 5 hours, 4 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes or 1 minute following change of state. The change in state may be a change in one or more of the following conditions: pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, and / or electrolyte concentration from that in at least one site in a subject. In some embodiments, the change in state is a higher concentration of blood, serum, or plasma compared to normal physiological conditions at the at least one site in the subject.

[0214] In one embodiment, the pathogenic microorganism is a MRSA. MRSA is a variant subgroup of Staphylococcus aureus. MRSA strains typically include a mecA cassette that allows production of an alternate penicillin binding protein that render them resistant to treatment with most beta-lactam and other first-line antibiotics. Staphylococcus aureus as a whole (including MRSA) is present as part of the normal microbiome of approximately 30% of the total human population. As part of the microbiome Staphylococcus aureus lives most commonly on the surface of the skin and in the anterior nasal vestibules, but can also be found in smaller amounts in the deep oropharynx and gastrointestinal tract and as part of the normal vaginal flora in some individuals.

[0215] In the vast majority of individuals Staphylococcus aureus remains a non-invasive commensal bacterium merely occupying an ecologic niche and not causing disease. The colonization state is far more common than that of invasive disease - some researchers estimate this ratio to be on the order of 1000 to one. Laupland et al., J Infect Dis (2008) 198:336. However, in a fraction of those colonized this bacterium can cause disease either opportunistically or as a result of increased tendencies toward invasion due to the acquisition of genetic cassettes coding for virulence protein products that allow such strains to more effectively invade through the epidermal or mucosal tissue layers initiating deep infection. In both above circumstances, the presence of the mecA cassette limits the treatment options for these patients and a number of studies have documented the increased mortality rate associated with MRSA when compared to MSSA in bacteremia, endovascular infection and pneumonia.Definitions

[0216] The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0217] The term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0218] The terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflicting terminology, the present specification is controlling.

[0219] The term "pathogen" or "pathogenic microorganism" refers to a microorganism that is capable of causing disease. A pathogenic microorganism may colonize a site on a subject and may subsequently cause systemic infection in a subject. The pathogenic microorganism may have evolved the genetic ability to breach cellular and anatomic barriers that ordinarily restrict other microorganisms. Pathogens may inherently cause damage to cells to forcefully gain access to a new, unique niche that provides them with less competition from other microorganisms, as well as with a ready new source of nutrients. Falkow, Stanley, 1998 Emerging Infectious Diseases, Vol. 4, No. 3, 495-497. The pathogenic microorganism may be a drug-resistant microorganism.

[0220] The term "virulent" or "virulence" is used to describe the power of a microorganism to cause disease.

[0221] The term "commensal" refers to a form of symbioses in which one organism derives food or other benefits from another organism without affecting it. Commensal bacteria are usually part of the normal flora.

[0222] The term "suppress" or "decolonize" means to substantially reduce or eliminate the original undesired pathogenic microorganism by various means (frequently referred to as "decolonization"). Substantially reduce refers to reduction of the undesirable microorganism by greater than 90%, 95%, 98%, 99%, or greater than 99.9% of original colonization by any means known in the art.

[0223] The term "replace" refers to replacing the original pathogenic microorganism by introducing a new microorganism (frequently referred to as "recolonization") that "crowds out" and occupies the niche(s) that the original microorganism would ordinarily occupy, and thus preventing the original undesired microorganism from returning to the microbiome ecosystem (frequently referred to as "interference" and "non-co-colonization").

[0224] The term "durably replace", "durably exclude", "durable exclusion", or "durable replacement", refers to detectable presence of the new synthetic microorganism for a period of at least 30 days, 60 days, 84 days, 120 days, 168 days, or 180 days after introduction of the new microorganism to a subject, for example, as detected by swabbing the subject. In some embodiments, "durably replace", "durably exclude", "durable exclusion", or "durable replacement" refers to absence of the original pathogenic microorganism for a period of at least 30 days, 60 days, 84 days, 120 days, 168 days, or 180 days after introduction of the new synthetic microorganism to the subject, for example, absence as detected over at least two consecutive plural sample periods, for example, by swabbing the subject.

[0225] The term "promote", or "promoting", refers to activities or methods to enhance the colonization and survival of the new organism, for example, in the subject. For example, promoting colonization of a synthetic bacteria in a subject may include administering a nutrient, prebiotic, and / or probiotic bacterial species.

[0226] The terms "prevention", "prevent", "preventing", "prophylaxis" and as used herein refer to a course of action (such as administering a compound or pharmaceutical composition of the present disclosure) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition. Such preventing and suppressing need not be absolute to be useful.

[0227] The terms "treatment", "treat" and "treating" as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.

[0228] The term "in need of treatment" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method, compound or pharmaceutical composition of the disclosure.

[0229] The term "in need of prevention" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a condition that is preventable by a method, compound or pharmaceutical composition of the disclosure.

[0230] The term "individual", "subject" or "patient" as used herein refers to any animal, including birds or mammals, such as mice, Norway rats, cotton rats, gerbils, cavies, hamsters, other rodents, rabbits, dogs, cats, swine, cattle, sheep, goat, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female. In one aspect, the patient is an adult human. In another aspect, the patient is a non-neonate human infant. In another aspect, the patient is a human toddler, child, or adolescent. In some embodiments, the subject is found to be colonized with a pathogenic strain of a microorganism prior to a systemic infection, or the subject may have been colonized or infected by a nosocomial (hospital-acquired) strain or community-acquired strain of a pathogenic microorganism.

[0231] The term "neonate", or newborn, refers to an infant in the first 28 days after birth. The term "non-neonate" refers to an animal older than 28 days.

[0232] The term "effective amount" as used herein refers to an amount of an agent, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state or condition. Such effect need not be absolute to be beneficial.

[0233] The term "measurable average cell death" refers to the inverse of survival percentage for a microorganism determined at a predefined period of time after introducing a change in state compared to the same microorganism in the absence of a change in state under defined conditions. The survival percentage may be determined by any known method for quantifying live microbial cells. For example, survival percentage may be calculated by counting cfus / mL for cultured synthetic microorganism cells and counting cfus / mL of uninduced synthetic microorganism cells at the predefined period of time, then dividing cfus induced / mL by cfus / mL uninduced x 100 = x % survival percentage. The measurable average cell death may be determined by 100%- x% survival percentage = y% measurable average cell death. For example, wherein the survival percentage is 5%, the measurable average cell death is 100%-5%= 95%. Any method for counting cultured live microbial cells may be employed for calculation of survival percentage including cfu, OD600, flow cytometry, or other known techniques. Likewise, an induced synthetic strain may be compared to a wild-type target microorganism exposed to the same conditions for the same period of time, using similar calculations to determine a "survival rate" wherein 100%-survival rate = z % "reduction in viable cells".

[0234] The term "including" as used herein is non-limiting in scope, such that additional elements are contemplated as being possible in addition to those listed; this term may be read in any instance as "including, but not limited to."

[0235] The term "animal" refers to the animal kingdom definition.

[0236] The term "substantial identity" or "substantially identical," when referring to a nucleotide or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleotide (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleotide molecule having substantial identity to a reference nucleotide molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleotide molecule.

[0237] The term "derived from" when made in reference to a nucleotide or amino acid sequence refers to a modified sequence having at least 50% of the contiguous reference nucleotide or amino acid sequence respectively, wherein the modified sequence causes the synthetic microorganism to exhibit a similar desirable atttribute as the reference sequence of a genetic element such as promoter, cell death gene, antitoxin gene, virulence block, or nanofactory, including upregulation or downregulation in response to a change in state, or the ability to express a toxin, antitoxin, or nanofactory product, or a substantially similar sequence, the ability to transcribe an antisence RNA antitoxin, or the ability to prevent or diminish horizontal gene transfer of genetic material from the undesirable microorganism. The term "derived from" in reference to a nucleotide sequence also includes a modified sequence that has been codon optimized for a particular microorganism to express a substantially similar amino acid sequence to that encoded by the reference nucleotide sequence. The term "derived from" when made in reference to a microorganism, refers to a target microorganism that is subjected to a molecular modification to obtain a synthetic microorganism.

[0238] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide or protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0239] The term "conservative amino acid substitution" refers to wherein one amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties, such as charge or hydrophobicity. In general, a conservative amino acid substitution will not substantially change the functional properties of the, e.g., toxin or antitoxin protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0240] Polypeptide sequences may be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see, e.g., Pearson, W.R., Methods Mol Biol 132: 185-219 (2000), herein incorporated by reference). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., J Mol Biol 215:403-410 (1990) and Altschul et al., Nucleic Acids Res 25:3389-402 (1997).

[0241] Unless otherwise indicated, nucleotide sequences provided herein are presented in the 5' - 3' direction.

[0242] All pronouns are intended to be given their broadest meaning. Unless stated otherwise, female pronouns encompass the male, male pronouns encompass the female, singular pronouns encompass the plural, and plural pronouns encompass the singular.

[0243] The term "systemic administration" refers to a route of administration into the circulatory system so that the entire body is affected. Systemic administration can take place through enteral administration (absorption through the gastrointestinal tract, e.g. oral administration) or parenteral administration (e.g., injection, infusion, or implantation).

[0244] The term "topical administration" refers to application to a localized area of the body or to the surface of a body part regardless of the location of the effect. Typical sites for topical administration include sites on the skin or mucous membranes. In some embodiments, topical route of administration includes enteral administration of medications or compositions.

[0245] The term "undesirable microorganism" refers to a microorganism which may be a pathogenic microorganism, drug-resistant microorganism, antibiotic-resistant microorganism, irritation-causing microorganism, odor-causing microorganism and / or may be a microorganism comprising an undesirable virulence factor.

[0246] The term "synthetic microorganism" refers to an isolated microorganism modified by any means to comprise at least one element imparting a non-native attribute. For example, the synthetic microorganism may be engineered to include a molecular modification comprising an addition, deletion and / or modification of genetic material to incorporate a non-native attribute. In some embodiments, the synthetic microorganism is not an auxotroph.

[0247] The term "detectable presence" of a microorganism refers to a confirmed positive detection in a sample of a microorganism genus, species and / or strain by any method known in the art. Confirmation may be a positive test interpretation by a skilled practitioner and / or by repeating the method.

[0248] The term "microbiome" or "microbiomic" or "microbiota" as used herein refers to microbiological ecosystems. These ecosystems are a community of commensal, symbiotic and pathogenic microorganisms found in and on all animals and plants.

[0249] The term "microorganism" as used herein refers to an organism that can be seen only with the aid of a microscope and that typically consists of only a single cell. Microorganisms include bacteria, protozoans and fungi.

[0250] The term "niche" and "niche conditions" as used herein refers to the ecologic array of environmental and nutritional requirements that are required for a particular species of microorganism. The definitions of the values for the niche of a species defines the places in the particular biomes that can be physically occupied by that species and defines the possible microbial competitors.

[0251] The term "colonization" as used herein refers to the persistent detectable presence of a microorganism on a body surface, e.g., a dermal or mucosal surface, without causing disease in the individual.

[0252] The term "co-colonization" as used herein refers to simultaneous colonization of a niche in a site on a subject by two or more strains, or variants within the same species of microorganisms. For example, the term "co-colonization" may refer to two or more strains or variants simultaneously and non-transiently occupying the same niche. The term non-transiently refers to positive identification of a strain or variant at a site in a subject over time at two or more time subsequent points in a multiplicity of samples obtained from the subject at least two weeks apart.

[0253] The term "bacterial replacement" or "non-co-colonization" as used herein refers to the principle that only one variant / strain of one species can occupy any given niche within the biome at any given time.

[0254] The term "kill switch" or "KS" as used herein refers to an intentional molecular modification of a synthetic microorganism, the molecular modification comprising a cell death gene operably linked to a regulatory region comprising an inducible promoter, genetic element or cassette, wherein induced expression of the cell death gene in the kill switch causes cell death, arrest of growth, or inability to replicate, of the microorganism in response to a specific state change such as a change in environmental condition of the microorganism. For example, in the synthetic microorganism comprising a kill switch, the inducible first promoter may be activated by the presence of blood, serum, plasma, and / or heme, wherein the upregulation and transcription / expression of the operably associated cell death gene results in cell death of the microorganism or arrested growth of the microorganism so as to improve the safety of the synthetic microorganism.

[0255] The term "cell death gene" refers to a gene that when induced causes a cell to enter a state where it either ceases reproduction, alters regulatory mechanisms of the cell sufficiently to permanently disrupt cell viability, induces senescence, or induces fatal changes in the genetic or proteomic systems of the cell. For example, the cell death gene may be a toxin gene encoding a toxin protein or toxin peptide.

[0256] The term "antitoxin gene" refers to a gene encoding an antitoxin RNA antisense molecule or an antitoxin protein or another antitoxin molecule specific for a cell death gene or a product encoded thereby

[0257] The term "virulence block" or "V-block" refers to a molecular modification of a microorganism that results in the organism have decreased ability to accept foreign DNA from other strains or species effectively resulting in the organism having decreased ability to acquire exogenous virulence or antibiotic resistance genes.

[0258] The term "nanofactory" as used herein refers to the molecular modification of a microorganism that results in the production of a product - either primary protein, polypeptide, amino acid or nucleic acid or secondary products of these modifications to beneficial effect.

[0259] The term "toxin protein" or "toxin peptide" as used herein refers to a substance produced internally within a synthetic microorganism in an effective amount to cause deleterious effects to the microorganism without causing deleterious effects to the subject that it colonizes.

[0260] The term "molecular modification" or "molecularly engineered" as used herein refers to an intentional modification of the genes of a microorganism using any gene editing method known in the art, including but not limited to recombinant DNA techniques as described herein below, NgAgo, mini-Cas9, CRISPR-Cpf1, CRISPR-C2c2, Target-AID, Lambda Red, Integrases, Recombinases, or use of phage techniques known in the art. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more elements, e.g., regulatory regions, promoters, toxin genes, antitoxin genes, or other domains into a suitable configuration, or to introduce codons, delete codons, optimize codons, create cysteine residues, modify, add or delete amino acids, etc. Molecular modifcation may include, for example, use of plasmids, gene insertion, gene knock-out to excise or remove an undesirable gene, frameshift by adding or subtracting base pairs to break the coding frame, exogenous silencing, e.g., by using inducible promoter or constitutive promoter which may be embedded in DNA encoding, e.g. RNA antisense antitoxin, production of CRISPR-cas9 or other editing proteins to digest, e.g., incoming virulence genes using guide RNA, e.g., linked to an inducible promoter or a constitutive promoter, or a restriction modification / methylation system, e.g., to recognize and destroy incoming virulence genes to increase resistance to horizontal gene transfer. The molecular modification (e.g. kill switch, expression clamp, and / or v-block) may be durably incorporated to the synthetic microorganism by inserting the modification into the genome of the synthetic microorganism.

[0261] The synthetic microorganism may further comprise additional molecular modifications, (e.g., a nanofactory), which may be incorporated directly into the bacterial genome, or into plasmids, in order to tailor the duration of the effect of, e.g., the nanofactory production, and could range from short term (with non-replicating plasmids for the bacterial species,) to medium term (with replicating plasmids without addiction dependency) to long term (with direct bacterial genomic manipulation).

[0262] The molecular modifications may confer a non-native attribute desired to be durably incorporated into the host microbiome, may provide enhanced safety or functionality to organisms in the microbiome or to the host microbiome overall, may provide enhanced safety characteristics, including kill switch(s) or other control functions. In some embodiments the safety attributes so embedded may be responsive to changes in state or condition of the microorganism or the host microbiome overall.

[0263] The molecular modification may be incorporated to the synthetic microorganism in one or more, two or more, five or more, 10 or more, 30 or more, or 100 or more copies, or no more than one, no more than three, no more than five, no more than 10, no more than 30, or in no more than 100 copies.

[0264] The term "recurrence" as used herein refers to re-colonization of the same niche by a decolonized microorganism.

[0265] The term "pharmaceutically acceptable" refers to compounds, carriers, excipients, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0266] The term "pharmaceutically acceptable carrier" refers to a carrier that is physiologically acceptable to the treated subject while retaining the integrity and desired properties of the synthetic microorganism with which it is administered. Exemplary pharmaceutically acceptable carriers include physiological saline or phosphate-buffered saline. Other physiologically acceptable carriers and their formulations are provided herein or are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0267] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0268] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0269] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.Vectors and Target Microorganisms

[0270] Also described herein are vectors comprising polynucleotide molecules, as well as target cells transformed with such vectors. Polynucleotide molecules described herein may be joined to a vector, which include a selectable marker and origin of replication, for the propagation host of interest. Target cells are genetically engineered to include these vectors and thereby transcribe RNA and express polypeptides. Vectors herein include polynucleotides molecules operably linked to suitable transcriptional or translational regulatory sequences, such as those for microbial target cells. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences as described herein are operably linked when the regulatory sequences herein functionally relate to, e.g., a cell death gene encoding polynucleotide.

[0271] Typical vehicles include plasmids, shuttle vectors, baculovirus, inactivated adenovirus, and the like. In certain examples described herein, the vehicle may be a modified pIMAY, pIMAYz, or pKOR integrative plasmid, as dicussed herein.

[0272] A target microorganism may be selected from any microorganism having the ability to durably replace a specific undesirable microorganism after decolonization. The target microorganism may be a wild-type microorganism that is subsequently engineered to enhance safety by methods described herein. The target microorganism may be selected from a bacterial, fungal, or protozoal target microorganism. The target microorganism may be a strain capable of colonizing a dermal and / or mucosal niche in a subject. The target microorganism may be a wild-type microorganism, or a synthetic microorganism that may be subjected to further molecular modification. The target microorganism may be selected from a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas. The target microorganism may be selected from the group consisting of Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissimum, Cutibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Escherichia coli, Streptococcus pyogenes, Streptococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcal intermedius, Streptococcus agalactiae, Streptococcus mutans, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

[0273] The target microorganism may be of the same genus and species as the undesirable microorganism, but of a different strain. For example, the undesirable microorganism may be an antibiotic-resistant Staphylococcus aureus strain, such as an MRSA strain. The antibiotic-resistant Staphylococcus aureus stain may be a pathogenic strain, which may be known to be involved in dermal infection, mucosal infection, bacteremia, and / or endocarditis. Where the undesirable microorganism is a Staphylococcus aureus strain, e.g., an MRSA, the target microorganism may be, e.g., a less pathogenic strain which may be an isolated strain such as Staphylococcus aureus target cell such as an RN4220 or 502a strain, and the like. Alternatively, the target cell may be of the same strain as the undesirable microorganism. In another example, the undesirable microorganism is an Escherichi coli strain, for example, a uropathogenic E. coli type 1 strain or p-fimbriated strain, for example, a strain involved in urinary tract infection, bacteremia, and / or endocarditis. In another example, the undesirable strain is a Cutibacterium acnes strain, for example a strain involved in acnes vulgaris, bacteremia, and / or endocarditis. In another example, the undesirable microorganism is a Streptococcus mutans strain, for example, a strain involved in S. mutans endocarditis, dental caries.Model Antibiotic-Susceptible Target Microorganism

[0274] The target microorganism may be an antibiotic-susceptible microorganism of the same species as the undesirable microorganism. In one embodiment, the undesirable microorganism is an MRSA strain and the replacement target microorganism is an antibiotic susceptible Staphylococcus aureus strain. The antibiotic susceptible microorganism may be Staphylococcus aureus strain 502a ("502a"). 502a is a coagulase positive, penicillin sensitive, nonpenicillinase producing staphylococcus, usually lysed by phages 7, 47, 53, 54, and 77. Serologic type (b)ci. Unusual disc antibiotic sensitivity pattern is exhibited by 502a because this strain is susceptible to low concentrations of most antibiotics except tetracycline; resistant to 5 µg, but sensitive to 10 µg of tetracycline. In some embodiments, the 502a strain may be purchased commercially as Staphylococcus aureus subsp. Aureus Rosenbach ATCC ®< 27217 ™< .

[0275] Unfortunately, even an antimicrobial agent-susceptible target microorganism may cause systemic infection. Therefore, as provided herein, the target microorganism is subjected to molecular modification to incorporate regulatory sequences including, e.g., an inducible first promoter for expression of the cell death gene, v-block, or nanofactory, in order to enhance safety and reduce the likelihood of pathogenic infection as described herein.

[0276] The target microorganism and / or the synthetic microorganism comprises (i) the ability to durably colonize a niche in a subject following decolonization of the undesirable microorganism and administering the target or synthetic microorganism to a subject, and (ii) the ability to prevent recurrence of the undesirable microorganism in the subject for a period of at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 24 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks after the administering step.Selection of a Target Microorganism for MRSA

[0277] Selection of the target microorganism may be performed by decolonizing the target microorganism and replacing with a putative target microorganism, as described herein. For example, the undesirable microorganism Methicillin-Resistant Staphylococcus aureus (MRSA) is the cause of a disproportionate amount of invasive bacterial infections worldwide. The colonization state for Staphylococcus aureus is regarded as a required precondition for most invasive infections. However, decolonization with standard antiseptic regimens has been studied as a method for reducing MRSA colonization and infections with mixed results. In one example provided herein, the feasibility and durability of a novel decolonization approach to undesirable microorganism MRSA by using intentional recolonization with a different Staphylococcus aureus strain as a candidate target microorganism was performed in hopes of improving duration of effect versus standard decolonization. Example 1 discloses the study in which a total of 765 healthy volunteers were screened for Staphylococcus aureus colonization. The overall MRSA rate for the screened population was 8.5%. A cohort of 53 MRSA colonized individuals participated in a controlled study of a decolonization / recolonization therapy using Staphylococcus aureus 502a WT strain BioPlx-01 vs. a control group of standard decolonization alone. Duration of MRSA absence from the colonization state as well as persistence of the intentional MSSA recolonization was monitored for 6 months. The control group (n=15) for the efficacy portion of the MRSA decolonization protocol showed MRSA recurrence of 60% at the 4 week time point. The test group employing the BioPlx-01WT protocol (n=34) showed 0% MRSA recurrence at the 8 week primary endpoint and continued to show no evidence of MRSA recurrence out to 26 weeks. Instead these participants exhibited surprising persistence of colonization with MSSA likely indicating ongoing colonization with the Staphylococcus aureus 502a BioPlx-01WT strain product out to 26 weeks. In addition, the components of the BioPlx-01WT in a phosphate buffered saline composition used in the decolonization / recolonization therapy showed no evidence of dermal irritation in a separate cohort of 55 participants. Therefore, target strain Staphylococcus aureus 502a BioPlx-01WT decolonization / recolonization protocol provides longer durability of decolonization from MRSA strains than standard decolonization and shows no observed negative dermal effects.Methods for determining detectable presence of a microorganism

[0278] Any method known in the art may be employed for determination of the detectable presence of a microorganism genus, species and strain. An overview of methods may be found in Aguilera-Arreola MG. Identification and Typing Methods for the Study of Bacterial Infections: a Brief Review and Mycobacterial as Case of Study. Arch Clin Microbiol. 2015, 7:1,

[0279] The detectable presence of a genus, species and / or strain of a bacteria may be determined by phenotypic methods and / or genotypic methods. Phenotypic methods may include biochemical reactions, serological reactions, susceptibility to anti-microbial agents, susceptibility to phages, susceptibility to bacteriocins, and / or profile of cell proteins. One example of a biochemical reaction is the detection of extracellular enzymes. For example, staphylococci produce many different extracellular enzymes including DNAase, proteinase and lipases. Gould, Simon et al., 2009, The evaluation of novel chromogenic substrates fro detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups. FEMS Microbiol Let 297; 10-16. Chomogenic substrates may be employed for detection of extracellular enzymes. For example, CHROMager ™< MRSA chromogenic media (CHROMagar, Paris, France) may be employed for isolation and differentiation of Methicillin Resistant Staphylococcus aureus (MRSA) including low level MRSA. Samples are obtained from, e.g., nasal, perineal, throat, rectal specimens are obtained with a possible enrichment step. If the agar plate has been refrigerated, it is allowed to warm to room temperature before inoculation. The sample is streaked onto plate followed by incubation in aerobic conditions at 37 °C for 18-24 hours. The appearance of the colonies is read, wherein MRSA colonies appear as rose to mauve colored, Methicillin Susceptible Staphylococcus aureus (MSSA) colonies are inhibited, and other bacteria appear as blue, colorless or inhibited colonies. Definite identification as MRSA requires, in addition, a final identification as Staphylococcus aureus. For example, CHROMagar ™< Staph aureus chromogenic media may be employed where S. aurues appears as mauve, S. saprophyticus appears turquoise blue, E. coli, C. albicans and E. faecalis are inhibited. For detection of Group B Streptococcus(GBS) (S. agalactiae), CHROMagar ™< StrepB plates may be employed, wherein Streptococcus agalactiae (group B) appear mauve, Enterococcus spp. and E. faecalis appear steel blue, Lactobacilli, leuconostoc and lactococci appear light pink, and other microorganisms are blue, colorless or inhibits. For detection of various Candida spp., CHROMager ™< Candida chromogenic media may be employed. Candida species are involved in superficial oropharyngeal and urogenital infections. Although C. albicans remains a major species involved, other types such as C. tropicalis, C. krusai, or C. glabrata have increased as new antifungal agents have worked effectively against C. albicans. Sampling and direct streaking of skin, sputum, urine, vaginal specimens samples and direct streaking or spreading onto plate, followed by incubation in aerobic conditions at 30-37 °C for 48 hours, and reading of plates for colony appearance where C. albicans is green, C. tropicalis is metallic blue, C. krusei is pink and fuzzy, C. kefyr and C. glabrata are mauve-brown, and other species are white to mauve.

[0280] Genotypic methods for genus and species identification may include hybridization, plasmids profile, analysis of plasmid polymorphism, restriction enzymes digest, reaction and separation by Pulsed-Field Gel Electrophoresis (PFGE), ribotyping, polymerase chain reaction (PCR) and its variants, Ligase Chain Reaction (LCR), Transcription-based Amplification System (TAS), or any of the methods described herein.

[0281] Identification of a microbe can be performed, for example, by employing Galileo ™< Antimicrobial Resistance (AMR) detection software (Arc Bio LLC, Menlo Park, CA and Cambridge, MA) that provides annotations for gram-negative bacterial DNA sequences.

[0282] The microbial typing method may be selected from genotypic methods including Multilocus Sequence Typing (MLST) which relies on PCR amplification of several housekeeping genes to create allele profiles; PCR-Extragenic Palindromic Repetitive Elements (rep-PCR) which involves PCR amplification of repeated sequences in the genome and comparison of banding patterns; AP-PCR which is Polymerase Chain Reaction using Arbitrary Primers; Amplified Fragment Length Polymorphism (AFLP) which involves enzyme restriction digestion of genomic DNA, binding of restriction fragments and selective amplification; Polymorphism of DNA Restriction Fragments (RFLP) which involves Genomic DNA digestion or of an amplicon with restriction enzymes producing short restriction fragments; Random Amplified Polymorphic DNA (RAPD) which employs marker DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence; Multilocus Tandem Repeat Sequence Analysis (MLVA) which involves PCR amplification of loci VTR, visualizing the polymorphism to create an allele profile; or Pulsed-Fields Gel Electrophoresis (PFGE) which involves comparison of macro-restriction fragments. PFGE method of electrophoresis is capable of separating fragments of a length higher than 50 kb up to 10 Mb, which is not possible with conventional electrophoresis, which can separate only fragments of 100 bp to 50 kb. This capacity of PFGE is due to its multidirectional feature, changing continuously the direction of the electrical field, thus, permitting the re-orientation of the direction of the DNA molecules, so that these can migrate through the agarose gel, in addition to this event, the applied electrical pulses are of different duration, fostering the reorientation of the molecules and the separation of the fragments of different size. One PFGE apparatus may be the Contour Clamped Homogeneous Electric Fields (CHEF, BioRad). Pulsed-filed gel electrophoresis (PFGE) is considered a gold standard technique for MRSA typing, because of its high discriminatory power, but its procedure is complicated and time consuming. The spa gene encodes a cell wall component of Staphylococcus aureus protein A, and exhibits polymorphism. The sequence based-spa typing can be used as a rapid test screen. Narukawa et al 2009 Tohoku J Exp Med 2009, 218, 207-213.

[0283] Methods and compositions are provided herein for suppressing (decolonizing) and replacing an undesirable microorganism with a new synthetic microorganism in order to durably displace and replace the undesirable microorganism from the microbiological ecosystem with a new microorganism so as to prevent the recurrence of the original undesirable organism (referred to here as niche or ecological interference).

[0284] In some embodiments, methods are provided to prevent colonization, prevent infection, decrease recurrence of colonization, or decrease recurrence of a pathogenic infection of a undesirable microorganism in a subject, comprising decolonization and administering a synthetic strain comprising a molecular modification that decreases the ability of the synthetic microorganism to cause disease to the subject relative to the wild type target strain where the microorganism is selected from the group consisting of Acinetobacterjohnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissimum, Cutibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Streptococcus pyogenes, Streptococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcal intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

[0285] In some embodiments, a method is provided to prevent transmission by a subject, or recurrence of colonization or infection, of a pathogenic microorganism in a subject, comprising suppressing the pathogenic microorganism in the subject, and replacing the pathogenic microorganism by topically administering to the subject a composition comprising a benign microorganism of the same species, different strain. The method may further comprise promoting the colonization of the benign microorganism. In some embodiments, the benign microorganism is a synthetic microorganism having at least one molecular modification comprising a first cell death gene operably linked to a first regulatory region comprising a first promoter, wherein the first promoter is activated in the presence of human serum or blood. In some embodiments, the first promoter is not activated during colonization of dermal or mucous membranes in a human subject.

[0286] In some embodiments, method is provided to prevent transmission by a subject, or recurrence of colonization or infection, of a methicillin-resistant Staphylococcus aureus (MRSA) in a subject, comprising suppressing the MRSA in the subject, and replacing the MRSA by topically administering to the subject a methicillin susceptible Staphylococcus aureus (MSSA) of the same species, different strain. The method may further comprise promoting the colonization of the MSSA in the subject.

[0287] A method is provided to prevent transmission by a subject, or recurrence of colonization or infection, of a undesirable microorganism in a subject, comprising suppressing the undesirable microorganism in the subject, and replacing the undesirable microorganism by administering to the subject a second microorganism of the same species, different strain. The method may further comprise promoting the colonization of the second microorganism. In some embodiments, the undesirable microorganism is a drug-resistant pathogenic microorganism. In some embodiments, the second microorganism is a drug-susceptible microorganism. In some embodiments, the second microorganism is a synthetic microorganism.Suppression / Decolonization

[0288] An undesirable microorganism may be supressed, or decolonized, by topically applying a disinfectant, antiseptic, or biocidal composition directly to the skin or mucosa of the subject, for example, by spraying, dipping, or coating the affected area, optionally the affected area and adjacent areas, or greater than 25%, 50%, 75%, or greater than 90% of the external or mucosal surface area of the subject with the disinfectant, antiseptic, or biocidal composition. In some embodiments, the affected area, or additional surface areas are allowed to air dry or are dried with an air dryer under gentle heat, or are exposed to ultraviolet radiation or sunlight prior to clothing or dressing the subject. In one embodiment, the suppression comprises exposing the affected area, and optionally one or more adjacent or distal areas of the subject, with ultraviolet radiation. In various embodiments, any commonly employed disinfectant, antiseptic, or biocidal composition may be employed. In one embodiment, a disinfectant comprising chlorhexidine or a pharmaceutically acceptable salt thereof is employed.

[0289] In some embodiments, the bacteriocide, antiseptic, astringent, and / or antibacterial agent is selected from the group consisting of alcohols (ethyl alcohol, isopropyl alcohol), aldehydes (glutaraldehyde, formaldehyde, formaldehyde-releasing agents (noxythiolin = oxymethylenethiourea, tauroline, hexamine, dantoin), o-phthalaldehyde), anilides (triclocarban = TCC = 3,4,4'-triclorocarbanilide), biguanides (chlorhexidine, alexidine, polymeric biguanides (polyhexamethylene biguanides with MW> 3,000 g / mol, vantocil), diamidines (propamidine, propamidine isethionate, propamidine dihydrochloride, dibromopropamidine, dibromopropamidine isethionate), phenols (fentichlor, p-chloro-m-xylenol, chloroxylenol, hexachlorophene), bis-phenols (triclosan, hexachlorophene), quaternary ammonium compounds (cetrimide, benzalkonium chloride, cetyl pyridinium chloride), silver compounds (silver sulfadiazine, silver nitrate), peroxy compounds (hydrogen peroxide, peracetic acid), iodine compounds (povidone-iodine, poloxamer-iodine, iodine), chlorine-releasing agents (sodium hypochlorite, hypochlorous acid, chlorine dioxide, sodium dichloroisocyanurate, chloramine-T), copper compounds (copper oxide), botanical extracts (Malaleuca spp. (tea tree oil), Cassia fistula Linn, Baekea frutesdens L., Melia azedarach L., Muntingia calabura, Vitis vinifera L, Terminalia avicennioides Guill & Perr., Phylantus discoideus muel. Muel-Arg., Ocimum gratissimum Linn., Acalypha wilkesiana Muell-Arg., Hypericum pruinatum Boiss.&Bal., Hypericum olimpicum L. and Hypericum sabrum L., Hamamelis virginiana (witch hazel), Eucalyptus spp., rosemarinus officinalis spp.(rosemary), Thymus spp.(thyme), Lippia spp. (oregano), Cymbopogon spp. (lemongrass), Cinnamomum spp., Geranium spp., Lavendula spp.), and topical antibiotic compounds (bacteriocins; mupirocin, bacitracin, neomycin, polymyxin B, gentamicin).

[0290] Suppression of the undesirable microorganism also may be performed by using photosensitizers instead of or in addition to, e.g., topical antibiotics. For example, Peng Zhang et al., Using Photosensitizers Instead of Antibiotics to Kill MRSA, GEN News Highlights, August 20, 2018; 48373, developed a technique using light to activate oxygen, which suppresses to microbial growth. Photosensitizers, such as dye molecules, become excited when illuminated with light. The photosensitizers convert oxygen into reactive oxygen species that kill the microbes, such as MRSA. In order to concentrate the photosensitizers to improve efficacy, water-dispersible, hybrid photosensitizers were developed by Zhang et al., comprising noble metal nanoparticles decorated with amphiphilic polymers to entrap molecular photosensitizers. The hybrid photosensitizers may be applied to a subject, for example, on a dermal surface or wound, in the form of a spray, lotion or cream, then illuminated with red or blue light to reduce microbial growth.

[0291] A decolonizing composition may be in the form of a topical solution, lotion, or ointment form comprising a disinfectant, biocide photosensitizer or antiseptic compound and one or more pharmaceutically acceptable carriers or excipients. In one specific example, an aerosol disinfectant spray is employed comprising chlorhexidine gluconate (0.4%), glycerin (10%), in a pharmaceutically acceptable carrier, optionally containing a dye to mark coverage of the spray. In one embodiment, the suppressing step comprises administration to one or more affected areas, and optionally one or more surrounding areas, with a spray disinfectant as disclosed in U.S. Pat. Nos. 4,548,807 and / or 4,716,032. The disinfectant spray may be commercially available, for example, Fight Bac ®< , Deep Valley Farm, Inc., Brooklyn, CT. Other disinfectant materials may include chlorhexidine or salts thereof, such as chlorhexidine gluconate, chlorhexidine acetate, and other diguanides, ethanol, SD alcohol, isopropyl alcohol, p-chloro-o-benzylphenol, o-phenylphenol, quaternary ammonium compounds, such as n-alkyl / dimethyl ethyl benzyl ammonium chloride / n-alkyl dimethyl benzyl ammonium choride, benzalkonium chloride, cetrimide, methylbenzethonium chloride, benzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, dofanium chloride, domiphen bromide, peroxides and permanganates such as hydrogen peroxide solution, potassium permanganate solution, benzoyl peroxide, antibacterial dyes such as proflavine hemisulphate, triphenylmethane, Brilliant green, Crystal violet, Gentian violet, quinolone derivatives such as hydroxyquinoline sulphate, potassium hydroxyquinoline sulphate, chlorquinaldol, dequalinium chloride, di-iodohydroxyquinoline, Burow's solution (aqueous solution of aluminum acetate), bleach solution, iodine solution, bromide solution. Various Generally Recognized As Safe (GRAS) materials may be employed in the disinfectant or biocidal composition including glycerin, and glycerides, for example but not limited to mono- and diglycerides of edible fat-forming fatty acids, diacetyl tartaric acid esters of mono- and diglycerides, triacetin, acettooleins, acetostearins, glyceryl lactopalmitate, glyceryl lactooleate, and oxystearins.

[0292] The suppression step -or decolonization- may be performed comprising administering 1-3 times daily, over a period of from 1 to 10 days; for example, on one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen days. In other embodiments, the suppression step may be administered from two, three, four, five, or six times, each administration from 6 to 48 hours, 8 to 40 hours, 18 to 36 hours, or about 20 to 28 hours apart. In specific embodiments, the suppression step is administered once per day from one to five, or three to four consecutive days. In some embodiments, the suppression step does not include systemic administration of antimicrobial agents. In some embodiments, the suppression step does not include systemic administration of antibiotic, antiviral, or antifungal agents. In other embodiments, the suppression step includes systemic administration of antimicrobial agents. In some embodiments, the suppression step may include systemic administration of one or more antibiotic, antiviral, or antifungal agents.Replace

[0293] Methods are provided wherein an undesirable microorganism is durably replaced with a synthetic microorganism. The synthetic microorganism has the ability to fill the same ecological niche and / or may be of the same species, different strain, as the pathogenic microorganism. By using same species, different strain, (or even the same strain) the environmental niche of the pathogenic microorganism may be filled, or durably replaced, with the benign synthetic microorganism.Synthetic Microorganism

[0294] In some embodiments, the undesirable pathogenic microorganism is replaced with a synthetic microorganism. For example, the replacement strain may be a synthetic microorganism that is a molecularly modified strain of the same species as the undesirable or pathogenic microorganism or the same strain as the undesirable or pathogenic microorganism.

[0295] In some embodiments, a synthetic microorganism comprising a "kill switch" is provided exhibiting rapid and complete cell death on exposure to blood or serum, but exhibits normal metabolism and colonization function in other environments. In some embodiments, the synthetic microorganism comprises stable and immobile kill switch genes. The minimal kill switch (KS) components include a regulatory region (RR) containing operator, promoter and translation signals, that is strongly activated in response to blood or serum exposure, a kill switch gene expressing a toxic protein or RNA, and a means of transcription termination. Chromosomal integration of the KS is preferred. The chromosomal locus may be in a transcriptionally inactive region, for example, an intergenic region (IR) between a seryl-tRNA synthetase and an amino acid transporter. Insertions here do not affect transcription of flanking genes (Lei et al., 2012). Preferably, no known sRNAs are present in the IR. Any other inert loci may be selected.The synthetic microorganism comprising a kill switch

[0296] In a particular embodiment, the pathogenic microorganism is an antimicrobial-resistant microorganism, and the replacement microorganism is a synthetic microorganism of the same species as the pathogenic microorganism. The synthetic microorganism may be a molecularly-modified, antibiotic-susceptible microorganism.

[0297] The synthetic microorganism may comprise one or more, two or more, or three or more molecular modifications comprising a first cell death gene operably linked to a first regulatory region comprising an inducible first promoter. Optionally, the synthetic microorganism further comprises a second cell death gene operably linked to the first regulatory region comprising the first promoter or a second regulatory region comprising an inducible second promoter. The first promoter, and optionally the second promoter, is activated (induced) by a change in state in the microorganism environment compared to the normal physiological conditions at the at least one site in the subject. For example, the change in state may be selected from one or more changes in pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, and electrolyte concentration. In some embodiments, the change in state is a higher concentration of blood, serum, or plasma compared to normal physiological conditions at the at least one site in the subject.

[0298] In one specific embodiment, the pathogenic microorganism is a MRSA and the replacement microorganism is a synthetic microorganism that is a molecularly modified Staphylococcus aureus coagulase positive strain. The synthetic microorganism may be a molecularly modified Staphylococcus aureus 502a, as described herein.

[0299] The use of live Staphylococcus aureus as a therapeutic platform raises safety concerns because this pathogen can cause serious disease if it gains access to the circulatory system. In one embodiment, the synthetic microorganism is molecularly engineered to comprise a "kill switch" (KS) and an inducible promoter that induces rapid bacterial death upon exposure to whole blood or serum. The kill switch may be composed of DNA encoding 3 main components: i) "control region", containing a promoter and other regulatory sequences, that is strongly activated by blood or serum; ii) a toxic RNA or polypeptide, whose expression is driven by the control region, and; iii) a transcription terminator. A cassette composed of these elements maybe integrated into the Staphylococcus aureus chromosome at a site(s) amenable to alteration without adversely affecting bacterial function.

[0300] It is desirable that basal or "leaky" expression of the control region is minimized or avoided. For example, if significant mRNA production occurs before exposure to blood or serum, the strain could be weakened during manufacturing or skin colonization and may accumulate mutations that bypass or escape the KS. To address this, candidates are screened to find those that are strongly induced in serum, but also have very low or undetectable mRNA expression in standard growth media in vitro. Despite this effort, some leaky expression may be observed, which may be controlled by further comprising a iv) "expression clamp" to prevent untimely toxin production.Recombinant Approach to Synthetic Microorganism

[0301] A synthetic microorganism is provided which comprises a recombinant nucleotide comprising at least one molecular modification (e.g., a kill switch) comprising (i) a cell death gene operatively associated with (ii) a first regulatory region comprising a first inducible promoter which is induced by a change in state in the environment of the synthetic microorganism. The synthetic microorganism may further comprises at least a second molecular modification (expression clamp) comprising (iii) an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is operably associated with (iv) a second regulatory region comprising a second promoter which is active (e.g., constitutive) upon dermal or mucosal colonization or in a media, and preferably is downregulated by change in state of the environment of the synthetic microorganism.

[0302] In some embodiments, a synthetic microorganism is provided comprising at least one molecular modification (e.g., a kill switch) comprising a first cell death gene operably linked to a first regulatory region comprising a first promoter, wherein the first promoter is activated (induced) by a change in state in the microorganism environment compared to the normal physiological conditions at the at least one site in the subject, optionally wherein cell death of the synthetic microorganism occurs within 30, 60, 90, 120, 180, 360 or 240 minutes following change of state. The change in state may be selected from one or more conditions of pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, heme concentration, sweat concentration, sebum concentration, metal concentration, chelated metal concentration, change in composition or concentration of one or more immune factors, mineral concentration, and electrolyte concentration. In some embodiments, the change in state is a higher concentration of blood, serum, or plasma compared to normal physiological conditions at the at least one site in the subject.Inducible promoters

[0303] A synthetic microorganism is provided which may comprise a recombinant nucleotide comprising at least one molecular modification (e.g., a kill switch) comprising (i) a cell death gene operatively associated with (ii) a first regulatory region comprising a first inducible promoter which exhibits conditionally high level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma, of at least two fold, at least three fold, at least 10-fold, at least 20 fold, at least 50 fold, at least 100-fold increase of basal productivity.

[0304] The inducible first promoter may be activated (induced) upon exposure to an increased concentration of blood, serum, plasma, or heme after a period of time, e.g., after 15 minutes, 30 minutes, 45 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 360 minutes, or any time point in between, to increase transcription and / or expression at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 300-fold, or at least 600-fold compared to transcription and / or expression in the absence of blood, serum, plasma or heme (non-induced).

[0305] The blood or serum inducible first promoter may be selected by a process comprising selecting a target microorganism, selecting one or more first promoter candidate genes in the target microorganism, growing the microorganism in a media, obtaining samples of the microorganism at t= 0 min, adding serum or blood to the media, obtaining samples at t=n minutes, where n= 1-240 min or more, 15-180 min, or 30-120 min, performing RNA sequencing of the samples, and comparing RNA sequencing read numbers for candidate first promoter in samples obtained at obtained at t= 0 min, and t=n minutes after exposure to blood or serum for the candidate first promoter gene. Alternatively, samples obtained after t=n minutes after exposure to blood or serum may be compared to t=n minutes in media without blood or serum for the candidate first promoter. Candidate first promoters may be selected from those that exhibit upregulation by RNA sequencing after target cell growth at t=n min in blood or serum of greater than about 10-fold, greater than about 20-fold, greater than about 50-fold, greater than about 100-fold, or greater than about 500-fold, when compared to the candidate promoter in the target cell at t=0, or when compared to the candidate promoter in the target cell at t=n in media without serum or blood.

[0306] Several serum responsive promoter candidate genes in Stapylocooccus aureus 502a were upregulated by greater than 20-fold after exposure to serum for 30 minutes as determined by RNA sequencing as compared to t=0 including isdB gene CH52_00245 (479-fold), sbnB gene CH52_05135 (158-fold), isdC gene CH52_00235 (93-fold), sbnA gene CH52_05140 (88-fold), srtB gene CH52_00215 (73-fold), sbnE gene CH52_05120 (70-fold), sbnD gene CH52_05125 (66-fold), isdI gene CH52_00210 (65-fold), heme ABC transporter 2 gene CH52_00225 (65-fold), sbnC gene CH52_05130 (63-fold), heme ABC transporter gene CH52_00230 (60-fold), isd ORF3 gene CH52_00220 (51-fold), sbnF gene CH52_05115 (43 fold), alanine dehydrogenase gene CH52_11875 (43-fold), HarA gene CH52_10455 (43-fold), sbnG gene CH52_05110 (42-fold), diaminopimelate decarboxylase gene CH52_05105 (32-fold), iron ABC transporter gene CH52_05145 (31-fold), threonine dehydratase gene CH52_11880 (24-fold), and isdA gene CH52_00240 (21-fold).

[0307] Several serum responsive promoter candidate genes in target micoorganism Stapylocooccus aureus 502a were found to be upregulated by greater than 20-fold after exposure to serum for 30 minutes as determined by RNAseq compared to TSB at 30 minutes including isdB gene CH52_00245 (471-fold), isdC gene CH52_00235 (56-fold), isdI gene CH52_00210 (53-fold), sbnD gene CH52_05125 (52-fold), sbnC gene CH52_05130 (51-fold), sbnE gene CH52_05120 (50-fold), srtB gene CH52_00215 (47-fold), sbnB gene CH52_05135 (44-fold), sbnF gene CH52_05115 (44- fold), heme ABC transporter 2 gene CH52_00225 (43-fold), isdA gene CH52_00240 (40-fold), heme ABC transporter gene CH52_00230 (40-fold), sbnA gene CH52_05140 (37-fold), isd ORF3 gene CH52_00220 (35-fold), sbnG gene CH52_05110 (34-fold), HarA gene CH52_10455 (28-fold), diaminopimelate decarboxylase gene CH52_05105 (25-fold), sbnI gene CH52_05100 (22-fold), and alanine dehydrogenase gene CH52_11875 (20-fold). Iron ABC transporter gene CH52_05145 was upregulated (19-fold) after 30 min of exposure to serum compared to 30 min in TSB. Threonine dehydratase gene CH52_11880 was upregulated (14-fold) after 30 min of exposure to serum compared to 30 min in TSB.

[0308] Several serum responsive promoter candidate genes in target micoorganism Stapylocooccus aureus 502a were upregulated by greater than 50-fold after exposure to serum after 90 minutes as determined by RNAseq compared to t=0 including isdB gene CH52_00245 (2052-fold), sbnB gene CH52_05135 (310-fold), alanine dehydrogenase gene CH52_11875 (304-fold), sbnE gene CH52_05120 (190-fold), sbnD gene CH52_05125 (187-fold), isdC gene CH52_00235 (173-fold), sbnC gene CH52_05130 (162-fold), sbnA gene CH52_05140 (143-fold), srtB gene CH52_00215 (143-fold), sbnG gene CH52_05110 (133-fold), sbnF gene CH52_05115 (129- fold), heme ABC transporter gene CH52_00230 (125-fold), heme ABC transporter 2 gene CH52_00225 (117-fold), isdI gene CH52_00210 (115-fold), HarA gene CH52_10455 (114-fold), diaminopimelate decarboxylase gene CH52_05105 (102-fold), sbnI gene CH52_05100 (101-fold), isd ORF3 gene CH52_00220 (97-fold), SAM dep Metrans gene CH52_04385 (75-fold). Iron ABC transporter gene CH52_05145 (44-fold), isdA gene CH52_00240 (44-fold), and siderophore ABC transporter gene CH52_05150 (33-fold) were also upregulated after 90 min exposure to serum compared to t=0.

[0309] Several serum responsive promoter candidate genes in target micoorganism Stapylocooccus aureus 502a were found to be upregulated by greater than 50-fold after exposure to serum after 90 minutes as determined by RNA sequencing compared to growth in TSB at 90 minutes including isdB gene CH52_00245 (1240-fold), sbnD gene CH52_05125 (224-fold), heme ABC transporter gene CH52_00230 (196-fold), sbnE gene CH52_05120 (171-fold), srtB gene CH52_00215 (170-fold), isdC gene CH52_00235 (149-fold), sbnC gene CH52_05130 (147-fold), diaminopimelate decarboxylase gene CH52_05105 (141-fold), heme ABC transporter 2 gene CH52_00225 (135-fold), sbnB gene CH52_05135 (130-fold), sbnF gene CH52_05115 (127- fold), bnG gene CH52_05110 (120-fold), isd ORF3 gene CH52_00220 (119-fold), isdI gene CH52_00210 (118-fold), HarA gene CH52_10455 (117-fold), isdA gene CH52_00240 (115-fold), sbnA gene CH52_05140 (93-fold), and sbnI gene CH52_05100 (89-fold). Iron ABC transporter gene CH52_05145 (47-fold), siderophore ABC transporter gene CH52_05150 (37-fold), and SAM dep Metrans gene CH52_04385 (25-fold) were also upregulated after 90 min exposure to serum compared to TSB at t=90 min.

[0310] The blood or serum inducible first promoter genes for use in a Staphylococcus aureus synthetic microorganism may be selected from or derived from a gene selected from isdA (iron-regulated surface determinant protein A), isdB (iron-regulated surface determinant protein B), isdG (heme-degrading monooxygenase), hlgA (gamma-hemolysin component A), hlgA1 (gamma-hemolysin), hlgA2 (gamma-hemolysin), hlgB (gamma-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosynthesis protein), sbnE (lucA / lucC family siderophore biosynthesis protein), lrgA (murein hydrolase regulator A), lrgB (murein hydrolase regulator B), ear (Ear protein), fhuA (ferrichrome transport ATP-binding protein fhuA), fhuB (ferrichrome transport permease), hlb (phospholipase C), splF (serine protease SplF), splD (serine protease SplD), dps (general stress protein 20U), SAUSA300_2617 (putative cobalt ABC transporter, ATP-binding protein), SAUSA300_2268 (sodium / bile acid symporter family protein), SAUSA300_2616 (cobalt family transport protein), srtB (Sortase B), sbnA (probable siderophore biosynthesis protein sbnA), leuA (2-isopropylmalate synthase amino acid biosynthetic enzyme), sstA (iron transport membrane protein), sirA (iron ABC transporter substrate-binding protein), IsdA (heme transporter), and Spa (Staphyloccocal protein A), HlgA (gamma hemolysin), leuA(amino acid biosynthetic enzyme), sstA (iron transporter), sirA (iron transport), spa (protein A), or IsdA (heme transporter) , or a substantially identical gene. The first promoter genes also may be selected from the group consisting of SAUSA300_0119 (Omithine cyclodeaminase family protein), IrgA (Murein hydrolase transporter), and bioA (Adenosylmethionine-8-amino-7-oxononanoate aminotransferase), or a substantially identical gene.

[0311] The blood or serum blood or serum inducible first promoter genes for use in a Staphylococcus aureus synthetic microorganism may be selected from or derived from a gene selected from isdB gene CH52_00245, sbnD gene CH52_05125, heme ABC transporter gene CH52_00230, sbnE gene CH52_05120, srtB gene CH52_00215, isdC gene CH52_00235, sbnC gene CH52_05130, diaminopimelate decarboxylase gene CH52_05105, heme ABC transporter 2 gene CH52_00225, sbnB gene CH52_05135, sbnF gene CH52_05115, bnG gene CH52_05110, isd ORF3 gene CH52_00220, isdI gene CH52_00210, HarA gene CH52_10455, isdA gene CH52_00240, sbnA gene CH52_05140, and sbnI gene CH52_05100, iron ABC transporter gene CH52_05145, siderophore ABC transporter gene CH52_05150, and SAM dep Metrans gene CH52_04385.

[0312] The blood or serum indicible first promoter gene for use in a Staphylococcus aureus synthetic microorganism may be derived from or comprise a nucleotide sequence selected from 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, and 163, or a substantially identical sequence.

[0313] In one embodiment, the synthetic microorganism is a molecularly modified Staphylococcus aureus 502a. Raw sequences of first ORF in the operon that follows each regulatory region, from start codon to stop codon, used for design of real time PCR probes are shown in Table 2.

[0314] Table 2. Staphylococcus aureus strain 502a, raw sequences of first ORF in the operon that follows each regulatory region used for design of real time PCR probes. Staphylococcus aureus strain 502a, spa ORF of 502aStaphylococcus aureus strain 502a, sirA ORF of 502aStaphylococcus aureus strain 502a, sstA of 502aStaphylococcus aureus strain 502a, hlgA ORF of 502aStaphylococcus aureus strain502a, isdA ORF of 502aStaphylococcus aureus strain 502a, leuA of 502a

[0315] As discussed herein below, the synthetic microorganism may include an expression clamp molecular modification that prevents expression of the cell death gene, wherein the expression clamp comprises an antitoxin gene specific for the cell death gene operably associated with a second promoter which is active upon dermal or mucosal colonization or in TSB media, and is preferably downregulated in blood, serum or plasma, for example, the second promoter may comprise a clfB gene (clumping factor B), for example as shown in Table 3. Table 3. Other Sequences Used for Design of Real time PCR probesclfB ORF of 502a (to drive antitoxin for "expression clamp")gyrA ORF of 502a (internal housekeeping gene)

[0316] Additional oligonucleotides used in the recombinant approach to preparing the synthetic microorganism molecularly modified Staphylococcus aureus 502a are shown in Table4A shown in FIG. 3A-C, and promoter sequences are shown below.Cell Death Genes

[0317] The synthetic microorganism may contain a kill switch molecular modification comprising cell death gene operably associated with an inducible first promoter, as described herein. The cell death gene may be selected from any gene, that upon overexpression results in cell death or significant reduction in the growth of the synthetic microorganism within a predefined period of time, preferably within 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 240 minutes, or 360 minutes of induction.

[0318] Cell death genes, toxin genes, or kill switch genes, have been developed in other contexts.

[0319] WO 2016 / 210373, Jonathan Kotula et al., assigned to Synlogic, Inc. discloses a recombinant bacterial cell that is an auxotroph engineered for biosafety, for example, that comprises a repression based kill switch gene that comprises a toxin, an anti-toxin and an arabinose inducible promoter and depends on the presence of an inducer (e.g., arabinose) to keep cells alive.

[0320] US 8,975,061, Bielinski, discloses regulation of toxin and antitoxin genes for biological containment for preventing unintentional and / or uncontrolled spread of the microorganisms in the environment.

[0321] WO 1999 / 058652, Gerdes, discloses cytotoxin based biological containment and kill systems including E. coli relBE locus and similar systems found in Gram-negative and Gram-positive bacteria and Archae.

[0322] US 20150050253, Gabant, discloses controlled growth of microorganisms and controlling the growth / spread of other exogenous recombinant or other microbes.

[0323] WO 2017 / 023818 and WO 2016 / 210384, Falb, disclose bacteria engineered to treat disorders involving propionate metabolism.

[0324] US 20160333326, Falb, discloses bacteria engineered to treat diseases associated with hyperammonemia.

[0325] US 9101597, Garry, discloses immunoprotective primary mesenchymal stem cells and methods and a proaptoptotic kill switch is described for use in mesenchymal stem cells.

[0326] US 20160206666, Falb, discloses bacteria engineered to treat diseases that benefit from reduced gut inflammation and / or tighten gut mucosal barrier.

[0327] In some embodiments, synthetic microorganisms are provided that comprise one or more of SprA1 (Staphylococcus aureus), Sma1 (Serratia marcescens), RelF (E. coli), KpnI (K. pneumoniae) and / or RsaE (Staphylococcus aureus) toxin genes.

[0328] In the present disclosure, various cell death toxin genes were tested in combinations with previously identified optimal control regions: i) a 30 amino acid peptide (PepA1) that forms pores in the cell membrane, impairing its function; ii) a restriction enzyme (Kpn1 or other) that rapidly digests the bacterial chromosome; iii) a small RNA (RsaE) that impairs central biochemical metabolism by inhibiting translation of 2 essential genes; iv) a restriction endonuclease (Sma1) derived from Serratia marcescens; and v) a toxin gene derived from E. coli (RelF). Some toxins are more potent than others and the ideal combination of control region induction strength and toxin potency may result in a strain that is healthy at baseline and that rapidly dies in the circulatory system.

[0329] sprA1 (Staphylococcus aureus) toxin gene (encoding PepA1 peptide) is described in WO 2013 / 050590, Felden, B, and Sayed, N, disclosing use of PepA1 as an antimicrobial, but the focus is on using the peptide as purified exogenous therapeutic to be delivered into the body.

[0330] relF (E. coli) toxin gene is described in EP 20090168998, Gerdes, disclosing kill switches for the purpose of biocontainment and focuses on revolve around killing of Gram-negative bacteria.

[0331] relF toxin gene is described in US 8852916, Hyde and Roderick, disclosing mechanisms of triggering cell death of microorganisms (programmed cell death). The main application is to use RelF in environmental biocontainment.

[0332] relF is described in US 8682619, Amodei, prophetically discloses RelF to regulate bacterial population growth.

[0333] The synthetic microorganism may be derived from a Staphylococcus aureus target microorganism by insertion of a kill switch molecular modification comprising a regulatory region comprising an inducible promoter operably linked to a cell death gene which may be a toxin gene.

[0334] The cell death gene may be selected from or derived from a sprA1 gene (encoding a peptide toxin that forms pores in cell membrane), sprA2 gene, sprG gene, sma1 gene (a restriction endonuclease), kpn1 gene (restriction enzyme that rapidly digests bacterial chromosome), rsaE gene (a small RNA that impairs central metabolism by inhibiting translation of 2 essential genes), a relF gene (E. coli), yoeB gene, mazF gene, yefM gene, or lysostaphin toxin gene. The synthetic Staphylococcus aureus may include a kill switch molecular modification comprising a cell death gene having a nucleotide sequence selected from SEQ ID NOs: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, or 317, or a substantially identical nucleotide sequence.

[0335] In a specific embodiment, a synthetic Staphylococcus aureus is provided having a molecular modification comprising a blood or serum inducible first promoter operably associated with a cell death gene comprising or derived from a SprA1 gene.Multiple kill switches

[0336] One KS may be sufficient to equip the synthetic microorganism with the desired characteristics, but more than one KS may further enhance the strain by: i) dramatically reducing the rate of KS-inactivating mutations, and; ii) killing the cell by more than one pathway, which could cause faster cell death (a product-enhancing feature). The cell death gene may comprise one or more of the DNA sequences (7) downstream of promoters that are shown below. Base pair numbers correspond to pCN51 vector location. 1. The sprA1 gene sequence between restriction sites PstI and EcoRI is shown below. The sequence was synthesized by DNA 2.0(Atum) and ligated into a vector, which can be transformed into E.coli cells for replication. The sprA1 gene was restriction cut at PstI and EcoRI sites and isolated by gel elecrophoresis. Full sequence between restriction sites with possible start and stop sites italicized. 2. The DNA sequence for the regulatory RNA sprA1sprA1 AS (sprA1sprA1 antisense) under the ClfB promoter (which is cloned in reverse behind the sprA1 gene, including the antisense regulatory RNA). This DNA sequence produces a non-coding antisense regulatory RNA, which acts as an antitoxin by regulating the translation of sprA1 outside of the environmental factors of serum and / or blood. Below is the sprA1sprA1 AS DNA sequence. 3. The SmaI DNA sequence between restriction sites PstI and EcoRI. Sequence was synthesized by DNA 2.0(Atum) and ligated into a vector that can be transformed into E. coli cells for replication. SmaI gene was restriction cut at PstI and EcoRI sites and isolated by gel electrophoresis. Full sequence between restriction sites with start and stop sites italicized. 4. The rsaE DNA sequence between restriction sites PstI and EcoRI. Sequence was synthesized by DNA 2.0(Atum) and ligated into a vector that can be transformed into E. coli cells for replication. RsaE small regulatory RNA (sRNA) was restriction cut at PstI and EcoRI sites and isolated by gel electrophoresis. This contains a 5' run-in and the mature RNA is processed out starting at the bold GAAATTAA and ending at the stretch of Ts after the ACG. 5. A variant can be used for RsaE sRNA which may express the sRNA more highly which may work more effectively. This variant would start with the GAAATTAA at the 5' end. 6. The relF (E. coli) DNA sequence. This potential kill gene will be tested and cloned. 7. The KpnI (restriction enzyme from K. pneumoniae) DNA sequence will be tested and cloned.

[0337] A synthetic Staphylococcus aureus 502a is provided herein comprising at least one molecular modification (kill switch) comprising a first cell death gene operably linked to a first regulatory region comprising a first promoter, optionally wherein the first cell death gene comprises a nucleotide sequence selected from SEQ ID NO: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or a substantially identical nucleotide sequence

[0338] Although kill switches (KSs) have been described for other purposes, the present KS has the unique features: i) it responds to being exposed to blood or serum; ii) it is endogenously regulated, meaning that the addition or removal of small molecules is not needed to activate or tune the KS (not an auxotroph); and iii) useful combinations of control region / toxin, and of multiple such cassettes may be used to achieve superior performance.Expression Clamp

[0339] A synthetic microorganism is provided which comprises kill switch molecular modification comprising (i) a cell death gene operatively associated with (ii) a first regulatory region comprising a first inducible promoter which is induced by exposure to blood or serum. In order for the synthetic microorganism to durably occupy a dermal or mucosal niche in the subject, the kill switch preferably should be silent (not expressed) in the absence of blood or serum.

[0340] In order to avoid "leaky expression" of the cell death gene, the synthetic microorganism may further comprise at least a second molecular modification (expression clamp) comprising (iii) an antitoxin gene specific for the cell death gene, wherein the antitoxin gene is operably associated with (iv) a second regulatory region comprising a second promoter which is active (e.g., constitutive) upon dermal or mucosal colonization or in a media (e.g., TSB), and preferably is downregulated by exposure to blood, serum or plasma.

[0341] The basal level of gene expression (the expression observed when cells are not exposed to blood or serum, e.g., in TSB (tryptic soy broth)) in the KS strain should ideally be very low because producing the toxin prior to contact with serum would kill or weaken the strain prematurely. Even moderate cell health impairment is unacceptable because: 1) escape mutations in the KS would accumulate (KS instability) - a known phenomenon that must be avoided, and / or; 2) the natural efficacy observed with our strain in preliminary trials could be reduced or lost. To understand if leaky expression is a problem, both the absolute level of baseline expression and the fold change in serum are being measured and closely considered in the selection of the optimal control region to drive the KS.

[0342] Awareness of leaky expression does not fix the problem and the reality is that even widely used "tightly controlled" rheostatic promoters such as P CUP1 and P Gal7 , and P Tet -on / off variants produce measurable mRNA transcription in the absence of specific induction. In some embodiments, an "expression clamp" is employed in which the KS cassette contains not only the serum-responsive control region that drives toxin expression, but also encodes a "translation blocking" RNA under control of a Staphylococcus aureus promoter (P clfB etc) that is normally strongly active in Staphylococcus aureus during colonization of the skin, and in downregulated in blood.

[0343] The clfB gene promoter (P clfB ) will be cloned to drive expression of the sprA1sprA1 AS RNA and the cassette will be incorporated into the same expression module as is used for expression of the sprA1 toxin from a serum-responsive promoter (eg, P isdB , P hlgA etc). In this strain, serum / blood exposure activates the toxin (e.g., up to 350-fold or more) but not the antitoxin, and growth in TSB or on the skin activates antitoxin but not toxin. A representative diagram of an exemplary molecular modification of a synthetic strain is shown in FIG. 1.An alternate approach to a synthetic microorganism: KO method

[0344] An alternative way to create a kill-switch-like phenotype in the synthetic microorganism is to disrupt ("knock-out") one or more genes that are required for survival in blood and / or for infection of organs but that are not required (or important) for growth in media or on the skin. In some embodiments, one or more, or two or more, of the 6 genes shown in Table 5 may be employed in the KO method. Table 5: Candidates for gene knockout to create an attenuated strain:ReferenceType of mutagenesisGenes required for survival in blood or infection of organsReported gene functionBenton et al (2004) Large-Scale Identification of Genes Required for Full Virulence of Staphylococcus aureus. J. bact. 186(24): 8478-8489. DOI 10.1128 / JB.186.2 4.8478-8489.2004Transposon insertionPycA; AspB; GabP. Mutation of these causes up to 1000-fold reduction in rate of organ infection in vivoPycA: Pyruvate carboxylaseAspB: Aspartate aminotransferaseGabP: Gamma-aminobutyrate permeaseValentino et al (2014). Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies. mBio5(5):e01729 - 14.doi:10.1128 / m Bio.01729-14.Transposon insertionGenes essential for in vitro survival in blood but not needed for growth in BHI liquid or agar:SAOUHSC_01216 : succinyl CoA-synthetase subunit b.SAOUHSC_00686: Unknown hypothetical protein- SAOUHSC_01216 -SAOUHSC_00378: Unknown hypothetical proteinSAOUHSC_00686 -SAOUHSC_00378

[0345] In one embodiment, a synthetic microorganism is provided comprising replacement of one or more of the genes in Table 5 with unmodified or expression-clamped KS, using allelic exchange. This may further enhance the death rate of the synthetic microorganism in blood. Alternatively, the need to integrate two KSs is diminished by having one KO and one KS. In a further embodiment, a synthetic microorganism may comprise a combination of more than one KO that may have synergistic effects.Kill Switch Regulatory Region

[0346] A synthetic microorganism comprising a kill switch is provided. The kill switch comprises a cell death gene operably linked to a regulatory region (RR) comprising an inducible promoter, as described herein.

[0347] Development of a synthetic microorganism involves identification and characterization of optimal regulatory regions (RRs) in order to drive kill switch genes; a list of serum responsive loci are chosen; RRs are identified; and Serum activation response is verified, and basal expression is investigated.Identification and characterization of optimal regulatory regions to drive kill switch candidates.

[0348] This important phase of KS strain construction involves identifying genes that are strongly upregulated in response to human serum and / or whole heparinized blood. Once the genes are identified, their RRs, which contain the promoter and other upstream elements, are identified and annotated. In one approach, any known serum- and blood-responsive gene in Staphylococcus aureus may be employed that is known in the literature.

[0349] A RR includes the upstream regulatory sequences needed for activation (or repression) of mRNA transcription in response to stimuli. The motifs include "up" elements, -35, and -10 consensus elements, ribosome binding sites ("shine-dalgarno sequence") and "operator" sequences which bind protein factors that strongly influence transcription. In practice for eubacteria, harnessing a 200 bp region of DNA sequence upstream of the start codon is usually adequate to capture all of these elements. However, it is preferred to deliberately identify these sequences to ensure their inclusion.

[0350] Six Staphylococcus aureus genes that are strongly upregulated by exposure to human blood or serum are shown in Table 6. Table 6: Identification of candidate RRs and serum or blood inducible promoters to drive kill switch components for driving the toxin.GeneFunctionFirst author, yearFold change in serum or bloodTime of exposure to blood or serumSA strain used in studyCommentsspaStaphylocco cal Protein A; Ig binding; monocistron ic geneMalachowa 2011~ 45 fold90 minUSA300 and mu50Wang 2004 predicts the monocistronic gene structure. Both experimental& computational evidence of this structure existsirSir ABC; iron transportMalachowa 2011 and Wang 200481 fold in serum; 68-fold in blood (sirA; first ORF in operon)30 to 120 minutesUSA300 and mu50High induction at earliest timepoint. Experimental and predicted operon structure matchsstSstABCD operon; Iron transportMalachowa 2011 and Wang 200425-fold in serum; 15 fold in blood;30 to 120 minuteUSA300 and mu50High induction at earliest timepoint. Experimental and predicted operon structure matchGamma hemolysin hlgArbc lysisMalachowa 2011;~ 350-fold (Fig 4b)90 minUSA300Operon structure characterized by Cooney 1993sai-1 (seg 7 surface29 kd cell surfaceWiltshire 200150-fold in serum;24-fold in16h (O / N plating assay)8325-4Serum agar and solution phase assays, separateprotein). Also called isdAprotein; heme transporter;blood. IsdB from the same operon is upregulated240-fold in serum and 140-fold in bloodpubs. Serum was sufficient for induction in Wiltshire 2001 & Malachowa 2011.leuA2-isopropylma late synthaseMalachowa 2011-6 fold downreg. in TSB;15 fold upreg in serum; 12 fold upreg in blood30 to 120 minUSA300Attractive b / c of downreg. in TSB but the fold upreg. in serum might be insufficientSAUSA300 _0119Ornithine cyclodeami nase family proteinMalachowa 201150 fold upreg. in serum, 27 fold in blood; no upreg in TSB compared to time 0 in TSB30 to 120 minUSA300Different category of gene than above and also seemingly tightly regulated in TSBlrgAMurein hydrolase transporterMalachowa 2011-3.3 fold downreg in TSB; 12 fold upreg in serum; 17 fold upreg in blood30 to 120 minUSA300Attractive b / c it is down- regulated in TSBbioAAdenosylme thionine-8-amino-7-oxononanoa te aminotransf eraseMalachowa 2011107 fold upreg in serum; 56 fold upreg in blood; no reg in TSB30 to 120 minUSA300Attractive b / c very strong upreg and a lesser known metabolic gene

[0351] The full genes in each operon and the flanking sequences from strain BioPlx-01 are obtained from Genbank and annotated based on the literature plus known motif-identifying algorithms. Transcription terminators have been identified through a combination of published experiments and predictive tools.

[0352] Additional Literature evidence of expression of serum responsive promoters in TSB (or similar media) was investigated. For example, spa gene and isdA gene are disclosed in Ythier et al 2012, Molecular & Cellular Proteomics, 11:1123-1139, 2012. The sirA gene is disclosed in Dale et al, 2004 J Bacteriol 186(24) 8356-8362. The sst gene is disclosed in Morrissey et al. 2000. The hlgA gene is disclosed in Flack et al 2014, PNAS E2037-E2045. www.pnas.org / cgi / doi / 10.1073 / pnas.1322125111. The leuA gene is disclosed in Lei et al 2015, Virulence 6:1, 75-84.

[0353] Since these data come from many different strains and experimental systems, the entire collection may be assessed for expression in a single standardized assay system with quantitative gene expression measurements made by using real time PCR. Importantly, the basal "leaky" level of gene expression (the expression observed when cells are not exposed to blood or serum, e.g., in TSB) should be very low because producing the toxin prior to contact with serum would kill / weaken the BioPlx-XX strain (synthetic microorganism comprising a kill switch) prematurely. Even moderate cell health impairment is unacceptable because: 1) escape mutations in the KS would accumulate (KS instability) - a known phenomenon that must be avoided, and / or 2) the natural efficacy observed with BioPlx-01 could be reduced or lost. Thus, both the absolute level of baseline expression and the fold change in serum may be measured and closely considered in the selection of the optimal RRs to drive the KS. It is noted that leuA is downregulated in TSB (6-fold) and upregulated in serum (15-fold) making its RR particularly interesting candidate to control KS expression.

[0354] In some embodiments, the synthetic microorganism having a kill switch may further comprise an "expression clamp" in which the KS cassette contains not only the serum-responsive RR that drives toxin expression, but also encodes a "translation blocking" RNA antitoxin under control of a promoter that is normally active on the skin or nasal mucosa during colonization. The kill switch may encode an antitoxin that is capable of suppressing the negative effects of the cell death toxin gene.

[0355] In some embodiments, the synthetic microorganism is a Staphylococcus aureus having a molecular modification comprising a kill switch which further comprises an "expression clamp" in which the KS cassette contains not only the serum-responsive RR that drives toxin expression, but also encodes a "translation blocking" RNA antitoxin under control of a Staphylococcus aureus promoter (P clfB etc.) that is normally active on the skin during colonization, for example, as shown in Table 7.

[0356] From those promoters listed on Table 6 plus real time PCR data, two or more RRs with the best mix of low basal expression and high response to serum / blood may be selected to drive KS expression. These RRs may be paired with 3 different KS genes as described herein, generating a panel of KS candidate strains for testing. The panel will include an "expression clamp" candidate as described next.Expression clamp to block toxin expression when the KS strain is on the skin or nasal epithelia

[0357] The synthetic microorganism may comprise an expression clamp. Genes involved in Staphylococcus aureus colonization of human nares are shown in Table 7 may be employed as a second promoter for use in an expression clamp further comprising an antitoxin gene to block leaky toxin expression when the synthetic strain is colonized on skin or mucosal environments. The second promotermay be a constituitive promoter, such as a houskeeping gene. The second promoter or may be preferably downregulated in the presence of blood or serum. Table 7. Genes involved in Staphylococcus aureus colonization of human naresGeneKnown or Putative roleReferenceCommentsclfB (Clumping factor B) (ClfB)AdhesionWertheim HF, Walsh 2008; also Burian 201010 fold higher than Gyr in vivo; same high expression as gyr in vitro. Also, expression in rodent models and in humans is important for nasal colonization. It isexpressed in exponential phase in vitro. Gene is downregulated 3-fold in human serum (Malachowa 2011)autolysin (seeD) (exoprotein D)Lytic transglycosylaseStapleton MR, Horsburgh MJ 2007expressed in exponential phase in vitrowalKR (virulence regulator)essential master regulator of virulenceBurian 2010In vivo expression at time zero and at year 1 is on par with gyrAatlA (Major autolysin)major autolysin; Bifunctional peptidoglycan hydrolaseBurian 2010Similar characteristics as walKR but expression is higher (5 fold above gyr)oatA (O-acetyltransferas e A)O-acetylation of peptidoglycan; renders Staphylococcus aureus cells resistant to lysozymeBurian 2010Similar to WalKR

[0358] In some embodiment, a synthetic microorganism is provided having a molecular modification comprising a kill switch and further comprising an expression clamp comprising an antitoxin gene driven by a second promoter that is normally active on the skin or nasal mucosa during colonization, optionally wherein the second promoter is selected from a gene selected from or derived from clumping factor B (clfB), autolysin (sceD; exoprotein D), walKR (virulence regulator), atlA (Major autolysin), and oatA (O-acetyltransferase A), as shown in Table 7. The constitutive second promoter may alternatively be selected from or derived from a housekeeping gene, for example, gyrB, sigB, or rho, optionally wherein the second promoter comprises a nucleotide sequence of SEQ ID NO: 324, 325, or 326, respectively, or a substantially identical sequence.

[0359] The second promoter for use in the expression clamp may be selected from a gene identified in the target microorganism that has been recognized as being downregulated upon exposure to blood or serum.

[0360] The second promoter for use in an expression clamp molecular modification should be a constitutive promoter that is preferably downregulated upon exposure to blood or serum after a period of time, e.g., after 15 minutes, 30 minutes, 45 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 360 minutes, or any time point in between, to decrease transcription and / or expression of the cell death gene, by at least 2-fold, 3-fold, 4-fold, 5-fold, or at least 10-fold, compared to transcription and / or expression in the absence of blood or serum.

[0361] The second promoter may be selected by a process comprising selecting a target microorganism, selecting one or more second promoter candidate genes in the target microorganism, growing the microorganism in a media, obtaining samples of the microorganism at t= 0 min, adding serum or blood to the media, obtaining samples at t=n minutes, where n= 1-240 min or more, 15-180 min, or 30-120 min, performing RNA sequencing of the samples, and comparing RNA sequencing read numbers for candidate first promoter in samples obtained at obtained at t= 0 min, and t=n minutes after exposure to blood or serum for the candidate first promoter gene. Alternatively, samples obtained after t=n minutes after exposure to blood or serum may be compared to t=n minutes in media without blood or serum for the candidate second promoter. Candidate second promoters may be selected from those that exhibit downregulation by RNA sequencing after target cell growth at t=n min in blood or serum, when compared to the candidate promoter in the target cell at t=0, or when compared to the candidate promoter in the target cell at t=n in media without serum or blood.

[0362] The second promoter may be selected from or derived from a promoter candidate gene identified herein for potential use in an expression clamp in Stapylocooccus aureus 502a that were found to be downregulated by at least 2-fold after exposure to serum for 30 minutes as determined by RNA sequencing as compared to t=0 including phosphoribosylglycinamide formyltransferase gene CH52_00525 (-4.30 fold), phosphoribosylaminoimidazole synthetase gene CH52_00530 (-4.27 fold), amidophosphoribosyltransferase gene CH52_00535(-4.13 fold), phosphoribosylformylglycinamidine synthase gene CH52_00540 (-4.04 fold), phosphoribosylformylglycinamidine synthase gene CH52_00545 (-3.49 fold), phosphoribosylaminoimidazole-succinocarboxamide gene CH52_00555 (-3.34 fold), trehalose permease IIC gene CH52_03480 (-3.33 fold), DeoR faimly transcriptional regulator gene CH52_02275(-2.55 fold), phosphofructokinase gene CH52_02270 (-2.46 fold), and PTS fructose transporter subunit IIC gene CH52_02265 (- 2.04 fold).

[0363] The second promoter may be selected from or derived from phosphoribosylglycinamide formyltransferase gene CH52_00525, trehalose permease IIC gene CH52_03480, DeoR faimly transcriptional regulator gene CH52_02275, phosphofructokinase gene CH52_02270, or PTS fructose transporter subunit IIC gene CH52_02265.

[0364] The second promoter may be a P clfB (clumping factor B) gene; optionally wherein the second promoter comprises a nucleotide sequence of SEQ ID NO: 7, 117, 118, 129 or 130, or a substantially identical sequence.

[0365] In one specific example, one of the KS constructs (sprA1) is equipped with an expression clamp comprising an antitoxin (sprA1 AS ) driven from the Clumping factor B (clfB) promoter. This promoter is one choice to drive the clamp because it is strongly expressed in TSB and during nasal / skin colonization (10 fold higher than the abundant housekeeping gene gyrA) (Burian 2010). This is directly relevant to manufacturing and use of the product, respectively. The Clumping factor B (clfB) promoter is also downregulated 3 fold in blood (Malachowa 2011), favoring clamp inactivity when. Complete inactivity in blood may not be needed because the serum-responsive promoters driving is so robustly activated in the blood.

[0366] The Clumping factor B (clfB) promoter is also stably expressed over at least 12 months during nasal colonization in humans and was also identified in rodent and in vitro models of colonization (Burian 2010).

[0367] In one example of an expression clamp, clfB is selected as a constitutive promoter for use in an expression clamp after confirmation of strong expression in TSB, and lower levels of expression in blood or serum (real time PCR), to determine its characteristics in target strain Staphylococcus aureus 502a. The clfB regulatory region is cloned to drive expression of the sprA1 antisense (antitoxin) RNA (see Table 3, first entry), and the cassette is incorporated into the same expression shuttle vector as is used for expression of the sprA1 toxin gene from a serum-responsive promoter. It is desirable that the serum / blood exposure may strongly activate the toxin but not the antitoxin, and TSB or skin / nasal epithelial exposure activates antitoxin but not toxin. This concept may be applied to the other KS genes in Table 3 below. An alternative possibility for using the clamp is for the restriction enzyme KpnI (toxin) approach for which the antitoxin may be an RNA aptamer that was recently developed as a potent inhibitor of this enzyme (Mondragon, 2015) as a means of imparting metabolic stability to the aptamer.

[0368] Awareness of leaky expression does not fix the problem and the reality is that even widely used "tightly controlled" rheostatic promoters such as P CUP1 and P Gal7 , and P Tet -on / off variants produce measurable mRNA transcription in the absence of specific induction.

[0369] The expression clamp comprises a second promoter operably linked to an antitoxin gene. For example, the antitoxin gene is specific for the cell death toxin gene in the kill switch in order to be effective. Under normal physiological conditions, the expression clamp acts to prevent leaky expression of the cell death gene. When exposed to blood or serum, the second promoter operably linked to the antitoxin is downregulated, allowing expression of the cell death gene.

[0370] The synthetic microorganism may contain an expression clamp comprising an antitoxin gene which is specific for silencing the cell death gene. The antitoxin may be selected or derived from any antitoxin specific for the cell death gene in the kill switch molecular modification that is known in the art. The antitoxin gene may encode an antisense RNA specific for the cell death gene or an antitoxin protein specific for the cell death gene.

[0371] The antitoxin gene may be a sprA1 antitoxin gene, or sprA1(AS). The sprA1 antitoxin gene may comprise a nucleotide sequence of TATAATTGAGATAA CGAAAATAAGTATTTACTTATACACCAATCCCCTCACTATTTGCGGTAGTGA GGGGATTT (SEQ ID NO: 311), or a substantially identical sequence, or CCCCTCACTA CCGCAAATAGTGAGGGGATTGGTGTATAAGTAAATACTTATTTTCGTTGT(S EQ ID NO: 273), or a substantially identical sequence.

[0372] The antitoxin gene may be a sprA2 antitoxin, or sprA2(AS), and may comprise a nucleotide sequence of or a substantially identical sequence; or TATAATTAATTACATAATAAATTGAACATCTAAAT ACACCAAATCCCCTCACTACTGCCATAGTGAGGGGATTTATTTAGGTGTTGG TTA (SEQ ID NO: 312), or a substantially identical sequence.

[0373] The antitoxin gene may be a sprG antitoxin gene, also known as sprF, and may comprise a nucleotide sequence of (5'-3') ATATATAGAAAAAGGG CAACATGCGCAAACATGTTACCCTAATGAG CCCGTTAAAAAGACGGTGGCTATTTTAGATTAAAGATTAAATTAATAACCA TTTAACCATCGAAACCAGCCAAAGTTAGCGATGGTTATTTTTT (SEQ ID NO: 307), or a substantially identical sequence. Pinel-Marie, Marie-Laure, Régine Brielle, and Brice Felden. "Dual toxic-peptide-coding Staphylococcus aureus RNA under antisense regulation targets host cells and bacterial rivals unequally." Cell reports 7.2 (2014): 424-435.

[0374] The antitoxin gene may be a yefM antitoxin gene which is specific for silencing yoeB toxin gene. The yefM antitoxin gene may comprise a nucleotide sequence of MIITSPTEARKDFYQLLKNVNNNHEPIYISGNNAENNAVIIGLEDWKSIQETIYLE STGTMDKVREREKDNSGTTNIDDIDWDNL (SEQ ID NO: 314), or a substantially identical nucleotide.

[0375] The antitoxin gene may be a lysostaphin antitoxin gene specific for a lysostaphin toxin gene. The lysostaphin antitoxin may comprise a nucleotide sequence of TATAATTGAGATATGTTCATGTGTTATTTACTTATACACCAATCCCCTCACT ATTTGCGGTAGTGAGGGGATTTTT (SEQ ID NO: 319), or a substantially identical nucleotide sequence.

[0376] The antitoxin gene may be a mazE antitoxin gene that targets mazF. The mazE toxin gene may comprise a nucleotide sequence of ATGTTATCTTTTAGTCAAAAT AGAAGTCATAGCTTAGAACAATCTTTAAAAGAAGGATATTCACAAATGGCT GATTTAAATCTCTCCCTAGCGAACGAAGCTTTTCCGATAGAGTGTGAAGCA TGCGATTGCAACGAAACATATTTATCTTCTAATTC (SEQ ID NO: 322), or a substantially identical sequence.

[0377] The antitoxin gene may alternatively be designed as follows. In Staphylococcus aureus, there are two main methods used for gene silencing. In one style of gene silencing, which is exemplified by sprA1, antisense RNA binds to the 5' UTR of the targeted gene, blocking translation of the gene and causing premature mRNA degradation. Another style of gene silencing is used for genes that do not have a transcriptional terminator located close to the stop codon. Translation can be controlled for these genes by an antisense RNA that is complementary (~3-10 bases) to the 3' end of the targeted gene. The antisense RNA will bind to the mRNA transcript covering the sequence coding for the last couple codons and creating double stranded RNA which is then targeted for degradation by RNaseIII.

[0378] Since there are many examples of RNA silencing in Staphylococcus aureus that have been identified with demonstrated ability to control their target genes, these regions and sequences may be used as a base for designing the toxin / antitoxin cassettes. This approach requires only small changes in the DNA sequences.

[0379] In the present disclosure, the antitoxin for a cell death gene may be designed to involve antisense binding to 5'UTR of targeted gene. The toxin gene may be inserted into the PepA1 reading frame, and the 12bp in the endogenous sprA1 antisense is swapped out for a sequence homologous to 12bp towards the beginning of the heterologous toxin gene.

[0380] In one example, Holin inserted into the sprA1 location can be controlled by the antisense RNA fragment encoded by (12 bp Holin targeting sequence in BOLD )= TATA ATTGAGAT AGTTTCATTAGC TATTTACTTATACACCAATCCCCTCA CTATTT GCGGTAGTGA GGGGATTTTT (SEQ ID NO: 308).

[0381] In another example, 187-lysK inserted into the sprA1 location can be controlled by the antisense RNA fragment encoded by (12 bp 187-lysK targeting sequence in BOLD ) TATAATTGAGAT TTTAGGCAGTGC TATTTACTTATACACCAA TCCCCTCA CTATTTGCGGT AGTGAGGGGATTTTT (SEQ ID NO: 309).

[0382] The antitoxin specific for the cell death gene may involve antisense binding to the 3' UTR of the toxin gene. This method involves inserting the heterologous toxin in the place of sprG in the genome of Staphylococcus aureus, and adding an additional lysine codon (AAA) before the final stop codon. The last 6 bases of the coding region (AAAAAA) plus the stop codon (TAA) overlap with the 3' region of the endogenous sprF antitoxin. When the sprF RNA is transcribed at a rate of 2.5 times greater than the heterologous toxin gene, it will form a duplex with the 3'UTR region of the toxin transcript, initiating degradation by RNaseIII and blocking the formation of a functional peptide. Since the 3' end of both of the heterologous toxins were manipulated in the same manner to overlap with the sprF sequence (adding the codon AAA in front of the TAA stop codon), which is also the same as the endogenous sprG 3' end, the sequence of the antitoxin will remain the same for all three of these toxin genes. For example, the sprG antitoxin gene (sprF) may comprise the nucleotide sequence ATATATAGAAAAA GGGCAACATGCGCAAACATGTTACCCTAATGAGCCC GTTAAAAAGACGGTGGCTATTTTAGATTAAAGATTAAATTAATAACCATTT AACCATCGAAACCAGCCAAAGTTAGCGATGGTTATTTTTT (SEQ ID NO: 310).

[0383] The antitoxin gene may comprise a nucleotide sequence selected from any of SEQ ID NOs: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319, or 322, or a substantially identical sequence thereof.

[0384] The antitoxin gene may or may not encode an antitoxin peptide. Wherein the synthetic microorganism is derived from a Staphylococcus aureus strain, the antitoxin peptide may be specific for the toxin peptide encoded by the cell death gene. For example, when the toxin gene is a yoeB toxin gene, e.g., encoding a toxin peptide comprising an amino acid sequence of SEQ ID NO: 316, the antitoxin gene may encode a yefM antitoxin protein comprising the amino acid sequence of MIITSPTEARKDFYQLLKNVNNNHEPI YISGNNAENNA VIIGLEDWKSIQETIYLESTGTMDKVREREKDNSGTTNIDDIDWDNL (SEQ ID NO: 314), or a substantially similar sequence. As another example, wherein the antitoxin gene is a mazF toxin gene, e.g., encoding a toxin peptide comprising an amino acid sequence of SEQ ID NO: 321, the antitoxin gene may be an mazE antitoxin gene, e.g., encoding an antitoxin protein comprising an amino acid sequence of MLSFSQNRSHSLEQSLKEGYSQ MADLNLSLANEAFPIECEACDCNETYLSSNSTNE (SEQ ID NO: 323), or a substantially similar sequence.

[0385] Three KS candidate genes were selected as being of particular interest because they elicit cell death in 3 disparate ways. In some embodiments, the synthetic microorganism comprises one or more, two or more or each of sprA1, kpnI or rsaE to achieve maximal death rates as early data instruct. The sprA1 mechanism of action is a loss of plasma membrane integrity / function by expression of a pore-forming peptide. the kpnI mechanism of action involves destruction of the Staphylococcus aureus genome with a restriction enzyme. The rsaE mechanism of action involves impairment of central metabolism including TCA cycle and tetrahydrfolate biosynthesis.

[0386] In some embodiments, the synthetic microorganism comprises regulatory region comprising a first promoter operably linked to a cell death gene, wherein the cell death gene encodes a toxin peptide or protein, and wherein the first promoter is upregulated upon exposure to blood or serum. The cell death gene may be a sprA1 gene. SprA1 encodes toxin peptide PepA1 as described in Sayed et al., 2012 JBC VOL. 287, NO. 52, pp. 43454-43463, December 21, 2012. PepA1 induces cell death by membrane permeabilization. PepA1 has amino acid sequence: MLIFVHIIAPVISGCAIAFFSYWLSRRNTK SEQ ID NO: 104. Related antimicrobial peptides include MMLIFVHIIAPVISGCAIAFFSYWLSRRNTK (SEQ ID NO: 105), ATAFFSYWLSRRNTK (SEQ ID NO: 106), IAFFSYWLSRRNTK (SEQ ID NO: 107), AFFSYWLSRRNTK (SEQ ID NO: 108), FFSYWLSRRNTK (SEQ ID NO: 109), FSYWLSRRNTK (SEQ ID NO: 110), SYWLSRRNTK (SEQ ID NO: 111), or YWLSRRNTK (SEQ ID NO: 112), as described in WO 2013 / 050590, The cell death gene may be an sprA2 gene. The sprA2 gene may encode a toxin MFNLLINIMTSALSGCLVAFFAHWLRTRNNKKGDK (SEQ ID NO: 305). The cell death gene may be a Staphylococcus aureus yoeB gene which may encode a yoeB toxin having the amino acid sequence of MSNYTVKIKNSAKSDLRKIKHSYLKKSFLEIVETLKND PYKITQSFEKLEPKYLERYSRRINHQHRVVYTVDDRNKEVLILSAWSHYD (SEQ ID NO: 316), or a substantially similar sequence. The cell death gene may be a Staphylococcus simulans gene which may encode a metallopeptidase toxin gene having an amino acid sequence of MTHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKI VEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSG STGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWK TNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDG HVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK (SEQ ID NO: 318), or a substantially similar sequence. The cell death gene may be a mazF toxin gene that encodes a mazF toxin comprising an amino acid sequence of MIRRGDVYLADLSPVQGSEQGGVRPVVIIQNDTGNKYSPTVIVAAITGRINKAK IPTHVEIEKKKYKLDKDSVILLEQIRTLDKKRLKEKLTYLSDDKMKEVDNALMI SLGLNAVAHQKN (SEQ ID NO: 321), or a substantially similar sequence.

[0387] The cell death gene may encode a toxin peptide or protein comprising an amino acid sequence of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 285, 287, 289, 291, 305, 316, 318, or 321, or a substantially similar amino acid sequence. Preferably, the first promoter is silent, is not active, or is minimally active, in the absence of blood or serum.

[0388] PepA1 is a toxic pore forming peptide that causes Staphylococcus aureus death by altering essential cell membrane functions. Its natural role is unknown but speculated to be altruistic assistance to the Staphylococcus aureus population / culture by killing of cells that are adversely affected by environmental conditions. By over-expressing this gene a rapid and complete cell death occurs in the presence of serum. Of note, sprA1 mRNA translation is repressed by an antisense RNA called sprA11 (SprA1 antisense). The cis-encoded SprA1 AS RNA operates in trans to downregulate the sprAl-encoded peptide expression in vivo, as described in WO 2013 / 050590, The antisense RNA may in fact be a convenient safeguard to minimize "leaky" toxicity. It will be driven from a promoter that is expressed in Staphylococcus aureus on the human skin and nasal epithelia during colonization. Advantages of sprA1 include the expression of a small peptide, having known structure and activity.

[0389] In a particular embodiment, a synthetic microorganism is provided comprising a first cell death gene sprA1 operably linked to a first regulatory region comprising a blood and / or serum inducible first promoter comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163. The first promoter may be upregulated greater than 5-fold, greater than 10-fold, greater than 50-fold, greater than 100-fold, greater than 300-fold, or greater than 600-fold after 15, 30, 45, 60, 90, 120, 180 or 240 minutes of incubation in blood or serum. The first promoter may be upregulated greater than 5-fold after 90 minutes of inclubation in serum and may be selected from fhuA, fhuB, isdI, isdA, srtB, isdG, sbnE, sbnA, sbnC, and isdB. The first promoter may be upregulated greater than 100-fold after 90 minutes of incubation in serum and may be selected from isdA, srtB, isdG, sbnE, sbnA, sbnC, and isdB.

[0390] The cell death gene may encode an antimicrobial peptide comprising an amino acid sequence of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 285, 287, 289, 291, 305, 316, 318, or 321, or a substantially similar amino acid sequence thereof.

[0391] The cell death gene may be selected from any known Staphylococcus spp. toxin gene. The cell death gene may be selected from a sprA1 toxin gene, sprA2 toxin gene, 187-lysK toxin gene, holin toxin gene, sprG toxin gene, yoeB toxin gene, lysostaphin toxin gene, metallopeptidase toxin gene, or mazF toxin gene, or a substantially identical toxin gene. The toxin gene may comprise a nucleotide sequence of SEQ ID NO: 274, 275, 284, 286, 288, 290, 304, 315, 317, or 320, or a substantially identical nucleotide sequence thereof.

[0392] The cell death gene may be sprA1 which encodes the antimicrobial peptide PepA1. In some embodiments, the synthetic microorganism further comprises an antitoxin gene SprA1-AS operably linked to a second regulatory region comprising a second promoter comprising a nucleotide sequence of clfB comprising a nucleotide sequence of SEQ ID NO: 7, 117, 118, 129 or 130, or a substantially identical sequence.

[0393] In some embodiments, the synthetic microorganism comprises a restriction enzyme KpnI (Klebsiella pnemoniae) gene. KpnI protects bacterial genomes against invasion by foreign DNA. High-level expression of (eg) 6-bp recognition restriction enzyme KpnI will efficiently cleave the Staphylococcus aureus genome. In some embodiments, the expression vector (below) will be engineered to lack cleavage recognition sites by (eg) adjustment of codon usage. The 6-base recognition sequence occurs once every 4096 bp, cutting the 2.8 MB genome of Staphylococcus aureus into ~684 fragments. KpnI has the advantage of rapid activity. In some embodiments, "leaky" expression problem may be managed by expressing an RNA aptamer as the clamp as described above for sprA1.

[0394] In some embodiments, the synthetic microorganism comprises a rsaE gene. The rsaE gene is a small RNA (93 nt) that coordinately inhibits 2 different metabolic pathways by targeting translation initiation of certain housekeeping mRNAs encoding enzymes of THF biosynthesis pathway and citric acid cycle; high-level expression is toxic. By over-expressing RseE growth impairment occurs due to inhibition of essential housekeeping enzymes. This occurs by binding to the Opp3A and OppB mRNAs in the ribosome-binding site and start codon region, preventing translation. Both genes encode components of the ABC peptide transporter system and affect the supply of essential nitrogen / amino acids in the cell, impairing central biochemical metabolism directly and indirectly. Advantages include severe growth inhibition (10,000 fold over empty vector controls), and efficient multifunctionality because a single sRNA impairs expression of multiple essential biochemical pathways. Geissman et al. 2009 and Bohn et al. 2010 report on the natural function of RsaE.

[0395] Creation of a panel of serum-activated kill switch (KS) plasmid candidates for expression in Staphylococcus aureus is performed wherein serum responsive RRs are sub-cloned to Staphylococcus aureus shuttle vectors; cell death genes are inserted downstream of RRs, and sequenced; feasibility of leaky expression repressor "expression clamp" is tested; and candidate strains are completed and evaluated to select lead candidate(s) that exhibit rapid and complete death, and good baseline viability.

[0396] Chromosomal integration of optimal kill switch candidates is important for long-term stable expression. In addition, comparison of death rate extent and stability of strains in vitro is performed. Insertion of up to 3 optimal kill switch cassettes alone and in 3 combinations of two, for a total of up to 6 strains is performed. This achievement may require a multistep cloning in E. coli to build the constructs. For example, E. coli strain DC10B may be employed. DC10B is an E. coli strain that is only DCM minus (BEI product number NR-49804). This is one way to generate DNA that can be readily transfected into most Staphylococcus aureus strains. To this end, stable integrants are obtained, and plasmid vector is excised during counter selection. The rate and extent of serum-induced cell death is confirmed and characterized, and genetic stability is determined for all 6 strains. A non-human functional test of preferred KS strain candidates is performed including a functional test of strain death in vivo; and a functional test of colonization-skin discs.

[0397] In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided comprising (1) producing a shuttle vector pCN51 in mid-scale in E. coli, (2) cloning cell death genes into pCN51 in E. coli under Cd-inducible promoter P cad , (3) replacing P cad with serum-responsive promoters and optionally inserting expression clamp, (4) verifying constructs by sequencing KS cassettes, (5) electroporating into Staphylococcus aureus RN4220 and selecting transformants on erythromycin plates (this strain is restriction minus and generates the right methylation pattern to survive in BioPlx-01), (6) preparing plasmid from RN4220 and restriction digest to confirm identification, (7) electroporating plasmids into BioPlx-01 and select on erythromycin plates, and (8) isolating strains. Stains produced in this fashion are ready for performance testing and serum experimentation. The method is further described in detail herein.

[0398] In some embodiments, a method for performance testing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided comprising (1) growing in TSB plus antibiotic as selective pressure for plasmid, (2) comparing growth to WT BioPlx-01 optionally generating a growth curve, (3a) for Cd-promoter variants, washing and shifting cells to Cd-medium (control is BioPlx-01 containing empty vector with no cell death gene) - or - (3b) for KS variants, washing and shifting cells to serum (control is WT BioPlx-01 containing empty vector with no cell death gene), and (4) monitoring growth using OD 630 nm with plate reader , optionally for extended period with monitoring for escape mutants. For whole blood test, the method is only performed on preferred candidates and using colony forming units (CFUs) on TSA as death readout. If colonies form on kill switch bearing strains after they have been exposed to blood, the plasmid should be sequenced to check for mutations. If there are escape mutants, shuttle plasmid out to E. coli and sequence whole plasmid.Method for creation of serum-activated kill switch (KS) plasmid candidates for expression in Staphylococcus aureus (SA)

[0399] Methods are provided for evaluation of cell death induction comprises recombinant construction of the synthetic microorganism comprising cloning the genes into an E. coli-SA shuttle vector and transfecting this vector into BioPlx-01 for evaluation.Step 1: Request Shuttle Vector PCN51

[0400] A commercially available shuttle vector is obtained such as PCN51 (available through BEI) is one excellent choice as it contains: i) a cadmium-inducible promoter that can be used in positive control strains to prove the toxins are expressed and functional; ii) a universal Transcription terminator (TT) that will apply to all of our constructs; and, iii) well-established replicons for E. coli and Staphylococcus aureus. A schematic of commercially available shuttle vector pCN51 (BEI cat # NR-46149) is shown in FIG. 2. Genetic elements shown of pCN51 shuttle plasmid are shown in Table 8. Table 8. Elements of pCN51 Shuttle VectorShuttle Plasmid pCN51 (BEI cat # NR-46149)ElementPurposepT181cop-WT repCSA replication machineryermCerythromycin resistanceAmpbeta-lactamase; confers resistance to ampicillin in E coliColE1 OriOrigin of replication for E coliPcad-cadCCadmium-inducible promoterMCS (black box)Multiple Cloning Site; unique sites for cloning our KS.TTblaZ transcription terminator

[0401] Promoter sequences (7) used in development are shown below, the base pair numbers in leuA, hlgA and Cadmium promoters correspond to pCN51 vector location. 1. leuA promoter (P leuA ) sequence between restriction sites SphI and PstI (underlined) amplified from genomic BioPlx-01 (502a) DNA. 2. hlgA promoter (P hlgA ) sequence between restriction sites SphI and PstI amplified from genomic BioPlx-01 (502a) DNA. 3. Cadmium promoter (P cad ) sequence between restriction sites SphI and PstI. This promoter is used for controls and is part of the original pCN51 vector from BEI Resources (https: / / www.beiresources.org / ). 4. clfB promoter (P clfB ) to drive the antisense regulatory RNA sprA1 AS . This is the forward sequence with EcoRI and BamHI sites. This sequence is put in reverse to drive the sprA1 AS to potentially act as a clamp to keep the sprAI gene regulated in the absence of blood. Underlined represents EcoRI and BamHI sites, respectively. P clfB as it is cloned in pCN51 vector with EcoRI and BamHI reversed. 5. The sirA promoter (P sirA ) as found in the NCBI 502a complete genome. This sequence was taken 300 base pairs upstream of the sirA start codon as shown underlined below. 6. The sstA promoter (P sstA ) as found in the NCBI 502a complete genome. This sequence was taken 300 base pairs upstream of the sstA start codon as shown underlined below. 7. The isdA promoter (P isdA ). This sequence was taken 300 base pairs upstream of the SstA start site as shown underlined below from the NCBI 502a complete genome.

[0402] In some embodiments, a plasmid, vector, or synthetic microorganism is provided comprising a molecular modification comprising a cell death gene operably linked to an inducible blood or serum responsive first promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, and 163, or a substantially identical nucleotide sequence. In some embodiments, the molecular modification further comprises an expression clamp comprising an antitoxin gene operably linked to a second promoter comprising a nucleotide sequence selected from SEQ ID NO: 7, 117, 118, 129 or 130.Step 2: Cloning best two serum-responsive RRs into the shuttle vector (E. coli host)

[0403] Cloning of candidate serum-responsive RRs into the shuttle vector (E. coli host) comprises: (a) PCR amplification of the best two preferred serum-responsive RRs from BioPlx-01 genomic DNA (gDNA); and (b) replacing the Cadmium-inducible promoter with these RR fragments in pCN51 to create two new plasmids (RR1 and RR2), and (3) selecting clones in E. coli DH10B (or DH5 alpha) and sequencing of insertions.

[0404] The following KS genes are obtained from Staphylococcus aureus gDNA or by de novo synthesis: (i) sprA1 / sprA1 AS : synthetic; (ii) RsaE: Staphylococcus aureus genomic DNA. And (iii) KpnI: synthetic. For genes amplified from gDNA, PCR primers are used with relevant restriction enzymes for cloning. For synthetic genes, the cloning sites will be included at synthesis and any undesirable sites removed during construction. For example, KpnI sites will be removed from the kpn1 cassette to prevent auto-digestion. The KS genes are inserted downstream of serum-responsive RRs in plasmids RR1 and RR2, generating all constructs listed below. Insert the KS genes downstream of Cd-inducible promoter in pCN51 to create positive control constructs. See additional relevant sequences and primer sequences provided herein useful for these steps, for example, Tables 2, 3 and 4. Sequencing of promoters and inserts of all constructs is performed to ensure that mutations have not accumulated in the construction process

[0405] A list of Plasmid constructs to be produced is shown below. All but 2, 4, 8 and 11 will be transfected into Staphylococcus aureus. 1. Cd-inducible promoter-sprA1 2. Cd-inducible promoter-reverse orientation sprA1 3. Serum responsive RR1- sprA1 4. Serum responsive RR1-reverse orientation sprA1 5. Serum responsive RR1- sprA1 + P clfB -sprA1 AS 6. Serum responsive RR2- sprA1 7. Serum responsive RR1-rsaE 8. Serum responsive RR1- rsaE -reverse orientation 9. Serum responsive RR2- rsaE 10. Serum responsive RR1-kpnI 11. Serum responsive RR1- kpnI reverse orientation 12. Serum responsive RR2- kpnI

[0406] The reverse orientation constructs are being created in the process, because if a cell death gene has some basal toxicity even in growth medium, it may not be possible to obtain the forward orientation construct. Such a negative result is not conclusive unless the reverse orientation construct is readily obtained in side-by-side fashion.

[0407] Step 3: Transfect plasmids into intermediate Staphylococcus aureus RN4220 (to obtain correct DNA methylation pattern). There is no need to transfect reverse orientation constructs; but transfection of pCN51 empty vector is performed as follows: A. Electroporate into RN4220; B. Select transformants on plates containing erythromycin; and C. Isolate and confirm plasmid ID with restriction digests. Step 4: Transfect into BioPlx-01

[0408] A. Electroporate plasmids from step 3C into competent BioPlx-01; B. Select transformants by erythromycin resistance; and C. Isolate and confirm plasmid ID with restriction digests; save stocks of 9 strains. Step 5: Test KS expression and extent and rate of death in response to serum and blood exposure

[0409] A. Qualitative test of expression of kill genes with real time PCR pre- and post-blood / serum exposure. This will: i) confirm the strain construction; ii) correlate onset of toxin production with onset of death, and iii) determine promoter "leakiness" in the context of the KS; B. Cell death induction curves in serum / blood compared to TSB (killing extent and kinetics by CFU); and C. Simple growth rate comparison of BioPlx-01 containing empty vector vs. BioPlx-01 with the KS plasmids. Step 6: Measure the rate of KS mutation

[0410] Count colonies that grow on serum or blood agar plates and / or in serum containing liquid media over several hundred generations via serial passaging. Determine if mutation rate is acceptable. It has been reported that the rate of functional KS loss is 10 -6< for one copy of a KS gene, but as low as 10 -10< for two copies of the same or different KS genes from two different promoters (Knudsen 1995; reporting on actual mutation rate assay measurements).Step 7: Analysis and interpretation

[0411] The best KS strain(s) are those with unaffected growth rates (and colonization potential); and that show rapid and complete death in response to blood and / or serum; and that have stable molecular modifications.Step 8: Determine need for inserting multiple KS cassettes

[0412] If the molecular stability of one KS is deemed inadequate, a second and different functional KS from the list of 9 candidates (if another functional one exists) will be added to the plasmid and a re-test of killing and stability will be performed. A dramatic improvement in KS stability is anticipated on the basis of Knudsen 1995 and theoretical calculations.Method for chromosomal integration of optimal kill switch(es), for long-term stable expression

[0413] The optimal serum / blood responsive KS construct(s) will be integrated into the chromosome precisely at a pre-selected location known to tolerate insertions without notably altering the cell's biology.Step 1: Obtain an Integrative vector for use in Staphylococcus aureus.

[0414] After careful consideration to the optimal integrative vector, plasmids pKOR1 or pIMAY may be employed because they provide the ability to choose the integration site, allowing us to avoid perturbing biologically critical regions of the genome that can occur with other methods. Both vectors possess a convenient means for counter-selection (secY) so that the plasmid backbone and its markers can be excised from the genome after the KS has been integrated. A genetic map of pKOR1 is shown in FIG. 5A and the features are described in Bae et al. 2006 Plasmid 55, pp. 58-63, and briefly described in Table 9. An advantage of pKOR is the ability to clone inserts without the limits of specific restriction enzymes. Table 9. Purpose of elements in pKOR integrative plasmidIntegrative Plasmid pKORElementPurposeAmpRbeta-lactamase; confers resistance to ampicillin in E. coli (but not in Staphylococcus aureus)Ori (-)E. coli origin of replicationAttp1 and 2Recombine with AttB elements of DNA insertsCcdBE. coli gyrase inhibitor protein; growth of cells containing non-recombinant plasmid are inhibited by this proteinCat- and Cat +Chloramphenicol resistance genes for use in gram neg and gram + bacteria respectivelySecY570570 nt encoding essential N terminus of secY; its antisense is expressed from the ATc-indicible pxyl / tetO promoter; growth in the presence of Atc means the plasmid backbone has been lostRepFReplication gene for Staphylococcus aureus

[0415] A Genetic map of pIMAY is shown in FIG. 5B from Monk, IR et al., mBio 2012; doi:10.1128 / mBio.00277-11. FIG. 12A-12C shows nucleotide sequence (SEQ ID NO: 131) of pIMAY Integrative Plasmid. (accession number JQ62198). The E. coli / staphylococcal temperature-sensitive plasmid pIMAYz comprises the low-copy-number E. coli origin of replication (p15A), an origin of transfer for conjugation (oriT), the pBluescript multiple cloning site (MCS), and the highly expressed cat gene (Phelp-cat) derived from pIMC. The temperature-sensitive replicon for Gram-positive bacteria (repBCAD) and the anhydrotetracycline-inducible antisense secY region (anti-secY) may be amplified from pVE6007 and pKOR1, respectively. The restriction sites listed are unique. Primers (IM151 / 152) bind external to the MCS of pIMAY and are used to screen clones in E. coli (amplify 283 bp without a cloned insert) and to determine the presence of a replicating plasmid in staphylococci. Advantages of pIMAYz are smaller size, blue white screening, and a lower nonpermissive temperature, which has been reported to avoid mutations that can occur in the integration process. Thus, the plasmid may be made by de novo gene synthesis at a contract vendor firm.Step 2. Review selectable markers in BioPlx-01.

[0416] BioPlx-01 is sensitive to ampicillin (50 µg / mL and 100 µg / mL), chloramphenicol (10 µg / mL), and erythromycin (Drury 1965). In one embodiment, the chloramphenicol (cat+) gene is used to select for transformants on chloramphenicol plates during the integration process.Step 3. Generate the DNA fragment to be integrated.

[0417] Prepare a plasmid in shuttle vector pTK1 that contains the following elements in tandem: [aTTB2]-[1 Kb of sequence upstream of target region to be replaced]-[KS cassette-AmpR]-[1 Kb of sequence downstream of target region] ATTB1 according to a modification of Bae et al., 2006. Drop the fragment out of this plasmid with restriction enzymes and isolate it. The "KS cassette" may actually be one or two copies of a KS, pending the outcome of genetic stability testing.Step 4. Insert KS cassette(s) to pKOR plasmid.

[0418] Perform in vitro recombination of the fragment from step 3 with the plasmid PKOR1 and then transfect the recombination mixture into DH5 alpha and obtain desired plasmid construct by standard screening methods in E. coli, using restriction mapping to verify construction.Step 5. Obtain the KS strain-containing integration plasmid, in BioPlx-01

[0419] Electroporate the plasmid into RN4220; isolate plasmid DNA from the thus transfected RN4220, and electroporate this DNA into BioPlx-01 and select transformants on TSA plates containing chloramphenicol (10 µg / mL).Step 6. Plasmid integration to chromosome.

[0420] Shift the strains to the non-permissive temperature (43°C) to promote plasmid integration to the target site, and select a colony on a chloramphenicol plate (10 µg / mL).Step 7. Counter selection to evict plasmid backbone

[0421] Grow the colony isolate from step 6 at the permissive temperature (30°C) to favor plasmid excision and plate on 2 µg / mL and 3 µg / mL anhydrotetracycline (aTc) agar to obtain colonies in which the target gene has integrated and the plasmid has been excised and lost (the counterselection step). Any colonies that grow on plates containing ≥2 µg / mL aTc do not contain the plasmid because the plasmid backbone contains the lethal aTc-derepressible SecY antisense gene.Step 8. Confirm integrated allele sequence

[0422] Isolate genomic DNA from the KS strain and confirm the knock-in cassette and flanking structure by PCR (and sequencing of the PCR amplicon).Step 9. Check serum-induced cell death

[0423] Once confirmed, conduct cell death rate assays by growing the cells first in TSB, then shifting to human blood or serum and determining the rate of death by CFU plating assays in TSA (10 days).Step 10. Verify expression of KS mRNA

[0424] Confirm expression changes of the target gene in blood, serum, and in TSB.Step 11. Prepare frozen banks

[0425] Animal studies may be performed with synthetic microorganisms BioPlx-XX created by these methods. In vivo functional studies to test kill switch strain function may be performed. Possible studies include a mouse study to show difference in pathogenicity of intravenous or intraperitoneal injection of wt BioPlx-01 vs. KS strain. An in vitro skin colonization test may also be performed. Additional tests may include, in mouse: LD 50 test, BioPlx-01 vs. BioPlx-XX is performed. As another example, in rat or other: colonization test, BioPlx-01 vs. BioPlx-XX is performed.CRISPR-Cas induced homology directed repair to direct insertion of optimal kill switch candidates for long term stable expression

[0426] In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided comprising use of CRISPR-Cas induced homology directed repair to direct insertion of optimal KS candidates for long-term stable expression. In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided comprising (1) obtaining competent cells, (2) design and testing of CRISPR guide RNA (gRNA) sequences and simultaneously testing pCasSA, (3) designing and testing homology dependent repair templates using a fluorescent reporter controlled by a constitutive reporter, (4) checking KS promoters with fluorescent reporter, (5) inserting KS into BioPlx-01 and verifying incorporation, and (6) testing for efficacy and longevity. Optionally, inserting additional KS cassettes in alternative locations within BioPlx-01 genome is performed.

[0427] FIG. 10 shows cassette for integration via CRISPR and layout of the pCasSA vector. PcaplA is a constitutive promoter controlling gRNA transcription. Target seq is targeting sequence, for example, with 10 possible cutting targets (1.1, 1.2 etc.). gRNA is single-strand guide RNA (provides structural component). Xbal and Xho1 are two restriction sites used to add the homology arms (HAs) to the pCasSA vector. HAs are homology arms to use as templates for homology directed repair (200 - 1000 bp). P rpsL -mCherry is a constitutive promoter controlling the "optimized" mCherry. P rpsL -Cas9 is a constitutive promoter controlling Cas9 protein expression.

[0428] FIG. 11 shows vectors for various uses in the present disclosure. A is a vector used for promoter screen with fluorescence using pCN51. B is a vector for promoter screen with cell death gene. C is a vector for chromosomal integration using CRISPR. D is a vector for chromosomal integration using homologous recombination. L & R HA: homology arms to genomic target locus, CRISPR targeting: RNA guide to genomic locus, mCherry: fluorescent reporter protein, Cas9 protein: CRISPR endonuclease, kanR: kanamycin resistance, oriT: origin of transfer (for integration), and Sma1: representative kill gene (restriction endonuclease).Administration and Compositions

[0429] In some embodiments, compositions are provided comprising a synthetic microorganism and an excipient, or carrier. The compositions can be administered in any method suitable to their particular immunogenic or biologically or immunologically reactive characteristics, including oral, intravenous, buccal, nasal, mucosal, dermal or other method, within an appropriate carrier matrix. In one embodiment, compositions are provided for topical administration to a dermal site, and / or a mucosal site in a subject. Another specific embodiment involves the oral administration of the composition of the disclosure.

[0430] In some embodiments, the replacing step comprises topically administering of the synthetic strain to the dermal or mucosal at least one host subject site and optionally adjacent areas in the subject no more than one, no more than two, or no more than three times. The administration may include initial topical application of a composition comprising at least 10 6< , at least 10 7< , at least 10 8< , at least 10 9< , or at least 10 10< CFU of the synthetic strain and a pharmaceutically acceptable carrier to the at least one host site in the subject. The initial replacing step may be performed within 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days of the final suppressing step.

[0431] The composition comprising a synthetic microorganism may be administered to the dermal and / or mucosal at least one site in the subject, and optionally adjacent sites at least once, for example, from one to 30 times, one to 20 times, one to ten times, one to six times, one to five times, one to four times, one to three times, or one to two times, or no more than once, twice, three times, 4 times, 5 times, 6 times, 8 times per month, 10 times, or no more than 12 times per month. Subsequent administration of the composition may occur after a period of, for example, one to 30 days, two to 20 days, three to 15 days, or four to 10 days after the first administration.

[0432] Colonization of the synthetic microorganism may be promoted in the subject by administering a composition comprising a promoting agent selected from a nutrient, prebiotic, stabilizing agent, humectant, and / or probiotic bacterial species. The promoting agent may be administered to a subject in a separate promoting agent composition or may be added to the microbial composition.

[0433] In some embodiments, the promoting agent may be a nutrient, for example, selected from sodium chloride, lithium chloride, sodium glycerophosphate, phenylethanol, mannitol, tryptone, and yeast extract. In some embodiments, the prebiotic is selected from the group consisting of short-chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid), glycerol, pectin-derived oligosaccharides from agricultural by-products, fructo-oligosaccarides (e.g., inulin-like prebiotics), galacto-oligosaccharides (e.g., raffinose), succinic acid, lactic acid, and mannan-oligosaccharides.

[0434] In some embodiments, the promoting agent may be a probiotic. The probiotic may be any known probiotic known in the art. Probiotics are live microorganisms that provide a health benefit to the host. In methods provided herein, probiotics may be applied topically to dermal and mucosal microbiomes, and / or probiotics may be orally administered to provide dermal and mucosal health benefits to the subject. Several strains of Lactobacillus have been shown to have systemic anti-inflammatory effects. Studies have shown that certain strains of Lactobacillus reuteri induce systemic anti-inflammatory cytokines, such as interleukin (IL)-10. Soluble factors from Lactobacillus reuteri inhibit production of pro-inflammatory cytokines. Lactobacillus paracasei strains have been shown to inhibit neutrogenic inflammation in a skin model Kober at al., 2015, Int J Women's Dermatol 1(2015) 85-89. In human dermal fibroblasts and hairless mice models, Lactobacillus Plantarum has been shown to inhibit UVB-induced matrix metalloproteinase 1 (MMP-1) expression to preserve procollagen expression in human fibroblasts. Oral administration of L. plantarum in hairless mice histologic samples demonstrated that L. plantarum inhibited MMP-13, MMP-2, and MMP-9 expression in dermal tissue.

[0435] Clinically, the topical application of probiotics has also been shown to modify the barrier function of the skin with a secondary increase in antimicrobial properties of the skin. Streptococcus thermophiles when applied topically has been shown to modify the barrier function of the skin with a secondary increase in antimicrobial properties of the skin. Streptococcus thermophiles when applied topically has been shown to increase ceramide production both in vitro and in vivo. Ceramides trap moisture in the skin, and certain ceramide sphingolipids, such as phytosphingosine (PS), exhibit direct antimicrobial activity against P. acnes. Kober at al., 2015, Int J Women's Dermatol 1(2015) 85-89.

[0436] Two clinical trials of topical preparations of probiotics have assessed their effect on acne. Enterococcus fecalis lotion applied to the face for 8 weeks resulted in a 50% reduction of inflammatory lesions was noted compared to placebo. A reduction in acne count, size, and associated erythema was noted during a clinical study of Lactobacillus plantarum topical extract. Kober at al., 2015, Int J Women's Dermatol 1(2015) 85-89.

[0437] Clinical trials of topical probiotics have evaluated their effect on mucosal systems. In one study, Streptococcus salivarius was administered by nasal spray for the prevention of acute otitis media (AOM). If the nasopharynx was successfully colonized, there was significant effect on reducing AOM. Marchisio et al. (2015). Eur. J. Clin. Microbiol. Infect. Dis. 34, 2377-2383. In another trial, sprayed application of S. sanguinis and L. Rhamnosus decreased middle ear fluid in children with secretory otitis media. Skovbjerg et al. (2008). Arch. Dis. Child. 94, 92-98.

[0438] The probiotic may be a topical probiotic or an oral probiotic. The probiotic may be, for example, a different genus and species than the undesirable microorganism, or of the same genus but different species, than the undesirable microorganism. The probiotic species may be a different genus and species than the target microorganism. The probiotic may or may not be modified to comprise a kill switch molecular modification. The probiotic may be selected from a Lactobacillus spp, Bifidobacterium spp. Streptococcus spp., or Enterococcuss spp. The probiotic may be selected from Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, Streptococcus thermophiles, Streptococcus salivarius, or Enterococcus fecalis.

[0439] The promoting agent may include a protein stabilizing agent such as those disclosed in U.S. Pat. No. 5,525,336 is included in the composition. Non-limiting examples include glycerol, trehelose, ethylenediaminetetraacetic acid, cysteine, a cyclodextrin such as an alpha-, beta-, or gamma-cyclodextrin, or a derivative thereof, such as a 2-hydroxypropyl betacyclodextrin, and proteinase inhibitors such as leupeptin, pepstatin, antipain, and cystatin.

[0440] The promoting agent may include a humectant. Non-limiting examples of humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, and dibutylphthalate.Compositions

[0441] Compositions are provided comprising a synthetic microorganism and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetening agent, flavoring agent, buffer, thickener, wetting agent, or absorbent.

[0442] Pharmaceutically acceptable diluents or carriers for formulating the composition are selected from the group consisting of water, saline, phosphate buffered saline, or a solvent. The solvent may be selected from, for example, ethyl alcohol, toluene, isopropanol, n-butyl alcohol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulphoxide, dimethyl formamide and tetrahydrofuran. The carrier or diluent may further comprise one or more surfactants such as i) Anionic surfactants, such as metallic or alkanolamine salts of fatty acids for example sodium laurate and triethanolamine oleate; alkyl benzene sulphones, for example triethanolamine dodecyl benzene sulphonate; alkyl sulphates, for example sodium lauryl sulphate; alkyl ether sulphates, for example sodium lauryl ether sulphate (2 to 8 EO); sulphosuccinates, for example sodium dioctyl sulphonsuccinate; monoglyceride sulphates, for example sodium glyceryl monostearate monosulphate; isothionates, for example sodium isothionate; methyl taurides, for example Igepon T; acylsarcosinates, for example sodium myristyl sarcosinate; acyl peptides, for example Maypons and lamepons; acyl lactylates, polyalkoxylated ether glycollates, for example trideceth-7 carboxylic acid; phosphates, for example sodium dilauryl phosphate; Cationic surfactants, such as amine salts, for example sapamin hydrochloride; quartenary ammonium salts, for example Quaternium 5, Quaternium 31 and Quaternium 18; Amphoteric surfactants, such as imidazol compounds, for example Miranol; N-alkyl amino acids, such as sodium cocaminopropionate and asparagine derivatives; betaines, for example cocamidopropylebetaine; Nonionic surfactants, such as fatty acid alkanolamides, for example oleic ethanolamide; esters or polyalcohols, for example Span; polyglycerol esters, for example that esterified with fatty acids and one or several OH groups; Polyalkoxylated derivatives, for example polyoxy:polyoxyethylene stearate; ethers, for example polyoxyethe lauryl ether; ester ethers, for example Tween; amine oxides, for example coconut and dodecyl dimethyl amine oxides. In some embodiments, more than one surfactant or solvent is included.

[0443] The composition may include a buffer component to help stabilize the pH. In some embodiments, the pH is between 4.5-8.5. For example, the pH can be approximately 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, including any value in between. In some embodiments, the pH is from 5.0 to 8.0, 6.0 to 7.5, 6.8 to 7.4, or about 7.0. Non-limiting examples of buffers can include ACES, acetate, ADA, ammonium hydroxide, AMP (2-amino-2-methyl-1-propanol), AMPD (2-amino-2-methyl-1,3-propanediol), AMPSO, BES, BICINE, bis-tris, BIS-TRIS propane, borate, CABS, cacodylate, CAPS, CAPSO, carbonate (pK1), carbonate (pK2), CHES, citrate (pK1), citrate (pK2), citrate (pK3), DIPSO, EPPS, HEPPS, ethanolamine, formate, glycine (pK1), glycine (pK2), glycylglycine (pK1), glycylglycine (pK2), HEPBS, HEPES, HEPPSO, histidine, hydrazine, imidazole, malate (pK1), malate (pK2), maleate (pK1), maleate (pK2), MES, methylamine, MOBS, MOPS, MOPSO, phosphate (pK1), phosphate (pK2), phosphate (pK3), piperazine (pK1), piperazine (pK2), piperidine, PIPES, POPSO, propionate, pyridine, pyrophosphate, succinate (pK1), succinate (pK2), TABS, TAPS, TAPSO, taurine (AES), TES, tricine, triethanolamine (TEA), and Trizma (tris). Excipients may include a lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, phosphate buffer, or any other ingredient of the similar nature alone or in a suitable combination thereof.

[0444] The microbial composition may include a binder may, for example, a gum tragacanth, gum acacia, methyl cellulose, gelatin, polyvinyl pyrrolidone, starch or any other ingredient of the similar nature alone or in a suitable combination thereof; excipients selected from the group consisting of agar-agar, calcium carbonate, sodium carbonate, silicates, alginic acid, corn starch, potato tapioca starch, primogel or any other ingredient of the similar nature alone or in a suitable combination thereof; lubricants selected from the group consisting of a magnesium stearate, calcium stearate, talc, solid polyethylene glycols, sodium lauryl sulfate or any other ingredient of the similar nature alone; glidants selected from the group consisting of colloidal silicon dioxide or any other ingredient of the similar nature alone or in a suitable combination thereof; a stabilizer selected from the group consisting of such as mannitol, sucrose, trehalose, glycine, arginine, dextran, or combinations thereof; an odorant agent or flavoring selected from the group consisting of peppermint, methyl salicylate, orange flavor, vanilla flavor, or any other pharmaceutically acceptable odorant or flavor alone or in a suitable combination thereof; wetting agents selected from the group consisting of acetyl alcohol, glyceryl monostearate or any other pharmaceutically acceptable wetting agent alone or in a suitable combination thereof; absorbents selected from the group consisting of kaolin, bentonite clay or any other pharmaceutically acceptable absorbents alone or in a suitable combination thereof; retarding agents selected from the group consisting of wax, paraffin, or any other pharmaceutically acceptable retarding agent alone or in a suitable combination thereof.

[0445] The microbial composition may comprise one or more emollients. Non-limiting examples of emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl mono stearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpolysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arrachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate.

[0446] The microbial composition may include a thickener, for example, where the thickener may be selected from hydroxyethylcelluloses (e.g. Natrosol), starch, gums such as gum arabic, kaolin or other clays, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose or other cellulose derivatives, ethylene glycol monostearate and sodium alginates. The microbial composition may include preservatives, antiseptics, pigments or colorants, fragrances, masking agents, and carriers, such as water and lower alkyl, alcohols, such as those disclosed in U.S. Pat. No. 5,525,336 are included in compositions.

[0447] The microbial compositions for topical administration may be provided in liquid, solution, suspension, cream, lotion, ointment, gel, or in a solid form such as a powder, tablet, or troche for suspension immediately prior to administration. The compositions for topical use may also be provided as hard capsules, or soft gelatin capsules, wherein the benign and / or synthetic microorganism is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules for dissolution in a conventional manner using, e.g., a mixer, a fluid bed apparatus, lyophilization or a spray drying equipment. A dried microbial composition may administered directly or may be for suspension in a carrier. When the composition is in a powder form, the powders may include chalk, talc, fullers earth, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl and / or trialkyl aryl ammonium smectites and chemically modified magnesium aluminum silicate in a carrier. When the composition is in a powder form, the powders may include chalk, talc, fullers earth, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl and / or trialkyl aryl ammonium smectites and chemically modified magnesium aluminum silicate

[0448] The microbial composition may exhibit a stable CFU losing less than 30%, 20%, 10% or 5% cfu over at least one, two, three months, six months, 12 months 18 months, or 24 months when stored at frozen, refrigerated or preferrably at room temperature.Kits

[0449] Any of the above-mentioned compositions or synthetic microorganisms may be provided in the form of a kit. In some embodiments, a kit comprises a container housing live bacteria or a container housing freeze-dried live bacteria. Kits can include a second container including media. Kits may also include one or more decolonizing agents. Kits can also include instructions for administering the composition. In certain embodiments, instructions are provided for mixing the bacterial strains with other components of the composition. In some embodiments, a kit further includes an applicator to apply the microbial composition to a subject.Dose

[0450] In certain embodiments, a composition is provided for topical administration that is a solution composition, or for reconstitution to a solution composition. In one embodiment, composition may include from about 1 x 10 5< to 1 x 10 12< cfu / ml, 1 x 10 6< to 1 x 10 10< cfu / ml, or 1.2 x 10 7< to 1.2 x 10 9< CFU / mL of the synthetic microorganism in an aqueous solution, such as phosphate buffered saline (PBS). Lower doses may be employed for preliminary irritation studies in a subject.

[0451] Preferably, the subject does not exhibit recurrence of the undesirable microorganism as evidenced by swabbing the subject at the at least one site after at least 2, 3, 4, 6, 10, 15, 22, 26, 30 or 52 weeks after performing the initial administering step.Nanofactory

[0452] In some embodiments, methods are provided to create production of a desired substance a...

Claims

1. A synthetic microorganism, comprising a recombinant nucleotide comprising at least one kill switch molecular modification comprising a first cell death gene operatively associated with a first regulatory region comprising an inducible first promoter, wherein the first inducible promoter exhibits conditionally high level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma of at least three fold increase of basal productivity, wherein the at least one molecular modification comprises a cell death gene nucleotide sequence that is integrated to a chromosome of the synthetic microorganism, and measurable average cell death of the synthetic microorganism occurs within at least a preset period of time following induction of the first promoter.

2. The synthetic microorganism of claim 1, wherein the synthetic microorganism further comprises at least a second molecular modification (expression clamp) comprising an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is operably associated with a second regulatory region comprising a second promoter which is active (constitutive) upon dermal or mucosal colonization or in a complete media, but is not induced, induced less than 1.5-fold, or is repressed after exposure to blood, serum or plasma for at least 30 minutes, and wherein the antitoxin gene encodes an antisense RNA sequence capable of hybridizing with at least a portion of the first cell death gene.

3. The synthetic microorganism of claim 1 or 2, wherein the synthetic microorganism is derived from a target microorganism having the same genus and species as an undesirable microorganism.

4. The synthetic microorganism of claim 1 or 2, wherein the first promoter is upregulated by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold within at least 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, or at least 360 min following exposure to blood, serum, or plasma, preferably wherein the first promoter is not induced, induced less than 1.5 fold, or is repressed in the absence of blood, serum, heme, or plasma.

5. The synthetic microorganism of claim 2, wherein the second regulatory region comprising a second promoter is active upon dermal or mucosal colonization or in TSB media, but is repressed at least 2 fold upon exposure to blood, serum or plasma after a period of time selected from the group consisting of the group consisting of at least 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, and at least 360 min.

6. The synthetic microorganism of any one of claims 1 to 5, wherein the measurable average cell death occurs within at least a preset period of time selected from the group consisting of_within at least 1, 5, 15, 30, 60, 90, 120, 180, 240, 300, and 360 minutes following exposure to blood, serum, heme, or plasma, and wherein the measurable average cell death is at least a 50% cfu, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% cfu count reduction following the preset period of time, preferably wherein the kill switch molecular modification reduces or prevents infectious growth of the synthetic microorganism under systemic conditions in a subject.

7. The synthetic microorganism of claim 1, wherein the target microorganism is selected from a bacterial, fungal, or protozoal target microorganism, optionally wherein the target microorganism is a bacterial species capable of colonizing a dermal and / or mucosal niche and is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Escherichia, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas, optionally wherein the target microorganism is selected from the group consisting of Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissimum, Cutibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Escherichia coli, Streptococcus pyogenes, Streptococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcal intermedius, Streptococcus agalactiae, Streptococcus mutans, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens, optionally wherein the target microorganism is susceptible to at least one antimicrobial agent.

8. The synthetic microorganism of claim 7, wherein synthetic microorganism is derived from a Staphylococcus aureus strain, wherein the cell death gene is selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM, or lysostaphin toxin gene, optionally wherein the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or a substantially identical nucleotide sequence.

9. The synthetic microorganism of claim 8, wherein the inducible first promoter comprises or is derived from a gene selected from the group consisting of isdA (iron-regulated surface determinant protein A), isdB (iron-regulated surface determinant protein B), isdG (heme-degrading monooxygenase), hlgA (gamma-hemolysin component A), hlgA1 (gamma-hemolysin), hlgA2 (gamma-hemolysin), hlgB (gamma-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosyntheis protein), sbnD, sbnl, sbnE (lucA / lucC family siderophore biosynthesis protein), isdl, lrgA (murein hydrolase regulator A), lrgB (murein hydrolase regulator B), ear (Ear protein), fhuA (ferrichrome transport ATP-binding protein fhuA), fhuB (ferrichrome transport permease), hlb (phospholipase C), heme ABC transporter 2 gene, heme ABC transporter gene, isd ORF3, sbnF, alanine dehydrogenase gene, diaminopimelate decarboxylase gene, iron ABC transporter gene, threonine dehydratase gene, siderophore ABC transporter gene, SAM dep Metrans gene, HarA, splF (serine protease SplF), splD (serine protease SplD), dps (general stress protein 20U), SAUSA300_2617 (putative cobalt ABC transporter, ATP-binding protein), SAUSA300_2268 (sodium / bile acid symporter family protein), SAUSA300_2616 (cobalt family transport protein), srtB (Sortase B), sbnA (probable siderophore biosynthesis protein sbnA), sbnB, sbnG, leuA (2-isopropylmalate synthase amino acid biosynthetic enzyme), sstA (iron transport membrane protein), sirA (iron ABC transporter substrate-binding protein), isdA (heme transporter), and spa (Staphyloccocal protein A), optionally wherein the first promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 114, 115, 119, 120, 121, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, and 163, or a substantially identical nucleotide sequence thereof.

10. The synthetic microorganism of claim 8 or 9, wherein the antitoxin gene is selected from the group consisting of a sprA1 antitoxin gene, sprA2 antitoxin gene, sprG antitoxin gene or sprF, holin antitoxin gene, 187-lysK antitoxin gene, yefM antitoxin gene, lysostaphin antitoxin gene, or mazE antitoxin gene, kpn1 antitoxin gene, sma1 antitoxin gene, relF antitoxin gene, rsaE antitoxin gene, or yoeB antitoxin gene, respectively, and optionally wherein the antitoxin gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319, or 322, or a substantially identical nucleotide sequence.

11. The synthetic microorganism of claim 10, wherein the second promoter comprises or is derived from a gene selected from the group consisting of clfB (Clumping factor B), sceD (autolysin, exoprotein D), walKR(virulence regulator), atlA (Major autolysin), oatA (O-acetyltransferase A); phosphoribosylglycinamide formyltransferase gene, phosphoribosylaminoimidazole synthetase gene, amidophosphoribosyltransferase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylformylglycinamidine synthase gene, phosphoribosylaminoimidazole-succinocarboxamide gene, trehalose permease IIC gen, DeoR faimly transcriptional regulator gene, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkylhydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein disaggregation chaperon gene, alkylhydroperoxidase gene, phosphofructokinase gene, gyrB, sigB, and rho, optionally wherein the second promoter is a PclfB (clumping factor B) and comprises a nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a substantially identical nucleotide sequence thereof.

12. The synthetic microorganism according to any one of claims 1 to 11, further comprising a molecular modification selected from the group consisting of a virulence block molecular modification, and nanofactory molecular modification, wherein the virulence block molecular modification prevents horizontal gene transfer of genetic material from the undesirable microorganism, and wherein the nanofactory molecular modification comprises an insertion of a gene that encodes, a knock-out of a gene that encodes, or a genetic modification of a gene that encodes a product selected from the group consisting of an enzyme, amino acid, metabolic intermediate, and a small molecule.

13. A composition comprising an effective amount of the synthetic microorganism of any one of claims 1 to 12, and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetening agent, flavoring agent, wetting agent, preservative, buffer, or absorbent, or a combination thereof, optionally further comprising a nutrient, prebiotic, commensal, and / or probiotic bacterial species.

14. A single dose unit comprising the composition of claim 13, comprising at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 CFU, or at least 1011 of the synthetic strain and a pharmaceutically acceptable carrier, optionally formulated for topical administration.

15. The synthetic microorganism of any one of claims 1 to 12, the composition of claim 13, or the single dose unit of claim 14 for use in a method for eliminating and preventing the recurrence of a undesirable microorganism in a subject hosting a microbiome, comprising: a. decolonizing the host microbiome; and b. durably replacing the undesirable microorganism with the synthetic microorganism comprising at least one element imparting a non-native attribute, wherein the synthetic microorganism is capable of durably integrating to the host microbiome, and occupying the same niche in the host microbiome as the undesirable microorganism, wherein the decolonizing is performed on at least one site in the subject to substantially reduce or eliminate the detectable presence of the undesirable microorganism from the at least one site, optionally wherein the niche is a dermal or mucosal environment that allows stable colonization of the undesirable microorganism at the at least one site, wherein the ability to durably integrate to the host microbiome is determined by detectable presence of the synthetic microorganism at the at least one site for a period of at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, wherein the ability to durably replace the undesirable microorganism is determined by the absence of detectable presence of the undesirable microorganism at the at least one site for a period of at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, and wherein the ability to occupy the same niche is determined by absence of co-colonization of the undesirable microorganism and the synthetic microorganism at the at least one site, optionally wherein the absence of co-colonization is determined at least one week, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least 12 weeks, at least 16 weeks, at least 26 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks, and preferably wherein the at least one element imparting the non-native attribute is durably incorporated to the synthetic microorganism.

16. A kit comprising in at least one container, the synthetic microorganism according to any one of claims 1 to 12, the composition according to claim 13, or the single dose unit of claim 14, and optionally at least a second container comprising a decolonizing agent, a sheet of instructions, at least a third container comprising a promoting agent, and / or an applicator.