Methods and compositions for preventing microbial infections

Abstract

The invention relates to methods and compositions for preventing microbial infections. Methods and compositions for permanently affecting the microbial ecosystem (microbiome) in a subject to prevent infection and reduce recurrence of microbial infections are provided. In some embodiments, compositions and methods are provided for the production and use of molecularly modified bacterial strains having potential to prevent infection with a variety of microorganisms.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of December 4, 2018, application number 201880088486.5, and invention name “Methods and compositions for preventing microbial infections”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application was filed as a PCT International Patent Application on December 4, 2018, and claims priority to U.S. Provisional Application No. 62 / 594,943, filed on December 5, 2017, which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing in electronic format as a file entitled "Sequence-Listing" created on December 3, 2018, and the file size is 128,065 kilobytes (KB). The contents of the txt file "Sequence-Listing" are incorporated herein by reference. Technical Field

[0006] Provided are methods and compositions for durably influencing the microbial ecosystem (microbiome) in a subject to combat infection and reduce recurrence of infection with undesirable microorganisms by decolonization and durably replacing them with synthetic microorganisms. Provided are synthetic microorganisms that can durably replace undesirable microorganisms under dermal or mucosal conditions and contain molecular modifications intended to enhance safety (e.g., by self-destruction upon exposure to systemic conditions, by reducing the likelihood of acquiring virulence or antibiotic resistance genes, and / or by producing desired products at the site of the subject's ecosystem). Background Art

[0007] Healthcare-associated or community-associated infections are often caused by colonizing microorganisms that overcome the patient's defenses. Inappropriate use of antibiotics can lead to microbiome mismanagement. A key unintended consequence of microbiome mismanagement is the emergence of antibiotic-resistant microorganisms.

[0008] Each person harbors tens of trillions of microorganisms, potentially comprising 10,000 different species, types, and strains. These "commensal" organisms reside both externally (such as the dermis) and internally (such as the gastrointestinal tract) and are essential for human survival. "Colonization" occurs automatically through continuous interaction with the environment.

[0009] Microbial fermentation constitutes the "biome," a dynamic, structured biological system that is crucial for health in many contexts and aspects. The biome structure is created by an extensive combinatorial network between the host, the environment, and the components of the biome. The human microbiome is an ecosystem. This ecosystem has a dynamic yet enduring structure—one that is "resilient" and has a normal, basic state of "health."

[0010] However, in some cases, the microbiome can be invaded and colonized by pathogenic microorganisms. This "colonization" can be a precursor to "infection." This disruption of the microbiome can lead to serious, even life-threatening, diseases.

[0011] One unintended consequence of improper biome management is the emergence of "antibiotic resistance." This occurs when antibiotics and antiseptics fail to completely eliminate the target microorganisms. The few survivors that exhibit resistance to these materials then preferentially grow ("recolonize") back into an open environment (or vacant "niche") that has been cleared of competing organisms. The survivors then dominate the space, often maintaining resistance to their offspring. If exposed to new killing agents, the survivors also tend to develop resistance. In just a few generations, these microorganisms can become resistant to many or all known antibiotics, becoming the well-known "superbugs" of today and creating enormous new global health problems in the process.

[0012] The phenomenon known as "relapse" is central to the process of developing antibiotic resistance. While there are methods to treat pathogenic infections, there are virtually no methods to prevent relapse.

[0013] Bacterial infections are at the root of the emerging "superbug" phenomenon. The overuse and misuse of antibiotics has led to strains of many pathogenic bacteria becoming resistant to a growing number of antibiotic therapies, creating a massive global public health problem. With each new antibiotic variant used to treat these superbugs, the inevitable process of selecting for resistant strains begins anew, and resistant variants of pathogens rapidly develop. Unfortunately, bacteria are developing resistance faster than new antibiotics can be developed.

[0014] In addition to fostering antibiotic resistance and often causing adverse health effects in recipients, antibiotic treatment has the undesirable effect of disrupting the entire microbiome (comprising both beneficial and harmful bacteria). This often creates new problems, such as opening the microbiome to colonization by adventitious pathogens after treatment.

[0015] However, bacteria have less leeway to adapt to different resource requirements because these requirements are more fundamental at the molecular level and inherently defined in the genome. This makes the microbiome ecology behave more like an "ideal" system, resulting in identical strains of competitors being excluded from their niches.

[0016] The community of organisms that colonizes the human body is called the microbiome. Microbiome analysis often subdivides it geographically (i.e., skin microbiome versus gastrointestinal microbiome) or systematically (i.e., bacterial microbiome versus fungal or native microbiome).

[0017] Antibiotics are lifesaving medications, but they can also alter, imbalance, and disrupt the microbiome. The microbiome is the naturally occurring community of bacteria found inside and on the skin, in the intestines, mouth, respiratory tract, and urinary tract. A healthy microbiome helps prevent infection. Antibiotics disrupt the microbiome, eliminating both "good" and "bad" bacteria. Drug-resistant bacteria, such as MRSA, CRE, and Clostridium difficile, can exploit this disruption and thrive. This overgrowth of resistant bacteria sets the body up for infection. Once a person is colonized with resistant bacteria, they can easily spread to others. See "Antibiotic Resistance (AR) Solutions Initiative: Microbiome, CDC Microbiome Fact Sheet 2016" at www.cdc.gov / drugresistance / solutions-initiative / innovations-to-slow-AR.html.

[0018] According to the Centers for Disease Control (CDC), the major resistance threats to the United States include Neisseria gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, methicillin-resistant Staphylococcus aureus (MRSA), and drug-resistant Streptococcus pneumoniae. 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.

[0019] Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted disease that can cause discharge and inflammation of the urethra, cervix, pharynx, or rectum. Approximately 820,000 cases of gonorrhea occur annually. Of these, approximately 246,000 cases are drug-resistant: 188,600 are tetracycline-resistant, 11,489 have reduced susceptibility to cefixime, 3,280 have reduced susceptibility to ceftriaxone, and approximately 2,460 have reduced susceptibility to azithromycin.

[0020] Acinetobacter is a gram-negative bacteria that can cause pneumonia or bloodstream infections in critically ill patients. Many of these bacteria have become highly resistant to antibiotics. Each year, there are approximately 12,000 cases of Acinetobacter infections, including approximately 7,300 cases of multi-resistant Acinetobacter infections and 500 deaths.

[0021] Candidiasis is a fungal infection caused by yeast from the genus Candida. There are over 20 species of Candida yeast 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 organisms can lead to symptoms. The symptoms of candidiasis vary depending on the part of the body infected. Candida is the fourth most common cause of healthcare-associated bloodstream infections in the United States. In some hospitals, Candida is the most common cause. These infections often occur in the sickest patients. Each year, approximately 46,000 Candida infections occur, including approximately 3,400 fluconazole-resistant Candida infections and 220 deaths.

[0022] Staphylococcus aureus is a common type of bacteria found on the skin. During medical procedures, when patients require catheters or ventilators or undergo surgical procedures, S. 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, which can lead to sepsis and death. Staphylococci (including MRSA) are one of the most common causes of health care-associated infections. Each year, there are approximately 80,461 serious MRSA infections and approximately 11,285 deaths caused by MRSA. When S. aureus becomes resistant to vancomycin, there are few treatments available because the vancomycin-resistant S. aureus identified to date are also resistant to methicillin and other classes of antibiotics. Since 2002, at least 13 cases of vancomycin-resistant S. aureus have been reported in four states.

[0023] Streptococcus pneumoniae (S. pneumoniae or pneumococcus) is the leading cause of bacterial pneumonia and meningitis in the United States. It is also a leading cause of bloodstream infections and ear and sinus infections. Each year, approximately 1.2 million antibiotic-resistant infections are diagnosed, resulting in approximately 19,000 additional hospitalizations and 7,000 deaths.

[0024] 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). In a given year, there are approximately 100,000 to 2.6 million cases of strep throat, including approximately 1,300 cases of drug-resistant GAS infections, and approximately 160 deaths.

[0025] Group B Streptococcus (GBS) is a bacteria that can cause serious illness in people of all ages, ranging from bloodstream infections (sepsis) and pneumonia to meningitis and skin infections. Group B Streptococcus is the leading cause of serious microbial infections in newborns. In 2011, there were approximately 27,000 cases of severe GBS, including approximately 7,600 infections with drug-resistant Group B Streptococcus, and approximately 440 deaths.

[0026] Prior art methods for preventing infection and spread of drug-resistant microorganisms in colonized individuals include screening and isolating drug-resistant microorganisms, decolonizing the drug-resistant microorganisms, and / or recolonizing with drug-sensitive microorganisms.

[0027] Existing art methods that employ suppression (decolonization) alone, such as the use of antibiotics and antimicrobials, often fail because they suffer from high recurrence rates. When used alone, decolonization is often insufficient to effectively prevent the recurrence and / or spread of drug-resistant microorganisms.

[0028] Among the pathogens causing healthcare-associated infections, methicillin-resistant Staphylococcus aureus (MRSA) is a priority due to its virulence and disease spectrum, multidrug-resistant properties, and prevalence in healthcare settings. MRSA is the most common cause of ventilator-associated pneumonia and surgical site infections and the second most common cause of central line-associated bloodstream infections.

[0029] Strategies that involve screening new hospital admissions for MRSA and isolating those who carry MRSA (with or without decolonization) have been shown to be somewhat effective in reducing transmission. However, this type of treatment is quite expensive and requires additional residential facilities with special containment and sanitation procedures.

[0030] Decolonization alone has been used in hospital patients in an attempt to reduce the spread of S. aureus carriers and prevent disease. Decolonization may involve a multiday regimen of antibiotics and / or antiseptics, such as intranasal mupirocin and chlorhexidine baths. Universal decolonization is an approach adopted by some hospitals in which all intensive care unit (ICU) inpatients are washed daily with chlorhexidine and intranasal mupirocin, but MRSA infection rates in the United States have not changed significantly since its widespread use. In addition, microorganisms may develop resistance to chlorhexidine and mupirocin after repeated exposure.

[0031] When used alone, decolonization may not be persistent because vacant niches may be recolonized by pathogenic or resistant microorganisms.

[0032] For example, Shinefield et al. (1963, American Journal of Child Diseases 105, June 1963, pp. 146-154) observed that newborn infants were colonized with Staphylococcus aureus strains having the 52 / 52a / 80 / 81 phage complex through contact with carriers, after which the infants and their families became frequently ill. Shinefield also observed that control measures using antiseptics or antimicrobials on infants resulted in the colonization of abnormal flora, which was primarily composed of highly resistant coagulase-negative Staphylococci and Gram-negative bacteria (such as Pseudomonas and Proteus). Shinefield attempted to address this problem by artificially colonizing newborns with Staphylococcus aureus strain 502a through nasal and / or umbilical cord inoculation. 502a is a coagulase-positive strain of Staphylococcus aureus with low toxicity that is susceptible to penicillin and cannot induce the production of β-lactamase. The results showed that the presence of other Staphylococci interfered with the acquisition of 502a. After 6 months to a year, the persistence of colonization was at most 35%.

[0033] Boris M. et al. ("Bacterial Interference: Protection Against Recurrent Intrafamilial Staphylococcal Disease" Am J Pediatr Dis 115 (1968): 521-29) intentionally colonized approximately 4,000 infants with S. aureus 502a within the first hours of life (nares and umbilical cord stump). Almost complete protection of 80 / 81 infants from infection was observed (infants were monitored for 1 year after vaccination). Although tiny therapeutic vesicles developed in 5-15% of infants, these resolved spontaneously within the first 3 days after treatment. Existing decolonization increased the persistence of 502a by 5-fold compared to placebo (saline) n=63. A controlled study of recurrent furunculosis showed that decolonization with systemic antibiotics + nasal antimicrobial followed by 502a reduced disease in 80% of patients.

[0034] Recolonization with drug-susceptible strains may be unsafe because drug-susceptible strains may still cause systemic infection.

[0035] Shinefield et al. (1973, " Microbiol Immunol ", Vol. 1, 541-547) reported the use of bacterial replacement (including decolonization) to treat patients with recurrent furunculosis. Chronic staphylococcal carriers were treated with antibiotic therapy (including systemic antibiotics and antimicrobial cream for nasal mucosa). In the initial study, 31 patients received antibiotic therapy only, and the recurrence rate of the original strain reached 74%. 18 patients received antibiotic therapy first and then underwent 502a inoculation, and the recurrence rate of the original strain was 27%. A larger study for 587 patients resulted in a recurrence rate of 21% for the original strain after 12 months. However, the recurrence rate in patients with diabetes, eczema or acne is very high. Diseases associated with 502a were found in 11 patients.

[0036] Aly et al. (1974 J Infect Dis 129(6) pp. 720-724) studied bacterial interference in S. aureus carriers. Carriers were treated with antibiotics and antibacterial soap and challenged with strain 502a. Specifically, decolonization involved oral dicloxacillin for 8 days; intranasal neosporin for 8 days, and trichlorocabanilide. It was found that adequate decolonization was required to achieve good uptake. Uptake was 100% on day 7, but this decreased to 60% to 80% on day 23. Persistence at 23 weeks was 73% in well-decolonized subjects, while persistence was only 17% in partially decolonized subjects. Co-colonization was found in 5 / 12 subjects on day 3, in 2 / 12 subjects on day 10, and in 1 / 12 subjects on days 35 and 70.

[0037] Decolonization followed by recolonization with microorganisms of the same genus but different species may not be sustainable because the vacant niche cannot be fully filled by a different species.

[0038] WO2009117310A2 (George Liu), assigned to Cedars-Sinai Medical Center, discloses methods for treating and preventing methicillin-resistant and methicillin-sensitive Staphylococcus aureus (MSSA) using a decolonization / recolonization approach. In one example, mice were treated with antibiotics to eradicate the existing flora (including MRSA), and the newly cleared surface area was colonized with bacteria of the same genus but different species (e.g., Staphylococcus epidermidis). No specific data on recurrence were provided.

[0039] Attempts to administer probiotics to treat infections with pathogenic microorganisms may be ineffective and may not be long-lasting because the probiotics may not permanently colonize the subject.

[0040] U.S. Patent No. 6,660,262 (Randy McKinney), assigned to Bovine Healthcare Products, Inc., discloses a broad-spectrum antimicrobial composition for treating microbial infections in animals comprising certain minerals, vitamins, cobalt amino acids, algae, and Lactobacillus species. Field trials were conducted in cattle and horses, but no infectious bacterial strain or other infectious agent was identified.

[0041] U.S. Patent No. 6,905,692 (Sean Farmer), assigned to Ganeden Biotechnology, Inc., discloses topical compositions for application to the skin or mucous membranes of mammals to inhibit certain bacteria, yeasts, fungi, and viruses, the topical compositions comprising certain combinations of probiotic Bacillus bacteria, spores, and extracellular products. Compositions comprising Bacillus coagulens spores or culture supernatants of Bacillus species and Pseudomonas lindbergii culture supernatants in a vehicle such as emu oil are provided. The disclosure indicates that because probiotics do not permanently colonize the host, regular ingestion or application of the probiotics is required for any health-promoting properties to persist.

[0042] U.S. Patent No. 6,461,607 (Sean Farmer), assigned to Ganeden Biotechnologies, Inc., discloses lactic acid-producing bacteria, preferably strains of Bacillus coagulans, for controlling gastrointestinal pathogens in mammals. Methods for selectively breeding and isolating probiotic lactic acid-producing bacteria strains that are resistant to antibiotics are disclosed. Methods for treating infections using compositions comprising antibiotic-resistant lactic acid-producing bacteria and an antibiotic are disclosed.

[0043] U.S. Patent No. 8,906,668, assigned to Seres Therapeutics, provides a binary combination of cytotoxic bacteria from two or more distinct operational taxonomic units (OTUs) to persistently exclude pathogenic bacteria. OTUs are identified by comparing sequences between organisms (e.g., sequences that share at least 95% sequence identity of the 16S ribosomal RNA gene, at least in the hypervariable region).

[0044] Existing methods that simply replace the original pathogen (recolonization) are susceptible to the colonization rate of new microorganisms. If the recolonization is not done correctly, the process may fail. Effective recolonization is critical, but it is not sufficient alone to prevent recurrence.

[0045] Prior art methods involving suppression of the original pathogenic microorganism (decolonization) and replacement with new microorganisms (recolonization) may result in variable recurrence of the pathogenic microorganism depending on the specific method.

[0046] Rather than waging futile warfare against commensal pathogenic or drug-resistant microorganisms, a better approach may be to manage the microbiome: actively promoting the dynamics of "good bacteria" and their support systems while selectively inhibiting the recurrence of specific pathogenic organisms. Improved methods are needed to safely and durably prevent and reduce the recurrence of infections with undesirable microorganisms, such as virulent pathogens and / or drug-resistant microorganisms. Summary of the Invention

[0047] Methods and compositions are provided for safely and durably influencing the microbial ecosystem (microbiome) in a subject to perform various functions, including, for example, reducing the risk of infection by undesirable microorganisms, such as virulent pathogenic and / or drug-resistant microorganisms.

[0048] Provided herein are methods for preventing or reducing the risk of colonization, infection, recurrence of colonization, or recurrence of pathogenic infection by undesirable microorganisms in a subject, the methods comprising: decolonizing at least one site of the subject with an undesirable microorganism, thereby reducing or eliminating the presence of the undesirable microorganism from the site; durably replacing the undesirable microorganism by administering a synthetic microorganism to at least one site of the subject, wherein the synthetic microorganism can durably integrate with the host microbiome by occupying an ecological niche previously occupied by the undesirable microorganism; and optionally promoting colonization of the synthetic microorganism in the subject.

[0049] The present disclosure provides a method for eliminating and preventing the recurrence of undesirable microorganisms in a subject hosting a microbiome, the method 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 conferring a non-natural property, wherein the synthetic microorganism is capable of durably integrating into the host microbiome and occupying the same ecological niche in the host microbiome as the undesirable microorganism.

[0050] In some embodiments, decolonization is performed on at least one site of the subject such that the detectable presence of the undesirable microorganism is substantially reduced or eliminated from the at least one site.

[0051] In some embodiments, the detectable presence of an undesirable or synthetic microorganism is determined by a method comprising a phenotypic method and / or a genotypic method, optionally wherein the phenotypic method is selected from the group consisting of: a biochemical reaction, a serological reaction, sensitivity to antimicrobial agents, sensitivity to bacteriophages, sensitivity to bacteriocins, and / or properties of cellular proteins. In some embodiments, the genotypic method is selected from hybridization techniques, plasmid mapping, plasmid polymorphism analysis, restriction enzyme digestion, reaction and separation by pulsed field gel electrophoresis (PFGE), ribotyping, polymerase chain reaction (PCR) and variants thereof, ligase chain reaction (LCR), and transcription-based amplification systems (TAS).

[0052] In some embodiments, the niche is a dermal or mucosal environment that permits stable colonization of the undesirable microorganism at at least one site of the subject.

[0053] In some embodiments, the ability to durably integrate into the host microbiome is determined by the 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 after the administering step.

[0054] In some embodiments, the ability to durably replace the undesirable organisms 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 after the administering step.

[0055] In some embodiments, after the administering step, the ability to occupy the same ecological niche is determined by the absence of co-colonization of the undesirable microorganism and the synthetic microorganism at the at least one site. In one embodiment, the absence of co-colonization is determined for 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 after the administering step.

[0056] In some embodiments, the synthetic microorganism comprises at least one element that confers a non-natural property, and the element is permanently incorporated into the synthetic microorganism. In some embodiments, the at least one element that confers a non-natural property is permanently incorporated into the host microbiome via the synthetic microorganism.

[0057] In some embodiments, the at least one element that imparts non-natural properties is a kill switch molecule modification, a virulence blocking molecule modification, or a nanofactory molecule modification. In some embodiments, the synthetic microorganism includes a molecule modification that is integrated into the chromosome of the synthetic microorganism. In some embodiments, the synthetic microorganism includes a virulence blocking molecule modification that prevents horizontal gene transfer from the genetic material of the undesirable microorganism.

[0058] In some embodiments, after the change in state, the measurable average cell death of the synthetic microorganism occurs within at least a predetermined time period after induction of the first promoter. In some embodiments, after the change in state, the measurable average cell death occurs within at least a predetermined time period selected from the group consisting of: at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes. In some embodiments, after the predetermined time period, the measurable average cell death is at least a 50% reduction in 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 a 99.9% reduction in cfu count. In some embodiments, the change in state is selected from one or more of: pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, metal concentration, chelated metal concentration, compositional changes, or concentrations of one or more immune factors, mineral concentrations, and electrolyte concentrations. In some embodiments, the altered state is a higher concentration of blood, serum, or plasma and / or an altered composition of the blood, serum, or plasma compared to normal physiological (niche) conditions at at least one site of the subject.

[0059] In some embodiments, the undesirable microorganism is a strain of Staphylococcus aureus, and wherein the detectable presence is measured by a method comprising obtaining a sample from at least one site of the subject, contacting chromogenic agar with the sample, incubating the contacted agar, and counting positive cfu of the bacterial species after a predetermined period of time.

[0060] In some embodiments, a method is provided comprising a decolonization step comprising topically administering a decolonizing agent to at least one site of the subject, thereby reducing or eliminating the presence of the undesirable microorganism from the at least one site.

[0061] In some embodiments, the decolonization step comprises topically administering a decolonizing agent without concurrently administering a systemic antimicrobial agent. In some embodiments, a systemic antimicrobial agent is not administered within one week, two weeks, three weeks, one month, two months, three months, six months, or one year prior to, concurrently with, and / or following the first topical administration of the decolonizing agent or the administration of the synthetic microorganism. In some embodiments, the decolonizing agent is selected from the group consisting of a disinfectant, a sterilant, an antiseptic, an astringent, and an antimicrobial agent. In some embodiments, the decolonizing agent is selected from the group consisting of: alcohols (ethanol, isopropanol), aldehydes (glutaraldehyde, formaldehyde, formaldehyde releasers (hydroxymethyl methylthiourea = formaldehyde thiourea, taurolidine, hexamine, dantoin), o-phthalaldehyde), anilides (triclocarban = TCC = 3,4,4'-trichlorocarbamide), biguanides (chlorhexidine, Alexidine, polymeric biguanides (polyhexamethylene biguanide with MW>3,000 g / mol, vantocil), bisamidines (propamidine, propoxybenzamide isethionate, propoxybenzamide dihydrochloride, dibromopropamidine, dibromopropamidine isethionate), phenols (dichlorophene, parachloromethane, chlorodiphenylmethanesulfonate), cresols, hexachlorophene), bisphenols (triclosan, hexachlorophene), chloroxylenol (PCMX), quaternary ammonium compounds (cetrimonium bromide, benzalkonium chloride, cetylpyridinium chloride), silver compounds (silver sulfadiazine, silver nitrate), peroxides (hydrogen peroxide, peracetic acid, benzoyl peroxide), iodine compounds (povidone-iodine, poloxamer-iodine, iodine), chlorine-releasing agents (sodium hypochlorite, hypochlorous acid, chlorine dioxide, sodium dichloroisocyanurate, chloramine-T), copper compounds (copper oxide), isotretinoin, sulfur compounds, plant extracts (Melaleuca alternifolia (tea tree oil), Vaccinium myrtles (e.g., A-type proanthocyanidins), Ficus microcarpa, Pinus lanceolata, Azadirachta indica, Mending fruit, Grape, Terminalia avicennioides Guill & Perr, Phyllanthus discoideus muel.Muel-Arg, Clove basil, Golden-edged red mulberry, Hypericum pruinatum Boiss. & Bal, Hypericum olimpicum L. and Hypericum sabrum L, Hamamelis virginiana (Witch hazel), Clove oil, Eucalyptus spp., Rosmarinus officinalis (Rosemary), Thymus vulgaris (Thyme), Origanum vulgare (Oregano), Cymbopogon spp., Cinnamomum spp., Geranium spp., Lavandula angustifolia), aminolevulinic acid, and topical antibiotic compounds (bacteriocins; mupirocin, bacitracin, neomycin, polymyxin B, gentamicin).

[0062] In some embodiments, the antimicrobial agent is selected from the group consisting of: vancomycin, cefazolin, cephalosporin, cephalexin, linezolid, daptomycin, clindamycin, lincomycin, mupirocin, bacitracin, neomycin, polymyxin B, gentamicin, prulifloxacin, ulifloxacin, fidaxomicin, minocycline hydrochloride, metronidazole, metronidazole, sulfamethoxazole, ampicillin, trimethoprim, ofloxacin, norfloxacin, tinidazole, Norfloxacin, ornidazole, levofloxacin, nalidixic acid, ceftriaxone, azithromycin, cefixime, ceftriaxone, cephalexin, ceftriaxone, rifaximin, ciprofloxacin, norfloxacin, ofloxacin, levofloxacin, gatifloxacin, gemifloxacin, prulifloxacin, ulifloxacin, moxifloxacin, nystatin, amphotericin B, flucytosine, ketoconazole, posaconazole, clotrimazole, voriconazole, griseofulvin, miconazole nitrate, and fluconazole.

[0063] In some embodiments, the decolonization comprises topically administering the decolonizing agent at least one, two, three, four, five, or six or more times prior to the replacing step. In some embodiments, the decolonization step comprises administering the decolonizing agent to at least one host site of the subject one to six or more times or two to four times at intervals of between 0.5 hours and 48 hours, and wherein the replacing step is performed after the final decolonization step.

[0064] In some embodiments, a method is provided comprising decolonizing and replacing undesirable microorganisms with synthetic microorganisms, the method comprising topically administering to at least one host site of the subject a composition comprising at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 or at least 10 11CFU of synthetic strain and pharmaceutically acceptable carrier. In some embodiments, the initial replacement step is performed within 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days of the decolonization step or between 0.5-10 days, 1-7 days or 2 to 5 days. In some embodiments, after the final decolonization step, the replacement step is repeated at intervals of no more than once every two weeks to once every six months. In some embodiments, the decolonization step and the replacement step are repeated at intervals of no more than once every two weeks to once every six months or once every three weeks to once every three months. In some embodiments, the replacement comprises applying at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times the synthetic microorganism to the at least one position. In some embodiments, the replacement comprises applying no more than once, no more than twice, no more than three times or no more than four times the synthetic microorganism to the at least one position per month.

[0065] In some embodiments, the method of decolonizing the undesirable microorganism and replacing it with a synthetic microorganism further comprises promoting colonization of the synthetic microorganism in the subject. In some embodiments, promoting colonization of the synthetic microorganism in the subject comprises administering to the subject an accelerator, optionally wherein the accelerator is a nutrient, a prebiotic, a symbiotic, a stabilizer, a moisturizer, and / or a probiotic species. In some embodiments, the accelerator comprises administering to the subject 10 5 to 10 10 cfu, 10 6 to 10 9 cfu or 10 7 to 10 8 cfu of probiotic species.

[0066] In some embodiments, the nutrient is selected from sodium chloride, lithium chloride, sodium glycerophosphate, phenylethyl alcohol, mannitol, pancreatic protein, peptides, 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, oligofructose (e.g., inulin-like prebiotics), oligogalactose (e.g., raffinose), succinic acid, lactic acid, and manno-oligosaccharides.

[0067] In some embodiments, the probiotic is selected from the group consisting of 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 ...obacillus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus case lactis), Streptococcus thermophiles, and Enterococcus fecalis.

[0068] In some embodiments, the commensal 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, pyogenes), Staphylococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcus intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

[0069] In certain embodiments, the undesirable microorganism is an antimicrobial resistant microorganism. In certain embodiments, the antimicrobial resistant microorganism is an antibiotic resistant bacterium. In certain embodiments, the antibiotic resistant bacterium is a gram-positive bacterial species selected from the group consisting of: Streptococcus spp., Cutibacterium spp., and Staphylococcus spp. In certain embodiments, the streptococcus is selected from the group consisting of: Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus pyogenes, and Streptococcus agalactiae. In certain embodiments, the propionibacterium is selected from the group consisting of: Propionibacterium acnes subsp. acnes, Propionibacterium acnes defendens subspecies, and Propionibacterium acnes subsp. elongatum. In some embodiments, the staphylococcus is selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. In some embodiments, the undesirable microorganism is a methicillin-resistant Staphylococcus aureus (MRSA) strain containing a staphylococcal chromosomal cassette (type I-III SCCmec), the cassette encoding an antibiotic resistance gene (type I SCCmec) or multiple antibiotic resistance genes (type II and type III SCCmec) and / or produces a toxin. In some embodiments, the toxin is selected from the group consisting of: Panton-Valentine albumin binding protein (PVL) toxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal α-hemolysin toxin, Staphylococcal β-hemolysin toxin, Staphylococcal γ-hemolysin toxin, Staphylococcal δ-hemolysin toxin, enterotoxin A, enterotoxin B, enterotoxin C, enterotoxin D, enterotoxin E, and coagulase toxin.

[0070] In some embodiments, a subject treated with the methods according to the present disclosure does not exhibit recurrence or colonization of the undesirable microorganisms for 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 following said administering step, as evidenced by swabbing said subject at said at least one site.

[0071] The present disclosure provides a synthetic microorganism for permanently replacing undesirable microorganisms in a subject. The synthetic microorganism includes molecular modifications intended to enhance safety by reducing the risk of systemic infection. In one embodiment, the molecular modifications result in a significant reduction in the growth or cell death of the synthetic microorganism in response to blood, serum, or plasma. The synthetic microorganism can be used in methods and compositions for preventing or reducing the recurrence of dermal or mucosal colonization or recolonization of undesirable microorganisms in a subject.

[0072] The present disclosure provides synthetic microorganisms for use in compositions and methods for treating, preventing, reducing the risk of, or reducing the likelihood of colonization or recolonization, systemic infection, bacteremia, or endocarditis caused by undesirable microorganisms in a subject.

[0073] The present disclosure provides a synthetic microorganism comprising a recombinant nucleotide comprising at least one kill switch molecule modification, the kill switch molecule modification comprising a first cell death gene operably associated with a first regulatory region comprising an inducible first promoter, wherein the first inducible promoter exhibits conditional high-level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma, with at least a three-fold increase in basal productivity. In some embodiments, the inducible first promoter exhibits a gene, comprising, derived from, or selected from a gene, that exhibits at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold upregulation within at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or at least 360 minutes after exposure to blood, serum, or plasma.

[0074] In some embodiments, the synthetic microorganism comprises a kill switch molecule modification comprising a first cell death gene operably linked to a first regulatory region comprising an inducible first promoter, wherein the first promoter is activated (induced) by an altered state of the microbial environment as compared to normal physiological environmental (niche) conditions at at least one site of the subject.

[0075] In some embodiments, the synthetic microorganism further comprises an expression clamp molecule modification comprising an antitoxin gene specific for the first cell death gene or its product, wherein the antitoxin gene is operably associated with a second regulatory region comprising a second promoter that is constitutively active upon colonization of the dermis or mucosa or in complete culture medium, but is not induced, is induced less than 1.5-fold, or is inhibited after exposure to blood, serum, or plasma for at least 30 minutes. In some embodiments, the second promoter is active upon colonization of the dermis or mucosa or in TSB medium, but is inhibited by at least 2-fold upon exposure to blood, serum, or plasma after a period of at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or at least 360 minutes.

[0076] In some embodiments, the synthetic microorganisms exhibit a measurable average cell death of at least 50% reduction in cfu within at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes after exposure to blood, serum, or plasma. In some embodiments, the synthetic microorganisms exhibit a 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% reduction in cfu count within at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes after exposure to blood, serum, or plasma.

[0077] In some embodiments, the synthetic microorganism comprises a kill switch molecule modification that reduces or prevents infectious growth of the synthetic microorganism under a systemic condition in a subject.

[0078] In some examples, the synthetic microorganism includes at least one molecular modification integrated into the chromosome of the synthetic microorganism.

[0079] In some embodiments, the synthetic microorganism is derived from a target microorganism of the same genus and species as the undesirable microorganism. In some embodiments, the target microorganism is sensitive to at least one antimicrobial agent. In some embodiments, the target microorganism is selected from a bacterial, fungal, or protozoan target microorganism. In certain embodiments, the target microorganism is capable of colonizing the dermis and / or mucosal niche.

[0080] In some embodiments, the target microorganism has the ability to biointegrate with a 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 protozoan target microorganism.

[0081] In some embodiments, the target microorganism is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Cutibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas.

[0082] In some embodiments, the target microorganism is a bacterial species capable of colonizing the dermal and / or mucosal niches and is a member of a genus selected from the group consisting of Acinetobacter, Corynebacterium, Propionibacterium, 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 wolleri, staphylococcus saprophyticus, corynebacterium acnes, corynebacterium striatum, corynebacterium diphtheriae, corynebacterium minimus, propionibacterium acnes, propionibacterium acnes, propionibacterium granulosum, streptococcus pyogenes, staphylococcus aureus, streptococcus mitis, viridans streptococcus, streptococcus pneumoniae, streptococcus anginosus, streptococcus constellatus, streptococcus intermedius, streptococcus agalactiae, pseudomonas aeruginosa, pseudomonas oryzae, pseudomonas stutzeri, pseudomonas putida and pseudomonas fluorescens. In a specific embodiment, a synthetic microorganism derived from a staphylococcus aureus strain is provided. In some embodiments, the target strain is a staphylococcus aureus 502a strain or an RN4220 strain.

[0083] In some embodiments, the synthetic microorganism comprises a kill switch molecule modification comprising a cell death gene selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM, or a lysostaphin toxin gene. In some embodiments, the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or substantially identical nucleotide sequences.

[0084] In some embodiments, the inducible first promoter is a blood, serum and / or plasma responsive promoter. In some embodiments, after exposure to human blood, serum or plasma, the first promoter is raised by at least 1.5 times, at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times within a time period selected from 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. In some embodiments, in the absence of a change in state, the first promoter is not induced, induced less than 1.5 times or suppressed. In some embodiments, in the presence of serum, blood or plasma, the first promoter is induced at least 1.5 times, 2 times, 3 times, 4 times, 5 times or at least 6 times over a period of time. In some embodiments, at the at least one site, under the normal physiological (niche) conditions, the first promoter is not induced, induced less than 1.5 times or suppressed.

[0085] 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 (γ-hemolysin component A), hlgA1 (γ-hemolysin), hlgA2 (γ-hemolysin), hlgB (γ-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosynthesis 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 (ferrochrome transport ATP-binding protein fhuA), fhuB (ferrochrome 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 genes, 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 transporter), srtB (sortase B), sbnA (possible 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 (staphylococcal 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 nucleotide sequence substantially identical thereto.

[0086] In some embodiments, the synthetic microorganism comprises an expression clamp molecule modification comprising a second promoter operably associated with an antitoxin gene, wherein the antitoxin gene encodes an antisense RNA sequence capable of hybridizing to at least a portion of a first cell death gene. In some embodiments, the antitoxin gene encodes an antisense RNA sequence capable of hybridizing to at least a portion of a first cell death gene. In some embodiments, the antitoxin gene is selected from the group consisting of a sprA1 antitoxin gene, a sprA2 antitoxin gene, a sprG antitoxin gene or a sprF antitoxin gene, a holin antitoxin gene, a 187-lysK antitoxin gene, a yefM antitoxin gene, a lysostaphin antitoxin gene or a mazE antitoxin gene, a kpn1 antitoxin gene, a sma1 antitoxin gene, a relF antitoxin gene, a rsaE antitoxin gene, or a yoeB antitoxin gene. In some embodiments, the antitoxin gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319, or 322, or a substantially identical nucleotide sequence.

[0087] In some embodiments, the second promoter comprises or is derived from a gene selected from the group consisting of: clfB (clustering factor B), sceD (autolysin, extracellular protein D), walKR (virulence regulator), atlA (major autolysin), oatA (O-acetyltransferase A); a phosphoribosylglycinamide formyltransferase gene, a phosphoribosylaminoimidazole synthetase gene, an amidophosphoribosyltransferase gene, a phosphoribosylformylglycinamide synth ... gene, phosphoribosylaminoimidazole-succinamide gene, trehalose permease IIC gene, DeoR family transcriptional regulator gene, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkyl hydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein degradation chaperone gene, alkyl hydroperoxidase gene, phosphofructokinase gene, gyrB, sigB and rho. In some embodiments, the second promoter is P clfB (aggregation factor B), which comprises the nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a substantially identical nucleotide sequence thereof.

[0088] In some embodiments, the synthetic microorganism comprises a virulence blocking molecule modification and / or a nanofactory molecule modification. In some embodiments, the virulence blocking molecule modification prevents horizontal gene transfer of genetic material from the undesirable microorganism.

[0089] In some embodiments, the nanofactory molecular modification comprises insertion of a gene, knockout of a gene, or genetic modification of a gene encoding a product selected from the group consisting of an enzyme, an amino acid, a metabolic intermediate, and a small molecule.

[0090] The present disclosure provides a composition comprising an effective amount of a synthetic microorganism according to the present disclosure and a pharmaceutically acceptable carrier, diluent, emollient, adhesive, excipient, lubricant, sweetener, flavoring, wetting agent, preservative, buffer or absorbent or a combination thereof. In some embodiments, the composition further comprises a facilitator. In some embodiments, the facilitator is selected from a nutrient, a prebiotic, a symbiotic and / or a probiotic species.

[0091] The present disclosure provides a single dose unit comprising a composition or synthetic microorganism of the present disclosure. In some embodiments, the single dose unit comprises 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 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.

[0092] The present disclosure provides synthetic microorganisms according to the present disclosure, compositions for use in the manufacture of a medicament for use in a method of eliminating, preventing, or reducing the risk of recurrence of an undesirable microorganism in a subject.

[0093] The present disclosure provides a kit for preventing or reducing the recurrence of dermal or mucosal colonization or recolonization by undesirable microorganisms in a subject, the kit comprising at least one container comprising a synthetic microorganism, composition or single dose of the present disclosure, and optionally one or more additional components selected from a second container comprising a decolonizing agent, an instruction sheet, at least a third container comprising a facilitating agent, and / or an applicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1A diagram of representative molecular modifications inserted into Staphylococcus aureus (e.g., BioPlx-01) to generate synthetic microbial strains of BioPlx is shown. The cassette comprising the molecular modifications includes a kill switch and an expression clamp comprising an expression clamp (e.g., ClfB) promoter cloned to drive expression of SprA1 antisense (antitoxin) RNA, wherein the cassette is incorporated into the same expression module with the kill switch comprising a serum-responsive promoter (e.g., P) operably associated with the SprA1 toxin gene. hlgA In this strain, serum / blood exposure activates the toxin (e.g., up to 350-fold or more) but not the antitoxin, whereas growth in TSB or on the skin activates the antitoxin but not the toxin.

[0095] Figure 2 Shown is the shuttle vector PCN51 used for cloning genes into the E. coli-S. aureus pass-through strain (IMO8B), which was then transfected into BioPlx-01 for evaluation.

[0096] Figure 3A 、 Figure 3B and Figure 3C Table 4A with primer sequences used for recombinant construction of synthetic S. aureus from strain BioPlx-01 is shown.

[0097] Figure 4A 、 Figure 4B 、 Figure 4C and Figure 4D Shown is Table 4B with primer sequences for CRISPR construction of S. aureus synthesized by strain BioPlx-01.

[0098] Figure 5A The genetic map of the pKOR1 integrative plasmid is shown, depicting repF (the replication gene of pE194ts), secY570 (the N-terminal 570 nucleotides of secY, containing the ribosome binding site), cat (chloramphenicol acetyltransferase), attP (attachment site for lambda), ori(-) (the replication origin of the ColE1 plasmid), and bla (beta-lactamase). (+) or (-) indicates function in Gram-positive (+) or Gram-negative (-) bacteria. The Pxyl / tetO promoter and the transcriptional direction of the promoter are indicated by arrows.

[0099] Figure 5B The genetic map of the pIMAY integrative plasmid is shown (Accession No. JQ62198).

[0100] Figure 6Shown is the fold induction of HlgA (gamma hemolysin) promoter candidates in methicillin-sensitive Staphylococcus aureus strain BioPlx-01 by incubation with human serum. Expression was normalized to a housekeeping gene (gyrB) and compared to expression in logarithmically growing cells in liquid TSB medium.

[0101] Figure 7 Shown is the fold induction of SstA (iron transport) promoter candidates in methicillin-sensitive S. aureus strain BioPlx-01 by incubation with human serum. Expression was normalized to a housekeeping gene (GyrB) and compared to expression in logarithmically growing cells in liquid TSB medium.

[0102] Figure 8 Shown are the intergenic regions of CRISPR gRNA target sites identified between 1,102,100 and 1,102,700 bp in the S. aureus 502a genome (GenBank: CP007454.1).

[0103] Figure 9 Shown are representative screenshots of CRISPRScan used to find putative gRNAs for use in CRISPR methods.

[0104] Figure 10 The layout of the cassette and pCasSA vector for integration via CRISPR is shown. Cap1A is a constitutive promoter that controls gRNA transcription. Target seq is the target sequence, for example, with 10 possible cleavage targets (1.1, 1.2, etc.). sgRNA is a single-stranded guide RNA (provides structural components). Xba1 and Xho1 are two restriction sites used to add HA to the pCasSA vector. HA is a homology arm that can be used as a template for homology-directed repair (usually 200–1000 bp). P rpsL -mCherry is a constitutive promoter that controls the "optimal" mCherry. rpsL -Cas9 is a constitutive promoter that controls the expression of Cas9 protein.

[0105] Figure 11Vectors used in the present disclosure are shown. A is a vector for screening promoters by fluorescence using pCN51. B is a vector for screening promoters by cell death genes. C is a vector for chromosomal integration using CRISPR. D is a vector for chromosomal integration using homologous recombination. Left and right (or upstream and downstream) HA: homology arms of the target locus, CRISPR targeting: RNA guide to the locus, mCherry: fluorescent reporter protein, Cas9 protein: CRISPR nuclease, kanR: kanamycin resistance, oriT: transfer origin (for integration) and sma1: representative killing gene (restriction endonuclease).

[0106] Figure 12A 、 Figure 12B and Figure 12C The nucleotide sequence of the pIMAY integrative plasmid (SEQ ID NO: 131) is shown (Accession No. JQ62198).

[0107] Figure 13A Shown are the activities of promoter candidates isdA, isdB, hlgA2, hrtAB, isdG, sbnE, lrgA, lrgB, fhuA, fhuB, ear, hlb, splF, splD, dps, and SAUSA300_2617 at 1, 15, and 45 minutes in serum, and the fold changes in gene expression relative to culture medium by qPCR.

[0108] Figure 13B Shown are the activities of promoter candidates isdA, isdB, hlgA2, hrtAB, isdG, sbnE, lrgA, lrgB, fhuA, fhuB, ear, hlb, splF, splD, dps, and SAUSA300_2617 at 1 minute, 15 minutes, and 45 minutes in blood, and the fold changes in gene expression by qPCR relative to culture medium.

[0109] Figure 13C Shown is gene expression of promoter candidates hlgB, ear, fnb, splF, splD, clfA, CH52_360, CH52_305, CH52_1670, hlb, lrgB, lrgA, emp, fhuA, fhuB, isdI, isdA, srtB, isdG, sbnE, sbnA, sbnC, and isdB in serum at T=90 minutes compared to TSB by qPCR.

[0110] Figure 14Inducible inhibition of cell growth is shown for synthetic microbial pTK1 cells containing a cell death toxin gene (sprA1) behind a cadmium promoter on a pCN51 plasmid (pTK1) that has been transformed into Staphylococcus aureus RN4220 cells. OD (630 nm) was read 2 hours after induction. Wild-type 4220 cells showed good cell growth 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 the presence of 500 nM and 1 uM cadmium 2 hours after induction.

[0111] Figure 15A The plasmid map of p174 (pRAB11_Ptet-sprA1) is shown, with an enlarged view of the region of the plasmid containing the Ptet-sprA cassette.

[0112] Figure 15B The entire p174 (pRAB11_Ptet-sprA1) plasmid is shown in its native circular form.

[0113] Figure 15C Shown is a photo of a plate dilution of the synthetic microorganism Staphylococcus aureus 502a p174 cells at 6 hours, which includes a cell death toxin gene (sprA1) behind the anhydrotetracycline promoter on the pRAB11-2 plasmid (p174) that has been transformed into Staphylococcus aureus 502a cells. The p174 plasmid contains the missing spra1 antisense (Das). The plate dilution of the uninduced 502a p174 (tet-spra1Das) cells (left) and the induced cells (right) at 10e-5 on BHI chlor10 after 6 hours of induction is shown. The left plate (uninduced) cannot be counted at 10e-5, but about 720 colonies are counted at 10e-6. The induced plate on the right produces 16 colonies at 10e-5. 6 hours after induction, the survival percentage of the induced cells was 0.22%.

[0114] Figure 16Shown are the cell growth before and after induction of four synthetic strains derived from Staphylococcus aureus 502a, which have a plasmid-based inducible expression system that includes four different cell death gene candidates, sprA1, 187-lysK, Holin, and sprG. The candidate cell death genes have been cloned behind a tetracycline-inducible promoter on the pRAB11 plasmid and transformed into Staphylococcus aureus 502a cells. Calculated OD600 readings of 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) were taken at T = 0, 30, 60, 120 and 240 minutes after induction of the AtC-induced (+) strain (indicated by the dotted line (------)) and the uninduced (-) strain (indicated by the solid line (______)) and compared with BP_001 (502a wt) in BHI medium. Each of the induced (+) strains BP_068 (sprA1), BP_069 (187lysK), and BP_070 (holin) exhibited (i) good cell growth before induction and (ii) significant inhibition of cell growth after induction. BP_068 (+) exhibited the best inhibition of cell growth at each time point after induction, T=30, T=60, T=60, T=120, and T=240. Therefore, the sprA1 gene was selected for preliminary further development of the kill switch in S. aureus 502a.

[0115] Figure 17The bar graph shows the difference in colony forming units (cfu) / mL between T = 0 (grey) and 240 minutes (black) for the uninduced (-) strain and the anhydrotetracycline-induced (+) strains 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) compared to BP_001 (502a wt) in BHI medium. Each of the induced (+) strains BP_068 (sprA1), BP_069 (187lysK), and BP_070 (holin) exhibited (i) good cell growth before induction and (ii) significant inhibition of cell growth after induction. BP_068 showed the best inhibitory effect on cell growth at 240 min after induction, so the sprA1 gene was selected for preliminary further development of the kill switch in S. aureus 502a.

[0116] Figure 18 Shown are the fold changes in GFP expression for induced (+) and uninduced (-) subcultures of S. aureus strains BP_001, BP_055, and BP_076.

[0117] Figure 19 The genomic map of strain BP_076 (SA502a, ΔsprA1 ::Ptet-GFP) is shown.

[0118] Figure 20 Shown is a map of the plasmid constructed for genomic integration in S. aureus.

[0119] Figure 21 A map of the PsbnA-sprA1 kill switch in the genome of Staphylococcus aureus 502a is shown. The serum and blood responsive promoter PisdB is operably linked to the sprA1 toxin cell death gene.

[0120] Figure 22 A diagram shows a kill switch configuration that uses the serum and blood responsive promoter PisdB operably linked to the sprA1 toxin cell death gene and an expression clamp including 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 was incorporated into the S. aureus 502a genome.

[0121] Figure 23Shown are growth curves for three strains exposed to human serum compared to TSB: 502a—S. aureus wild-type, S. aureus BP_011—502aΔsprA1-sprA1(AS), and S. aureus BP_084—502aΔPsprA::PsbnA, in which the kill switch is integrated into the genome of S. aureus 502a. The dashed line represents strains grown in serum, while the solid line represents strains grown in TSB. After 180 minutes, strain BP_084, harboring the integrated kill switch, exhibited a significantly reduced growth curve compared to both the wild-type strain in serum and the kill switch in complex medium. After three hours of exposure to human serum, S. aureus BP_084 (502aΔPsprA::PsbnA) cells exhibited a measurable average cell death of 98.84% compared to the same BP_084 cells grown in TSB. DETAILED DESCRIPTION

[0122] The present disclosure is based on a principle known as "bacterial displacement" or "niche exclusion," in which one microorganism displaces and excludes another. In ecology, competitive exclusion, or Gauss's law, states that two species competing for exactly the same resources cannot coexist stably. This is due to the fact that one competitor will have some slight advantage over the other, which in the long run can lead to the extinction of the smaller competitor. In higher-order organisms, this often results in the smaller competitor adapting to a slightly different ecological niche.

[0123] Provided are methods and compositions for persistently managing a subject's microbiome. In an embodiment, the microbiome is a dermal and / or mucosal microbiome (Exobiome). Although methods exist for treating infections caused by pathogenic microorganisms, methods for preventing recurrence do not exist.

[0124] Infectious Agents—Staphylococcus aureus (MSSA and MRSA)

[0125] Classified since the early 20th century as the most deadly of all pathogenic organisms, Staphylococcal infections, primarily caused by Staphylococcus aureus, affect approximately 500,000 patients annually in US hospitals. In the US, up to 50,000 deaths annually are linked to S. aureus infections. S. aureus is found on the skin or in the nostrils of 40–44% of healthy humans. S. aureus is also sometimes found in the mouth, gastrointestinal tract, genitourinary tract, and upper respiratory tract. Some studies suggest even higher colonization rates. For example, Eriksen et al. suggest that a higher proportion of transient or intermittent carriers may contribute to the prevalence, which can sometimes exceed 75%.

[0126] Staphylococcus aureus 502a WT and other replacement and blocking strains

[0127] In Example 1, the The Staphylococcus aureus 502a WT strain, a naturally occurring "wild-type" organism known to be relatively non-infectious and with no known side effects, has been shown to be highly effective in this intended application (occupying and blocking the desired microbial niche to prevent MRSA recurrence) in BioPlx clinical studies.

[0128] The method of the present invention prevents infection by permanently replacing a poorly colonizing strain of Staphylococcus aureus (typically virulent and antibiotic-resistant) 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 organisms developed by BioPlx.

[0129] Provided herein are other replacement strains, such as synthetic strains, that are fully capable of colonizing properly prepared skin and mucosal surfaces and occupying the niche used by that bacterial species, thereby blocking other variants from recolonizing that niche.

[0130] There are a large number of S. 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.

[0131] Methicillin-resistant Staphylococcus aureus (MRSA) refers to a class of antibiotic-resistant variants of this common human commensal and sometimes pathogenic bacterium. They differ from wild-type strains (MSSA-sensitive S. aureus) in that they carry a mecA cassette that allows MRSA strains to produce an alternative penicillin-binding protein (PBP2A), rendering them resistant to most β-lactams and many other first-line antibiotics.

[0132] Methicillin-resistant Staphylococcus aureus (MRSA) and highly toxic methicillin-sensitive Staphylococcus aureus (vMSSA) are toxic invasive variants of Staphylococcus aureus that are colonized in many humans and can further cause superficial soft tissue infections and serious systemic infections. Colonization of MRSA or vMSSA is typically a necessary precursor to active staphylococcal infection. Infection is caused by colonies of bacteria on the skin or mucous membranes that penetrate external immune barriers and invade tissue or bloodstream through wounds, incisions, acupuncture points, or other breaches in the skin. This can lead to bacteremia and other systemic infections, and mortality is very high.

[0133] The present disclosure uses a generally passive strain of Staphylococcus aureus to replace MRSA or vMSSA and exclude them from their usual position in the dermal / mucosal microbiome. It is well known that the wild-type, interfering Staphylococcus aureus used by BioPlx is poor at causing systemic disease, but virtually any microorganism, regardless of its level of mutation or invasiveness, can become an "accidental pathogen" through natural or accidental inoculation. This is particularly true in the case of Staphylococcus aureus.

[0134] The decolonization and administration methods developed by BioPlx and the BioPlx01 strain provide the strains presented herein with a significant numerical and positional competitive advantage. This approach provides a prolonged MRSA decolonization effect compared to simple bactericidal destruction of virulent MRSA strains. Early studies have shown that the decolonization / recolonization process with BioPlx products for BioPlx01 MRSA has a total exclusion effect of greater than 6 months, whereas previous literature showed that recurrence rates of S. aureus nasal colonization were 45% at 4 weeks and 60% at 12 weeks using standard decolonization methods alone.

[0135] Overview of Indications

[0136] Staphylococcus aureus infections are a serious problem in both hospitals and community health facilities. Methicillin-resistant Staphylococcus aureus (MRSA) is genetically different from other Staphylococcus aureus strains, and its genetic factors confer resistance to methicillin and other (usually β-lactam) antibiotics commonly used to treat Staphylococcus aureus infections. MRSA strains carry a mecA expression cassette that causes the MRSA strain to produce a replacement penicillin-binding protein (PBP2A), and it is this mutation that confers resistance. Due to this resistance, MRSA is difficult to treat, which makes it a life-threatening problem in many cases. MRSA is often infected in hospitals or other types of medical facilities (hospital-associated [HA]). These infections usually occur when invasive procedures (such as surgery, intravenous catheterization, intubation, or artificial joint placement) are performed. Community-associated (CA) MRSA is usually transmitted through skin-to-skin contact, and the initial symptoms are often large boils on the skin.

[0137] The BioPlx approach, using the BioPlx strains, is not a treatment for invasive MRSA disease and is not intended for use in patients during an invasive disease state. The benefit of the BioPlx approach can be demonstrated in the following patient groups: 1) at high risk for invasive disease, 2) with elevated morbidity and mortality due to this increased risk, who demonstrate a significant clinical benefit and who have no other effective options for preventing invasive S. aureus disease. These characteristics define a subset of patients tracked by the Centers for Disease Control since 2005 who have experienced invasive MRSA disease—72,444 cases—according to 2014 ABC surveillance data.

[0138] The surfaces of human skin and mucous membranes, where Staphylococcus aureus resides in a colonized state, have very different levels of required nutrients and a different environmental quality than the interior of the human body. It is well known that for bacteria to successfully invade, they must be able to adjust their needs and responses between the colonized and invasive states. This is achieved by the bacteria detecting changes between these environments and switching on or off certain gene cassettes, allowing the production of proteins more adapted to the new invasive state.

[0139] The BioPlx method (and specifically, the BioPlx01 strain) exploits this requirement by rearranging the molecular instructions to cause the death of the organism in the operon of one or more of these specific cassettes. This creates a "maintainer strain" of colonizing S. aureus that is unable to cause disease in an introduced patient, but also does not allow other circulating S. aureus strains that might normally colonize the human population to colonize the patient. This occurs through the ecological premise of competitive exclusion.

[0140] The current “standard of care” for patients colonized with MRSA is inconsistent. There are no guidelines for the management of staphylococcal colonization in patients at high risk for recurrent disease. The IDSA’s 2011 Clinical Practice Guideline for the Treatment of MRSA Infections in Adults and Children provides only Class C-III (Minimum—No Data, Expert Opinion) support for decolonization procedures in patients with recurrent community-acquired skin and soft tissue infections and does not address the role of decolonization in preventing invasive MRSA disease. Some hospitals have implemented extensive screening and isolation programs for all admitted patients, but this approach has not been shown to be effective due to poor sustainability of efficacy and the low baseline risk of the average hospitalized patient (i.e., the MRSA outbreak at the University of California, Irvine). Consequently, other hospitals have reduced their focus to patients admitted to the ICU and cardiothoracic surgery settings. This strategy has been shown to reduce MRSA clinical isolates and bloodstream infections from any pathogen. However, these are short-term, scenario-based strategies designed to reduce the risk of MRSA infection in the immediate future.

[0141] For the U.S. and other parts of the world, MRSA disease and colonization are complex epidemiological problems. From asymptomatic colonization to invasive disease states, MRSA has a wide range of manifestations, thereby giving the system high mortality and high costs. Obviously, MRSA patients who have experienced invasive disease are medically different. The mortality rate of these patients is higher than any other MRSA subgroup. These patients have a higher treatment failure rate. Compared with other groups of patients, the risk of another invasive MRSA event occurring in these patients is much higher. This makes the group become the appropriate orphan group that the BioPlx method should be directed to, and the appropriate orphan group will benefit from the use of the method.

[0142] It can be concluded that decolonization is largely ineffective in permanently clearing MRSA colonization and leads to high relapse rates. It has been found that only decolonization combined with active recolonization can provide long-term conversion from one organism (variant) to another.

[0143] Recurrent invasive MRSA as a clinically distinct disease

[0144] Another indication is "prevention of recurrent invasive MRSA." Patients who have already experienced an invasive MRSA infection have a greatly increased susceptibility to subsequent invasive MRSA infections. The BioPlx technology provided herein works by occupying an ecological niche in the microbiome that might normally be occupied by virulent forms of MRSA.

[0145] Systemic infections caused by invasive MRSA:

[0146] SA (including the variant MRSA) can coexist harmlessly on the skin and mucosal surfaces of at least 40% of the human population, so the bacteria themselves do not describe the disease; rather, the clinical manifestations of the bacteria are definitive.

[0147] All national and international authorities that define and monitor the condition agree that invasive MRSA infection is a separate and distinct disease from other conditions caused by the bacteria.

[0148] Simple colonization with any type of S. aureus should not be considered a disease state. Indeed, people with a skin and mucosal biome characterized by nutrients and an environment suitable for S. aureus to inhabit must harbor some of these niche occupants as part of their microbiome to achieve a stable equilibrium "resting state" of their biome. The goal of any approach is to durably replace MRSA strains in high-risk patients with the product strain (in this case, antibiotic-susceptible S. aureus modified to be unable to survive in an invasive state in the human body).

[0149] To establish an invasive infectious disease, MRSA must abandon its passive commensal state and breach the dermal / mucosal barrier to enter the subcutaneous interstitial (interstitial fluid) or circulatory (blood, serum, plasma) regions. This "state change" triggers a new disease state through new biological behaviors and relationships with the host.

[0150] Staphylococcus aureus bacteremia (SAB) is a significant example of this type of infection, with an incidence ranging from 20 to 50 cases per 100,000 population per year (range, 64,600 to 161,500 cases per year). Between 10% and 30% of these patients will die from SAB. The mortality rate from invasive systemic MRSA bacteremia is approximately 20%. By comparison, this number of deaths is greater than that from AIDS, tuberculosis, and viral hepatitis combined.

[0151] The CDC-US National Case Estimates for Invasive MRSA Disease (2014) reported 72,444 cases. This suggests that fewer than 200,000 patients develop the disease annually, potentially allowing for orphan drug designation. MRSA can affect patients at three different levels: 1) colonization, 2) superficial infection—skin and soft tissue, and 3) systemic invasive infection.

[0152] 1) Colonization. Staphylococcus aureus is a normal commensal organism that permanently colonizes approximately one-third of the human population, while transient colonization occurs in approximately another one-third. MRSA variants of the microorganism occupy an ecological niche in the microbiome and colonize approximately 2% of the U.S. population (highly variable depending on location and occupancy). MRSA colonization establishes a permanent reservoir of potentially infectious organisms directly on the outer layer of the immune / defense system, posing a constant risk to patients.

[0153] 2) Superficial infections—skin and soft tissue infections. Of the two main disease state categories, skin-associated MRSA, or skin and soft tissue infections, are the most common. They typically begin with swelling, pus or discharge, boils, and sometimes fever, which can be painful and burning. If left untreated, these boils can become abscesses, requiring surgical drainage. For MRSA confined to the skin, surgical drainage of the abscess may be the only necessary treatment, and antibiotics are not recommended. Skin and soft tissue infections can be treated by surgically draining the boil, with antibiotics administered only when absolutely necessary.

[0154] 3) Systemic invasive infection. MRSA bacteremia (invasive MRSA) is a systemic MRSA infection defined as the presence of MRSA in sterile sites, including blood, cerebrospinal fluid, joint fluid, bone, lower respiratory tract, and other body fluids. Compared to MRSA infections confined to the skin, MRSA bacteremia has a much worse prognosis, with 20% of cases resulting in death. Differences in prognosis, site of infection, and clinical symptoms distinguish it clinically from MRSA infections of the skin and soft tissues. MRSA bacteremia can lead to a variety of complications not seen in skin and soft tissue infections, including infective endocarditis, septic arthritis, and osteomyelitis. For invasive MRSA, daptomycin and vancomycin are recommended for treatment in the United States. Vancomycin is relatively slow-acting and cannot penetrate certain tissues. Daptomycin has been shown to be effective, but in addition to the problem of vancomycin causing daptomycin resistance in MRSA, daptomycin insensitivity in emergency treatment is also a problem. The differences in clinical symptoms and treatment of invasive MRSA provide clear evidence that invasive MRSA is a clinically distinct entity from MRSA skin and soft tissue infections.

[0155] BioPlx technology works by preventing the recurrence of invasive MRSA infections in already colonized people (including those who have experienced invasive MRSA infections) and those who have undergone decolonization procedures. As a decolonization / recolonization microbiology approach, BioPlx technology will not be used to "treat" patients while they have systemic MRSA infections. The BioPlx technology will be applied after the systemic MRSA infection has been cleared (and systemic decolonization has been completed).

[0156] The established principle of medical terminology is that disease or condition is not simply synonymous with pathogen. In the present case, MRSA-mediated systemic bacteremia (or other names of invasive systemic disease) is clearly different from other superficial skin and mucosal conditions that may be caused by MRSA or other Staphylococcus aureus strains or are associated with it. Invasive systemic MRSA-mediated disease has significantly different diagnosis, pathology, treatment and prognosis characteristics.

[0157] Notably, based on the mechanism of action of the BioPlx01 strain, as opposed to treating invasive MRSA itself, it may prevent patients from subsequently developing systemic MRSA infections. Therefore, “prevention of recurrent systemic MRSA infections” would be the most accurate description of the indication for the BioPlx01 strain.

[0158] The target population of patients with invasive MRSA infections have been successfully cleared of the organism, typically by standard antibiotic intervention (eg, vancomycin), but remain at high risk (incidence) of MRSA recolonization, relapse, and the associated increased risk of MRSA systemic reinfection.

[0159] International and U.S. recognized disease names:

[0160] The Centers for Disease Control and Prevention (CDC) has a clear definition for this illness, as it has been actively monitoring the condition in the United States since 2005. The agency conducts this surveillance through its Emerging Infections Program (EIP) using an active bacterial core surveillance system. In this context, a case is defined by the isolation of MRSA from a normally sterile body site. These normally sterile sites include blood, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid, joint / synovial fluid, bone, internal body sites (lymph nodes, brain, heart, liver, spleen, vitreous humor, kidneys, pancreas, or ovaries), or other normally sterile sites.

[0161] The CDC also created the National Healthcare Safety Network (NHSN), a tracking system for more than 16,000 healthcare facilities in the United States, to provide data to guide prevention efforts. The Centers for Medicare and Medicaid Services (CMS) and other payers use this data to determine financial incentives for healthcare facility performance. The system tracks MRSA bloodstream infections as a marker for invasive disease for epidemiological purposes.

[0162] In ICD9 and ICD10 systems, the invasive disease state mediated by MRSA is also coded by a set of symptom conditions, each of which has its own digital code for the pathogen MRSA. For example, sepsis owing to MRSA is coded as A41.02, and pneumonia owing to MRSA is coded as J15.212. This further illustrates the following difference characterization: the invasive MRSA disease due to the same cause of disease is listed in parallel with superficial skin and soft tissue disease - code L03.114 (left upper limb example), and its subsequent code B95.6 MRSA is the cause of the disease classified elsewhere, and the code is attached to various other infection codes to indicate that MRSA is the cause of the disease condition.

[0163] The European Centre for Disease Control (ECDC), an arm of the EU, also monitors invasive S. aureus isolates using a similar definition to the NHSN and tracks the percentage that are methicillin-resistant, but reporting requirements do not produce an EU estimate of total annual cases.

[0164] In contrast, simple MRSA colonization is not generally considered a disease in itself, unlike systemic pathologies. However, colonization is considered a prerequisite for most invasive diseases, as evidenced by studies showing, for example, that nasal Staphylococcus aureus isolates are often identical to the strains that subsequently cause clinical infection. This persistent colonization state reflects the ecological stability of the bacterium on skin and mucosal surfaces.

[0165] This colonization status is recorded in the ICD10 system Z22.322 under the Z subheading, which is reserved for factors that affect health status and contact with health services, but not for the disease or injury itself.

[0166] Target orphan disease groups:

[0167] The orphan disease colony of BioPlx non-recurrence method targeting is previously by the crowd (known as sensitive population) of MRSA invasive infection (systemic infection), and continues to suffer from the crowd that MRSA continues to colonize. U.S. Centers for Disease Control and Prevention (CDC) monitors all invasive MRSA infection cases in the U.S. A plurality of researchers have described this medically unique colony---the patient who has suffered from a kind of invasive MRSA infection event of definition. Due to the sensitivity that they show and lasting colonization, the risk that described colony suffers from life-threatening invasive disease increases.

[0168] In some embodiments, a method for preventing recolonization or preventing recurrence of systemic invasive bacteremia caused by MRSA is provided, the method comprising preventing primary requisite MRSA colonization (or preventing recurrence of said MRSA colonization) by:

[0169] 1) Decolonization of MRSA from the mucosal and dermal microbiomes, and

[0170] 2) These microbial groups are re-colonized with synthetic Staphylococcus aureus (e.g., BioPlx01 strains). The method is effective through the niche dynamics within the target dermal / mucosal microbial group ecosystem by the effect of bacterial interference, because synthetic Staphylococcus aureus (e.g., BioPlx01 strains) plays the role of occupying a specific niche and therefore occupies / prevents the re-colonization (preventing recurrence) of MRSA. The evidence of principle research provided herein has clearly demonstrated the effectiveness of the method.

[0171] S. aureus (SA) is present as part of the normal microbiome in over 40% of the general population. Colonization with MSSA is common. MRSA variants account for approximately 1-2% of the US population, but levels may be much higher in certain regions or demographics. MRSA is believed to have the ability to colonize anyone in a population susceptible to S. aureus. S. aureus most commonly resides on the skin and in the anterior nasal vestibule, but small numbers can also be found in the deep oropharynx and gastrointestinal tract of some individuals, as well as in the normal vaginal flora.

[0172] In colonized individuals, S. aureus typically remains a non-invasive commensal bacterium that simply occupies an ecological niche without causing disease. However, in a subset of colonized individuals, the bacterium may either cause disease at the appropriate time or, due to specific mutations, increase its likelihood of invasion, thereby causing disease.

[0173] Approximately 23% of persistent MRSA carriers developed a discrete MRSA infection within a year of being identified as a carrier.

[0174] Many variants of Staphylococcus aureus have acquired genetic cassettes encoding virulence protein products that allow such strains to more efficiently invade the epidermal or mucosal tissue layers and subsequently initiate deep or systemic infections. The presence of the mecA cassette in colonization or infection limits treatment options for these patients, and many studies have demonstrated increased mortality associated with MRSA compared with MSSA in bacteremia, intravascular infection, and pneumonia.

[0175] Because it is impossible to determine in advance whether an individual colonized with MRSA will ultimately develop invasive disease, it is particularly important to identify and treat the entire patient population at well-documented increased risk for invasive MRSA disease.

[0176] MRSA-Mediated Invasive Disease Statistics:

[0177] MRSA was identified by British scientists in 1961, and the first clinical case in the United States was recorded in 1968. For the next 25 years, MRSA was largely considered a problem in local hospitals, and incidence rates were on the rise. However, starting in the mid-to-late 1990s, community-associated MRSA incidence increased, primarily manifesting as superficial skin and soft tissue infections. Of greatest concern to the medical community was the rise in invasive infections caused by MRSA. The incidence of invasive MRSA disease continued to rise throughout the 1990s, peaking in 2005.

[0178] The CDC tracks the incidence of invasive MRSA disease through the National Health Service Network (NHSN) and the Emerging Infections Program—Active Bacterial Core surveillance system, which also began in 2005. Data from 2015 indicate that the overall incidence of invasive MRSA disease decreased by almost 50%, from 37.56 to 18.8 cases, compared to 2005. This reduction in incidence is largely attributed to costly and labor-intensive infection control interventions implemented in hospitals in response to this public health crisis, as the majority of gains were seen in healthcare-associated cases, not community-associated cases. Despite progress over the past decade, invasive MRSA infections remain a public health priority. These infections can be difficult to treat, and among patients who receive appropriate treatment, nearly 25% experience treatment failure. Predicting which healthcare-experienced patients are at risk for invasive MRSA infection is a challenging problem. Risk factors such as MRSA colonization, the presence of chronic open wounds, and the use of invasive devices have been identified.

[0179] The presence of these features alone cannot predict which patient will ultimately develop invasive disease. However, one of the most predictive risk factors for a patient to develop an invasive MRSA infection is having had a prior invasive MRSA infection. In the 2004-2005 data from the Active Bacterial Core Surveillance (ABC), it was noted that almost 13% of invasive cases went on to develop a second invasive MRSA infection during the 18 months of retrospective data evaluation. Another investigation of EIP-ABC data from calendar year 2011 found that 8% of these patients had an interval of at least 30 days with more than one invasive MRSA infection. The long-term risk of recurrent invasive MRSA infection is certainly still higher because these estimates will miss early infections in these patients before the study period, as well as late infections that occurred after the end date. Since Huang and Platt (2003) found that 29% of hospitalized patients with known MRSA colonization or infection went on to develop a second MRSA infection (usually severe) within 18 months, targeting this group to prevent recurrence of invasive disease states could prevent approximately 17,500 subsequent invasive MRSA infections (using the most recent CDC data).

[0180] Invasive MRSA and MRSA skin and soft tissue infections are both caused by the same pathogen. However, orphan designation is granted based on the association of the drug and the disease. MRSA is a pathogen, not a disease state. However, it can cause infection, and treatment for these different infectious diseases is ongoing. Compared to MRSA confined to the skin or colonized with MRSA alone, invasive MRSA carries a much more serious prognosis and presents differently. Approximately 40% of the US population is colonized with Staphylococcus aureus, typically found in the nose or skin. Typically, there are no signs of infection that would be considered a "disease state." However, systemic MRSA infection manifests with high fever, chills, dizziness, chest pain, swelling of the affected area, headache, rash, cough, and other systemic symptoms. The two conditions are managed differently. Skin and soft tissue infections are typically treated by incision and drainage of the furuncle commonly associated with skin and soft tissue infections. Antibiotics and decolonization are only used when systemic disease or signs of severe illness have spread to multiple sites.

[0181] The incidence of invasive MRSA is 20 to 50 cases per 100,000 population per year. 6a The current U.S. population is 326,199,002 (accessed on November 2, 2017, from www.census.gov / popclock), which means that there are conservatively 163,100 cases of invasive MRSA infection in the United States each year, which is lower than the 200,000 patient criterion for FDA orphan designation. Other sources of prevalence reports were searched to confirm that the most conservative estimates for this patient population had been calculated. Hassoun et al. reported an incidence of 72,444 invasive MRSA cases in the United States in 2014, a decrease from 111,261 cases in 2005. 7a Based on this incidence rate and assuming the population will continue to decline, it can be assumed that the prevalence of 163,029 invasive MRSA patients in the United States in 2017 is a very conservative estimate. According to the Centers for Disease Control and Prevention (CDC), there were more than 80,000 invasive MRSA infections and 11,285 related deaths in 2011.

[0182] To address this problem, the inventors have developed the BioPlx01 strain, which is a molecularly altered strain of Staphylococcus aureus that cannot cause disease but can reside in the microbiome niche that MRSA may occupy. The reason for the BioPlx01 strain's lack of invasiveness is that the operon opens upon contact with blood or plasma, triggering the organism's death. Prior to administering the BioPlx01 strain, patients who have tested positive for MRSA and experienced systemic symptoms would undergo systemic decolonization, allowing the strain to occupy the position that MRSA had previously occupied in the patient's microbiome. By preventing virulent strains of MRSA from occupying the niche, these virulent strains are unable to colonize and subsequently invade sterile tissue sites. The BioPlx01 strain is able to prevent recurrent systemic MRSA infections.

[0183] In one embodiment, a method for treating a Staphylococcus aureus lung infection in a patient with cystic fibrosis is provided.

[0184] In one embodiment, a method for treating invasive bacteremia is provided. Using the criteria adopted by the CDC (Centers for Disease Control and Prevention), invasive bacteremia is indicated by isolating bacteria from a normally sterile body part. These body parts may include blood, CSF, joint fluid, bone samples, lower respiratory tract samples, and other sterile body fluids. This condition is related to, but distinct from, simple bacterial colonization and bacterial-mediated skin and soft tissue infections. It is recognized that, in most cases, a colonized state is a prerequisite for invasive disease.

[0185] Invasive (systemic) bacterial infections mediated by MRSA and v-MSSA

[0186] In the present invention, MRSA is induced by the method for the treatment of MRSA infection.Under the mediation of staphylococcus aureus, MRSA invasive bacterial infection also can be commonly referred to as or be referred to as in the document: MRSA bacteremia or septicemia, systemic MRSA infection, MRSA bloodstream infection, invasive MRSA infection.The systemic symptom range of specific MRSA induction is from osteomyelitis, septic arthritis, pneumonia, endocarditis, bacteremia, toxic shock syndrome to septic shock.The exploitation of the method for preventing or reducing invasive MRSA disease recurrence in high-risk population by the colonization mechanism of persistently interfering with bad bacterial strain will make significant progress in preventing invasive MRSA infection aspect the commonly required symptom, and will reduce the probability that these patients suffer from MRSA invasive infection subsequently.

[0187] One objective of this disclosure is to evaluate the ability of BioPlx-01 WT material to prevent MRSA recurrence in active, healthy adult healthcare workers. This population is at particularly high risk for MRSA infection and has the highest MRSA colonization rate of any population. Successfully demonstrating protection in this group will validate the efficacy of BioPlx-01 WT in effectively preventing MRSA recurrence in all at-risk individuals.

[0188] "Relapse" simply means "the bacteria reappears." Relapse is crucial to the development and control of a disease. With relapse, a pathogen reappears again and again, and each time it goes through a survival cycle, it "learns" to become increasingly resistant to the antibiotics it encounters. Without relapse, once a pathogen disappears, it disappears—that's it. Without relapse, there would be no pressure to develop antibiotic resistance.

[0189] In various embodiments, the subject can be colonized with one or more pathogenic microorganisms. In certain embodiments, the undesirable microorganisms are drug-resistant pathogenic microorganisms. The drug-resistant pathogenic microorganisms can be selected from Neisseria gonorrhoeae, fluconazole-resistant Candida species, 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.

[0190] In one embodiment, the undesirable microorganism may be drug-resistant pathogenic Staphylococcus aureus.

[0191] Staphylococcus is the most abundant bacterial genus colonizing the skin and is the most important cause of both nosocomial and community-associated skin infections. S. aureus species may cause fulminant infections, whereas infections caused by other Staphylococcus species are mostly subacute. Colonization is often a prerequisite for infection. Otto 2010, Expert Rev Dermatol 2010 Apr;5(2):183-195. However, not all invasive S. aureus infections are preceded by colonization with the same strain. The unrelated fraction may be explained by the “direct inoculation” or “direct wound inoculation” theory, such as events occurring during surgery in secondary carriers, or incomplete detection of all these patients’ S. aureus strains in colonization or invasive strain colonization in the period after the initial colonization surveillance.

[0192] S. aureus (SA) is a common human commensal organism that is present (colonized) in 30% to 50% of the US population and is usually asymptomatic. Although domestic animals, livestock, and mites can serve as secondary reservoirs, humans asymptomatically carry S. aureus as the primary natural reservoir.

[0193] There are many different Staphylococcus aureus strains, many of which can also serve as serious pathogens. The symptoms of Staphylococcus aureus infection may vary, from asymptomatic to mild symptoms, from skin and soft tissue infection to life-threatening invasive systemic diseases (such as intravascular infection, pneumonia, septic arthritis, endocarditis, osteomyelitis, foreign body infection, sepsis, toxic shock and endocarditis). Traditionally, it is believed that the prenasal mucosa is the most common position for detecting Staphylococcus aureus colonization of healthy carriers. Some positions may be colonized asymptomatically, and the positions include nostrils, throat, axilla, perineum, inguinal area and rectum.

[0194] MRSA isolates were once primarily confined to hospitals and other health care settings and to patients who frequently used these facilities. However, since the mid-1990s, there has been a surge in reported MRSA infections in populations lacking risk factors for exposure to the health care system. The increase in MRSA infection rates has been associated with the identification of novel MRSA clones, termed community-associated MRSA (CA-MRSA). CA-MRSA strains differ from older health care-associated MRSA strains; they infect different types of patients, they cause different clinical syndromes, they have different antimicrobial susceptibility patterns, they spread rapidly among healthy people in the community, and they often cause infections in health care settings. David, Michael, et al., 2010, Clin Microbiol Rev 23(3):616-687.

[0195] It is unknown why recurrent CA-MRSA skin and soft tissue infections are common. The mechanism by which recurrence occurs is unclear. Possible scenarios include reinfection due to persistent asymptomatic CA-MRSA carriage, reinfection after acquisition of MRSA from the environment, or reinfection after acquisition of a novel MRSA species through close contact with humans or animals. Skin and soft tissue infections caused by MSSA can also recur, but less frequently than infections caused by MRSA.

[0196] Under constant antibiotic pressure, many S. aureus variants have developed antibiotic resistance. Penicillin resistance is now virtually universal in S. aureus, and common β-lactam resistance and the related multi-antibiotic (methicillin) resistance are now widespread, giving rise to an important new class of antibiotic-resistant "superbugs."

[0197] The pathogenic Staphylococcus aureus can be a drug-resistant Staphylococcus aureus, such as MRSA or a vancomycin-resistant strain (such as VISA or VRSA). Alternatively, the pathogenic Staphylococcus aureus can be a highly toxic methicillin-sensitive Staphylococcus aureus (v-MSSA). v-MSSA is a highly virulent cause of life-threatening invasive infections. MRSA and v-MSSA are very prevalent and the human cost is very high.

[0198] MRSA has become a serious public health problem in hospitals, clinics, prisons, military barracks, and even gyms and health clubs worldwide. MRSA is a common cause of hospital-acquired infections (500,000 US patients per year) and is increasingly becoming a major cause of community-acquired infections. Invasive systemic disease—20% of cases result in death. MRSA is the most important of the new antibiotic-resistant "superbugs." While there are treatments for S. aureus infections, there are virtually no methods to prevent recurrence. Recurrence of MRSA skin infections is seen in 31% to 45% of subjects.

[0199] One effort to prevent recurrence involves decolonization. The first (and currently only) widely practiced step to prevent recurrence is decolonization. Unfortunately, simple decolonization is poorly effective in preventing recurrence. Physicians can initially treat microbial colonization or infection (e.g., MRSA or v-MSSA colonization / infection) with topical chemicals (e.g., chlorhexidine) or antibiotics. In many cases, treatment with antibiotics can "clinically" eliminate the disease. Antiseptics and astringents can 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. The most commonly used systemic antibiotics for MRSA include vancomycin, first-generation antibiotics such as cefazolin, cephalosporins, or cephalexin; and newer-generation antibiotics such as linezolid or daptomycin. In less severe cases of MRSA, clindamycin or lincomycin can be used. Despite this, MRSA and v-MSSA pathogens typically relapse or regrow nearly half the time with decolonization alone. This level of performance naturally leads to skepticism about the efficacy of simple decolonization in preventing relapse.

[0200] 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 eliminate the Staphylococcus carrier state. Several drugs can temporarily interrupt Staphylococcus carriage, but no drug is effective in preventing skin infections caused by MRSA. Creech et al. Infect Dis Clin North Am. 2015 Sep;29(3):429–464.

[0201] Both in the literature and in the hands of the present inventors, the quality of decolonization has been found to correlate with observed relapse rates, but simple decolonization rarely leads to durable, successful outcomes.

[0202] The present disclosure provides methods and compositions directed to preventing recurrence by effectively and durably modifying the microbiome.

[0203] Methods have been developed for preventing or reducing recurrence of pathogenic microbial infection that include inhibiting microbial infection or colonization.

[0204] A method for reducing the recurrence of pathogenic infection or reducing undesirable microbial colonization in a subject is provided, the method comprising: decolonizing the undesirable microorganism at at least one site of the subject, thereby significantly reducing or eliminating the presence of the undesirable microorganism at the site; and replacing the undesirable microorganism by administering to the subject a synthetic second microorganism of the same genus and species as the undesirable microorganism.

[0205] Methods and compositions for preventing recurrence include replacing pathogenic microorganisms by filling the biome niche occupied by the pathogen with specially designed synthetic microorganisms (or "beneficial bacteria"). By occupying the same biome niche, the "beneficial bacteria" crowd out the pathogen, thereby preventing the pathogen from recolonizing or migrating into (or back into) its preferred ecological community. One way to ensure that the same biome niche is filled is by designing a synthetic microorganism of the same genus and species as the pathogenic microorganism.

[0206] Methods and compositions for preventing relapse include promoting or supporting synthetic microorganisms ("beneficial bacteria") by reestablishing a critical nutritional, chemical, or symbiotic environment that further promotes preferred organisms and inhibits recolonization by pathogens. For example, symbiotic clusters can provide an additional layered defense to prevent pathogens from migrating back to their old niches—which can help prevent relapse.

[0207] The BioPlx approach is enabled by state-of-the-art methods / techniques encompassing microbiology, systems and computational biology; environmental interactions (colonization and signaling); proprietary organisms (selected and modified); and variant and strain replacement strategies.

[0208] Provided herein is a replacement microorganism comprising (1) " variant”—a wild-type Staphylococcus aureus 502a microorganism with established avirulence and passive colonization history that has been isolated, validated, and prepared for field trials using the strain cluster, as described in Example 1; (2) “ Engineered variants”, a synthetic strain of Staphylococcus aureus that has been designed to enhance safety by knocking out specific virulence genes; (3) “ The invention relates to an engineered variant of a synthetic Staphylococcus aureus strain that incorporates a molecularly programmed cell death trigger to prevent invasive virulence. In some embodiments, the synthetic microorganism acts purely as a replacement for the pathogenic strain, without "new" infection or colonization.

[0209] An extensive proprietary library of well-characterized S. aureus cultures (strains and variants) has been developed for use in replacement biological sources; for durability and competition analysis; for comparative genotype / phenotype analysis; for cluster modeling of the virulence genome / transcriptome; and for cluster modeling of the signaling genome / transcriptome.

[0210] Libraries of controlled symbionts are being developed to The variants were co-administered together as potential variant clusters.

[0211] Methods are also being developed for computational microbiology, encompassing machine learning; modeling of complex, dynamic microbial systems; genome / transcriptome / proteome (phenotype) relationships; virulence factor genetics and promoters; modeling resilience and changes over time / conditions; n-dimensional niche formation relationships; and high-dimensional clustering relationships.

[0212] Central to our model's anti-relapse approach is the principle of "non-cocolonization," meaning that only one species, or one variant of that species, can occupy the relevant skin or mucosal biome niche at any given time. This simple and testable phenomenon underlies many deeper, generative principles that collectively underlie the emerging science of microbiome science. Although broadly generalizable, the discussion of non-cocolonization in this section specifically addresses Staphylococcus aureus colonization.

[0213] Non-cocolonization

[0214] The principle of non-cocolonization (also known as "bacterial replacement") states that at any given time, only one variant / strain of a species can occupy any given ecological niche within a biome.

[0215] The central empirical phenomenon of non-cocolonization represents a combined effect: the result of the interplay of a large number of forces that can be found in complex systems and that has only today been well characterized and mathematically characterized.

[0216] The species-specific bacterial niches in the human biome are the basis of this technology. Without specificity in the biological niches occupied, it would be impossible to achieve intentional strain exchange, as demonstrated by the bacterial replacement phenomenon.

[0217] The expectation of non-co-colonization is important for the persistence of the methods of the present invention for preventing pathogenic colonization or recurrence of infection. Non-co-colonization of variants has been experimentally demonstrated in the literature by strain / variant replacement (e.g., Shinefield et al., 1963, for the transformation of Staphylococcus aureus 80 / 81 to 502a) and has been confirmed in current clinical studies, as shown in Example 1.

[0218] Persistent niche occupation across species is questionable, as a careful review of the literature suggests low durability and little in vivo evidence. Transient occupation may occur but is not considered an important outcome, as we focused solely on persistence outcomes.

[0219] The failure of persistent interspecies substitution demonstrates that specific niche filling requires “close variant” substitution. This is important because only persistent biomes can display the functional properties (such as resilience) required for effective non-recurrent technologies / products.

[0220] In the case of variant to variant replacement (such as replacement seen in the present disclosure about MRSA anti-recurrence materials), there is no evidence from the literature to indicate whether the replacement requires a "biome-destructive event" (such as accidentally or intentionally decolonizing by antimicrobials, antibiotics, etc.) or whether the replacement can occur by "slow competitive replacement" (one organism competes with another organism for resources, growth, etc.). However, in the vast majority of human skin biomes, only one strain is "completely" colonized in humans, which indicates that slow competitive replacement has occurred. Further, the success rate of the anti-MRSA non-colonization method is 55%, which indicates that "biome-destructive events" can also induce lasting biome changes. Both phenomena are manifestations of non-co-colonization.

[0221] Non-cocolonization occurs naturally, for example, in most cases only one S. aureus variant was detected in a single biome (over 95% of the time, where the equilibrium was likely caused by a “transient state”).

[0222] In specifying and evaluating the persistence (efficacy) of non-cocolonization, it is necessary to recognize three different levels of outcome: (1) short-term—immediately following recolonization, (2) early stabilization—after expulsion of excess organisms, and (3) long-term—after establishment of a stable “new” biome.

[0223] In the short term (immediately following recolonization), the decolonized biome is dominated by organisms "overdosed" during the recolonization process—creating a serendipitous and temporary combination (like spreading peanut butter). Testing during this period can only confirm that biome application has occurred. Duration = several days, followed by replacement of excess organisms.

[0224] During the early stabilization phase (after the excess organisms have been eliminated), the biome itself is reestablishing its equilibrium state, but it appears to be replacement organisms rather than pre-existing pathogens. Confidence in this result is primarily due to the large ratio of new organisms to pathogens that survive decolonization (approximately one million to one). The expectation is that this will become a stable colonization with a high level of durability. Testing during this period will confirm that MRSA or vMSSA has been eliminated and that the replacement strain has recolonized. Duration = weeks to months.

[0225] In the long term (after a stable "new" biome is established), not only will the ability of an organism to occupy or "capture" the niche be demonstrated, but also its ability to "maintain" that niche. In some embodiments, the phase is used to assess the competitiveness of the replacement strain or synthetic microorganism against the current generation of new biome invaders (such as USA300). This question refers to the ability of the "new" replacement organism to compete over time with slower competing replacements, as well as external forces that may cause damage to the biome over time (such as exposure to antibiotics or preservatives or frequent exposure to pathogens) - especially when the strains are differentially sensitive to such disruptors.

[0226] Characterizing the phenomenon of non-cocolonization of microbial variants, variant-to-variant niche occupation, is important, and empirical evidence has been developed demonstrating that this phenomenon exists and is a powerful force in the dermal biome ecosystem.

[0227] The law of "competitive exclusion" refers to situations where only one organism dominates an ecological niche.

[0228] A historical mistake in understanding this phenomenon has been to assume that it is a binary system, conceptually driven by one or two variants. In reality, at any given time, and over time, there can be a large number of different microorganisms, such as various strains of Staphylococcus aureus, present in the environment.

[0229] It follows that, if non-cocolonization is absent, virtually all “staphylococcal-competent” biomes are essentially a highly variable, mixed, heterogeneous “soup” of multiple variants. Table 1 lists the various possibilities.

[0230] Table 1. Niche compatibility and expected outcomes for Staphylococcus aureus (SA)

[0231]

[0232]

[0233] In Table 1, cases 2 and 4 can be eliminated because cocolonization occurs in less than 5% of cases (in the literature), and even in these cases, the vast majority of observed instances of cocolonization involve only one other organism. In the few cases (less than 5%) that may be related to incomplete or non-overlapping niches with the footprints of the replacement organisms, case 3 can be considered possible.

[0234] There is no direct evidence from the literature to show that the replacement of a variant observed to another variant (such as the acquisition of MRSA) is caused by a biome destructive event or by slow competitive replacement. However, it is clear from experience that only one strain tends to colonize any single biome (completely) at a time. Biogeographically distinct and distant positions in a given biome often have the same variant, and this occurs in the absence of any observable systemic decolonization and replacement process, which shows that a rule-driven competitive replacement process has occurred. The observation of competitive replacement is another manifestation of the non-total colonization principle.

[0235] In hypothetical situations where the replacement variants do not completely fill the niche, there may be a tendency to weaken co-colonization. In these cases, clusters of variants can be used to "fill the gaps" with replacements, so that co-colonization favors the synthetic replacement microorganisms over the original pathogen. Although this may involve using different replacement microorganisms, this is not relapse—it is further blocking relapse.

[0236] Current evidence for non-cocolonization

[0237] A large study investigated the incidence of co-colonization in 3,197 positive S. aureus samples collected from healthy patients in Oxfordshire, England, followed longitudinally for up to two years; the point prevalence of multiple S. aureus strains in nasal samples ranged from 3.4% to 5.8%. Votintseva et al., 2014 J Clin Microbiol, 52(4):1192-1200. Among S. aureus carriers who submitted swabs almost every two months for two years, 11% had transient co-carriage. The study used a validated spa typing protocol that allows for the detection of even a low proportion of co-colonizing strains using sensitive procedures. Interpretation of this dataset suggests that S. aureus colonization is a dynamic process, with a low probability that multiple S. aureus strains compete for presence in the same niche over time. A simple calculation can determine that the observed results are not simply due to independent occupation of a nonspecific niche. In this case, 1,000 patients were screened and 360 were found to be positive for S. aureus. In the case of a non-specific niche, co-colonization would be expected to occur in .36 x .36 or 13% (130 persons); however, at the primary site, only 3.9% (14 persons) of 360 carriers were actually co-colonized, indicating strain specificity of the microbiome niche for S. aureus.

[0238] A small percentage of S. aureus carriers can be transiently colonized with two different strains of S. aureus at any given time point. As mentioned above, Votintseva et al. studied all variants within MSSA and MRSA and reported a point incidence of this phenomenon ranging from 3.4% to 5.8%. Papers that studied only mixtures of MRSA and MSSA (which would only find species that differed at the mecA locus) had a predictably lower point incidence of 2.3%. If co-colonization were a stable state, nearly all samples would contain a mixture of S. aureus species. This was not observed.

[0239] Another study looked at 680 patients undergoing any type of hospitalization. At the time, the National Health Service screened all admitted patients for MRSA. Dall'Antonia, M. et al., 2005, J Hospital Infect 61, 62-67. During this evaluation, the protocol was refined to identify patients with MSSA, MRSA, and co-colonization with MRSA and MSSA. Only MSSA was found in 115 patients (16.9%), only MRSA was found in 56 patients (8.2%), and co-colonization was found in 4 patients (0.58%), reaffirming the idea of ​​strain-specific niches for S. aureus in the microbiome. This supports the concept that one S. aureus strain can prevent the establishment of another S. aureus strain. The results showed that, as predicted by the null hypothesis, the percentage of co-colonized carriers was lower, indicating that prior colonization with a single S. aureus strain confers a significant protective effect against colonization with a single S. aureus strain. The statistical significance was p < 0.01. The calculated protective effect of MSSA colonization against MRSA colonization was 78% (CI: 29-99%).

[0240] A further study examined non-discordant S. aureus isolates in a population of HIV-infected IV drug users at a methadone clinic. Of the 121 baseline-positive S. aureus samples, four showed significant discordance in three colonies evaluated by PFGE. However, reevaluation of these four samples revealed that two of the four samples were concordant at the second evaluation. Reevaluation of the 18 samples initially found to be concordant revealed no discordance. Thus, 1.7–3.3% of this population was found to be co-colonized at a single time point. Cespedes C. et al., J Infect Dis 2005;191:444-52.

[0241] Historical evidence from decolonization / recolonization studies also shows evidence of non-cocolonization. This principle was previously partially demonstrated in the famous 80 / 81 to 502a "bacterial interference" studies and clinical applications in the 1960s and 1970s. The lack of co-colonization was shown in early bacterial interference papers from the 1960s and 1970s, which also clearly demonstrated "competitive exclusion" in regulating co-colonization. Mixed cultures of 80 / 81 as the resident strain and 502A as the donor strain were not observed, experimentally demonstrating that non-cocolonization is a stable condition in the microbiome (Shinefield et al., 1963; Shinefield et al., 1966; Shinefield et al., 1973; Aly et al., 1974; Boris et al., 1964; Light et al., 1967; Fine et al., 1967).

[0242] In the absence of "non-equivalence" and "competitive exclusion," microbiome niches will always be populated by multiple strains of the same bacterial species. Isolating a substantially pure culture of a S. aureus strain from the nasal passages would be a rare event, if never seen before. The population dynamics in this state would produce a heterogeneous "soup" of many variants of S. aureus, determined by accidental or random exposure to the environment. Any strain that the host has ever been exposed to would have an equal chance of colonizing the space without competing with or interfering with any other strain variant (polyclonal colonization). The absence of this empirical result suggests "competitive exclusion."

[0243] However, the exclusion principle is not so strict that once a niche is occupied, other variants cannot usurp its place. These observations demonstrate an exclusion principle that is powerful, but allows outside species to challenge the occupying species by briefly sharing the niche, while ultimately competing for dominance in the space. In some cases, the "new" strain outcompetes the previous resident strain and establishes a new dominant resident strain. In other cases, the interloper is rejected, the resident strain repels the replacement attempt and re-establishes exclusive dominance. In both cases, the state of co-colonization is short-lived and unstable; the frequency of occurrence is low.

[0244] Microbial system

[0245] Provided are methods and compositions for permanently and safely preventing recurrence of pathogenic microorganism infection in a subject, the methods comprising: inhibiting the pathogenic microorganism; replacing it with a synthetic microorganism capable of occupying the same ecological niche to permanently exclude the pathogenic microorganism; and promoting the synthetic microorganism to persist in the ecological niche. As described above, the method is referred to as In some embodiments, the subject is found to be colonized with a pathogenic microorganism prior to the inhibiting step.

[0246] To successfully work within the microbiome to promote colonization by desired organisms in a way that produces lasting protective outcomes, one needs to know the “rules” of the microbiome: as discussed in more detail in the following sections.

[0247] A non-cocolonization model was developed to provide context and establish the characteristics of the target product. The fundamental principles of the present technology are based on the microbiological paradigm (biomes / ecosystems / niche) and the identification of microbial communities with certain persistent and verifiable characteristics. The key discoverable indicators were based on co-colonization statistics from the literature, which were modified by detailed descriptions of decolonization, testing, and other relevant conditions, followed by direct observations from the clinical study of Example 1.

[0248] The skin microbiome in a 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 can be defined as a system or set of interconnected elements formed by the interaction of a community of organisms with their environment. The skin microbiome ecosystem has a basic state of "health" or "normal". The biome can be "healthy" or "sick" (dysbiosis) and can be invaded by pathogenic organisms - in other words, the microbiome can be invaded by "bad bacteria" (such as MRSA), or it can be infected or contaminated by undesirable organisms or variants (dysbiosis). Dysbiosis is a term used for an imbalance or maladaptation of microorganisms in or on the body, such as an impaired microbiome.

[0249] The skin microbiome has a structure created by an extensive combinatorial network between the host and all components of the biome. The microbiome or biome is a complex system with a dynamic structure and is a "resilient" ecosystem.

[0250] The skin microbiome has a dynamic yet persistent structure—for example, it is "resilient" even in the presence of massive cell death (e.g., washing, use of ethanol, hand sanitizers, etc.), with the biome regenerating in a similar form.

[0251] Resilience

[0252] The human microbiome is resilient, meaning that mild perturbations tend to recalibrate toward a previously established baseline of species mixture and concentrations. However, members of each niche can successfully challenge their position in the stable mixture due to acute external disruptive events (i.e., application of antimicrobials or preservatives) or due to slow competitive replacement.

[0253] In ecology, resilience is the ability of an ecosystem to respond to perturbations or disturbances by resisting damage and recovering quickly. Resilience refers to the stability of an ecosystem and its ability to withstand disturbances and restore itself.

[0254] In the literature, the main mathematical definition of resilience is based on dynamical systems theory, and more specifically, on attractors and domains of attraction. The human microbiome operates in various ways, as a multidomain complex system. It can change states or domains, but resilience then 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.

[0255] The microbiome operates in many ways, like a complex system of multiple attractors—it can change the state or domain of an attractor, but then the resilience associated with that attractor stabilizes that state.

[0256] Ecological resilience is defined as the ability of a system to absorb disturbances and reorganize itself while undergoing change, while still maintaining essentially the same functions, structures, properties, and feedbacks. Mitra, C. et al., 2015, “An integrative quantifier of multistability in complex systems based on ecological resilience,” Nature, Scientific Reports, 5, 1–12.

[0257] The "competitive exclusion principle" stipulates that perfect competitors do not exist. The "inequality axiom" states that no two things or processes in the real world are completely equal. Hardin, 1960, Science, Vol. 131, 1292-1297, p. 1292. According to Hardin's "inequality axiom" and the competitive exclusion principle, long-term persistence can only be achieved by replacing similar variants, but species replacement is impossible. For example, MRSA and MSSA can co-colonize briefly—just as any other variant of Staphylococcus aureus can co-colonize briefly. See Dall'Antonia, M. et al., 2005, Journal of Hospital Infection, 61, 62-67, which discloses a study of 680 patients undergoing any type of hospitalization and screening all admitted patients for MRSA. During this evaluation process, the protocol was refined to identify patients with MSSA, patients with MRSA, and patients who co-colonized with MRSA and MSSA. Only MSSA was found in 115 patients (16.9%), only MRSA was found in 56 patients (8.2%), and co-colonization was found in 4 patients (0.58%), reaffirming the idea of ​​strain-specific niches for S. aureus in the microbiome. This supports the concept that one S. aureus strain can prevent the establishment of another S. aureus strain.

[0258] Resilience may lead to relapse (an observed natural phenomenon) as the existing (MRSA-contaminated) biome attempts to protect itself.

[0259] However, as shown by the methods and compositions provided herein, resilience can also prevent MRSA recurrence. By suppressing pathogenic microorganisms, such as MRSA ("bad bacteria"), that colonize a subject's body and replacing them with safe synthetic microorganisms of the same species ("good bacteria"), it has been determined that the "good bacteria" can durably prevent the recurrence of the "bad bacteria" (preventing MRSA from invading again).

[0260] A historical example of resilience resulting in a long-lasting, sustained replacement has been observed in S. aureus carriers, who were replaced with strain 502a. Aly et al. (1974 J. Infect. 129(6) pp. 720-724) studied bacterial interference in S. aureus carriers. Carriers were treated with antibiotics and antibacterial soaps and challenged with S. aureus strain 502a. It was found that adequate decolonization was required to obtain good colonization of 502a. Uptake was 100% on day 7, but this dropped to 60% to 80% on day 23. Persistence data for subjects who were well decolonized was 73% at 23 weeks. Therefore, long-term persistence can only be achieved by replacement with tight variants. Symbiotic flora (normal flora, native microbiome) can help with recolonization dynamics, but they cannot meet strict persistence requirements. The inventors have devised a method for obtaining a "passive" form of the organism or pathogen (same species) to be "replaced" or "excluded".

[0261] Relative stability in microbial ecosystems of adults in the absence of severe perturbations has been proposed, and long-term stability of human communities is maintained not by inertia but by the action of resilience within dynamic systems. Relman, DA, 2012, Nutr Rev., 70(Suppl 1):S2–S9.

[0262] Functional resilience is an inherent property of microbial communities, and it has been proposed that state changes in response to environmental changes may be a key mechanism driving functional resilience in microbial communities. Song et al., 2015, Frontiers in Microbiology, 6, 1298. Seeking an integrative concept applicable to all microbial communities, Song et al. compared engineered resilience and ecological resilience, reconciling the two by arguing that resilience is an inherent property of complex adaptive systems that recover their system-level functions and interactions with the environment after a disturbance, rather than their endogenous state.

[0263] Therefore, biome ecosystems exhibit a dynamic yet stable elastic-plastic equilibrium, attempting to restore this equilibrium upon disturbance. At any given moment, biome ecosystems maintain a "base state" of equilibrium. Even after extensive cell death (e.g., washing, ethanol use, hand sanitizer use, etc.), biome regeneration is often observed in a similar fashion.

[0264] Microbiome ecosystems have "niche"s defined by their structure and internal and external interactions. A "fact" or "principle" of any biome structure is that different organisms occupy different "niche"s within the biome, as defined / allowed by the relationship structure. An "ecological niche" in ecology is the role and position a species has in its environment; how a species meets its needs for food and shelter, how it survives, and how it reproduces. A species' niche encompasses all of its interactions with the biotic and abiotic factors in its environment. A biome "niche" has specific environmental factors and conditions, including, for example, pH, temperature, osmotic pressure, osmolality, oxygen levels, nutrient concentrations, blood concentrations, plasma concentrations, serum concentrations, and electrolyte concentrations.

[0265] Different organisms occupy different "niche" within a biome, as defined / allowed by the relational structure. A niche is a persistent characteristic of a biome's ecosystem. Each niche has boundary conditions; a virtual shape or "footprint" that reflects that shape, which will be discussed in the context of the "Hutchinsonian niche."

[0266] Hutchinson's niche is an n-dimensional hypervolume, where the dimensions are the environmental conditions and resources that define the requirements for an individual or species to practice "its" lifestyle (more specifically, for its population to persist). The "hypervolume" defines the multidimensional space of resources (e.g., light, nutrients, structures, etc.) available to (and used exclusively by) organisms, and "all species other than the one under consideration are considered to be part of the coordinate system."

[0267] An ecological niche is a very specific portion of ecological space occupied by an individual species. Given the assumption that no two species are identical in all respects (i.e., Hardin's "inequality axiom") and the competitive exclusion principle, some resource or fitness dimension will provide each species with a specific ecological niche.

[0268] Ecological niches are exclusive. Each organism competes for that niche with similar organisms, and the successful organism fills the niche. Two organisms will not / cannot fill the same niche because, over time, one organism will outcompete the other. Therefore, the longer two organisms coexist in the same biome, the less likely they are to occupy the same niche.

[0269] Once an ecological niche becomes vacant, other organisms can fill it. This is because one species does not have the same footprint as another species, so one species cannot fill the same ecological niche as another species. Successful replacement requires using the same organism (e.g., the same species or a close variant) to fill or permanently replace the ecological niche. It has been recognized that some competition exists in the form of short-term colonization / infection and is an observable phenomenon.

[0270] Partial competition for a single niche can occur. One organism can "narrow" the "niche width" of another organism by partial competition. This may be the case with Staphylococcus epidermidis and Staphylococcus aureus. Staphylococcus epidermidis is a commensal bacterium that, when used in sufficiently high concentrations, secretes a serine protease that is able to break down pre-formed S. aureus biofilms. Sugimoto et al., J Bacteriol, 195(8): 1645-1655. However, there is an important distinction between organisms that are carriers of the expression of a toxic phenotype (temporarily overloaded by administration at the site) and organisms that are persistent residents of a niche that shrinks or outcompetes the pathogen.

[0271] Interspecies co-colonization is a phenomenon distinct from the ability to persistently fill and block niches. For example, Shu et al. (2013) demonstrated that glycerol fermentation with Propionibacterium acnes (C. acnes), a skin commensal bacterium, to form short-chain fatty acids (SCFAs) inhibited the growth of USA300, the most prevalent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Shu demonstrated that SCFAs produced by P. acnes under anaerobic conditions inhibited the growth of S. aureus at high concentrations. Shu et al., 2013 PLoS ONE 8(2):e55380. However, this fermentation capacity of these bacteria and P. acnes is already present in the normal human skin biome and has not effectively eliminated or reduced the pathogenicity of S. aureus. There is no reason to believe that supraphysiological application of these substrates will achieve the goal of reducing S. aureus colonization or disease incidence.

[0272] Decolonization / recolonization

[0273] A method for preventing or reducing recurrence of pathogenic infection by an undesirable microorganism in a subject is provided, the method comprising the steps of: (i) inhibiting (decolonizing) the undesirable microorganism at at least one site of the subject, thereby reducing or eliminating the presence of the undesirable microorganism at the site; and (ii) replacing the undesirable microorganism by administering a synthetic second microorganism of the same genus and species as the undesirable microorganism to the subject at the at least one site. Optionally, the method further comprises (iii) promoting colonization of the synthetic microorganism, for example, at the site of administration.

[0274] In some embodiments, the undesirable microorganism is a pathogenic microorganism, and the term inhibit (S) refers to the process of inhibiting, reducing, or eliminating the pathogenic microorganism at one or more, two or more, three or more, or four or more sites of the subject. For example, the undesirable microorganism can be subjected to a nasal, mucosal, and / or dermal decolonization regimen.

[0275] The term "replace" (R) refers to replacing a pathogenic microorganism with a synthetic microorganism that is benign, drug-sensitive, and / or incapable of causing systemic or pathogenic infection in a subject. The replacement microorganism can be a molecularly modified synthetic microorganism of the same species as the pathogenic microorganism. The synthetic microorganism can be a molecularly modified microorganism of a different strain of the same species as the pathogenic microorganism, such that the synthetic microorganism can colonize a site in the subject but cannot cause systemic infection in the subject. By filling the vacant ecological niche of the pathogenic microorganism, the synthetic microorganism can eliminate the recolonization of the pathogenic microorganism in the subject, thereby reducing or eliminating the recurrence of the pathogenic infection.

[0276] The term promote (P) refers to methods and compositions that promote and support a new biome comprising the synthetic microorganisms, such as by employing prebiotics and biome management (eg, by employing a biome regulator) to promote replacement synthetic microorganisms in a subject.

[0277] These approaches broadly define platform technologies (SRPs) and develop tailor-made solutions to address specific medical conditions (e.g., MRSA). If the S, R, and P processes are chosen correctly—opening a specific biome niche, then filling and maintaining that niche—a "durable," persistent biome will be created that is resistant to pathogenic colonization.

[0278] A method for reducing the recurrence or chance of systemic infection by a pathogenic microorganism in a subject is provided, the method comprising: inhibiting a pathogenic microorganism on the subject, thereby significantly reducing or eliminating the detectable presence of the pathogenic microorganism; replacing the pathogenic microorganism by administering a synthetic microorganism to the subject, wherein the synthetic microorganism is capable of occupying the same ecological niche as the pathogenic microorganism, as demonstrated by: (1) the synthetic microorganism has the same genetic background or genus and species as the pathogenic microorganism; and / or (2) the synthetic microorganism exhibits a persistent detectable presence to the subject for at least 60 days after replacement. The method may comprise promoting the colonization of the synthetic microorganism on at least one site of the subject. In some cases, it may be found that the subject has become colonized by the pathogenic microorganism.

[0279] Typically, pathogenic microorganisms colonize the subject before the subject becomes systemically infected with the pathogen. For example, a significant proportion of cases of Staphylococcus aureus bacteremia appear to be endogenous, as they may originate from colonies of the nasal mucosa. For example, in a multicenter study of Staphylococcus aureus bacteremia, 180 of 219 patients (82.2%) had blood isolates that were identical to those from the anterior nares. In a second study, 14 of 1278 patients colonized with Staphylococcus aureus nasally subsequently developed Staphylococcus aureus bacteremia. In 12 of these 14 patients (86%), isolates obtained from the nares were clonally identical to isolates obtained from the blood 1 day to 14 months later. See von Eiff et al., 2001, The New England Journal of Medicine (NEJM), Vol. 344, No. 1, 11-16. Another study showed that nasal carriers had a several-fold increased relative risk of S. aureus bacteremia compared with noncarriers, reporting an 80% match between invasive isolates and previously identified colonizing strains. Wertheim et al., Lancet 2004;364:703-705.

[0280] In some embodiments, the subject is found to be colonized with pathogenic strains of microorganisms prior to systemic infection. In other embodiments, the subject may have been colonized or infected with nosocomial (hospital-acquired) or community-acquired strains of pathogenic microorganisms.

[0281] The pathogenic microorganism may be a wild-type microorganism and / or a pathogenic microorganism that can colonize or be detectably present in at least one site of the subject. The site may be a dermal or mucosal site of the subject. The one or more colonization sites may include skin and soft tissue, including but not limited to the nostrils, throat, perineum, inguinal area, vagina, nasal cavity, groin, perirectal area, between the fingers, forehead, pharynx, axilla, hands, chest, abdomen, head, and / or between the toes.

[0282] The pathogenic microorganism can be a drug-resistant microorganism. The Centers for Disease Control (CDC) recently released a report outlining 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 species, 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.

[0283] In some embodiments, the pathogenic microorganism is MRSA.

[0284] The synthetic microorganism (a) must be capable of populating an ecological niche in at least one site of the subject so as to durably exclude the undesirable microorganism after 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 environmental conditions compared to normal physiological conditions in at least one site of the subject.

[0285] The synthetic microorganism can be of the same genus and species as the undesirable microorganism in order to enhance the ability to fill a niche and permanently exclude the undesirable microorganism from at least one site of the subject.

[0286] In certain embodiments, the present disclosure provides a kind of synthetic microorganism, described synthetic microorganism is not pathogenic and cannot become accidental pathogen, because it does not have the ability of infecting experimenter when state changes (for example, when being exposed to blood or serum).Described synthetic microorganism comprises at least one molecular modification, and described molecular modification comprises the first cell death gene that is operably connected to the first regulatory region that comprises the first promoter, and described first promoter is activated (induced) by the change of environmental state compared with the normal physiological condition in at least one position of experimenter.For example, if the position in experimenter is dermis or mucosal position, then being exposed to blood or serum is the change of state, and it causes the cell death of synthetic microorganism.For example, the average cell death of 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 after state changes.State change can be the change of one or more of the following conditions relative to the condition at at least one position of experimenter: pH, temperature, osmotic pressure, weight osmotic molar concentration, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration and electrolyte concentration. In some embodiments, the altered state is a higher concentration in blood, serum, or plasma compared to normal physiological conditions at at least one site of the subject.

[0287] In one embodiment, the pathogenic microorganism is MRSA. MRSA is a variant subgroup of Staphylococcus aureus. MRSA strains typically contain a mecA box that allows the production of alternative penicillin-binding proteins, making the strains resistant to treatment with most beta-lactams and other first-line antibiotics. Staphylococcus aureus as a whole (including MRSA) exists as part of the normal microbiome, accounting for approximately 30% of the total population. As part of the microbiome, Staphylococcus aureus most often lives on the surface of the skin and in the anterior nasal vestibule, but small amounts of Staphylococcus aureus may also be found in the deep oropharynx and gastrointestinal tract of some individuals and as part of the normal vaginal flora.

[0288] In the vast majority of individuals, S. aureus remains a non-invasive commensal bacterium that merely occupies an ecological niche without causing disease. Colonization is far more common than invasive disease—some researchers estimate the ratio to be approximately 1,000 to one. Laupland et al., Journal of Infectious Diseases (2008) 198:336. However, in a subset of individuals who become colonized, this bacterium may cause disease either in due time or due to an increased tendency to invade, due to the acquisition of a gene cassette encoding a virulence protein product that enables this strain to more effectively invade the epidermal or mucosal tissue layers, thereby causing deep infections. In both cases, the presence of the mecA cassette limits treatment options for these patients, and numerous studies have demonstrated an increased mortality rate associated with MRSA compared to MSSA in bacteremia, intravascular infections, and pneumonia.

[0289] definition

[0290] Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include the plural forms as well.

[0291] The term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0292] 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 specified, all terms used in this specification, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of a conflict in terms, this specification shall prevail.

[0293] The term "pathogen" or "pathogenic microorganism" refers to a microorganism that is capable of causing disease. Pathogenic microorganisms may colonize a site in a subject and may subsequently cause systemic infection in the subject. Pathogenic microorganisms may have evolved genetic abilities to break through cellular and anatomical barriers that normally restrict other microorganisms. Pathogens may inherently damage cells to forcefully gain access to new, unique ecological niches, thereby reducing competition with other microorganisms and providing new nutrients. Falkow, Stanley, 1998, Emerging Infectious Diseases, Vol. 4, No. 3, 495-497. Pathogenic microorganisms may be drug-resistant microorganisms.

[0294] The terms "virulent" or "virulence" are used to describe the ability of a microorganism to cause disease.

[0295] The term "commensal" refers to a form of symbiosis in which one organism obtains food or other benefits from another without affecting it. Commensal bacteria are often part of the normal flora.

[0296] The term "inhibit" or "decolonization" refers to substantially reducing or eliminating the original undesirable pathogenic microorganisms (commonly referred to as "decolonization") by various means. Substantially reducing means reducing the original colonization of undesirable microorganisms by greater than 90%, 95%, 98%, 99% or greater than 99.9% by any means known in the art.

[0297] The term "replacement" refers to the replacement of original pathogenic microorganisms by the introduction of new microorganisms (often referred to as "recolonization"), which "crowd out" and occupy the ecological niches that the original microorganisms would normally occupy, and thus prevent the original undesirable microorganisms from returning to the microbiome ecosystem (often referred to as "disturbance" and "non-cocolonization").

[0298] The terms "durably replace" or "durably exclude" or "durably exclude" refer to the presence of the newly synthesized microorganism, e.g., as detected by swabbing the subject, for a period of at least 30, 60, 84, 120, 168, or 180 days after the introduction of the new microorganism into the subject. In some embodiments, "durably replace" or "durably exclude" refers to the absence of the original pathogenic microorganism, e.g., as detected by swabbing the subject, for a period of at least 30, 60, 84, 120, 168, or 180 days after the introduction of the new synthetic microorganism into the subject for at least two consecutive sampling periods.

[0299] The term "promote" or "promoting" refers to an activity or method that enhances the colonization and survival of a neoplastic organism in a subject. For example, promoting colonization of a neoplastic organism in a subject can comprise administering nutrients, prebiotics, and / or probiotic species.

[0300] As used herein, the term "prevention" refers to a course of action (such as administering a compound or pharmaceutical composition of the present disclosure) that begins before the onset of clinical manifestations of a disease state or condition to prevent or reduce such clinical manifestations of the disease state or condition. Such prevention and inhibition are not necessarily absolute to be useful.

[0301] As used herein, the terms "treatment," "treat," and "treating" refer to a course of action (such as administration of a compound or pharmaceutical composition) initiated after the onset of clinical manifestations of a disease state or condition to eliminate or reduce such clinical manifestations of the disease state or condition. Such treatment is not necessarily absolute to be useful.

[0302] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's expertise, but includes the recognition that the patient is ill or will become ill due to a condition treatable by the methods, compounds, or pharmaceutical compositions of the present invention.

[0303] As used herein, the term "in need of prevention" refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is based on a variety of factors within the caregiver's expertise, but includes the recognition that the patient will become ill or is likely to become ill due to a condition that can be prevented by the methods, compounds, or pharmaceutical compositions of the present invention.

[0304] As used herein, the terms "individual," "subject," or "patient" refer to any animal, including birds or mammals, such as mice, Norway rats, cotton rats, gerbils, fish, hamsters, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, goats, horses, or primates, and humans. The terms can specify male or female, or both, or exclude male or female. In one aspect, the patient is an adult. In another aspect, the patient is a non-neonatal human infant. In another aspect, the patient is a human child, child, or adolescent. In some embodiments, the subject was found to be colonized with a pathogenic strain of a microorganism prior to systemic infection, or the subject may have been colonized or infected with a nosocomial (hospital-acquired) strain or a community-acquired strain of a pathogenic microorganism.

[0305] The term "neonate" refers to infants in the first 28 days of life. The term "non-neonate" refers to animals older than 28 days.

[0306] As used herein, the term "effective amount" refers to an amount of an agent that can be used alone or as part of a pharmaceutical composition, which is capable of having any detectable positive effect on any symptom, aspect or characteristic of a disease state or condition. Such an effect need not necessarily be absolute beneficial.

[0307] The term "measurable average cell death" refers to the reciprocal of the percent survival of a microorganism determined within a predetermined time period after the introduction of a state change, compared to the same microorganism under defined conditions without the state change. The percent survival can be determined by any known method for quantifying viable microbial cells. For example, the percent survival can be calculated by counting the cfus / mL of cultured synthetic microbial cells and counting the cfus / mL of uninduced synthetic microbial cells over a predetermined time period, then dividing the induced cfus / mL by the uninduced cfus / mL x 100 = x% percent survival. The measurable average cell death can be determined by the following formula: 100% - x% percent survival = y% measurable average cell death. For example, where the percent survival is 5%, the measurable average cell death is 100% - 5% = 95%. The percent survival can be calculated using any method for counting viable cultured microbial cells, including cfu, OD600, flow cytometry, or other known techniques. Likewise, the induced synthetic strain can be compared to the wild-type target microorganism exposed to the same conditions for the same period of time and a similar calculation method can be used to determine "survival rate", where 100% - survival rate = z% "reduction in viable cells".

[0308] As used herein, the term "comprising" is non-limiting in scope such that additional elements other than the listed elements may be contemplated; the term should in any case be understood as "including, but not limited to."

[0309] The term "animal" refers to the definition of the animal kingdom.

[0310] The term "substantial identity" or "substantially identical" when referring to nucleotides or fragments thereof means that the nucleotide sequence identity is at least about 95% of the nucleotide bases when optimally aligned with another nucleotide (or its complement) by appropriate nucleotide insertions or deletions, and more preferably, at least about 96%, 97%, 98% or 99%, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleotide molecule having substantial identity to a reference nucleotide molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to the polypeptide encoded by the reference nucleotide molecule.

[0311] When referring to a nucleotide or amino acid sequence, the term "derived from" refers to a modified sequence having at least 50% contiguous reference nucleotide or amino acid sequence, respectively, wherein the modified sequence causes the synthetic microorganism to exhibit similar desired properties as a reference sequence of a genetic element such as a promoter, cell death gene, antitoxin gene, virulence blocker, 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 antisense RNA antitoxins; or the ability to prevent or reduce horizontal gene transfer of genetic material from an undesirable microorganism. The term "derived from" with respect to a nucleotide sequence also includes a modified sequence that has been codon-optimized for a specific microorganism to express an amino acid sequence that is substantially similar to the amino acid sequence encoded by the reference nucleotide sequence. When referring to a microorganism, the term "derived from" refers to the target microorganism that has been molecularly modified to obtain the synthetic microorganism.

[0312] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide or protein sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0313] A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (charge or hydrophobicity). Typically, a conservative amino acid substitution does not substantially alter the functional properties of, for example, a toxin or antitoxin protein. Examples of amino acid groups having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0314] Peptide sequences can be compared using FASTA using default or recommended parameters, which is a program in version 6.1 GCG. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the best overlapping region between the query sequence and the search sequence (see, e.g., Pearson, WR, Methods Mol Biol 132: 185-219 (2000), which is incorporated herein by reference). When comparing the sequence of the present disclosure with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al., J Mol Biol 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res 25: 3389-402 (1997).

[0315] Unless otherwise indicated, nucleotide sequences provided herein are presented in the 5'-3' direction.

[0316] All pronouns are intended to be given their broadest meanings. Unless otherwise indicated, the feminine pronoun includes the masculine, the masculine pronoun includes the feminine, the singular pronoun includes the plural, and the plural pronoun includes the singular.

[0317] The term "systemic administration" refers to an administration route that enters the circulatory system so that the entire body is affected. Systemic administration can be performed by enteral administration (absorption through the gastrointestinal tract, such as oral administration) or parenteral administration (eg, injection, infusion or implantation).

[0318] The term "topical administration" refers to application to a localized area of ​​the body or the surface of a body part, regardless of the site of action. Typical sites of topical administration include sites on the skin or mucous membranes. In some embodiments, the topical administration route comprises enteral administration of the drug or composition.

[0319] The term "undesirable microorganisms" refers to microorganisms that may be pathogenic microorganisms, drug-resistant microorganisms, antibiotic-resistant microorganisms, irritation-causing microorganisms, odor-causing microorganisms, and / or may be microorganisms that include undesirable virulence factors.

[0320] The term "synthetic microorganism" refers to an isolated microorganism that has been modified by any means to include at least one element that confers a non-natural property. For example, a synthetic microorganism can be engineered to include molecular modifications including additions, deletions, and / or modifications of genetic material to incorporate the non-natural property. In some embodiments, the synthetic microorganism is not an auxotrophic organism.

[0321] The term "detectable presence" of a microorganism refers to a positive test confirmed in a sample of a microorganism genus, species and / or strain by any method known in the art. Confirmation can be by interpretation of a positive test obtained by a skilled practitioner and / or by repetition of the method.

[0322] As used herein, the term "microbiome" or "microbiomic" or "microbiota" refers to microbial ecosystems. These ecosystems are communities of symbiotic, commensal, and pathogenic microorganisms found in and on all plants and animals.

[0323] As used herein, the term "microorganism" refers to an organism that can be seen only with the aid of a microscope and typically consists of only a single cell. Microorganisms include bacteria, protozoa, and fungi.

[0324] As used herein, the terms "niche" and "niche profile" refer to the ecological array of environmental and nutritional requirements required by a particular microbial species. The definition of a species' niche value defines the location within a particular biome that the species can physically occupy and defines possible microbial competitors.

[0325] As used herein, the term "colonization" refers to the persistent, detectable presence of a microorganism on a body surface (eg, the dermis or a mucosal surface) without causing disease in an individual.

[0326] As used herein, the term "co-colonization" refers to the simultaneous colonization of an ecological niche at a site on a subject by two or more strains or variants of the same microbial species. For example, the term "co-colonization" can refer to the simultaneous and non-transient occupation of the same ecological niche by two or more strains or variants. The term "non-transiently" refers to the positive identification of a strain or variant at a site on a subject at two or more subsequent time points over time in multiple samples obtained from the subject at least two weeks apart.

[0327] As used herein, the term "bacterial replacement" or "non-cocolonization" refers to the principle that only one variant / strain of a species can occupy any given ecological niche within a biome at any given time.

[0328] As used herein, the term "kill switch" or "KS" refers to an intentional molecular modification of a synthetic microorganism that includes a cell death gene operably linked to a regulatory region that includes an inducible promoter, genetic element, or cassette, wherein the expression of the cell death gene induced in the kill switch causes cell death, cessation of growth, or the inability of the microorganism to replicate in response to a specific change in state (e.g., a change in the environmental conditions of the microorganism).

[0329] For example, in a synthetic microorganism comprising a kill switch, the inducible first promoter can be activated by the presence of blood, serum, plasma, and / or heme, wherein upregulation and transcription / expression of an operably bound cell death gene results in cell death of the microorganism or arrested growth of the microorganism, thereby improving the safety of the synthetic microorganism.

[0330] The term "cell death gene" refers to a gene that, when induced, causes the cell to enter a state in which the gene ceases cell proliferation, alters the cell's regulatory mechanisms sufficiently to permanently destroy cell viability, induces senescence, or induces lethal changes in the cell's genetic or proteomic systems. For example, a cell death gene can be a toxin gene that encodes a toxin protein or toxin peptide.

[0331] The term "antitoxin gene" refers to a gene or product encoded by a gene encoding an antitoxin RNA antisense molecule or an antitoxin protein or another antitoxin molecule specific for a cell death gene.

[0332] The term "virulence block" or "v-block" refers to the molecular modification of a microorganism that results in a reduced ability of the organism to accept foreign DNA from other strains or species, effectively resulting in a reduced ability of the organism to acquire exogenous virulence or antibiotic resistance genes.

[0333] As used herein, the term "nanofactory" refers to the molecular modifications of microorganisms that result in the production of products—primary proteins, polypeptides, amino acids, or nucleic acids or secondary products of these modifications that produce a beneficial effect.

[0334] As used herein, the term "toxin protein" or "toxin peptide" refers to a substance produced within a synthetic microorganism in an effective amount that causes a deleterious effect on the microorganism without causing a deleterious effect on the subject in which it is colonized.

[0335] As used herein, the term "molecular modification" or "molecular engineering" refers to the intentional modification of microbial genes using any gene editing method known in the art, including but not limited to recombinant DNA technology, NgAgo, mini-Cas9, CRISPR-Cpf1, CRISPR-C2c2, Target-AID, Lambda Red, integration, recombinase, or the use of phage technology known in the art as described below. DNA can 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, generate cysteine ​​residues, modify, add or delete amino acids, etc. Molecular modifications can include, for example, the use of plasmids, gene insertions, gene knockouts to excise or remove undesirable genes; frameshifting by adding or subtracting base pairs to disrupt the coding frame; exogenous silencing, for example, by using an inducible promoter or constitutive promoter that can be embedded in DNA encoding, for example, RNA antisense antitoxins; producing CRISPR-cas9 or other editing proteins to digest, for example, incoming virulence genes using, for example, guide RNAs or restriction modification / methylation systems linked to inducible promoters or constitutive promoters (e.g., for identifying and destroying incoming virulence genes), thereby increasing resistance to horizontal gene transfer. Molecular modifications (e.g., kill switches, expression clamps, and / or v-blocks) can be permanently incorporated into synthetic microorganisms by inserting the modifications into their genomes.

[0336] The synthetic microorganisms can further include additional molecular modifications (e.g., nanofactories) that can be incorporated directly into the bacterial genome or plasmids to adjust, for example, the duration of action of nanofactory production, and can range from short-term (using non-replicating plasmids for bacterial species) to medium-term (using replicating plasmids for situations without addiction dependencies) to long-term (direct bacterial genome manipulation).

[0337] Molecular modifications can confer non-natural properties that are desired to be permanently incorporated into the host microbiome, can provide enhanced safety or functionality to organisms in the microbiome or the host microbiome as a whole, can provide enhanced safety features, including kill switches(s) or other control functions. In some embodiments, such embedded safety properties can be responsive to changes in the state or condition of the microorganism or the host microbiome as a whole.

[0338] The molecular modification can be incorporated into 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 in no more than one, no more than three, no more than five, no more than 10, no more than 30, or no more than 100 copies.

[0339] As used herein, the term "relapse" refers to the recolonization of the same ecological niche by decolonized microorganisms.

[0340] The term "pharmaceutically acceptable" refers to compounds, carriers, excipients, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0341] The term "pharmaceutically acceptable carrier" refers to a physiologically acceptable carrier for the treated subject while retaining the integrity and desired properties of the synthetic microorganism to be administered therewith. Exemplary pharmaceutically acceptable carriers include physiological saline or phosphate buffered saline. Other physiologically acceptable carriers and formulations thereof are provided herein, or are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A.R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania.

[0342] As used herein, numerical ranges are intended to include every number and subset of numbers contained within that range, whether or not specifically disclosed. Further, these numerical ranges should be interpreted as providing support for claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges of 2 to 8, 3 to 7, 5 to 6, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0343] All patents, patent publications, and peer-reviewed publications cited herein (i.e., "references") are expressly incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the present disclosure and an incorporated reference, the present disclosure controls.

[0344] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. As used herein, when used to refer to a specific recited value, the term "about" means that the numerical value may differ 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 ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0345] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0346] Vector and target microorganisms

[0347] This paper also describes the carrier that comprises polynucleotide molecule, and the target cell transformed with this carrier.Polynucleotide molecule as herein described can be combined with carrier, and described carrier comprises the selection marker and replication origin for the breeding host of being paid close attention to.Target cell is carried out genetic engineering transformation to comprise these carriers, thereby transcribes RNA and expresses polypeptide.Carrier herein comprises the polynucleotide molecule that is operably connected to suitable transcriptional or translational regulatory sequence (as the regulatory sequence for microbial target cell).The example of regulatory sequence comprises transcription promoter, operator or enhancer, mRNA ribosome bind site and the appropriate sequence of control transcription and translation.When the regulatory sequence herein functionally relates to the polynucleotide of for example encoding cell death gene, nucleotide sequence as herein described is operably connected.

[0348] Typical vectors include plasmids, shuttle vectors, baculovirus, inactivated adenovirus, etc. In certain examples described herein, the vector can be a modified pIMAY, pIMAYz, or pKOR integrative plasmid described herein.

[0349] The target microorganism can be selected from any microorganism that can replace specific bad microorganisms durably after going to colonization.The target microorganism can be a wild-type microorganism, which is subsequently engineered to enhance safety by the methods described herein.The target microorganism can be selected from bacteria, fungi or protozoa target microorganisms.The target microorganism can be a strain that can colonize dermis and / or mucosal niches in the subject.The target microorganism can be a wild-type microorganism or a synthetic microorganism that can carry out additional molecular modification.The target microorganism can be selected from the genus selected from the group consisting of: Acinetobacter, Corynebacterium, Propionibacterium (Cutibacterium), Staphylococcus, Streptococcus, Propionibacterium (Propionibacterium) and Pseudomonas. The target microorganism is selected from the group consisting of: Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minimus, Propionibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, Streptococcus agalactiae, Streptococcus mutans, Pseudomonas aeruginosa, Pseudomonas oryzae, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

[0350] The target microorganism may belong to the same genus and species as the undesirable microorganism, but to a different strain. For example, the undesirable microorganism may be an antibiotic-resistant Staphylococcus aureus strain, such as a MRSA strain. The antibiotic-resistant Staphylococcus aureus strain may be a pathogenic strain that may be associated with skin infections, mucosal infections, bacteremia, and / or endocarditis. When the undesirable microorganism is a Staphylococcus aureus strain (e.g., MRSA), the target microorganism may be, for example, a strain with lower pathogenicity, which may be an isolated strain, such as a Staphylococcus aureus target cell (e.g., RN4220 or 502a strain). Alternatively, the target cell may belong to the same strain as the undesirable microorganism. In another example, the undesirable microorganism is an Escherichia coli strain, such as a uropathogenic Escherichia coli type 1 strain or a beta-fibrosis strain, such as a strain involved in urinary tract infections, bacteremia, and / or endocarditis. In another example, the undesirable strain is a Propionibacterium acnes strain, such as a strain involved in acne vulgaris, bacteremia, and / or endocarditis. In another example, the undesirable microorganism is a strain of Streptococcus mutans, eg, a strain involved in Streptococcus mutans endocarditis or dental caries.

[0351] Model antibiotic susceptibility target microorganisms

[0352] The target microorganism can be an antibiotic-sensitive microorganism of the same species as the undesirable microorganism. In one embodiment, the undesirable microorganism is a MRSA strain, and the replacement target microorganism is an antibiotic-sensitive Staphylococcus aureus strain. The antibiotic-sensitive microorganism can be Staphylococcus aureus strain 502a ("502a"). 502a is a coagulase-positive, penicillin-sensitive, non-penicillinase-producing Staphylococcus that is typically lysed by phages 7, 47, 53, 54, and 77. Serological type (b) ci. 502a exhibits an unusual intervertebral antibiotic sensitivity pattern in that the strain is sensitive to low concentrations of most antibiotics except tetracycline; it tolerates 5 μg of tetracycline but is sensitive to 10 μg of tetracycline. In some embodiments, the 502a strain can be a Staphylococcus aureus subspecies Aureus Rosenbach. 27217 TM Commercially available.

[0353] Unfortunately, even target microorganisms that are sensitive to antimicrobial agents may cause systemic infection. Therefore, as provided herein, target microorganisms are molecularly modified to incorporate regulatory sequences (including, for example, an inducible first promoter for expressing a cell death gene, v-block, or nanofactory) to enhance safety and reduce the likelihood of pathogenic infection as described herein.

[0354] The target microorganism and / or synthetic microorganism comprises (i) the ability to persistently colonize an ecological niche in a subject after decolonization of the undesirable microorganism and administration of the target microorganism or synthetic microorganism to the 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.

[0355] Selection of target microorganisms for MRSA

[0356] As described herein, selection of a target microorganism can be performed by decolonizing the target microorganism and replacing it with a hypothetical target microorganism. For example, undesirable methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a disproportionate number of invasive bacterial infections worldwide. The colonization state of Staphylococcus aureus is considered to be a necessary prerequisite for most invasive infections. However, decolonization using standard antiseptic regimens has been studied as a method for reducing MRSA colonization and infection, with mixed results. In an example provided herein, the feasibility and durability of a new decolonization method for the undesirable microorganism MRSA was investigated by intentionally recolonizing the target microorganism using different Staphylococcus aureus strains as candidate target microorganisms, with the aim of extending the duration of effect compared to standard decolonization. Example 1 discloses a study in which 765 healthy volunteers were screened for Staphylococcus aureus colonization. The overall MRSA rate in the screened population was 8.5%. A cohort of 53 MRSA-colonized individuals participated in a controlled study of decolonization / recolonization treatment with BioPlx-01, a Staphylococcus aureus 502a WT strain, versus a control group that underwent standard decolonization alone. The duration of MRSA disappearance from the colonized state and the persistence of intended MSSA recolonization were monitored for 6 months. For the efficacy portion of the MRSA decolonization regimen, the control group (n=15) showed 60% MRSA recurrence at the 4-week time point. The test group (n=34) using the BioPlx-01WT regimen showed 0% MRSA recurrence at the primary endpoint of 8 weeks and showed no evidence of MRSA recurrence until 26 weeks. In contrast, these participants showed a surprising persistence of MSSA colonization, which may indicate that the Staphylococcus aureus 502a BioPlx-01WT strain product persisted for 26 weeks. Additionally, the BioPlx-01WT component of the phosphate-buffered saline composition used in the decolonization / recolonization treatment did not show signs of skin irritation in a separate cohort of 55 participants. Thus, the decolonization / recolonization regimen of the target strain S. aureus 502a BioPlx-01WT provided longer persistence of MRSA strain decolonization than standard decolonization, and no negative skin effects were observed.

[0357] Methods for determining the detectable presence of microorganisms

[0358] Any method known in the art can be used to determine the detectable presence of microbial genera, species, and strains. An overview of the methods can 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, which is incorporated herein by reference.

[0359] The detectable presence of the genus, species and / or strain of bacteria can be determined by phenotypic and / or genotypic methods. Phenotypic methods can include biochemical reactions, serological reactions, sensitivity to antimicrobial agents, sensitivity to bacteriophages, sensitivity to bacteriocins and / or properties of cellular proteins. An example of a biochemical reaction is the detection of extracellular enzymes. For example, Staphylococcus produces many different extracellular enzymes, including DNAase, protease and lipase. Gould, Simon et al., 2009, "The evaluation of novel chromogenic substrates for detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups" FEMS Microbiol Let 297; 10-16. Chromogenic substrates can be used to detect extracellular enzymes. For example, CHROMager TMMRSA chromogenic medium (CHROMagar, Paris, France) is used to isolate and differentiate methicillin-resistant Staphylococcus aureus (MRSA), including low levels of MRSA. Samples are obtained from, for example, the nose, perineum, throat, and rectal samples through a possible enrichment step. If the agar plates have been refrigerated, they should be warmed to room temperature before inoculation. The samples are streaked onto the plates and then incubated at 37°C under aerobic conditions for 18-24 hours. The appearance of the colonies is read, where MRSA colonies appear rose to lavender, methicillin-sensitive Staphylococcus aureus (MSSA) colonies are inhibited, and other bacteria appear blue, colorless, or inhibited colonies. In addition, to clearly identify it as MRSA, the final identification as Staphylococcus aureus is also required. For example, CHROMagar can be used TM A chromogenic medium for Staphylococcus aureus, in which Staphylococcus aureus appears lavender, Staphylococcus saprophyticus appears turquoise blue, and Escherichia coli, Candida albicans, and Enterococcus faecalis are inhibited. For the detection of Group B Streptococcus (GBS) (Streptococcus agalactiae), CHROMagar can be used. TM StrepB plates, in which Streptococcus agalactiae (Group B) is pale purple, Enterococci and Enterococcus faecalis are steel blue, Lactobacillus, Leuconostoc and Lactococcus are light pink, while other microorganisms are blue, colorless or inhibited. To detect various Candida species, a CHROMager TM Candida chromogenic medium. Candida species are implicated in superficial oropharyngeal and urogenital tract infections. Although C. albicans remains the predominant species, other species such as C. tropicalis, C. krusai, or C. glabrata have increased in number due to new antifungal agents effective against C. albicans. Samples of skin, sputum, urine, or vaginal specimens are sampled and streaked directly onto plates, either directly streaked or spread onto plates, and then incubated at 30-37°C under aerobic conditions for 48 hours. The plates are then read for colony appearance: C. albicans is green, C. tropicalis is metallic blue, C. krusai is pink and fuzzy, C. kefyr and C. glabrata are lavender-brown, and other species are white to lavender.

[0360] Genotyping methods for genus and species identification can comprise hybridization, plasmid mapping, plasmid polymorphism analysis, restriction enzyme digestion, 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 method described herein.

[0361] This can be accomplished, for example, by using Galileo, which provides annotations for Gram-negative bacterial DNA sequences.TM Antimicrobial resistance (AMR) detection software (Arc Bio LLC, Menlo Park, CA and Cambridge, MA) was used for identification of microorganisms.

[0362] Microbial typing methods can be selected from the following genotyping methods: multilocus sequence typing (MLST), which relies on PCR amplification of several housekeeping genes to generate an allelic map; PCR-extragenic palindromic repeat elements (rep-PCR), which involves PCR amplification of repetitive sequences in the genome and comparison of banding patterns; AP-PCR, which is a polymerase chain reaction using arbitrary primers; amplified fragment length polymorphism (AFLP), which involves enzymatic restriction digestion of genomic DNA, binding of restriction fragments and selective amplification; polymorphism of DNA restriction fragments (RFLP), which involves genomic DNA digestion or amplicons with restriction enzymes that produce short restriction fragments; randomly amplified polymorphic DNA (RAPD), which uses marker DNA fragments obtained by PCR amplification of random fragments of genomic DNA by single primers of arbitrary nucleotide sequence; multilocus tandem repeat analysis (MLVA), which involves PCR amplification of locus VTRs, visualization of polymorphisms to create an allelic map; or pulsed-field gel electrophoresis (PFGE), which involves comparison of macroscopic restriction fragments. The PFGE electrophoresis method is able to separate fragments greater than 50 kb and up to 10 Mb in length, which is not possible with conventional electrophoresis, which can only separate fragments between 100 bp and 50 kb. This ability of PFGE is attributed to its pleiotropic characteristic, which continuously changes the direction of the electric field, allowing the reorientation of DNA molecules so that, in addition to this event, these molecules can migrate through the agarose gel. The applied electric pulses have different durations, which promote the reorientation of the molecules and the separation of fragments of different sizes. A PFGE device can be a uniform electric field with a fixed profile (CHEF, BioRad). Pulsed-field gel electrophoresis (PFGE) is considered the gold standard technique for MRSA typing because of its high discrimination, but the procedure is complex and time-consuming. The spa gene encodes a cell wall component of S. aureus protein A and exhibits polymorphism. Sequence-based spa typing can be used as a rapid test screen. Narukawa et al. 2009. Tohoku J Exp Med 2009, 218, 207-213.

[0363] Provided herein are methods and compositions for inhibiting and removing undesirable microorganisms (decolonization) and replacing them with new, synthetic microorganisms to permanently displace and replace undesirable microorganisms in a microbial ecosystem with new microorganisms, thereby preventing the recurrence of the original undesirable microorganisms (referred to herein as niche or ecological disturbance).

[0364] In some embodiments, a method for preventing colonization of an undesirable microorganism, preventing infection by an undesirable microorganism, reducing recurrence of colonization by an undesirable microorganism, or reducing recurrence of pathogenic infection by an undesirable microorganism in a subject is provided, the method comprising decolonization and administering a synthetic strain comprising a molecular modification, wherein the molecular modification reduces the ability of the synthetic microorganism to cause disease in the subject relative to a wild-type target strain, wherein the microorganism is selected from the group consisting of: Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis Staphylococcus, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minimus, Propionibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzae, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

[0365] In some embodiments, a method for preventing the spread of pathogenic microorganisms in a subject or preventing the colonization or recurrence of infection of the pathogenic microorganisms is provided, the method comprising inhibiting the pathogenic microorganisms in the subject and replacing the pathogenic microorganisms by topically administering to the subject a composition comprising benign microorganisms of the same species but different strains. The method may further comprise promoting the colonization of the benign microorganisms. In some embodiments, the benign microorganism is a synthetic microorganism having at least one molecular modification, the 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 the dermis or mucosa of a human subject.

[0366] In some embodiments, a method is provided for preventing the spread of methicillin-resistant Staphylococcus aureus (MRSA) in a subject or preventing colonization or recurrence of infection of the methicillin-resistant Staphylococcus aureus in the subject, the method comprising inhibiting the MRSA in the subject and replacing the MRSA by topical administration of a different strain of methicillin-sensitive Staphylococcus aureus (MSSA) of the same species to the subject. The method may further comprise promoting colonization of MSSA in the subject.

[0367] A method for preventing the spread of undesirable microorganisms in a subject or preventing colonization or recurrence of infection by the undesirable microorganisms is provided, the method comprising inhibiting the undesirable microorganisms in the subject and replacing the undesirable microorganisms by administering a second microorganism of the same species but a different strain to the subject. The method may further comprise promoting 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-sensitive microorganism. In some embodiments, the second microorganism is a synthetic microorganism.

[0368] Suppression / decolonization

[0369] Undesirable microorganisms can be inhibited by topical application of a disinfectant, antiseptic or bactericidal composition directly to the skin or mucous membrane of the subject (e.g., by spraying, immersing or coating the affected area, optionally the affected area and adjacent areas, or greater than 25%, 50%, 75% or greater than 90% of the outer surface area or mucosal surface area of ​​the subject with the disinfectant, antiseptic or bactericidal composition. In some embodiments, the affected area or other surface area is air-dried or dried with an air dryer under mild heat, or exposed to ultraviolet light or sunlight before dressing or dressing the subject. In one embodiment, inhibition comprises exposing the affected area (and optionally one or more adjacent or distal areas of the subject) to ultraviolet radiation. In various embodiments, any conventional disinfectant, antiseptic or bactericidal composition can be used. In one embodiment, a disinfectant comprising chlorhexidine or a pharmaceutically acceptable salt thereof is used.

[0370] In some embodiments, the antiseptic, antiseptic, astringent and / or antimicrobial agent is selected from the group consisting of alcohols (ethanol, isopropyl alcohol), aldehydes (glutaraldehyde, formaldehyde, formaldehyde releasers (hydroxymethyl methylthiourea = formaldehyde thiourea, taurolidine, hexamine, dantoin), o-phthalaldehyde), anilides (triclocarban = TCC = 3,4,4'-trichlorocarbamide), biguanides (chlorhexidine, alexidine, polymeric biguanides (polyhexamethylene biguanide with MW>3,000 g / mol, vantocil), bisamidines (propamidine, propamidine isethionate, propamidine dihydrochloride, propamidine dibromide, Bisbromopropamidine isethionate), phenols (dichlorophene thiophene, parachloro-m-xylenol, chloroxylenol, hexachlorophene), bisphenols (triclosan, hexachlorophene), quaternary ammonium compounds (cetrimonium bromide, benzalkonium chloride, cetylpyridinium chloride), silver compounds (silver sulfadiazine, silver nitrate), peroxides (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), plant extracts (melaleuca alternifolia (tea tree oil), jasmine, pine, chinaberry, grape, Terminalia avicennioides Guill & Perr, Phyllanthus discoideusmuel.Muel-Arg, Clove Basil, Golden Edged Red Mulberry, Hypericum pruinatum Boiss. & Bal, Hypericumolimpicum L. and Hypericum sabrum L, Hamamelis virginiana (Witch Hazel), Eucalyptus spp., Rosmarinus officinalis (Rosemary), Thymus vulgaris (Thyme), Origanum vulgare (Oregano), Cymbopogon dahurica (Lemongrass), Cinnamomum spp., Geranium spp., Lavandula angustifolia), and topical antibiotic compounds (bacteriocins; mupirocin, bacitracin, neomycin, polymyxin B, gentamicin).

[0371] It is also possible to suppress undesirable microorganisms by using a photosensitizer instead of, for example, a topical antibiotic or using a photosensitizer in addition to a topical antibiotic. For example, Peng Zhang et al. ("Using photosensitizers instead of antibiotics to kill MRSA", GEN News Highlights, August 20, 2018; 48373) have developed a technology that uses light to activate oxygen, and the reactive oxygen species can inhibit the growth of microorganisms. Photosensitizers such as dye molecules become excited under light. Photosensitizers convert oxygen into reactive oxygen species that kill microorganisms (such as MRSA). In order to concentrate photosensitizers to improve efficacy, Zhang et al. have developed a water-dispersible hybrid photosensitizer, which includes precious metal nanoparticles modified with amphiphilic polymers to capture molecular photosensitizers. Hybrid photosensitizers can be applied to, for example, the skin surface or wound of a subject in the form of a spray, lotion or cream, and then irradiated with red or blue light to reduce the growth of microorganisms.

[0372] The decolonization composition may be in the form of a topical solution, lotion, or ointment comprising a disinfectant, a biocide photosensitizer, or an antiseptic compound and one or more pharmaceutically acceptable carriers or excipients. In one particular example, an aerosol disinfectant spray is used comprising chlorhexidine gluconate (0.4%), glycerol (10%) in the form of a pharmaceutically acceptable carrier, the carrier optionally containing a dye to mark the coverage of the spray. In one embodiment, the inhibiting step comprises applying to one or more affected areas (and optionally, one or more surrounding areas) a spray disinfectant disclosed in U.S. Patent Nos. 4,548,807 and / or 4,716,032, each of which is incorporated herein by reference in its entirety. Disinfectant sprays are commercially available, for example, from Deep Valley Farm, Inc. of Brooklyn, Connecticut. Other disinfectant materials may include chlorhexidine or its salts (such as chlorhexidine gluconate, chlorhexidine acetate and other biguanides), ethanol, SD alcohol, isopropyl alcohol, para-chloro-o-benzylphenol, o-phenol, quaternary ammonium compounds (such as n-alkyl / dimethylethylbenzyl ammonium chloride / n-alkyldimethylbenzyl ammonium chloride, benzalkonium chloride, cetrimide bromide, methylbenzethonium chloride, benzethonium chloride, cetylpyridinium chloride, dofaconium chloride), doxorubicin, peroxides and permanganates (such as hydrogen peroxide solution, potassium permanganate solution, benzoyl peroxide), antibacterial dyes (such as proflavine hemisulfate, triphenylmethane, brilliant green, crystal violet, gentian violet), quinolone derivatives (such as hydroxyquinoline sulfate, hydroxyquinoline potassium sulfate, chloroquinaldol, dequalinium chloride, diiodohydroxyquinoline), Burow's solution (aqueous solution of aluminum acetate), bleach, iodine solution, bromide solution. Various generally recognized as safe (GRAS) materials can be used in the disinfectant or sterilizing composition, including glycerol and glycerides, such as, but not limited to, mono- and diglycerides of edible fat-forming fatty acids, diacetyl tartaric acid esters of mono- and diglycerides, triacetin, acettooleins, acetylstearin, lactopalmitin, lactoolein, and oxystearins.

[0373] Performing the inhibition step (or decolonization) can include applying 1-3 times per day over a period of 1 to 10 days; for example, on the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth day. In other embodiments, the inhibition step can be applied twice, three times, four times, five times, or six times, with each application interval being 6 to 48 hours, 8 to 40 hours, 18 to 36 hours, or about 20 to 28 hours. In a specific embodiment, the inhibition step is applied once per day for one to five days or three to four days in a row. In some embodiments, the inhibition step does not include systemic administration of an antimicrobial. In some embodiments, the inhibition step does not include systemic administration of an antibiotic, antiviral, or antifungal. In other embodiments, the inhibition step includes systemic administration of an antimicrobial. In some embodiments, the inhibition step can include systemic administration of one or more antibiotics, antivirals, or antifungals.

[0374] replace

[0375] Provided are methods for durably replacing undesirable microorganisms with synthetic microorganisms. Synthetic microorganisms have the ability to fill the same ecological niche as pathogenic microorganisms and / or can be of the same species or different strains as the pathogenic microorganisms. By using the same species or different strains (or even the same strain), the environmental niche of the pathogenic microorganism can be filled or durably replaced with a benign synthetic microorganism.

[0376] synthetic microorganisms

[0377] In certain embodiments, undesirable pathogenic microorganisms are replaced by synthetic microorganisms. In certain embodiments, for example, the replacement strain can be a synthetic microorganism, which is a molecularly modified strain of the same species as the undesirable microorganism or pathogenic microorganism, or a strain identical with the undesirable microorganism or pathogenic microorganism.

[0378] In some embodiments, there is provided a synthetic microorganism comprising a "kill switch" that exhibits rapid and complete cell death when exposed to blood or serum, but exhibits normal metabolism and colonization function in other environments. In some embodiments, the synthetic microorganism comprises a stable and immovable kill switch gene. The minimal kill switch (KS) component comprises: a regulatory region (RR) containing an operator, a promoter, and a translation signal that is strongly activated in response to blood or serum exposure; a kill switch gene that expresses a toxic protein or RNA; and transcription termination. Chromosomal integration of KS is preferred. The chromosomal locus can be located in a transcriptionally inactive region, for example, an intergenic region (IR) between a seryl-tRNA synthetase and an amino acid transporter. Insertion herein will not affect the transcription of flanking genes (Lei et al., 2012). Preferably, there is no known sRNA in the IR. Any other inert locus can be selected.

[0379] Synthetic microbes that include a kill switch

[0380] In certain embodiments, 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 can be a molecularly modified microorganism that is antibiotic-sensitive.

[0381] The synthetic microorganism can include 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 the second regulatory region comprising an inducible second promoter. The first promoter and the optional second promoter are activated (induced) by a change in state in the microbial environment compared to the normal physiological condition at at least one site of the subject. For example, the change in state can be selected from one or more changes in: pH, temperature, osmotic pressure, osmotic molarity, 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 the normal physiological condition at at least one site of the subject.

[0382] In a specific embodiment, the pathogenic microorganism is MRSA, and the replacement microorganism is a synthetic microorganism that is a molecularly modified coagulase-positive strain of Staphylococcus aureus. As described herein, the synthetic microorganism can be a molecularly modified Staphylococcus aureus 502a.

[0383] The use of live Staphylococcus aureus as a therapeutic platform raises safety concerns because if the pathogen enters the circulatory system, it can cause severe disease. In one embodiment, the synthetic microorganism is molecularly engineered to include a "kill switch" (KS) and an inducible promoter that induces rapid bacterial death when exposed to whole blood or serum. The kill switch can be composed of DNA encoding the following three main components: i) a "control region" containing a promoter and other regulatory sequences that are 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 consisting of these elements can be integrated into the S. aureus chromosome at an easily modifiable site without adversely affecting the function of the bacteria.

[0384] It is desirable to minimize or avoid basal expression or "leakage" expression in the control zone. For example, if mRNA is produced in large quantities before exposure to blood or serum, the strain may become weak during manufacturing or during skin colonization and may accumulate mutations that bypass or escape KS. To address this issue, candidates are screened to find those that are strongly induced in serum but have very low or undetectable mRNA expression in standard in vitro growth media. Despite this effort, some leaky expression may still be observed, which can be controlled by further including iv) an "expression clamp" to prevent inappropriate toxin production.

[0385] Recombinant methods for synthesizing microorganisms

[0386] Provided is a synthetic microorganism comprising a recombinant nucleotide comprising at least one molecular modification (e.g., a kill switch) comprising (i) a cell death gene operably associated with (ii) a first regulatory region comprising a first inducible promoter, wherein the first inducible promoter is induced by a change in the state of the synthetic microorganism environment. The synthetic microorganism may further comprise at least one 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, wherein the second promoter is active (e.g., constitutively) upon colonization of the dermis or mucosa or in culture medium and is preferably downregulated by a change in the state of the synthetic microorganism environment.

[0387] In some embodiments, a synthetic microorganism is provided that includes 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 of the microbial environment compared to normal physiological conditions at at least one site of the subject, optionally wherein cell death of the synthetic microorganism occurs within 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 360 minutes, or 240 minutes after the change in state. The change in state can be selected from one or more of the following: 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, compositional changes, or concentrations of one or more immune factors, mineral concentrations, and electrolyte concentrations. In some embodiments, the change in state is a higher concentration of blood, serum, or plasma compared to normal physiological conditions at at least one site of the subject.

[0388] Inducible promoter

[0389] Provided is a synthetic microorganism comprising a recombinant nucleotide comprising at least one molecular modification (e.g., a kill switch) comprising (i) a cell death gene operably associated with (ii) a first regulatory region comprising a first inducible promoter that exhibits conditional high-level gene expression of the recombinant nucleotide in response to exposure to blood, serum, or plasma, resulting in an increase in basal productivity of at least two-fold, at least three-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold.

[0390] 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 therebetween), upon exposure to increasing concentrations of blood, serum, plasma, or heme, the inducible first promoter can be activated (induced) such that transcription and / or expression is increased by 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).

[0391] A blood- or serum-inducible first promoter can be selected by the following method: selecting a target microorganism; selecting one or more first promoter candidate genes in the target microorganism; growing the microorganism in a culture medium; adding serum or blood to the culture medium at t=0 minutes; obtaining a sample at t=n minutes (n=1-240 minutes or longer, 15-180 minutes, or 30-120 minutes); performing RNA sequencing on the sample; and comparing RNA sequencing reads of the candidate first promoter in samples obtained at t=0 minutes and t=n minutes after the candidate first promoter gene is exposed to blood or serum. Alternatively, in a culture medium without blood or serum, for the candidate first promoter, a sample obtained t=n minutes after exposure to blood or serum can be compared with a sample at t=n minutes. The candidate first promoter can be selected from promoters that exhibit 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 upregulation by RNA sequencing following growth of target cells at t=n minutes in blood or serum when compared to the candidate promoter in target cells at t=0, or compared to the candidate promoter in target cells at t=n in medium without serum or blood.

[0392] As determined by RNA sequencing, several serum-responsive promoter candidate genes in S. aureus 502a were upregulated by more than 20-fold after 30 minutes of serum exposure 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_00237 (73-fold), and sbnA gene CH52_05141 (88-fold). 00225 (65 times), sbnC gene CH52_05130 (63 times), heme ABC transporter gene CH52_00230 (60 times), isdORF3 gene CH52_00220 (51 times), sbnF gene CH52_05115 (43 times), alanine dehydrogenase gene CH52_11875 (43 times), HarA gene CH52_10455 (43 times), sbnG gene CH52_05110 (42 times), diaminopimelate decarboxylase gene CH52_05105 (32 times), iron ABC transporter gene CH52_05145 (31 times), threonine dehydratase gene CH52_11880 (24 times) and isdA gene CH52_00240 (21 times).

[0393] As determined by RNAseq, several serum-responsive promoter candidate genes in the target microorganism Staphylococcus aureus 502a were found to be upregulated by more than 20-fold after 30-min exposure to serum compared with TSB at 30 min, 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_05135 (53-fold), and isdI gene CH52_00217 (53-fold). 0 (51 times), sbnE gene CH52_05120 (50 times), srtB gene CH52_00215 (47 times), sbnB gene CH52_05135 (44 times), sbnF gene CH52_05115 (44 times), heme ABC transporter 2 gene CH52_00225 (43 times), isdA gene CH52_00240 (40 times), heme ABC transporter gene CH52_00230 (40 times), sbnA gene CH52_05140 (37 times), isd ORF3 gene CH52_00220 (35 times), sbnG gene CH52_05110 (34 times), HarA gene CH52_10455 (28 times), diaminopimelate decarboxylase gene CH52_05105 (25 times), sbnI gene CH52_05100 (22 times) and alanine dehydrogenase gene CH52_11875 (20 times). Compared with being exposed to TSB for 30 minutes, iron ABC transporter gene CH52_05145 was upregulated (19 times) after being exposed to serum for 30 minutes. Compared with being exposed to TSB for 30 minutes, threonine dehydratase gene CH52_11880 was upregulated (14 times) after being exposed to serum for 30 minutes.

[0394] As determined by RNAseq, several serum-responsive promoter candidate genes in the target microorganism Staphylococcus aureus 502a were upregulated by more than 50 times after 90 minutes of exposure to serum compared with t=0, including isdB gene CH52_00245 (2052 times), sbnB gene CH52_05135 (310 times), alanine dehydrogenase gene CH52_11875 (304 times), sbnE gene CH52_05120 (190 times), sbnD gene CH52_05125 (187 times), isdC gene CH52_00235 (173 times), sbnC gene CH52_05130 (162 times), sbnA gene CH52_05140 (143 times), srtB gene CH52_00215 (143 times), sbnG gene CH52_05110 (133 times), sbnF gene CH52_05115 (129 times), heme ABC transporter gene CH52_00230 (125 times), heme ABC transporter 2 gene CH52_00225 (117 times), isdI gene CH52_00210 (115 times), HarA gene CH52_10455 (114 times), diaminopimelate decarboxylase gene CH52_05105 (102 times), sbnI gene CH52_05100 (101 times), isd ORF3 gene CH52_00220 (97-fold), SAMdep 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 minutes of serum exposure compared to t=0.

[0395] As determined by RNA sequencing, several serum-responsive promoter candidate genes in the target microorganism Staphylococcus aureus 502a were found to be upregulated by more than 50-fold after 90 minutes of serum exposure 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), s rtB gene CH52_00215 (170 times), isdC gene CH52_00235 (149 times), sbnC gene CH52_05130 (147 times), diaminopimelate decarboxylase gene CH52_05105 (141 times), heme ABC transporter 2 gene CH52_00225 (135 times), sbnB gene CH52_05135 (130 times), sbnF gene CH52_05115 (127 times), bnG gene CH52_05110 (120 times), 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). Compared to TSB at t = 90 minutes, 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 minutes of serum exposure.

[0396] The blood or serum inducible first promoter gene for the Staphylococcus aureus synthetic microorganism can be selected from or derived from the following genes: isdA (iron-regulated surface determinant protein A), isdB (iron-regulated surface determinant protein B), isdG (heme-degrading monooxygenase), hlgA (γ-hemolysin component A), hlgA1 (γ-hemolysin), hlgA2 (γ-hemolysin), hlgB (γ-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 (ferrochrome transport ATP-binding protein fhuA), fhuB (ferrochrome 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 / The first promoter gene may also be selected from the group consisting of: SAUSA300_0119 (ornithine cyclodeaminase family protein), lrgA (murein hydrolase transporter) and bioA (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) or substantially the same gene.

[0397] The blood or serum inducible first promoter gene for the synthetic microorganism of Staphylococcus aureus can be selected from or derived from the following genes: 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.

[0398] The blood or serum-inducible first promoter gene for the Staphylococcus aureus synthetic microorganism can be derived from or include a nucleotide sequence selected from the group consisting of 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.

[0399] In one embodiment, the synthetic microorganism is a molecularly modified Staphylococcus aureus 502a. Table 2 shows the original sequence (from the start codon to the stop codon) of the first ORF in the operon following each regulatory region used for real-time PCR probe design.

[0400] Table 2. S. aureus strain 502a, original sequence of the first ORF in the operon following each regulatory region used for real-time PCR probe design.

[0401]

[0402]

[0403]

[0404]

[0405] As described below, the synthetic microorganism can include an expression clamp molecule modification that prevents the expression of a cell death gene, wherein the expression clamp includes an antitoxin gene specific for the cell death gene, and the antitoxin gene is operably associated with a second promoter, which is active when colonized in the dermis or mucosa or in TSB medium and is preferably downregulated in blood, serum or plasma. For example, the second promoter can include the clfB gene (clustering factor B), as shown in Table 3.

[0406] Table 3. Other sequences used for real-time PCR probe design

[0407]

[0408]

[0409] Additional oligonucleotides used in the recombinant method to prepare the synthetic microorganism Staphylococcus aureus 502a with molecular modifications are shown in Figure 3A 、 Figure 3B and Figure 3C are shown in Table 4A, and the promoter sequences are shown below.

[0410] cell death genes

[0411] As described herein, the synthetic microorganism can contain a kill switch molecular modification comprising a cell death gene operably associated with an inducible first promoter. The cell death gene can be selected from any gene whose overexpression results in cell death or a significant reduction in growth of the synthetic microorganism within a predetermined time period (preferably, within 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 240 minutes, or 360 minutes of induction).

[0412] In other cases cell death genes, toxin genes, or kill switch genes have been developed.

[0413] WO 2016 / 210373 (Jonathan Kotula et al.), assigned to Synlogic, Inc., discloses a recombinant bacterial cell that is an auxotrophic organism engineered for biosafety, for example, comprising an inhibition-based kill switch gene comprising a toxin-inducible promoter, an antitoxin-inducible promoter, and an arabinose-inducible promoter, and relying on the presence of an inducer (e.g., arabinose) for cell survival.

[0414] US 8,975,061 to Bielinski discloses the regulation of toxin and antitoxin genes for biocontainment to prevent the unintended and / or uncontrolled spread of microorganisms in the environment.

[0415] WO 1999 / 058652 to Gerdes discloses a cytotoxin-based biocontainment and killing system comprising the E. coli relBE locus and similar systems found in Gram-negative and Gram-positive bacteria and archaea.

[0416] US20150050253 to Gabant discloses the controlled growth of microorganisms and the control of the growth / spread of other exogenous recombinant or other microorganisms.

[0417] WO 2017 / 023818 and WO 2016 / 210384 to Falb disclose bacteria engineered to treat disorders involving propionate metabolism.

[0418] US20160333326 to Falb discloses bacteria engineered to treat diseases associated with hyperammonemia.

[0419] US Pat. No. 9,101,597 to Garry discloses immunoprotective primary mesenchymal stem cells and methods, and describes a pro-apoptotic kill switch for use with mesenchymal stem cells.

[0420] US20160206666 to Falb discloses bacteria engineered to treat diseases that benefit from reduced intestinal inflammation and / or a tightened intestinal mucosal barrier.

[0421] In some embodiments, synthetic microorganisms are provided that include one or more of the following: SprA1 (S. aureus), Sma1 (Serratia marcescens), RelF (E. coli), KpnI (Klebsiella pneumoniae), and / or RsaE (S. aureus) toxin genes.

[0422] In this disclosure, a variety of cell death toxin genes were tested in combination with the previously identified optimal control region: i) a 30-amino acid peptide (PepA1) that forms pores in the cell membrane, thereby impairing cell membrane function; ii) a restriction enzyme (Kpn1 or others) that rapidly digests bacterial chromosomes; iii) a small RNA (RsaE) that impairs central biochemical metabolism by inhibiting the translation of two essential genes; iv) a restriction endonuclease (Sma1) derived from Serratia marcescens; and v) a toxin gene (RelF) from Escherichia coli. Some toxins are more potent than others, and an ideal combination of control region induction strength and toxin potency may result in a strain that is healthy at baseline but rapidly dies in the circulatory system.

[0423] The sprA1 (Staphylococcus aureus) toxin gene (encoding the PepA1 peptide) is described in WO 2013 / 050590 by Felden, B and Sayed, N, which discloses the use of PepA1 as an antimicrobial agent but focuses on the use of the peptide as a purified exogenous therapeutic for delivery into the body.

[0424] The relF (E. coli) toxin gene is described in EP 20090168998 to Gerdes, which discloses a kill switch for biocontainment purposes and focuses on the killing of gram-negative bacteria.

[0425] The relF toxin gene is described in US 8852916 to Hyde and Roderick, which discloses a mechanism for triggering cell death (programmed cell death) in microorganisms. A major application is the use of ReIF in environmental biocontainment.

[0426] relF is described in US Pat. No. 8,682,619 to Amodei, which prophetically discloses ReIF to regulate the growth of bacterial populations.

[0427] The synthetic microorganism can be derived from a Staphylococcus aureus target microorganism by inserting a kill switch molecule modification comprising a regulatory region comprising an inducible promoter operably linked to a cell death gene, which can be a toxin gene.

[0428] The cell death gene can be selected from or derived from the sprA1 gene (encoding a peptide toxin that forms pores in the cell membrane), the sprA2 gene, the sprG gene, the sma1 gene (restriction endonuclease), the kpn1 gene (a restriction enzyme that rapidly digests bacterial chromosomes), the rsaE gene (a small RNA that impairs central metabolism by inhibiting the translation of two essential genes), the relF gene (Escherichia coli), the yoeB gene, the mazF gene, the yefM gene, or the lysostaphin toxin gene. The synthetic Staphylococcus aureus can comprise a kill switch molecule modification comprising a cell death gene having a nucleotide sequence selected from SEQ ID NO: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, or 317, or a substantially identical nucleotide sequence.

[0429] In a specific embodiment, a synthetic S. 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 the SprA1 gene.

[0430] Multiple kill switches

[0431] A single KS may be sufficient to impart the desired properties to a synthetic microorganism, but more than one KS may further enhance the strain by: i) greatly reducing the rate of mutations that inactivate the KS; and ii) killing cells through more than one pathway, which may result in more rapid cell death (a function of enhancing the product). The cell death gene may include one or more DNA sequences downstream of the promoter as shown below (7). The base pair numbers correspond to the positions in the pCN51 vector.

[0432] 1. The sprA1 gene sequence between the restriction sites PstI and EcoRI is shown below. The sequence was synthesized using DNA 2.0 (Atum) and ligated into a vector that can be transformed into E. coli cells for replication. The sprA1 gene was restricted at the PstI and EcoRI sites and separated by gel electrophoresis. The complete sequence between the restriction sites, with possible start and end sites, is italicized.

[0433]

[0434] 2. Regulatory RNA sprA1sprA1 under the ClfB promoter AS The DNA sequence of sprA1sprA1 antisense is cloned in reverse order behind the sprA1 gene and contains the antisense regulatory RNA. This DNA sequence produces non-coding antisense regulatory RNA that acts as an antitoxin by regulating the translation of sprA1 in addition to environmental factors in serum and / or blood. AS DNA sequence.

[0435] EcoRI

[0436] GAATTC AGTCAAGGATCCACAACGAAAATAAGTATTTACTTATACACCATCCCCTCACTATT

[0437] TGCGGTAGTGAGGGGATTTTTATTGGTGCGGCTATATGTCACCTATTTTGTATTGCGTCTACT

[0438] TAGCCAATAAGAAAAAAACGCAATGGCACAGCCACTGATGACTGGTGCTATGATGTGAACGAAAATAAGCATCACCTTATACACCTCCTCTCTGCGGTACC CTGCAG SEQ ID NO:123

[0439] PsI

[0440] 3. SmaI DNA sequence between the restriction sites PstI and EcoRI. The sequence was synthesized using DNA 2.0 (Atum) and ligated into a vector that can be transformed into E. coli cells for replication. The SmaI gene was restricted at the PstI and EcoRI sites and separated by gel electrophoresis. The complete sequence between the restriction sites, start site, and end site are in italics.

[0441]

[0442] 4. The rsaE DNA sequence between the restriction sites PstI and EcoRI. The sequence was synthesized by DNA 2.0 (Atum) and ligated into a vector that can be transformed into E. coli cells for replication. The RsaE small regulatory RNA (sRNA) was restricted at the PstI and EcoRI sites and separated by gel electrophoresis. It contains a 5' insertion, and the mature RNA is processed starting from the bold GAAATTAA and ends with the Ts extension after the ACG.

[0443]

[0444] 5. A variant could be used for the RsaE sRNA that would allow for higher expression of the sRNA, which would work more efficiently. This variant would start with GAAATTAA at the 5' end.

[0445]

[0446] 6. relF (E. coli) DNA sequence. This potential killer gene will be tested and cloned.

[0447] ATGAAGCAGC AAAAGGCGAT GTTAATCGCC CTGATCGTCA TCTGTTTAAC CGTCATAGTG

[0448] ACGGCACTGG TAACGAGGAA AGACCTCTGC GAGGTACGAA TCCGAACCGG CCAGACGGAGGTCGCTGTCT TCACAGCTTA CGAACCTGAG GAGTAA SEQ ID NO:127

[0449] 7. KpnI (restriction enzyme from Klebsiella pneumoniae) DNA sequence will be tested and cloned.

[0450]

[0451] Provided herein is a synthetic Staphylococcus aureus 502a 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 the group consisting of SEQ ID NOs: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315, and 317, or substantially identical nucleotide sequences.

[0452] Although kill switches (KS) have been described for other purposes, the KS of the present invention have unique features: i) they respond to exposure to blood or serum; ii) they are endogenously regulated, meaning that no small molecules need to be added or removed to activate or regulate the KS (no auxotrophic organisms); and iii) useful combinations of control regions / toxins, as well as useful combinations of multiple such cassettes, can be used to achieve superior performance.

[0453] Expression clamp

[0454] Provided is a synthetic microorganism comprising a kill switch molecule modification, the kill switch molecule modification comprising (i) a cell death gene operably associated with (ii) a first regulatory region comprising a first inducible promoter that is induced by exposure to blood or serum. In order for the synthetic microorganism to persistently occupy a dermal or mucosal niche in a subject, the kill switch is preferably silenced (not expressed) in the absence of blood or serum.

[0455] To avoid "leaky expression" of the cell death gene, the synthetic microorganism can further include at least one second molecular modification (expression clamp), which includes (iii) an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is operably associated with (iv) a second regulatory region including a second promoter, which is active (e.g., constitutively) upon colonization of the dermis or mucosa or in culture medium (e.g., TSB) and is preferably downregulated by exposure to blood, serum or plasma.

[0456] Ideally, the basal level of gene expression in a KS strain (expression observed when cells are not exposed to blood or serum, for example, expression observed when in TSB (tryptic soy broth)) should be very low, because toxins produced before exposure to serum would prematurely kill or weaken the strain. Even a moderate degree of cell health impairment is unacceptable because: 1) escape mutations in KS will accumulate (KS instability) - a known phenomenon that must be avoided, and / or; 2) the natural efficacy of the strain observed in preliminary trials may be reduced or lost. To understand whether leaky expression is a problem, the absolute level of baseline expression and the fold change in serum are measured and carefully considered when selecting the optimal control region to drive KS.

[0457] Recognizing that leaky expression is not the answer, and the reality is that even the widely used “tightly controlled” transposition promoters (e.g., P CUP1 and P Gal7 and P Tet -on / off variants) also produce measurable mRNA transcription. In some embodiments, an "expression clamp" is used in which the KS cassette contains not only a serum responsive control region driving toxin expression, but also a promoter under the Staphylococcus aureus promoter (P clfB The promoter encodes a "translation-blocking" RNA under the control of S. aureus, a promoter that is normally highly active in S. aureus during skin colonization and is downregulated in the blood.

[0458] The clfB gene promoter (P clfB ) to drive sprA1sprA1 AS RNA expression and the cassette is integrated into the same region as that for expression by a serum-responsive promoter (e.g., P isdB 、P hlgA In this strain, serum / blood exposure activates the toxin (e.g., up to 350-fold or more) but not the antitoxin, whereas growth in TSB or on the skin activates the antitoxin but not the toxin. Representative diagrams of exemplary molecular modifications of the synthetic strain are shown in Figure 1 Shown in.

[0459] An alternative approach to synthetic microorganisms: the KO approach

[0460] An alternative approach to generating a kill switch-like phenotype in a synthetic microorganism is to disrupt ("knock out") one or more genes required for survival in the blood and / or infection of organs, but which are not required (or not essential) for growth in culture or growth on the skin. In some embodiments, one or more of the six genes shown in Table 5, or two or more genes, can be used in the KO approach.

[0461] Table 5: Candidates for gene knockout to generate attenuated strains:

[0462]

[0463] In one embodiment, a synthetic microorganism is provided that includes replacing one or more genes listed in Table 5 with an unmodified or fixed expression KS using allelic exchange. This can further increase the mortality rate of the synthetic microorganism in blood. Alternatively, by having one KO and one KS, the need for integrating two KSs can be reduced. In other embodiments, the synthetic microorganism can include a combination of more than one KO that has a synergistic effect.

[0464] Kill switch adjustment area

[0465] Provided is a synthetic microorganism comprising a kill switch. As described herein, the kill switch comprises a cell death gene operably linked to a regulatory region (RR) comprising an inducible promoter.

[0466] Development of the synthetic microbe involved identifying and characterizing the optimal regulatory region (RR) to drive the kill switch gene; selecting a list of serum-responsive loci; identifying the RRs; validating the serum activation response; and studying basal expression.

[0467] Identification and characterization of optimal regulatory regions to drive kill switch candidates.

[0468] This important stage of KS strain construction involves identifying genes that are strongly upregulated in response to human serum and / or fully heparinized blood. After identifying the genes, the RRs containing the promoter and other upstream elements are identified and annotated. In one approach, any known serum- and blood-responsive gene in S. aureus known in the literature can be used.

[0469] The RR contains upstream regulatory sequences required for activating (or inhibiting) mRNA transcription in response to stimuli. The motif includes an "up" element, a -35 and -10 consensus element, a ribosome binding site ("shine-dalgarno sequence"), and an "operator" sequence, which bind protein factors that strongly influence transcription. In practice, for eubacteria, a DNA sequence region 200 bp upstream of the start codon is generally sufficient to capture all of these elements. However, it is preferred to intentionally identify these sequences to ensure they are included.

[0470] Table 6 shows six S. aureus genes that are strongly upregulated by exposure to human blood or serum.

[0471] Table 6: Identification of candidate RR and serum or blood-inducible promoters to drive kill switch components and thus toxins.

[0472]

[0473]

[0474] The complete genes in each operon and the flanking sequences of strain BioPlx-01 were obtained from Genbank and annotated according to the literature and known motif recognition algorithms. Transcription terminators were identified using a combination of published experiments and prediction tools.

[0475] Other literature evidence for expression of serum-responsive promoters in TSB (or similar media) has been investigated. For example, the spa gene and the 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, Journal of Bacteriology, 186(24): 8356–8362. The sst gene is disclosed in Morrissey et al., 2000. The hlgA gene is disclosed in Flack et al., 2014, Proceedings of the National Academy of Sciences (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.

[0476] Because these data are derived from many different strains and experimental systems, quantitative gene expression measurements using real-time PCR can be used to assess expression across the entire collection in a single standardized assay system. Importantly, basal "leakage" levels of gene expression (expression observed when cells are not exposed to blood or serum, e.g., expression observed in TSB) should be very low, as toxin production prior to serum exposure would prematurely kill / weaken the BioPlx-XX strain (a synthetic microorganism containing a kill switch). Even moderate impairment of cell health is unacceptable because: 1) escape mutations in KS could accumulate (KS instability)—a known phenomenon that must be avoided, and / or 2) the observed natural efficacy of BioPlx-01 could be reduced or lost. Therefore, both the absolute level of baseline expression and the fold change in serum can be measured and carefully considered when selecting the optimal RR to drive KS. Of note, leuA is downregulated in TSB (6-fold) and upregulated in serum (15-fold), making its RR a particularly attractive candidate for controlling KS expression.

[0477] In some embodiments, synthetic microorganisms with a kill switch can further include an "expression clamp," wherein the KS cassette contains not only a serum-responsive RR that drives toxin expression, but also encodes a "translation-blocking" RNA antitoxin under the control of a promoter typically active on the skin or nasal mucosa during colonization. The kill switch can encode an antitoxin capable of suppressing the negative effects of the cell death toxin gene.

[0478] In some embodiments, the synthetic microorganism is a Staphylococcus aureus having a molecular modification comprising a kill switch, wherein the molecular modification further comprises an "expression clamp" wherein the KS cassette contains not only a serum responsive RR driving toxin expression, but also a KS cassette under a Staphylococcus aureus promoter (P clfB The promoters encoding "translation blocking" RNA antitoxins are typically active in the skin during colonization, e.g., as shown in Table 7.

[0479] From the promoters listed in Table 6, combined with real-time PCR data, two or more RRs with the optimal combination of low basal expression and high response to serum / blood can be selected to drive KS expression. As described herein, these RRs can be paired with three different KS genes to generate a panel of KS candidate strains for testing. This panel will include "expression clamp" candidates, as described below.

[0480] When KS strains are localized on the skin or nasal epithelium, the expression clamp blocks toxin expression

[0481] The synthetic microorganism can include an expression clamp. Table 7 lists genes involved in Staphylococcus aureus colonization of the human nares. These genes can be used as a second promoter in the expression clamp, which can further include an antitoxin gene to block leaked toxin expression when the strain colonizes the skin or mucosal environment. The second promoter can be a constitutive promoter, such as a housekeeping gene. Alternatively, the second promoter can be preferentially downregulated in the presence of blood or serum.

[0482] Table 7. Genes involved in Staphylococcus aureus colonization of the human nares

[0483]

[0484] In some embodiments, 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, the second promoter typically being active on the skin or nasal mucosa during colonization, optionally wherein the second promoter is selected from the genes shown in Table 7, the genes being selected from or derived from clumping factor B (clfB), autolysin (sceD; exoprotein D), walKR (virulence regulator), atlA (major autolysin), and oatA (O-acetyltransferase A). Alternatively, the constitutive second promoter can be selected from or derived from a housekeeping gene, such as gyrB, sigB, or rho, optionally wherein the second promoter comprises the nucleotide sequence of each of SEQ ID NOs: 324, 325, or 326, or a substantially identical sequence.

[0485] The second promoter used to express the clamp can be selected from genes identified in the target microorganism that have been identified as being downregulated upon exposure to blood or serum.

[0486] The second promoter used to express the clamp molecule 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, after 30 minutes, after 45 minutes, after 90 minutes, after 120 minutes, after 180 minutes, after 240 minutes, after 360 minutes, or any time point therebetween) such that transcription and / or expression of the cell death gene is reduced 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.

[0487] The second promoter can be selected by the following method: selecting a target microorganism; selecting one or more second promoter candidate genes in the target microorganism; growing the microorganism in culture; adding serum or blood to the culture at t=0 minutes; obtaining a sample at t=n minutes (n=1-240 minutes or longer, 15-180 minutes, or 30-120 minutes); performing RNA sequencing on the sample; and comparing RNA sequencing reads of the candidate first promoter in samples obtained at t=0 minutes and t=n minutes after the candidate first promoter gene is exposed to blood or serum. Alternatively, for the candidate second promoter, the sample obtained t=n minutes after exposure to blood or serum can be compared to the sample at t=n minutes in culture medium without blood or serum. The candidate second promoter can be selected from a promoter that exhibits downregulation by RNA sequencing after growth of target cells in blood or serum at t=n minutes when compared to the candidate promoter in target cells at t=0, or when compared to the candidate promoter in target cells at t=n in culture medium without serum or blood.

[0488] The second promoter can be selected from or derived from the promoter candidate genes identified herein that may be used in the expression clamp of Staphylococcus aureus 502a, which were found to be downregulated by at least 2-fold after 30 minutes of serum exposure compared to t=0 as determined by RNA sequencing, including the phosphoribosylglycinamide formyltransferase gene CH52_00525 (-4.30-fold), phosphoribosylaminoimidazole synthetase gene CH52_00530 (-4.27-fold), amidophosphoribosyltransferase gene CH52_00535 (-4.13-fold), phosphoribosylaminoimidazole synthetase gene CH52_00540 (-4.16-fold), amidophosphoribosyltransferase gene CH52_00541 (-4.20-fold), phosphoribosylaminoimidazole synthetase gene CH52_00543 (-4.26-fold), amidophosphoribosyltransferase gene CH52_00544 (-4.13-fold), phosphoribosylaminoimidazole synthetase gene CH52_00545 (-4.27-fold), amidophosphoribosyltransferase gene CH52_00547 (-4.14-fold), phosphoribosylaminoimidazole synthetase gene CH52_00548 (-4.16-fold), amidophosphoribosyltransferase gene CH52_00549 (-4.28-fold), amidophosphoribosyltransferase gene CH52_00550 (-4.29-fold), amidophosphoribosyltransferase gene CH52_00551 (-4.29-fold), amidophosphoribosyltransferase gene CH52_00552 (-4.29-fold), amidophosphoribosyltransferase gene CH52_00553 (- -formylglycinamide synthase gene CH52_00540 (-4.04 times), phosphoribosylformylglycinamide synthase gene CH52_00545 (-3.49 times), phosphoribosylaminoimidazole-succinamide gene CH52_00555 (-3.34 times), trehalose permease IIC gene CH52_03480 (-3.33 times), DeoR family transcriptional regulator gene CH52_02275 (-2.55 times), phosphofructokinase gene CH52_02270 (-2.46 times) and PTS fructose transporter subunit IIC gene CH52_02265 (-2.04 times).

[0489] The second promoter may be selected from or derived from phosphoribosylglycinamide formyltransferase gene CH52_00525, trehalose permease IIC gene CH52_03480, DeoR family transcriptional regulator gene CH52_02275, phosphofructokinase gene CH52_02270 or PTS fructose transporter subunit IIC gene CH52_02265.

[0490] The second promoter can be P clfB (aggregation factor B) gene; optionally, wherein the second promoter comprises the nucleotide sequence of SEQ ID NO: 7, 117, 118, 129 or 130, or a substantially identical sequence.

[0491] In a specific example, one of the KS constructs (sprA1) was equipped with an expression clamp comprising an antitoxin (sprA1) driven by the clumping factor B (clfB) promoter. ASThis promoter is a candidate for driving clamping 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 product manufacturing and use, respectively. The clustering factor B (clfB) promoter is also downregulated 3-fold in blood (Malachowa 2011), which favors clamping inactivity. Complete inactivity in blood may not be necessary because serum-responsive promoters are so strongly activated in blood.

[0492] The clustering factor B (clfB) promoter is also stably expressed for at least 12 months during nasal colonization in humans and has also been characterized in rodent and in vitro colonization models (Burian 2010).

[0493] In one example of an expression clamp, after confirming strong expression in TSB and lower expression levels in blood or serum (real-time PCR), clfB was selected as a constitutive promoter for use in an expression clamp to determine its properties in the target strain, Staphylococcus aureus 502a. The clfB regulatory region was cloned to drive expression of sprA1 antisense (antitoxin) RNA (see Table 3, first entry), and the cassette was integrated into the same expression shuttle vector used to express the sprA1 toxin gene from a serum-responsive promoter. It is expected that serum / blood exposure would strongly activate the toxin without activating the antitoxin, while TSB or skin / nasal epithelium exposure would activate the antitoxin without activating the toxin. This concept can be applied to the other KS genes listed in Table 3 below. Another possibility for using a clamp is the restriction enzyme KpnI (toxin) approach, where the antitoxin could be an RNA aptamer, which has recently been developed as a potent inhibitor of this enzyme (Mondragon, 2015), as a means of conferring metabolic stability to the aptamer.

[0494] Recognizing that leaky expression is not the answer, and the reality is that even the widely used “tightly controlled” transposition promoters (e.g., P CUP1 and P Gal7 and P Tet -on / off variants) also produce measurable mRNA transcription.

[0495] The expression clamp includes a second promoter operably linked to an antitoxin gene. For example, to be effective, the antitoxin gene must be specific for the cell death toxin gene in the kill switch. Under normal physiological conditions, the expression clamp prevents leaky expression of the cell death gene. Upon exposure to blood or serum, the second promoter operably linked to the antitoxin is downregulated, thereby expressing the cell death gene.

[0496] The synthetic microorganism can contain an expression clamp comprising an antitoxin gene specific for a silenced cell death gene. The antitoxin can be selected from or derived from any antitoxin known in the art that is specific for the cell death gene in the kill switch molecule modification. The antitoxin gene can encode an antisense RNA specific for the cell death gene or an antitoxin protein specific for the cell death gene.

[0497] The antitoxin gene can be a sprA1 antitoxin gene or sprA1 (AS). The sprA1 antitoxin gene can include the nucleotide sequence of TATAATTGAGATAACGAAAATAAGTATTTACTTATACACCAATCCC CTCACTATTTGCGGTAGTGAGGGGATTT (SEQ ID NO: 311) or a sequence substantially identical thereto, or the nucleotide sequence of CCCCTCACTACCGCAAATAGTGAGGGGATTGGTGTA TAAGTAAATACTTATTTTCGTTGT (SEQ ID NO: 273) or a sequence substantially identical thereto.

[0498] The antitoxin gene can be sprA2 antitoxin or sprA2(AS) and can include the nucleotide sequence of TATAATTAATTACATAATAAATTGAACATCTAAATACACCAAATCCCCTCACT ACTGCCATAGTGAGGGGATTTATT (SEQ ID NO:306), or a sequence substantially identical; or TATAATTAATTACATAATAAATTGAACATCTAAATACACCAAATCCCCTCACTACTGCCATAGTGAGGGGATTTATTTAGGTGTTGGTTA (SEQ ID NO:312), or a sequence substantially identical.

[0499] The antitoxin gene can be a sprG antitoxin gene (also known as sprF) and can include the nucleotide sequence of (5'-3')ATATATAGAAAAAGGGCAACATGCGCAAACATGTTACCCTAATGAGCCCGT TAAAAAGACGGTGGCTATTTTAGATTAAAGATTAAATTAATAACCATTTAAC CATCGAAACCAGCCAAAGTTAGCGATGGTTATTTTTT (SEQ ID NO: 307), or a sequence substantially identical. 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.

[0500] The antitoxin gene can be a yefM antitoxin gene that is specific for the silent yoeB toxin gene. The yefM antitoxin gene can include the nucleotide sequence of MIITSPTEARKDFYQLLKNVNNNHEPIYISGNNAE NNAVIIGLEDWKSIQETIYLESTGTMDKVREREKDNSGTTNIDDIDWDNL (SEQ ID NO: 314) or substantially identical nucleotides.

[0501] The antitoxin gene can be a lysostaphin antitoxin gene specific for a lysostaphin toxin gene. The lysostaphin antitoxin can comprise the nucleotide sequence of TATAATTGAGATATGTTCATGTGT TATTTACTTATACACCAATCCCCTCACTATTTGCGGTAGTGAGGGGATTTTT (SEQ ID NO: 319) or a substantially identical nucleotide sequence.

[0502] The antitoxin gene can be a mazE antitoxin gene that targets mazF. The mazE toxin gene can comprise the nucleotide sequence of ATGTTATCTTTTAGTCAAAATAGAAGTCATAGCTTAGAACAATCTTTAAAAGAAGGATATTCACAAATGGCTGATTTAAATCTCTCCCTAGCGAACGAAGCTTTTCCGATAGAGTGTGAAGCATGCGATTGCAACGAAACATATTTATCTTCTAATTC (SEQ ID NO: 322), or a sequence substantially identical thereto.

[0503] Alternatively, antitoxin genes can be designed as follows. In Staphylococcus aureus, there are two main methods for gene silencing. In one type of gene silencing, taking sprA1 as an example, antisense RNA binds to the 5'UTR of the target gene, thereby blocking the translation of the gene and causing premature degradation of the mRNA. Another type of gene silencing is used for genes that do not have a transcription terminator positioned near the stop codon. The translation of these genes can be controlled by antisense RNA that is complementary to the 3' end of the target gene (about 3-10 bases). The antisense RNA will bind to the sequence that covers the last pair of codons encoding the mRNA transcript and produce double-stranded RNA, which is then targeted for RNaseIII degradation.

[0504] Because there are many examples of RNA silencing in Staphylococcus aureus that have been shown to control their target genes, these regions and sequences could be used as a basis for designing toxin / antitoxin cassettes. This approach only requires minor changes to the DNA sequence.

[0505] In the present disclosure, antitoxins to cell death genes can be designed to involve antisense binding to the 5'UTR of the target gene. The toxin gene can be inserted into the PepA1 reading frame and the 12 bp in the endogenous sprA1 antisense are replaced with a sequence homologous to the 12 bp toward the beginning of the heterologous toxin gene.

[0506] In one example, Holin inserted into the sprA1 position can be controlled by an antisense RNA fragment consisting of (12 bp Holin targeting sequence in bold)

[0507] coding.

[0508] In another example, the 187-lysK inserted into the sprA1 position can be controlled by an antisense RNA segment consisting of (12 bp 187-lysK targeting sequence in bold)

[0509] coding.

[0510] Antitoxins specific for cell death genes can involve antisense binding to the 3'UTR of the toxin gene. The method involves inserting a heterologous toxin into the genome of Staphylococcus aureus in the position of sprG 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 transcription rate of sprF RNA is 2.5 times higher than that of the heterologous toxin gene, it will form a duplex with the 3'UTR region of the toxin transcript, thereby initiating RNaseIII degradation and preventing the formation of functional peptides. Since the 3' ends of the two heterologous toxins are manipulated in the same way to overlap with the sprF sequence (adding an AAA codon before the TAA stop codon) - which is also the same as the 3' end of the endogenous sprG, the antitoxin sequences of all three toxin genes will remain the same. For example, the sprG antitoxin gene (sprF) can comprise the nucleotide sequence ATATATAGAAAAAGGGCAACATGCGCAAACATGTTACCCTAATGAGCCCGTTAAAAAGACGGTGGCTATTTTAGATTAAAGATTAAATTAATAACCATTTAACCATCGAAACCAGCCAAAGTTAGCGATGGTTATTTTTT (SEQ ID NO: 310).

[0511] The antitoxin gene may comprise a nucleotide sequence selected from any one of SEQ ID NOs: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319 or 322, or a sequence substantially identical thereto.

[0512] The antitoxin gene may or may not encode an antitoxin peptide. Wherein the synthetic microorganism is derived from a strain of Staphylococcus aureus, the antitoxin peptide can 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 the amino acid sequence of SEQ ID NO: 316), the antitoxin gene can encode a yefM antitoxin protein comprising the amino acid sequence of MIITSPTEARKDFYQLLKNVNNNHEPIYISGNNAENNA VIIGLEDWKSIQETIYLESTGTMDKVREREKDNSGTTNIDDIDWDNL (SEQ ID NO: 314) or a substantially similar sequence. As another example, where the antitoxin gene is a mazF toxin gene (e.g., encoding a toxin peptide comprising the amino acid sequence of SEQ ID NO: 321), the antitoxin gene can be a mazE antitoxin gene (e.g., encoding an antitoxin protein comprising the amino acid sequence of MLSFSQNRSHSLEQSLKEGYSQMADLNLSLANEAFPIECEACDCNETYLSSNS TNE (SEQ ID NO: 323) or a substantially similar sequence).

[0513] Three KS candidate genes of particular interest were selected because they cause cell death in three distinct ways. In some embodiments, synthetic microorganisms include one or more, two or more, or each of sprA1, kpnI, or rsaE to achieve the maximum mortality indicated by earlier data. The mechanism of action of sprA1 is loss of plasma membrane integrity / function through expression of a pore-forming peptide. The mechanism of action of kpnI involves disruption of the S. aureus genome with restriction enzymes. The mechanism of action of rsaE involves impairment of central metabolism, including the TCA cycle and tetrahydrofolate biosynthesis.

[0514] In some embodiments, the synthetic microorganism comprises a 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 can be the sprA1 gene. sprA1 encodes the toxin peptide PepA1, as described in Sayed et al., 2012, Journal of Biological Chemistry (JBC), Vol. 287, No. 52, pp. 43454–43463, December 21, 2012. PepA1 induces cell death by membrane permeabilization. PepA1 has the amino acid sequence: MLIFVHIIAPVISGCAIAFFSYWLSRRNTK SEQ ID NO: 104. Related antimicrobial peptides include MMLIFVHIIAPVISGCAIAFFSYWLSRRNTK (SEQ ID NO: 105), AIAFFSYWLSRRNTK (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, which is incorporated herein by reference. The cell death gene can be the sprA2 gene. The sprA2 gene can encode the toxin MFNLLINIMTSALSGCLVAFFAHWLRTRNNKKGDK (SEQ ID NO: 305). The cell death gene can be a Staphylococcus aureus yoeB gene, which can 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 can be a Staphylococcus mimetic gene, which can encode a metallopeptidase toxin gene having the amino acid sequence of MTHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK (SEQ ID NO: 318), or a substantially similar sequence. The cell death gene can be a mazF toxin gene, which can encode a mazF toxin comprising the amino acid sequence of MIRRGDVYLADLSPVQGSEQGGVRPVVIIQNDTGNKYSPTVIVAAITGRINKA KIPTHVEIEKKKYKLDKDSVILLEQIRTLDKKRLKEKLTYLSDDKMKEVDNAL MISLGLNAVAHQKN (SEQ ID NO: 321) or a substantially similar sequence.

[0515] The cell death gene may encode a toxin peptide or protein comprising the 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, inactive or minimally active in the absence of blood or serum.

[0516] PepA1 is a toxic pore-forming peptide that causes the death of S. aureus by altering basic cell membrane functions. The natural role of PepA1 is unknown, but it is speculated that it provides beneficial benefits to S. aureus populations / cultures by killing cells adversely affected by environmental conditions. By overexpressing this gene, rapid and complete cell death occurs in the presence of serum. Notably, sprA1 mRNA translation is inhibited by an antisense RNA called sprA11 (SprA1 antisense). As described in WO 2013 / 050590, the cis-encoded SprA11 mRNA is expressed in the presence of serum. ASRNA operates in trans in vivo to downregulate expression of the peptide encoded by sprA1, which is incorporated herein by reference. Indeed, antisense RNA could be a convenient protective agent to minimize "leakage" toxicity. It would be driven by a promoter expressed in S. aureus during colonization on human skin and nasal epithelium. The advantages of sprA1 include the expression of a small peptide with known structure and activity.

[0517] In a specific embodiment, a synthetic microorganism is provided that includes a first cell death gene sprA1 operably linked to a first regulatory region comprising a blood and / or serum-inducible first promoter, wherein the first promoter includes a nucleotide sequence of any one of SEQ ID NO: 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. After 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes of incubation in blood or serum, the first promoter may be upregulated by more than 5-fold, more than 10-fold, more than 50-fold, more than 100-fold, more than 300-fold, or more than 600-fold. After 90 minutes of incubation in serum, the first promoter may be upregulated by more than 5-fold and may be selected from fhuA, fhuB, isdI, isdA, srtB, isdG, sbnE, sbnA, sbnC, and isdB. After 90 minutes of incubation in serum, the first promoter may be upregulated by more than 100-fold and may be selected from isdA, srtB, isdG, sbnE, sbnA, sbnC, and isdB.

[0518] The cell death gene may encode an antimicrobial peptide comprising the 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.

[0519] The cell death gene can be selected from any known Staphylococcus toxin gene. The cell death gene can be selected from the 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 substantially identical toxin genes. The toxin gene can include the nucleotide sequence of SEQ ID NO: 274, 275, 284, 286, 288, 290, 304, 315, 317 or 320, or substantially identical nucleotide sequences thereof.

[0520] The cell death gene can be sprA1 encoding 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, the second promoter comprising the nucleotide sequence of clfB, the nucleotide sequence of clfB comprising the nucleotide sequence of SEQ ID NO: 7, 117, 118, 129 or 130, or a substantially identical sequence.

[0521] In some embodiments, the synthetic microorganism includes a restriction enzyme KpnI (Klebsiella pneumoniae) gene. KpnI protects the bacterial genome from invasion by foreign DNA. (For example) high-level expression of the 6-bp recognition restriction enzyme KpnI will effectively cut the Staphylococcus aureus genome. In some embodiments, by (for example) regulating codon usage, the expression vector (as follows) is designed to lack a cutting recognition site. The recognition sequence of 6 bases occurs once every 4096bp, thereby cutting the 2.8MB Staphylococcus aureus genome into about 684 fragments. KpnI has the advantage of rapid activity. In some embodiments, the "leakage" expression problem can be handled by expressing the RNA aptamer as a clamp as described above for sprA1.

[0522] In some embodiments, the synthetic microorganism includes the rsaE gene. The rsaE gene is a small RNA (93 nt) that inhibits two different metabolic pathways by targeting the translation initiation of housekeeping mRNAs encoding the THF biosynthetic pathway and citric acid cycle enzymes; high levels of expression are toxic. Overexpression of RseE leads to growth impairment due to inhibition of essential housekeeping enzymes. This occurs by binding to Opp3A and OppB mRNAs in the ribosome binding site and start codon regions, thereby preventing translation. Both genes encode components of the ABC peptide transport system and affect the supply of basic nitrogen / amino acids in the cell, thereby directly or indirectly impairing central biochemical metabolism. Advantages include severe growth inhibition (10,000 times greater than the empty vector control) and effective versatility, as a single sRNA can impair the expression of multiple essential biochemical pathways. Geissman et al. 2009 and Bohn et al. 2010 "Report on the natural function of RsaE".

[0523] A panel of serum-activated kill switch (KS) plasmid candidates for expression in S. aureus was created, in which a serum-responsive RR was subcloned into a S. aureus shuttle vector; a cell death gene was inserted downstream of the RR and sequenced; the feasibility of a leaky expression repressor "expression clamp" was tested; and candidate strains were completed and evaluated to select one or more lead candidates that exhibited rapid and complete lethality and good baseline viability.

[0524] Chromosomal integration of the best kill switch candidates is important for long-term stable expression. In addition, a comparison of the mortality range and stability of the strains was performed. Up to 3 best kill switch cassettes were inserted individually and in 3 combinations of two strains, for a total of 6 strains. Completion of the insertion may require multi-step cloning in E. coli to construct the construct. For example, E. coli strain DC10B can be used. DC10B is a DCM-only E. coli strain (BEI product number NR-49804). This is a method for producing DNA that can be easily transfected into most Staphylococcus aureus strains. To this end, stable integrants were obtained and the plasmid vector was excised during the counter selection process. The rate and extent of serum-induced cell death were determined and characterized, and the genetic stability of all 6 strains was determined. The preferred KS candidate strains were subjected to non-human functional testing, including a functional test for in vivo strain death; and a functional test for colonization of the skin and intervertebral disc.

[0525] In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided, the method comprising (1) producing the shuttle vector pCN51 on a large scale in E. coli; (2) expressing the shuttle vector pCN51 under the Cd-inducible promoter P cad The cell death gene was cloned into pCN51 in E. coli; (3) the serum-responsive promoter was used to replace the P cad , and optionally insert an expression cassette; (4) verify the construct by sequencing the KS cassette; (5) electroporate into Staphylococcus aureus RN4220 and select transformants on erythromycin plates (this strain is restriction-resistant and produces the correct methylation pattern to survive BioPlx-01); (6) prepare plasmids from RN4220 and perform restriction digests to confirm identification; (7) electroporate the plasmids into BioPlx-01 and select on erythromycin plates; and (8) isolate the strains. Strains generated in this manner can be used for performance testing and serum experiments. The methods are described in further detail herein.

[0526] In some embodiments, a method for performance testing a synthetic S. aureus strain from BioPlx-01 is provided, the method comprising (1) growing in TSB plus antibiotics as a selective pressure for the plasmid; (2) comparing growth to that of WT BioPlx-01, optionally generating a growth curve; (3a) for Cd-promoter variants, washing and transferring the cells to Cd-medium (the control is BioPlx-01 containing an empty vector without a cell death gene) - or - (3b) for KS variants, washing and transferring the cells to serum (the control is WT BioPlx-01 containing an empty vector without a cell death gene); and (4) using OD 630 nm Growth is monitored using a microplate reader, optionally over an extended period to monitor for escape mutants. For whole blood testing, the method is performed only on preferred candidates, and colony forming units (CFU) on TSA are used as a readout of mortality. If colonies form on a strain carrying a kill switch exposed to blood, the plasmid should be sequenced to check for mutations. If an escape mutant is present, the plasmid is shuttled into E. coli and the entire plasmid is sequenced.

[0527] Methods for generating serum-activated kill switch (KS) plasmid candidates expressed in Staphylococcus aureus (SA)

[0528] A method for evaluating cell death induction is provided, comprising synthesizing a recombinant construction of a microorganism comprising cloning a gene into an E. coli-SA shuttle vector and transfecting the vector into BioPlx-01 for evaluation.

[0529] Step 1: Request shuttle carrier PCN51

[0530] Obtaining a commercially available shuttle vector such as pCN51 (available through BEI) is a good option because it contains: i) a cadmium-inducible promoter that can be used in a positive control strain to demonstrate that the toxin is expressed and functional; ii) a universal transcription terminator (TT) for all constructs; and iii) established E. coli and S. aureus replicons. A schematic diagram of the commercially available shuttle vector pCN51 (BEI catalog number NR-46149) is provided in Figure 2 Table 8 shows the genetic elements of the pCN51 shuttle plasmid.

[0531] Table 8. Elements of the pCN51 shuttle vector

[0532]

[0533] The promoter sequences used in development (7) are shown below, with base pair numbering corresponding to positions in the pCN51 vector for the leuA, hlgA, and cadmium promoters.

[0534] 1. The leuA promoter (P) between the restriction sites SphI and PstI was amplified from the genomic BioPlx-01 (502a) DNA. leuA ) sequence (underlined).

[0535]

[0536] 2. The hlgA promoter (P hlgA )sequence.

[0537]

[0539] 3. The cadmium promoter (P cad ) sequence. This promoter was used for control and was part of the original pCN51 vector from BEI Resources (https: / / www.beiresources.org / ).

[0540]

[0541] 4. Used to drive antisense regulatory RNAsprA1 AS clfB promoter (P clfB). It is a forward sequence with EcoRI and BamHI sites. This sequence is reversed to drive sprA1 AS Potentially acting as a clamp, thereby maintaining regulation of the sprAI gene in the absence of blood. The underlined sites represent the EcoRI and BamHI sites, respectively.

[0542] EcoRI

[0543] GAATTC AGGTGATGAAAAATTTAGAACTTCTAAGTTTTGAAAGTAAAAAAATTTGTAATAGTGTAAAAATAGTATATTGATTTTTGCTAGTTAACAGAAAATTTTAAGTTATATAAATAGGAAGAAAACAAATTTTACGTAATTTTTTTCGAAAAGCAATTGATATAATTCTTATTTCATTATACAATTTAGACTAATCTAGAAATTGAAATGGAGTAATATT GGATCC SEQ ID NO:117

[0544] P cloned into pCN51 vector clfB , where EcoRI and BamHI are in reverse order.

[0545] BamHI

[0546] GGATCC AAATATTACTCATTTCAATTTCTAGATTAGTCTAAATTGTATAATGAAATAAGAATTATATCAATTGCTTTTCGAAAAAAATTACGTAAAATTTGTTTTCTTCCTATTTATATAACTTAAAATTTTCTGTTAACTAGCAAAAATCAATATACTATTTTTACACTATTACAAATTTTTTACTTTTCAAAAACTTAGAAGTTCTAAATTTTTCCATCACCT GAATTC

[0547] SEQ ID NO:118

[0548] 5. The sirA promoter (P sirA ). This sequence is taken from 300 base pairs upstream of the sirA start codon and is underlined.

[0549] ttagaaagatttacttttatatatgaagagactggattaaatacttttattgacgtaaaaattcacttttgaaccgttcaatatcttgccgatttttatataacagctacaaataaaatataacagtttgattttacagcctcggtaaat cgtcttgacaaacaaaaattttgtgctatcacaacatttgcaacgtcttaacaagtcatctataaacatttctaaatatttaacattacttatgcgtcatttattgctaaaattattgtattaaaatatacatagaattgatggggatatc ATG SEQ ID NO:119

[0550] 6. The sstA promoter (P sstA ). This sequence is taken from 300 base pairs upstream of the sstA start codon and is underlined.

[0551] acgaaaaattaattaacatcgcattgtttatattactgcaactattacagcattggtagtggtgactgttggaacattaccgttcttaggactagtaataccaaatattatttcaatttatcgaggtgatcatttgaaaaatgctatccctc atacgatgatgttaggtgccatctttgtattattttctgatatagttggcagaattgttgtttatccatatgaaataaatattggtttaacaataggtgtatttggaacaatcattttccttatcttgcttatgaaaggtaggaaaaatt ATG SEQ ID NO:120

[0552] 7.isdA promoter (P isdA ). This sequence was taken from the 300 base pairs upstream of the SstA start site of the complete genome from NCBI 502a, as underlined.

[0553] CTATCTGCGGCATTTGCAGAATTACTGAATGTCGCGATGATGATAATTAACGCTAAAATCGTTGTATTAAAAACTTTTAAAATATTTTTCAAAACATAATCCTCCTTTTTATGATTGCTTTTTAAGTCTTTAGTAAAATCATAAATAATAA TGATTATCATTGTCAATATTATTTTATAATCAATTTATTATTGTTATACGGAAATAGATGTGCTAGTATAATTGATAACCATTATCAATTGCAATGGTTAATCATCTCATATAACAACACATAATTTGTATCCTTAGGAGGAAAACAAC ATG SEQ ID NO: 121.

[0554] In some embodiments, a plasmid, vector or synthetic microorganism is provided that comprises 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 substantially the same 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.

[0555] Step 2: Cloning of the best two serum-responsive RRs into a shuttle vector (E. coli host)

[0556] Cloning of candidate serum-responsive RRs into a shuttle vector (E. coli host) involved: (a) PCR amplification of the best two preferred serum-responsive RRs from BioPlx-01 genomic DNA (gDNA); (b) replacing the cadmium-inducible promoter with these RR fragments in pCN51, thereby creating two new plasmids (RR1 and RR2); and (3) selecting clones in E. coli DH10B (or DH5α) and sequencing the inserts.

[0557] The following KS genes were 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 for cloning together with the relevant restriction enzymes. For synthetic genes, cloning sites will be included in the synthesis and any undesirable sites will be removed during the construction process. For example, the KpnI site will be removed from the kpn1 box to prevent automatic digestion. The KS gene was inserted downstream of the serum-responsive RR in plasmids RR1 and RR2 to generate all the constructs listed below. The KS gene was inserted downstream of the Cd-inducible promoter in pCN51 to create a positive control construct. See additional relevant sequences and primer sequences provided herein that can be used for these steps, for example, Tables 2, 3 and 4. The promoter and insert sequences of all constructs were sequenced to ensure that mutations had not accumulated during the construction process.

[0558] The list of plasmid constructs to be generated is shown below. Except for 2, 4, 8 and 11, all other genes will be transfected into S. aureus.

[0559] 1. Cd-inducible promoter—sprA1

[0560] 2. Cd-inducible promoter—reverse orientation sprA1

[0561] 3. Serum responsiveness RR1-sprA1

[0562] 4. Serum-responsive RR1—reverse orientation sprA1

[0563] 5. Serum responsiveness RR1-sprA1+P clfB -sprA1 AS

[0564] 6. Serum responsiveness RR2-sprA1

[0565] 7. Serum responsiveness RR1-rsaE

[0566] 8. Serum responsiveness RR1-rsaE-reverse orientation

[0567] 9. Serum responsiveness RR2-rsaE

[0568] 10. Serum responsiveness RR1-kpnI

[0569] 11. Serum-responsive RR1-kpnI reverse orientation

[0570] 12. Serum responsiveness RR2-kpnI

[0571] The reverse orientation construct is being generated during this process because if the cell death gene has some basal toxicity even in growth medium, then the forward orientation construct may not be available. Unless the reverse orientation construct can be readily obtained in a side-by-side fashion, such a negative result is not conclusive.

[0572] Step 3: Transfect the plasmid into the intermediate S. aureus RN4220 (to obtain correct DNA methylation model). It is not necessary to transfect the construct in the reverse orientation; however, transfection of the empty pCN51 vector is performed as follows:

[0573] A. Electroporation into RN4220;

[0574] B. Select transformants on plates containing erythromycin; and

[0575] C. Isolate and confirm plasmid ID using restriction digestion.

[0576] Step 4: Transfection into BioPlx-01

[0577] A. Insert the electroporated plasmid from step 3C into competent BioPlx-01;

[0578] B. selecting transformants by erythromycin resistance; and

[0579] C. Isolate and confirm the plasmid ID using restriction digestion; save 9 strains.

[0580] Step 5: Testing for KS expression and extent of mortality and mortality in response to serum and blood exposure

[0581] A. Qualitative testing of killing gene expression by real-time PCR before and after blood / serum exposure. This will: i) confirm strain construction; ii) correlate the onset of toxin production with the onset of mortality, and iii) determine promoter "leakiness" in the context of KS.

[0582] B. Cell death induction curves (extent and kinetics of CFU killing) in serum / blood compared to TSB; and

[0583] C. Simple growth rate comparison of BioPlx-01 containing empty vector and BioPlx-01 carrying KS plasmid.

[0584] Step 6: Measure the rate of KS mutation

[0585] Count colonies grown on serum or blood agar plates and / or in serum-containing liquid media over several hundred generations by serial passage. Determine whether the mutation rate is acceptable. The loss of functional KS in one copy of the KS gene has been reported to be 10 -6, while the KS loss rate of two copies of the same or different KS genes from two different promoters is as low as 10 -10 (Knudsen 1995; Reporting on actual mutation rate assay measurements).

[0586] Step 7: Analysis and Interpretation

[0587] The most preferred KS strain(s) are those that: have an unaffected growth rate (and colonization potential); exhibit rapid and complete death in response to blood and / or serum; and possess stable molecular modifications.

[0588] Step 8: Determine if you need to insert multiple KS boxes

[0589] If the molecular stability of one KS is deemed insufficient, a second and different functional KS from the list of nine candidates (if another functional KS exists) is added to the plasmid and retested for killing and stability. Based on Knudsen 1995 and theoretical calculations, it is expected that KS stability will be significantly improved.

[0590] Chromosomal integration method for optimal kill switch for long-term stable expression

[0591] The optimal serum / blood responsive KS construct or constructs are precisely integrated into the chromosome at a pre-selected location known to tolerate the insertion without significantly altering the cell biology.

[0592] Step 1: Obtain an integration vector for S. aureus.

[0593] After careful consideration of the optimal integration vector, plasmids pKOR1 or pIMAY can be used because they offer the ability to select integration sites, thus avoiding interference with biologically critical regions of the genome that may occur with other methods. Both vectors have a convenient counter-selection (secY) mechanism to allow excision of the plasmid backbone and its marker from the genome after integration of KS. The genetic map of pKOR1 is shown in Figure 5A The pKOR constructs are described in and characterized by Bae et al. 2006 Plasmids 55, pp. 58-63 and briefly described in Table 9. One advantage of pKOR is the ability to clone inserts without being restricted by specific restriction enzymes.

[0594] Table 9. Purpose of elements in the pKOR integrative plasmid

[0595]

[0596] The genetic map of pIMAY is shown in Figure 5B From Monk, IR, et al., Microbiology (mBio) 2012; doi:10.1128 / mBio.00277-11. Figure 12A 、 Figure 12B and Figure 12C The nucleotide sequence of the pIMAY integrative plasmid (SEQ ID NO: 131) is shown. (Accession number JQ62198). The E. coli / Staphylococcus temperature-sensitive plasmid pIMAYz includes a low-copy number E. coli replication origin (p15A), a conjugated transfer origin (oriT), a pBluescript multiple cloning site (MCS), and a highly expressed cat gene (Phelp-cat) from pIMC. The temperature-sensitive replicon (repBCAD) of Gram-positive bacteria and the anhydrotetracycline-inducible antisense secY region (anti-secY) can be amplified from pVE6007 and pKOR1, respectively. The restriction sites listed are unique. Primers (IM151 / 152) bind outside the MCS of pIMAY and are used to screen clones in E. coli (amplifying 283bp, no cloned insert) and determine whether there is a replicating plasmid in Staphylococcus. The advantages of pIMAYz are smaller size, blue-white screening, and lower non-permissive temperature, which are reported to avoid mutations that may occur during the integration process. Therefore, plasmids can be prepared by de novo gene synthesis at a contract sales company.

[0597] Step 2. Check for selectable markers in BioPlx-01.

[0598] BioPlx-01 is sensitive to ampicillin (50 μg / mL and 100 μg / mL), chloramphenicol (10 μg / mL) and erythromycin (Drury 1965). In one embodiment, during the integration process, transformants were selected on chloramphenicol plates using the chloramphenicol (cat+) gene.

[0599] Step 3. Produce the DNA fragment to be integrated.

[0600] A plasmid containing the following elements in tandem: [aTTB2]-[1 kb of sequence upstream of the target region to be replaced]-[KS cassette-AmpR]-[1 kb of sequence downstream of the target region]ATTB1 was prepared in the shuttle vector pTK1, modified according to Bae et al. (2006). The fragment was removed from the plasmid using restriction enzymes and isolated. In practice, the "KS cassette" may contain one or two copies of KS, pending the results of gene stability testing.

[0601] Step 4. Insert one or more KS cassettes into the pKOR plasmid.

[0602] The fragment from step 3 was recombined with plasmid PKOR1 in vitro, and the recombinant mixture was then transfected into DH5α. The construction was verified using restriction mapping, and the desired plasmid construct was obtained in E. coli by standard screening methods.

[0603] Step 5. Obtaining the integrated plasmid containing KS strain in BioPlx-01

[0604] Plasmids were electroporated into RN4220; plasmid DNA was isolated from transfected RN4220 and electroporated into BioPlx-01, and transformants were selected on TSA plates containing chloramphenicol (10 μg / mL).

[0605] Step 6. The plasmid integrates into the chromosome.

[0606] The strain was shifted to a non-permissive temperature (43°C) to promote integration of the plasmid into the target site, and colonies were selected on chloramphenicol plates (10 μg / mL).

[0607] Step 7. Counterselection to expel the plasmid backbone

[0608] Colony isolates from step 6 were grown at a permissive temperature (30°C) to facilitate plasmid excision and plated on 2 μg / mL and 3 μg / mL anhydrotetracycline (aTc) agar to obtain colonies in which the target gene had integrated and the plasmid had been excised and lost (counter-selection step). Any colonies grown on plates containing ≥ 2 μg / mL aTc did not contain the plasmid because the plasmid backbone contained the lethal aTc-suppressive SecY antisense gene.

[0609] Step 8. Confirm the integrated allele sequence

[0610] Genomic DNA was isolated from the KS strain, and the knock-in cassette and flanking structure were confirmed by PCR (and sequencing of the PCR amplicons).

[0611] Step 9. Examination of serum-induced cell death

[0612] Once confirmed, cell mortality assays were performed by first growing cells in TSB, then transferring cells to human blood or serum and determining mortality by CFU plate assay in TSA (10 days).

[0613] Step 10. Verify KS mRNA expression

[0614] Confirm the expression changes of target genes in blood, serum and TSB.

[0615] Step 11. Preparation of frozen libraries

[0616] Animal studies can be conducted using the synthetic microorganism BioPlx-XX produced by these methods. In vivo functional studies can be performed to test the functionality of the kill switch strain. Possible studies include mouse studies to demonstrate differences in pathogenicity between the wt BioPlx-01 strain and the KS strain following intravenous or intraperitoneal injection. In vitro skin colonization tests can also be performed. Additional testing may include performing LD comparisons of BioPlx-01 and BioPlx-XX in mice. 50 As another example, in rats or other animals: perform colonization tests of BioPlx-01 and BioPlx-XX.

[0617] CRISPR-Cas-induced homology-directed repair for direct insertion of optimal kill switch candidates for long-term stable expression

[0618] In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided, the method comprising using CRISPR-Cas induced homology-directed repair to directly insert the best KS candidate for long-term stable expression. In some embodiments, a method for preparing a synthetic Staphylococcus aureus strain from BioPlx-01 is provided, the method comprising (1) obtaining competent cells; (2) designing and testing CRISPR guide RNA (gRNA) sequences and simultaneously testing pCasSA; (3) designing and testing homology-dependent repair templates using fluorescent reporters controlled by constitutive reporters; (4) checking the KS promoter with fluorescent reporters; (5) inserting KS into BioPlx-01 and verifying its incorporation; and (6) testing efficacy and longevity. Optionally, additional KS cassettes are inserted at other locations within the BioPlx-01 genome.

[0619] Figure 10 The layout of the cassette and pCasSA vector for integration via CRISPR is shown. Pcap1A is a constitutive promoter that controls gRNA transcription. Target seq is the target sequence, for example, with 10 possible cleavage targets (1.1, 1.2, etc.). gRNA is a single-stranded guide RNA (provides structural components). Xba1 and Xho1 are two restriction sites used to add homology arms (HA) to the pCasSA vector. HA is a homology arm that can be used as a template for homology-directed repair (200–1000 bp). P rpsL -mCherry is a constitutive promoter that controls the "optimal" mCherry. P rpsL -Cas9 is a constitutive promoter that controls the expression of Cas9 protein.

[0620] Figure 11Vectors used for various purposes in the present disclosure are shown. A is a vector for screening promoters by fluorescence using pCN51. B is a vector for screening promoters by cell death genes. C is a vector for chromosomal integration using CRISPR. D is a vector for chromosomal integration using homologous recombination. Left and right HA: homology arms of the target locus, CRISPR targeting: RNA guide to the locus, mCherry: fluorescent reporter protein, Cas9 protein: CRISPR nuclease, kanR: kanamycin resistance, oriT: transfer origin (for integration) and Sma1: representative killing gene (restriction endonuclease).

[0621] Administration and composition

[0622] In certain embodiments, compositions comprising a synthetic microorganism and an excipient or carrier are provided. The compositions can be administered in an appropriate carrier matrix in any manner suitable for their specific immunogenicity or biological or immunological response characteristics (including oral, intravenous, buccal, nasal, mucosal, dermal or other methods). In one embodiment, compositions for topical application to a subject's dermal and / or mucosal sites are provided. Another specific embodiment relates to compositions of the present disclosure for oral administration.

[0623] In some embodiments, the replacing step comprises topically administering the synthetic strain to the dermis or mucosa at at least one host subject site and optionally adjacent areas within the subject, no more than once, no more than twice, or no more than three times. 6 , at least 10 7 , at least 10 8 , at least 10 9 or at least 10 10 The composition of the synthetic strain of CFU and a pharmaceutically acceptable carrier is topically administered to at least one host site of the subject. The initial replacement step can 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 inhibition step.

[0624] The composition comprising the synthetic microorganism can be applied to the dermis and / or mucosa of at least one site and optionally adjacent sites of the subject at least once (e.g., 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 per month, or no more than once, twice, three times, four times, five times, six times, eight times, ten times per month, or no more than 12 times per month). Subsequent administration of the composition can 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.

[0625] By administering a composition comprising a promoter selected from nutrients, prebiotics, stabilizers, moisturizers and / or probiotic species, colonization of a synthetic microorganism in a subject can be promoted. The promoter can be administered to the subject in the form of a separate promoter composition or can be added to the microbial composition.

[0626] In some embodiments, the promoter can be a nutrient, for example, selected from sodium chloride, lithium chloride, sodium glycerophosphate, phenylethyl alcohol, 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, oligosaccharides derived from pectin of agricultural by-products, oligofructose (e.g., inulin-like prebiotics), oligogalactose (e.g., raffinose), succinic acid, lactic acid and manno-oligosaccharides.

[0627] In some embodiments, the promoter can be a probiotic. The probiotic can be any probiotic known in the art. Probiotics are live microorganisms that provide health benefits to the host. In the methods provided herein, probiotics can be topically applied to the dermis and mucosal microbiome, and / or probiotics can be orally administered to provide health benefits to the dermis and mucosa to the subject. Several strains of Lactobacillus have been shown to have systemic anti-inflammatory effects. Studies have shown that certain Lactobacillus reuteri strains induce systemic anti-inflammatory cytokines, such as interleukin (IL) -10. Soluble factors from Lactobacillus reuteri inhibit the production of pro-inflammatory cytokines. In a skin model, it has been shown that Lactobacillus paracasei strains can inhibit neutrophil inflammation (Kober et al., 2015, "International Journal of Women's Dermatol (Int J Women's Dermatol)" 1 (2015) 85-89). In human dermal fibroblasts and a hairless mouse model, Lactobacillus plantarum has been shown to inhibit UVB-induced expression of matrix metalloproteinase 1 (MMP-1), thereby preserving procollagen expression in human fibroblasts. Oral administration of Lactobacillus plantarum to hairless mouse histological samples showed that Lactobacillus plantarum inhibited the expression of MMP-13, MMP-2, and MMP-9 in dermal tissue.

[0628] Clinically, topical application of probiotics has also been shown to alter the barrier function of the skin, which in turn increases the skin's antimicrobial properties. Topical application of Streptococcus thermophilus has been shown to alter the barrier function of the skin, which in turn increases the skin's antimicrobial properties. Topical application of Streptococcus thermophilus has been shown to increase ceramide production both in vitro and in vivo. Ceramides absorb moisture from the skin, and certain ceramide sphingolipids, such as phytosphingosine (PS), exhibit direct antimicrobial activity against Propionibacterium acnes. Kober et al., 2015, International Journal of Women's Dermatology 1 (2015) 85-89.

[0629] Two clinical trials of topical probiotics have evaluated their effects on acne. An 8-week application of an Enterococcus faecalis wash to the face resulted in a 50% reduction in inflamed lesions compared with a placebo. In a clinical study of a topical Lactobacillus plantarum extract, the number, size, and associated erythema of acne were reduced. (Kober et al., 2015, International Journal of Women's Dermatology 1 (2015) 85-89).

[0630] Clinical trials of topical probiotics have evaluated their effects on the mucosal system. In one study, Streptococcus salivarius was administered via nasal spray to prevent acute otitis media (AOM). If the nasopharynx is successfully colonized, it has a significant effect on reducing AOM. Marchisio et al. (2015). European Journal of Clinical Microbiology and Infectious Diseases (Eur. J. Clin. Microbiol. Infect. Dis.) 34, 2377-2383. In another trial, spraying Streptococcus sanguinis (S. sanguinis) and Lactobacillus rhamnosus on children with secretory otitis media reduced middle ear effusion. Skovbjerg et al. (2008). Archives of Child Microbiology (Arch. Dis. Child.) 94, 92-98.

[0631] Probiotics can be topical probiotics or oral probiotics. Probiotics can be, for example, genus and species different from undesirable microorganisms, or have the same genus but different species from undesirable microorganisms. Probiotic species can be genus and species different from target microorganisms. Probiotics can be modified to include kill switch molecules or can not be modified. Probiotics can be selected from lactobacillus, bifidobacterium, streptococcus or enterococcus. Probiotics can 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 thermophilus, streptococcus salivarius or enterococcus faecalis.

[0632] The promoter may include a protein stabilizer, such as those disclosed in U.S. Pat. No. 5,525,336, incorporated by reference, which is included in the composition. Non-limiting examples include glycerol, trehalose, ethylenediaminetetraacetic acid, cysteine, cyclodextrins (such as α-, β-, or γ-cyclodextrin) or derivatives thereof (such as 2-hydroxypropyl β-cyclodextrin), and protease inhibitors (such as leupeptin, pepstatin, antiproteases, and cystatins).

[0633] The accelerator may include a humectant. Non-limiting examples of humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, and dibutyl phthalate.

[0634] Composition

[0635] Compositions comprising a synthetic microorganism and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetener, flavoring, buffer, thickener, wetting agent, or absorbent are provided.

[0636] The pharmaceutically acceptable diluent or carrier for formulating the composition is selected from the group consisting of water, saline, phosphate buffered saline or a solvent. The solvent can be selected from, for example, ethanol, toluene, isopropanol, n-butanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide and tetrahydrofuran. The carrier or diluent may further include one or more surfactants, such as: i) anionic surfactants, such as metal salts or alkanolamine salts of fatty acids, for example sodium laurate and triethanolamine oleate; alkylphenyl sulfones, for example triethanolamine dodecylbenzenesulfonate; alkyl sulfates, for example sodium lauryl sulfate; alkyl ether sulfates, for example sodium lauryl ether sulfate (2 to 8 EO); sulfosuccinates, for example sodium dioctylsulfosuccinate; monoglyceride sulfates, for example sodium monostearate glyceryl monosulfate; isothiosulfates, for example sodium isothiosulfate; methyltaurates, for example Igepon T; acyl sarcosinates, for example sodium myristyl sarcosinate; acyl peptides, for example Maypons and Lamepons; acyl lactylates, polyalkoxylated ether glycolates, for example trideceth-7 carboxylic acid; phosphates, for example sodium dilauryl phosphate; cationic surfactants, such as amine salts, for example sabamin hydrochloride; quaternary ammonium salts, for example Quaternium 5, Quaternium 31 and Quaternium 18; amphoteric surfactants such as imidazole compounds, such as Miranol; N-alkyl amino acids, such as sodium cocamidopropionate and asparagine derivatives; betaines, such as cocamidopropyl betaine; nonionic surfactants, such as fatty acid alkanolamides, such as oleic ethanolamide; esters or polyols, such as Span; polyglycerol esters, such as polyglycerol esters esterified with fatty acids and one or more OH groups; polyalkoxylated derivatives, such as polyoxyethylene stearate; ethers, such as polyoxyethylene lauryl ether; ester ethers, such as Tween; amine oxides, such as coconut and dodecyl dimethylamine oxide. In some embodiments, more than one surfactant or solvent is included.

[0637] The composition can include a buffer component that helps stabilize the pH. In some embodiments, the pH is between 4.5 and 8.5. For example, the pH value can be about 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 therebetween. In some embodiments, the pH is 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), (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, trimethylglycine, triethanolamine (TEA) and trishydroxymethylaminomethane (tris). Excipients may include lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, phosphate buffer or any other similar component (presented alone or in suitable combination thereof).

[0638] The microbial composition may comprise: a binder such as gum tragacanth, gum arabic, methylcellulose, gelatin, polyvinyl pyrrolidone, starch or any other similar component (presented alone or in suitable combination); an excipient selected from the group consisting of: agar, calcium carbonate, sodium carbonate, silicate, alginic acid, corn starch, potato tapioca starch, gelatin or any other similar component (presented alone or in suitable combination); a lubricant selected from the group consisting of: magnesium stearate, calcium stearate, talc, solid polyethylene glycol, sodium lauryl sulfate or any other similar component (present alone); a glidant selected from the group consisting of: colloidal silicon dioxide or any other similar component (presented alone or in suitable combination); a glidant selected from the group consisting of: colloidal silicon dioxide or any other similar component (presented alone or in suitable combination); a glidant selected from the group consisting of: Stabilizers such as mannitol, sucrose, trehalose, glycine, arginine, dextran or a combination thereof; flavoring agents or flavoring agents selected from the group consisting of: mint, methyl salicylate, orange flavoring, vanilla flavoring or any other pharmaceutically acceptable flavoring agents or flavoring agents (presented 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 agents (presented alone or in a suitable combination thereof); absorbents selected from the group consisting of: kaolin, bentonite or any other pharmaceutically acceptable absorbents (presented alone or in a suitable combination thereof); retardants selected from the group consisting of: wax, paraffin or any other pharmaceutically acceptable retardants (presented alone or in a suitable combination thereof).

[0639] The microbial compositi...

Claims

1. A synthetic microorganism comprising a recombinant nucleotide comprising at least one kill switch molecule modification, the kill switch molecule modification comprising a first cell death gene, wherein the first cell death gene is A first regulatory region comprising an inducible first promoter is operably associated with In response to exposure to blood, serum or plasma, the first inducible promoter exhibits conditional high level gene expression of the recombinant nucleotide with at least a three-fold increase in basal productivity.

2. The synthetic microorganism according to claim 1, wherein the synthetic microorganism further comprises At least one second molecular modification (expression clamp), said second molecular modification comprising an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is A second regulatory region comprising a second promoter is operably associated, the second promoter being active (constitutive) upon dermal or mucosal colonization or in complete culture medium but not induced, induced less than 1.5-fold, or repressed after exposure to blood, serum, or plasma for at least 30 minutes.

3. The method of claim 1 or 2, wherein the synthetic microorganism is derived from a target microorganism of the same genus and species as the 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 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or at least 360 minutes after exposure to blood, serum, or plasma.

5. The synthetic microorganism of claim 1 or 2, wherein in the absence of blood, serum or plasma, the first promoter is not induced, is induced less than 1.5-fold or is repressed.

6. The synthetic microorganism of claim 2, wherein the second regulatory region comprises a second promoter that is active upon dermal or mucosal colonization or in TSB medium, but is inhibited at least 2-fold upon exposure to blood, serum, or plasma after a time period selected from 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.

7. The synthetic microorganism of any one of claims 1 to 6, wherein measurable average cell death of the synthetic microorganism occurs within at least a predetermined period of time after induction of the first promoter.

8. The synthetic microorganism of claim 7, wherein the measurable average cell death occurs within at least a predetermined time period selected from the group consisting of at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes after exposure to blood, serum, or plasma.

9. A synthetic microorganism according to claim 8, wherein after the preset time period, the measurable average cell death is at least 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.

10. The synthetic microorganism of any one of claims 1 to 9, wherein the kill switch molecule modification reduces or prevents infectious growth of the synthetic microorganism under a systemic condition in the subject.

11. The synthetic microorganism of claim 1 or 2, wherein the at least one molecular modification is integrated into the chromosome of the synthetic microorganism.

12. The synthetic microorganism of claim 3, wherein the target microorganism is sensitive to at least one antimicrobial agent.

13. The synthetic microorganism of claim 12, wherein the target microorganism is selected from a bacterial, fungal or protozoan target microorganism.

14. The method of claim 13, wherein the target microorganism is a bacterial species capable of colonizing the dermal and / or mucosal niches and is a member of a genus selected from the group consisting of: Acinetobacter, Corynebacterium, Cutibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas.

15. The synthetic microorganism of claim 14, wherein the synthetic microorganism is derived from a strain of Staphylococcus aureus.

16. The synthetic microorganism of claim 15, wherein the cell death gene is selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM, or a lysostaphin toxin gene.

17. The synthetic microorganism of claim 16, wherein the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315 and 317, or substantially identical nucleotide sequences.

18. The synthetic microorganism of claim 16 or 17, 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 (γ-hemolysin component A), hlgA1 (γ-hemolysin), hlgA2 (γ-hemolysin), hlgB (γ-hemolysin component B), hrtAB (heme-regulated transporter), sbnC (luc C family siderophore biosynthesis 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 (ferrochrome transport ATP-binding protein fhuA), fhuB (ferrochrome 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 genes, 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 transporter), srtB (sortase B), sbnA (possible 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 (staphylococcal protein A).

19. The synthetic microorganism of claim 18, 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 nucleotide sequence substantially identical thereto.

20. The synthetic microorganism of any one of claims 15 to 19, wherein the antitoxin gene encodes an antisense RNA sequence capable of hybridizing to at least a portion of the first cell death gene.

21. A synthetic microorganism according to claim 20, wherein the antitoxin gene is selected from the group consisting of: 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.

22. A synthetic microorganism according to claim 21, wherein the antitoxin gene comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319 or 322, or a substantially identical nucleotide sequence.

23. A synthetic microorganism according to claim 21 or 22, wherein the second promoter comprises or is derived from a gene selected from the group consisting of: clfB (clustering factor B), sceD (autolysin, extracellular protein D), walKR (virulence regulator), atlA (major autolysin), oatA (O-acetyltransferase A); a phosphoribosylglycinamide formyltransferase gene, a phosphoribosylaminoimidazole synthetase gene, an amidophosphoribosyltransferase gene, a phosphoribosylformylglycinamide synthase gene, a phosphoribosylaminoimidazole synthetase gene, a ... Formylglycinamide synthase gene, phosphoribosylaminoimidazole-succinamide gene, trehalose permease IIC gene, DeoR family transcriptional regulator genes, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkyl hydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein degradation chaperone gene, alkyl hydroperoxidase gene, phosphofructokinase gene, gyrB, sigB and rho.

24. The synthetic microorganism of claim 23, wherein the second promoter is P clfB (aggregation factor B), and includes the nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a nucleotide sequence substantially identical thereto.

25. The synthetic microorganism of any one of claims 1 to 24, further comprising a molecular modification selected from the group consisting of: a virulence blocking molecule modification and a nanofactory molecule modification.

26. The synthetic microorganism of claim 25, wherein the virulence blocking molecule modification prevents horizontal gene transfer of genetic material from the undesirable microorganism.

27. The synthetic microorganism of claim 25, wherein the nanofactory molecular modification comprises insertion of a gene, knockout of a gene, or genetic modification of a gene encoding a product selected from the group consisting of an enzyme, an amino acid, a metabolic intermediate, and a small molecule.

28. A composition comprising an effective amount of a synthetic microorganism according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier, diluent, emollient, binder, excipient, lubricant, sweetener, flavoring agent, wetting agent, preservative, buffer or absorbent or a combination thereof.

29. The pharmaceutical composition of claim 28, further comprising nutrients, prebiotics, commensals and / or probiotic species.

30. A single dosage unit comprising the composition according to claim 28 or 29.

31. The single dose unit according to claim 30, comprising at least 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 a synthetic strain or and a pharmaceutically acceptable carrier.

32. The dosage unit of claim 31 formulated for topical administration.

33. A synthetic microorganism according to any one of claims 1 to 27 or a composition according to claim 28 or 29 for use in the manufacture of a medicament for eliminating and preventing the recurrence of undesirable microorganisms in a subject.

34. A method for eliminating and preventing the recurrence of undesirable microorganisms in a subject hosting a microbiome, the method comprising: a. decolonizing the host microbiome; as well as b. durably replacing the undesirable microorganism by administering to the subject a synthetic microorganism comprising at least one element that imparts a non-natural property, wherein the synthetic microorganism is capable of durably integrating into the host microbiome and occupying the same ecological niche in the host microbiome as the undesirable microorganism.

35. The method of claim 34, wherein decolonization is performed on at least one site of the subject, thereby substantially reducing or eliminating the detectable presence of the undesirable microorganism from the at least one site.

36. The method of claim 35, wherein the detectable presence of the undesirable microorganism is determined by a method comprising a phenotypic method and / or a genotypic method, optionally wherein the phenotypic method is selected from the group consisting of: a biochemical reaction, a serological reaction, sensitivity to antimicrobial agents, sensitivity to bacteriophages, sensitivity to bacteriocins and / or properties of cellular proteins, and optionally wherein the genotyping method is selected from the group consisting of hybridization, plasmid mapping, plasmid polymorphism analysis, restriction enzyme digestion, reaction and separation by pulsed field gel electrophoresis (PFGE), ribotyping, polymerase chain reaction (PCR) and its variants, ligase chain reaction (LCR) and transcription-based amplification system (TAS).

37. The method of claim 35, wherein the ecological niche is a dermal or mucosal environment that allows stable colonization of the undesirable microorganisms at the at least one site.

38. The method of claim 37, wherein the ability to durably integrate into the host microbiome is determined by the 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 after the administering step.

39. The method of claim 38, 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 after the administering step.

40. The method of claim 39, wherein after the step of administering, the ability to occupy the same ecological niche is determined by the absence of co-colonization of the undesirable microorganism with the synthetic microorganism at the at least one site, Optionally, 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 after the administering step.

41. The method of claim 34, wherein the at least one element conferring a non-natural property is durably incorporated into the synthetic microorganism.

42. The method of claim 41, wherein the at least one non-natural property-conferring element is durably incorporated into the host microbiome via the synthetic microorganism.

43. The method of claim 42, wherein the at least one non-natural property-conferring element is selected from the group consisting of: a kill switch molecule modification, a virulence blocking molecule modification, a metabolic modification, and a nanofactory molecule modification.

44. The method of claim 43, wherein the molecular modification is integrated into the chromosome of the synthetic microorganism.

45. The method of claim 43, wherein the synthetic microorganism includes a virulence blocking molecule modification that prevents horizontal gene transfer of genetic material from the undesirable microorganism.

46. ​​The method of claim 43, wherein the synthetic microorganism comprises a kill switch molecule modification that reduces or prevents infectious growth of the synthetic microorganism under a systemic condition in the subject.

47. The method of claim 43, wherein the synthetic microorganism is derived from a target microorganism of the same genus and species as the undesirable microorganism.

48. The method of claim 43, wherein the synthetic microorganism is derived from a target microorganism having the ability to biointegrate with the decolonized host microbiome.

49. The method of claim 43, wherein the synthetic microorganism is derived from a target microorganism isolated from the host microbiome.

50. The method of claim 48 or 49, wherein the target microorganism is sensitive to at least one antimicrobial agent.

51. The method of claim 50, wherein the target microorganism is selected from a bacterial, fungal or protozoan target microorganism.

52. The method of claim 51, wherein the target microorganism is a bacterial species capable of colonizing the dermal and / or mucosal niches and is a member of a genus selected from the group consisting of: Acinetobacter, Corynebacterium, Propionibacterium, Staphylococcus, Streptococcus, Propionibacterium, and Pseudomonas.

53. The method of claim 52, 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, acnes), Propionibacterium granulosum, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosis, Steptococcus constellatus, Streptococcus intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas stutzeri, Pseudomonas putida putida and Pseudomonas fluorescens, optionally wherein the target strain is Staphylococcus aureus 502a strain or RN4220 strain.

54. The method of claim 46, wherein the synthetic microbial kill switch molecule modification comprises a first cell death gene operably linked to a first regulatory region comprising a first inducible promoter.

55. The method of claim 54, wherein the first promoter is activated (induced) by a change in conditions in the microbial environment compared to normal physiological (niche) conditions at at least one site of the subject.

56. The method of claim 55, wherein the measurable average cell death of the synthetic microorganism occurs within at least a predetermined period of time after induction of the first promoter.

57. The method of claim 56, wherein after the state change, the measurable average cell death occurs within at least a predetermined time period selected from the group consisting of: at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes.

58. The method of claim 57, wherein after the preset time period, 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% reduction in cfu count.

59. The method of claim 55, wherein the change in state is selected from one or more of: pH, temperature, osmotic pressure, osmolality, oxygen level, nutrient concentration, blood concentration, plasma concentration, serum concentration, metal concentration, chelated metal concentration, changes in the composition or concentration of one or more immune factors, mineral concentration, and electrolyte concentration.

60. The method of claim 59, wherein the altered state is a higher concentration and / or altered composition of blood, serum or plasma compared to normal physiological (niche) conditions at at least one site of the subject.

61. The method of claim 60, wherein the first promoter is a blood, serum, plasma and / or heme responsive promoter.

62. The method of any one of claims 55 to 61, wherein after the state change, 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 time period selected from 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.

63. The method of claim 62, wherein in the absence of the change in state, the first promoter is not induced, is induced less than 1.5-fold, or is repressed.

64. The method of claim 63, wherein in the presence of serum or blood, the first promoter is induced at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or at least 6-fold over a period of time.

65. The method of claim 64, wherein at the at least one site, under the normal physiological (niche) conditions, the first promoter is not induced, induced less than 1.5-fold, or repressed.

66. The method of claim 64, wherein in the absence of blood, serum, plasma or heme, the first promoter is not induced, is induced less than 1.5-fold or is repressed.

67. The method of any one of claims 54 to 66, wherein the synthetic microorganism is derived from a target microorganism that is a strain of Staphylococcus aureus, and wherein the first promoter 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 biosynthesis 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 (ferrochrome transport ATP-binding protein fhuA), fhuB (ferrochrome 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 genes, 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 transporter), srtB (sortase B), sbnA (possible 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 (staphylococcal protein A).

68. The method of claim 67, wherein the first promoter is derived from or 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 nucleotide sequence substantially identical thereto.

69. The method of any one of claims 54 to 68, wherein the undesirable microorganism is a strain of Staphylococcus aureus, and wherein the detectable presence is measured by a method comprising obtaining a sample from at least one site of the subject, contacting a chromogenic agar with the sample, incubating the contacted agar, and counting positive cfu of the bacterial species after a predetermined period of time.

70. The method of any one of claims 54 to 69, wherein the cell death gene is selected from a toxin gene selected from the group consisting of sprA1, sprA2, kpn1, sma1, sprG, relF, rsaE, yoeB, mazF, yefM and a lysostaphin toxin gene.

71. The method of claim 70, wherein the cell death gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 122, 124, 125, 126, 127, 128, 274, 275, 284, 286, 288, 290, 315 and 317, or substantially identical nucleotide sequences.

72. The method of any one of claims 54 to 71, wherein the synthetic microorganism further comprises an expression clamp molecule modification comprising an antitoxin gene specific for the first cell death gene, wherein the antitoxin gene is operably linked to a second regulatory region comprising a second promoter, the second promoter being active upon dermal or mucosal colonization or in TSB medium, but being inhibited by at least 2-fold upon exposure to blood, serum or plasma after a time period selected from 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.

73. The method of claim 72, wherein the antitoxin gene encodes an antisense RNA sequence capable of hybridizing to at least a portion of the first cell death gene.

74. The method of claim 73, wherein the antitoxin genes are selected from the group consisting of: 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.

75. The method of claim 74, wherein the antitoxin gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 273, 306, 307, 308, 309, 310, 311, 312, 314, 319 or 322, or a substantially identical nucleotide sequence.

76. The method of any one of claims 72 to 75, wherein the second promoter is derived from a gene selected from the group consisting of: clfB (clustering factor B), sceD (autolysin, extracellular protein D), walKR (virulence regulator), atlA (major autolysin), oatA (O-acetyltransferase A); a phosphoribosylglycinamide formyltransferase gene, a phosphoribosylaminoimidazole synthetase gene, an amidophosphoribosyltransferase gene, a phosphoribosylformylglycinamide synthase gene, a phosphoribosylmethyltransferase gene Acylglycinamide synthase gene, phosphoribosylaminoimidazole-succinamide gene, trehalose permease IIC gene, DeoR family transcriptional regulator gene, phosphofructokinase gene, PTS fructose transporter subunit IIC gene, galactose-6-phosphate isomerase gene, NarZ, NarH, NarT, alkyl hydroperoxidase gene, hypothetical protein gene, DeoR trans factor gene, lysophospholipase gene, protein degradation chaperone gene, alkyl hydroperoxidase gene, phosphofructokinase gene, gyrB, sigB and rho.

77. The method of claim 76, wherein the second promoter is P clfB (aggregation factor B), and includes the nucleotide sequence of SEQ ID NO: 117, 118, 129 or 130, or a substantially identical nucleotide sequence thereof.

78. The method of any one of claims 43 to 77, wherein the decolonization step comprises topically administering a decolonizing agent to at least one site of the subject, thereby reducing or eliminating the presence of the undesirable microorganisms from the at least one site.

79. The method of claim 78, wherein the decolonization step comprises topically administering the decolonizing agent without concurrently administering a systemic antimicrobial agent.

80. The method of claim 78 or 79, wherein no systemic antimicrobial agent is administered within one week, two weeks, three weeks, one month, two months, three months, six months, or one year of the first topical administration of the decolonizing agent.

81. The method of any one of claims 40 to 42, wherein the decolonizing agent is selected from the group consisting of a disinfectant, a bactericide, an antiseptic, an astringent, and an antimicrobial agent.

82. The method of claim 81, wherein the decolonizing agent is selected from the group consisting of: alcohols (ethanol, isopropanol), aldehydes (glutaraldehyde, formaldehyde, formaldehyde releasers (hydroxymethyl methylthiourea = formaldehyde thiourea, taurolidine, hexamine, dantoin), o-phthalaldehyde), anilides (triclocarban = TCC = 3,4,4'-trichlorocarbamide), biguanides (chlorhexidine, Alexidine, polymeric biguanides (polyhexamethylene biguanide with MW>3,000 g / mol, vantocil), bisamidines (propamidine, propoxybenzamide isethionate, propoxybenzamide dihydrochloride, dibromopropamidine, dibromopropamidine isethionate), phenols (dichlorophenthiophene, para-chloro-m-xylenesulfonate, Phenol, chloroxylenol, hexachlorophene), bisphenols (triclosan, hexachlorophene), chloroxylenol (PCMX), quaternary ammonium compounds (cetrimonium bromide, benzalkonium chloride, cetylpyridinium chloride), silver compounds (silver sulfadiazine, silver nitrate), peroxides (hydrogen peroxide, peracetic acid, benzoyl peroxide), iodine compounds (povidone-iodine, poloxamer-iodine, iodine), chlorine-releasing agents (sodium hypochlorite, hypochlorous acid, chlorine dioxide, sodium dichloroisocyanurate, chloramine-T), copper compounds (copper oxide), isotretinoin, sulfur compounds, plant extracts (Melaleuca (tea tree oil), Vaccinium (e.g., A-type proanthocyanidins), Ligusticum chuanxiong, Pinus lanceolata, Azadirachta indica, Vitis vinifera, Terminalia avicennioides Guill & Perr, Phylanthus discoideus muel.Muel-Arg, Clove basil, Golden edge red mulberry, Hypericum pruinatum Boiss. & Bal, Hypericum olimpicum L. and Hypericumsabrum L, Hamamelis virginiana (witch hazel), Clove oil, Eucalyptus spp., Rosmarinus officinalis (rosemary), Thymus vulgaris (thyme), Origanum vulgaris (oregano), Cymbopogon spp., Cinnamomum spp., Geranium spp., Lavandula angustifolia), aminolevulinic acid, and topical antibiotic compounds (bacteriocins; mupirocin, bacitracin, neomycin, polymyxin B, gentamicin).

83. The method of claim 81 or 82, wherein the antimicrobial agent is selected from the group consisting of: vancomycin, cefazolin, cephalosporin, cephalexin, linezolid, daptomycin, clindamycin, lincomycin, mupirocin, bacitracin, neomycin, polymyxin B, gentamicin, prulifloxacin, ulifloxacin, fidaxomicin, minocycline hydrochloride, metronidazole, metronidazole, sulfamethoxazole, ampicillin, trimethoprim, ofloxacin, norfloxacin, floxacin, tinidazole, norfloxacin, ornidazole, levofloxacin, nalidixic acid, ceftriaxone, azithromycin, cefixime, ceftriaxone, cephalexin, ceftriaxone, rifaximin, ciprofloxacin, norfloxacin, ofloxacin, levofloxacin, gatifloxacin, gemifloxacin, prulifloxacin, ulifloxacin, moxifloxacin, nystatin, amphotericin B, flucytosine, ketoconazole, posaconazole, clotrimazole, voriconazole, griseofulvin, miconazole nitrate, and fluconazole.

84. The method of any one of claims 78 to 83, wherein the decolonization comprises topically applying the decolonization agent at least once, twice, three times, four times, five times, or six times or more prior to the replacing step.

85. The method of claim 84, wherein the decolonization step comprises administering the decolonizing agent to at least one host site of the subject one to six or more times or two to four times at intervals between 0.5 hours and 48 hours, and wherein the replacement step is performed after the final decolonization step.

86. The method of claim 85, wherein the replacing step comprises initially topically administering to the at least one host site of the subject a composition comprising at least 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 synthetic strains and pharmaceutically acceptable carriers.

87. The method of claim 86, wherein the initial replacement step is performed within 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days of the decolonization step.

88. The method of claim 86 or 87, wherein after the final decolonization step, the replacement step is repeated at intervals no more than once every two weeks to once every six months.

89. The method of claim 86 or 87, wherein the decolonization step and the replacement step are repeated at intervals no more than once every two weeks to once every six months.

90. The method of any one of claims 86 to 89, wherein the replacing comprises applying the synthetic microorganism to the at least one site at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.

91. The method of claim 90, wherein the replacing comprises applying the synthetic microorganism to the at least one site no more than once, no more than twice, no more than three times, or no more than four times per month.

92. The method according to any one of claims 43 to 91, further comprising Promoting colonization of the synthetic microorganism in the subject.

93. The method of claim 92, wherein promoting colonization of the synthetic microorganism in the subject comprises administering to the subject a promoting agent, optionally wherein the promoting agent is a nutrient, a prebiotic, a commensal, a stabilizer, a humectant and / or a probiotic species.

94. The method of claim 93, wherein the promoting comprises administering to the subject 10 6 to 10 10 cfu or 10 7 to 10 9 cfu of probiotic species.

95. The method of claim 93, wherein the nutrient is selected from the group consisting of sodium chloride, lithium chloride, sodium glycerophosphate, phenylethyl alcohol, mannitol, trypsin, peptides, and yeast extract.

96. The method of claim 93, wherein 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 byproducts, fructooligosaccharides (e.g., inulin-like prebiotics), galacto-oligosaccharides (e.g., raffinose), succinic acid, lactic acid, and manno-oligosaccharides.

97. The method of claim 93, wherein the probiotic is selected from the group consisting of 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 ... lactis), Streptococcus thermophiles, and Enterococcus fecalis.

98. The method of claim 93, wherein the commensal is selected from the group consisting of: Acinetobacter johnsonii, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus saprophyticus, Corynebacterium acnes, Corynebacterium striatum, Corynebacterium diphtheriae, Corynebacterium minutissima, Propionibacterium acnes, Propionibacterium acnes, Propionibacterium granulosum, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus mitis, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, Streptococcus agalactiae, Pseudomonas aeruginosa, Pseudomonas oryzae, Pseudomonas stutzeri, Pseudomonas putida, and Pseudomonas fluorescens.

99. The method of any one of claims 43 to 98, wherein the undesirable microorganism is an antimicrobial resistant microorganism.

100. The method of claim 99, wherein the antimicrobial-resistant microorganism is an antibiotic-resistant bacteria.

101. The method of claim 99, wherein the antibiotic resistant bacteria is a Gram-positive bacterial species selected from the group consisting of Streptococcus, Propionibacterium, and Staphylococcus.

102. The method of claim 101, wherein the Streptococcus is selected from the group consisting of: Streptococcus pneumoniae, Steptococcus mutans, Streptococcus sobrinus, Streptococcus pyogenes, and Streptococcus agalactiae.

103. The method of claim 101, wherein the Propionibacterium spp. is selected from the group consisting of Cutibacterium acnes subsp. acnes, Cutibacterium acnes subsp. defendens, and Cutibacterium acnes subsp. elongatum.

104. The method of claim 101, wherein the Staphylococcus spp. is selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.

105. The method of claim 104, wherein the undesirable microorganism is a methicillin-resistant Staphylococcus aureus (MRSA) strain containing a staphylococcal chromosomal cassette (type I-III SCCmec) encoding one (type I SCCmec) or more antibiotic resistance genes (type II and type III SCCmec) and / or produces toxins.

106. The method of claim 105, wherein the toxin is selected from the group consisting of: Panton-Valentine albumin-binding protein (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 coagulase toxin.

107. The method of any one of claims 43 to 106, wherein the subject does not exhibit recurrence of the undesirable microorganism for 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 30 weeks, at least 36 weeks, at least 42 weeks, or at least 52 weeks after the administering step, as evidenced by swabbing the subject at the at least one site.

108. A kit comprising in at least one container a synthetic microorganism according to any one of claims 1 to 27, a composition according to any one of claims 28 or 29, or a single dose according to any one of claims 30 to 32, and optionally at least a second container comprising a decolonizing agent, an instruction sheet, at least a third container comprising a facilitating agent, and / or an applicator.

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