Anti-fungal methods and Anti-fungal compounds derived from chromobacterium vaccinii

Abstract

The present invention relates to a method for preventing and / or treating fungal infections in human subjects. The method involves obtaining a subject with a fungal infection, a subject susceptible to such infections, or a subject suspected of having a fungal infection. Healing bacteria are cultivated in proximity to the subject, allowing the subject to be exposed to volatile compounds and / or agents released by the bacteria. These volatile compounds and / or agents are contained to ensure continued contact with the subject, thereby preventing and / or treating the fungal infection. The method leverages the therapeutic properties of the volatile compounds and / or agents released by the healing bacteria to effectively manage fungal infections in human subjects.

Description

Docket No.: 405002-558001WO ANTI-FUNGAL METHODS AND ANTI-FUNGAL COMPOUNDS DERIVED FROM CHROMOBACTERIUM VACCINII CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application No.: 63 / 635,599, filed 17-April-2024, the entire disclosure of which is incorporated by reference as if fully set forth herein in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates to methods, devices and compositions for treating and / or for preventing infections from micro-organisms and human pathogens. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable (N / A). BACKGROUND OF THE INVENTION

[0004] Traditional approaches to preventing and treating fungal infections in humans have primarily relied on the use of older antifungal medications, which can be administered topically, orally, or intravenously. These medications often target the cell membrane or cell wall of the fungus, disrupting its growth and replication. However, the effectiveness of these treatments can be limited by the development of drug resistance, potential (serious) side effects in humans, and the need for prolonged treatment durations. Additionally, some antifungal drugs may not penetrate well into certain tissues, reducing their efficacy in treating infections in those areas.

[0005] Another approach has involved the use of natural remedies and alternative therapies, such as essential oils, homeopathic medicines and herbal extracts, which are believed to possess antifungal properties. These treatments are often used as complementary therapies alongside conventional antifungal medications. While some studies have shown promising results, the variability in the composition of natural products and the lack of standardized dosing regimens can lead to inconsistent outcomes or even danger for the subject being treated. Furthermore, the mechanisms by which these natural compounds exert their antifungal effects are not always well understood, which can complicate their application in clinical settings.

[0006] None of these approaches have provided a comprehensive solution that combines theDocket No.: 405002-558001WO features described in this disclosure. What is urgently needed are new devices, treatments and methods for infections including drug-resistant infections and treatments for immune compromised individuals. BRIEF SUMMARY OF THE INVENTION

[0007] The following brief summary presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify all key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0008] Fungal infections are a significant health concern affecting millions of people worldwide. These infections can range from superficial conditions, such as athlete's foot, nail fungus and ringworm, to more severe systemic infections that can be life-threatening, particularly in immunocompromised individuals. The increasing prevalence of fungal infections is partly due to the growing number of people with weakened immune systems, such as those undergoing chemotherapy, COVID-19 infection, organ transplant recipients, and individuals with HIV / AIDS. Traditional antifungal treatments often involve the use of topical or systemic antifungal medications, which can have limited efficacy and may lead to drug-resistance over time.

[0009] In an example, a method for preventing and treating fungal infections in humans involves cultivating healing bacteria near the subject to release volatile compounds or agents that contact the subject. This approach is effective against drug-resistant and treatment-resistant fungal infections, particularly in immune-compromised individuals. The healing bacteria can include various species such as Bacillus subtilis and Lactobacillus spp., which release substances like dimethyl disulfide and bacteriocins; many more examples are provided herein. Additionally, an anti-fungal composition can be created by culturing microbes near fungi, confining the released substances, and using them to inhibit fungal growth. Devices are also described for implementing these methods, featuring air chambers and HEPA filters to facilitate the contact of healing bacteria with the subject.

[0010] The need for innovative approaches to prevent and treat fungal infections is critical, as the current antifungal drugs can be associated with side effects and the emergence of drug- resistant strains. Additionally, the environmental and biological diversity of fungi presents challenges in developing broad-spectrum antifungal agents. Herein we are exploring alternativeDocket No.: 405002-558001WO methods, such as utilizing beneficial microorganisms, to combat fungal infections. These methods aim to harness the natural antagonistic properties of certain bacteria against fungi, offering a potentially effective and sustainable solution to this growing health issue.

[0011] Historically, one of the most significant breakthroughs in this field was the discovery of antibiotics, which are naturally occurring compounds produced by bacteria and fungi that can kill or inhibit the growth of other microorganisms. The discovery of penicillin, a groundbreaking antibiotic derived from the fungus Penicillium notatum, highlighted the potential of microbial interactions in medical applications. This discovery paved the way for further research into bacterial metabolites with antifungal properties.

[0012] Herein we are starting to be able to identify the genes responsible for the production of antifungal compounds and manipulate these genes to enhance the antifungal properties of bacterial strains. This genetic engineering approach opened new avenues for the development of biocontrol agents that could be used in agriculture and medicine.

[0013] The potential applications of bacterial cultures in inhibiting fungal growth are vast. In agriculture, biocontrol agents derived from bacterial cultures are being developed to protect crops from fungal pathogens, reducing the reliance on chemical fungicides. In medicine, researchers are exploring the use of probiotic bacteria to prevent and treat fungal infections in humans, particularly in immunocompromised individuals.

[0014] The technology disclosed herein can be applied to vast spaces, to buildings and to treatments beyond human subjects. The technology is discussed, though, from the perspective of saving human lives.

[0015] Recent advancements herein explore the use of beneficial bacteria to combat fungal infections. The concept is based on the idea that certain bacteria can inhibit fungal growth through competitive exclusion or by producing antifungal compounds. This approach has been investigated in the context of gut health and skin microbiome management, where maintaining a balanced microbial environment is crucial for preventing infections. However, the application of probiotics specifically for treating fungal infections in humans is still in its early stages, and more research is needed to identify the most effective strains and delivery methods.

[0016] Keeping in mind possible combination therapies and the above discussion, as an additional brief summary or to provide discussion points for a brief summary, some example features of the technology disclosed herein can be briefly summarized by the following list of features, any of which can be inter-combined or discussed optionally with any other feature,Docket No.: 405002-558001WO Figure, Drawing, detail, embodiment, aspect, or example disclosed herein:

[0017] Feature 1: A method of preventing and / or treating a fungal infection in a human subject, the method comprising the steps of: (1) obtaining a subject with a fungal infection, as subject susceptible to a fungal infection or a subject suspected of having a fungal infection; (2) cultivating a healing bacteria in the vicinity of the subject so that the subject will be contacted with one or more volatile compounds and / or one or more agents released from the healing bacteria; and (3) containing the one or more volatile compounds and / or one or more agents released from the healing bacteria such that one or more volatile compounds and / or one or more agents will continue to contact the subject; whereby one or more volatile compounds and / or one or more agents prevents and / or treats the fungal infection in the subject.

[0018] Feature 2: The method of feature 1, wherein the fungal infection is a drug resistant and / or a treatment resistant fungal infection and / or wherein the subject is immune compromised.

[0019] Feature 3: The method of feature 1, wherein the substances released by the healing bacteria comprise one or more volatile compounds including: dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and / or sulfur compounds.

[0020] Feature 4: The method of feature 1, wherein the healing bacteria is a bacterium selected from the group consisting of: Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, StreptomycesDocket No.: 405002-558001WO aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and / or Chromobacterium vaccinii BUC-1.

[0021] Feature 5: The method of feature 1, wherein the method further comprises administering another anti-fungal agent to the subject and wherein the method is a combination therapy.

[0022] Feature 6: The method of feature 1, wherein the substances released by the healing bacteria comprise one or more agents including: bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and / or metal chelators.

[0023] Feature 7: The method of feature 1, wherein containing the substances optionally comprises containing the one or more volatile compounds released from the healing bacteria, containing the one or more agents released from the healing bacteria, containing the substances such that the substances will continue to contact the subject, the substances released by the healing bacteria prevent the fungal infection in the subject, and / or the substances released by the healing bacteria treat the fungal infection in the subject.

[0024] Feature 8: A method of making an anti-fungal composition for inhibiting a fungal infection in a subject, the method comprising: culturing a microbe near a fungus, wherein the microbe releases substances that inhibit growth of the fungus; and confining the substances released by the microbe to a container; wherein the substances in the container are including the anti-fungal composition.

[0025] Feature 9: The method of feature 8, wherein the fungal infection comprises: Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, Saccharomyces cerevisiae, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and / or Paracoccidioides spp., and wherein the fungal infection can be resistant to fluconazole, amphotericin B, echinocandins, and / or an azole resistance.

[0026] Feature 10: The method of feature 8, wherein the microbe is a bacterium.

[0027] Feature 11: The method of feature 8, wherein the microbe is a bacterium selected fromDocket No.: 405002-558001WO the group consisting of: Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and / or Chromobacterium vaccinii BUC-1.

[0028] Feature 12: The method of feature 8, wherein the substances include at least one volatile compound including: dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and / or sulfur compounds.

[0029] Feature 13: The method of feature 8, wherein the substances include at least one agent including: bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and / or metal chelators.

[0030] Feature 14: The method of feature 12, wherein confining the substances includes confining the at least one volatile compound released from the microbe.

[0031] Feature 15: The method of feature 13, wherein confining the substances includes confining the at least one agent released from the microbe.

[0032] Feature 16: The method of feature 8, wherein the anti-fungal composition prevents the fungal infection in the subject.Docket No.: 405002-558001WO

[0033] Feature 17: The method of feature 8, wherein the anti-fungal composition treats the fungal infection in the subject.

[0034] Feature 18: A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air chamber around the human subject; a culture of the healing bacteria inside the air chamber; and a HEPA filter over the culture of the healing bacteria; wherein cultivating the healing bacteria in the vicinity of the subject so that the subject will be contacted with one or more volatile compounds and / or one or more agents released from the healing bacteria prevents and / or treats the fungal infection in the subject.

[0035] Feature 19: The method of feature 8, further comprising a device to carry out the method, the device comprising: an enclosure surrounding the fungus; a culture of the microbe within the enclosure; and an air filter covering the culture of the microbe.

[0036] Feature 20: A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air chamber configured to surround the human subject; a culture of healing bacteria disposed inside the air chamber; and a HEPA filter positioned over the culture of healing bacteria.

[0037] Any of the devices can include sterilization. For example, irradiation of the air around a subject or other treatment. Additional example bacteria are Neisseriaceae (Neisseria sicca, N. lactamica, Kingella kingae, Eikinella corrodens), Aquaspirillaceae (Aquaspirillum polymorphum), and Chitinibacteraceae (Iodobacter fluviatilis). Also neighbors from other genera within Chromobacteriaceae, including Vogesella indigofera, Aquitalea palustris, and / or Pseudogulbenkiania.

[0038] Additional example fungi are Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and / or Paracoccidioides spp.

[0039] Any of the features, methods and / or details herein can be provided in a kit. While the summary examples disclosed above provide some introduction to embodiments of the invention, other implementations are also contemplated, described, and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description, the example claims, and a review of the associated drawings. It is to be understood that both theDocket No.: 405002-558001WO foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] For the purpose of illustration, certain discernable embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, data, dimensions, and illustrations shown. In the examples of the drawings:

[0041] FIG.1A, FIG.1B and FIG.1C show photos of 100mm petri dishes cultured herein.

[0042] FIG.1D shows a comparison of colony areas for each condition being displayed at percentage of maximum growth, with maximum growth being average area (mm2) of colonies on C.

[0043] FIG.1E and FIG.1F show Candida albicans SC5314 growth is strongly inhibited by the lawn of 2 different C. vaccinii.

[0044] FIG.2A shows images of an inhibition experiment. FIG.2B shows daily colony area measurements.

[0045] FIG.3A and FIG.3B show electron microscopy images with scale bar 10 microns.

[0046] FIG.4A shows the schematic of ALI cultures where A549 cells (at right,) were exposed to VOCs or VCs produced by the bacterial culture 330.

[0047] FIG.4B shows results of a resazurin viability assay, reported in relative fluorescence units (RFUs).

[0048] FIG.5A and FIG.5B show plots of viability up to 6 days after inoculation. FIG.5C shows images of control vs. pVS1-SAH2.

[0049] FIG.6A shows images of control, LAC300 and C. vaccinii. FIG.6B shows a plot of percent of maximal growth. FIG.6C shows images of control, LAC300 and C. vaccinii under differing conditions. FIG.6D shows a plot of percent of maximal growth under the differing conditions. FIG.6E shows images including a comparison of wild type and KMB + kanamycin.

[0050] FIG.7 shows images comparing KMB with KMB + kanamycin.

[0051] FIG.8A shows an example of phylogenetic distance including Gammaproteobacteria, Alphaproteobacteria and Betaproteobacteria. FIG.8B shows and example of genetic distancesDocket No.: 405002-558001WO among Neisseriaceae, Chromobacteriaceae, Aquaspirillaceae and Chitinibacteraceae

[0052] FIG.9 shows an example of the Chromobacterium antifungal phenotype.

[0053] FIG.10 shows example images of control, C. vaccinii, C. substugae WT and C. subtsugae Var.

[0054] FIG.11 shows an example diagram of locating candidates.

[0055] FIG.12 shows images supporting S. cerevisiae growth is strongly inhibited by Chromobacterium VCs.

[0056] FIG.13A is showing images of growth inhibition of a Mucor species in one iteration (visually in panel of FIG.13A) and the statistics across 3 replicates are shown in FIG.13B.

[0057] FIG.14 shows a life-saving device 500 configured to treat a single human or to treat an entire building or an area.

[0058] FIG.15 is a flowchart illustrating a method in steps including obtaining a subject with a fungal infection (suspected and / or susceptible), according to an embodiment.

[0059] FIG.16 is a flowchart illustrating a method in step forms for inhibiting fungal growth through the release of substances by a microbe, according to an embodiment. FIG.17 shows percent maximal growth for more additional tests.

[0060] It should be understood that while illustrations can sometimes be used in the example figures above to describe different embodiments and different aspects of the technology, any aspect from any figure can be optionally inter-combined with an aspect from any other figure or text. Any example disclosed herein can be inter-combined with any other. All trademarks, images, likenesses, words, and depictions that could be construed in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of an urgent need to prevent injuries and to treat subjects as further discussed in more detail below. DETAILED DESCRIPTION OF THE INVENTION

[0061] The subject innovation is now described, in some examples with reference to the drawings, wherein examples can used to refer to the aspects of the breadth of concepts of the invention. In the following description, for purposes of explanation, specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. It is to be appreciatedDocket No.: 405002-558001WO that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. DEFINITIONS

[0062] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention can be determined by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0063] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0064] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0065] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] As used herein, a “range” may be provided. A statement may include “in the range from about A to about B”. All points from A to B are subsumed by the range, and all those points can define preferred ranges. Within said range, any range subsumed therein means any range that is within the stated range. Endpoints within the range can define a new range. For example,Docket No.: 405002-558001WO the following are all subsumed within the range of about 10 to about 50.10 to 20; 15 to 35; 23 to 40; or 50 to 31; or any other range or set of ranges within the stated range. As such, within the range any set of endpoints subsumed therein can be used as an exemplary range.

[0067] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Any method described herein can be claimed and / or described as a composition and vice versa.

[0068] The term "consisting of" as it is known in the practice refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0069] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. In specific examples, “consisting essentially of” can be explained herein for each example or can be defined broadly, for example, by stating that an administration to a subject (in a method herein) does not include any other active pharmaceutical ingredient or therapeutic agent in addition to the one specified. In another example, the term “consisting essentially of” can be utilized to indicate a nanocarrier and a therapeutic agent with no other ingredients that are listed in a claim and yet including any other ingredients that are not specifically listed.

[0070] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference. The term “feature” and the term “detail” can be interchanged with a “claim”. Any list of features, details, examples, embodiments, and / or aspects herein can be placed into a “claim”.

[0071] As used herein, the term "subject" refers to a mammal, bird, or the like, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), racing mammals, laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. In another example, the term “subject” can refer to a connective tissue culture, and the methods disclosed herein, while claimed towards subjects, contemplate use in the laboratory in synthetic tissue(s). As used herein, a female cell can refer to a cell with 2X chromosomes; a male cell can refer to a cell with 1X and 1Y chromosome.Docket No.: 405002-558001WO

[0072] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to modulate a treatment or prevent or ameliorate a manifestation of disease or medical condition, such as a connective tissue condition or a risk of a connective tissue injury. Such a condition (or risk) may not be readily discernable and may take years, statistical analysis, and / or machine learning to determine a prevention, treatment, or amelioration. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the condition and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0073] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, manage, modulate, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect (undesirable characteristic) or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a condition or decay, delay or slowing of a progression and / or risk of injury, and an increased lifespan / enjoyment as compared to that expected in the absence of treatment.

[0074] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. The therapeutic agent or drug may refer to a formulation, composition, or agent. The formulation can be changed to a fresh formulation during administration. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-Docket No.: 405002-558001WO acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

[0075] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by any technique known in the art that is subsequently applied to a subject, topical application, absorption, injection, ingestion, transfusion, implantation or transplantation. In an example embodiment, compositions are applied as a tablet or drug in capsule. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subdermal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. It is known in the art that therapeutic agents can be rapidly deployed through the skin and directly into joint / ligaments by use of DMSO (dimethyl sulfoxide) as a carrier solvent applied (with the therapeutic agent) to the skin near to or surrounding a joint. While DMSO is rarely used anymore for these purposes because of its nature as a universal solvent and its tendency to carry any residual chemicals present on the skin into the bloodstream (along with the intended agent), the technology contemplates such uses. In one contemplated embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tissue, lymph node, or site of treatment. In another example, administration is provided in the form of a natural product, vitamin, supplement, food, aerosol, inhalation, vapor, or drink. Formulations disclosed herein can be ready made or require mixing just before administration.

[0076] Any of the methods disclosed herein can be carried out in part or completely by including a dietary change, a food, natural product, precursor, or prodrug of a therapeutic agent. As used herein, a precursor or a prodrug is intended to encompass compounds or therapeutic agents which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound for any of the present claims or features). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or of carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the small-molecule chemical structures selectedDocket No.: 405002-558001WO from this disclosure can be replaced with the corresponding suitable prodrug, for example, wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. A common method of making a precursor / prodrug that can be used herein is to use a carrier / nanocarrier (e.g., mesoporous silica particles). The precursor / prodrug can be released from a carrier to form the active therapeutic agent. A precursor or prodrug can be metabolized to the active parent compound (therapeutic agent) in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid). No argument can be made that the term “prodrug” is not enabled herein based on an assertion that actual prodrugs were not made and tested.

[0077] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0078] The terms: “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0079] The term “healing bacteria” as used herein can refer to any micro-organism suitable for treating infections in humans as disclosed herein. As used herein, the term: “small molecule”Docket No.: 405002-558001WO refers to a molecule that has a molecular weight < 1000. As used herein, the term: “large molecule” refers to a molecule that has a molecular weight > 1000, and the term includes biologics such as the examples of oligonucleotides, peptides, antibodies, linkers, oligosaccharides, polymers, DNA chains, and RNA chains. The term: “therapeutic agent” may refer to small molecule, element, large molecule, biologic, formulation, composition, agent, or a combination thereof. PHARMACEUTICAL COMPOSITIONS

[0080] The compositions and methods of the present invention may be utilized to prevent a need for other treatment, to provide benefit when other treatment(s) fail, or to treat an individual in need thereof. In some embodiments, the individual is suspected of needing treatment. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. A compound can represent a combination therapy herein. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. Compositions can be in gas forms. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0081] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologicallyDocket No.: 405002-558001WO acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0082] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0083] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible compositions employed in pharmaceutical formulations.

[0084] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally, for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue; absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermal administration (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or sprayDocket No.: 405002-558001WO applied to the skin). The compound may also be formulated for inhalation. Inhalation can include inhalation of a liquid (droplets or aerosol). Inhalation can include a micronized powder adhered to carrier particles or can be without carrier particles. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, all of which are incorporated herein by reference in their entireties, as well as in patents cited therein.

[0085] In the embodiments discussed and in any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0086] Other terms are defined herein within the description of the various aspects of the invention or are used as would be understood by an ordinary person. ANTI-FUNGAL METHODS AND ANTI-FUNGAL COMPOUNDS DERIVED FROM CHROMOBACTERIUM VACCINII

[0087] Invasive fungal infections (IFIs) cause significant illness and mortality. Historically associated with immune suppression such as organ transplantation and diabetes, the COVID-19 pandemic demonstrated normal hosts can become susceptible to these infections. Climate change is felt to be exacerbating IFIs as fungi adapt to warmer, i.e. mammalian, temperatures. Candida auris is an example of this adaptation contributing to pathogenesis. Limited numbers of antifungal agents and increasing resistance to these medications have created a significant need for novel antifungal therapies. A significant barrier to developing new antifungals, and antimicrobials in general, is the limited availability of new chemical compounds that can serve as lead compounds or that identify important antimicrobial targets in pathogens. Volatile compounds (VCs) are increasingly recognized for their complex biological effects. Prior research has shown cross-kingdom interactions between environmental bacteria and fungi mediated by bacterial VCs. We have isolated a new strain of Chromobacterium vaccinii with potent antifungal VCs and have shown for the first time that these compound(s) inhibit the growth of fungi that are human pathogens, including C. auris and Aspergillus fumigatus.

[0088] Our overarching technology is that volatile compounds are an underexplored chemical source of antimicrobial compounds. The objective of this research program is to delineate theDocket No.: 405002-558001WO mechanism of Chromobacterium VC production and the mechanism of action on pathogenic fungi that is broadly active against yeasts and molds. We propose complementary computational, genomic, and genetic studies to investigate these mechanisms. An expanded collection of fungal strains are also tested, including clinical isolates, isolates with preexisting resistance to antifungals, and a broader assortment of filamentous fungi, including Mucorales. The outcome of this research further pioneers the foundation for chemical identification of the relevant antifungal VCs. Defining the mechanism of action in fungi will identify systems and pathways that could also be targeted using other approaches. In addition, this research will develop a suite of genetic tools for the Chromobacterium research community. The ultimate objective is to develop new antifungal agents for clinical use.

[0089] VCs could be deployed as an inhaled antifungals and can serve as lead compounds for developing agents delivered in aqueous or liposomal formulations. This program generates early-stage, translational data on drug discovery that will be used for the development of novel antifungal therapeutics. In some embodiments, the technology herein uses the discovery that C. vaccinii produces substance(s) that inhibit the growth of a wide range of fungi, from yeasts to molds, that are pathogenic to humans. This phenotype has never been reported while using it for human pathogens. The phenotype is observed when C. vaccinii and fungi grow on separate plates but share the same air space, implicating a volatile compound. The compound(s) block growth that creates dysmorphic fungal elements when viewed by electron microscopy. The compound(s) are not cytotoxic to human cells. This finding also provides a guide to identifying a new drug target in fungi, which could be used to screen for other anti-fungal compounds.

[0090] This invention provides a new approach to treating patients infected with fungi, yeasts or molds. Physicians who treat patients with fungal infections can use this, particularly in patients with immunosuppression such as organ transplant recipients or patients with prolonged neutropenia because they are at higher risk for more serious, invasive fungal infections.

[0091] We have tested these methods using, for example, Candida albicans, Aspergillus fumigatus, Fusarium. Surprisingly these methods have worked on all three pathogens. We are currently trying Cryptococcus neoformans, Candida auris, and Cladophialophera bantiana. We have shown complete inhibition of Cryptococcus and strong inhibition of C. auris in our data, which are in, for example, in the figures such as FIGs.6A-6E. There might be a subtle phenotype on other bacteria, and this will need to be fleshed out. The anti-fungal methods herein are unexpected and striking.

[0092] For example, in FIGs. 1A-1D are sandwich plate assays with C. albicans andDocket No.: 405002-558001WO Chromobacterium vaccinii BUC-1 and MWU328. FIG.1A shows a 100mm petri dish containing C. albicans plated on SDA which was exposed to C. vaccinii BUC-1 photographed using a light box. FIG.1B shows a 100mm petri dish containing C. albicans plated on SDA which was exposed to C. vaccinii MWU 328 photographed using a light box. FIG.1C shows a 100mm petri dish containing C. albicans plated on SDA grown under standard conditions photographed using a light box. FIG.1D shows a comparison of colony areas for each condition being displayed at percentage of maximum growth, with maximum growth being average area (mm2) of colonies on C. albicans control plates (1.84 mm2). Results represent averages of three independent trails, and an ordinary one-way ANOVA with Tukey post hoc was utilized to compare conditions. **** p<0.0001.

[0093] The data demonstrate that Chromobacterium vaccinii BUC-1 and MWU328 inhibit the growth of Aspergillus fumigatus. FIG.2A shows images of an A. fumigatus culture during 6 days of growth. The culture in the row labeled “A. fumigatus” was grown under standard conditions, SDA media incubated at 30ºC and serves as a control. The culture in the row labeled “+ BUC-1” is the same starting culture of A. fumigatus but was co-cultured with BUC-1 in the sandwich plate assay. FIG.2B shows daily colony area measurements of A. fumigatus cultures are displayed as percentage of maximum growth, with maximum growth being an average of measured colonyarea (mm2) of A. fumigatus control plates on day 6 (6,220.7 mm2). Results represent averages of4 independent trails where 100% growth was the area measured in the control plate on day 6. Areas on each day were compared using a repeated measures two-way ANOVA with Tukey post hoc. Differences in colony area between control A. fumigatus cultures and A. fumigatus cultures exposed to BUC-1 and MWU328 were significant (p≤0.05) beginning at day two of growth.

[0094] We have acquired scanning electron microscope images of C. albicans and are setting up to generate data on A. fumigatus. Candida albicans shows altered ultrastructure on scanning electron microscopy after co-culture with C. vaccinii. FIG.3A shows C. albicans grown on SDA for 48 hours at 30ºC (scale bar, 10 μm). FIG.3B shows C. albicans grown on SDA for 48 hours at 30ºC in sandwich-plate assay with C. vaccinii BUC-1 (scale bar, 10 μm).

[0095] The data shows that co-culture with human cells at an air-liquid interface does not lead to eukaryotic cell death (resazurin is a viability dye, the more viable cells the more conversion to the fluorescent substrate). In FIG.4A, the schematic shows the example setup of an airtight box 320 containing open cultures of both human A549 cells in what's called a Transwell insert 315 that is in each well 310 of the 96-well plate 305. In a novel example, human lung epithelial cells were not adversely affected by co-culture with C. vaccinii. FIG.4A shows the schematic of ALIDocket No.: 405002-558001WO cultures where A549 cells (at right, 325) were exposed to VOCs produced by C. vaccinii BUC-1 in an air-tight box 320 (at left). A549 cells 325 were grown on the upper surface of a Transwell tissue culture membrane 315 (right) and alongside an open petri dish(es) 330 with a lawn of C. vaccinii BUC-1. Both cultures were open to the air in the chamber, which was incubated for 24 hours at 37°C. FIG.4B shows results of a resazurin viability assay, reported in relative fluorescence units (RFUs). “Control” measurements represent RFU measurements from A549 cells cultured in ALI conditions but not exposed to C. vaccinii. “Exposure” measurements represent RFU measurements from A549 cells cultured in ALI conditions while being exposed to C. vaccinii VOCs. Results represent averages of 3 independent trials each of which contained XXX technical replicate wells. Student’s T test was used to compare conditions.

[0096] These data are relevant for example activity against Candida auris and Cryptococcus neoformans. In addition, BUC-1 (our strain, see FIG.5A and FIG.5B) had a greater effect than a C. vaccinii strain MWU (FIG.5A) though not as dramatic as, for example, the Aspergillus fumigatus in FIG.2A.

[0097] The methods herein can be used to prevent or treat fungal infections in humans by obtaining a subject with a fungal infection or at risk of one, cultivating healing bacteria near the subject, and ensuring the subject is exposed to volatile compounds or agents from the bacteria. These compounds or agents can continue to contact the subject to prevent or treat the infection.

[0098] The methods may also apply to drug-resistant or treatment-resistant fungal infections, and it can be used for subjects with compromised immune systems. The substances released by the healing bacteria may include volatile compounds such as dimethyl disulfide, methanethiol, and hydrogen sulfide, among others. The healing bacteria may be selected from a variety of species, including Bacillus subtilis, Pseudomonas aeruginosa, and Lactobacillus spp., among some examples. The method may also involve administering some additional anti-fungal agents to the subject, making it a combination therapy. The substances released by the healing bacteria may include agents like bacteriocins, lipopeptides, and enzymes, among some examples. Containing the substances may involve ensuring that the volatile compounds or agents continue to contact the subject, thereby preventing or treating the fungal infection. A method may be used to create an anti-fungal composition by culturing a microbe near a fungus, releasing substances that inhibit fungal growth, and confining these substances in a container. The fungal infection may include species like Candida albicans and Aspergillus fumigatus, and it can be resistant to some treatments like fluconazole.

[0099] The microbe used in the method may be a bacterium, with examples including BacillusDocket No.: 405002-558001WO subtilis and Lactobacillus spp. The substances in the anti-fungal composition may include volatile compounds like dimethyl disulfide and hydrogen sulfide. The substances may also include agents such as bacteriocins and lipopeptides. Confining the substances may involve containing the volatile compounds released from the microbe. Confining the substances may also involve containing the agents released from the microbe. The anti-fungal composition may prevent fungal infections in subjects. The anti-fungal composition may also treat fungal infections in subjects.

[0100] A device may be used to prevent or treat fungal infections in humans, comprising an air chamber around the subject, a culture of healing bacteria inside, and a HEPA filter over the culture. The method may also involve a device with an enclosure surrounding the fungus, a culture of the microbe within, and an air filter covering the culture. A device may be used to prevent or treat fungal infections in humans, featuring an air chamber to surround the subject, a culture of healing bacteria inside, and a HEPA filter positioned over the culture.

[0101] FIG.14 shows a device configured to treat a single human or to treat an entire building or area. In a prophetic example, the methods herein are tested on humans using a device shown in FIG.14. The device includes an air or atmosphere containment structure 410, a culture of micro-organisms 415 with a filter 420 as described herein. The micro-organisms release VCs, VOCs, and / or agents 455 that inhibit and / or terminate a fungal infection 440 associated with the human 425. The air and / or atmosphere 450 surrounding the human 425 includes the VCs, VOCs, and / or agents 455 and contacts the cells of the human to prevent and / or to treat the fungal infection 440.

[0102] In another example, the technology includes a method of preventing and / or treating a fungal infection in a human subject, the method comprising the steps of: (1) obtaining a subject with a fungal infection, as subject susceptible to a fungal infection or a subject suspected of having a fungal infection; and / or a device configured for (2) and (3): (2) cultivating a healing bacteria in the vicinity of the subject so that the subject will be contacted with one or more volatile compounds and / or one or more agents released from the healing bacteria; and (3) containing the one or more volatile compounds and / or one or more agents released from the healing bacteria such that one or more volatile compounds and / or one or more agents will continue to contact the subject; whereby one or more volatile compounds and / or one or more agents prevents and / or treats the fungal infection in the subject.

[0103] In some inter-changeable and / or optional examples, a fungal infection herein can optionally include Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida auris, Candida dubliniensis, Candida guilliermondii, Candida kefyr,Docket No.: 405002-558001WO Candida lusitaniae, Candida rugosa, Candida stellatoidea, Candida utilis, Candida viswanathii, Candida zeylanoides, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus ustus, Aspergillus clavatus, Aspergillus glaucus, Aspergillus oryzae, Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus uniguttulatus, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton interdigitale, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton concentricum, Microsporum canis, Microsporum gypseum, Microsporum audouinii, Microsporum nanum, Microsporum persicolor, Epidermophyton floccosum, Malassezia globosa, Malassezia restricta, Malassezia sympodialis, Malassezia furfur, Malassezia pachydermatis, Malassezia slooffiae, Malassezia obtusa, Malassezia japonica, Malassezia yamatoensis, Malassezia dermatis, Malassezia equina, Malassezia caprae, Malassezia cuniculi, Malassezia nana, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Fusarium graminearum, Fusarium dimerum, Fusarium fujikuroi, Penicillium marneffei, Penicillium chrysogenum, Penicillium citrinum, Penicillium digitatum, Penicillium italicum, Penicillium expansum, Penicillium glabrum, Penicillium roqueforti, Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Paracoccidioides lutzii, Sporothrix schenckii, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana, and / or a combination thereof.

[0104] In some inter-changeable and optional examples, the healing bacteria can be comprising: Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus curvatus, Lactobacillus pentosus, Lactobacillus coryniformis, Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus gallinarum, Lactobacillus graminis, Lactobacillus mucosae, Lactobacillus oris, Lactobacillus panis, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus vaginalis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium thermophilum, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium catenulatum, Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium gallicum, BifidobacteriumDocket No.: 405002-558001WO indicum, Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacterium minimum, Bifidobacterium pseudolongum, Bifidobacterium pullorum, Bifidobacterium ruminantium, Bifidobacterium saeculare, Bifidobacterium scardovii, Bifidobacterium subtile, Bifidobacterium thermacidophilum, Streptococcus thermophilus, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus mutans, Streptococcus sobrinus, Streptococcus downei, Streptococcus criceti, Streptococcus rattus, Streptococcus ratti, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus cereus, Bacillus circulans, Bacillus firmus, Bacillus lentus, Bacillus smithii, Bacillus thuringiensis, Propionibacterium acidipropionici, Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium shermanii, Propionibacterium thoenii, Propionibacterium acnes, Propionibacterium avidum, Propionibacterium cyclohexanicum, Propionibacterium granulosum, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus gallinarum, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus sulfureus, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc carnosum, Leuconostoc fallax, Leuconostoc gelidum, Leuconostoc inhae, Leuconostoc kimchii, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces kudriavzevii, Escherichia coli Nissle 1917, and / or a combination thereof.

[0105] In some embodiments, this disclosure provides a method for preventing and / or treating fungal infections in human subjects by utilizing healing bacteria. The method may involve obtaining a subject with a fungal infection, a subject susceptible to a fungal infection, or a subject suspected of having a fungal infection. Healing bacteria can be cultivated in the vicinity of the subject, allowing the subject to be exposed to volatile compounds and / or agents released from the bacteria. These substances may include a variety of volatile compounds such as dimethyl disulfide, methanethiol, and hydrogen sulfide, as well as agents like bacteriocins and enzymes. The volatile compounds and agents can be contained to ensure continuous contact with the subject, thereby preventing and / or treating the fungal infection.

[0106] The method may also include selecting specific bacteria from a group, such as BacillusDocket No.: 405002-558001WO subtilis or Lactobacillus spp., to optimize the treatment. Additionally, the method can be part of a combination therapy with other anti-fungal agents. The disclosure may also encompass a device comprising an air chamber, a culture of healing bacteria, and a HEPA filter to facilitate the treatment process. In the context of the system, a bacterium may be selected from a specified group to ensure the effectiveness of the treatment. This selection process may involve choosing bacteria that are known for their ability to release volatile compounds and agents that can inhibit fungal growth. The selected bacterium may then be cultured near a fungus, which may facilitate the release of substances that inhibit the growth of the fungus.

[0107] This process may be part of a broader method that includes cultivating healing bacteria in the vicinity of a subject. The cultivation may be designed to ensure that the subject is contacted with one or more volatile compounds and / or agents released from the healing bacteria. The system may include a device comprising an air chamber, which may be configured to surround the subject. This air chamber may serve as a controlled environment where the healing bacteria can be cultivated effectively. The device may also include a HEPA filter positioned over the culture of healing bacteria. The HEPA filter may play a role in maintaining the purity of the air within the chamber, ensuring that the volatile compounds and agents can contact the subject without contamination. The method may further involve containing the one or more volatile compounds and / or agents released from the healing bacteria. This containment may ensure that the substances continue to contact the subject, thereby enhancing the potential for preventing and / or treating the fungal infection.

[0108] The containment process may involve confining the volatile compounds and agents within a specific area, possibly using an enclosure or similar structure. The substances released by the healing bacteria may include a variety of volatile compounds and agents, such as dimethyl disulfide, methanethiol, and bacteriocins, among others. These substances may provide treatment agents and compounds that contribute to the prevention and / or treatment of the fungal infection. The method may also involve contacting the subject with these volatile compounds and / or agents, which may be a step in the treatment process. Overall, the system may be designed to provide an approach to preventing and / or treating fungal infections by leveraging the properties of healing bacteria and the controlled environment of the device. The actions and components described may work together to create a treatment environment, ensuring continuous contact with the subject and maximizing the potential for successful treatment. In the context of preventing and / or treating a fungal infection, a method may be employed that involves obtaining a subject who may have a fungal infection, is susceptible to such an infection, or isDocket No.: 405002-558001WO suspected of having one.

[0109] This method may include cultivating healing bacteria in proximity to the subject, allowing the subject to be contacted with volatile compounds and / or agents released from the bacteria. The volatile compounds and / or agents may be contained to ensure continuous contact with the subject, potentially preventing and / or treating the fungal infection. The anti-fungal composition may include volatile compounds such as dimethyl disulfide, methanethiol, and hydrogen sulfide, as well as agents like bacteriocins and lipopeptides. The method may also involve identifying the subject for treatment, which may be for targeting the appropriate individuals. The cultivation of healing bacteria may ensure that the subject is exposed to the necessary volatile compounds and / or agents, which may be for the treatment process. The containment of these substances may ensure that they continue to contact the subject, thereby maintaining their potential therapeutic effect. The method may also include the administration of another anti-fungal agent, suggesting a combination therapy approach.

[0110] This approach may enhance the treatment efficacy by utilizing multiple mechanisms to combat the fungal infection. The overall process may be designed to provide a strategy for addressing fungal infections, particularly in cases where the infection is resistant to conventional treatments. FIG.15 is a flowchart illustrating a method in step 100 for obtaining a subject with a fungal infection, according to an embodiment. At step 100, a subject may be identified as having a fungal infection, being susceptible to a fungal infection, or being suspected of having a fungal infection. This identification may involve assessing the subject's symptoms, medical history, or risk factors that suggest a potential fungal infection. The process may involve a healthcare professional evaluating the subject to determine the likelihood of a fungal infection. The identification of the subject sets the stage for subsequent steps in the method aimed at preventing or treating the fungal infection. The subject's condition may be documented, and relevant data may be collected to facilitate the next steps in the treatment process. This step may serve as the foundation for implementing a targeted approach to manage the fungal infection effectively. The identification process may be supported by diagnostic tools or tests that provide additional information about the subject's health status.

[0111] The outcome of this step may guide the selection of appropriate treatment strategies tailored to the subject's specific needs. In the context of Step 102 (FIG.15), the process may involve cultivating healing bacteria in the vicinity of a subject. This cultivation may be intended to ensure that the subject is contacted with one or more volatile compounds and / or agents released from the healing bacteria. The healing bacteria may be selected from a diverse group, potentiallyDocket No.: 405002-558001WO including Bacillus subtilis, Pseudomonas aeruginosa, and various species of Lactobacillus, among others. The volatile compounds released by these bacteria may include dimethyl disulfide, methanethiol, and benzothiazole, which may serve as treatment agents. The cultivation of these bacteria may be strategically performed to expose the subject to these healing agents, thereby potentially preventing or treating a fungal infection. The process may be designed to ensure that the volatile compounds and / or agents maintain continuous contact with the subject, which may be for the effectiveness of the treatment. This step may be part of a broader method aimed at preventing and / or treating fungal infections in human subjects.

[0112] The cultivation of healing bacteria and the subsequent release of volatile compounds and / or agents may be a component of this method, potentially offering an approach to addressing fungal infections. At step 104 (FIG.15), the process may involve containing the volatile compounds and / or agents released from the healing bacteria. This containment may be designed to ensure that these substances continue to contact the subject, thereby potentially preventing or treating the fungal infection. The containment of these substances may involve various methods to maintain their proximity to the subject, allowing for continuous interaction. The volatile compounds and agents, which may include a range of chemical substances, can be released by the healing bacteria and may play a role in the therapeutic process. Additionally, the method may incorporate the administration of another anti-fungal agent, suggesting a combination therapy approach. This combination therapy may enhance the effectiveness of the treatment by utilizing multiple mechanisms to combat the fungal infection.

[0113] The containment strategy may ensure that the therapeutic agents remain effective over time, providing a sustained treatment effect. The process may also involve selecting specific healing bacteria known for their ability to release beneficial volatile compounds and agents, further optimizing the treatment strategy. The overall approach may be aimed at maximizing the therapeutic contact between the subject and the healing agents, thereby increasing the likelihood of preventing or treating the fungal infection effectively.

[0114] FIG.16 is a flowchart illustrating a method in step 200 for inhibiting fungal growth through the release of substances by a microbe, according to an embodiment. At step 200, a microbe may be cultured near a fungus, where the microbe may release substances that potentially inhibit the growth of the fungus. The microbe may be identified as a bacterium, which may be selected from a specified group of bacteria. The substances released by the microbe may include volatile compounds and agents, which may be confined to a container to ensure their continuous contact with the fungus. The volatile compounds may include a variety of chemicalDocket No.: 405002-558001WO compounds such as dimethyl disulfide, methanethiol, and benzothiazole, among others. The agents may include bacteriocins, lipopeptides, and enzymes, which may contribute to the inhibition of fungal growth. The anti-fungal composition, comprising these substances, may prevent or treat the fungal infection in the subject. Additionally, a device may be employed to carry out the method, which may include an enclosure surrounding the fungus, a culture of the microbe within the enclosure, and an air filter covering the culture of the microbe. This method may provide a controlled environment for the microbe to release its substances, thereby potentially enhancing the effectiveness of the anti-fungal treatment.

[0115] In the context of Step 202 (FIG.16), the process may involve confining the substances released by the microbe to a container. This step may ensure that the volatile compounds and agents released from the healing bacteria are effectively contained. The containment may facilitate continuous contact with the subject, which may be essential for the prevention and treatment of fungal infections. The substances confined may include at least one volatile compound, such as dimethyl disulfide, dimethyl trisulfide, or methanethiol, among others. Additionally, the agents confined may include bacteriocins, lipopeptides, or polypeptides, which may contribute to the therapeutic effect. The containment process may be designed to maintain the efficacy of these compounds and agents by preventing their dissipation into the environment, thereby ensuring that they remain in proximity to the subject. This approach may enhance the potential for the volatile compounds and agents to exert their intended effects on the fungal infection.

[0116] The method may also involve the use of a device that comprises an enclosure surrounding the fungus, a culture of the microbe within the enclosure, and an air filter covering the culture. This configuration may support the containment of the substances, thereby optimizing the conditions for the treatment of the fungal infection. The overall process may be aimed at providing a controlled environment where the healing bacteria can release their therapeutic agents in a manner that maximizes their contact with the subject, thereby potentially improving the treatment outcomes for fungal infections. At step 204 (FIG.16), the anti-fungal composition may be utilized to prevent the fungal infection in the subject. The anti-fungal composition may also treat the fungal infection in the subject. The method may further comprise a device to carry out the method, which may include an enclosure surrounding the fungus, a culture of the microbe within the enclosure, and an air filter covering the culture of the microbe. The anti-fungal composition may be designed to ensure that the substances released by the microbe, such as volatile compounds and agents, are confined within the enclosure. This confinement may facilitateDocket No.: 405002-558001WO continuous contact with the subject, thereby potentially enhancing the effectiveness of the treatment. The device may be configured to maintain an environment for the microbe to release the necessary substances, which may include volatile compounds like dimethyl disulfide and agents such as bacteriocins. The air filter may serve to regulate the flow of these substances, ensuring that they remain in contact with the subject for an extended period. This method may offer an approach to managing fungal infections, particularly in cases where traditional treatments may be less effective.

[0117] In a discussion, study or a reading of the details, features, embodiments, aspects, FIGs., and / or examples of the technology disclosed herein, any of the features, embodiments, aspects, and / or examples herein can be optionally inter-combined (or inter-discussed) with the example details listed below, and any portion (or aspect) of any detail below can be inter- combined with any portion of any feature or example herein:

[0118] Detail 1: A method of preventing and / or treating a fungal infection in a human subject, the method comprising the steps of: (1) obtaining a subject with a fungal infection optionally caused by Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, and / or Saccharomyces cerevisiae, wherein the fungal infection is resistant to fluconazole, amphotericin B, echinocandins, and / or an azole antifungal agent, and wherein the subject is immunocompromised due to a condition selected from the group consisting of HIV / AIDS, cancer, organ transplantation, stem cell transplantation, and prolonged corticosteroid therapy; (2) cultivating a healing bacteria optionally selected from the group consisting of Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, BifidobacteriumDocket No.: 405002-558001WO infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1 in the vicinity of the subject, wherein the healing bacteria is cultivated in a growth medium comprising a carbon source selected from the group consisting of glucose, fructose, sucrose, lactose, glycerol, mannitol, and sorbitol, a nitrogen source selected from the group consisting of peptone, tryptone, yeast extract, meat extract, malt extract, casein hydrolysate, soybean meal, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea, and an inorganic salt selected from the group consisting of dipotassium phosphate, monopotassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride, and wherein the healing bacteria is cultivated under aerobic conditions with shaking at a temperature in the range of 25-40°C and a pH in the range of 6.0-8.0 for a period of 24-72 hours; and (3) containing the one or more volatile compounds and / or one or more agents released from the healing bacteria in an air-tight chamber surrounding the subject, wherein the air-tight chamber is equipped with a HEPA filter to prevent the escape of the healing bacteria and the volatile compounds and / or agents, and wherein the one or more volatile compounds and / or one or more agents are allowed to contact the subject for a period of 1-12 hours per day for 7-14 days; whereby the one or more volatile compounds selected from the group consisting of dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and sulfur compounds and / or one or more agents selected from the group consisting of bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and metal chelators prevents and / or treats the fungal infection in the subject.

[0119] Detail 2: The method of detail 1, wherein the fungal infection is a drug-resistant fungal infection caused by a fungal strain exhibiting resistance to one or more of the following antifungal drugs: fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, nystatin, flucytosine, caspofungin, micafungin, and anidulafungin, and / or wherein the subject is immunocompromised due to a condition selected from the group consisting of diabetes, chronic obstructive pulmonary disease, asthma, cystic fibrosis, cystic fibrosis, bronchiectasis, bronchopulmonary aspergillosis, and recurrent mycoses.Docket No.: 405002-558001WO

[0120] Detail 3: The method of detail 1, wherein the substances released by the healing bacteria comprise one or more volatile compounds including: dimethyl disulfide at a concentration of 0.1-100 μg / ml, dimethyl trisulfide at a concentration of 0.1-100 μg / ml, dimethyl tetrasulfide at a concentration of 0.1-100 μg / ml, methanethiol at a concentration of 0.1-100 μg / ml, hydrogen sulfide at a concentration of 0.1-100 μg / ml, benzothiazole at a concentration of 0.1-100 μg / ml, one or more ketones at a total concentration of 0.1-100 μg / ml, one or more aldehydes at a total concentration of 0.1-100 μg / ml, one or more esters at a total concentration of 0.1-100 μg / ml, one or more alcohols at a total concentration of 0.1-100 μg / ml, one or more terpenoids at a total concentration of 0.1-100 μg / ml, one or more benzenes at a total concentration of 0.1-100 μg / ml, one or more heterocycles at a total concentration of 0.1-100 μg / ml, ammonia at a concentration of 0.1-100 μg / ml, one or more amines at a total concentration of 0.1-100 μg / ml, one or more pyrazines at a total concentration of 0.1-100 μg / ml, one or more acids at a total concentration of 0.1-100 μg / ml, and one or more sulfur compounds at a total concentration of 0.1-100 μg / ml.

[0121] Detail 4: The method of detail 1, wherein the healing bacteria is a bacterium selected from the group consisting of: Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 15692, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus fermentum ATCC 14931, Lactobacillus plantarum ATCC 8014, Lactobacillus brevis ATCC 367, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus helveticus ATCC 15009, Lactobacillus johnsonii ATCC 33200, Lactobacillus paracasei ATCC 25302, Lactobacillus reuteri ATCC 23272, Lactobacillus rhamnosus ATCC 53103, Lactobacillus salivarius ATCC 11741, Bifidobacterium animalis subsp. lactis ATCC 27536, Bifidobacterium bifidum ATCC 29521, Bifidobacterium breve ATCC 15700, Bifidobacterium infantis ATCC 15697, Bifidobacterium longum subsp. longum ATCC 15707, Streptomyces griseus ATCC 10137, Streptomyces coelicolor A3(2), Streptomyces lividans TK24, Streptomyces avermitilis MA-4680, Streptomyces albus J1074, Streptomyces aureofaciens ATCC 10762, Streptomyces hygroscopicus subsp. hygroscopicus ATCC 53653, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ATCC 10712, Streptomyces virginiae NRRL B-1446, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1.

[0122] Detail 5: The method of detail 1, wherein the method further comprises administering another anti-fungal agent to the subject, wherein the anti-fungal agent is selected from the group consisting of fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, nystatin, flucytosine, caspofungin, micafungin, anidulafungin, terbinafine, and griseofulvin, and wherein the anti-fungal agent is administered orally, intravenously, subcutaneously,Docket No.: 405002-558001WO intramuscularly, topically, or by inhalation at a dosage of 0.01-1000 mg / kg body weight per day for 7-14 days.

[0123] Detail 6: The method of detail 1, wherein the substances released by the healing bacteria comprise one or more agents including: bacteriocins at a concentration of 0.1-100 μg / ml, lipopeptides at a concentration of 0.1-100 μg / ml, polypeptides at a concentration of 0.1-100 μg / ml, enzymes at a concentration of 0.1-100 U / ml, polyketides at a concentration of 0.1-100 μg / ml, terpenes at a concentration of 0.1-100 μg / ml, fatty acids at a concentration of 0.1-100 μg / ml, organic acids at a concentration of 0.1-100 μg / ml, alkanes at a concentration of 0.1-100 μg / ml, alkenes at a concentration of 0.1-100 μg / ml, organosulfur compounds at a concentration of 0.1- 100 μg / ml, organophosphorus compounds at a concentration of 0.1-100 μg / ml, organohalogen compounds at a concentration of 0.1-100 μg / ml, phenolic compounds at a concentration of 0.1- 100 μg / ml, quinones at a concentration of 0.1-100 μg / ml, and metal chelators at a concentration of 0.1-100 μg / ml.

[0124] Detail 7: The method of detail 1, wherein containing the substances optionally comprises: (i) containing the one or more volatile compounds released from the healing bacteria in an air-tight chamber equipped with a charcoal filter to adsorb the volatile compounds and prevent their escape; (ii) containing the one or more agents released from the healing bacteria in an air-tight chamber equipped with a 0.22 μm filter to prevent the escape of the agents; (iii) containing the substances in an air-tight chamber and continuously circulating the air within the chamber to ensure even distribution of the substances; (iv) the substances released by the healing bacteria prevent the fungal infection in the subject by inhibiting the growth and / or proliferation of the fungal pathogen; and / or (v) the substances released by the healing bacteria treat the fungal infection in the subject by killing or inducing programmed cell death in the fungal pathogen.

[0125] Detail 8: A method of making an anti-fungal composition for inhibiting a fungal infection in a subject, the method comprising: culturing a microbe selected from the group consisting of Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium,Docket No.: 405002-558001WO Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1 in a growth medium comprising a carbon source selected from the group consisting of glucose, fructose, sucrose, lactose, glycerol, mannitol, and sorbitol at a concentration of 0.1-10% (w / v), a nitrogen source selected from the group consisting of peptone, tryptone, yeast extract, meat extract, malt extract, casein hydrolysate, soybean meal, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea at a concentration of 0.1- 5% (w / v), and an inorganic salt selected from the group consisting of dipotassium phosphate, monopotassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride at a concentration of 0.01-1% (w / v), and wherein the microbe is cultured under aerobic conditions with shaking at 100-300 rpm at a temperature in the range of 25-40°C and a pH in the range of 6.0- 8.0 for a period of 24-72 hours in the presence of a fungus selected from the group consisting of Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, Saccharomyces cerevisiae, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp., wherein the fungus is resistant to one or more of the following antifungal drugs: fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, nystatin, flucytosine, caspofungin, micafungin, and anidulafungin, and wherein the microbe releases substances that inhibit the growth of the fungus; and confining the substances released by the microbe to a sealed and sterilized container, wherein the sealed and sterilized container is selected from the group consisting of a vial, a bottle, a flask, a syringe, a bag, and a pouch, and wherein the container is made of a material selected from the group consisting of glass, plastic, metal, and ceramic; wherein the substances confined in the sealed and sterilized container constitute the anti-fungal composition, and wherein the anti-Docket No.: 405002-558001WO fungal composition comprises one or more volatile compounds selected from the group consisting of dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and sulfur compounds at a total concentration of 0.1-1000 μg / ml and / or one or more agents selected from the group consisting of bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and metal chelators at a total concentration of 0.1-1000 μg / ml.

[0126] Detail 9: The method of detail 8, wherein the fungal infection is caused by a fungus selected from the group consisting of Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, Saccharomyces cerevisiae, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp., and wherein the fungal infection is resistant to one or more of the following antifungal drugs: fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, nystatin, flucytosine, caspofungin, micafungin, and anidulafungin.

[0127] Detail 10: The method of detail 8, wherein the microbe is a bacterium selected from the group consisting of Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 15692, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus fermentum ATCC 14931, Lactobacillus plantarum ATCC 8014, Lactobacillus brevis ATCC 367, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus helveticus ATCC 15009, Lactobacillus johnsonii ATCC 33200, Lactobacillus paracasei ATCC 25302, Lactobacillus reuteri ATCC 23272, Lactobacillus rhamnosus ATCC 53103, Lactobacillus salivarius ATCC 11741, Bifidobacterium animalis subsp. lactis ATCC 27536, Bifidobacterium bifidum ATCC 29521, Bifidobacterium breve ATCC 15700, Bifidobacterium infantis ATCC 15697, Bifidobacterium longum subsp. longum ATCC 15707, Streptomyces griseus ATCC 10137, Streptomyces coelicolor A3(2), Streptomyces lividans TK24, Streptomyces avermitilis MA-4680, Streptomyces albus J1074, Streptomyces aureofaciens ATCC 10762, Streptomyces hygroscopicus subsp. hygroscopicus ATCC 53653, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ATCC 10712, Streptomyces virginiae NRRL B-1446, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1.Docket No.: 405002-558001WO

[0128] Detail 11: The method of detail 8, wherein the microbe is a bacterium selected from the group consisting of Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 15692, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus fermentum ATCC 14931, Lactobacillus plantarum ATCC 8014, Lactobacillus brevis ATCC 367, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus helveticus ATCC 15009, Lactobacillus johnsonii ATCC 33200, Lactobacillus paracasei ATCC 25302, Lactobacillus reuteri ATCC 23272, Lactobacillus rhamnosus ATCC 53103, Lactobacillus salivarius ATCC 11741, Bifidobacterium animalis subsp. lactis ATCC 27536, Bifidobacterium bifidum ATCC 29521, Bifidobacterium breve ATCC 15700, Bifidobacterium infantis ATCC 15697, Bifidobacterium longum subsp. longum ATCC 15707, Streptomyces griseus ATCC 10137, Streptomyces coelicolor A3(2), Streptomyces lividans TK24, Streptomyces avermitilis MA-4680, Streptomyces albus J1074, Streptomyces aureofaciens ATCC 10762, Streptomyces hygroscopicus subsp. hygroscopicus ATCC 53653, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ATCC 10712, Streptomyces virginiae NRRL B-1446, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1, and wherein the microbe is cultured in a growth medium comprising glucose at a concentration of 1% (w / v), peptone at a concentration of 0.5% (w / v), yeast extract at a concentration of 0.3% (w / v), dipotassium phosphate at a concentration of 0.1% (w / v), magnesium sulfate at a concentration of 0.05% (w / v), and sodium chloride at a concentration of 0.05% (w / v), and wherein the microbe is cultured under aerobic conditions with shaking at 200 rpm at a temperature of 30°C and a pH of 7.0 for a period of 48 hours.

[0129] Detail 12: The method of detail 8, wherein the substances released by the microbe comprise one or more volatile compounds selected from the group consisting of dimethyl disulfide at a concentration of 1-50 μg / ml, dimethyl trisulfide at a concentration of 1-50 μg / ml, dimethyl tetrasulfide at a concentration of 1-50 μg / ml, methanethiol at a concentration of 1-50 μg / ml, hydrogen sulfide at a concentration of 1-50 μg / ml, benzothiazole at a concentration of 1-50 μg / ml, one or more ketones at a total concentration of 1-50 μg / ml, one or more aldehydes at a total concentration of 1-50 μg / ml, one or more esters at a total concentration of 1-50 μg / ml, one or more alcohols at a total concentration of 1-50 μg / ml, one or more terpenoids at a total concentration of 1-50 μg / ml, one or more benzenes at a total concentration of 1-50 μg / ml, one or more heterocycles at a total concentration of 1-50 μg / ml, ammonia at a concentration of 1-50 μg / ml, one or more amines at a total concentration of 1-50 μg / ml, one or more pyrazines at a total concentration of 1-50 μg / ml, one or more acids at a total concentration of 1-50 μg / ml, and one or more sulfur compounds at a total concentration of 1-50 μg / ml.Docket No.: 405002-558001WO

[0130] Detail 13: The method of detail 8, wherein the substances released by the microbe comprise one or more agents selected from the group consisting of bacteriocins at a concentration of 1-50 μg / ml, lipopeptides at a concentration of 1-50 μg / ml, polypeptides at a concentration of 1- 50 μg / ml, enzymes at a concentration of 1-50 U / ml, polyketides at a concentration of 1-50 μg / ml, terpenes at a concentration of 1-50 μg / ml, fatty acids at a concentration of 1-50 μg / ml, organic acids at a concentration of 1-50 μg / ml, alkanes at a concentration of 1-50 μg / ml, alkenes at a concentration of 1-50 μg / ml, organosulfur compounds at a concentration of 1-50 μg / ml, organophosphorus compounds at a concentration of 1-50 μg / ml, organohalogen compounds at a concentration of 1-50 μg / ml, phenolic compounds at a concentration of 1-50 μg / ml, quinones at a concentration of 1-50 μg / ml, and metal chelators at a concentration of 1-50 μg / ml.

[0131] Detail 14: The method of detail 12, wherein confining the substances released by the microbe comprises passing the culture medium containing the microbe and the substances through a 0.22 μm filter to remove the microbe and collecting the filtrate containing the substances in the sealed and sterilized container, and wherein the filtrate is concentrated by evaporation, lyophilization, or ultrafiltration to increase the concentration of the volatile compounds to 1-1000 μg / ml prior to being collected in the sealed and sterilized container.

[0132] Detail 15: The method of detail 13, wherein confining the substances released by the microbe comprises passing the culture medium containing the microbe and the substances through a 0.22 μm filter to remove the microbe and collecting the filtrate containing the substances in the sealed and sterilized container, and wherein the filtrate is concentrated by evaporation, lyophilization, or ultrafiltration to increase the concentration of the agents to 1-1000 μg / ml prior to being collected in the sealed and sterilized container.

[0133] Detail 16: The method of detail 8, wherein the anti-fungal composition prevents the fungal infection in the subject by inhibiting the growth and / or proliferation of the fungal pathogen when administered to the subject at a dosage of 0.01-10 mg / kg body weight per day for 7-14 days.

[0134] Detail 17: The method of detail 8, wherein the anti-fungal composition treats the fungal infection in the subject by killing the fungal pathogen or inducing programmed cell death in the fungal pathogen when administered to the subject at a dosage of 0.01-10 mg / kg body weight per day for 7-14 days.

[0135] Detail 18: A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air-tight chamber configured to surround the human subject, whereinDocket No.: 405002-558001WO the air-tight chamber is equipped with an air inlet and an air outlet, and wherein the air-tight chamber is made of a material selected from the group consisting of plastic, glass, metal, and ceramic; a culture of a healing bacteria selected from the group consisting of Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1 disposed inside the air-tight chamber, wherein the healing bacteria is cultured in a growth medium comprising a carbon source selected from the group consisting of glucose, fructose, sucrose, lactose, glycerol, mannitol, and sorbitol at a concentration of 0.1-10% (w / v), a nitrogen source selected from the group consisting of peptone, tryptone, yeast extract, meat extract, malt extract, casein hydrolysate, soybean meal, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea at a concentration of 0.1-5% ( w / v), and an inorganic salt selected from the group consisting of dipotassium phosphate, monopotassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride at a concentration of 0.01-1% (w / v), and wherein the healing bacteria is cultured under aerobic conditions with shaking at 100-300 rpm at a temperature in the range of 25-40°C and a pH in the range of 6.0-8.0; and a HEPA filter disposed over the culture of the healing bacteria, wherein the HEPA filter has a pore size of 0.3 μm or less and is configured to prevent the passage of the healing bacteria while allowing the passage of the one or more volatile compounds and / or one or more agents released by the healing bacteria; wherein the air inlet is equipped with a fan configured to draw air into the air-tight chamber and the air outlet is equippedDocket No.: 405002-558001WO with a fan configured to expel air out of the air-tight chamber, and wherein the fans are configured to operate at a flow rate of 1-10 liters per minute; whereby cultivating the healing bacteria in the air-tight chamber in the vicinity of the subject allows the one or more volatile compounds and / or one or more agents released by the healing bacteria to contact the subject and prevent and / or treat the fungal infection in the subject, wherein the fungal infection is caused by a fungus selected from the group consisting of Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, Saccharomyces cerevisiae, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp., and wherein the fungal infection is resistant to one or more of the following antifungal drugs: fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, nystatin, flucytosine, caspofungin, micafungin, and anidulafungin.

[0136] Detail 19: The method of detail 8, further comprising a device for carrying out the method, the device comprising: a sealed and sterilized container configured to confine the substances released by the microbe, wherein the sealed and sterilized container is selected from the group consisting of a vial, a bottle, a flask, a syringe, a bag, and a pouch, and wherein the container is made of a material selected from the group consisting of glass, plastic, metal, and ceramic; an air-tight enclosure configured to surround the fungus, wherein the air-tight enclosure is equipped with an air inlet and an air outlet, and wherein the air-tight enclosure is made of a material selected from the group consisting of plastic, glass, metal, and ceramic; a culture of the microbe disposed within the air-tight enclosure, wherein the microbe is cultured in a growth medium comprising a carbon source selected from the group consisting of glucose, fructose, sucrose, lactose, glycerol, mannitol, and sorbitol at a concentration of 0.1-10% (w / v), a nitrogen source selected from the group consisting of peptone, tryptone, yeast extract, meat extract, malt extract, casein hydrolysate, soybean meal, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea at a concentration of 0.1-5% (w / v), and an inorganic salt selected from the group consisting of dipotassium phosphate, monopotassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride at a concentration of 0.01-1% (w / v), and wherein the microbe is cultured under aerobic conditions with shaking at 100-300 rpm at a temperature in the range of 25-40°C and a pH in the range of 6.0-8.0; and an air filter disposed over the culture of the microbe, wherein the air filter has a pore size of 0.22 μm or less and is configured to prevent the passage of the microbe while allowing the passage of the substances released by the microbe; whereinDocket No.: 405002-558001WO the air inlet is equipped with a fan configured to draw air into the air-tight enclosure and the air outlet is equipped with a fan configured to expel air out of the air-tight enclosure, and wherein the fans are configured to operate at a flow rate of 1-10 liters per minute; whereby culturing the microbe in the air-tight enclosure in the presence of the fungus allows the substances released by the microbe to contact and inhibit the growth of the fungus, and whereby the substances can be confined in the sealed and sterilized container to produce the anti-fungal composition.

[0137] Detail 20: A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air-tight chamber configured to surround the human subject, wherein the air-tight chamber is equipped with an air inlet and an air outlet, and wherein the air-tight chamber is made of a material selected from the group consisting of plastic, glass, metal, and ceramic; a culture of a healing bacteria selected from the group consisting of Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and Chromobacterium vaccinii BUC-1 disposed inside the air-tight chamber, wherein the healing bacteria is cultured in a growth medium comprising glucose at a concentration of 1% (w / v), peptone at a concentration of 0.5% (w / v), yeast extract at a concentration of 0.3% (w / v), dipotassium phosphate at a concentration of 0.1% (w / v), magnesium sulfate at a concentration of 0.05% (w / v), and sodium chloride at a concentration of 0.05% (w / v), and wherein the healing bacteria is cultured under aerobic conditions with shaking at 200 rpm at a temperature of 30°C and a pH of 7.0; and a HEPA filter disposed over the culture of the healingDocket No.: 405002-558001WO bacteria, wherein the HEPA filter has a pore size of 0.3 μm and is configured to prevent the passage of the healing bacteria while allowing the passage of the one or more volatile compounds and / or one or more agents released by the healing bacteria; wherein the air inlet is equipped with a fan configured to draw air into the air-tight chamber at a flow rate of 5 liters per minute and the air outlet is equipped with a fan configured to expel air out of the air-tight chamber at a flow rate of 5 liters per minute; whereby cultivating the healing bacteria in the air-tight chamber in the vicinity of the subject allows the one or more volatile compounds selected from the group consisting of dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and sulfur compounds at a total concentration of 10-500 μg / ml and / or the one or more agents selected from the group consisting of bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and metal chelators at a total concentration of 10-500 μg / ml released by the healing bacteria to contact the subject for 2-8 hours per day for 7-14 days and prevent and / or treat a fungal infection caused by a fungus selected from the group consisting of Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, and Saccharomyces cerevisiae, wherein the fungal infection is resistant to one or more of the following antifungal drugs: fluconazole, amphotericin B, caspofungin, and itraconazole, and wherein the subject is immunocompromised due to a condition selected from the group consisting of HIV / AIDS, cancer, organ transplantation, stem cell transplantation, and prolonged corticosteroid therapy.

[0138] Detail 21: A method of preventing and / or treating a fungal infection in a subject, the method comprising: cultivating a healing bacteria in the vicinity of the subject to release one or more volatile compounds and / or one or more agents; and containing the one or more volatile compounds and / or the one or more agents to continue contact with the subject; whereby the one or more volatile compounds and / or the one or more agents prevent and / or treat the fungal infection in the subject.

[0139] Detail 22: The method of detail 21, wherein the subject has a fungal infection caused by a pathogenic fungus selected from the group consisting of Candida species, Aspergillus species, Cryptococcus species, Trichophyton species, Microsporum species, Epidermophyton species, and combinations thereof, is susceptible to a fungal infection due to a compromised immune system resulting from a condition selected from the group consisting of HIV / AIDS,Docket No.: 405002-558001WO cancer, diabetes, organ transplantation, and combinations thereof, or is suspected of having a fungal infection based on presenting symptoms including skin rashes, itching, burning, redness, swelling, discharge, and combinations thereof.

[0140] Detail 23: The method of detail 21, wherein cultivating the healing bacteria comprises cultivating the healing bacteria selected from the group consisting of Lactobacillus species, Bifidobacterium species, Bacillus species, Streptomyces species, and combinations thereof, such that the subject is contacted with the one or more volatile compounds and / or the one or more agents released from the healing bacteria, wherein the one or more volatile compounds are selected from the group consisting of short-chain fatty acids, terpenes, aromatic compounds, sulfur compounds, and combinations thereof, and the one or more agents are selected from the group consisting of antimicrobial peptides, bacteriocins, enzymes, organic acids, and combinations thereof.

[0141] Detail 24: The method of detail 21, wherein containing the one or more volatile compounds and / or the one or more agents comprises containing the one or more volatile compounds and / or the one or more agents released from the healing bacteria in a smart enclosure equipped with sensors for monitoring environmental conditions and the subject's vital signs, and actuators for controlling the release and concentration of the volatile compounds and / or agents to optimize their antifungal efficacy while minimizing any potential adverse effects on the subject.

[0142] Detail 25: The method of detail 21, further comprising obtaining the subject prior to cultivating the healing bacteria, wherein obtaining the subject comprises: a) collecting a detailed medical history of the subject including any prior fungal infections, underlying health conditions, medications, and lifestyle factors; b) conducting a thorough physical examination of the subject to identify any signs and symptoms suggestive of a fungal infection; c) performing appropriate diagnostic tests such as skin scrapings, nail clippings, blood tests, and imaging studies to confirm the presence and type of fungal infection; and d) assessing the subject's suitability and willingness to undergo the healing bacteria-based treatment.

[0143] Detail 26: The method of detail 25, wherein the subject obtained has a confirmed diagnosis of a fungal infection based on the presence of fungal elements such as hyphae, pseudohyphae, or yeast cells in the collected clinical specimens as determined by microscopic examination, fungal culture, or molecular methods such as polymerase chain reaction (PCR).

[0144] Detail 27: The method of detail 25, wherein the subject obtained is susceptible to a fungal infection due to the presence of one or more risk factors such as a weakened immuneDocket No.: 405002-558001WO system, prolonged antibiotic use, poor hygiene, occlusive clothing or footwear, exposure to contaminated soil or surfaces, and genetic predisposition, as determined by the comprehensive medical history and physical examination.

[0145] Detail 28: The method of detail 25, wherein the subject obtained is suspected of having a fungal infection based on the presence of characteristic clinical manifestations such as scaly, pruritic, erythematous skin lesions, discolored or dystrophic nails, oral thrush, vaginal discharge, or pulmonary infiltrates, in the absence of a definitive laboratory diagnosis.

[0146] Detail 29: The method of detail 21, wherein the healing bacteria is cultivated in the vicinity of the subject in a state-of-the-art bioreactor system that provides optimal growth conditions such as temperature, pH, oxygen levels, and nutrient availability, and is equipped with advanced monitoring and control features to ensure the consistent production and release of the desired volatile compounds that exert potent antifungal activities through multiple mechanisms such as disrupting fungal cell membranes, inhibiting fungal enzymes, and modulating the host immune response.

[0147] Detail 30: The method of detail 21, wherein the healing bacteria is cultivated in the vicinity of the subject in a specially designed probiotic formulation that contains the live bacteria along with prebiotic substrates, micronutrients, and other bioactive compounds that enhance their growth, survival, and functionality, and is administered to the subject through various routes such as oral, topical, or inhalation, to deliver the beneficial agents that combat fungal infections by producing antifungal metabolites, competing for nutrients and adhesion sites, and stimulating the host's natural defenses.

[0148] Detail 31: The method of detail 21, wherein containing the one or more volatile compounds comprises containing the one or more volatile compounds released from the healing bacteria in an airtight chamber or suit that surrounds the subject and is equipped with filters, adsorbents, and catalysts that capture, retain, and degrade the volatile compounds, allowing for their prolonged and targeted delivery to the subject's skin, mucous membranes, and respiratory tract, where they exert their antifungal effects by inducing fungal cell apoptosis, inhibiting fungal biofilm formation, and promoting the growth of commensal microbes.

[0149] Detail 32: The method of detail 21, wherein containing the one or more agents comprises containing the one or more agents released from the healing bacteria in a smart bandage or patch that is applied directly to the affected area of the subject's skin or nails and is composed of a multilayered matrix that incorporates the agents along with other therapeuticDocket No.: 405002-558001WO compounds such as antioxidants, anti-inflammatory drugs, and growth factors, and provides sustained and controlled release of the agents through various mechanisms such as diffusion, erosion, or stimulus-responsive activation, to achieve localized and synergistic antifungal activity.

[0150] Detail 33: The method of detail 21, wherein the one or more volatile compounds prevent the fungal infection in the subject by creating an inhospitable environment for fungal growth and survival through various mechanisms such as lowering the pH, generating reactive oxygen species, and disrupting fungal cell wall synthesis, as well as by priming the subject's innate and adaptive immune responses to recognize and eliminate fungal pathogens more effectively.

[0151] Detail 34: The method of detail 21, wherein the one or more volatile compounds treat the fungal infection in the subject by exerting direct fungicidal or fungistatic activities through various mechanisms such as binding to fungal cell surface proteins, inhibiting fungal metabolic pathways, and inducing fungal programmed cell death, as well as by modulating the subject's immune system to enhance phagocytosis, cytokine production, and T-cell activation against fungal antigens.

[0152] Detail 35: The method of detail 21, wherein the one or more agents prevent the fungal infection in the subject by forming a protective barrier on the subject's skin or mucous membranes that prevents the adhesion and invasion of fungal cells, as well as by stimulating the production of the subject's own antimicrobial peptides and immunoglobulins that neutralize fungal virulence factors and toxins.

[0153] Detail 36: The method of detail 21, wherein the one or more agents treat the fungal infection in the subject by selectively targeting and killing fungal cells through various mechanisms such as permeabilizing fungal cell membranes, inhibiting fungal protein synthesis, and inducing fungal mitochondrial dysfunction, as well as by enhancing the subject's cell-mediated immunity to promote the clearance of fungal-infected cells and the resolution of inflammation.

[0154] Detail 37: The method of detail 21, wherein the fungal infection is prevented in the subject as evidenced by the absence of any clinical signs and symptoms of fungal infection, the negative results of fungal diagnostic tests, and the maintenance of a healthy and diverse skin and mucosal microbiome, following the administration of the healing bacteria-based treatment.

[0155] Detail 38: The method of detail 21, wherein the fungal infection is treated in the subject as evidenced by the complete or partial resolution of the clinical manifestations of the fungal infection, the reduction or elimination of fungal burden as determined by diagnostic tests, and theDocket No.: 405002-558001WO restoration of the normal structure and function of the affected tissues, following the administration of the healing bacteria-based treatment.

[0156] Detail 39: The method of detail 21, wherein the subject is a human subject selected from the group consisting of infants, children, adolescents, adults, and the elderly, who may be immunocompromised or immunocompetent, and may have acute or chronic fungal infections affecting various body sites such as the skin, hair, nails, mucous membranes, and internal organs.

[0157] Detail 40: The method of detail 21, wherein the healing bacteria releases the one or more volatile compounds and / or the one or more agents in a pulsatile or continuous manner, at concentrations and durations that are optimized for each subject based on factors such as the type and severity of the fungal infection, the subject's age, weight, and health status, and the presence of any concomitant medications or treatments, as determined by a personalized algorithm that integrates data from the subject's medical records, real-time monitoring devices, and predictive models of fungal growth and host response.

[0158] Detail 41: The method of detail 21, wherein the healing bacteria is genetically engineered to overexpress one or more genes encoding for the biosynthesis of the volatile compounds and / or agents, or to express heterologous genes derived from other antifungal microbes or plants, using cutting-edge molecular biology techniques such as CRISPR-Cas9, metabolic engineering, and synthetic biology, to enhance the potency, specificity, and safety of the healing bacteria-based treatment.

[0159] Detail 42: The method of detail 21, wherein the healing bacteria is formulated into a smart probiotic that is encapsulated in a pH-responsive, mucoadhesive, and self-healing hydrogel that protects the bacteria from the harsh environmental conditions of the human body, targets them to the site of fungal infection, and releases them in a controlled and sustained manner, along with other bioactive compounds that potentiate their antifungal efficacy, such as enzymes that degrade fungal biofilms, antibodies that neutralize fungal antigens, and nanoparticles that deliver antifungal drugs.

[0160] Detail 43: The method of detail 21, wherein the one or more volatile compounds and / or the one or more agents are purified and concentrated from the healing bacteria culture using advanced separation and purification technologies such as supercritical fluid extraction, nanofiltration, and affinity chromatography, and are formulated into a range of dosage forms such as creams, lotions, sprays, gels, ointments, and aerosols, that are customized for each subject based on their preferences, compliance, and response to the treatment.Docket No.: 405002-558001WO

[0161] Detail 44: The method of detail 21, wherein the efficacy and safety of the healing bacteria-based treatment is monitored and validated using a combination of clinical, microbiological, and immunological endpoints, such as the resolution of fungal infection symptoms, the reduction of fungal burden in clinical specimens, the improvement of skin and mucosal barrier function, the modulation of inflammatory and antimicrobial responses, and the absence of adverse events or unintended effects on the subject's health and quality of life.

[0162] Detail 45: The method of detail 21, wherein the healing bacteria-based treatment is used as a standalone therapy or as an adjunct to conventional antifungal medications such as azoles, echinocandins, and polyenes, to improve their efficacy, reduce their side effects, and prevent the development of fungal resistance, and is personalized for each subject based on their individual characteristics, preferences, and responses, using a holistic and integrative approach that combines the best of natural and synthetic antifungal strategies.

[0163] Any of the discussion points above can be inter-combined with any aspect(s) of the Examples presented further below or any other feature or aspect herein.

[0164] Example life-saving problem to be addressed: Insufficient anti-fungal therapies. Fungal diseases are emerging as an underappreciated cause of morbidity and mortality. In October of 2022, the World Health Organization made the first global effort to address fungal disease by releasing the Fungal Pathogens Priority List (FPPL) [1]. This list systematically classifies 19 fungi based on their need for research and development, as well as impacts on public health. Members of the Candida, Aspergillus, and Cryptococcus genera were assigned the most critical priority [1]. The FPPL comes in response to rising global presence of invasive fungal infections (IFIs), where fungi are responsible for around 13 million serious infections and over 1.5 million deaths on an annual basis [2]. The CDC issued similar warnings in its 2019 report on antibiotic resistance threats in the United States [3].

[0165] In the US alone, IFIs account for $11.5 billion of economic burden annually [4]. IFIs disproportionately impact immunocompromised patients, experiencing a mortality rate that is 50% higher than non-immunocompromised IFI patients [5]. The SARS-CoV-2 (COVID-19) pandemic showcased the insidious nature of fungal pathogens, even among immunocompetent patients. In the United States, around 15% of COVID-19 patients admitted to the intensive care unit experienced concurrent Aspergillus infections [6], increasing to 20% of patients when looking globally [7, 8]. These patients subsequently experienced a 50% higher mortality than other COVID-19 patients admitted to the intensive care unit [7].Docket No.: 405002-558001WO

[0166] Current treatments for IFIs have limitations. Amphotericin B is a common agent utilized in patients with serious fungal infections. Despite improvements in the formulation, amphotericin B has long been associated with toxicity that sometimes warrants discontinuation of therapy [9]. Most common antifungal agents are associated with less toxicity but have been impacted by the challenge of increasing antimicrobial resistance. Concerns for resistance are illustrated by the emergence of Candida auris on several continents over the last decade, with isolates demonstrating resistance to fluconazole, amphotericin B, and echinocandins

[0010] . An increase in resistance to echinocandins has been widely documented in Candida glabrata isolates

[0011] , with some evidence of resistance emerging among Candida albicans

[0012] .

[0167] Azole resistance amongst Aspergillus spp., with specific emphasis on the common pathogen Aspergillus fumigatus, has been documented in the U.S. and around the globe over the last several decades

[0013] . Rates of selection for resistance among Aspergillus species have been increasing across the globe over time [14-16], posing concerns for treatment failure. Numerous studies have demonstrated increased mortality in patients with azole-resistant invasive Aspergillosis [13, 14, 17-19]. Further adding to the complexity of the problem is the observation than fungi themselves have been adapting to human hosts. Specifically, climate change promotes structural and metabolic adaptations, leading to resistance against many stressors including antifungal agents and host defenses

[0020] . Therefore, treatment alternatives must be investigated.

[0168] Antifungal therapeutic development has not risen to meet the needs demonstrated by the emergence of resistance and limitations of current therapies

[0021] . Over the last ten years only one new agent from the known classes of antifungals has been approved, while there have been no new classes of antifungals introduced over the last twenty years

[0022] . There have been some encouraging developments in the antifungal pipeline though unmet needs remain, especially with molds

[0021] . The U.S. Centers for Disease Control and Prevention has highlighted the need for new therapeutics and taken efforts to encourage collaborations between academia and industry

[0023] . Given the toxicity of current treatments and the need for innovative therapies, particularly in immunocompromised patients, additional research is required into new agents and delivery mechanisms, such as inhaled therapies

[0023] .

[0169] The long-term objective of this technology is to develop new antifungal compounds using a novel chemical source. The primary objective of this technology is to identify and characterize the production and the target(s) of antifungal compound(s) that are responsible for the pronounced antifungal activity that we have characterized in our preliminary data. When the proposed aims of this project are completed successfully, we will have defined the biosyntheticDocket No.: 405002-558001WO machinery, identified the fungal target, and assessed activity against a broad range of fungi.

[0170] Example Summary of Significance: The incidence of invasive fungal infections is rising as immune suppression, intercurrent illnesses, and invasive procedures continue to increase in humans. Antifungal resistance is also increasing, such as the emergence of resistant organisms like C. auris. New antifungal compounds with novel mechanisms of action are needed. EXAMPLES

[0171] Example Impact statement: further completion of the projects disclosed herein will create foundational knowledge in developing new antifungal, change concepts in the field of antimicrobial discovery, and generate technical advances in Chromobacterium research. INTRODUCTION EXAMPLE. TESTING ANTI-FUNGAL COMPOUNDS DERIVED FROM CHROMOBACTERIUM VACCINII IN VIEW OF CONCEPTUAL INTRODUCTIONS: NOVEL CHEMICAL SPACE

[0172] At the outset, we have discovered for the first time that C. vaccinii produces substance(s) that inhibit the growth of a wide range of fungi, from yeasts to molds, that are pathogenic to humans. This phenotype has been reported in the past looking at environmental molds, but never human pathogens. The phenotype is observed when C. vaccinii and fungi grow on separate plates but share the same air space, implicating a volatile compound. The compound(s) block growth that creates dysmorphic fungal elements when viewed by electron microscopy. The compound(s) are not cytotoxic to human cells. This finding also provides a guide to identifying a new drug target in fungi, which could be used to screen for other anti-fungal compounds. Note, we have not yet isolated the compound(s) released from the C. vaccinii and therefore do not have chemical structure(s) to patent.

[0173] This invention provides a new approach to treating patients infected with fungal infections, yeasts or molds. Physicians who treat patients with fungal infections will use this, particularly in patients with immunosuppression such as organ transplant recipients or patients with prolonged neutropenia because they are at higher risk for more serious, invasive fungal infections.

[0174] Mining for novel antifungal agents is limited, in part, by the availability of chemical compounds to test. This research proposal develops a new, relatively unexplored chemical space of volatile compounds. There are multiple reports of bacteria which can inhibit the growth of fungi through emission of volatile compounds (VCs) [24-27]. These bacteria produce a variety of different chemical compounds, which in turn have distinct mechanisms against select fungi

[0028] . Chromobacterium vaccinii is one such bacterium that produces VCs capable of inhibiting the growth of environmental molds

[0029] . These bacteria have been found to produce many VCs, andDocket No.: 405002-558001WO may even change VC production in order to inhibit the growth of molds

[0029] . The antifungal activity of bacterial VCs against environmental molds has been explored as a potential biocontrol agent

[0028] . To our knowledge, investigation of VC activity against human fungal pathogens has been lacking until now. Our data show how Chromobacterium species, C. vaccinii in particular, inhibits the growth of a wide range of human fungal pathogens. Of particular interest to this RFA-AI-24- 065 is that our preliminary data show inhibition of both Aspergillus fumigatus and Candida auris.

[0175] Technical: new genetic tools applied to Chromobacterium. C. violaceum is a recognized human pathogen and research has been conducted to understand its virulence mechanisms

[0030] . Genetic tools to study Chromobacterium species are, however, limited. This technology investigates the genetic elements responsible for the production of antifungal VCs. In doing so, we have developed and are refining a suite of tools to manipulate the genomes of Chromobacterium species. These tools will be a contribution to the general Chromobacterium research community.

[0176] 1. Antifungal activity and preliminary data supporting it. This technology builds on our observations that a VC from C. vaccinii inhibits fungal growth. Our model uses a “sandwich” plate assay to create a closed system of co-culture that allowed free gas exchange without contact between microorganisms. Two agar plates are joined with petri dish sealing tape (Petri-Seal™, RPI Corp), one being a bacterial agar plate on the bottom and an agar plate for fungal growth on the top. King’s media B (KMB) agar is used to grow a lawn of Chromobacterium organisms, and a Sabouraud dextrose agar (SDA) plate is inoculated with the fungus being tested. FIG.1E and FIG.1F show Candida albicans SC5314 growth is strongly inhibited by the lawn of 2 different C. vaccinii isolates in the sandwich plate assay. The plates (FIG.1E) need not be taped directly together because plates cultivated in an airtight box (Mitsubishi Gas Chemical Co., Inc.) show that C. vaccinii inhibits yeast growth (not shown). In contrast, a sheet of aluminum foil positioned between the two plates blocks the antifungal activity in the sandwich plate assay (not shown). The antimicrobial effect is restricted to antifungal activity because bacterial growth, e.g. E. coli, is minimally affected in a sandwich plate assay with C. vaccinii (not shown).

[0177] 2. Broad spectrum antifungal activity. We have tested C. vaccinii against other human fungal pathogens, including two relevant to RFA-AI-24-065. Two additional yeasts have been tested; the growth of both Cryptococcus neoformans (FIG.6A, FIG.6B) and Candida auris (FIG.6C, FIG.6D) are significantly inhibited by C. vaccinii in the sandwich plate assay. FIG.2A and FIG.2B show the growth of Aspergillus fumigatus growth is profoundly inhibited in sandwich plate assays with C. vaccinii. In addition, we have seen that A. niger growth is inhibited by C.Docket No.: 405002-558001WO vaccinii (data not shown). We acquire additional fungal strains with drug resistance and test Mucorales as described in the Approach section below. To our knowledge, this is the first instance where volatile compounds (VCs or VOCs) have demonstrated activity against human pathogens

[0178] 3. Antifungal specificity. While we have observed activity against all fungi tested to date, the effects of C. vaccinii VCs appear restricted to fungi. As mentioned, other bacteria have limited responses to C. vaccinii in sandwich plate assays (data not shown). We have also tested mammalian cells for susceptibility to C. vaccinii VCs and not seen an effect. Here, human A549 cells grown for 10 days on the apical surface of a transwell were co-cultured with C. vaccinii in an airtight box, the same conditions that inhibit C. albicans growth. The viability of the A549 cells, however at the air-liquid interface was not affected (FIG.4B).

[0179] 4. New genetic tools for Chromobacterium. Limited genetic work has been performed on Chromobacterium species. For example, a published plasmid is no longer available due to a PI’s retirement

[0031] . A protocol available online for transposon mutagenesis does not appear to have been published

[0032] . Allelic exchange has, however, been described in C. violaceum using standard methodology

[0033] . In our preliminary studies we have found a plasmid that propagates in C. vaccinii and that expresses a trans-gene, the antibiotic resistance marker, using the endogenous promoter (FIG.5C). It is also maintained in E. coli (not shown). Shuttle vectors like this are central to complementation studies in bacterial genetics and will be used to verify that phenotypes associated with site-directed mutagenesis are due to the mutation that was introduced (e.g.

[0034] ).

[0180] Site-directed mutagenesis requires a suicide plasmid that cannot be propagated in the target organism [35, 36]. Our studies have identified four different plasmids in our collection that will serve as suicide vectors in C. vaccinii (data not shown). Importantly, they are equipped with an oriT element to transfer the plamid by conjugation, a strategy that we have found central to increasing the number of transformants

[0036] . Using a suicide vector and conjugation, we have successfully created an insertion mutant with stable expression of antibiotic resistance in C. vaccinii (FIG.6E). We have recently taken this one step further by inserting an internal sequence of the vioE gene into the same suicide vector. After triparental mating, growth on selective media showed white colonies compatible with vioE inactivation (FIG.7). We have since verified the stable heritability of the resistance and the white phenotype, along with confirming the white colonies are Chromobacterium. Genomic analysis and complementation studies are ongoing.

[0181] Innovation: a new chemistry source for antimicrobials, the volatilome, represents a new field of research. The antifungal activity from C. vaccinii is specific for yeasts and molds butDocket No.: 405002-558001WO not bacteria or mammalian cells. New genetic tools for Chromobacterium are being refined, which will facilitate genetics research into this genus. EXAMPLE 1. EXPERIMENTAL APPROACH, SPECIFIC AIM 1, DEFINE THE BIOSYNTHESIS OF CHROMOBACTERIUM ANTIFUNGAL VCS

[0182] Overall Strategy. The strategy outlined below is designed to identify the mechanism of production and mechanism of action of the antifungal present in Chromobacterium VCs. The two-pronged approach investigates the source of the VCs in Chromobacterium using a combined -omics and bacterial genetics experiments. The second prong uses -omics and genetic screens to characterize where the VCs act. The third aim is designed to test an array of fungal pathogens and verify our preliminary results of a broad spectrum of activity.

[0183] Scientific Premise, Rigor of Prior Research, and Review of Relevant Literature: The scientific premise is embodied in our overarching hypothesis: the volatilome is an understudied source of antifungal compounds, and in bioactive compounds in general. The volatilome is increasingly recognized for its complexity and biological activity

[0037] . Recent work has demonstrated certain bacteria can inhibit the growth of environmental fungi that are non- pathogenic to humans [29, 38, 39]. Some prior work has identified volatile compounds from cultures with antifungal properties but not isolated the specific bioactive components. Moreover, the prior work has not performed mechanistic studies to define specific biosynthetic pathways in bacteria responsible for the production of antifungal VCs. Similarly, the mechanism of how VCs affect fungi is poorly characterized. Importantly, this prior work has not studied VC effects on human fungal pathogens.

[0184] The PI’s laboratory has substantial experience with developing genetic tools and genetic modifications in bacteria [35, 36, 40-43]. The laboratory has a history of transcription analysis [35, 44-46], with more recent work in RNA-seq together with our collaborators

[0047] .

[0185] Aim 1: Define the biosynthesis of Chromobacterium antifungal VCs

[0186] The objective of this aim is to identify and mutate key genes / pathways that are associated with C. vaccinii’s antifungal phenotype. We have established that all five Chromobacterium species we have tested (vaccinii, violaceum, subtsugae, alticapitis, sinusclupearum), inhibit the growth of C. albicans (FIG.1E and FIG.1F, additional data not shown). We also know that Staphylococcus aureus MRSA LAC300, the E. coli type strain MG1655, and Pseudomonas aeruginosa model strain PA-14 are unable to inhibit the growth of C. albicans (data not shown). We refine this search for other bacteria demonstrating antifungal activity by selecting near neighbors of the Chromobacterium genus. Antifungal phenotypes willDocket No.: 405002-558001WO be correlated with genotypes in a genomics analysis. We plan to leverage other observations about Chromobacterium species and exploit transcriptomics to identify relevant genes / operons / pathways responsible for VC synthesis. Site-directed mutagenesis will then be used to confirm the correlations made using genomics and transcriptomics.

[0187] Experimental Approach; Aim 1.1. Genomics analysis (R21): Our objective is to identify genetic features in the Chromobacterium genus that correlate with antifungal phenotype. While E. coli does not inhibit fungal growth, its phylogenetic distance from the Chromobacterium genus is great (FIG.8A). Comparing their genomes using zDB

[0048] , there are several hundred genomic loci that could account for the Chromobacterium antifungal phenotype (FIG.9), which would be too many to study using site-directed mutagenesis.

[0188] To reduce the number of possibilities, we continue our collaboration with other laboratories to compare genomes of bacteria more closely related to the Chromobacterium genus. The ultimate goal is to identify genes / operons that correlate with the antifungal phenotype. The Chromobacterium genus is in the Chromobacteriaceae family within the Neisseriales order of betaproteobacteria (FIG.8B). Therefore, our five Chromobacterium genomes will be compared in silico with other Neisseriales representatives.

[0189] We have selected organisms representing other families within the Neisseriales order, including Neisseriaceae (Neisseria sicca, N. lactamica, Kingella kingae, Eikinella corrodens), Aquaspirillaceae (Aquaspirillum polymorphum), and Chitinibacteraceae (Iodobacter fluviatilis). Closer neighbors are to be selected from the other genera within Chromobacteriaceae, including Vogesella indigofera, Aquitalea palustris, and Pseudogulbenkiania sp. We will compare genomes using zDB

[0048] to identify genus-specific orthologous groups found in Chromobacterium spp. but not in the other related organisms. Secondary analyses of Pfam, Cluster of Orthologs Genes (COG), and KEGG annotations of the Chromobacterium-specific orthologous groups within zDB will be used to prioritize candidate genes / pathways for future study

[0048] . Bacterial strains will be purchased from ATCC to empirically test their anti-fungal phenotypes. Iterative genomic comparisons and phenotype testing will be used to narrow the list of possible genomic features that account for the antifungal phenotype.

[0190] Aim 1.2. RNA-seq and transcriptome analysis (R21+R33): Along with a genomic approach to identify genes that correlate with antifungal activity, we identify transcriptional activity that is associated with the antifungal phenotype.

[0191] We conduct RNA-seq of Chromobacterium species under conditions that have moreDocket No.: 405002-558001WO or less antifungal activity. Our studies have shown C. vaccinii lawns that are immature are less effective at inhibiting C. albicans (data not shown), which implicates density of organisms and quorum sensing in the antifungal phenotype. Therefore, we perform a kinetics analysis of the C. vaccinii transcriptome using serial sampling to measure genes / pathways induced over time. C. vaccinii RNA will be harvested from KMB agar plates using RNAzol (Molecular Research Center) and RNA quality will be confirmed with our Bioanalyzer (Agilent). Controls include C. vaccinii grown on SDA and lysogeny broth (LB) agar plates, which elicit less antifungal activity (data not shown). Library construction and sequencing will be performed by Azenta.

[0192] We plan to work closely with other laboratories to process raw RNA-seq data and perform gene expression comparisons using standard methodologies [49, 50], along with additional approaches for kinetics analysis (e.g.

[0051] ) including weighted gene coexpression network analysis (WGCNA) [52, 53], which we have used successfully

[0045] . As a complementary approach, we will adapt MaASlin2

[0054] , a tool that applies linear or mixed models to determine multivariable associations between quantitative measurements like relative antifungal activity. This will statistically identify genes or gene pathways that change in a manner correlated with antifungal activity over all the kinetic time points.

[0193] A second transcriptomics approach will leverage spontaneous variants we have discovered in C. subtsugae. We have found a spontaneous variant that has lost its ability to inhibit C. albicans growth (FIG.10). From this culture we have also isolated a revertant that regained antifungal activity. Wild type, variant, and revertant C. subtsugae will be compared by RNA-seq to identify the genes / pathways that correlate with antifungal activity. These experiments will also provide insight into this phenotypic switching in Chromobacterium. RNA will be extracted from cultures using our standard methodology as we have done previously [35, 44]. After RNA samples will be processed as described above.

[0194] Aim 1.3 Targeted gene deletions (R21): We will validate our genetic tools (FIG.5C, FIG.6E, FIG.7) in the R21 phase by targeting two well-defined systems using homologous recombination with either single or double cross-over reactions as we have done in Francisella [35, 36]. First, we will continue targeting the biosynthesis of Chromobacterium’s pigment, violacein. Violacein has a number of biological properties, including antimicrobial activity

[0055] and its synthesis is well-characterized

[0056] . Violacein is unlikely to mediate the phenotypes we have observed because its large molecular weight and high boiling point make it non-volatile. Inactivating its synthesis, however, will be necessary to transition our work to liquid phase cultures where we must eliminate confounding antifungal compounds (Aim 1.5). We will continue toDocket No.: 405002-558001WO characterize the vioE disruption strains that we have generated by the single cross-over strategy (FIG.7). Importantly, vioE is not in an operon so the white phenotype we have observed is likely due to the insertion mutation; this is currently being confirmed by genetic complementation in trans using an intact copy of vioE. Once the insertion mutation is validated using genomic PCR and complementation, we will delete the gene using our existing suicide vector

[0036] . This two-step process results in strains with markerless deletions that no longer require antibiotic selection in future experiments

[0036] .

[0195] Quorum sensing (QS) will also be targeted in this subaim. QS regulates virulence among other important functions

[0030] . cviI and cviR have been identified in C. violaceum as homologs to luxI and luxR of V. fischeri

[0030] . We have identified the cviI and cviR orthologs in C. vaccinii, which we will target using homologous recombination. Disruption of quorum sensing will directly test the possibility that this system regulates antifungal VC production (see Aim 1.2 on the relationship between density of C. vaccinii growth and antifungal activity).

[0196] Aim 1.4 Targeted gene deletions informed by -omics studies (R33): In the second phase of this research program, we target candidate genes / pathways responsible for the antifungal phenotype that are identified by the genomics and transcriptomics studies described above. Targets will be prioritized if are present only in Chromobacterium genomes and are expressed when the phenotype is present, e.g. only in wild-type C. subtsugae but not in the variant. Selection will be guided further by functional annotations of candidate genes / pathways. Initial testing will use single-crossover insertion mutations to accelerate discovery (FIG.6E and FIG.7). Mutations that abrogate antifungal activity will be studied further using double-crossover, markerless deletion mutations and trans complementation using the pVS1 plasmid (FIG.5C).

[0197] Aim 1.5 Testing conditioned media from broth cultures (R33): It will be valuable to determine if antifungal activity is present in a liquid phase of Chromobacterium cultures because the volatile component may represent only a fraction of the total amount of antifungal compound(s) that are produced. Testing conditioned media from Chromobacterium cultures, however, is complicated by the fact that other compounds are produced with known antifungal activity, e.g. violacein

[0055] . Two other antimicrobial compounds, which are also structurally too complex to be VCs, are also produced by this genus

[0057] . Our initial attempts to characterize antifungal activity in conditioned media were confounded by violacein. Removing confounding compounds will allow testing for antifungal activity in liquid phase.

[0198] An outcome of this aim is that we create a collection of markerless deletion constructs that target the biosynthesis of the confounding compounds, e.g. violacein. By iterativelyDocket No.: 405002-558001WO introducing markerless deletions in the same C. vaccinii isolate, we will generate a strain that is incapable of making compounds that might have antifungal activity in liquid cultures. Conditioned media from this multi-deletion strain will then be investigated for antifungal activity and will be compared to deletion strain(s) from Aim 1.4 that have lost antifungal VCs. Antifungal activity of conditioned media will be tested using co-culture with C. albicans and in disc diffusion assays. Conditioned media will be tested for VCs by incubating with C. albicans in our airtight box (FIG.4B). If conditioned medium from the multi-deletion strain retains antifungal activity, additional genes will be deleted using the constructs from Aim 1.4 to validate the same gene(s) / pathway(s) contribute to fungal inhibition in both liquid and VCs.

[0199] The genomics comparison and antifungal phenotype testing (Aim 1.1), generation of RNA-seq data and initiation of analysis, and mutation of candidate genes (Aim 1.3) will be performed during the R21 phase. Mutation of the candidate genes (Aim 1.3) will validate our methodology in preparation for Aim 1.4. We will integrate the -omics data to create a short list of genes / pathways that contribute to the antifungal phenotype in Chromobacterium (FIG.11). An alternative outcome of studying the genomics of other members of the Neisseriales order is that we identify non-Chromobacterium species that have a similar antifungal phenotype. In that situation we will look for genes / pathways shared between the Chromobacterium and non- Chromobacterium species. If none overlap, we will explore the non-Chromobacterium species for unique genetic loci that are responsible for an antifungal phenotype. This outcome is especially notable because it would support our overarching hypothesis that the volatilome is an understudied source of new compounds that could be investigated for new biological activities.

[0200] An important future direction is to identify the chemical compound(s) that mediate the antifungal phenotype. We have an ongoing collaboration with a Brown University researcher who is a global leader in characterizing VC, but it is premature to pursue this line of investigation. The chemical space is vast and it is impractical to attempt to identify all possible VCs without some prior knowledge to guide the chemical analysis. Identification of relevant biosynthetic pathway(s) will provide initial insight into the makeup of the relevant VCs. Deletion mutations on an isogenic background, which will be created in this aim, will reduce variables that could confound the chemical detection; this will enhance the specificity of the chemical analysis. Defining the antifungal activity in conditioned media will also simplify subsequent chemical isolation. Since the resources needed for a thorough chemical analysis exceeds the budget of this funding mechanism, we will pursue dedicated funding for chemistry studies once we have foundational studies to guide the chemical analysis. Once the chemical structure(s) of these lead compoundsDocket No.: 405002-558001WO are defined, additional investigation will advance compound development using medicinal chemistry approaches to modify compounds and to investigate alternative delivery mechanisms in aqueous solutions or in liposomal preparations.

[0201] Another future direction will involve moving the relevant functional genomic region(s) into a model organism where VC production can be controlled for industrial production. Engineering E. coli to produce the Chromobacterium VCs can be achieved using standard methodology [58, 59]. Overproduction of Chromobacterium VCs in a model organism would also facilitate the chemical identification described above.

[0202] The approaches described above have been successfully performed on a pilot / preliminary basis. Technical challenges, therefore, are unlikely. The representatives of the Neisseriales order described above were selected based on the availability of strains and genomic sequences. It is possible that these genomes will have too many differences compared to Chromobacterium, therefore identifying too many candidates. The candidate list, however, will be narrowed further by including additional genomes of other phylogenetic neighbors, thereby reducing the list of genomic loci that are specific to the Chromobacterium genus. Integrating the transcriptomics with this narrowed list of genomic loci will further reduce the number of genes / pathways that correlate with the antifungal phenotype (FIG.7).EXAMPLE 2. EXPERIMENTAL APPROACH, SPECIFIC AIM 2, IDENTIFY THE FUNGAL TARGET(S) OFCHROMOBACTERIUM VCS

[0203] The objective of this aim is to identify the systems or pathways in fungi that are being targeted by the VCs generated by Chromobacterium species. Characterizing the target of an antimicrobial agent is often performed by identifying spontaneous resistance to the agent [60, 61]. We have not, however, seen spontaneous resistance in C. albicans grown in Chromobacterium VCs (data not shown). Therefore, we will use alternative approaches to evaluate the VC mechanism of action (MOA), including genetic screens, morphology, and transcriptomics

[0060] .

[0204] Experimental approach: Aim 2.1. Screen for resistance in S. cerevisiae (R21): S. cerevisiae is a model yeast useful because of its genetic tractability, the similarity of its biochemical and metabolic pathways compared to other fungi, and the tools to perform high- throughput screens [62-64]. S. cerevisiae has been used successfully in the past to characterize antifungal VCs from another bacterium

[0039] . We have also found that S. cerevisiae growth is strongly inhibited by Chromobacterium VCs (FIG.12). In this subaim we will screen an insertion mutant library looking for S. cerevisiae strain(s) that are able to grow in the presence of C. vaccinii VCs. A commercially available, gene knockout library will be used to screen more than 6,000 S.Docket No.: 405002-558001WO cerevisiae genes (Dharmacon). Gene inactivation in S. cerevisiae is a proven strategy to identify MOA of antifungal compounds (e.g. [62, 65]). The library will be replicate plated on to rectangular SDA plates and incubated with C. vaccinii in our airtight chambers (see FIG.4B describing the methodology). The strains that are resistant to VCs will be subcultured to confirm the resistant phenotype and the identity of the inactivated gene will be confirmed by genomic PCR. Independent mutant strains will be purchased to validate the results. Inactivated genes conferring resistance will be analyzed for shared ontology, e.g. Saccharomyces Genome Database or GOrilla

[0066] .

[0205] Aim 2.2. Morphologic analysis (R21): Antimicrobial agents can cause characteristic morphological changes in their target organisms and comparison of these changes with a collection of agents can provide insight into the MOA of a new antimicrobial

[0060] . We have found that the scant growth of C. albicans exposed to Chromobacterium VCs show aberrant morphology compared to control cells, including narrowing consistent with pseudohyphae formation (FIG.3A, FIG.3B)

[0067] . We will pursue this finding by performing fungal cytological profiling of C. albicans as has been done previously

[0068] . C. albicans will be grown alone, or in the presence of VCs or subinhibitory concentrations of antifungals (caspofungin, fluconazole, amphotericin B, 5- flucytosine) on solid agar media to mimic culture conditions of the VCs. The organisms will be evaluated by fluorescence microscopy for membrane integrity and structure using Sytox and FM 4-26, DNA with DAPi, and additional EM imaging. Morphological changes induced by Chromobacterium VCs will be compared to the other antifungals to identify overlapping phenotypes. Similarities in appearance will indicate a similar MOA.

[0206] Aim 2.3. RNA-seq analysis of response to VCs (R33): Transcriptome profiling is a complementary approach to inform our understanding of an antimicrobial agent’s MOA

[0060] . Similar to the morphologic analysis described above (Aim 2.2), gene expression signatures generated by a collection of known antimicrobials have been used to classify the response to a new agent

[0060] . In this approach, it is the gene signature and not the specific genes that define the response since there are indirect and secondary gene expression changes that confound identification of the primary effect of the antibiotic

[0060] . This approach has already been applied successfully to characterize Candida responses to azoles, echinocandins, and amphotericin B [69, 70]. In this subaim we will perform a time series analysis of transcription responses to the antifungal agents described above (Aim 2.2) and the RNA changes induced by Chromobacterium VCs. The transcription signatures observed with RNA-seq will then be compared. C. albicans cultured under control conditions or exposed to antifungals or VCs will be harvested and RNADocket No.: 405002-558001WO extracted.

[0207] After validation of RNA integrity with our Bioanalyzer, library preparation and sequencing will be performed through Azenta. Our co-investigator, Dr. Alger Fredericks, will lead the analysis of the RNA-seq data with established approaches that he has used for other studies [47, 71]. Briefly, RNA sequencing reads will be aligned to the C. albicans genome using STAR aligner

[0072] and mapped reads will be separated from unmapped reads using SAMtools

[0073] . Mapped reads will then be filtered for differential gene expression (DGE) using publicly available DGE analysis software

[0074] , which utilizes the negative binomial distribution as a model to compare over-dispersion across the dataset. Values will subsequently be adjusted using a quantile maximum likelihood estimator to establish a Fisher’s exact test with improved performance, separating biological from technical variation.

[0208] Aim 2.4. Screen for resistance in C. albicans (R33): In this subaim we will apply functional genomics approaches to study MOA of Chromobacterium VCs

[0075] . Similar to S. cerevisiae, loss of function mutations in C. albicans can cause antifungal resistance, such as erg3 / erg3 strain of C. albicans that is resistant to azoles [76, 77]. Conversely, gain of function or overexpression can also generate resistance, such as overexpression of efflux pumps [76, 78]. In this subaim we will take a functional genomics approach using C. albicans libraries to screen for resistance to Chromobacterium VCs. Similar to the approach in Aim 2.1, we will screen the heterozygous deletion library from Merck, available through the Candida Genome Database, for VC resistance due to haploinsufficiency. This approach, termed genome-wide fitness test or C. albicans Fitness Test (CaFT)

[0079] , uses a collection of 5,470 heterozygous deletion strains representing 5,157 distinct genes. Each strain has unique barcodes in the up- and downstream regions of the deleted allele. Originally used to identify strains hypersensitive to antifungals

[0079] , we will query for loss of function conveying resistance. The library is arrayed in 96-well plates, which will be replica-plated on to rectangular SDA plates and co-incubated with c. vaccinii in airtight boxes. Strains showing growth will be subcultured to confirm the phenotype and relevant gene(s) will be identified by their barcode.

[0209] It is possible that haploinsufficiency does not confer resistance because of the residual, functioning allele. This will be addressed using a second Merck library available from the CGD: the “gene replacement and conditional expression” (GRACE) collection

[0080] . The 2,357 mutant strains in this collection have one allele deleted and the remaining wild-type allele under control of tet-off transcription repression system

[0080] . We will again replica-plate from the 96-well format on to rectangular SDA plates with and without tetracycline, looking for growth in SDA platesDocket No.: 405002-558001WO with tetracycline when co-cultured with C. vaccinii VCs.

[0210] A complementary approach will be used to investigate C. albicans resistance to C. vaccinii VC by exploiting overexpression, similar to prior work

[0081] . We will again collaborate with another lab to utilize a new strain collection they have helped to generate (Mahto et al. in preparation). There are ~300 genes encoding transcription factors (TFs) in the C. albicans genome; in this collection the strains have individual TF genes regulated by the tet-on system. In the presence of tetracycline there is overexpression of the TF with a corresponding change in the TF regulon. We will screen for growth of strains on SDA with tetracycline during co-culture with C. vaccinii VCs. Overexpression of a particular TF’s regulon implicates those genes in resistance to antifungals. A similar approach identified the role of Adr1 in resistance to azoles

[0082] . Our approach is a more comprehensive screen of C. albicans TFs than the previous study because all TFs are targeted in the new collection (Mahto et al. in preparation).

[0211] The morphologic and transcriptome analyses will complement and inform the functional genomics studies. Specialized, mutagenized strains or other manipulations are not required to perform the experiments in Aims 2.2 and 2.3. The results will permit rapid determination of whether C. vaccinii VCs exert stresses similar to existing antifungals. If the morphology or gene expression induced by VCs overlaps with existing antifungals, we will test existing strains that are known to be resistant to that antifungal agent. Because of the activity is mediated by the volatilome, however, we anticipate little to no phenotypic overlap with other antifungals. If there are similar responses on morphology or RNA-seq but C. albicans strains resistant to the antifungal are still susceptible to VCs, we will conclude that the morphologic or transcription similarities are due to a shared stress pathway.

[0212] Genetic screening is labor-intensive, which is why we will begin with S. cerevisiae, which has a smaller genome and therefore fewer genes to screen in the R21 phase. A positive result, i.e. restoration of S. cerevisiae growth in C. vaccinii VCs, will be translated to C. albicans. With the assistance of another researcher, homologs in C. albicans will be targeted using homologous recombination. A stable haploid line of C. albicans that this researcher helped to create and that is available in his laboratory will facilitate genetic analysis using auxotrophic haploid strains for one-step deletions

[0083] . With a larger genome, however, it is possible that other mechanisms will be present in C. albicans. Therefore, we will also pursue the C. albicans screens. If the loss of function S. cerevisiae library yields hits, i.e. mutations allow for growth in the presence of VCs, we will first screen the C. albicans double bar coded, i.e. loss of function, library.

[0213] The GRACE library covers fewer genes but may be necessary if haploinsufficiencyDocket No.: 405002-558001WO fails to identify resistant strains. If the S. cerevisiae library fails to identify candidates, we will prioritize screening the C. albicans gain of function / TF overexpression library for VC resistance. Differential gene expression measured by RNA-seq will be used to characterize the relevant TF’s regulon; the genes in that regulon will be leading candidates to explain resistance to Chromobacterium VCs. Positive findings from any of the C. albicans screens will be validated by subculturing the relevant strain from the library. In addition, phenotypes will be confirmed by purchasing individual strains from suppliers, which reduces the chances of a cross-contamination causing a false positive result. Definitive validation will be performed by creating independent mutations in C. albicans described above with guidance from another researcher.

[0214] Future directions include expanding the MOA studies to additional organisms. A. fumigatus will be a priority here because of its profound susceptibility to C. vaccinii VCs, yet there is minimal residual fungus at the inoculation site that will permit morphologic and gene expression studies (FIG.2A, FIG.2B). A. fumigatus is also phylogenetically distinct from C. albicans, allowing us to determine if the MOA is similar across a broad spectrum of fungi.

[0215] It is contemplated that genetic screens can give false positive results due to cross contamination. This will be minimized by using a library copier system with disposable pins (V&P Scientific, Inc.). This is also why positive hits will be validated by confirming the mutation in the strain, re-purchasing strains, and making an independent strain with a mutation at the same genetic locus. Morphologic and transcription profiling will be performed using subinhibitory concentrations of antifungals to permit minimal growth on agar. RNA yields may be compromised due to the antifungals. Alternatives include using higher inoculums and culturing with in broth are alternatives. EXAMPLE 3. EXPERIMENTAL APPROACH, SPECIFIC AIM 3, TEST CHROMOBACTERIUM VCS AGAINST CLINICAL ISOLATES AND DRUG-RESISTANT STRAINS

[0216] Our experiments thus far have been conducted with an established laboratory strain of C. albicans and an antifungal susceptible strain of C. auris provided by another researcher. The A. fumigatus and C. neoformans experiments were conducted using clinical isolates. It will be important, however, to expand the scope of testing to determine if C. vaccinii VCs are active against a broader array of clinical isolates and organisms with pre-existing drug resistance. While VCs have profound antifungal activity against A. fumigatus (FIG.2A, FIG.2B), testing additional molds, including Mucorales, will directly address the objectives stipulated in RFA-AI-24-065.

[0217] Experimental approach: Aim 3.1. Measure Chromobacterium VC activity on clinical Candida isolates (R21 & R33): The C. albicans experiments thus far have been performed withDocket No.: 405002-558001WO the sequenced, reference strain SC5314. The Biorepository Core of our COBRE Center for Antimicrobial Resistance and Therapeutic Discovery has a collection of over 100 Candida isolates derived from sterile sites in patients, typically candidemia. C. albicans is the predominant strain in this collection, while at least 10% of strains have reduced susceptibility to fluconazole. Our objective in this subaim will be to test this collection for their susceptibility to VCs, targeting 20% of the isolates during the R21 phase. The clinical isolates will be grown from frozen stocks, subcultured overnight in broth, and then standardized inoculums will be placed on SDA agar for the sandwich assays. Strains will be selected to represent species diversity (albicans, glabrata, tropicalis, parapsilosis) and reduced fluconazole susceptibility. The R33 phase will test a greater fraction of the COBRE collection. The total number tested will depend on the variation in responses: uniform inhibition of, for example, 30-40% of the collection will suggest diminishing returns on continued testing of the Candida collection. In this case we will focus our efforts on higher yield experiments in the following subaims. Isolates will be tested using our standard sandwich plate assay in three iterations to evaluate robustness and reproducibility.

[0218] Aim 3.2. Test Chromobacterium VCs against drug resistant strains (R33): With antifungal resistance a rising concern [10-12], it will be important to establish VC activity against strains that are resistant to standard antifungals. Activity against C. auris is promising (FIG.6A, FIG.6B, FIG.6C, FIG.6D), but strains with bona fide resistance must be studied. For this investigation, we will obtain three collections from the CDC Antimicrobial Resistance Isolate Bank: Aspergillus panel, resistant Candida panel, C. auris panel. Together, these three panels contain 99 distinct isolates. To enhance our efficiency, we will prioritize isolates that have distinct mechanisms of resistance within each panel. Priority will also be given to strains with multi-drug resistance, which will be especially enriched in the C. auris collection. We will perform sandwich plate assays using our standard methodology for yeasts (FIG.1E, FIG.1F, FIG.6A, FIG.6B, FIG.6C, FIG.6D) and Aspergillus (FIG.2A, FIG.2B). Isolates will be tested in three independent iterations to ensure reproducibility of the results.

[0219] Aim 3.3. Test Chromobacterium VCs against filamentous fungi including Mucorales (R21 and R33): Fungi of the Mucorales order are among the priority pathogens for the WHO

[0084] , and are specified in this RFA-AI-24-065. Molds are less commonly encountered in patients than, for example, Candida species. We will therefore work with our clinical microbiology lab to identify isolates of Mucor and Rhizopus, two of the more commonly encountered genera in this order

[0084] . Our clinical laboratory stocks strains from patients indefinitely when they are tested for antifungal susceptibility. We will therefore work with our clinical mycology lab to identify appropriateDocket No.: 405002-558001WO candidates from the frozen stocks and monitor samples for new isolates. Sandwich assays will be performed as we have done previously for A. fumigatus (FIG.2A, FIG.2B) using SDA plates for the Mucorales and C. vaccinii lawns on KMB. Images of fungal growth will be taken daily for measurement using Cell Profiler. Our objective in the R21 phase is to test five Mucorales isolates. Importantly, we will know the susceptibility results for standard antifungals, allowing us to compare susceptibility / resistance with C. vaccinii VCs.

[0220] The objective of this aim is to expand the range of organisms tested for susceptibility or resistance to C. vaccinii VCs. The MOA of VCs are expected to be different than existing antifungals (Aim 2), therefore it is unknown how broad is the VC activity. Based on the strong anti- yeast responses that we have observed thus far, we anticipate the clinical strains of Candida will be susceptible. If the VC MOA is different than existing antifungals, we anticipate VCs will inhibit the drug-resistant fungal strains from the CDC panels. An alternative outcome is that we find some drug-resistant strains are also resistant to VCs. If a group of isolates that share a common resistance mechanism are also resistant to VCs, it will implicate a shared molecular mechanism of resistance to VCs, and possibly the same fungal target. The inhibition of Aspergillus growth we have seen is encouraging and suggests there will be activity against other molds. Future directions include expanding the study of filamentous fungi beyond Mucorales, such as Fusarium species. In addition, Brown University is currently completing a new science research facility that includes BSL-3 laboratories; therefore, future experiments can also include Coccidiodes.

[0221] The methodology for this aim is a straightforward application of our existing sandwich plate assay. Technical difficulties are therefore not expected. Identification of suitable Mucorales may be challenging because of their infrequent isolation relative to other organisms. If Mucorales isolates not readily available from patient specimens, our alternative is to purchase strains from ATCC. These strains, however, may not be isolated from patients or may have been passaged such that their virulence and antifungal resistance is altered. Moreover their antifungal susceptibility pattern would be unknown. Therefore, we will first focus on strains recently isolated in our clinical laboratory.

[0222] Statistical Plan; Sandwich Plate Assays: Three independent iterations of sandwich plate assays are performed, i.e. three independent subcultures of the fungal stocks and C. vaccinii. Fungal growth on the control and test plates are measured using Cell Profiler analysis of plate photographs

[0085] . The percent maximum growth is calculated within one iteration: % maximum = (growth with VC / control growth without VC) * 100. This normalization approach allows for statistical analysis across multiple iterations, e.g. FIG.6A, FIG.6B, FIG.6C, and FIG.6D.Docket No.: 405002-558001WO Typically, ANOVA analysis is applied with post-hoc testing using Prism (GraphPad).

[0223] RNA-seq Analysis: As described above in the corresponding sections, statistical testing is a component of the dedicated analysis tools that will be deployed for analysis of RNA- seq data, e.g. WGCNA and MaASlin2.

[0224] Our research strategy uses complementary approaches to validate findings, thereby enhancing the robustness of our analysis. For example, the genomics and transcriptomics analysis in Aim 1 are complementary, with the site-directed mutagenesis validating the role of genomic loci that are identified by the -omics experiments. Fungal strains will be run using technical and biological replicates to ensure reproducibility. Gender as a biological variable is not applicable to this research program as there are no animal studies or human subjects. EXAMPLE 4. BROAD ANTIFUNGAL ACTIVITY OF THE SUBSTANCE(S) RELEASED BY CHROMOBACTERIUM VACCINII BUC-1

[0225] Example images from this experiment are shown in FIG.13A, top agar and bottom agar with “control” and “BUC-1”, and the growth was measured by a simple surface area (ultimately it will be measured using a different methodology but that is time intensive). The FIG.13A is showing growth inhibition of a Mucor species in one iteration (visually in panel of FIG.13A) and the statistics across 3 replicates (FIG.13B). Of particular interest with this fungus is that it grew rapidly on the bottom agar of the control (FIG.13A, bottom) but there was no fungal growth on the bottom agar with the lawn of BUC-1 bacteria. This illustrates broad antifungal activity of the substance(s) released by Chromobacterium vaccinii BUC-1.

[0226] FIG.17 shows percent maximal growth for additional tests that were conducted. The experiment was conducted to compare different strains of Chromobacterium for the phenotype of fungal inhibition. Our base strain, BUC-1, is the C. vaccinii in FIG.17. The other species were obtained from the American Type Culture Collection (ATCC). The results show a range of phenotypes with C. violaceum having the greatest inhibitory effect. We will use these differences in our other experiments to identify the genes, biochemical pathways, and chemical composition of the volatile compound(s) that are responsible for the anti-fungal phenotype. The quantification of growth inhibition is underway in detail.

[0227] The invention contemplates that any of the above-described compounds, methods, devices or combinations can be derivatized or can be structurally altered to further save lives, for example, by addition or substitution of one or more atoms using a radioisotope or using a different element (e.g., B or boron in place of C or carbon), by removal of an ester or by addition of a salt form, an amino acid, a sugar, or a peptide. Hydrates and / or solvates can be formed by 1)Docket No.: 405002-558001WO dissolving the molecule in water and / or solvent and slowly drying, whereby water and / or solvent remain hydrogen bonded with OH groups in the molecule or associated with the molecule. A formation of a hydrate or solvate can typically be confirmed by an attenuated total reflection (ATR) infrared spectrum acquired from the solid-state sample. The ATR spectrum of a hydrate or solvate will typically show increased broad bands (indicating hydrogen bonding) above about 3200cm-1, as compared to the non-hydrate or non-solvate solid sample. In some embodiments, the above- described compounds are attached to or associated with a targeting moiety. In some embodiments, the targeting moiety is a particle or an antibody with affinity for a specific type of cell. While various crystal structures are contemplated, these investigations will also be initiated. REFERENCES: 1. WHO, WHO fungal priority pathogens list to guide research, development and public health action.2022, Geneva: World Health Organization.48. 2. Bongomin, F., et al., Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. J Fungi (Basel), 2017.3(4). 3. CDC, Antibiotic Resistance Threats in the United States, 2019.2019, Centers for Disease Control and Prevention: Atlanta. p.150. 4. Benedict, K., H.K. Whitham, and B.R. 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Biotechniques, 2007.42(1): p.71-5.

Claims

Docket No.: 405002-558001WO CLAIMS We claim:

1. A method of preventing and / or treating a fungal infection in a human subject, the method comprising the steps of: (1) obtaining a subject with a fungal infection, as subject susceptible to a fungal infection or a subject suspected of having a fungal infection; (2) cultivating a healing bacteria in the vicinity of the subject so that the subject will be contacted with one or more volatile compounds and / or one or more agents released from the healing bacteria; and (3) containing the one or more volatile compounds and / or one or more agents released from the healing bacteria such that one or more volatile compounds and / or one or more agents will continue to contact the subject; whereby one or more volatile compounds and / or one or more agents prevents and / or treats the fungal infection in the subject.

2. The method of claim 1, wherein the fungal infection is a drug resistant and / or a treatment resistant fungal infection and / or wherein the subject is immune compromised.

3. The method of claim 1, wherein the substances released by the healing bacteria comprise one or more volatile compounds including: dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes, heterocycles, ammonia, amines, pyrazines, acids, and / or sulfur compounds.

4. The method of claim 1, wherein the healing bacteria is a bacterium selected from the group consisting of: Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, PseudomonasDocket No.: 405002-558001WO chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and / or Chromobacterium vaccinii BUC-1.

5. The method of claim 1, wherein the method further comprises administering another anti- fungal agent to the subject and wherein the method is a combination therapy.

6. The method of claim 1, wherein the substances released by the healing bacteria comprise one or more agents including: bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and / or metal chelators.

7. The method of claim 1, wherein containing the substances optionally comprises containing the one or more volatile compounds released from the healing bacteria, containing the one or more agents released from the healing bacteria, containing the substances such that the substances will continue to contact the subject, the substances released by the healing bacteria prevent the fungal infection in the subject, and / or the substances released by the healing bacteria treat the fungal infection in the subject.

8. A method of making an anti-fungal composition for inhibiting a fungal infection in a subject, the method comprising: culturing a microbe near a fungus, wherein the microbe releases substances that inhibit growth of the fungus; and confining the substances released by the microbe to a container; wherein the substances in the container are including the anti-fungal composition.Docket No.: 405002-558001WO 9. The method of claim 8, wherein the fungal infection comprises: Candida albicans, Candida auris, Aspergillus fumigatus, Mucorales, Fusarium, Cryptococcus neoformans, Cladophialophera bantiana, Aspergillus niger, Saccharomyces cerevisiae, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and / or Paracoccidioides spp., and wherein the fungal infection can be resistant to fluconazole, amphotericin B, echinocandins, and / or an azole resistance.

10. The method of claim 8, wherein the microbe is a bacterium.

11. The method of claim 8, wherein the microbe is a bacterium selected from the group consisting of: Bacillus subtilis, Pseudomonas aeruginosa, Lactobacillus spp., Bifidobacterium spp., Streptomyces spp., Actinomyces spp., Staphylococcus epidermidis, Escherichia coli, Mycobacterium vaccae, Rhodococcus spp., Arthrobacter spp., Corynebacterium spp., Brevibacterium spp., Micrococcus spp., Alcaligenes spp., Acinetobacter spp., Enterobacter spp., Erwinia spp., Flavobacterium spp., Nocardia spp., Pseudonocardia spp., Micromonospora spp., Streptosporangium spp., Thermoactinomyces spp., Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Bacillus cereus, Bacillus megaterium, Bacillus clausii, Bacillus firmus, Bacillus coagulans, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas stutzeri, Pseudomonas putida, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Streptomyces griseus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces avermitilis, Streptomyces albus, Streptomyces aureofaciens, Streptomyces hygroscopicus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces virginiae, Chromobacterium vaccinii, Chromobacterium vaccinii MWU 328, and / or Chromobacterium vaccinii BUC-1.

12. The method of claim 8, wherein the substances include at least one volatile compound including: dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, methanethiol, hydrogen sulfide, benzothiazole, ketones, aldehydes, esters, alcohols, terpenoids, benzenes,Docket No.: 405002-558001WO heterocycles, ammonia, amines, pyrazines, acids, and / or sulfur compounds.

13. The method of claim 8, wherein the substances include at least one agent including: bacteriocins, lipopeptides, polypeptides, enzymes, polyketides, terpenes, fatty acids, organic acids, alkanes, alkenes, organosulfur compounds, organophosphorus compounds, organohalogen compounds, phenolic compounds, quinones, and / or metal chelators.

14. The method of claim 12, wherein confining the substances includes confining the at least one volatile compound released from the microbe.

15. The method of claim 13, wherein confining the substances includes confining the at least one agent released from the microbe.

16. The method of claim 8, wherein the anti-fungal composition prevents the fungal infection in the subject.

17. The method of claim 8, wherein the anti-fungal composition treats the fungal infection in the subject.

18. A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air chamber around the human subject; a culture of the healing bacteria inside the air chamber; and a HEPA filter over the culture of the healing bacteria; wherein cultivating the healing bacteria in the vicinity of the subject so that the subject will be contacted with one or more volatile compounds and / or one or more agents released from the healing bacteria prevents and / or treats the fungal infection in the subject.

19. The method of claim 8, further comprising a device to carry out the method, the device comprising: an enclosure surrounding the fungus; a culture of the microbe within the enclosure; and an air filter covering the culture of the microbe.Docket No.: 405002-558001WO 20. A device for preventing and / or treating a fungal infection in a human subject, the device comprising: an air chamber configured to surround the human subject; a culture of healing bacteria disposed inside the air chamber; and a HEPA filter positioned over the culture of healing bacteria.

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