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WO2011085367A2 - Micro-organismes non-réplicatifs en tant que post-biotiques - Google Patents

Micro-organismes non-réplicatifs en tant que post-biotiques Download PDF

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Publication number
WO2011085367A2
WO2011085367A2 PCT/US2011/020826 US2011020826W WO2011085367A2 WO 2011085367 A2 WO2011085367 A2 WO 2011085367A2 US 2011020826 W US2011020826 W US 2011020826W WO 2011085367 A2 WO2011085367 A2 WO 2011085367A2
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Prior art keywords
inactivated
microorganisms
microorganism
radioactive phosphorus
microorganism populations
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PCT/US2011/020826
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English (en)
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WO2011085367A3 (fr
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Jeffrey Daniel Hillman
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Oragenics, Inc.
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Publication of WO2011085367A2 publication Critical patent/WO2011085367A2/fr
Publication of WO2011085367A3 publication Critical patent/WO2011085367A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria

Definitions

  • TITLE Non-Replicating Microorganisms as Post-Biotics
  • a host's endogenous (autochthonous) microbial flora help to protect it from deleterious microbial infections.
  • the mechanisms responsible for this phenomenon are complex, dynamic and not completely understood despite over 100 years of research in this field. It is clear that competition by endogenous species can inhibit the colonization and/or growth of a pathogenic microorganism, thereby preventing or mollifying the disease caused by the pathogen.
  • the competition can occur for attachment sites on susceptible surfaces of the host and/or for essential nutrients.
  • excreted metabolic products or specific antibiotic-like molecules (bacteriocins) produced by the endogenous flora can inhibit colonization and/or growth of the pathogen.
  • the presence of endogenous species may alter physical aspects of the environment, such as oxidation reduction potential, oxygen tension, pH, etc., to render the susceptible sites of the host inhospitable to the pathogen.
  • endogenous species may alter physical aspects of the environment, such as oxidation reduction potential, oxygen tension, pH, etc., to render the susceptible sites of the host inhospitable to the pathogen.
  • one enthetic (allochthonous) strain of a pathogen would inhibit colonization or growth of another strain of the same or closely related species since they likely would occupy the same habitat and niche.
  • the mechanisms that engender this inhibition are predicted to fall into the same categories as those seen with inhibition caused by endogenous bacteria.
  • the ability of any particular microorganism to inhibit the colonization or growth of another microorganism of the same or different species is referred to as bacterial interference.
  • compositions and methods are needed in the art for effective and safe microbial interference treatments.
  • One embodiment of the invention provides a method of amelioration or prophylaxis of a disease, infection or colonization in a subject or on a surface caused by a first microorganism.
  • the method comprises administering one or more radioactive phosphorus-inactivated second microorganism populations to the subject or surface, wherein the disease, infection, or colonization is ameliorated or prevented.
  • the radioactive phosphorus-inactivated second microorganism populations can be obtained by growing the microorganism populations in the
  • the radioactive phosphorus-inactivated second microorganism populations can be the same or similar microorganisms as the first microorganism.
  • the radioactive phosphorus-inactivated second microorganism populations can be different microorganisms from the first microorganism.
  • the radioactive phosphorus- inactivated second microorganism populations can be administered topically, orally, intravenously, intramuscularly, intrapulmonary, intradermally, intraperitoneally, subcutaneously, via aerosol, intranasally, via infusion pump, via suppository, or mucosally.
  • the first microorganism can be a virus, alga, bacterium, yeast, fungus, or protozoan.
  • the radioactive phosphorus-inactivated second microorganism populations can be algae, bacteria, yeast, fungi, or protozoa that are metabolically active, but are substantially unable to replicate; or viruses that are substantially unable to replicate.
  • Another embodiment of the invention provides a method of ameliorating or preventing a biofouling condition or a biofilm condition, caused by one or more first microorganisms.
  • the method comprises administering one or more radioactive phosphorus-inactivated second microorganism populations to the biofouling condition or biofilm condition, wherein the biofouling condition or biofilm condition is ameliorated or prevented.
  • the one or more radioactive phosphorus-inactivated second microorganism populations can be obtained by growing the microorganisms
  • the one or more radioactive phosphorus-inactivated second microorganism populations can comprise microorganisms that are the same or similar to the one or more microorganisms that cause the biofouling condition or the biofilm condition.
  • the one or more radioactive phosphorus-inactivated second microorganism populations can comprise microorganisms that are different from the one or more microorganisms that cause the biofouling condition or the biofilm condition.
  • Yet another embodiment of the invention provides a method of inducing an immune response to first microorganisms in a subject.
  • the method comprises administering one or more radioactive phosphorus-inactivated second microorganism populations to the subject, wherein the radioactive phosphorus- inactivated second microorganism populations are the same or similar to the first microorganisms, and wherein an immune response is induced in the subject.
  • the radioactive phosphorus-inactivated second microorganism populations can be
  • the radioactive phosphorus-inactivated second microorganism populations can be administered topically, orally, intravenously, intramuscularly, intrapulmonary, intramuscularly, intradermally, intraperitoneally, subcutaneously, via aerosol, intranasally, via infusion pump, via suppository, or mucosally.
  • the first microorganisms can be viruses, algae, bacteria, yeast, fungi, or protozoa.
  • Even another embodiment of the invention provides a therapeutic composition comprising one or more radioactive phosphorus-inactivated microorganisms and an acceptable carrier.
  • the therapeutic composition can further comprise an adjuvant.
  • the radioactive phosphorus-inactivated microorganisms can be viruses, algae, bacteria, yeast, fungi, or protozoa.
  • the composition can be a pharmaceutically acceptable composition and the carrier can be a pharmaceutically acceptable carrier.
  • kits comprising a container comprising one or more radioactive phosphorus-inactivated microorganism populations; an applicator for the one or more radioactive phosphorus-inactivated microorganism populations; and optionally, one or more buffers or diluents.
  • Yet another embodiment of the invention provides a method for preparing a therapeutic or prophylactic composition of one or more radioactive phosphorus- inactivated microorganism populations.
  • the method comprises growing one or more microorganism populations in the presence of 32 P in a medium; harvesting and washing the microorganisms; freezing or cooling the microorganisms until the
  • the medium can be a phosphate- limited cultivation medium.
  • the invention provides methods and compositions useful to ameliorate or prevent of a disease, infection, colonization, biofilm, or biofouling condition in a subject or on a surface caused by one or more microorganisms.
  • Figure 1 shows the effect of heat killed S. aureus strain 502A on the growth of S. aureus strain Smith and the possible role of nutrient and carbon limitation.
  • Figure 2 shows the effect of heat killed E. coli on the growth of S. aureus strain Smith.
  • FIG. 3 shows that heat treatment inhibits the glycolytic activity of S. aureus strain 502 A.
  • Figure 4 shows the effect of medium pre-conditioned by the growth of S. aureus strain 502A on S. aureus strain Smith.
  • Figure 5 shows the inhibition of attachment of strain Smith to glass surfaces previously treated with different numbers of heat killed cells of strain 502A.
  • Figure 6 shows the inhibition of biofilm formation by strain Smith on glass surfaces when co-incubated with heat killed cells of strain 502A.
  • Figure 7 shows the effect on a preformed biofilm of strain Smith on glass surfaces by co-incubation with heat killed cells of strain 502A.
  • Figure 8 shows the effect of P0 4 on the survival of Escherichia coli as a function of time ( 32 P suicide).
  • Figure 9A-B show data and a semi-log plot of DPM/mL as a function of incubation time at -80 °C for a control (no 32 P added) and cell samples incubated with differing levels of 32 P.
  • Figure 10A-B show a semi-log plot of viable CFU as a function of incubation time at -80 °C for a control and cell samples incubated with differing levels of 32 P.
  • Figure 1 1 A-B show average lactic acid production capability as a function of incubation time at -80 °C following exposure to 32 P at different levels.
  • S. aureus 502A are described to serve as therapeutic and prophylaxis agents for a variety of diseases, infections, and colonizations caused by, e.g., S. aureus.
  • the same approach can be used for the prevention and/or treatment of a wide variety of infectious diseases, and to control the colonization or growth of microorganisms in a wide variety of applications, such as biofouling.
  • the use of P suicide-induced non-replicating cells obviates the potential for adverse events caused in live cells by spontaneous mutations resulting in increased pathogenicity, acquisition of virulence traits by genetic exchange, and residual pathogenic potential.
  • Microorganisms that are inactivated and cannot replicate can be used to induce microbial interference. That is, the colonization or presence of one or more types of non-replicating microorganisms prevents or reduces the colonization or presence of one or more other types of microorganisms. Microbial interference can occur without host immune stimulation.
  • a non-replicating microorganism of the invention can be delivered to a subject and interfere with or reduce the amount of wild-type microorganisms present in or on the subject without the help of the subject's immune system.
  • the effectiveness of post-biotics logically depends on the method used to kill the post-biotic cells: it cannot disrupt or otherwise damage the mechanism(s) used by the post-biotic to interfere with colonization and/or outgrowth of the wild-type microorganism that is causing a disease, infection, colonization or biofilm.
  • heat, antibiotic, gamma irradiation or other methods of killing a microorganism may affect the surface adhesion molecules essential for the attachment process, and thus diminish their efficacy.
  • any of these methods of killing are likely to inactivate proteins involved in metabolism, such that interference caused by excreted metabolic products, specific antibiotic-like molecules (bacteriocins), or alteration of the environment would be eliminated.
  • Methods of the invention provide for the prevention or treatment of a disease using a post-biotic, which is a strain of a potentially pathogenic microorganism that has been inactivated so that it is non-replicating, in a fashion that minimally affects its structure and physiology, thereby optimizing its ability to compete with live, virulent strains of the pathogen.
  • a post-biotic which is a strain of a potentially pathogenic microorganism that has been inactivated so that it is non-replicating, in a fashion that minimally affects its structure and physiology, thereby optimizing its ability to compete with live, virulent strains of the pathogen.
  • the ideal post-biotic is one that retains all of the properties of the live cell but has completely and irrevocably lost the ability to replicate itself. In this fashion, the post-biotic would maintain its ability to temporarily colonize the host and interfere with the target wild-type pathogen. Assuming that the starting strain for the post- biotic is carefully chosen to have negligible pathogenic potential (e.g., S. aureus 502A), the likelihood for adverse events is essentially eliminated. Post-biotics of the invention are inactivated using a protocol based on the decay of radioactive isotopes such as radioactive phosphorus ( P).
  • P radioactive phosphorus
  • 32 P is incorporated into the backbone of the cell's chromosomal deoxyribonucleic acid or affects other cellular components important in cell replication.
  • the approach is referred to as 32 P suicide.
  • the label is incorporated into the gamma ( ⁇ ) position of ATP, and then into the backbone of the cell's DNA.
  • Non-replicating, inactive microorganisms of the invention can be metabolically active. That is, for non-virus microorganisms, catabolism and anabolism occur in the microorganism; however, the microorganism is substantially unable to replicate. In the case of viruses, they are able to infect and/or enter host cells, but they are not substantially able to replicate.
  • Microorganisms can be inactivated using, e.g., P suicide. Fuerst & Stent, J. Gen. Physiol. 40:73 (1956); Miller, J. Virol., 5:533 (1970).
  • radioactive isotopes other than P can be used such as H, S, I, C or combinations of thereof.
  • Microorganisms subjected to P suicide or other radioactive isotope inactivation or combinations thereof according to the invention are inactive and cannot substantially replicate. Microorganisms do not substantially replicate when none or less than about 3.0, 2.0, 1 .0, 0.75, 0.5, 0.25, 0.1 , or 0.01 percent of the population can replicate.
  • microorganisms do not substantially replicate when they are only able to complete one or two rounds of replication and are then not able to further replicate.
  • Microorganisms can be grown in media comprising P at a specific activity of about 1 .0, 5.0, 5.8, 10, 20, 30, 40, 50, 100, 200, 300, 400 or 500 (or any range between about 1 .0 and 500) mci/mg of total phosphorus.
  • the conditions for 32 P incorporation into the backbone of the microorganism's DNA (and RNA in the case of RNA viruses) can be optimized by using a phosphate-limited cultivation medium.
  • Microorganisms can then be incubated in such a phosphate- limited medium for a period of time (e.g.
  • Bacteriophage and viruses can be prepared and used to infect cells, which are then incubated, as described in, e.g., Miller.
  • cells can be washed, centrifuged, and resuspended in medium or buffer for storage at about -196, -80, - 60, -40, -20, 0, or 4°C (or any range between about -196 and about 4°C) until the level of radioactivity remaining is about one times or two times background or less.
  • the cells may also be lyophilized or treated in some other fashion that preserves their physiological integrity during prolonged storage and does not interfere with P decay-induced disruption of the microorganisms genomic material.
  • P suicide does not significantly alter or destroy surface molecules or the metabolic machinery of the microorganism, making the microorganisms especially useful in the methods of the invention because they do not substantially replicate, but are metabolically active and are otherwise identical to the wild-type untreated microorganism for some period following their recovery from storage.
  • the addition of 32 P0 4 to the medium of actively growing microorganisms results in its incorporation into the DNA backbone.
  • decay of the 32 P leads to cell death, presumably because of irreparable breakage of the DNA at multiple sites or effects of the 32 P on other cellular components important in cell replication.
  • a semi-log plot of survival as a function of time can be biphasic (as evidenced in Example 8); it will have a linear phase, which then plateaus, approaching a linear asymptote.
  • the first phase is likely to be the result of P suicide in wild-type cells that efficiently incorporated the radionuclide, which then decayed during storage.
  • the second phase is likely to be due the presence of mutants, which are unable, for whatever reasons, to grow and metabolize P in the medium in which the exposure to P0 4 was performed.
  • significant amounts of the radionuclide are not incorporated into their DNA or other cellular components.
  • These survivors would only be identified if samples of the treated cells were placed in or on medium that is permissive for the mutations that prevented 32 P incorporation.
  • the post-biotic should have negligible cells capable of replicating in or on the treated host or surface.
  • different growth conditions can be used to find the one that yields the highest killing percentage during the initial linear phase of viability testing.
  • the variables of how much P to incorporate in the medium, how long the incubation in the presence of the 32 P should continue, and how long the harvested, washed and frozen cells should be stored can be experimentally determined to obtain optimal yields and efficiency using no more than routine experimentation and ordinary skill in the art.
  • the level of 32 P decay in the sample used to treat a host to prevent or cure a disease should be no more than twice background for it to be considered safe for use as a post-biotic.
  • Non-replicating microorganisms of the invention can be used to treat, ameliorate, or prevent a disease, infection, or colonization.
  • a disease is a pathological condition of a part, organ, or system of an organism resulting from infection and characterized by an identifiable group of signs and symptoms.
  • An infection is invasion by and multiplication of pathogenic microorganism in a bodily part or tissue, which may produce a subsequent tissue injury and progress to overt disease through a variety of cellular or toxic mechanisms.
  • Colonization is the act or process of a microorganism of establishing a colony or colonies. Colonization may produce a subsequent biofilm or biofouling condition as described below.
  • Non- replicating microorganisms of the invention can be used prophylactically to prevent disease, infection or colonization or to prevent the spread of a disease, infection or colonization to additional bodily parts or tissues, additional surfaces, or to different subjects.
  • Non-replicating microorganisms of the invention can also be used to reduce the number of pathogenic microorganisms on or in a subject or on a surface.
  • the invention provides method of treatment, amelioration or prevention of a disease, infection, or colonization in a subject or on a surface caused by a first microorganism comprising: administering one or more radioactive phosphorus-inactivated second microorganism populations to the subject, wherein the disease, infection, or colonization is treated, ameliorated or prevented.
  • the second microorganism population can be one or more radioactive phosphorus- inactivated microorganisms (e.g., 1 , 2, 3, 4, 5, 6, or more different types of microorganism populations).
  • the second microorganism population can cause bacterial interference with the microorganism causing the disease, infection or colonization.
  • the radioactive phosphorus-inactivated second microorganism population can be the same or similar (e.g., same species, but a different strain) microorganism as the first microorganism.
  • the radioactive phosphorus- inactivated second microorganism population can be a different microorganism from the first microorganism.
  • normal pharyngeal flora e.g., alpha-hemolytic streptococci
  • endocervical flora including certain streptococci, staphylococci, and lactobacilli, can inhibit the growth of Neisseria gonorrhoeae. See, Saigh ei al., Infect. Immun. 19:704 (1978).
  • a non-replicating microorganism of the invention can be administered to a mammal, such as a mouse, rabbit, guinea pig, macaque, baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or to a non-mammalian animal such as a chicken, duck, or fish.
  • Non-replicating microorganisms of the invention can also be administered to plants.
  • Non-replicating microorganisms of the invention can be, for example, bacteria, such as Pseudomonas sp., e.g., Pseudomonas aeruginosa, Streptococcus sp., e.g., and Streptococcus pyogenes, Legionella sp., e.g. L. pneumophila, Staphylococcus sp., e.g., S.
  • bacteria such as Pseudomonas sp., e.g., Pseudomonas aeruginosa, Streptococcus sp., e.g., and Streptococcus pyogenes, Legionella sp., e.g. L. pneumophila, Staphylococcus sp., e.g., S.
  • Neisseria sp. e.g., Neisseria gonorrhoeae
  • Propionibacterium sp. e.g., Propionibacterium acnes
  • Porphyromonas sp. e.g., Porphyromonas gingivalis
  • Actinomyces sp. Escherichia coli
  • Bordetella sp. e.g., Bordetella pertussis
  • Helicobactor sp. e.g., Helicobactor pylori
  • Yersinia sp. Haemophilus sp., e.g., Haemophilus influenzae, Klebsiella sp., e.g., Klebsiella pneumoniae
  • a fungi or yeast such as Candida sp., e.g., Candida albicans, Aspergillus sp., Acremonium
  • a non-replicating, post-biotic microorganism of the invention is administered to an animal in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises a therapeutically effective amount of the non-replicating microorganism.
  • Administration of the non-replicating, post-biotic microorganisms of the invention can be by any means known in the art, including intramuscular, intravenous, intrapulmonary, intramuscular, intradermal, intraperitoneal, or subcutaneous injection, aerosol, intranasal, infusion pump, suppository, mucosal, topical, and oral.
  • a non-replicating microorganism can be accompanied by a carrier for oral administration.
  • a combination of administration methods can also be used.
  • non-replicating microorganisms are administered at a daily dose of about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 ,10 10 , 10 11 , or 10 12 , microorganisms (or any range between about 10 3 and 10 12 microorganisms).
  • microorganisms or any range between about 10 3 and 10 12 microorganisms.
  • microorganisms/cm 2 can be applied for biofilm or biofouling conditions.
  • the virus can be harvested and purified from their host cells prior to administration.
  • Non-replicating microorganisms can be administered for a certain period of time (e.g., 1 day, 3 days, 1 week, 1 month, 2 months, 3 months, 6 months, 1 year or more) or can be administered in maintenance doses for long periods of time to prevent or reduce disease, infection, colonization, biofilms or biofouling conditions.
  • a certain period of time e.g., 1 day, 3 days, 1 week, 1 month, 2 months, 3 months, 6 months, 1 year or more
  • maintenance doses for long periods of time to prevent or reduce disease, infection, colonization, biofilms or biofouling conditions.
  • Carriers such as pharmaceutically acceptable carriers and diluents for therapeutic use are well known in the art and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. (1985)).
  • the carrier should not induce the production of antibodies harmful to the host.
  • Such carriers include, but are not limited to, large, slowly metabolized, macromolecules, such as proteins, polysaccharides such as latex functionalized SEPHAROSE®, agarose, cellulose, cellulose beads and the like, polylactic acids, polyglycolic acids, polymeric amino acids such as polyglutamic acid, polylysine, and the like, amino acid copolymers, peptoids, lipitoids, and inactive, avirulent virus particles or bacterial cells.
  • Liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesives can also be used as a carrier for a composition of the invention.
  • Water and phosphate buffered saline can also be used as carriers or diluents. Carriers and diluents should not inhibit microbial metabolism or interfere with binding of the microorganisms to tissues or other surfaces.
  • Pharmaceutically acceptable salts can also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates.
  • compositions of the invention can also contain liquids or excipients, such as water, saline, phosphate buffered saline, Ringer's solution, Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, tonicity adjusting agents, detergent, or pH buffering agents. Additional active agents, such as bacteriocidal agents can also be used.
  • compositions of the invention can be formulated into ingestible tablets, buccal tablets, troches, capsules, aerosols, elixirs, suspensions, syrups, wafers, injectable formulations, mouthwashes, dentrifices, and the like.
  • the percentage of one or more non-replicating microorganisms of the invention in such compositions and preparations can vary from about 0.1 % to about 90% (or any range between about 0.1 % and about 90%) of the weight of the unit.
  • Non-replicating microorganisms or combinations thereof can be administered either to an animal that is not infected or colonized with a wild-type microorganism or wild-type microorganisms or can be administered to a wild-type microorganism infected or colonized animal.
  • Non-replicating microorganisms can be the same or substantially the same (e.g., the same species, but a different strain) microorganism as the microorganism that is targeted for reduction.
  • the non-replicating microorganism can be different from the microorganism that is targeted for reduction (e.g., a different genus and/or a different species).
  • a therapeutically effective amount means the administration of that amount to an individual, either in a single dose or as part of a series, which is effective for treatment, amelioration, or prevention of wild-type microorganism infection or colonization.
  • a therapeutically effective amount is also an amount effective in alleviating or reducing the symptoms of an infection or in reducing the amount of wild-type microorganisms in or on a subject.
  • non-replicating microorganisms of the invention cause interference with other microorganism populations without inducing a host immune response.
  • non-replicating microorganisms of the invention can be present in an immunogenic composition and used to elicit an immune response in a host.
  • An immunogenic composition or immunogen is capable of inducing an immune response in an animal.
  • An immunogenic non-replicating microorganism composition of the invention is particularly useful in sensitizing an immune system of an animal such that, as one result, an immune response is produced that ameliorates or prevents the effect of an infection caused by the wild-type microorganism.
  • the elicitation of an immune response in animal model can be useful to determine, for example, optimal doses or administration routes.
  • Elicitation of an immune response can also be used to treat, prevent, or ameliorate a disease, infection, colonization, or biofilm caused by wild-type microorganisms.
  • An immune response includes humoral immune responses or cell mediated immune responses, or a combination thereof.
  • An immune response can also comprise the promotion of an innate host response, e.g., by promoting the production of defensins.
  • co-stimulatory molecules which improve immunogen presentation to lymphocytes, such as B7-1 or B7-2, or cytokines such as MIP1 a, GM-CSF, IL-2, and IL-12, can be included in a composition of the invention.
  • adjuvants can also be included in a composition.
  • Adjuvants are substances that can be used to nonspecifically augment a specific immune response.
  • an adjuvant and cells or viruses of the invention are mixed prior to presentation to the immune system, or presented separately, but are presented into the same site of the animal.
  • Adjuvants can include, for example, oil adjuvants (e.g. Freund's complete and incomplete adjuvants) mineral salts (e.g.
  • polynucleotides i.e. Poly IC and Poly AU acids
  • certain natural substances e.g. wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis and members of the genus Brucella).
  • Adjuvants which can be used include, but are not limited to MF59-0, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637), referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE, and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN® 80 (polysorbate)
  • non-replicating microorganisms of the invention can elicit an immune response in the subject that lasts for at least 1 week, 1 month, 3 months, 6 months, 1 year, or longer.
  • an immune response can be maintained in an animal by providing one or more booster injections or administrations of the non- replicating microorganism at 1 month, 3 months, 6 months, 1 year, or more after the primary injection or administration.
  • co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions.
  • Biofouling is the undesirable accumulation of microorganisms, algae, plants, animals or a combination thereof on structures exposed to solvent. Biofouling can occur, for example on the hulls of ships, in membrane systems, such as membrane bioreactors and reverse osmosis spiral wound membranes, water cooling systems of large industrial equipment and power stations, and oil pipelines carrying, e.g., used oils, cutting oils, soluble oils or hydraulic oils.
  • a biofilm can cause biofouling and is an aggregate of organisms wherein the organisms are adhered to each other, to a surface, or a combination thereof.
  • a biofilm can comprise one or more species of bacteria, fungi, filamentous fungi, yeasts, algae, cyanobacteria, viruses, and protozoa and combinations thereof.
  • Microorganisms present in a biofilm can be embedded within a self-produced matrix of extracellular polymeric substances. When a microorganism switches to a biofilm mode of growth, it can undergo a phenotypic shift in behavior wherein large suites of genes are differentially regulated. Nearly every species of microorganism can form biofilms. Biofilms can be found on or in living organisms or in or on non-living structures.
  • Biofilms can be present on structures contained in naturally occurring bodies of water or man-made bodies of water, on the surface of water, surfaces exposed to moisture, interiors of pipes, cooling water systems, marine systems, boat hulls, on teeth, on plant surfaces, inside plants, on human and animal body surfaces, inside humans and animals, on contact lenses, on catheters, prosthetic cardiac valves, other prosthesis, intrauterine devices, and other structures/devices.
  • Biofilms can cause corrosion of metal surfaces, inhibit vessel speed, cause dental decay and gum disease, cause plant diseases, and can cause human and animal diseases.
  • Biofilms are involved in human and animal infections, including, for example, urinary tract infections, catheter infections, middle-ear infections, dental plaque, gingivitis, endocarditis, infections in cystic fibrosis, chronic sinusitis, and infections of permanent indwelling devices such as joint prostheses and heart valves.
  • Biofilms can also impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds.
  • Some microorganisms that can form biofilms, cause biofouling and/or cause disease in humans and animals include, for example, bacteria, fungi, yeast, algae, protozoa, and viruses as described above.
  • Biofilms can be treated in living organisms as described above.
  • Biofilms and biofouling conditions on non-living surfaces can be treated by applying the non-replicating, post-biotic microorganisms of the invention onto the non-living surface or to the area surrounding the surface.
  • Non-replicating, post-biotic microorganisms can also be added to the water, oil, or other fluid surrounding and in contact with the non-living surface.
  • the invention provides methods of ameliorating or preventing a biofouling condition or a biofilm condition, caused by one or more first microorganisms.
  • the methods comprise administering one or more radioactive isotope-inactivated second microorganism populations to the biofouling condition or biofilm condition, wherein the biofouling condition or biofilm condition is ameliorated.
  • the radioactive isotope can be 32 P.
  • the one or more radioactive isotope-inactivated second microorganism populations can be obtained by growing the microorganisms in the presence of a radioactive isotope such as 32 P.
  • the second microorganism populations can cause bacterial interference with the microorganism causing the biofilm or biofouling condition.
  • the one or more radioactive isotope-inactivated second microorganism populations can comprise microorganisms that are the same or similar to the one or more microorganisms that cause the biofouling condition or the biofilm condition.
  • the one or more radioactive isotope-inactivated second microorganism populations can comprise microorganisms that are different from the one or more microorganisms that cause the biofouling condition or the biofilm condition.
  • the one or more radioactive isotope-inactivated second microorganism populations can be administered to a surface that has a biofilm or biofouling condition or can be administered to a surface as a prophylactic measure.
  • the one or more radioactive isotope-inactivated second microorganism populations can be in a dried form (e.g., lyophilized or tablet form) or a liquid solution or suspension form.
  • the dried or liquid forms can be swabbed, poured, sprayed, flushed through the surface (e.g. , pipes or membranes) or otherwise applied to the surface.
  • the artificial surface can be contacted with the one or more radioactive isotope-inactivated microorganism populations prior to insertion into the human or animal.
  • the one or more radioactive phosphorus-inactivated microorganism populations can be delivered to the surface after the artificial surface is inserted into the human or animal. Kits
  • compositions of the invention can be present in a kit comprising a container of one or more radioactive isotope-inactivated microorganism populations, such as radioactive phosphorus-inactivated microorganism populations.
  • the radioactive isotope-inactivated microorganism populations can lyophilized and in the form of a lyophilized powder or tablet or can be in a solution or suspension optionally with buffers, diluents, adjuvants, therapeutically acceptable carriers, or pharmaceutically acceptable carriers.
  • a kit can also comprise one or more applicators for the one or more radioactive isotope-inactivated microorganism populations to a body part or tissue or surface.
  • the applicator can be, for example, a swab, a syringe (with or without a needle), a dropper, a sprayer, a surgical dressing, wound packing, or a bandage.
  • the kit can comprise one or more buffers, diluents, adjuvants, therapeutically acceptable carriers, or pharmaceutically acceptable carriers for reconstituting, diluting, or preparing the one or more radioactive phosphorus- inactivated microorganism populations.
  • Example 1 Heat-inactivated S. aureus strain 502A inhibits the growth of wild- type S. aureus strain Smith.
  • Staphylococcus aureus strain 502A (ATCC-27217) and S. aureus strain Smith (ATCC 19636) were cultured in Mueller Hinton Broth (MHB, Difco, Franklin Lakes, NJ), aerobically at 37°C until saturation (16 hours).
  • MHB Mueller Hinton Broth
  • PBS sterile phosphate-buffered saline
  • Resuspended S. aureus strain 502A was inactivated by heat treatment at 60°C for 60 min. Complete inactivation of this strain was confirmed by inoculating an aliquot of the treated cells on rich nutrient (tryptic soy broth, TSB) agar plates and demonstrating the complete absence of colonies following incubation at 37°C for 2 days. Inactivated S. aureus strain 502A was washed 3 times with 5 ml of sterile PBS and resuspended in a final volume of 1 ml of sterile PBS. 1 , 2, 5, and 10 ml aliquots (starting volume) of inactivated S. aureus strain 502A were added to the 30 ml of S.
  • aureus strain Smith sub-cultures. This mixture was further cultivated aerobically with mild (50 rpm) agitation at 37°C. Aliquots (1 ml) of the cultures were sampled at the indicated time intervals and their optical density measured at 600 nm using a Thermo spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA). To test whether the growth inhibition of S. aureus strain Smith by the inactivated S. aureus strain 502A was due to nutrient deprivation, the same experiment was repeated with 2.5x MHB broth (i.e., 2.5 times the recommended amount of broth powder per unit volume). In a separate experiment, MHB broth was supplemented with 5% dextrose to determine if this was the limiting nutrient (Thermo fisher Scientific, Pittsburgh, PA).
  • the inactivated S. aureus strain 502A inhibited the growth of S. aureus strain Smith under the conditions tested in a dose-dependent fashion. It was also found that the use of a concentrated growth medium or the addition of dextrose reversed this inhibition.
  • Example 2 Demonstration of the specificity of the growth inhibitory effect of S. aureus strain 502A on S. aureus strain Smith.
  • Escherichia coli ATCC-23225 was cultured in MHB aerobically at 37°C until saturation (for 16 hours). Cells were harvested by centrifugation (4,000X g, 20 min., 25°C), and resuspended in 1 ml of sterile PBS. Resuspended E. coli was inactivated by heat treatment at 60°C for 60 min. Complete inactivation of this strain was monitored by inoculating an aliquot of the inactivated cells on TSB agar plates. Inactivated E.
  • a mid-exponential phase culture S. aureus strain Smith was prepared in MHB aerobically at 37°C by inoculating fresh medium (1 :10) with an overnight culture as described in Example 1 .
  • An equal volume of E. coli and mid-exponential phase S. aureus strain Smith were mixed and further incubated at 37°C. Aliquots (1 ml) of the mixed cultures were sampled at time intervals and the optical density of the culture measured at 600 nm using a BioMateTM 5 spectrophotometer.
  • heat killed E. coli had no measurable growth inhibitory effect on S. aureus strain Smith. It was concluded that the heat killed E. coli did not inhibit the growth of S. aureus strain Smith to the same extent as heat killed S. aureus, suggesting that the basis for inhibition has an element of specificity.
  • Example 3 Determination of the metabolic capacity of the heat-inactivated S. aureus strain 502A.
  • Staphylococcus aureus strain 502A (ATCC-27217) was cultured in MHB, aerobically at 37°C until saturation was reached (16 hours). Ten (10) ml of cultures in stationary phase were harvested by centrifugation (4,000X g, 20 min., 25°C), and resuspended in 10 ml of sterile PBS. Resuspended S. aureus strain 502A was inactivated by heat treatment at 60°C for 60 min. Complete inactivation of this strain was confirmed by inoculating an aliquot of the inactivated cells on TSB agar plates as described in Example 1 . Inactivated S.
  • aureus strain 502A was washed 3 times with 50 ml of sterile PBS and resuspended in a final volume of 10 ml of sterile PBS containing 1 % dextrose, and incubated at 37°C overnight.
  • Replicate cultures of live (non-heat treated) 502A were prepared and treated by the addition of 5% sodium fluoride (NaF) or 5% sodium arsenate to inhibit glycolysis. Aliquots (1 ml) of the cell suspensions were sampled at various times, and the lactic acid content of the solution was determined enzymatically (lactic acid determination kit 826-B, Sigma- Aldrich).
  • Example 4 Demonstration of the effect of pre-conditioning cultivation medium with S. aureus 502A on the growth of S. aureus strain Smith.
  • Staphylococcus aureus strain 502A and S. aureus strain Smith were cultured in MHB aerobically at 37°C until saturation (16 hours). Three (3) ml of S. aureus strain Smith in stationary phase was inoculated into 30 ml of fresh MHB. Cell-free supernatants of S. aureus strain 502A were isolated by centrifugation (4,000X g, 20 min., 25°C), and 15 ml of the supernatants was added to an equal volume of mid-exponential phase S. aureus strain Smith. Aliquots (1 ml) of the supplemented cultures were sampled at time intervals and the optical density of the culture measured at 600 nm using a Thermo BioMateTM 5 spectrophotometer.
  • Example 5 Demonstration of the effect of heat-inactivated S. aureus strain 502A on the attachment of S. aureus strain Smith to a surface.
  • S. aureus strain 502A and S. aureus strain Smith were cultured in MHB aerobically at 37°C until saturation (16 hours). Three (3) ml of S. aureus strain Smith was subcultured into 30 ml of fresh MHB. Samples of S. aureus strain 502A were heat inactivated as described in Example 1 . Complete inactivation of this strain was confirmed by inoculating an aliquot of the inactivated cells on TSB agar plates. Inactivated S. aureus strain 502A was washed 3 times with 5 ml of sterile PBS and resuspended in a final volume of 1 ml of sterile PBS.
  • Example 6 Demonstration that co-incubation of heat-killed strain 502A cells can inhibit biofilm formation of S. aureus strain Smith.
  • Staphylococcus aureus strain 502A and S. aureus strain Smith were cultured in MHB aerobically at 37°C to saturation (16 hours). Cells of strain 502A were harvested, washed and killed by heat treatment as described in Example 1 . Heat killed cells at 1 -fold, 2-fold, 5-fold, and 10-fold concentration relative to the overnight culture were added to a 1 :10 subculture of strain Smith in MHB medium. Sterile glass slides were placed in the cultures, which were then incubated at 37°C for 24 hours. The slides were washed by immersion three times in sterile water, and cells that remained bound to the slide were removed and dispersed by sonication and vortexing as described in Example 5. Viable cell counts were determined as described in Example 5.
  • the presence of heat killed 502A had a marked effect on biofilm formation by strain Smith.
  • the effect increased as the number of 502A cells increased through the first three concentrations, and then leveled off or increased at the highest concentration. See Fig. 6.
  • the continuous presence of heat killed strain 502A cells significantly decreased biofilm formation by strain Smith. The effect is dose dependent and saturable.
  • Example 7 Effect of heat killed cells of 502A on a pre-formed biofilm of strain Smith.
  • Strain Smith was grown overnight in MHB and subcultured 1 :10 in fresh medium. Sterile glass slides were placed in vessels containing the cultures and incubated overnight at 37°C. The strain Smith-coated glass slides were washed three times by immersion in sterile PBS and placed in 20 ml of fresh MHB. Aliquots of heat killed S. aureus strain 502A equivalent to 0.1 -fold, 0.2-fold, 0.5-fold and 1 -fold dilution of the overnight culture were added to the vessels. The glass slides were incubated for an additional 16 hours at 37°C. Slides were rinsed three times by immersion in sterile PBS to dislodge loosely adhering cells, and placed in 20 ml of fresh PBS.
  • Adherent bacteria were dispersed using ultrasonic treatment and vortexing as described in Example 5, and serially diluted in PBS. Aliquots were inoculated onto TSB agar, and incubated at 37°C for 12-16 hours and colonies that arose were enumerated. The number of viable cells recovered from a preformed biofilm of strain Smith on glass slides were markedly decreased when incubation occurred in the presence of heat killed cells of strain 502A. See Fig. 7. At the concentrations of 502A used, a clear dose dependency was not observed.
  • strain 502A cells caused a reduction in the level of viable cells in preformed biofilms of strain Smith.
  • the reduction could have been due to inhibition of continued biofilm development or to desorption of Smith cells from the biofilm.
  • Example 8 32 P Suicide in Escherichia coli.
  • E. coli strain K10 was grown overnight in minimal medium containing 0.4% glucose and limiting ortho-phosphate under anaerobic conditions. After several doublings at 37°C, 32 P labeled NaH 2 P0 4 was added to give a specific activity of 5.8 Mci/mg of total phosphorus. After incubation at 37°C for 8.5 hours, the cells were washed by centrifugation with M63 buffer, the pellet resuspended in M63 containing 0.4% glucose, and divided into 1 ml aliquots which were stored at 4°C. At 24 hour intervals, samples were spread on TYE plates to monitor the extent of 32 P killing.
  • Example 9 Phosphate-limited Medium (PLP) to Enhance 32 P Uptake in S. aureus.
  • PLP medium A semidefined medium for the growth of S. aureus with limiting phosphate, called PLP medium, was developed by trial and error.
  • the recipe for this medium is as follows:
  • the pH is adjusted to 7.6 using 6N hydrochloric acid.
  • MEM Essential vitamin mixture (100x; Lonza, Walkerville, MD) is added to give a final concentration equal to 0.5x.
  • the medium is filter sterilized.
  • Sterile potassium phosphate (pH 7.2) to give a final concentration in the range of 0.1 -10.0 mM may be added as needed, depending on the yield of cells desired.
  • PLP is phosphate limited for S. aureus strain 502A
  • a sample of 502A was scraped from the surface of a Todd-Hewitt agar plate and used to inoculate 5 ml of PLP supplemented with 10 mM potassium phosphate (pH 7.2).
  • the culture was incubated overnight at 37°C aerobically with mild agitation (50 rpm in an environmental shaker). This starter culture was used to inoculate fresh medium supplemented with 0, 0.2, 0.6, 2.0, 6.0 or 20.0 mM potassium phosphate (pH 7.2).
  • the cells were incubated for 24 hours as described above and the optical densities of the cultures were determined spectrophotometncally and the viable cell counts were determined by plating serially diluted samples on Todd-Hewitt agar plates (Table 1 ).
  • Bacteria Strain 502A of Staphylococcus aureus was used throughout. This strain of S. aureus has naturally reduced pathogenic potential, but routine biosafety level 2 procedures and personal protection equipment were used when handling it.
  • a culture was provided as an agar stab/slant, which was streaked out and checked for purity on rich, permissive agar medium, namely Todd-Hewitt broth (THB) containing 1 .5% agar.
  • TTB Todd-Hewitt broth
  • a glycerol stab of 502A was made by scraping cells from an agar starter plate into cryovials containing sterile glycerohTHB using a ratio of 3 parts:7 parts (v/v), vortexed vigorously for 10 seconds, and stored at -80°C.
  • Fresh starter plates were made from glycerol stabs as needed by inserting a sterile loop or toothpick into the stab and transferring adherent material to a rich agar plate, which was then streaked and incubated.
  • a starter plate was used as a source of inocula for up to 1 month when stored at 4°C wrapped in parafilm to prevent drying.
  • PLM phosphate limited medium
  • 10 mM sodium phosphate by addition of a sterile 0.1 M stock solution, pH 7.6
  • the medium was vortexed for 5 sec at high speed and incubated overnight at 37 °C with gentle rocking or shaking (ca. 50 rpm) to keep the cells in suspension.
  • the culture was sampled and Gram stained to confirm presence of Gram positive cocci in clusters.
  • the culture was centrifuged (8,000xg for 10 minutes at 4°C) and the cells washed once with ice cold 10 ml of PLM (unsupplemented).
  • One tenth volume of sterile, unlabeled 1 M sodium phosphate (pH 7.6) was added and incubated for 15 min (cold chase step).
  • the tubes were centrifuged (8,000xg for 10 minutes at 4°C) and washed 3 times by centrifugation with ice cold PBS.
  • the cells were resuspended in each tube with 10.0 ml of ice cold PLM and vortexed vigorously.
  • Samples (0.7 mL) were aliquoted into 12 cryovials, each containing 0.3 mL of sterile glycerol. The cryovials were vortexed vigorously and snap frozen in dry ice/ethanol bath and stored at -80°C.
  • CFU Viable colony forming units
  • Metabolic activity 500 ⁇ samples of the thawed cell suspensions were placed in microfuge tubes on ice. The tubes were transferred to a 37°C water bath for 60 min. Cells were pelleted in a microfuge (14,000 x g for 5 min at 4°C). Supernatants were recovered into prechilled microfuge tubes and stored at -80°C until all time points were recovered, and lactic acid concentrations determined enzymatically as previously described (Hillman et al., A spontaneous lactate dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of dental caries. Journal of Applied Microbiology 107:1551 -8 (2009).
  • Sample number 4 was determined to have been contaminated during the 32 P incorporation step and was not further analyzed.
  • glycolytic activity of the samples was measured at each of the time points.
  • Cells were incubated at 37 °C for 60 min in the presence of orthophosphate and magnesium as described above. The cells were removed by centrifugation and the supernatants stored at -80 °C until all samples were collected at 207 days.
  • concentration of lactic acid was measured using an adaptation of the Sigma Diagnostic Procedure No. 826-UV, which measures the stoichiometric production of NADH by enzymatic (lactate dehydrogenase) oxidation of lactic acid.
  • the increased absorbance at 340 nm due to NADH formation becomes a measure of lactate originally present in the sample.
  • NAD nicotinamide adenine dinucleotide, Grade III
  • glycine buffer Sigma catalog No. 826-3, glycine, 0.6 mol/L, and hydrazine, pH 9.2 at 25 °C
  • 0.06 ml LDH Sigma catalog No. 826-6, LDH bovine heart suspension in ammonium sulfate, approximately 1 ,000 U/ml when prepared

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Abstract

L'invention concerne des micro-organismes post-biotiques non réplicatifs, pour la prévention ou le traitement sans danger de maladies, infections, colonisations, états de biofilms et états de bio-encrassement.
PCT/US2011/020826 2010-01-11 2011-01-11 Micro-organismes non-réplicatifs en tant que post-biotiques WO2011085367A2 (fr)

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WO2002045727A1 (fr) * 2000-12-08 2002-06-13 Plbio Co., Ltd. Bacteries lactiques permettant d'inhiber l'adhesion d'helicobacter pylori a la muqueuse gastrique
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US20040166094A1 (en) * 2000-06-09 2004-08-26 Darouiche Rabih O. Combination of antimicrobial agents and bacterial interference to coat medical devices
WO2002045727A1 (fr) * 2000-12-08 2002-06-13 Plbio Co., Ltd. Bacteries lactiques permettant d'inhiber l'adhesion d'helicobacter pylori a la muqueuse gastrique

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Publication number Priority date Publication date Assignee Title
US12163135B2 (en) 2017-12-05 2024-12-10 BioPlx, Inc. Methods and compositions to prevent microbial infection

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