WO2011085367A2 - Non-replicating microorganisms as post-biotics - Google Patents

Abstract

The invention provides non-replicating, post-biotic microorganisms for the safe prevention or treatment of diseases, infections, colonizations, biofilm conditions, and biofouling conditions.

Description

TITLE: Non-Replicating Microorganisms as Post-Biotics

PRIORITY

This application claims the benefit of U.S. Ser. No. 61 /293,884, filed on January 1 1 , 2010, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Infections caused by microorganisms resistant to frontline antibiotics have increased dramatically during the past decade, and have led to significant morbidity and mortality in human and animal populations. The ability of microorganisms to adapt to environmental situations that hinder or prevent their growth (such as the introduction of antibiotics) and the ability of antibiotic resistance genes to rapidly spread within a species and from species to species necessitate the discovery of new antibiotics to replace ones that have become outmoded. During the past decade, the number of new antibiotics has decreased, which may be due in part to the likelihood that there are finite such molecules in nature.

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. In addition, it is certain that excreted metabolic products or specific antibiotic-like molecules (bacteriocins) produced by the endogenous flora can inhibit colonization and/or growth of the pathogen. Finally, 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. In another instance, it is reasonable to presume that the presence of 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. In either case, 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.

There are numerous examples of bacterial interference in the literature (reviewed by Hillman, J.D. 2001 . Replacement therapy of dental caries. Oper. Dent. Suppl. 6:43-53; Hillman, J.D. 2002. Genetically modified Streptococcus mutans for l the prevention of dental caries. Antonie van Leeuwenhoek 82:361 -366.) For example, live cells of a naturally attenuated Staphylococcus aureus strain called 502A were used successfully in the 1960s and 70s to treat patients with recurrent furunculosis and to aid in the prevention of S. aureus infections of newborn infants' umbilical stumps and in patients with burns and wounds (Boris, M., T. F. Seller, H. F. Eichenwald, J. C. Ribble, and H. R. Shinefield. 1964. Bacterial interference. Am. J. Dis. Child. 108:252-261 .; Light, I. J., J. M. Sutherland, and J. E. Schott. 1965. Control of a staphylococcal outbreak in a nursery: use of bacterial interference. JAMA 193:699-704.; Shinefield, H. R., J. C. Ribble, M. Boris, and H. F. Eichenwald. 1963. Bacterial interference: its effect on nursery acquired infection with S. aureus. I. Preliminary observations on artificial colonization of newborns. Am. J. Dis. Child. 105:646-654.; Strauss, W. G., H. I. Maibach, and H. R. Shinefield. 1969. Bacterial interference treatment of recurrent furunculosis. JAMA 208:861-863.). Originally isolated from the nares of a healthy nurse, S. aureus 502A apparently prevented colonization by more virulent strains, presumably by competition for the binding sites in the nose. However, the exact mechanism for bacterial interference has never been elucidated (Aly, R., H. I. Maibach, H. R. Shinefield, A. Mandel, and W. G. Strauss. 1974. Bacterial interference among strains of Staphylococcus aureus in man. J. Infect. Dis. 129:720-724.; M. Dall'Antonia, P.G. Coen, M. Wilks, A. Whiley and M. Millar. 2005. Competition between methicillin-sensitive and -resistant Staphylococcus aureus in the anterior nares. Journal of Hospital Infection 61 :62-67). The basis for attenuation of pathogenicity in the 502A strain was also never definitely identified, although it was known to be a coagulase negative variant of S. aureus. Unfortunately, this approach was occasionally complicated by infections due to S. aureus 502A (Blair, E. B., and A. H. Tull. 1969. Multiple infections among newborns resulting from colonization with S. aureus 502A. Am. J. Clin. Pathol. 52: 42-49; Drutz, D. J., M. H. Van Way, W. Schaffner, and M. Glenn Koenig. 1966. Bacterial interference in the therapy of recurrent staphylococcal infections: multiple abscesses due to the implantation of the 502A strain of Staphylococcus. N. Engl. J. Med. 275:1 161-1 165.). The use of 502A as a therapeutic and prophylaxis measure was found to cause a variety of adverse events, including pustules, conjunctivitis, abscesses, and a single fatal infection was reported (reviewed by Houck, P. W., J. D. Nelson, and J. L. Kay. 1972. Fatal septicemia due to S. aureus 502A. Report of a case and review of the infectious complications of bacterial interference programs. Am. J. Dis. Child. 123:45-48.). The basis for the occurrence of adverse events may have been due to spontaneous mutations that occurred during production of 502A that increased its pathogenic potential or may have been a reflection of residual pathogenic potential of the wild-type strain that became evident when 502A achieved a threshold number of cells. Regardless, the result of these adverse events was the removal of 502A from the pharmacopeia.

Compositions and methods are needed in the art for effective and safe microbial interference treatments.

SUMMARY OF THE INVENTION

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

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presence of P. 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

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in the presence of P. 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

32 obtained by growing the microorganism populations in the presence of P. 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.

Still another embodiment of the invention provides a kit 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 ³²P in a medium; harvesting and washing the microorganisms; freezing or cooling the microorganisms until the

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microorganisms can no longer substantially replicate and the P levels are suitable for administration to a subject or to a surface. The medium can be a phosphate- limited cultivation medium.

Therefore, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

Figure 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 P0₄ on the survival of Escherichia coli as a function of time (³²P 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 ³²P added) and cell samples incubated with differing levels of ³²P.

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 ³²P.

Figure 1 1 A-B show average lactic acid production capability as a function of incubation time at -80 °C following exposure to ³²P at different levels.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a," "an", and "the" include plural referents unless the context clearly dictates otherwise.

In this application, the use of P suicide-induced non-replicating cells of, e.g.,

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. By extension, 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 (i.e., post-biotics) 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. For example 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. In instances where there is competition for binding to host surfaces, 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. Further, 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.

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). While not wishing to be bound to the specific mechanism of action, it is believed that the ³²P 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 ³²P suicide. Thus, when wild-type cells are grown in the presence of ³²P0₄, the label is incorporated into the gamma (γ) position of ATP, and then into the backbone of the cell's DNA. It was shown using a bacteriophage model that the ionizations produced by the hard ³²P β- electrons on their way out of the phage particle are not the principal cause of death, but rather the transmutation ( P → S) or the recoil energy imparted by the randomly decaying phosphorus atoms is thought to induce double-stranded breaks in DNA, with consequent irreparable lethal effect on the cell (Stent, G.S. and C.R. Fuerst. 1955. Inactivation of bacteriophage by decay of incorporated radioactive phosphorous. J. Gen. Physiol. 38:441 -58; Fuerst, C.R. and G.S. Stent. 1956. Inactivation of bacteria by decay of incorporated radioactive phosphorous. J. Gen. Physiol. 40:73-90.). ³²P suicide has been used in the study of intermediary metabolism (Gunsalus, I.C. and C.W. Shuster. 1961 . Energy yielding metabolism in bacteria. In: The Bacteria, eds. I.C. Gunsalus and R.Y. Stanier, pp. 1 -58. New York, Academic Press; Wood, W.A. 1961 . Fermentation of carbohydrates and related compounds. In: The Bacteria, vol. II, eds. I.C. Gunsalus and R.Y. Stanier, pp. 59- 149. New York, Academic Press; Wood, W.A. 1961 ) to show that essentially all of the ATP produced by Escherichia coli, and likely most facultative anaerobes such as streptococci and staphylocococci, growing anaerobically on glucose can be accounted for by the two glyolytic enzymes, phosphoglycerate kinase (PGK) and pyruvate kinase (PK), and the phosphoroclastic pathway enzyme, acetate kinase (AK). More recently, P suicide has been used to investigate the bacteriophage growth cycle (Miller RC Jr. 1970. J Virol. 5:533-5) and in comparison to ionizing radiation and ultraviolet light in wild-type and radiosensitive mutants of yeast (Hatzfeld J and Moustacchi E. 1969. Comparison of the lethal effect of ionizing or ultraviolet radiation and the suicide effect of P incorporated on Saccharomyces cerevisiae haploid and radiosensitive mutants. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 15:101 -13).

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.

This is the first disclosure of the idea of using ³²P suicide to create non- replicating microorganisms for use as post-biotics in the treatment and/or prevention of infectious diseases, biofilms and biofouling conditions. The method should be generally applicable to a wide range of diseases caused by bacteria, fungi, yeast, protozoa, algae, and viruses.

Radioactive Isotope Inactivation

Microorganisms can be inactivated using, e.g., P suicide. Fuerst & Stent, J. Gen. Physiol. 40:73 (1956); Miller, J. Virol., 5:533 (1970). Optionally, 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. Alternatively, 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 ³²P 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. about 30 minutes, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours (or any range between about 30 minutes and 10 hours)) in order for them to achieve the desired cell density before the addition of P in the form of orthophosphate or other metabolite. Incubation is then continued until one or more doublings (generations) of the microorganism has occurred in order to optimize incorporation of the radiolabeled phosphorous into the microorganism's DNA. Bacteriophage and viruses can be prepared and used to infect cells, which are then incubated, as described in, e.g., Miller. After incubation, 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 ³²P0₄ to the medium of actively growing microorganisms results in its incorporation into the DNA backbone. During storage of the culture, decay of the ³²P leads to cell death, presumably because of irreparable breakage of the DNA at multiple sites or effects of the ³²P on other cellular components important in cell replication. In one embodiment of the invention 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 P0₄ was performed. As a result, 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 ³²P incorporation. Ideally, after treatment, the post-biotic should have negligible cells capable of replicating in or on the treated host or surface. To reduce the potential for occurrence of this technical problem, different growth conditions can be used to find the one that yields the highest killing percentage during the initial linear phase of viability testing. In addition, the variables of how much P to incorporate in the medium, how long the incubation in the presence of the ³²P 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 ³²P 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.

Methods of Treatment, Amelioration, or Prevention (Prophylaxis) of a Disease, Infection or Colonization Caused by Microorganisms.

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.

In one embodiment, 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. That is, 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. Alternatively, the radioactive phosphorus- inactivated second microorganism population can be a different microorganism from the first microorganism. For example, normal pharyngeal flora (e.g., alpha-hemolytic streptococci) can inhibit the growth of group A streptococci. See, Sanders et al., Infect. Immun. 16:599 (1977); Sanders ei al., Infect. Immun. 13:808 (1976). Additionally, 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. aureas, 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 sp., Alternaria sp., Epicoccum sp., Cryptococcus sp., Penicillium sp., Phodotorula sp., Aureobasidium sp., Cladosporium sp., Mucor sp., Phoma sp., Stachybotrys sp., Pichia sp., Fusarium sp., Trichoderma sp.; algae, such as, Amphora sp., Cocconeis sp., Achnathes sp., Pleurococcus sp., Trentepohlia sp., Enteromorpha sp., Ectocarpus sp., Ulothrix sp., Cladophora sp., Navicula sp., protozoans such as Giardia, Cryptosporidium, Entamoeba, Idoamoeba, Dientamoeba, Trichomonas, Chilomastix, Balantidium, Isospora, Sarcocystis, Cyclospora, Enterocytozoon, Encephalitozoon, Plasmodium, Babesia, Toxoplasma, Sarcocystis, Leishmania, Trypanosoma, or viruses such as papillomaviruses, polyomaviruses, adenoviruses, herpesviruses, poxviruses, parvoviruses, picornaviruses (including rhinoviruses), paramyxoviruses, orthomyxoviruses, reoviruses, rhabdoviruses, togaviruses, flaviviruses, bunyaviridae, retroviruses, hepatitis viruses.

In one embodiment of the invention 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. In one embodiment of the invention non-replicating microorganisms are administered at a daily dose of about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, or 10¹², microorganisms (or any range between about 10³ and 10¹² microorganisms). For biofilm or biofouling conditions about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ , or 10¹² microorganisms/cm² can be applied. In the case of viruses, 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.

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. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art. 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.

The 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. Alternatively, 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).

The particular dosages of non-replicating microorganisms in a composition will depend on many factors including, but not limited to the species, age, gender, concurrent medication, general condition of the animal to which the composition is administered, and the mode of administration of the composition. An effective amount of the composition of the invention can be readily determined using only routine experimentation. 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. In general, the non-replicating microorganisms of the invention cause interference with other microorganism populations without inducing a host immune response. However, if desired, 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.

If desired, 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. Optionally, adjuvants can also be included in a composition. Adjuvants are substances that can be used to nonspecifically augment a specific immune response. Generally, 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. Alk(S0₄)₂; AINa(S0₄)₂, AINH₄(S0₄), Silica, Alum, AI(OH)₃, and Ca₃(P0₄)₂), polynucleotides (i.e. Poly IC and Poly AU acids), and 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) emulsion.

Administration of 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. Optionally, 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. If desired, co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions.

Methods of Amelioration or Prevention of a Biofouling Conditions or Biofilm Conditions

Methods of the invention can also be used to ameliorate, reduce, remove, or prevent biofouling or biofilms. 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 ³²P. 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 ³²P. 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.

Where the biofilm is present or potentially present on an artificial surface within a human or animal (e.g., a catheter or medical device), the artificial surface can be contacted with the one or more radioactive isotope-inactivated microorganism populations prior to insertion into the human or animal. Optionally, 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. Optionally, 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.

All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference in their entirety. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of may be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

Examples The following examples describe the production and testing of post-biotics for prevention or treatment of infections caused by methacillin resistant S. aureus, but could be adapted for the production of a wide variety of useful post-biotics.

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). Three ml of S. aureus strain Smith in stationary phase was inoculated into 30 ml of fresh MHB. Appropriate volumes of cultures (see below) of S. aureus strain 502A in stationary phase were harvested by centrifugation (4,000X g, 20 min., 25°C), and resuspended in 1 ml of sterile phosphate-buffered saline (PBS, pH 7.4, Thermo Fisher Scientific, Pittsburgh, PA). 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).

As shown in Fig. 1 , 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.

It was concluded that the heat killed S. aureus strain 502A inhibited the growth of S. aureus strain Smith by competition for nutrients, most likely fermentable carbohydrate.

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. coli was washed 3 times with 5 ml of sterile PBS and resuspended in a final volume of 1 ml of sterile PBS. 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 BioMate™ 5 spectrophotometer.

As shown in Fig. 2, 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).

As shown in Fig. 3, lactic acid could not be detected when S. aureus strain 502A was heat-inactivated, or treated with 5% sodium fluoride or 5% arsenate. Therefore, heat-treatment inhibits the glycolytic activity of S. aureus strain 502A. These results indicate that the basis for inhibition of strain Smith did not involve competition for glucose by heat killed cells of 502A since the glycolytic pathway in the heat killed cells was not functional, or at least did not result in the production of the common end product (lactic acid) for fermentation by this organism.

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 (25 ml each) 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 BioMate™ 5 spectrophotometer.

As shown in Fig. 4, the addition of S. aureus strain 502A pre-conditioned medium had no effect on the growth of S. aureus Smith strain. It was concluded that the growth inhibition effect of the inactivated S. aureus strain 502A on S. aureus strain Smith is not likely to be related to quorum sensing. The molecular basis for inhibition of growth of planktonic cells of strain Smith by heat killed 502A thus remains unknown.

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. Sterile (autoclaved) microscope slides were immersed in vessels containing undiluted, 2-fold or 4-fold diluted suspensions of heat-inactivated S. aureus strain 502A or sterile PBS (as a control) for 24 hours. Slides were transferred to duplicate mid-exponential phase cultures of S. aureus strain Smith and incubated for 1 hour. The slides were rinsed with by immersion 3 times in sterile water, and bacteria that remained attached to the slide were dispersed using ultrasonic treatment (Sonic Dismembrator, model 100, Thermo Fisher Scientific, Pittsburgh, PA). Dispersed bacteria were further homogenized by vortex treatment for 30 seconds and serially diluted in PBS. Aliquots were inoculated onto MHB agar, and incubated at 37°C for 12-16 hours. Viable cell counts were enumerated for dilutions that yielded 30 to 300 colonies. As shown in Fig. 5, pre-incubation of a glass surface with heat killed S. aureus strain 502A significantly inhibited the subsequent attachment of the S. aureus strain Smith after 1 hour. The inhibition of attachment was dose-dependent. These results indicate that heat killed strain 502A occupy attachment sites necessary for binding of strain Smith. The effect is dose dependent.

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.

The presence of heat killed 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. ³²P 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, ³²P labeled NaH₂P0₄ 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 ³²P killing.

After 10 days of incubations, approximately 5 logs of killing had occurred in the cultures treated with P0₄ whereas control cultures (injected with sterile water in place of ³²P labeled NaH₂P0₄) showed negligible decreases in viable counts. The rate of decrease in viable counts in the treated culture was linear until about day 5 and then appeared to decline though the end of the experiment at day 10. See Fig. 8.

Example 9. Phosphate-limited Medium (PLP) to Enhance ³²P Uptake in S. aureus.

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:

Combine 278 mM glucose, 31 mM thymine, 5% (w/v) Bacto™ casamino acids (Beckon, Dickinson and Co., Sparks, MD) and 42 mM magnesium sulfate in an appropriate glass vessel. After dissolving in nanopure water, the medium is treated by the addition of 1 % calcium hydroxide and stirred in an ice bath for 4 hours. The precipitate is removed by centrifugation at 7,500xg for 5 minutes at room temperature. The supernatant is sparged with carbon dioxide gas for 2 hours, and the precipitate is removed by centrifugation as described above. The pH is adjusted to 8.5 with 5 N sodium hydroxide, and the resulting precipitate is removed by centrifugation as described above. 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.

Preliminary demonstration that PLP is phosphate limited for S. aureus strain 502A was accomplished. 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 ).

Table 1

Additional buffering of the medium with Tris or MES did not improve the yields. Increased concentration of casamino acids is most likely to achieve this effect. The PLP medium is clearly phosphate-limited based on the results obtained in this and similar experiments. Example 10

Background

Herein, we describe the use of ³²P suicide-induced non-replicating cells of, e.g., S. aureus 502A to serve as a therapeutic and prophylaxis agent for a variety of diseases caused by, e.g., S. aureus. By extension, 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.

Experimental Procedure

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.

All incubations of 502A cultures were performed at 37°C in ambient atmosphere; overnight incubation was typically sufficient to achieve visible colonies on agar media and saturated growth in liquid media.

When possible, a Gram stain to confirm the presence of Gram positive cocci in clusters of representative colonies and samples of liquid cultures was performed with oil immersion microscopy.

For long term storage, 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. ³²P Incorporation

Fifty (50) mL of phosphate limited medium (PLM) supplemented with 10 mM sodium phosphate (by addition of a sterile 0.1 M stock solution, pH 7.6) was inoculated with a 3-5 colonies of 502A from a starter plate. 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). Cells were harvested and resuspended in PLM supplemented with 0.1 M sodium phosphate (pH 7.6) to give a final phosphate concentration of 6 mM. An amount of PLM was used to give an initial OD₆₀o approximately equal to 0.2 in a volume equal to 130 mL. The culture was incubated in a 37°C water bath. At hourly intervals, the OD₆₀o was measured. When the OD₆oo reached approximately 1 .0, the cell suspension was divided into 6 x 20 mL aliquots and placed into a 37 °C water bath. To each of the tubes a specified amount of ³²P0 was added, see Table 2.

The tubes were Incubated further in a 37°C water bath until OD₆oo = 1 -4. 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.

Measurements of ³²P Suicide, Radionuclide Decay, and Metabolic Activity

Three days following completion of the steps above, 1 aliquot of each of tubes 1 -6 were removed. The tubes were thawed in a 4°C water bath. The cells were washed twice by centrifugation (Microfuge, 14,000xg for 10 min at 4°C) with 1 ml ice cold PBS. The cells were resuspended in 0.7 ml ice cold PBS (pH 7.2) containing 1 % glucose and 1 mM magnesium chloride and vortexed vigorously to assure complete dispersion of pellets. The following tests were performed.

a. Viable colony forming units (CFU) - serial 10-fold dilutions (10^"1 through

10^"6) were prepared and 0.1 mL samples of appropriate dilutions were plated in triplicate on THB agar medium. The plates were incubated at 37°C overnight. Colonies were counted.

b. Specific activity - decays per minute (DPM) were determined using liquid scintillation counting using triplicate samples from 10^"1 dilution of tubes 1 -6.

c. Metabolic activity - 500 μΙ 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).

Additional cryovials stored at -80 °C were removed at indicated time points and tested as described above. These steps were repeated for each of tubes 1 -6 for decays per minute to determine the theoretical time to achieve background levels of radioactivity as shown in "Data Analysis" section (below). These steps were also repeated for viable CFU, and the theoretical time to achieve complete killing (CFU = 0) was determined by extrapolation of the data in "Data Analysis" section (below) for each of tubes 1 -6.

Data Analysis

Sample number 4 was determined to have been contaminated during the ³²P incorporation step and was not further analyzed.

The data and a semi-log plot of DPM/mL as a function of incubation at -80°C for a control (no ³²P added) and each of the cell samples incubated with differing levels of ³²P is shown in Figures 9A and B. The incorporation of ³²P correlated with the specific activity of the radionuclide used in each of the samples. The slopes of each curve were roughly comparable, indicating that the rate of decay of ³²P was the same in all samples. Based on the first three data points and the half-life of ³²P (ca. 14 days), it was calculated that the sample with the highest DPM (sample number 6) would reach background levels after approximately 207 days. The final time point, at 207 days, showed this be the case. The data and a semi-log plot of viable CFU as a function of incubation at - 80°C time for a control and each of the cell samples incubated with differing levels of ³²P is shown in Figures 10A and B. The viability of the control sample (number 1 , no added ³²P) remained constant throughout the experiment. The rate of decrease in viability for samples incubated with ³²P correlated with the specific activity of ³²P used during the ³²P0₄ incorporation step. Sample 6, in which 502A cells were exposed to the highest specific activity of ³²P during the ³²P0₄ incorporation step showed over 99% killing compared to the control (sample 1 ) after 207 days of incubation at -80 °C. The data indicate that improved killing could be achieved by increasing the specific activity of ³²P and/or increasing the length of exposure to ³²P during the ³²P0₄ incorporation step.

To determine if ³²P suicide had any effect on the cells' metabolic activity, 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. The 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.

Thirty (30) mg NAD (Sigma catalog No. 260-1 10, nicotinamide adenine dinucleotide, Grade III) was dissolved in 6 ml of glycine buffer (Sigma catalog No. 826-3, glycine, 0.6 mol/L, and hydrazine, pH 9.2 at 25 °C) on ice and 12 ml ice cold deioninzed water was added. 0.06 ml LDH (Sigma catalog No. 826-6, LDH bovine heart suspension in ammonium sulfate, approximately 1 ,000 U/ml when prepared) was added. The solution was mixed thoroughly by gentle inversion. 980 ul aliquots of cocktail were added to 1 .5 ml cuvettes (Plastibrand catalog No. 7591 -65). Twenty (20) ul of sample were added per cuvette. The cuvettes were covered with parafilm and incubated in 37°C water bath for 20 minutes. The OD₃₄₀ was read. A standard calibration curve using OD₃₄₀ as a function of lactic acid concentration was prepared using serial 2-fold dilutions of sodium lactate (0.2 to 6.4 mM). The results are shown in Figures 1 1A and B. The slopes of the trend lines for samples 1 (- 0.0002), sample 2 (-0.0029), sample 3 (+0.0015), sample 5 (+0.0024) and sample 6 (-0.000006) indicate that no significant loss of glycolytic activity was observed when cells were treated with varying specific activities of P and stored at -80 °C for 207 days. During that same period of time, over 99% killing of cells occurred in sample number 6, indicating that loss of replicative ability from ³²P suicide did not result in loss of metabolic activity.

Claims

CLAIMS I claim:

1 . A method of amelioration or prophylaxis 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 or surface, wherein the disease, infection, or colonization is ameliorated or prevented.

2. The method of claim 1 , wherein the radioactive phosphorus-inactivated second microorganism populations are obtained by growing the microorganism populations in the presence of P.

3. The method of claim 1 , wherein the radioactive phosphorus-inactivated second microorganism populations are the same or similar microorganisms as the first microorganism.

4. The method of claim 1 , wherein the radioactive phosphorus-inactivated second microorganism populations are different microorganisms from the first microorganism.

5. The method of claim 1 , wherein the radioactive phosphorus-inactivated second microorganism populations are administered topically, orally, intravenously, intramuscularly, intrapulmonary, intradermally, intraperitoneally, subcutaneously, via aerosol, intranasally, via infusion pump, via suppository, or mucosally.

6. The method of claim 1 , wherein the first microorganism is a virus, alga, bacterium, yeast, fungus, or protozoan.

7. The method of claim 6, wherein the radioactive phosphorus-inactivated second microorganism populations are algae, bacteria, yeast, fungi, or protozoa that are metabolically active, but are substantially unable to replicate; or viruses that are substantially unable to replicate.

8. A method of ameliorating or preventing a biofouling condition or a biofilm condition, caused by one or more first microorganisms comprising 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.

9. The method of claim 8, wherein the one or more radioactive phosphorus- inactivated second microorganism populations are obtained by growing the microorganisms in the presence of P.

10. The method of claim 8, wherein the one or more radioactive phosphorus- inactivated second microorganism populations comprise microorganisms that are the same or similar to the one or more microorganisms that cause the biofouling condition or the biofilm condition.

1 1 . The method of claim 8, wherein the one or more radioactive phosphorus- inactivated second microorganism populations comprise microorganisms that are different from the one or more microorganisms that cause the biofouling condition or the biofilm condition.

12. A method of inducing an immune response to first microorganisms in a subject comprising: 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.

13. The method of claim 12, wherein the radioactive phosphorus-inactivated second microorganism populations are obtained by growing the microorganism populations in the presence of P.

14. The method of claim 12, wherein the radioactive phosphorus-inactivated second microorganism populations are administered topically, orally, intravenously, intramuscularly, intrapulmonary, intramuscularly, intradermally, intraperitoneally, subcutaneously, via aerosol, intranasally, via infusion pump, via suppository, or mucosally.

15. The method of claim 12, wherein the first microorganisms are viruses, algae, bacteria, yeast, fungi, or protozoa.

16. A therapeutic composition comprising one or more radioactive phosphorus- inactivated microorganisms and an acceptable carrier.

17. The therapeutic composition of claim 16, further comprising an adjuvant.

18. The therapeutic composition of claim 16, wherein the radioactive phosphorus- inactivated microorganisms are viruses, algae, bacteria, yeast, fungi, or protozoa.

19. The therapeutic composition of claim 16, wherein the composition is a pharmaceutically acceptable composition and the carrier is a pharmaceutically acceptable carrier.

20. A kit comprising:

(a) a container comprising one or more radioactive phosphorus-inactivated microorganism populations;

(b) an applicator for the one or more radioactive phosphorus-inactivated microorganism populations; and (c) optionally, one or more buffers or diluents.

21 . A method for preparing a therapeutic or prophylactic composition of one or more radioactive phosphorus-inactivated microorganism populations comprising:

(a) growing one or more microorganism populations in the presence of ³²P in a medium;

(b) harvesting and washing the microorganisms;

(c) freezing the microorganisms until the microorganisms can no longer substantially replicate and the ³²P levels are suitable for administration to a subject or to a surface.

22. The method of claim 21 , wherein the medium is phosphate-limited cultivation medium.