Method and composition for sterilization

Abstract

The present invention relates to a method of treating tissue of a subject infected with a catalase-positive microorganism, the method comprising the steps of: applying light having a wavelength of about 400 nm to about 430 nm to the tissue of the subject infected with the catalase-positive microorganism, wherein catalase is inactivated, and contacting the tissue with a composition comprising a dilute peroxide solution. The present invention also relates to a device or light source for delivering light-activated pulsed light to treat tissue of a subject infected with a catalase-positive microorganism, wherein the light-activated pulsed light has a wavelength of about 400 nm to about 430 nm.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080022202.X, entitled "Sterilization Method and Composition", which entered the Chinese national phase of PCT international patent application PCT / US2020 / 016125, filed on January 31, 2020.

[0002] Claims for inventions made under federally funded research

[0003] This work was supported by grant number R01AI132638 from the National Institutes of Health. The government holds certain rights to this invention.

[0004] Related applications / patents

[0005] This application claims priority to U.S. Application No. 62 / 799,328, filed on January 31, 2019, as agent file number 701586-094400PL01, the contents of which are incorporated herein by reference. Technical Field

[0006] The present invention generally relates to sterilization methods and compositions, and particularly to the killing of catalase-positive microorganisms by applying pulsed light. Background Technology

[0007] Antibiotic resistance is estimated to cause 700,000 deaths worldwide each year, and studies predict that this number could rise to 10 million by 2050 if efforts are not made to reduce resistance (Willyard, CJNN The drug-resistant bacteria that pose the greatest health threats. 543, 15 (2017)). However, resistance is acquired from pathogen mutations at a rate that exceeds the rate at which new antibiotics are introduced clinically. There is an urgent need to develop unconventional approaches to combat resistance. Summary of the Invention

[0008] The lethal effect of some antibiotics is achieved through the generation of reactive oxygen species (ROS). Catalase, a key defense enzyme ubiquitous in most aerobic pathogens, is used to scavenge hydrogen peroxide, thereby preventing downstream oxidative damage. It has now been shown that catalase can be optimally photoinactivated by blue light at wavelengths from about 400 nm to about 430 nm, particularly about 410 nm. Photoinactivation of catalase makes broad-spectrum catalase-positive microbial pathogens highly sensitive to ROS-producing antimicrobial agents and / or immune cell attack. It has now been further determined that the antimicrobial effect of photoinactivation is significantly and unexpectedly increased after administration of low concentrations of H2O2 and / or ROS-generating agents.

[0009] On one hand, the present invention provides a method for treating tissues of a subject infected with catalase-positive microorganisms, the method comprising the steps of: applying light of a wavelength of about 400 nm to about 430 nm to the tissues of the subject infected with catalase-positive microorganisms, wherein catalase is inactivated, and contacting the tissues with a composition comprising a dilute peroxide solution, thereby treating the tissues of the subject infected with catalase-positive microorganisms.

[0010] In one implementation, the wavelength is approximately 410 nm.

[0011] In another embodiment, the light dose is approximately 5 J / cm². 2 Approximately 200 J / cm 2 .

[0012] In yet another implementation, the light dose is approximately 15 J / cm². 2 .

[0013] In yet another implementation, the catalase-positive microorganism is a fungal or bacterial microorganism and the light is provided by a pulsed nanosecond laser.

[0014] In yet another embodiment, the catalase-positive microorganism is a fungus or bacteria and the light is provided by a continuous-wave LED.

[0015] In yet another implementation, the dilute peroxide solution is a hydrogen peroxide solution.

[0016] In yet another embodiment, the hydrogen peroxide solution contains between about 0.03% and about 0.3% hydrogen peroxide.

[0017] In yet another embodiment, the method further includes administering a ROS-generating agent to the infected tissue of the subject.

[0018] In yet another implementation, the ROS-generating agent is tobramycin, silver cations, iodine tincture, gold nanoparticles, methylene blue, β-lactam antibiotics, aminoglycosides, fluoroquinolones, azoles, membrane-targeted polyene antifungals, or cell wall-targeted antifungals.

[0019] In yet another implementation, the tissue is skin, scalp, or nails.

[0020] In yet another implementation, catalase-positive microorganisms are eradicated.

[0021] On the other hand, the present invention provides a method for disinfecting a non-living surface contaminated with catalase-positive microorganisms, the method comprising the steps of: applying light of a wavelength of about 400 nm to about 430 nm to the non-living surface, wherein the catalase is inactivated, and contacting the non-living surface with a composition comprising a dilute peroxide solution, thereby disinfecting the non-living surface.

[0022] In one implementation, the wavelength is approximately 410 nm.

[0023] In another embodiment, the light dose is approximately 5 J / cm². 2 Approximately 200 J / cm 2 .

[0024] In yet another implementation, the light dose is approximately 15 J / cm². 2 .

[0025] In yet another implementation, the catalase-positive microorganism is a fungal or bacterial microorganism and the light is provided by a pulsed nanosecond laser.

[0026] In yet another embodiment, the catalase-positive microorganism is a fungus or bacteria and the light is provided by a continuous-wave LED.

[0027] In yet another implementation, the dilute peroxide solution is a hydrogen peroxide solution.

[0028] In yet another embodiment, the hydrogen peroxide solution contains between about 0.03% and about 0.3% hydrogen peroxide.

[0029] In yet another embodiment, the method further includes administering a ROS-generating agent to the infected tissue of the subject.

[0030] In yet another implementation, the ROS-generating agent is tobramycin, silver cations, iodine tincture, gold nanoparticles, methylene blue, β-lactam antibiotics, aminoglycosides, fluoroquinolones, azoles, membrane-targeted polyene antifungals, or cell wall-targeted antifungals.

[0031] In yet another implementation, inanimate surfaces include materials such as metal, plastic, fabric, rubber, stone, composite materials, or wood.

[0032] In yet another implementation, catalase-positive microorganisms are eradicated.

[0033] On the other hand, the present invention provides a method for treating tissue of a subject infected with catalase-positive microorganisms, the method comprising the steps of: applying light of a wavelength of about 400 nm to about 460 nm provided by a pulsed nanosecond laser to the tissue of the subject infected with catalase-positive microorganisms, wherein catalase is inactivated, and contacting the tissue with a composition comprising a ROS-generating agent, thereby treating the tissue of the subject infected with catalase-positive microorganisms.

[0034] In one implementation, the wavelength is approximately 410 nm.

[0035] In another embodiment, the light dose is approximately 5 J / cm². 2 Approximately 200 J / cm 2 .

[0036] In yet another implementation, the light dose is approximately 15 J / cm². 2 .

[0037] In yet another implementation, the catalase-positive microorganism is a fungus or a bacterium.

[0038] In yet another implementation, the dilute peroxide solution is a hydrogen peroxide solution.

[0039] In yet another embodiment, the hydrogen peroxide solution contains between about 0.03% and about 0.3% hydrogen peroxide.

[0040] In yet another implementation, the ROS-generating agent is tobramycin, silver cations, iodine tincture, gold nanoparticles, methylene blue, β-lactam antibiotics, aminoglycosides, fluoroquinolones, azoles, membrane-targeted polyene antifungals, or cell wall-targeted antifungals.

[0041] In yet another implementation, the tissue is skin, scalp, or nails.

[0042] In yet another implementation, catalase-positive microorganisms are eradicated.

[0043] On the other hand, the present invention provides a method for generating a synergistic antimicrobial effect in the tissues of a subject infected with catalase-positive microorganisms, the method comprising the steps of: applying light of a wavelength of about 400 nm to about 460 nm to the tissues of the subject infected with catalase-positive microorganisms, wherein catalase is inactivated, and contacting the tissues with a composition comprising a dilute peroxide solution, thereby generating a synergistic antimicrobial effect in the tissues of the subject infected with catalase-positive microorganisms.

[0044] In one implementation, the wavelength is approximately 410 nm.

[0045] In another embodiment, the light dose is approximately 5 J / cm². 2 Approximately 200 J / cm 2 .

[0046] In yet another implementation, the light dose is approximately 15 J / cm². 2 .

[0047] In yet another implementation, the catalase-positive microorganism is a fungal or bacterial microorganism and the light is provided by a pulsed nanosecond laser.

[0048] In yet another embodiment, the catalase-positive microorganism is a fungus or bacteria and the light is provided by a continuous-wave LED.

[0049] In yet another implementation, the dilute peroxide solution is a hydrogen peroxide solution.

[0050] In yet another embodiment, the hydrogen peroxide solution contains between about 0.03% and about 0.3% hydrogen peroxide.

[0051] In yet another embodiment, the method further includes administering a ROS-generating agent to the infected tissue of the subject.

[0052] In yet another implementation, the ROS-generating agent is tobramycin, silver cations, iodine tincture, gold nanoparticles, methylene blue, β-lactam antibiotics, aminoglycosides, fluoroquinolones, azoles, membrane-targeted polyene antifungals, or cell wall-targeted antifungals.

[0053] In yet another implementation, the tissue is skin, scalp, or nails.

[0054] In yet another implementation, catalase-positive microorganisms are eradicated. Attached Figure Description

[0055] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be understood in conjunction with the accompanying drawings, which are incorporated herein by reference.

[0056] Figure 1 The effect of ns-410 nm exposure on pure catalase solution was depicted. (a) Absorption spectrum of pure catalase solution under ns-410 nm exposure. Catalase solution: 3 mg / ml, filtered through a 0.2 μm filter. (b) Percentage of residual active catalase after different treatment regimens (different wavelengths at the same dose). Catalase was quantified using the AmplexRed Catalase kit. Data: mean ± standard deviation (N = 3).

[0057] Figure 2The effect of ns-410 nm exposure on the percentage of active catalase from MRSA USA300 and Pseudomonas aeruginosa was described. (ab). Percentage of active catalase retained in MRSA USA300 (a) and Pseudomonas aeruginosa (b) after different treatment regimens (different wavelengths at the same dose). Catalase was quantified using the Amplex Red Catalase kit. Data: mean ± standard deviation (N=3).

[0058] Figure 3 Resonance Raman spectra of bovine liver catalase powder with and without 410 nm exposure were depicted. 410 nm dose: 250 mW / cm² 2 Raman spectroscopy acquisition time: 25 seconds, 532 nm excitation. Data: Average ± SD from five spectra.

[0059] Figure 4 The photoinactivation effects of ns-410nm and CW-410nm exposures on pure catalase solution (a), catalase from MRSA USA300 (b), and catalase from Pseudomonas aeruginosa (c) were compared. Catalase quantification was obtained using the Amplex Red Catalase kit. Data: mean ± standard deviation (N = 3). Student's unpaired t-test, ***: p < 0.001; **: p < 0.01.

[0060] Figure 5 The study depicted the CFU / ml of methicillin-resistant Staphylococcus aureus (A), Pseudomonas aeruginosa (B), and Salmonella enterica (C) in the stationary phase of MRSA USA300 under various exposure conditions with and without combination of H2O2 treatment. -1 Data: mean ± standard deviation (N=3). Unpaired t-test for students, ***: p<0.001; **: p<0.01. 250 CFU: limit of detection. Figure 5 The synergistic effect of photoinactivation by catalase and low concentrations of hydrogen peroxide in eliminating stationary MRSAUSA300 and stationary Pseudomonas aeruginosa was further depicted. Left and right: CFU / ml of stationary MRSA and Pseudomonas aeruginosa under different treatment regimens, respectively. -1 N=3. Data: mean ± standard deviation. ***: statistically significant. p<0.001. 250 CFU: limit of detection.

[0061] Figure 6The efficacy of CW-410nm and ns-410nm combined with H2O2 against stationary MRSA USA300 and Pseudomonas aeruginosa was compared. Left and right: CFU / ml of stationary MRSA and Pseudomonas aeruginosa under different treatment regimens, respectively. -1 N=3. Data: mean ± standard deviation. ***: statistically significant. p<0.001. 250 CFU: limit of detection.

[0062] Figure 7 The CFU / ml of Escherichia coli BW25113 under different treatment regimens were described. -1 Tobramycin: 2 μg / ml, incubated for 4 hours. ***: p<0.001, student unpaired t-test.

[0063] Figure 8 The CFU / ml of Enterococcus faecalis NR-31970 under different treatment regimens were described. -1 Tobramycin: 2 μg / ml, incubate for 4 hours.

[0064] Figure 9 Confocal laser scanning microscopy (Cd) images of intracellular MRSA were depicted. (ac) Fluorescence images of live MRSA (a) and dead MRSA (b) in RAW 264.7 macrophages 1 hour after MRSA infection, along with transmission images (c). (df) Fluorescence images of live MRSA (d) and dead MRSA (e) in RAW 264.7 macrophages 1 hour after MRSA infection with ns-410-exposed MRSA, along with transmission images (f). (gh) Quantitative analysis of live / dead MRSA from the above two groups. Scalar bars = 10 μm.

[0065] Figure 10 The percentage of active catalase in various fungal strains with and without 410 nm exposure was depicted. Dosage: 410 nm, 150 mW / cm² 2 5 minutes. Fungal concentration: 10 6 Cells / mL. Candida albicans CASC5314: Wild-type Candida albicans.

[0066] Figure 11 The CFU results of Candida albicans CASC5314 after different treatment regimens are depicted. (a) Time-kill assay of CASC5314 after various treatment regimens. (b) Spread plates of CASC5314 after 1 hour of incubation under different treatment regimens.

[0067] Figure 12The synergistic effect of photoinactivation of catalase at different wavelengths and elimination of quiescent CASC5314 by low concentrations of hydrogen peroxide was depicted. CFU / ml of CASC5314 after treatment with H2O2 and various wavelength combinations were also described. -1 Dosage 40mW / cm 2 24J / cm 2 H2O2: 44mM, incubation for 1.5 hours. Data: mean ± SEM (N=3). ##: detection limit.

[0068] Figure 13 The PrestoBlue fluorescence signals from CASC5314 under various treatment schemes are depicted. (a, c) represent the stationary and logarithmic periods of CASC5314 treated with H2O2 alone, respectively. (b, d) represent the stationary and logarithmic periods of CASC5314 treated with 410 nm plus H2O2, respectively.

[0069] Figure 14 PrestoBlue fluorescence signals of three different Candida auris strains under different treatment regimens were depicted. (a, c, e). Group treated with Amp B alone. (b, d, f). Group treated with 410 nm plus Amp B.

[0070] Figure 15 Confocal laser scanning imaging depicts live / dead Candida albicans after infection with RAW264.7 macrophages.

[0071] Figure 16 The photoinactivation of catalase, which kills MRSA in combination with silver cations, is depicted. The images show MRSA USA300 spread on agar plates under different treatment regimens.

[0072] Figure 17 A comparison of the inactivation of catalase and elimination of *E. coli* BW25113 by CW-410 and ns-410 through synergistic interaction with silver cations was depicted. (ab). CFU / ml of *E. coli* BW25113 after different treatment regimens. -1 (a) 30 minutes, (b) 60 minutes. Dosage: 22 J / cm³ 2 Silver cation: 0.5 μM. Data: mean ± SEM (N = 3). ***: p < 0.001, statistically significant. Unpaired t-test performed by students. Detailed Implementation

[0073] definition

[0074] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, this application (including the definitions) shall prevail.

[0075] As used in this article, the phrase "treatment of infected tissue" refers to the clinical manifestation of curing, reducing, or partially stopping an infection or its complications. Treatment of infected tissue achieves a medically desirable outcome. In some cases, this is complete eradication of the infection. In other cases, it is an improvement in the symptoms of the infection.

[0076] "ROS generator" is any biological or chemical agent that produces reactive oxygen species (ROS). As defined herein, ROS generators do not include exogenous photosensitizers that have been photoactivated. "Photosensitizer" is a compound or its biological precursor that, upon photoactivation, exhibits phototoxicity or other biological effects on biomolecules.

[0077] "Subjects" are vertebrates, including any member of the class Mammalia, including humans, livestock and farm animals, as well as zoo, sport or pet animals, such as mice, rabbits, pigs, sheep, goats, cattle and higher primates.

[0078] "Microorganisms" are multicellular or single-celled microorganisms, including bacteria, protozoa, and some fungi and algae. As used herein, the term microorganisms include pathogenic microorganisms, such as bacteria, protozoa, or fungi.

[0079] The term "inanimate surface" refers to any non-biological surface.

[0080] The term "disinfection" refers to the destruction or elimination of pathogenic microorganisms that cause infection.

[0081] Unless specifically stated or clear from the context, as used herein, the term “about” is understood to mean within the normal tolerance range in the field, such as within 2 standard deviations of the mean. “About” is understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term “about”.

[0082] The ranges provided in this document should be understood as abbreviations of all values ​​within that range. The range 1 to 50 is understood to include any number, combination of numbers, or subrange that comes from groups of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (and their fractions, unless the context clearly specifies otherwise). For example, wavelengths from approximately 400 nm to approximately 460 nm include wavelengths 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, and 427. 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 554, 455, 456, 457, 458, 459, and 460 nm. Approximately 5 J / cm² 2 Approximately 200 J / cm 2The light includes numbers 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 15 8, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 and 200 J / cm 2 .

[0083] In this disclosure, terms such as “comprises,” “comprising,” “containing,” and “having” may have the meanings given to them under U.S. Patent Law, and may mean “includes,” “including,” etc.; “substantially constitutes” or “substantially comprises” also have the meanings given to them under U.S. Patent Law, and the term is open-ended, allowing for more content than the referenced content, as long as the essential or novel features of the referenced content are not altered by the presence of more content than the referenced content, but excluding prior art embodiments.

[0084] Other definitions appear in the context of this disclosure.

[0085] The compositions and methods of the present invention

[0086] Hydrogen peroxide (H2O2) is continuously produced within microorganisms through the auto-oxidation of oxidoreductases and rapidly diffuses into the intracellular environment, causing serious harmful effects (such as lipid peroxidation, DNA and protein damage) due to the Fenton reaction.

[0087] Fe²⁺ + H₂O₂ → Fe³⁺.OH + OH⁻

[0088] Fe3++H2O2→Fe2++.OOH+H+

[0089] Due to the lethal accumulation of ROS, photoinactivation of catalase produces an effective antimicrobial effect. Photoinactivation of catalase further helps immune cells eliminate intracellular pathogens. Neutrophils and macrophages are highly motile phagocytes and are the first line of defense of the innate immune system (Segal, AW, Annu Rev Immunol 23, 197-223, doi:10.1146 / annurev.immunol.23.021704.115653 (2005)). These cells play an important role in providing resistance to bacterial and fungal infections by releasing ROS bursts (e.g., superoxide, hydroxyl radicals, and singlet oxygen) (Hampton, MB, Blood 92, 3007-3017 (1998)). However, pathogens possess a range of sophisticated strategies to invade and survive within neutrophils or macrophages, thus acting as 'Trojan horses' responsible for further spread and recurrent infections (Lehar, SM et al., Nature 527, 323-328 (2015)). Catalase, encoded by the gene katA, provides essential resistance against antimicrobial agents or reactive oxygen species released by immune cells (Flannagan, R., Pathogens 4, 826-868 (2015)). Photoinactivation of catalase helps macrophages and neutrophils reduce intracellular and extracellular bacterial load.

[0090] In the method of this invention, photoinactivation of catalase is preferably performed using light with a wavelength of about 400 nm to about 430 nm in combination with the application of a low concentration of peroxide solution and / or ROS-generating agent. The method of this invention excludes the use of exogenous photosensitizers that have already been photoactivated.

[0091] Peroxide solutions include, but are not limited to, solutions containing hydroperoxides, metal peroxides, and organic peroxides. Hydroperoxides include, but are not limited to, peroxyacid, peroxymonosulfuric acid, peracetic acid, peroxydisulfuric acid, peroxynitric acid, peroxynitrite, perchloric acid, and phthalimide peroxyhexanoic acid. Metal peroxides include, but are not limited to, ammonium periodate, barium peroxide, sodium peroxide, sodium perborate, sodium persulfate, lithium peroxide, magnesium peroxide, magnesium perchlorate, and zinc peroxide. Organic peroxides include, but are not limited to, acetone peroxide, acetozone peroxide, alkenyl peroxide, arachidonic acid 5-hydroperoxide, and artelinic acid. Artemisinin, artemotil, arterolane, artesunate, ascaridole, benzoyl peroxide, bis(trimethylsilyl)peroxide, tert-butyl hydroperoxide-tert-butyl peroxybenzoate, CSPD ([3-(1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxane]-3'-yl)phenyl] phosphate dihydrogen ester), cumene hydroperoxide, di... tert-butyl peroxide, diacetyl peroxide, diethyl peroxide, dihydroartemisinin, dimethyl diepoxide, 1,2-dioxane, 1,2-dioxane, 1,2-dioxanedione, dioxirane, dipropyl peroxide, ergosterol peroxide, hexamethylene triperoxide, methyl ethyl ketone peroxide, nardosinone, hydroperoxide, p-menthol, phosphatidyl nitrate, peroxyacetyl nitrate, peroxyacyl nitrate, prostaglandin H2, 1,2,4-trioxane, and verruculogen.

[0092] Other peroxides include potassium disulfide peroxide, bis(trimethylsilyl) peroxide (Me3SiOOSiMe3), phosphorus oxide, ammonium peroxide, copper peroxide (II), sodium peroxide, cobalt peroxide (II), mercuric peroxide (I), iron peroxide (II), potassium peroxide, copper peroxide (I), rubidium peroxide, cesium peroxide, iron peroxide (III), beryllium peroxide, magnesium peroxide, nickel peroxide (II), cadmium peroxide, barium peroxide, benzoyl peroxide, calcium peroxide, diacetyl peroxide, cesium superoxide, lead peroxide (IV), lithium peroxide, gallium peroxide (II), chromium peroxide (III), mercuric peroxide (II), gold peroxide (I), strontium peroxide, zinc peroxide, potassium superoxide, and chromium peroxide (VI).

[0093] In other specific embodiments, the dilute peroxide solution is a hydrogen peroxide solution prepared with about 0.030% to about 0.3% hydrogen peroxide (which is converted to about 8.8 mM to about 88 mM hydrogen peroxide).

[0094] The photoinactivation of catalase and the application of peroxide solutions can also be provided in combination with ROS-generating agents, including antibiotics (e.g., tobramycin). Other ROS-generating agents include, but are not limited to, silver cations, iodine tincture, gold nanoparticles, methylene blue (non-photoactivated), β-lactam antibiotics, aminoglycosides, fluoroquinolones, antifungal azoles, membrane-targeting polyene antifungals (e.g., amphotericin B), and cell wall-targeting antifungals (e.g., caspofungin).

[0095] Typically, after photoinactivation, the peroxide solution can be applied to the site of infection for a duration of approximately 10 to 30 minutes. In alternative embodiments, the peroxide solution, the ROS-generating agent, and / or the photoinactivating light can be applied to the site of infection simultaneously or sequentially. For example, in a specific embodiment, the ROS-generating agent is applied before catalase photoinactivation. In other specific embodiments, the ROS-generating agent is applied after catalase photoinactivation. Preferably, the peroxide solution is applied topically (e.g., as a liquid or spray). The application of the ROS-generating agent can be based on all topical or systemic application modalities known in the art.

[0096] In one embodiment, the method of the present invention, which includes catalase photoinactivation, targets infected external tissues of the subject, including but not limited to skin, hair, and nails. In other embodiments, internal tissues, such as gastrointestinal organs or cavities (oral cavity, vaginal cavity, or nasal cavity), may also be targeted.

[0097] Peroxide solutions and / or ROS-generating agents may be applied alone or as components of a pharmaceutical formulation. The compounds may be formulated for administration to humans or veterinary medicines in any convenient manner. Wetting agents, emulsifiers, and lubricants, such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, release agents, and preservatives may also be present in the composition.

[0098] The pharmaceutical formulations of the present invention include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or vaginal administration. The formulations can be conveniently provided in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration (e.g., intradermal).

[0099] Formulations may include pharmaceutically acceptable carriers. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Formulations of the present invention may be administered parenterally, intraperitoneally, subcutaneously, topically, orally (e.g., ROS-generating agents), or by local administration (e.g., via aerosol or transdermal administration). Depending on the severity or site of infection and disease extent, each patient's general medical condition, and the resulting preferred method of administration, formulations may be administered in multiple unit dosage forms. Details of the formulation and administration techniques of the medicines are described in detail in scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA (“Remington's”).

[0100] The pharmaceutical formulations of the present invention can be prepared according to any method known in the art for manufacturing pharmaceuticals. The formulations can be mixed with suitable, non-toxic, pharmaceutically acceptable excipients. The formulations may contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and may be provided in forms such as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled-release formulations, tablets, pills, gels, patches, implants, etc.

[0101] In carrying out this invention, pharmaceutical formulations can be prepared as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jelly, coatings, powders, and aerosols for transdermal delivery via a local route. In specific embodiments, delivery can be mediated by transdermal patches, bandages, or dressings impregnated with a composition comprising a peroxide solution and / or a ROS-generating agent. Sustained release can be provided via transdermal patches for slow release at the site of infection.

[0102] The amount of a drug formulation sufficient to reduce or eradicate pathogenic microorganisms is the therapeutically effective dose. The effective dosage schedule and amount for this purpose, i.e., the dosing regimen, will depend on a variety of factors, including the stage of infection, the severity of the infection, the patient's general health condition, physical condition, age, etc. The mode of administration must also be considered when calculating the patient's dosing regimen.

[0103] The dosing regimen also takes into account well-known pharmacokinetic parameters in the art, namely the absorption rate, bioavailability, metabolism, and clearance of the active agent (see, for example, Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; and more recently, Remington, ibid.). The prior art allows clinicians to determine the dosing regimen for each individual patient, the active agent, and the disease or condition being treated. The guidelines provided for similar compositions used as medicines can be used as guidelines for determining dosing regimens, i.e., whether the dosage schedule and dosage levels applied to carry out the methods of the present invention are correct and appropriate.

[0104] The single or multiple administration of the pharmaceutical formulation of the present invention may depend on, for example, the patient's required and tolerable dose and frequency, the persistence of the infection, or the disappearance of the infection after each administration. The formulation should provide an adequate amount of peroxide solution to effectively treat, prevent, or improve the infection.

[0105] The method of this invention targets catalase-positive microorganisms that are associated with or may cause infection. Both Gram-negative and Gram-positive bacteria serve as infectious pathogens in vertebrates. Such catalase-positive Gram-positive bacteria include, but are not limited to, Staphylococcus species. Catalase-positive Gram-negative bacteria include, but are not limited to, Escherichia coli, Pasteurella species, Pseudomonas species (e.g., Pseudomonas aeruginosa), and Salmonella species. Specific examples of infectious catalase-positive bacteria include, but are not limited to, *Helicobacter pylori*, *Borelia burgdorferi*, *Legionella pneumophila*, and mycobacterial species (e.g., *Mycobacterium tuberculosis* complex, *Mycobacterium avium* complex, *Mycobacterium gordonae* clade, *Mycobacterium kansasii* clade, *Mycobacterium nonchromogenicum / terrae* clade, mycolactone-producing mycobacteria, *Mycobacterium simiae* clade, *Mycobacterium abscessus* clade, *Mycobacterium chelonae* clade, and *Mycobacterium fortuitum* clade). Mycobacterium clade), Mycobacterium mucogenicum clade, Mycobacterium parafortuitum clade, Mycobacterium vaccae clade, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Mycobacterium gilvum, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium spyr1, Mycobacterium kms, Mycobacterium mcs, Mycobacterium jls, Mycobacterium intracellulare, and Mycobacterium Gordon (M.Acinetobacter baumannii, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, pathogenic Campylobacter species, Haemophilus influenzae, Bacillus anthrax, Corynebacterium diphtheriae, Corynebacterium species, Erysipelothrix rhusiopathiae, Chlamydia trachomatis, Clostridium perfringens, Clostridium tetani *Tetani*, *Klebsiella pneumoniae*, *Pasteurella multocida*, *Bacteroides* species, *Fusobacterium nucleatum*, *Treponemapallidium*, *Treponema pertenue*, *Leptospira*, *Rickettsia*, and *Actinomyces israelli*. *Mycoplasma* and *Chlamydia* species.

[0106] Examples of catalase-positive fungi include, but are not limited to, Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida glabrata, Candida tropicalis, Candida parapsilosis and other catalase-positive Candida species, Candida auris and Trichophyton rubrum.

[0107] Light for photoactivating catalase can be generated and delivered to the site of infection by any suitable means known in the art.

[0108] While it has been established that photoinactivation of antimicrobial activity significantly and unexpectedly increases after the application of low concentrations of H₂O₂ and / or ROS-generating agents, the antimicrobial efficacy is also significantly enhanced when the light is provided by a pulsed nanosecond laser compared to continuous-wavelength LEDs. Therefore, in this specific embodiment, the light source is a pulsed nanosecond laser. Pulsed operation of a laser refers to any laser not classified as a continuous-wave laser, where the optical power appears in a pulse of a certain duration with a certain repetition rate. The nanosecond laser family ranges from ultraviolet (UV) to infrared (IR), with wavelengths up to 1064 nm, repetition rates up to 2 kHz, and pulse energies up to 20 mJ. Photoinactivation of catalase can be performed using light with wavelengths from about 400 nm to about 460 nm. In this specific embodiment, wavelengths from about 400 nm to about 430 nm are used, with a pulse energy of about 5 J / cm². 2 Approximately 200 J / cm 2 The dosage is applied, and in other specific embodiments, at approximately 14 J / cm². 2 Approximately 32 J / cm 2 The dose is applied. In other specific embodiments, the pulse duration is approximately 5 nanoseconds. Pulsed nanosecond lasers deliver light in this range that is clinically advantageous because thermal damage is minimal, temporary, or nonexistent. In a more specific embodiment, the wavelength is 410 nm (using approximately 15 J / cm²). 2 Delivery is made by means of a pulsed nanosecond laser, according to methods known in the art for operating such lasers.

[0109] The duration of exposure ranges from approximately 5 to 10 minutes and can be repeated weekly as needed, for example, approximately twice a week, for several months. In clinical practice, patients may receive treatment for 1 to 3 months or longer, depending on the decision of the practicing physician.

[0110] In other embodiments, photoactivating light can be delivered to the infection site via various optical waveguides such as optical fibers or implants. In some embodiments, photoinactivating light is delivered by an optical fiber device that directly illuminates the infection site. For example, light can be delivered via an optical fiber passing through a small-gauge hypodermic needle. Alternatively, light can be transmitted via percutaneous instruments using optical fibers or cannulated waveguides. For open surgical sites, suitable light sources include broadband conventional light sources, extensive LED arrays, and defocused laser beams. The light source can operate in continuous wave (CW) mode. Photoinactivation of catalase preferably uses wavelengths from about 400 nm to about 430 nm and a dose of about 5 J / cm². 2 Approximately 200 J / cm 2 In the specific implementation plan, it is approximately 14 J / cm. 2 Approximately 32 J / cm 2 The light is used. In other specific embodiments, the wavelength is 410 nm (using approximately 15 J / cm²). 2 (Delivery), applied by the CW LED according to methods known in the art for operating such LED sources. The length of exposure time for any light source ranges from about 5 to about 10 minutes.

[0111] In other embodiments of the invention, photoinactivation of catalase is carried out on inanimate surfaces, including but not limited to metals, plastics, fabrics, rubber, stone, composite surfaces, or wood. In specific embodiments, inanimate surfaces include objects such as instruments, conduits, medical and military equipment, furniture, handrails, textiles, fixtures (such as sink and pipe materials), building materials, industrial or electronic equipment, and food or food processing equipment. Photoinactivation of catalase on inanimate surfaces is preferably carried out using light with a wavelength of about 400 nm to about 430 nm, in conjunction with the application of a solution containing a low concentration of peroxide. In specific embodiments, a wavelength of 410 nm (at 15 J / cm²) is used. 2 (Delivery), applied by a pulsed nanosecond laser. The exposure time ranges from approximately 5 to approximately 10 minutes.

[0112] The following examples are provided for illustrative purposes only and are not intended to limit the scope of what the inventors consider to be their invention.

[0113] Example

[0114] The following materials and methods were used throughout Examples 1-4.

[0115] Bacterial strains: *Enterococcus faecalis* NR-31970, *Enterococcus faecalis* HM-325, *Escherichia coli* BW 25113, *Escherichia coli* ATCC 25922. *Klebsiella pneumoniae* ATCC BAA 1706. *Klebsiella pneumoniae* ATCC BAA 1705. *Salmonella enterica* ATCC 70720. *Salmonella enterica* ATCC 13076. *Acinetobacter baumannii* ATCC BAA 1605. *Acinetobacter baumannii* ATCC BAA-747. *Pseudomonas aeruginosa* ATCC 47085 (PAO-1). *Pseudomonas aeruginosa* 1133. *Pseudomonas aeruginosa* ATCC 15442. *Pseudomonas aeruginosa* ATCC 9027.

[0116] Catalase quantification using the Amplex Red Catalase kit: Quantification of catalase from pure catalase solution and bacteria was achieved using the Amplex Red Catalase kit. 25 μl of analyte was incubated with 25 μl of (40 μM H₂O₂) at room temperature for 30 min. Then, 50 μl of working solution (100 μM Amplex Red reagent containing 0.4 U / mL horseradish peroxidase) was added to the mixture, and the mixture was subsequently incubated in the dark for 30–60 min. Fluorescence was then recorded at 590 nm emission upon excitation at 560 nm.

[0117] Resonance Raman spectra of dried catalase films: Catalase is detected through its wavelengths in the 1300-1700 cm⁻¹ range. -1 The Raman peaks were measured using resonance Raman spectroscopy (1221, LABRAM HR EVO, Horiba) with a 40x objective (Olympus) and an excitation wavelength of 532 nm. The sample (dried 'coffee ring') was sandwiched between two glass coverslips (48393-230, VWR International) at a spatial distance of approximately 80 μm. To investigate photoinactivation (via a continuous-wave LED), the same sample was measured after each laser irradiation.

[0118] CFU experiments were conducted to test the potential synergistic effect between catalase photoinactivation and H2O2: overnight cultured bacteria were centrifuged, the supernatant was discarded, and the precipitate was resuspended in the same volume of PBS. The laser source used in the study was a nanosecond (ns) pulsed OPO laser, model Opolette HE355LD, purchased from OPOTEK Inc., with the following key specifications: wavelength range, 410-2400 nm; pulse repetition rate, 20 Hz; maximum pulse energy at 460 nm, 8 mJ; pulse duration, 5 nanoseconds; spectral linewidth, 4-6 cm⁻¹; and inter-pulse stability, <5%. For each bacterial strain, four groups were used: untreated group, ns-410 nm treated group, H2O2 (22 or 44 mM) treated group, and ns-410 nm plus H2O2 (22 or 44 mM) treated group. The dose used for ns-410 nm exposure was 15 J / cm². 2 H2O2 was incubated with the bacteria at 37°C with shaking at 200 rpm for 30 minutes. After incubation, the bacterial load from each group was serially diluted, inoculated onto TSA plates, and then counted by spotting these plates.

[0119] CFU assays were used to test the potential synergistic effect between catalase photoinactivation and ROS-generating antibiotics: Bacteria cultured overnight were centrifuged, the supernatant was discarded, and the culture was resuspended in the same volume of fresh TSB. The solution was then incubated with antibiotics (10 μg / ml) for 1 hour before any further treatment. Four groups were tested for each bacterial strain: untreated, ns-410nm treated, antibiotic (2 μg / ml) treated, and ns-410nm plus antibiotic (2 μg / ml) treated. The dose used for ns-410nm exposure was 15 J / cm². 2 The antibiotics and bacteria were incubated together at 37°C with shaking at 200 rpm for up to 6 hours. At each time interval, the bacterial load from each group was serially diluted, inoculated onto TSA plates, and then counted by spotting these plates.

[0120] Confocal imaging for intracellular bacterial assay: As described elsewhere (Yang, X. et al., International Journal of Nanomedicine 13, 8095 (2018)), mouse macrophages (RAW 264.7) were cultured in DMEM supplemented with 10% FBS at 37°C and CO2 (5%). Cells were exposed to MRSA USA300 or Salmonella enterica at a multiplicity of infection (MOI) of approximately 100:1 in serum-free DMEM medium (with / without ns-410nm exposure). One or two hours post-infection, RAW264.7 cells were washed with gentamicin (50 μg / mL, 1 h) to kill extracellular bacteria in DMEM + 10% FBS. Subsequently, RAW 264.7 cells were washed with gentamicin (50 μg / mL) followed by lysis with 0.1% Triton-X 100 for 3 min. After membrane permeabilization, infected RAW 264.7 cells were stained with Live / Dead staining agent for 15 minutes, and then the samples were fixed in 10% formalin for 10 minutes before confocal imaging.

[0121] Example 1: Pulsed blue laser effectively inactivates pure catalase and catalase in bacteria.

[0122] A pure catalase solution (bovine liver catalase, 3 mg / ml in PBS) was prepared in PBS using a previously published protocol to examine the effect of 410 nm exposure on the absorption spectrum of the catalase solution (Cheng, L., Photochemistry and Photobiology 34, 125-129 (1981)). Catalase showed significant absorption near 410 nm, and its absorption at this wavelength gradually decreased with increasing exposure time at 410 nm. Figure 1 a). This suggests that the secondary structure of catalase may change, particularly in the domain containing active heme. Furthermore, this photoinactivation effect was examined at different wavelengths using the Amplex Red Catalase kit. Figure 1 b). The photoinactivation trend is similar to that of catalase, with 410 nm being the most effective, where a 5-minute exposure consumes approximately 70% of the active catalase.

[0123] Since most aerobic and facultative anaerobic bacteria express catalase (Mishra, S. & Imlay, J. Arch Biochem Biophys 525, 145-160, doi:10.1016 / j.abb.2012.04.014(2012)), we examined whether we could in situ photoinactivate catalase from catalase-positive bacteria. MRSA USA300 and Pseudomonas aeruginosa (PAO-1) were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. Notably, from MRSA USA300 ( Figure 2 a) and Pseudomonas aeruginosa ( Figure 2 (b) Catalase was photoinactivated by exposure to blue light, particularly at 410 nm. The dose used was approximately 15 J / cm². 2 It is far below the ANSI safety limit of 200 J / cm³. 2 And the sample was a quiescent culture of bacteria (~10). 8 (cells / mL). ANSI is the American National Standard for Laser Safety Use; see ANSI Z136.1, American Laser Society 2014.

[0124] To further understand how 410 nm exposure leads to structural changes in catalase, we performed resonance Raman spectroscopy to capture the Raman features of the dried catalase film. Figure 3 Clearly, 410nm exposure significantly reduces the 750cm depth. -1 The Raman intensity at [location], and the Raman band range is 1300 cm⁻¹. -1 Up to 1700cm -1 This is a typical vibrational band of the heme ring of catalase (Chuang, W.-J., Heldt, J. & Van Wart, HJJoBC Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II. 264, 14209-14215 (1989)). These data further confirm the fact that exposure at 410 nm may lead to structural changes in catalase.

[0125] Furthermore, the inactivation efficiencies of ns-410nm and CW-410nm on catalase were compared. Compared to CW-410nm, ns-410nm showed better performance in pure solution form (…). Figure 4 a) or from MRSA USA300 ( Figure 4 b) and Pseudomonas aeruginosa ( Figure 4 In case c), it is significantly more effective. Furthermore, ns-410 exposure eliminates the need to heat tissues during future clinical studies.

[0126] Example 2: Photoinactivation of catalase enables various bacteria to respond to low concentrations of H+. 2O2 sensitive

[0127] Catalase is an essential detoxification enzyme for bacteria when encountering various endogenous or exogenous stresses (Nakamura, K. et al., Microbiology and Immunology 56, 48-55 (2012)). When the gene encoding catalase expression is a mutant, pathogens are more susceptible to environmental stress (Mandell, GL, J ClinInvest 55, 561-566, doi:10.1172 / jci107963 (1975)). The study investigated whether the exogenous addition of low concentrations of H2O2 could eliminate those 'traumatic' pathogens. Figure 5 As shown, photoinactivation of catalase alone (15 J / cm) 2 ) did not significantly reduce MRSA load ( Figure 5 A) Pseudomonas aeruginosa load ( Figure 5 B) and intestinal Salmonella load ( Figure 5 C). Furthermore, low concentrations of H2O2 (22 mM) did not exhibit any significant antimicrobial activity against MRSA and Pseudomonas aeruginosa. Figure 5 However, the application of low-concentration H2O2 after catalase photoinactivation significantly reduced the load of MRSA and Pseudomonas aeruginosa (≥3-log10 reduction). Figure 5 Interestingly, the relationship between bacterial killing tendency and irradiation wavelength is similar to that between photoinactivation of catalase and irradiation wavelength. Notably, low concentrations of H₂O₂ combined with 410 nm exposure (15 J / cm²) showed a significant effect. 2 Complete eradication of Pseudomonas aeruginosa was achieved. Figure 5 B).

[0128] Example 3: Synergistic effect of photoinactivation of catalase and low concentration of hydrogen peroxide

[0129] There is a synergistic effect between the photoinactivation of catalase and low concentrations of hydrogen peroxide, which can be used to eliminate stationary MRSAUSA300 and stationary Pseudomonas aeruginosa. Figure 5 A uses a bar chart to depict the collaborative results. (CFU ml) -1(Coronation forming units) represent bacterial load. 'Untreated' refers to pristine, stationary MRSA without any exogenous treatment. 'H2O2 (22 mM, 0.075%)' and 'ns light' refer to stationary MRSA treated with H2O2 and ns light alone, respectively. As shown in the figure, H2O2 and ns light alone did not exert any significant killing effect on MRSA; however, ns-410 nm combined with H2O2 reduced the bacterial load by approximately four orders of magnitude. The same phenomenon occurred at other wavelengths. Notably, ns-430 nm or ns-430 nm combined with H2O2 reduced the bacterial load by approximately 99% under the same conditions. ns-450 nm or ns-460 nm combined with H2O2 reduced the bacterial load by approximately 90%. ns-470 nm combined with H2O2 reduced the bacterial load by approximately 50%. ns-480 nm combined with H2O2 exerted almost no antimicrobial effect. In summary, the bactericidal effect of H2O2 was significantly enhanced by blue light photoinactivation of catalase, especially when using ns-410nm. Stationary Pseudomonas aeruginosa (a Gram-negative bacterium) Figure 5 Similar phenomena were observed in *Salmonella intestinale* (represented by B). By combining ns-410nm with H2O2 for *Salmonella intestinale*, an enhancement of approximately five orders of magnitude in killing effect was observed. Figure 5 C).

[0130] Furthermore, compared to CW-410nm binding H2O2, ns-410nm binding H2O2 is significantly more effective in eliminating microorganisms. Figure 6 ).

[0131] Example 4: Photoinactivation of catalase restores the activity of conventional antibiotics against a variety of bacteria.

[0132] In addition to H2O2, the study investigated whether the photoinactivation of catalase could synergize with conventional antibiotics, particularly those that generate downstream intracellular ROS. Tobramycin, a representative aminoglycoside, is one example. Tobramycin can induce downstream ROS bursts (Dwyer, DJ et al., Proceedings of the National Academy of Sciences 111, E2100-E2109, doi:10.1073 / pnas.1401876111(2014)), therefore, the combination of catalase photoinactivation and tobramycin administration was tested to see if an enhanced effect was observed.

[0133] Interestingly, an enhanced killing effect was observed in the combination therapy group. Figure 7The over 100-fold enhancement indicates that photoinactivation of catalase does indeed accelerate the antimicrobial activity of ROS-producing antibiotics. As a control, the same treatment protocol was tested on the catalase-negative enterococcus strain *Enterococcus faecalis* NR-31970, which did not produce any enhanced killing effect. Figure 7 In summary, this suggests that photoinactivation of catalase helps restore the effectiveness of conventional antibiotics against catalase-positive pathogens.

[0134] Example 5: Photoinactivation of catalase helps macrophages fight intracellular pathogens.

[0135] Neutrophils and macrophages are highly essential phagocytic cells, forming the first line of defense in the innate immune system (Segal, AW, Annu Rev Immunol 23, 197-223, doi:10.1146 / annurev.immunol.23.021704.115653 (2005)). Catalase, encoded by the gene katA, provides indispensable resistance to antimicrobial agents released by immune cells (Flannagan, R., Heit, B. & Heinrichs, D., Pathogens 4, 826-868 (2015)). Based on these facts, it is hypothesized that photoinactivation of catalase can help immune cells eliminate extracellular and intracellular pathogens. To test potential adjunctive effects, a fluorescence Live / Dead assay was used to visualize live / dead bacteria within cells after exposure to ns-410 nm. A higher percentage of dead MRSA was observed within cells. Figure 9 ).

[0136] In summary, photoinactivation of catalase significantly improved the efficacy of low-concentration H2O2, ROS-producing antibiotics, and immune cells against broad-spectrum bacteria, including notorious drug-resistant Gram-negative bacteria.

[0137] The following materials and methods were used throughout Examples 5-9.

[0138] Chemicals and fungal strains: DMSO (W387520, Sigma Aldrich), amphotericin B (A9528-100 MG, Sigma Aldrich), ergosterol (AC1178100050, 98%, ACROS Organics). YPD broth (Y1375, Sigma Aldrich). YPD agar (Y1500, Sigma Aldrich). PrestoBlue cell viability assay (A13262, ThermoFisher Scientific). Candida albicans SC5314; tests of the fungal strains used are shown in Table 1.

[0139] Table 1. Fungal strains used in amp-B imaging experiments.

[0140]

[0141]

[0142] Quantification of fungal catalase before and after 410 nm exposure: Quantification of catalase from pure catalase solution and fungal solution was achieved using the Amplex Red Catalase Kit. Essentially, 25 μl of analyte was incubated with 25 μl of (40 μM H₂O₂) at room temperature for 30 min. Then, 50 μl of working solution (100 μM Amplex Red reagent containing 0.4 U / ml horseradish peroxidase) was added to the mixture, and the mixture was subsequently incubated in the dark for 30–60 min. Afterward, fluorescence was recorded at 590 nm emission upon excitation at 560 nm.

[0143] CFU assay for quantitative therapeutic efficacy: Quantification of the antifungal treatment regimen was achieved as follows: Fungal samples cultured overnight were washed with sterile PBS. Logarithmic-phase fungal pathogens were prepared by diluting them 1:50 in fresh YPD broth and then incubating for 2–3 hours at 30°C and 200 rpm with shaking. Subsequently, the fungal concentration was adjusted to approximately 1 × 10⁻⁶ by centrifugation or further dilution with PBS. 8 Cells / mL. Expose 10 μl of the above fungal solution to 410 nm for 5 minutes (150 mW / cm²). 2 The exposed samples were then collected in 990 μl of sterile PBS, followed by replenishment of treatment agents. Subsequently, the CFU of fungal cells were counted by serial dilution and the samples were incubated on YPD agar plates for 48 hours.

[0144] PrestoBlue viability assay: Logarithmic-phase fungal pathogens were first prepared by diluting overnight cultured fungal pathogens in fresh YPD broth at a ratio of 1:50, and then incubated for 2-3 hours at 30°C and 200 rpm with shaking. Subsequently, the fungal concentration was adjusted to approximately 1 × 10⁻⁶ by centrifugation or further dilution with PBS. 8 Cells / mL. Expose 10 μl of the above fungal solution to 410 nm for 5 minutes (150 mW / cm²). 2The exposed samples were then collected in 990 μl of sterile PBS, followed by replenishment of the treatment agent. Aliquots of the above samples were prepared in 96-well plates, each containing 100 μl. Then, 100 μl of sterile YPD broth and 23 μl of PrestoBlue were added to the same well. Fluorescence signals at 590 nm were recorded for each well on a time-series basis (maximum 18 hours, 30-minute intervals) under 560 nm excitation. For each strain, to determine the exact quantity of fungal pathogen in each well, the corresponding fluorescence signal was recorded from a known quantity of fungal pathogen, without external treatment.

[0145] Macrophage-Candida albicans Interaction Revealed by Confocal Laser Scanning Microscopy: As described elsewhere (Yang, X. et al., International Journal of Nanomedicine 13, 8095 (2018)), mouse macrophages (RAW 264.7) were cultured in DMEM supplemented with 10% FBS plus penicillin and streptomycin at 37°C and CO2 (5%). Cells were exposed to Candida albicans SC5314 (with / without 410 nm exposure) in serum-free DMEM medium at a multiplicity of infection (MOI) of approximately 10:1. One or two hours post-infection, RAW264.7 cells were washed with gentamicin (50 μg / mL, 1 h) to kill the extracellular pathogen in DMEM + 10% FBS. Subsequently, RAW264.7 cells were washed with gentamicin (50 μg / mL) followed by lysis with 0.1% Triton-X 100 for 3 min. After membrane permeabilization, infected RAW 264.7 cells were stained with Live / Dead stain for 15 minutes, and then the samples were fixed in 10% formalin for 10 minutes. The formalin was washed off before confocal imaging.

[0146] Example 6: 410nm exposure reduces the amount of intracellular catalase.

[0147] It is well known that most fungal pathogens are catalase-positive (Hansberg, W. et al., ArchBiochem Biophys 525, 170-180 (2012)). To test whether 410 nm exposure leads to the loss of catalase activity, the intracellular catalase levels were quantified using the same method before and after 410 nm exposure via the Amplex Red Catalase Kit. Catalase from various fungal pathogens, both in logarithmic and quiescent phases, was significantly inactivated by 410 nm exposure (Hansberg, W. et al., ArchBiochem Biophys 525, 170-180 (2012)). Figure 10 It is noteworthy that catalase from the notorious Candida auris strain decreased by 60% after only 5 minutes of exposure at 410 nm.

[0148] Example 7: The photoinactivation of catalase binding to H+ was determined by CFU assay. 2O2 Achieved Candida albicans SC5314 Completely eradicate

[0149] Since catalase is effectively inactivated in various fungal strains, this study investigated whether photoinactivation of catalase could suscept fungal strains to external H2O2 attack. Following 410 nm exposure, further application of low-concentration H2O2, combined with treatment, achieved eradication. Figure 11 It is noteworthy that after catalase photoinactivation, the function of H2O2 was enhanced by more than five orders of magnitude. Figure 11 ).

[0150] A synergistic effect between photoinactivation of catalase and H2O2 in eliminating Candida albicans SC5314 was also observed. The results were obtained from... Figure 12 The scatter plot in CFU ml is shown. -1 (Coronation forming units) refers to the number of bacterial loads. 'Untreated' refers to the original stationary phase SC5314 without any exogenous treatment. 'H2O2 (44mM, 0.15%)' and 'ns light' refer to the stationary phase SC5314 treated with H2O2 and ns light alone, respectively. Figure 12 As shown, neither H2O2 alone nor ns light alone exerted a significant bactericidal effect on CASC5314. However, ns light combined with H2O2 reduced the bacterial load by approximately four orders of magnitude. In particular, ns-410 or ns-420, ns-430 combined with H2O2 achieved complete eradication. The amount of fungal load reduction by ns-450 or ns-480nm combined with H2O2 was similar to that of H2O2 alone. In summary, the bactericidal effect of H2O2 was significantly enhanced by photoinactivation of catalase using blue light, particularly ns-410-ns-430nm. Therefore, there is an effective synergistic effect between photoinactivation of catalase and H2O2 in the blue light range for the elimination of CASC5314.

[0151] Example 8: PrestoBlue assay showed that the photoinactivation of catalase bound to H... 2 O 2 Achieved broad-spectrum fungi Effective eradication of species

[0152] To further confirm the efficacy of this combination therapy against other fungal strains, we tested its feasibility against a wider range of clinical fungal strains. Unlike bacteria, fungal cells grow more slowly, with each colony forming approximately after 48 hours. Therefore, the high-throughput PrestoBlue viability assay was used to measure therapeutic efficacy. Figure 13As shown, PrestoBlue achieves the same killing effect as CFU assay. Interestingly, CASC5314 exhibits different behaviors in the logarithmic and quiescent phases in response to the combined killing, likely due to differences in metabolic activity between the two states. However, photoinactivation of catalase consistently enhances the killing effect with low concentrations of H2O2, regardless of whether the phase is logarithmic or quiescent. This synergistic therapy was tested in over 20 different clinical fungal isolates, consistently demonstrating significant killing effects.

[0153] Example 9: Candida auris strains are sensitive to 410nm exposure.

[0154] In addition to H2O2, the study investigated whether photoinactivation of catalase could synergize with conventional antifungal agents such as azoles or amphotericin B (amp B). Similar to some classes of antibiotics, amp B kills fungi partly due to oxidative damage (Belenky, P. et al., Fungicidal drugs induce a common oxidative-damage cellular death pathway. Cell Rep 3, 350-358, doi:10.1016 / j.celrep.2012.12.021(2013)). Therefore, to test our hypothesis, PrestoBlue assays were performed after photoinactivation of catalase followed by treatment with amp B targeting various clinical fungal isolates, including Candida auris strains.

[0155] Interestingly, some Candida auris strains exhibit resistance to AMP B in the absence of catalase photoinactivation. Figure 14 Nevertheless, with or without the addition of amp B, photoinactivation of catalase achieved complete eradication of Candida auris strains. Ten Candida auris strains were tested, and they all exhibited the same behavior. This suggests that Candida auris strains are abnormally sensitive to blue light exposure.

[0156] Example 10: Photoinactivation of catalase inhibits the formation of Candida albicans hyphae and helps macrophages phagocytose. bite

[0157] Host immune cells play a crucial role in combating pathogens that evade external pathogens. Catalase plays a vital role in the battle between Candida albicans and neutrophils or macrophages (Pradhan, A. et al., Elevated catalase expression in afungal pathogen is a double-edged sword. Plos Pathog 13, e1006405 (2017)). Therefore, we examined whether photoinactivation of catalase could aid macrophages in combating Candida albicans. To visualize this effect, RAW 264.7 cells were infected with Candida albicans at an MOI of 10 and exposed to Candida albicans at 410 nm, and labeled with live / dead fluorescent staining.

[0158] like Figure 15 As shown, untreated Candida albicans retains its hyphal form and penetrates macrophages. However, Candida albicans exposed to 410 nm remains in a dead 'yeast' form within the cell.

[0159] In summary, the photoinactivation of catalase combined with low concentrations of H2O2 provides an effective and novel approach to eliminate broad-spectrum fungi and fungal infections.

[0160] Example 11: Photoinactivation of catalase combined with ROS activator silver cations synergistically kills microorganisms

[0161] Electromagnetic energy at a wavelength of ns-410 nm combined with 10 μM silver cations eliminated approximately 90% of MRSA one hour after treatment, while using ns-410 nm alone or silver cations alone did not produce any significant antimicrobial effect. Figure 16 ).

[0162] The photoinactivation of catalase and low concentrations of silver cations also synergistically eliminated *E. coli* BW25113. The results were obtained from... Figure 17 The scatter plot in CFU ml is shown. -1 (Coronation forming unit) refers to the amount of bacterial load. 'Untreated' refers to untreated, untreated raw *E. coli* BW25113. '0.5 μM Ag + 'CW-410' or 'ns light' refer to the use of 0.5μM Ag alone. + E. coli BW25113 with ns-410. Used alone with 0.5 μM Ag. + CW-410 alone or ns-410 alone did not have a significant bactericidal effect on Escherichia coli. However, ns-410 combined with 0.5 μM Ag + It reduced the bacterial load by approximately 99%. Figure 17 The same phenomenon occurs at other wavelengths. It is noteworthy that, under the same conditions, CW-410 binds 0.5 μM Ag. + There was no significant reduction in bacterial load. Our results were consistent at 30 and 60 minutes post-treatment.

[0163] From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to suit various uses and conditions. Such embodiments also fall within the scope of the claims of this application. A description of a list of elements in any definition of a variable herein includes defining that variable as any single element or combination (or sub-combination) of the listed elements. A description of embodiments herein includes embodiments as any single embodiment or in combination with any other embodiment or part thereof.

[0164] References

[0165] All patents, patent applications and publications mentioned in this specification are incorporated herein by reference to the extent that each individual patent and publication is specifically and individually indicated as being incorporated by reference.

Claims

1. A method for disinfecting a non-living surface contaminated with catalase-positive microorganisms, the method comprising the following steps: A pulse of light with a wavelength of 400 nm to 430 nm is applied to the inanimate surface, wherein catalase is inactivated, and the inanimate surface is subsequently contacted with a composition containing a peroxide solution to disinfect the inanimate surface, wherein the dose of the pulsed light is at least 5 J / cm². 2 And the peroxide solution is a hydrogen peroxide solution containing at least 0.03% hydrogen peroxide.

2. The method according to claim 1, wherein the wavelength is 410 nm.

3. The method according to claim 1, wherein the dose of the pulsed light is 5 J / cm². 2 Up to 200 J / cm 2 .

4. The method according to claim 1, wherein the dose of the pulsed light is 15 J / cm². 2 .

5. The method of claim 1, wherein the catalase-positive microorganism is a fungus or bacteria and the pulsed light is provided by a pulsed nanosecond laser or an LED.

6. The method according to claim 1, wherein the hydrogen peroxide solution is 0.3% hydrogen peroxide.

7. The method of claim 1, further comprising applying a ROS-generating agent to the inanimate surface.

8. The method of claim 1, wherein the inanimate surface is a material including metal, plastic, fabric, rubber, stone, composite material surface or wood.

9. An apparatus for killing said catalase-positive microorganism after inactivating catalase, comprising: A source of light and a source of peroxide solution and / or ROS generator, said light source being used to deliver at least 5 J / cm at a wavelength of 400 nm to 430 nm. 2 The source of the pulsed light, the peroxide solution and / or ROS generator, is used to deliver the peroxide solution and / or ROS generator to the catalase-positive microorganism, wherein catalase inactivation makes the microorganism sensitive to being killed by the peroxide solution and / or ROS generator.

10. The apparatus of claim 9, wherein the wavelength is 410 nm.

11. The apparatus of claim 9, wherein the dose of the pulsed light is 5 J / cm². 2 Up to 200 J / cm 2 .

12. The apparatus of claim 9, wherein the dose of the pulsed light is 15 J / cm². 2 .

13. The apparatus of claim 9, wherein the peroxide solution and / or ROS generator are delivered after the delivery of the pulsed light.

14. The apparatus of claim 9, wherein the catalase-positive microorganism is a fungus or bacteria, and the pulsed light is provided by a pulsed nanosecond laser or an LED.

15. The apparatus of claim 9, wherein the peroxide solution and / or ROS generator is at least 0.3% hydrogen peroxide solution or silver cations.

16. The apparatus of claim 9, wherein the optical waveguide delivers the pulsed light.

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