WO2022087213A1

WO2022087213A1 - Methods and compositons for the treatment of catalase-positive microbes

Info

Publication number: WO2022087213A1
Application number: PCT/US2021/055967
Authority: WO
Inventors: Steven Jun QIAN; Ji-Xin Cheng
Original assignee: Pulsethera, Inc.
Priority date: 2020-10-22
Filing date: 2021-10-21
Publication date: 2022-04-28

Abstract

Methods of the present invention comprise photoinactivation of catalase in combination with low-concentration peroxide solutions and/or ROS generating agents to provide antibacterial effects.

Description

TITLE OF THE INVENTION

METHODS AND COMPOSITONS FOR THE TREATMENT OF CATALASE-POSITIVE MICROBES

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. AH32638 awarded by the National Institutes of Health. The government has certain rights to the invention.

BACKGROUND OF THE INVENTION

[0002] Antibiotic resistance kills an estimated 700,000 people each year worldwide, and studies predict that this number could rise to 10 million by 2050, if efforts are not made to curtail resistance (Willyard, C. J. N. N. The drug-resistant bacteria that pose the greatest health threats. 543, 15 (2017)). Yet, the pace of resistance acquisition from mutation in pathogens is faster than clinical introduction of new antibiotics. There is an urgent need to develop unconventional ways to combat the resistance for sixteen of the World Health Organization 2020 most serious microbial threats that are catalase- positive microbes.

SUMMARY OF THE INVENTION

[0003] The lethal effect of certain antibiotics occurs through the generation of Reactive Oxygen Species (ROS). Catalase, the ubiquitous key defense enzyme existing in most of the aerobic pathogens, is utilized to scavenge hydrogen peroxide, thus preventing downstream oxidative damage. The methods of the present invention can be practiced with regards to catalase-positive microbes (bacteria, fungus, or viruses) that can be located on a surface or internal to a surface, including in a fluid. The reduction of catalase-positive microbes that can be spread to other objects and people can alter physiological conditions and prevent disease. It has now been shown that catalase can be optimally photoinactivated by blue light having a wavelength of about 400 nm to about 460 nm, and specifically, a wavelength of about 410 nm. Photoinactivation of catalase renders broad-SPECTRUM catalase-positive microbial pathogens highly susceptible to ROS-generating antimicrobials and/or immune cell attack. It has now been further determined that the antimicrobial effect of photoinactivation is significantly and unexpectedly increased upon administration of a low-concentration of H2O2 and/or a ROS- generating agent.

[0004] The invention addresses methods of reducing the presence of a catalase-positive microbe in various environments by (i) the photoinactivation of catalase with various wavelengths of light and, optionally, (ii) contacting the microbe with hydrogen peroxide (“H2O2”) and/or an ROS generating agent.

[0005] In one aspect, the invention provides a method of reducing the presence of a catalasepositive microbe in or on a tissue or fluid of a subject.

[0006] In one embodiment, the invention provides a method of treating cells, in culture or that are part of a subject, a tissue of a subject or fluid, be it part of, from, or not associated with a subject, in contact with a catalase-positive microbe, said method comprising the steps of: applying light to the tissue of the subject infected with the catalase-positive microbe at a wavelength of about 400 nm to about 460 nm, wherein the catalase is inactivated, and contacting the tissue with a composition comprising a peroxide solution, thereby treating the cells, tissue or fluid of the subject in contact with the catalase-positive microbe.

[0007] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe in an indoor or outdoor setting. Such setting can be an agricultural environment, such as the treatment of seeds, plants, fruits and vegetables, commercial fungi (including, but not limited to, edible mushrooms), soil, nutrients, additives, or harvested parts of plants or commercial fungi. Such an outdoor setting can also be inanimate objects, including, but not limited to benches, water fountains, exercise equipment, and the exterior of structures, such as buildings or handrails.

[0008] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe with the light being generated in various forms, including, but not limited to, continuous wave or pulsed wave, from various sources, including, but not limited to, an incandescent lamp, a fluorescent lamp, a halogen lamp, a xenon lamp, a light emitting diode (’’LED”), and a laser or any other light creating device.

[0009] In one embodiment, the wavelength is within the range of 400 nm to 460 nm. [0010] In another embodiment, the wavelength is about 410 nm. [0011] In another embodiment, the dose of light is about 0.1 J/cm² to about 10,000 J/cm² and may be varied during exposure.

[0012] In another embodiment, the dose of the light is about 5 J/cm² to about 700 J/cm².

[0013] In yet another embodiment, the dose of the light is about 15 J/cm².

[0014] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a pulsed nanosecond laser.

[0015] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a continuous wave LED.

[0016] In yet another embodiment, the peroxide solution is a hydrogen peroxide solution.

[0017] In yet another embodiment, the hydrogen peroxide solution is between about 0.001% and about 35% hydrogen peroxide.

[0018] In yet another embodiment, the method further comprises administering a ROS generating agent for the treatment of the infected cells, tissue or fluid of the subject.

[0019] In yet another embodiment, the ROS generating agent may be, but is not limited to, tobramycin, silver cation, iodine tincture, a gold nanoparticle, methylene blue, a P-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, a membrane-targeting polyene antifungal or a cell-wall targeting antifungal.

[0020] In yet another embodiment, the tissue is skin, scalp or nails tissues or organs.

[0021] In yet another embodiment, the catalase-positive microbe is eradicated by the method of the invention.

[0022] In another aspect, the invention provides a method of disinfecting an inanimate surface contaminated with a catalase-positive microbe, said method comprising the steps of: applying light to the inanimate surface at a wavelength of about 400 nm to about 460 nm, wherein the catalase is inactivated, and contacting the inanimate surface with a composition comprising a diluted peroxide solution, thereby disinfecting the inanimate surface.

[0023] In one embodiment, the wavelength is within the range of 400 nm to 460 nm.

[0024] In one embodiment, the wavelength is about 410 nm.

[0025] In another embodiment, the dose of the light is about 0.1 J/cm² to about 10000 J/cm². [0026] In yet another embodiment, the dose of the light is about 15 J/cm². [0027] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a pulsed nanosecond laser.

[0028] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a continuous wave LED.

[0029] In yet another embodiment, the peroxide solution is a hydrogen peroxide solution.

[0030] In yet another embodiment, the hydrogen peroxide solution is between about 0.001% and about 0.3% hydrogen peroxide.

[0031] In yet another embodiment, the method further comprises administering a ROS generating agent.

[0032] In yet another embodiment, the ROS generating agent may be, but is not limited to, tobramycin, silver cation, iodine tincture, a gold nanoparticle, methylene blue, a P-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, a membrane-targeting polyene antifungal or a cell-wall targeting antifungal.

[0033] In yet another embodiment, the inanimate surface is a material comprising metal, plastic, fabric, rubber, stone, composite surfaces or wood. In yet another embodiment, the inanimate surface can be a gas, including, but not limited to, air in a ventilation system, confined space, or space of limited circulation.

[0034] In yet another embodiment, the catalase-positive microbe is eradicated by the method of the invention.

[0035] In yet another aspect, the invention provides a method of treating a tissue of a subject infected with a catalase-positive microbe, said method comprising the steps of: applying light from a pulsed nanosecond laser or any other photonic light generator to the tissue of the subject infected with the catalase-positive microbe at a wavelength of about 400 nm to about 460 nm, wherein the catalase is inactivated, and contacting the tissue with a composition comprising a diluted peroxide solution and/or a ROS generating agent, thereby treating the tissue of the subject infected with the catalase-positive microbe.

[0036] In one embodiment, the wavelength is about 410 nm.

[0037] In another embodiment, the dose of the light is about 5 J/cm² to about 700 J/cm².

[0038] In yet another embodiment, the dose of the light is about 15 J/cm². [0039] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe.

[0040] In yet another embodiment, the diluted peroxide solution is a hydrogen peroxide solution.

[0041] In yet another embodiment, the hydrogen peroxide solution is between about 0.03% and about 3.0% hydrogen peroxide.

[0042] In yet another embodiment, the ROS generating agent may be, but is not limited to, tobramycin, silver cation, iodine tincture, a gold nanoparticle, methylene blue, a P-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, a membrane-targeting polyene antifungal or a cell-wall targeting antifungal. Multiple ROS agents can be dispersed before, during or after the exposure of light.

[0043] In yet another embodiment, the tissue is skin, scalp or nail or tissue, organ in humans or animals.

[0044] In yet another embodiment, the catalase-positive microbe is eradicated by the method of the invention.

[0045] In yet another aspect, the invention provides a method of producing a synergistic antimicrobial effect in a tissue of a subject infected with a catalase-positive microbe, said method comprising the steps of: applying light to the tissue of the subject infected with the catalasepositive microbe at a wavelength of about 400 nm to about 460 nm, wherein the catalase is inactivated, and contacting the tissue with a composition comprising a diluted peroxide solution, thereby producing the synergistic antimicrobial effect in the tissue of the subject infected with the catalase-positive microbe.

[0046] In one embodiment, the wavelength is about 410 nm.

[0047] In another embodiment, the dose of the light is about 5 J/cm² to about 700 J/cm².

[0048] In yet another embodiment, the dose of the light is about 15 J/cm².

[0049] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a pulsed nanosecond laser.

[0050] In yet another embodiment, the catalase-positive microbe is a fungal or bacterial microbe and the light is provided by a continuous wave LED. The continuous wave maybe a constant or variable power and wavelength. [0051] In yet another embodiment, the diluted peroxide solution is a hydrogen peroxide solution.

[0052] In yet another embodiment, the hydrogen peroxide solution is between about 0.001% and about 0.3% hydrogen peroxide.

[0053] In yet another embodiment, the method further comprises administering a ROS generating agent to the infected tissue of the subject.

[0054] In yet another embodiment, the ROS generating agent may be, but is not limited to, tobramycin, silver cation, iodine tincture, a gold nanoparticle, methylene blue, a P-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, a membrane-targeting polyene antifungal or a cell-wall targeting antifungal. ROS agents can be combined or delivered in a sequence.

[0055] In yet another embodiment, the tissue is skin, scalp or nail.

[0056] In yet another embodiment, the catalase-positive microbe is eradicated by the method of the invention.

[0057] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe in contact with various cells and/or tissues or organs. Such cells and/or tissues or organs can include, but not be limited to, skin cells, skin tissue, blood cells, bladder cells, organs and anatomical structures, and a body cavity, such as, but not limited to, the vagina, intestine, oral cavity, nasal orifice, and ear canal.

[0058] In one embodiment, the cells and/or tissue can be skin.

[0059] In another embodiment, the cells and/or tissue can be a wound, burn, or a resultant sore from a catalase-positive microbe.

[0060] In another embodiment, the cells and/or tissue can be a skin ulcer.

[0061] In another aspect, the invention provides for the number of catalase-positive microbes in various sizes of area to be reduced.

[0062] In one embodiment, the size of an area can range from about a square femtometer to square meters.

[0063] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe with exposure for various amounts of time. Such amounts of time can range from nanoseconds to minutes and to hours. [0064] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe with exposure at a range of temperatures under which the population of the microbe can be reduced.

[0065] In another aspect, the invention provides a method of reducing the presence of a catalase-positive microbe under various pH conditions - ranging from acidic to basic conditions - or modifying the pH of the H2O2 and/or an ROS generating agent or the environment of the catalase-positive microbe. For, example, the pH of the H2O2 and/or an ROS generating agent can vary depending on the needed effect. Such variation can include a recent change of the pH just prior to contact with the catalase-positive microbe. The invention described herein also has the ability to be applied through a photonic-transparent material so that it deactivates the catalase under the material. ROS agents may be added or present at physiological or low concentration. [0066] In another aspect, the invention provides devices for carrying out the methods of the invention.

[0067] In any aspect of the invention, the methods and/or steps of said methods addressed herein reduce the presence of a catalase-positive microbe. Because there can be numerous variables associated with reducing the presence of a catalase-positive microbe, such as, but not limited to, the location of the microbe, such as, but not limited to, being in contact with a cell, an inanimate object, soil, and an agricultural agent, edible material, the environment of the microbe (including body cavities), such as, but not limited to the pH of the environment, the distance from the light source, and the effect of various light sources (and the power associated with those sources), the method of reducing the presence of the microbe may require variations in one or more steps of the invention, the invention can encompass various combinations of such aspects of the inventions as the light source, the wavelength, the nature of the light, the H2O2 and/or an ROS generating agent, and the time of contacting the microbe with any of the such elements. For example, the invention may require changes in the light wavelength to which different microbes are exposed. Further, the time of light exposure may be different due to the cell, tissue, or surface to be treated. Additionally, the time of exposure may vary due to the energy associated with the light generation. Also, the energy associated with the light generated may vary due to the distance between the light source and the catalase-positive microbe. Additionally, the concentration of the H2O2 and/or an ROS generating agent may vary based upon the environment of the catalase-positive microbe.

[0068] The aspects of the invention may also vary due to any other steps and/or methods performed with the invention described herein. Combination therapy with other treatments, such as, but not limited to, pharmaceuticals, topicals, other light treatments, surgeries, bandages, and medications, may require variations in the steps of the invention described herein. For example, the use of wavelengths from 200 to 800 nm when used in conjunction with blue light adds to the ability of invention to be a broader microbial disinfectant. All combinations of the aspects and embodiments described herein that lead to inactivation of a microbe’s catalase and reduction in the number or eradication of the microbe are envisioned by the inventors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying figures, incorporated herein by reference.

[0070] Figure 1 depicts the effect of ns-410 nm exposure on pure catalase solution. Panel (a). Absorption spectra of pure catalase solution under ns-410 nm exposure. Catalase solution: 3 mg/ml, filtered with a 0.2 pm filter. Panel (b). Percent of remaining active catalase after different treatment schemes (different wavelengths under the same dosage). Quantification of catalase was obtained by an Amplex Red Catalase kit. Data: Mean ± standard deviation (N=3).

[0071] Figure 2 depicts the effect of ns-410 nm exposure on active catalase percentages from MRSA USA300 and P. aeruginosa. (Panels a-b). Percent of active catalase remained inside MRSA USA300 (Panel a) and P. aeruginosa (Panel b) after different treatment schemes (different wavelengths under the same dosage). Quantification of catalase was obtained by an Amplex Red Catalase kit. Data: Mean ± standard deviation (N=3).

[0072] Figure 3 depicts Resonance Raman spectra of bovine liver catalase powder with and without 410 nm exposure. 410 nm dose: 250 mW/cm². Raman spectrum acquisition time: 25 s. 532 nm excitation. Data: Mean ± SD from five spectra.

[0073] Figure 4 depicts the comparison between ns-410 nm and CW-410 nm exposure on the catalase photoinactivation effect from pure catalase solution (Panel a), catalase from MRSA USA300 (Panel b), and catalase from P. aeruginosa (Panel c). Quantification of catalase was obtained by an Amplex Red Catalase kit. Data: Mean ± standard deviation (N=3). Student unpaired t-test, ***: p<0.001; **: p < 0.01.

[0074] Figure 5 depicts CFU ml-1 of stationary-phase MRSA USA300 methicillin-resistant Staphylococcus aureus (Panel a), Pseudomonas aeruginosa (Panel b), and Salmonella enterica (Panel c) under the treatment of 22 mM H2O2 with/without the combination with various light exposure. Data: Mean ± standard deviation (N=3). Student unpaired t-test, ***: p<0.001; **: p< 0.01. 250 CFUs: limit of detection. Figure 5 further depicts the synergistic effect between photoinactivation of catalase and low-concentration hydrogen peroxide to eliminate stationary- phase MRSA US A300 and stationary-phase Pseudomonas aeruginosa.

[0075] Figure 6 depicts the killing efficacy comparison between CW-410 nm and ns-410 nm combined with H2O2 in both stationary-phase MRSA US A300 and Pseudomonas aeruginosa. Left and right: CFU ml-1 of stationary-phase MRSA and P. aeruginosa under different treatment schemes, respectively. N=3. Data: Mean ± SD. ***: significant difference. p<0.001. 250 CFUs: detection of limit.

[0076] Figure 7 depicts CFU ml-1 of E. coli BW25113 under different treatment schemes. Tobramycin: 2 pg/ml, 4-hour incubation. ***: p<0.001, student unpaired t-test.

[0077] Figure 8 depicts CFU ml-1 of Enterococcus faecalis NR-31970 under different treatment schemes. Tobramycin: 2 pg/ml, 4-hour incubation.

[0078] Figure 9 depicts confocal laser scanning microscopy of intracellular MRSA. (Panels a- c). Fluorescence images of intracellular live MRSA (Panel a), and dead MRSA (Panel b), along with the transmission images (Panel c) after MRSA infecting RAW 264.7 macrophage cells for 1 hour. (Panels d-f). Fluorescence images of intracellular live MRSA (Panel d), and dead MRSA (Panel e), along with the transmission images (Panel f) after ns-410 exposed MRSA infecting RAW 264.7 macrophage cells for 1 hour. (Panels g-h). Quantitative analysis of live/dead MRSA from the above two groups. Scalar bar=10 pm.

[0079] Figure 10 depicts active catalase percent of various fungal strains with or without 410 nm light exposure. Dose: 410 nm, 150 mW/cm2, 5 min. Fungal concentration: 10⁶ cells/ml. C. albicans CASC5314: wild-type Candida albicans. [0080] Figure 11 depicts CFU results of C. albicans CASC5314 after different treatment schemes, (left panel). Time-killing assay of CASC5314 after various treatment schemes, (right panel). Spread plates of CASC5314 after 1-hour incubation at different treatment schemes.

[0081] Figure 12 depicts the synergistic effect between photoinactivation of catalase under various wavelengths and low-concentration hydrogen peroxide to eliminate stationary-phase CASC5314. CFU ml-1 of CASC5314 after treatments under the combination between H2O2 and various wavelengths. Dosage: 40 mW/cm², 24 J/cm². H2O2: 44 mM, 1.5-hour incubation. Data: Mean ± SEM (N=3). ##: detection limit.

[0082] Figure 13 depicts fluorescence signals of PrestoBlue from CASC5314 under various treatment schemes. (Panels a, c). EECh-alone treated stationary-phase CASC5314 and log-phase CASC5314, respectively. (Panels b, d). 410 nm plus H2O2 treated stationary-phase CASC5314 and log-phase CASC5314, respectively.

[0083] Figure 14 depicts fluorescence signals of PrestoBlue of three different C. auris strains under different treatment schemes, (top panels). Amp B alone-treated groups, (bottom panels). 410 nm plus amp B-treated groups.

[0084] Figure 15 depicts confocal laser scanning imaging of live/dead C. albicans after infecting RAW264.7 macrophage cells.

[0085] Figure 16 depicts photoinactivation of catalase in combination with silver cation kills MRS A. Shown in the images are spread agar plates of MRS A US A300 under different treatment schemes.

[0086] Figure 17 depicts the comparison between CW-410 and ns-410 to inactivate catalase and eliminate E. coli BW25113 by synergizing with silver cation. (Panels a-b). CFU ml'¹ of E. coli BW25113 after different treatment schemes: 30 min for (Panel a) and 60 min for (Panel b). Dose: 22 J/cm². Silver cation: 0.5 pM. Data: Mean ± SEM (N=3). ***: p<0.001, significant difference. Student unpaired t-test.

[0087] Figure 18 depicts the percent of sterilization of MSSA and S. aureus on smooth, dry, metal surfaces with CW-410 nm and 0.3% H2O2 from several times between 20 to 300 seconds with 15 J/cm² for both organisms and 15 J/cm² and 200 J/cm² for MSSA on a rough surface.

[0088] Figure 19 depicts the percent of sterilization of MSSA on a plastic surface with CW-410 nm and 0.09% H2O2 from 75 seconds with 15 J/cm². [0089] Figure 20 shows a device for delivering the photonic and ROS agent and any other affecter that can increase the effectiveness of treatment.

[0090] Figure 21 shows components that may be used in a device to deliver the methods described herein.

[0091] Figure 22 shows an embodiment of the light generating and delivery device claimed herein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control.

[0093] As used herein, the phrase “treating an infected tissue” refers to curing, alleviating or partially arresting the clinical manifestations of the infection or its complications. Treating an infected tissue achieves a medically desirable result. In some cases, this is a complete eradication of infection. In other cases, it is an improvement in the symptoms of the infection.

[0094] A “ROS-generating agent” is any biological or chemical agent that produces Reactive Oxygen Species (ROS). ROS-generating agents as defined herein, exclude exogenous photosensitizer agents that have been light-activated. A “photosensitizer” is a chemical compound, or a biological precursor thereof, that produces a phototoxic or other biological effect on biomolecules upon photoactivation.

[0095] A “subject” is a vertebrate, including any member of the class mammalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates.

[0096] A “microbe” is a multi-cellular or single-celled microorganism or virus, including, but not limited to, bacteria, protozoa, fungi, and algae. The term microbe, as used herein, includes pathogenic microorganisms such as bacterium, protozoan, fungus, or virus.

[0097] The term “inanimate surface” refers any non-living surface.

[0098] The term “disinfecting” refers to destroying or eliminating pathogenic microorganisms that cause infections. [0099] Unless specifically stated or clear from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” is understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0100] Ranges provided herein are understood to be shorthand for all of the values within the range. A range of 1 to 60 is understood to include any number, such as seconds or minutes, combination of numbers, or sub-range from the group consisting 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 (as well as fractions thereof unless the context clearly dictates otherwise). For example, the wavelengths from about 400 nm to about 460 nm include the 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, 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. The light from about 5 J/cm² to about 200 J/cm² includes 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, 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, 115, 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, 158, 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, 200 , 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,

229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,

248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,

267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,

286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,

324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,

343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,

362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,

381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,

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, 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, 454, 455, 456,

457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,

476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,

495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,

514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532,

533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551,

552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,

571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589,

590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,

609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627,

628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,

647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665,

666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684,

685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703,

704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, and 720 J/cm². [0101] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. [0102] Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention [0103] Hydrogen peroxide (H2O2) is continuously produced inside microbes from autoxidation of the redox enzyme, and it diffuses quickly into the intracellular environment, causing an acutely detrimental effect (e.g., lipid peroxidation, DNA and protein damage) as a result of the Fenton reaction: a. Fe2+ + H2O2 — Fe3+ + . OH + OH— b. Fe3+ + H2O2 — * Fe2+ + . OOH + H+

[0104] Photoinactivation of catalase creates potent antimicrobial effects due to a lethal accumulation of ROS. Photoinactivation of catalase further assists immune cells to eliminate intracellular pathogens. Neutrophils and macrophages are highly motile phagocytic cells that serve as the first line of defense of the innate immune system (Segal, A. W., Annu Rev Immunol 23, 197-223, doi: 10.1146/annurev.immunol.23.021704.115653 (2005)). These cells play an essential role in providing resistance to bacterial and fungal infections through releasing ROS burst (e.g., superoxide, hydroxyl radicals, and singlet oxygen (Hampton, M. B., Blood 92, 3007- 3017 (1998)). However, pathogens possess an array of elaborate strategies to invade and survive inside neutrophils or macrophages, thus acting as the ‘Trojan horses’ responsible for further dissemination and recurrent infections (Lehar, S. M. et al. Nature 527, 323-328 (2015)).

Catalase, which is encoded by the gene katA, confers indispensable resistance to antimicrobial agents or reactive oxygen species released by immune cells (Flannagan, R., Pathogens 4, 826- 868 (2015)). Photoinactivation of catalase assists macrophages and neutrophils to reduce the intracellular and extracellular bacterial burden. ROS agents maybe naturally occurring or introduced to the microbial environment.

[0105] In conducting the methods of the present invention, photoinactivation of catalase is preferably conducted with light having a wavelength of about 400 nm to about 460 nm, in combination with administration of a low-concentration peroxide solution and/or an ROS generating agent. Methods of the invention exclude the use of exogenous photosensitizing agents that have been activated by light.

[0106] Peroxide solutions include, but are not limited to solutions containing hydrogen peroxides, metal peroxides, and organic peroxides. Hydrogen peroxides include, but are not limited to, peroxy acids, peroxymonosulfuric acid, peracetic acid, peroxydisulfuric acid, peroxynitric acid, peroxynitrous acid, perchloric acid, and phthalimidoperoxycaproic 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, alkenyl peroxide arachidonic acid 5-hydroperoxide, artelinic acid, artemether, artemisinin, artemotil, arterolane, artesunate, ascaridole, benzoyl peroxide, bis(trimethylsilyl) peroxide, tert-butyl hydroperoxide tert-butyl peroxybenzoate, CSPD ([3-(l - chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate), cumene hydroperoxide, di-tert-butyl peroxide, diacetyl peroxide, diethyl ether peroxide, dihydroartemisinin, dimethyldi oxirane, 1,2-di oxane, 1,2-dioxetane, 1,2-dioxetanedione, dioxirane, dipropyl peroxydicarbonate, ergosterol peroxide, hexamethylene triperoxide diamine, methyl ethyl ketone peroxide, nardosinone, paramenthane hydroperoxide, perfosfamide, peroxyacetyl nitrate, peroxyacyl nitrates, prostaglandin h2, 1,2,4-trioxane, and verruculogen. [0107] Other peroxides include potassium peroxy di sulfate, bi s(trimethyl silyl) peroxide (MesSiOOSiMes), phosphorus oxides, ammonium peroxide, copper(II) peroxide, sodium peroxide, cobalt(II) peroxide, mercury(I) peroxide, iron(II) peroxide potassium peroxide, copper(I) peroxide, rubidium peroxide, cesium peroxide, iron(III) peroxide, beryllium peroxide, magnesium peroxide, nickel(II) peroxide, cadmium peroxide, barium peroxide, benzoyl peroxide, calcium peroxide, diacetyl peroxide, cesium superoxide, lead(IV) peroxide, lithium peroxide, gallium(II) peroxide, chromium(III) peroxide, mercury(II) peroxide, gold(I) peroxide, strontium peroxide, zinc peroxide, potassium superoxide, and chromium(VI) peroxide.

[0108] In other specific embodiments, the diluted peroxide solution is a hydrogen peroxide solution formulated with between about 0.001% and about 0.3% hydrogen peroxide (which converts to about 0.03 mM to about 88 mM hydrogen peroxide).

[0109] Photoinactivation of catalase and administration of the peroxide solution can also be provided in combination with ROS generating agents including antibiotics, such as tobramycin. Other ROS generating agents include, but are not limited to, silver cation (including silver sulfadiazine), iodine tincture, gold nanoparticles, methylene blue (non-photoactivated), P-lactam antibiotics, aminoglycosides, fluoroquinolones, antifungal azoles, membrane-targeting polyene antifungals, such as amphotericin B, and cell-wall targeting antifungals, such as caspofungin. [0110] Typically following photoinactivation, the peroxide solution can be administered to the site of the infection for a duration of about a fraction of a second to about 30 minutes. In alternate embodiments, the peroxide solution, the ROS generating agent and/or the photoinactivating light can be administered concomitantly or sequentially to the site of infection. Preferably, the peroxide solution is topically administered (e.g., as a liquid or a spray). Administration of the ROS generating agent can be according to all modes of local or systemic administration known in the art.

[OHl] In one embodiment, methods of the invention comprising photoinactivation of catalase are directed to an infected external tissue of a subject, including, but not limited to, skin, hair and nails. In other embodiments, internal tissues, such as gastrointestinal organs or cavities (oral, vaginal or nasal cavities), may be targeted as well.

[0112] Peroxide solutions and/or ROS generating agents can be administered alone or as a component of a pharmaceutical formulation. The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, and preservatives can also be present in the compositions and combination of ROS agents.

[0113] Pharmaceutical formulations of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration, e.g., intradermal.

[0114] The formulations can include a pharmaceutically acceptable carrier. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

Formulations of the invention can be administered parenterally, intraperitoneally, subcutaneously, topically, orally (e.g., the ROS generating agent) or by local administration, such as by aerosol or transdermally. Formulations can be administered in a variety of unit dosage forms depending upon the severity of the infection or the site of the infection and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA (“Remington’s”).

[0115] Pharmaceutical formulations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0116] In practicing this invention, the pharmaceutical formulations can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. In specific embodiments, delivery can be mediated by a transdermal patch, bandage or dressing impregnated with compositions comprising the peroxide solution and/or ROS generating agent. Sustained release can be provided by transdermal patches, for slow release at the site of infection.

[0117] The amount of pharmaceutical formulation adequate to reduce or eradicate pathogenic microbes is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the infection, the severity of the infection, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

[0118] The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., 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; the latest Remington’s, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

[0119] Single or multiple administrations of pharmaceutical formulations of the invention can be given depending on, for example: the dosage and frequency as required and tolerated by the patient, the persistence of infection, or lack thereof, after each administration, and the like. The formulations should provide a sufficient quantity of peroxide solution to effectively treat, prevent or ameliorate the infection.

[0120] Methods of the invention target catalase-positive microbes which are associated with, or may give rise to, infection. Both Gram-negative and Gram-positive bacteria serve as infectious pathogens in vertebrate animals. Such catalase-positive Gram-positive bacteria include, but are not limited to, Staphylococci species. Catalase positive Gram-negative bacteria include, but are not limited to, Escherichia coh. Pasteurella species, Pseudomonas species (e.g., P. aeruginosa), and Salmonella species. Specific examples of infectious catalase-positive bacteria include but are not limited to, Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g. M. tuberculosis complex, M. avium complex, M. gordonae clade, M. kansasii clade, M. nonchromogenicum/terrae clade, Mycolactone-producing mycobacteria, M. simiae clade, M. abscessus clade, M. chelonae clade, M. fortuitum clade, M. mucogenicum clade, M. parafortuitum clade, M. vaccae clade, M. ulcerans,M. vanbaalenii,M. gilvum,M. bovis,M. leprae, M. spyrl,M. kms,M. mcs,M. jls, M. intracellulare, and gordonae), Acinetobacter baumannii, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, pathogenic Campylobacter species, Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, Corynebacterium species, Erysipelothrix rhusiopathiae, Chlamydia trachomatis, Clostridium perfringers, Clostridium tetani, Klebsiella pneumoniae, Pasturella multocida, Bacteroides species, Fusobacterium nucleatum, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelii, Mycoplasma and Chlamydia species.

[0121] 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 parapsi losis, and other catalase-positive Candida spp, Candida auris. and Trichophyton rubrum.

[0122] The light for photoactivation of catalase can be produced and delivered to the site of infection or contamination by any suitable means known in the art. In specific embodiments, the light source is a is a pulsed nanosecond laser. Pulsed operation of lasers refers to any laser not classified as continuous wave, so that the optical power appears in pulses of some duration at some repetition rate. Nanosecond laser families can range from the UV to the IR with wavelengths up to 1064 nm, repetition rates up to 2 kHz, and pulse energy up to 20 mJ. Photoinactivation of catalase can be conducted with light having a wavelength of about 400 nm to about 460 nm. In specific embodiments, the wavelength is about 400 nm to about 430 nm, applied at a dosage of about 0.01 J/cm² to about 200 J/cm² and in other specific embodiments, about 14 J/cm² to about 32 J/cm². In other specific embodiments, the pulse duration is about 5 nanoseconds. Light delivered in this range by a pulsed nanosecond laser is clinically advantageous because thermal damage is minimal, temporary or otherwise non-existent. In more specific embodiments, the wavelength is 410 nm (delivered using about 15 J/cm²), applied by a pulsed nanosecond laser according to methods known in the art for operation of such lasers.

[0123] Exposure times range from about a fraction of a second to about 30 minutes in length, and can be repeated daily or weekly as needed, for example, about twice per week for several months. In clinical applications, patients may receive treatment for between one to 3 months or longer as determined by the practicing physician.

[0124] Photoactivating light can be delivered to the site of infection or contamination through various optical waveguides, such as an optical fiber or implant. In some embodiments, the photoinactivating light is delivered by optical fiber devices that directly illuminate the site of infection. For example, the light can be delivered by optical fibers threaded through small gauge hypodermic needles. In addition, light can be transmitted by percutaneous instrumentation using optical fibers or cannulated waveguides. For open surgical sites, suitable light sources include broadband conventional light sources, broad arrays of LEDs, and defocused laser beams. The light source can be operated in the Continuous Wave (CW) mode. Photoinactivation of catalase is preferably conducted with light having a wavelength of about 400 nm to about 430 nm and a dosage of about 0.01 J/cm² to about 700 J/cm², and in specific embodiments, about 14 J/cm² to about 32 J/cm² In other specific embodiments, the wavelength is 410 nm (delivered using about 15 J/cm²), applied by a CW LED according to methods known in the art for operation of such LED sources. Exposure times range from any light source range from about 10 seconds to about 30 minutes in length.

[0125] In other embodiments of the invention, the photoinactivation of catalase is performed on an inanimate surface including but not limited to metal, plastic, fabric, rubber, stone, composite surfaces or wood. In yet another embodiment, the inanimate surface can be a gas, including, but not limited to air in a ventilation system, confined space, or space of limited circulation. In specific embodiments, the inanimate surface comprises objects such as instruments, catheters, medical and military equipment, furniture, handrails, textiles, fixtures such as sinks and plumbing materials, building materials, industrial or electronic equipment, and food product or food processing equipment. Photoinactivation of catalase on inanimate surfaces is preferably conducted with light having a wavelength of about 400 nm to about 460 nm, and a dosage of about 0.01 J/cm² to about 10000 J/cm², and in combination with administration of a solution having a low-concentration of a peroxide. In specific embodiments, the wavelength is 410 nm (delivered at 15 J/cm²), applied by a pulsed nanosecond laser. Exposure times range from about a fraction of a second to about 30 minutes in length.

[0126] Embodiments of the invention can be practiced with a variety of light sources. Any light source that can generate the wavelength range described herein can be used. Such sources include, but are not limited to, an incandescent lamp, a fluorescent lamp, a halogen lamp, a xenon lamp, a light emitting diode (’’LED”), and a laser. The type of light generated can also vary. The light generated can be continuous wave or pulsed. Further, the energy associated with the light generation can range from about 0.01 J/cm² to about 10000 J/cm². Additionally, the area exposed to the light and/or the time of exposure can range from square femtometers to square meters and from fractions of a second to hours. This variability will allow for numerous applications of the invention from the minute, such as, but not limited to, treating a single catalase-positive microbe, to treating entire objects, such as, but not limited to, a bed or door. Large-scale treatments with the invention described herein, such as treating an entire room, are also envisioned by the inventors. Because distance between the light source and the catalasepositive microbe may vary, the light source, the wavelength, the type of light, the energy associated with the generated light, and the duration of exposure can also vary in order to inactivate the microbe’s catalase. For example, a shorter distance between the light source and the catalase-positive microbe could require less light energy and duration of treatment than a light source that is further away from a catalase-positive microbe.

[0127] Because the catalase-positive microbe can be present in various environments, the pH of the environment as well as the pH associated with aspects of the invention method may vary. For example, the pH of a subject’s skin or within a wound may need to be addressed by the invention and can be done so by varying the pH of the H2O2 and/or a ROS-generating agent. Overly acidic or basic H2O2 could damage the subject’s cells and/or tissue. Therefore, the invention described herein also encompasses varying the pH of the H2O2 to bring about differing activities and stability of the H2O2 (lower pH H2O2 is more stable but less active than that of H2O2 having a higher pH). A non-limiting example of this is envisioned by the inventors by using H2O2 having a higher pH, which would increase its activity and may need less volume or lower concentration for treatment when, for example, treating a subject’s cells, tissue (including skin), or a wound.

[0128] The surface on which a catalase-positive microbe is present and can be subject to the present invention can require variation in the claimed methods and formulations. For example, shadowing can be less of an issue when treating a smooth, inanimate surface. Therefore, the type of light used, light-associated energy, and duration of illumination can be different than that required for a rough surface. To better address possible shadowing on rougher surfaces, be it an inanimate surface or that of a subject, the light source can be moved with CW or pulsed light and may require additional light exposure or repeated pulses.

[0129] Additionally, the location of non-biological or organic surfaces to be treated is not limited. The surface can be the interior or exterior of a structure, such as a container, packaging, a room, an aircraft, a seafaring vessel, or a building. The surface is also not terrestrial bound in that practicing the present invention can occur above the Earth’s surface as in spaceships, capsules, space stations, or on a moon or another planet.

[0130] Another non-limiting application of the invention described herein is agriculture based. There are numerous possible applications associated with the soil, any additive to the soil, any crop, including, but not limited to, plants, consumable fungi, algae, consumable meat, etc., harvesting, processing, storage, and transportation of the crop. For example, light-associated variables and non-light components of the present invention can be optimized to decontaminate soil of any catalase-positive microbes. Additionally, the present invention can be incorporated into the packaging process for a crop prior to shipment to a consumer retailer. Such incorporation could entail treatment of consumable meat before the meat is packaged within plastic wrap to increase its shelf-life and then refrigerated or frozen.

[0131] Another aspect of the invention relates to devices for carrying out the methods of the invention. In some embodiments, the device can include a light source, e.g., a laser or LED. In some embodiments, the device can include a reservoir comprising an ROS agent for delivery to a surface, e.g., as part of a spray bottle or other means for delivering the ROS agent. In some embodiments, the device is a hand-held device. The device may be powered by alternating or direct current, and the needed power can be from such sources including a battery, generator, or a wall outlet. An embodiment of the device can be comprised of a light source, means to remove any excess heat generated by the light source, such as a heat sink, and components to deliver the light source to the desired target. This may involve using a single or series of lenses to manipulate the beam of light generated. An embodiment may include a number of light sources, such as more than one LED array. The energy generated by the light source can also be modified with the use of a driver. In an embodiment that uses an LED array, the LED driver can allow for different energy levels to be created by the array. The light energy generated by other embodiments that use a light source other than an LED can be similarly controlled.

Manipulation of the beam of light generated by each light source can occur with the use of a collimator and/or lenses, such as Fresnel lenses, and deliver the light by a light pipe, also known as a light guide, over varied distances, which may range from centimeters to meters. An embodiment of a light pipe can be a rod made of optical acrylic or polycarbonate that transmits light from the input, closer to the light source, to the output or exit surface. The geometry or shape of the light pipe as well as the exterior surface of the light pipe, which can include such characteristics as a smooth or mirrored surface, can affect light delivery efficiency of the light pipe. The light delivered with or without a light pipe can be further manipulated to adjust the area to which the light will be delivered. All of the components, except when delivering the light a distance from the light source via a light pipe, may be housed within an enclosure. The length of the light pipe can vary depending on the application to be achieved. If the distance is relatively close, the components may be enclosed within a hand-held device. Powering such a device can occur via a number of means.

[0132] The following examples are put forth for illustrative purposes only and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

[0133] The following materials and methods were employed throughout Examples 1-4.

[0134] 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.

[0135] Quantitation of catalase by Amplex red catalase kit: Quantification of catalase both from the pure catalase solution and bacteria was achieved by a fluorescent Amplex red catalase kit. 25 pl of analyte were incubated with 25 pl (40 pM of H2O2) for 30 min at room temperature. Then 50 pl of working solution (100 pM Amplex Red reagent containing 0.4 U/ml horseradish peroxidase) was added to the abovementioned mixture, and the subsequent mixture was incubated for another 30-60 min in the dark. After that, the fluorescence was recorded at an emission of 590 nm when excited at 560 nm.

[0136] Resonance Raman spectrum of dried catalase film: Catalase was measured by its Raman peaks at around 1300-1700 cm'¹ measured by resonance Raman spectroscopy (1221, LABRAM HR EVO, Horiba) with a 40*objective (Olympus) and an excitation wavelength of 532 nm. Samples (dried ‘coffee ring’) were sandwiched between two glass cover slides (48393-230, VWR international) with a spatial distance of ~80 pm. To study the photoinactivation (by a continuous-wave LED), the same samples were measured after each laser irradiation.

[0137] CFU experiments to test the potential synergy between photoinactivation of catalase and H2O2: Overnight-cultured bacteria was centrifuged, the supernatant was discarded, and the pellet was resuspended with the same amount of PBS. The laser source used in the study is nanosecond (ns) pulsed OPO laser purchased from OPOTEK Inc, model number Opolette HE355 LD, having 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 nanosecond; spectral linewidth, 4-6 cm-1; and pulse-to-pulse stability, <5%. For each bacterial strain there were four groups: untreated one, ns-410 nm -treated group, H2O2 (22 or 44 mM)-treated group, ns- 410 nm plus H2O2 (22 or 44 mM)-treated group. The dose for ns-410 nm exposure was 15 J/cm². H2O2 was incubated with bacteria for 30 min at 37°C with the shaking speed of 200 rpm. After incubation, bacterial burden from each group was serial diluted, inoculated onto TSA plates, then counted by enumeration of these plates.

[0138] CFU experiments to test the potential synergy between photoinactivation of catalase and ROS-generating antibiotics: Overnight-cultured bacteria was centrifuged, and then the supernatant was discarded and re-suspended with the same amount of fresh TSB. Then prior to any treatments, the above solution was incubated with antibiotics (10 pg/ml) for 1 hour. For each bacterial strain, four groups were tested: untreated one, ns-410 nm-treated group, antibiotic (2 pg/ml)-treated group, ns-410 nm plus antibiotic (2 pg/ml)-treated group. The dose for ns-410 nm exposure was 15 J/cm². Antibiotic was incubated with bacteria for up to 6 hours at 37°C with the shaking speed of 200 rpm. At each time interval, bacterial burden from each group was serial diluted, inoculated onto TSA plates, then counted by enumeration of these plates.

[0139] Confocal imaging of intracellular bacteria assay: As described elsewhere (Yang, X., et al. International journal of nanomedicine 13, 8095 (2018)), murine macrophage cells (RAW 264.7) were cultured in DMEM supplemented with 10% FBS at 37 degrees C with CO2 (5%). Cells were exposed to MRS A US A300 or Salmonella enterica (with/without ns-410 nm exposure) at a multiplicity of infection (MOI) of approximately 100: 1 at serum-free DMEM medium. 1 or 2-hour post-infection, RAW 264.7 cells were washed with gentamicin (50 pg/mL, for one hour) to kill extracellular bacteria in DMEM + 10% FBS. After that, RAW 264.7 cells were washed with gentamicin (50 pg/mL) and subsequently lysed using 0.1% Triton-X 100 for 3 min. After membrane permeabilization, infected RAW 264.7 cells were stained with Live/Dead stain for 15 min, then samples were fixed in 10% formalin for 10 min prior to confocal imaging. Example 1 : Pulsed Blue Laser Effectively Inactivates Pure Catalase and Catalase from Bacteria [0140] Pure catalase solution (bovine liver catalase, 3 mg/ml in the PBS) was prepared in PBS using a protocol previously published to examine the effect of 410-nm exposure on the absorption spectrum of catalase solution (Cheng, L., Photochemistry and Photobiology 34, 125- 129 (1981)). Catalase shows a pronounced absorption at around 410 nm, and its absorption at this wavelength gradually decreases as the 410-nm exposure elongates (Figure 1, panel a). This suggests that the secondary structure of catalase might be changed, especially in the active hemecontaining domain. In addition, this photoinactivation effect was examined by an Amplex Red Catalase kit at different wavelengths (Figure 1, panel b). The photoinactivation trend is similar to the absorption spectrum of catalase, with the 410 nm being the most effective, where 5-min exposure depleted -70% active catalase.

[0141] Since most of the aerobic bacteria and facultative anaerobes express catalase (Mishra, S. & Imlay, J. Arch Biochem Biophys 525, 145-160, doi: 10.1016/j.abb.2012.04.014 (2012)), whether one could photoinactivate catalase in situ from the catalase-positive bacteria was examined. MRS A US A300 and P. aeruginosa (PAO-1) were selected as the representative for Gram-positive and Gram-negative bacteria, respectively. Noteworthy, catalase from both MRSA USA300 (Figure 2, panel a) and P. aeruginosa (Figure 2, panel b) were photoinactivated by blue light exposure region, especially 410-nm exposure. The dose utilized was about 15 J/cm², well below the ANSI safety limit of 200 J/cm², and the specimens were stationary-phase cultured bacteria (-10⁸ cells/ml). ANSI is the American National Standard for Safe Use of Lasers, see ANSI Z136.1, Laser Institute of America 2014.

[0142] To further understand how 410 nm exposure could cause the structural change of catalase, Resonant Raman spectroscopy was performed to capture the Raman signature of dried catalase film (Figure 3). Apparently, 410 nm exposure significantly drops the Raman intensity at 750 cm'¹, and the Raman bands ranging from 1300 cm'¹ to 1700 cm'¹, which are typical vibrational bands of heme ring from catalase (Chuang, W.-J., Heldt, J. & Van Wart, H. J. J. o. B. C. 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 consolidate the fact that 410 nm exposure could cause structural change of catalase.

[0143] In addition, the efficacy between ns-410 nm and CW-410 nm to inactivate catalase was compared, ns-410 nm is significantly more effective both in the pure solution form (Figure 4, panel a), or from MRS A US A300 (Figure 4, panel b) and P. aeruginosa (Figure 4, panel c) compared to CW-410 nm. Moreover, ns-410 exposure eliminates the necessity of heating tissue during future clinical study.

Example 2: Photo-inactivation of Catalase Sensitizes a Wide Range of Bacteria to Low- Concentration H2O2

[0144] Catalase is an essential detoxifying enzyme in bacteria encountering various endogenous or exogenous stresses (Nakamura, K. et al. Microbiology and immunology 56, 48-55 (2012)). When the gene encoding the expression of catalase is mutant, pathogens are more susceptible to the environmental stress (Mandell, G. L., J Clin Invest 55, 561-566, doi: 10.1172/j ci 107963 (1975)). Whether exogenous addition of low-concentration H2O2 could eliminate those ‘traumatized’ pathogens was investigated. As shown in Figure 5, photoinactivation of catalase (15 J/cm²) alone didn’t reduce the MRSA burden (Figure 5, panel a), P. aeruginosa burden (Figure 5, panel b), and Salmonella enterica burden (Figure 5, panel c) significantly. Moreover, low-concentration H2O2 (22 mM) didn’t exert any significant antimicrobial effect against both MRSA and P. aeruginosa (Figure 5). However, subsequent administration of low-concentration H2O2 after photoinactivation of catalase significantly reduced the MRSA and P. aeruginosa burden (> 3-logl0 reduction, Figure 5). Interestingly, the bacterial killing trend versus irradiance wavelength is similar to that of photoinactivation of catalase versus irradiance wavelength. Noteworthy, low-concentration H2O2 combined with 410 nm exposure (15 J/cm²) achieved total eradication of P. aeruginosa (Figure 5, panel b).

Example 3, Photoinactivation of Catalase and Low-concentration Hydrogen Peroxide Create a Synergistic Effect

[0145] There is a synergistic effect between photoinactivation of catalase and low- concentration hydrogen peroxide to eliminate stationary-phase MRSA US A300 and stationary- phase Pseudomonas aeruginosa. Figure 5, panel a depicts the synergistic results in a bar-graph. CFU ml-1 (colony-forming unit) designates the bacterial burden. ‘Untreated’ refers to the original stationary-phase MRSA without any exogenous treatment. ‘H2O2 (22 mM, 0.075%)’ and ‘ns-light’ refer to stationary-phase MRSA with H2O2 and ns light alone, respectively. As shown in the graph, H2O2 alone and ns-light alone do not exert any significant killing effect on MRS A, however, ns-410 nm in combination with H2O2 reduces approximately four orders of magnitude of bacterial burden. The same phenomenon happens with other wavelengths as well. Noteworthy, ns-430 nm or ns-430 nm combined with H2O2 reduces around 99% of the bacterial burden under the same conditions, ns-450 or ns-460 nm combined with H2O2 together reduces around 90% of the bacterial burden, ns-470 nm combined with H2O2 together reduces around 50% of the bacterial burden, ns-480 nm combined with H2O2 barely exerts an antimicrobial effect. Altogether, the killing effect of H2O2 is significantly enhanced by blue light photoinactivation of catalase, especially when applied using ns-410 nm. A similar phenomenon occurred with stationary-phase Pseudomonas aeruginosa, which is a representative of Gramnegative bacteria (Figure 5, panel b) and Salmonella enterica. By employing ns-410 nm combined with H2O2 to Salmonella enterica, an enhanced killing effect of around five orders of magnitude was observed (Figure 5, panel c).

[0146] In addition, ns-410 nm combined with H2O2 is significantly more effective in eliminating microbes compared to CW-410 nm combined with H2O2 (Figure 6).

Example 4: Photoinactivation of Catalase Revives Conventional Antibiotics Against a Wide Range of Bacteria

[0147] Besides H2O2, whether photoinactivation of catalase could synergize with conventional antibiotics was investigated, especially for antibiotics that can generate the downstream intracellular ROS. Tobramycin, a representative of aminoglycoside, is an example. Tobramycin can induce downstream ROS burst (Dwyer, D. J. et al. Proceedings of the National Academy of Sciences 111, E2100-E2109, doi: 10.1073/pnas.1401876111 (2014)), thus the combination of photoinactivation of catalase and tobramycin administration, together, was tested to see whether an enhanced effect was observed.

[0148] Interestingly, enhanced killing effect was observed in the combination-treated group (Figure 7). More than 100 times enhancement suggests that photoinactivation of catalase indeed accelerates the antimicrobial effect of ROS-generating antibiotics. As a control, the same treatment schemes were tested on a catalase-negative Enterococcus strain, Enterococcus faecalis NR-31970, which did not produce any enhanced killing effect (Figure 7). Altogether, this indicates that photoinactivation of catalase helps to revive traditional antibiotics against catalasepositive pathogens.

Example 5: Photoinactivation of Catalase Assists Macrophage Cells Against Intracellular Pathogens

[0149] Neutrophils and macrophage cells are highly essential phagocytic cells that serve as the first line of defense of the innate immune system (Segal, A. W., Annu Rev Immunol 23, 197- 223, doi: 10.1146/annurev.immunol.23.021704.115653 (2005)). Catalase, which is encoded by the gene katA, confers indispensable resistance to the antimicrobial agents released by immune cells (Flannagan, R., Heit, B. & Heinrichs, D., Pathogens 4, 826-868 (2015)). Based on these facts, it was hypothesized that photoinactivation of catalase could assist immune cells to eliminate extracellular and intracellular pathogens. To test the potential assistance effect, a fluorescent Live/Dead assay was used to visualize the intracellular live/dead bacteria after ns-410 nm exposure. A higher percent of dead MRSA was observed intracellularly (Figure 9).

[0150] In conclusion, photoinactivation of catalase significantly boosts the efficacy of low- concentration H2O2, ROS-generating antibiotics, and immune cells against broad-spectrum bacteria, including the notorious drug-resistant gram-negative bacteria.

[0151] The following materials and methods were employed throughout Examples 5-9.

[0152] Chemicals and fungal strains: DMSO (W387520, Sigma Aldrich), amphotericin B (A9528-100 MG, Sigma Aldrich), ergosterol (AC 1178100050, 98%, ACROS Organics). YPD broth (Y1375, Sigma Aldrich). YPD agar (Y1500, Sigma Aldrich). PrestoBlue cell viability assay (A13262, Thermo Fisher Scientific). Candida albicans SC5314, the test of fungal strains used see Table 1.

Table 1. Fungal strains utilized for amp B imaging experiments.

[0153] Quantification of catalase from fungus before and after 410 nm exposure: Quantification of catalase both from the pure catalase solution and fungal solution were achieved by a fluorescent Amplex red catalase kit. Basically, 25 pl of analyte were incubated with 25 pl (40 pM of H2O2) for 30 min at room temperature. Then 50 pl of working solution (100 pM Amplex Red reagent containing 0.4 U/ml horseradish peroxidase) was added to the abovementioned mixture, and the subsequent mixture was incubated for another 30-60 min in the dark. After that, the fluorescence was recorded at an emission of 590 nm when excited at 560 nm. [0154] CFU test to quantify the treatment efficacy: Quantification of antifungal treatment schemes were achieved as follows: overnight cultured fungal specimen was washed by sterile PBS. Log-phase fungal pathogens were prepared by dilution into fresh YPD broth at a ratio of 1 :50 and cultured for another 2-3 hours at 30 °C with the shaking speed of 200 rpm. After that, the fungal concentration was adjusted to be around 1 x 10⁸ cells/ml by centrifuging or further dilution with PBS. 10 pl of the above fungal solution was exposed to 410 nm for 5 min (150 mW/cm²). After that, the exposed sample was collected into 990 pl of sterile PBS, then supplemented with treatment agents. Later, CFU of fungal cells was enumerated by serial dilution and cultured in YPD agar plates for 48 hours.

[0155] PrestoBlue viability assay: First log-phase fungal pathogens were prepared by diluting overnight-cultured fungal pathogens into fresh YPD broth at a ratio of 1 : 50 and cultured for another 2-3 hours at 30 °C with the shaking speed of 200 rpm. After that, the fungal concentration was adjusted to be around 1 x 10⁸ cells/ml by centrifuging or further dilution with PBS. 10 pl of the above fungal solution was exposed to 410 nm for 5 min (150 mW/cm²). After that, the exposed sample was collected into 990 pl of sterile PBS, then supplemented with treatment agents. Aliquots were made from the above sample into a 96-well plate, with each well containing 100 pl. Then 100 pl sterile YPD broth and 23 pl of PrestoBlue were added into the same well. Fluorescence signal at 590 nm from each well was recorded in a time-course (up to 18 hours with the interval of 30 min) manner at an excitation of 560 nm. For each strain, in order to know the exact number of fungal pathogens in each well, the corresponding fluorescence signals were recorded from fungal pathogens with known numbers, however no external treatments.

[0156] Macrophage-Q/ /t/a albicans interaction unveiled by confocal laser scanning microscopy: As described elsewhere (Yang, X., et al. International journal of nanomedicine 13, 8095 (2018)), murine macrophage cells (RAW 264.7) were cultured in DMEM supplemented with 10% FBS plus penicillin and streptomycin at 37 °C with CO2 (5%). Cells were exposed to Candida albicans SC5314 (with/without 410 nm exposure) at a multiplicity of infection (MOI) of approximately 10: 1 at serum-free DMEM medium. 1 or 2-hours post-infection, RAW 264.7 cells were washed with gentamicin (50 pg/mL, for one hour) to kill extracellular pathogens in DMEM + 10% FBS. After that, RAW 264.7 cells were washed with gentamicin (50 pg/mL) and subsequently lysed using 0.1% Triton- X 100 for 3 min. After membrane permeabilization, infected RAW 264.7 cells were stained with Live/Dead stain for 15 min, then samples were fixed in 10% formalin for 10 min. Formalin was washed away prior confocal imaging.

Example 6: 410 nm Exposure Reduces Intracellular Catalase Amount

[0157] It is known that most fungal pathogens are catalase positive (Hansberg, W., et al. Arch Biochem Biophys 525, 170-180 (2012)). To test whether 410 nm exposure could cause the loss of catalase activity, the same approach to quantify the intracellular catalase amount by the Amplex red catalase kit was utilized before and after 410 nm exposure. Catalase from various fungal pathogens, either log-phase or stationery -phase could be significantly inactivated by 410 nm exposure (Figure 10). Noteworthy, catalase from notorious Candida auris strain reduced by 60% after only 5-min 410 nm exposure.

Example 7: Photoinactivation of Catalase in Combination with H2O2 Achieved Total Eradication of C. albicans SC5314 by CFU Assay

[0158] Since catalase was effectively inactivated among various fungal strains, whether photoinactivation of catalase could sensitize fungal strains to external H2O2 attack was investigated. With further administration of low-concentration H2O2 after 410 nm exposure, eradication was achieved after combinational treatments (Figure 11). Noteworthy, there was more than five orders of magnitude enhancement of the function of H2O2 after photoinactivation of catalase (Figure 11).

[0159] Synergism between photoinactivation of catalase and H2O2 to eliminate Candida albicans SC5314 was also observed. The result is shown by a scatter plot in Figure 12. CFU ml- 1 (colony-forming unit) refers to the number of bacterial burden. ‘Untreated’ means the original stationary-phase SC5314 without any exogenous treatment. ‘H2O2 (44 mM, 0.15%) and ‘ns- light’ means stationary-phase SC5314 with H2O2 and ns-light alone, respectively. As shown in Figure 12, H2O2 alone and ns-light alone doesn’t exert significant killing effect on CASC5314, however, ns-light in combination with H2O2 reduces around four orders of magnitude of bacterial burden. Especially, ns-410 or ns-420, ns-430 combined with H2O2 achieved total eradication, ns- 450 or ns-480 nm combined with H2O2 reduced a similar amount of fungal burden as H2O2- alone. Altogether, the killing effect of H2O2 is significantly enhanced by photoinactivation of catalase by blue light, especially by ns-410-ns-430 nm. Therefore, an effective synergy exists between photoinactivation of catalase under the blue light range and H2O2 to eliminate CASC5314.

Example 8, Photoinactivation of Catalase in Combination with H2O2 Achieved Efficient Eradication of Broad-spectrum Fungal Species by PrestoBlue Assay

[0160] To further confirm that this combinational therapy works as well for other fungal strains, more clinical fungal strains were tested for feasibility of this synergistic therapy. Unlike bacteria, fungal cell growth is slower, with each colony forming after around 48 hours. Thus, a high-throughput method, PrestoBlue viability assay, was used to measure the treatment efficacy. As shown in Figure 13, the utilization of PrestoBlue could achieve the same killing effect as the CFU assay. Interestingly, log-phase and stationary-phase CASC5314 demonstrate different behavior towards the combinational killing, presumably because of the difference in metabolic activity between these two states. However, either log-phase or stationary-phase, photoinactivation of catalase always boosts the killing effect of low-concentration H2O2. This synergistic therapy was tested among more than twenty different clinical fungal isolates, and significant killing was consistently found among them.

Example 9, Candida auris Strains Are Sensitive to 410 nm Light Exposure

[0161] Apart from H2O2, whether photoinactivation of catalase was capable of synergizing with conventional antifungal agents, such as azoles or amphotericin B (amp B) was investigated. Similar to some classes of antibiotics, amp B kills fungi partly due to the 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, the PrestoBlue assay was conducted after the treatments of photoinactivation of catalase and subsequent addition of amp B against various clinical fungal isolates, including C. auris strains. [0162] Interestingly, without the assistance of photoinactivation of catalase, some C. auris strains were resilient to amp B (Figure 14). Nonetheless, photoinactivation of catalase achieved total eradication of C. auris strains regardless of the addition of amp B. Ten C. auris strains were tested and they demonstrated the same behavior. This means C. auris strains are exceptionally sensitive to blue light exposure.

Example 10. Photoinactivation of Catalase Inhibits the Formation of Hyphae of C. albicans, and Assists Macrophage Cells to Phagocytose

[0163] Host immune cells play important roles against external evasive pathogens. Catalase holds an essential role during the battle between C. albicans and neutrophils or macrophage cells (Pradhan, A. et al. Elevated catalase expression in a fungal pathogen is a double-edged sword of iron. Pios Pathog 13, el006405 (2017). Thus, whether photoinactivation of catalase could assist macrophage cells against C. albicans was examined. To visualize this effect, RAW 264.7 cells were infected with C. albicans and 410 nm-exposed C. albicans at a MOI of 10 and labeled with live/dead fluorescence stains.

[0164] As shown in Figure 15, untreated C. albicans stay as hyphae form and pierced through macrophage cells. Whereas 410 nm-exposed C. albicans remained as dead 'yeast' form intracellularly.

[0165] In summary, photoinactivation of catalase in combination with low-concentration H2O2 presents an effective and novel approach to eliminate broad-spectrum fungus and fungal infections.

Example 11. Photoinactivation of Catalase in Combination With ROS Activating Agent Silver Cation Synergistically Kills Microbes

[0166] Electromagnetic energy having a wavelength of ns-410 nm combined with 10 pM of silver cation eliminated about 90% of MRS A one hour after treatment, whereas ns-410 nm alone or silver cation alone does not exert any significant antimicrobial effect (Figure 16).

[0167] Photoinactivation of catalase and low-concentration silver cation synergistically eliminate E. coli BW25113 as well. The result is shown by scatter plots in Figure 17. CFU ml-1 (colony -forming unit) is designated as the amount of bacterial burden. ‘Untreated’ refers to the original E. coli BW25113 without any exogenous treatment. ‘0.5 pM Ag⁺ and ‘CW-410’ or ‘ns- light’ refers to E. coli BW25113 with 0.5 pM Ag⁺ and ns-410 alone, respectively. 0.5 pM Ag⁺ alone and CW-410 alone or ns-410 alone doesn’t exert any significant killing effect on E. coli, however, ns-410 in combination with 0.5 pM Ag⁺ reduces around 99% of bacterial burden (Figure 17). The same phenomenon happens at other wavelengths as well. Noteworthy, CW- 410 combined with 0.5 pM Ag⁺ didn’t significantly reduce bacterial burden under the same conditions. Our results are consistent for both 30 and 60 minutes after treatments.

Example 12, Photoinactivation of Catalase in Combination with H2O2 Achieved Efficient Eradication of MSSA and S. aureus on a metal surface.

[0168] To confirm that the combinational treatment works as well on inanimate, plastic surfaces, both smooth and rough surfaces contaminated with MSSA or S. aureus were treated with CW 400 nm to CW 460 nm light (15 - 200 J/cm²) for a number of times between 20 seconds and 30 minutes and 0.3% H2O2. Figure 18 shows that S. aureus or MSSA contamination on smooth aluminum was reduced but a different light frequency and greater energy exposure was needed for reduction of MSSA on rough aluminum.

Example 13, Photoinactivation of Catalase in Combination with H2O2 Achieved Efficient Eradication of MSSA on a plastic surface.

[0169] To confirm that the combinational treatment works as well on inanimate plastic surfaces, surfaces contaminated with MSSA were treated with CW 410 nm (15 J/cm²) for 75 second and 0.09% H2O2. Figure 19 shows MSSA contamination on plastic was reduced.

Example 14, Delivery Device

[0170] The device that can deliver the technology described herein can have numerous embodiments. For example, Figure 20 depicts a hand-held device that can emit light, continuous wave or pulsed, and an ROS agent, such as hydrogen peroxide, with the light source being a laser or LED. In order to meet the constraints of such a hand-held device, enough energy must be available to deliver the required light energy. For example, the device of Figure 20 could have a battery and capacitor in order to deliver the desired light energy. Other devices can be comprised of numerous components to deliver the technology described herein. For example, Figure 21 shows numerous components, such as an LED array, which is the source of the light generation and can be used alone or in combination with other arrays. To address any additional energy not associated with the light, a heat sink can safely address any such energy. Lenses may also be used to manipulate the beam of light in order to deliver the light in the intended field, be it the surface to be illuminated or to enter the light pipe via the light pipe input. Drivers may also be used to vary the light energy level of the delivered light. Based on the area to be treated and the distance of that surface from the device, a driver can vary the light energy to account for any change in the energy, such as distance between the light source and the area to be exposed, in order that the correct dose is delivered. Light pipes may be used to deliver and modulate the light described herein. For example, the light generating components may be in an enclosure and fixed or mounted on a cart with the area for exposure being some distance away. Light manipulated with devices including one or more collimator and/or lens may be delivered distances, ranging from centimeters to meters, via a light pipe, the efficiency of which may be varied due to the geometry or exterior surface of the light pipe. Once the light reaches the light pipe output, the light may be further modified and focused. Figure 22 shows an optical layout as to how components may be combined in an embodiment to practice the application of light in the claimed methodology. Specifically, continuous wave or pulsed light having variable associated energy can be generated by an LED array. In this example, the LED box can be a single array or multiple arrays. The energy may be modified with a driver, not shown, and any energy not associated with the light can be addressed with a heat sink. The generated light is manipulated with collimating and focusing lenses to deliver the light to the light pipe input. The light pipe may be solid or made up of numerous fibers or other material to deliver the light along the light pipe’s length, which can vary. For example, in a hand-held device, the light pipe may be a few centimeters in length. For a device supported by a cart or on a countertop, the light pipe can be at least a meter in length. The light exits the light pipe at the output and the area to be illuminated, such as an inanimate surface, such as a tabletop, or the skin of a human subject, can vary via the adjustable focus.

[0171] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

REFERENCES

[0172] All patents, patent applications and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A device for treating a surface contaminated with at least one catalase-positive microbe comprising a component that generates light having a wavelength of about 410 nm and a reservoir comprising a reactive oxygen species (ROS) agent, wherein the light delivered to the surface has an energy of at least 5 J/cm² and the ROS agent is applied to the microbe.

2. The device of claim 1, wherein the ROS agent is hydrogen peroxide.

3. The device of claim 1, wherein the surface is on a subject.

4. The device of claim 3, wherein the ROS agent is silver sulfadiazine.

5. The device of claim 1, wherein the surface is in a subject.

6. The device of claim 1, wherein the surface is an inanimate surface.

7. The device of claim 6, wherein the inanimate surface is metal, plastic, fabric, rubber, stone, composite surfaces or wood.

8. The device of claim 6, wherein the inanimate surface is air in a ventilation system.

9. The device of claim 1, wherein the energy is 20 J/cm² or greater.

10. The device of any of claims 1-9, wherein the component is a laser or an LED.

11. The device of any of the claims 1-10, wherein the light is pulsed.

12. The device of any of the claims 1-10, wherein the light is continuous wave.

37

13. A device for treating a surface contaminated with at least one catalase-positive microbe comprising a component that generates light having a wavelength of 410 nm and a reservoir comprising an ROS agent, wherein the light delivered to the surface is pulsed and has an energy of at least 5 J/cm² and the ROS agent is applied to the microbe.

14. A method of reducing the number of a catalase-positive microbe, regardless of its lifecycle stage, said method comprising the steps of: applyingjight to the microbe, wherein the catalase is inactivated, and contacting the microbe with a composition comprising a peroxide solution and/or a reactive oxygen species (ROS) generating agent.

15. A method of reducing the presence of a catalase-positive microbe in an environment, said method comprising the steps of: applying light to the environment, wherein the catalase is inactivated, and contacting the environment with a composition comprising a peroxide solution and/or an ROS generating agent.

16. A method of treating a cell, tissue, or fluid infected with a catalase-positive microbe, said method comprising the steps of: applyingjight to the cell, tissue, or fluid, wherein the catalase is inactivated, and contacting the cell, tissue, or fluid with a composition comprising a peroxide solution and/or an ROS generating agent.

17. A method of producing a synergistic antimicrobial effect in a cell, tissue, or fluid infected with a catalase-positive microbe, said method comprising the steps of: applyingjight to the cell, tissue, or fluid, wherein the catalase is inactivated, and contacting the cell, tissue, or fluid with a composition comprising a peroxide solution and/or an ROS generating agent.

18. The method of claim 16 or 17, wherein the cell, tissue, or fluid is skin, scalp, or nail.

19. A method of disinfecting an inanimate surface contaminated with a catalase-positive microbe in an environment, said method comprising the steps of: applying light to the inanimate

38 surface, wherein the catalase is inactivated, and contacting the inanimate surface with a composition comprising a peroxide solution and/or an ROS generating agent.

20. The method of any one of claims 14-19, wherein the light has a wavelength of about 400 nm to about 460 nm.

21. The method of claim 20, wherein the light has a wavelength of about 410 nm.

22. The method of any one of claims 14-21, wherein the dose of the light is about 0.1 J/cm² to about 10000 J/cm².

23. The method of claim 22, wherein the dose of the light is about 5 J/cm² to about 700 J/cm².

24. The method of claim 23, wherein the dose of the light is about 15 J/cm².

25. The method of any one of claims 14-24, wherein the light is provided by an incandescent lamp, a fluorescent lamp, a halogen lamp, a xenon lamp, a light emitting diode, or a laser.

26. The method of any one of claims 14-25, wherein the catalase-positive microbe is a fungal, bacterial, or viral microbe and the light is provided as a pulse.

27. The method of any one of claims 14-25, wherein the catalase-positive microbe is a fungal or bacterial microbe and the light is provided as a continuous wave.

28. The method of any one of claims 14-27, wherein the peroxide solution is a hydrogen peroxide solution.

29. The method of claim 28, wherein the hydrogen peroxide solution is between about 0.001% and about 3.0% hydrogen peroxide.

30. The method of any one of claims 14-29, wherein the ROS generating agent is tobramycin, silver cation, iodine tincture, a gold nanoparticle, methylene blue, a P-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, a membrane-targeting polyene antifungal or a cell-wall targeting antifungal.

31. The method of any one of claims 14-30, wherein the catalase-positive microbe is in proximity to other cells, tissues, organs, materials, vegetables, food solids, skin, fingernail, fluid, or an inanimate surface.

32. The method of claim 30, wherein the inanimate surface is a material comprising metal, plastic, fabric, rubber, stone, soil, composite surface, wood, or a gas.