WO2009117675A1

WO2009117675A1 - Low aspect ratio diffusing fiber tip

Info

Publication number: WO2009117675A1
Application number: PCT/US2009/037834
Authority: WO
Inventors: Eric Bornstein
Original assignee: NOMIR MEDICAL TECHNOLOGIES Inc
Current assignee: NOMIR MEDICAL TECHNOLOGIES Inc
Priority date: 2008-03-20
Filing date: 2009-03-20
Publication date: 2009-09-24
Anticipated expiration: 2010-09-20
Also published as: WO2009117672A3; US20110208274A1; US20150057598A1; WO2009117672A2

Abstract

A kit for treating an antimicrobial resistant biological contaminate at a treatment site is disclosed which includes a diffuser tip adapted to receive near infrared therapeutic light from a light delivery system and diffuse the light to illuminate at least a portion of the treatment site; a quantity of an antimicrobial; agent; instructions to use the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to treat the biological contaminate; and suitable packaging.

Description

LOW ASPECT RATIO DIFFUSING FIBER TIP

RELATED APPLICATIONS This application is a continuation in-part of applications U S S N l 1/930,941 and

U S S N l 1/981,486, both filed October 31 , 2007, and further claims pπoπty to U S S N 61/097,792 filed September 17, 2008 and 61/038,265 filed March 20, 2008, the contents of all of which are hereby incorporated herein by reference m their entireties

FIELD OF THE INVENTION

The present invention generally relates to methods and systems for generating infrared optical radiation in selected energies and dosimetries that will modify the bioenergetic steady-state trans-membrane and mitochondnal potentials of irradiated cells through a depolarization effect, and more particularly, relates to methods and systems for membrane depolarization to potentiate antibiotic compounds m bacteπal cells, and particularly antibiotic resistant bacterial cells

BACKGROUND OF THE INVENTION

The universal πse of bacteπa, fungi and other biological contaminants resistant to antimicrobial agents presents humanity with a grievous threat to its very existence Since the advent of sulfa drugs (sulfanilamide, first used in 1936) and penicillin (1942, Pfizer Pharmaceuticals), exploitation of significant quantities of antimicrobial agents of all kinds across the planet has created a potent environment for the materialization and spread of resistant contaminants and pathogens Certain resistant contaminants take on an extraordinary epidemiological significance, because of their predominance m hospitals and the general environment Widespread use of antibiotics not only prompts generation of resistant bacteπa, such as, for example, methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), but also creates favorable conditions for infection with the fungal organisms (mycosis), such as, Candida Given the increasing world's population and the prevalence of drug resistant bacteπa and fungi, the πse m incidence of bacterial or fungal infections is anticipated to continue unabated for the foreseeable future

Currently, available therapies for bacteπal infections include administration of antibacterial therapeutics or, in some instances, application of surgical debπdement of the mfected area Because antibacteπal therapies alone are rarely curative, especially in view of newly emergent drug resistant pathogens and the extreme morbidity of highly disfiguπng surgical therapies, it has been imperative to develop new strategies to treat or prevent microbial infections

Therefore, there exist a need for methods and systems that can reduce the risk of bacteπal mfections, in/at a given target site, without intolerable risks and/or intolerable adverse effects to the host organism (e g , mammalian tissues) other than the targeted microbial contaminants

SUMMARY OF THE INVENTION

The present invention provides an apparatus, systems and methods for microbial reduction using optical energy Specific near infrared wavelength ranges photodamage cell membranes, causing oxidative stress and membrane depolarization Bacteπa in the field of the optical beam are photodamaged m that ATP production is compromised, efflux pumps are inhibited, cell wall biosynthesis is disrupted, and the bacteria display increased sensitivity to antibiotics hi many cases, optical photodamage can reverse a drug resistance phenotype, permitting the (re)use of common antibiotics against even multiple drug resistance (MDR) sti ains

Accordingly, in a first aspect, the mvention mcludes a method of effectuating antimicrobial activity at a microbial colonization site m a subject, by applymg a redox modifying and membrane depolarizing dosage of near infrared energy to the site, the near infrared energy having a first wavelength of about 870 nm and a second wavelength of 930 nm, the dosage of near infrared energy being insufficient to cause thermolysis of subject tissues at the site, and applying one or more antimicrobial agents to the microbial colonization site, wherein at least a two-fold log reduction m microbial colonization is observed in the subject at the colonization site

In another aspect, the invention provides a method of inhibiting bacteπal viability at a microbial colonization site in a subject, by applying a peptidoglycan biosynthesis inhibiting dosage of near infrared energy to the site, the near infrared energy having a first wavelength of about 870 nm and a second wavelength of 930 nm, the dosage of near infrared energy being insufficient to cause thermolysis of subject tissues at the site, and applying one or more antimicrobial agents to the microbial colonization site wheπn at least one of the antimicrobial agents binds the active site of a bacteπal transpeptidase enzyme, wherein at least a two-fold log reduction m microbial colonization is observed m the subject at the colonization site In another aspect, the invention includes a method of inhibiting microbial viability at a microbial colonization site in a subject, comprising a) applymg a DNA replication and transcription inhibiting dosage of near infrared energy to the site, the near infrared energy having a first wavelength of about 870 nm and a second wavelength of 930 run, the dosage of near infrared energy being insufficient to cause thermolysis of subject tissues at the site, and b) applying one or more antimicrobial agents to the microbial colonization site, wherein at least a two-fold log reduction m microbial colonization is observed m the subject at the colonization site

Pn yet another aspect, the invention provides a method of reducing the number and viability of microbes at a microbial colonization site in a subject, comprising a) applymg a bacteπal phospholipid biosynthesis inhibiting dosage of near infrared energy to the site, the near infrared energy having a first wavelength of about 870 nm and a second wavelength of 930 nm, the dosage of near infrared energy being insufficient to cause thermolysis of subject tissues at the site, and b) applying one or more antimicrobial agents to the microbial colonization site, wherein at least a two-fold log reduction m microbial colonization is observed in the subject at the colonization site hi still yet another aspect, the invention provides a method of decontaminating an area of a subject, comprising a) identifying m or on a subject, a wound or infection site or a surgical location m need of a reduction m bacteπal colonization, b) applymg one or more photodamaging doses of optical radiation to the area without thermally damaging the area, c) applying an antimicrobial agent to the area hi another aspect diffuser tip assembly adapted to receive the distal end of optical fiber is disclosed The assembly includes a reflective cavity including a first reflector positioned proximal the distal end of the received fiber and including an aperture adapted to admit light emitted from the fiber into the cavity, and a second reflector positioned distal the first reflector The assembly also mcludes a diffuser tube positioned between the first and second reflectors about a cavity axis extending from the first reflector to the second reflector, the diffuser tube including an inner void surrounded by an outer portion including a diffusive scatteπng mateπal The cavity and diffuser tube are arranged such that at least a portion of light admitted into the cavity is scattered by the diffusive scattering material out of the tip assembly through the outer portion m a direction transverse to the cavity axis hi some embodiments, the cavity and diffuser tube are configured such that light admitted into the cavity is directed from the aperture towards the second reflector, a portion of the light directed towards the second reflector impinges upon the diffusive scatteπng mateπal and is scattered out of the tip assembly in a direction transverse to the axis, at least a portion of unscattered light impinges upon the second reflector and is reflected back towards the first reflector, and a portion of the light directed back towards the first reflector impinges upon the diffusive scatteπng mateπal and is scattered out of the tip assembly m a direction transverse to the axis In some embodiments,the cavity and diffuser tube are configured such that light admitted into the cavity travels multiple passes between the first and second reflectors, and on each pass, at least a portion of the light is scattered by the diffusive scatteπng mateπal out of the tip assembly m a direction transverse to the axis In some embodiments, the light scattered out of the tip assembly on each pass combine to produce a cumulative illumination pattern In some embodiments, the cumulative illumination pattern is characteπzed by substantially uniform axial intensity profile along at least a portion of the diffuser tube In some embodiments, the cumulative illumination pattern is characteπzed by a substantially uniform azimuthal illumination profile In some embodiments, the cumulative illumination pattern is characterized by substantially proscπbed illumination m the direction parallel to the axis In some embodiments,the cumulative illumination pattern is a substantially uniform cylindπcal illumination pattern emitted radially from the outer surface of the diffuser tube In some embodiments, the cumulative illumination pattern is determined by at least on at least one chosen from the list consistmg of a length of the diffuser tube, the diameter of the inner void of the diffuser tube, a numeπcal aperture associate with the aperture m the first reflector In some embodiments, the inner void is filled with a substantially transparent non-scatteπng mateπal

In some embodiments, the at least one of the first and second reflectors includes a curved reflector In some embodiments, the first reflector is a diffuse reflector and the second reflector is a specular reflector In some embodiments, the ratio of the distance between the first reflector and the second reflector along the cavity axis to the outer diameter of the diffusion tube is about 10 or less, about 1 or less, or even about 0 1 or less

In some embodiments, diffusive scatteπng material includes a plastic, a glass, a polymer, or a fluid In some embodiments, the diffusive scatteπng mateπal includes PTFE In some embodiments, the diffusive scatteπng mateπal is adapted to scatter light m the near infrared

In some embodiments,the reflective cavity and the diffuser tube are autoclavable Some embodiments include substantially transparent outer jacket adapted to contain the reflective cavity and the diffuser tube In some embodiments, the outer jacket is detachable from the reflective cavity and the diffuser tube The diffuser tip assembly of any of the preceding claims, where the tip assembly is adapted to scatter about 80% or more of the light delivered from the fiber while absorbmg about 20% or less of the light delivered from the fiber

In another aspect, a kit for treating an antimicrobial resistant biological contaminate at a treatment site is disclosed which includes a diffuser tip adapted to receive near infrared therapeutic light from a light delivery system and diffuse the light to illuminate at least a portion of the treatment site, a quantity of an antimicrobial agent, instructions to use the antimicrobial agent m conjunction with the therapeutic light to potentiate the antimicrobial agent to treat the biological contaminate, and suitable packaging In some embodiments, the diffuser tip is a diffuser tip assembly of the any of the types described herein

In some embodiments, the therapeutic light includes optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges In some embodiments,the diffuser tip assembly is adapted to provide substantially uniform illumination of the illuminated portion of the treatment site In some embodiments, the diffuser tip assembly is adapted to illuminate the illuminated portion of the treatment site at a power density and an energy density which potentiates the antimicrobial agent at the treatment site without causing substantial photothermal or photomechanical damage to the treatment site In some embodiments, the diffuser tip assembly is adapted to illuminate the illuminated portion of the treatment site at a power density of about 0 2 W/cm² to about 1 W/cm² and an energy density from about 100 J/cm² to about 400 J/cm² at the illuminated target region In some embodiments, the diffuser tip is adapted to operate for about 30 seconds or more at an operating temperature of 110° F or less

In some embodiments, the diffuser tip assembly is adapted for detachable connection to a distal end of an optical fiber which transmits the therapeutic light from a light source to the distal end of the fiber

In some embodiments, the quantity of antimicrobial agent includes a topical paste In some embodiments, the antimicrobial agent includes an antibiotic or a pharmacologically acceptable salt thereof, selected from the group consisting of /3-lactams, glycopeptides, cyclic polypeptides, macrolides, ketohdes, amlmouracils, lmcosamides, chloramphenicols, tetracyclines, aminoglycosides, bacitracins, cefazolms, cephalosporins, muprrocms, nitroimidazoles, qumolones and fluoroquinolones, novobiocins, polymixms, catiomc detergent antibiotics, oxazohdmones or other heterocyclic organic compounds, glycylcyclmes, hpopeptides, cyclic lipopeptides, pleuromutilms, and gramicidins, daptomycins, lmezolids, ansamycms, carbacephems, carbapenems, monobactams, platensimycms, streptogramms and tmidazoles

In some embodiments, the antimicrobial agent is ineffective for treating the antimicrobial resistant biological contaminate in the absence of the therapeutic light In some embodiments, the resistant biological contaminate includes MRSA or MSSA

In some embodiments, the biological contaminate includes an aberrant microbial colonization, and where the instructions mclude instruction to use the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to reduce the level of colonization at the treatment site In some embodiments, the an aberrant microbial colonization, prior to application of the treatment light, has a drug resistant phenotype with respect to the antimicrobial agent

In some embodiments, the diffuser tip is sterilized

In another aspect, q therapeutic system for treatment of a biological contaminant at a treatment site is disclosed including an optical radiation generation device configured and arranged to generate near infrared therapeutic light, a controller operatively connected to the optical radiation generation device for controlling dosage of the therapeutic light transmitted to the treatment site at a dosimetry sufficient to produce photodamage m the biological contaminant without causing substantial photothermal or photomechanical damage to biological tissue at the treatment site, a delivery assembly including an optical fiber which directs the therapeutic light to be transmitted to the treatment site, and a diffusei tip adapted to receive the therapeutic light from the delivery assembly and diffuse the therapeutic light to illuminate at least a portion of the treatment site with a prescribed illumination pattern

In some embodiments, the diffuser tip is the diffuser tip of any of the types described herein In some embodiments, where the therapeutic light includes optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges

In some embodiments, the diffuser tip assembly is adapted to provide substantially uniform illumination of at least a portion of the illuminated portion of the treatment site In some embodiments, the diffuser tip assembly is adapted to illuminate the portion of the treatment site at a power density and an energy density which potentiates an antimicrobial application at the treatment site In some embodiments, the diffuser tip assembly is adapted to illuminate the portion of the treatment site at a power density of about 0 3 W/cm² to about 7 W/cm² and an energy density from about 100 J/cm² to about 400 J/cm² at the illuminated portion of the treatment site In some embodiments, the diffuser tip is adapted to operate for about 30 seconds or more at an operating temperature of 110° F or less

In some embodiments, the diffuser tip assembly is adapted for detachable connection to a distal end of the optical fiber

In another aspect, a method treatment of a biological contaminant at a treatment site is disclosed which includes generating near infrared therapeutic light, controlling dosage of the therapeutic light transmitted to the treatment site at a dosimetry sufficient to produce photodamage in the biological contaminant without causing substantial photothermal or photomechanical damage to biological tissue at the treatment site, directing the therapeutic light to be transmitted to the treatment site, and using a diffuser tip, diffusing the therapeutic light to illuminate at least a portion of the treatment site with a prescribed illumination pattern

In some embodiments, the diffuser tip is a diffuser tip assembly of any of the types descπbed herein

In some embodiments, the therapeutic light includes optical radiation substantially m a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges

In some embodiments, the diffusing the therapeutic light includes providing substantially uniform illumination of at least a portion of the illuminated portion of the treatment site

Some embodiment further πnclude applying a quantity of antimicrobial agent the treatment site, and illuminating the portion of the treatment site at a power density and an energy density which potentiates the antimicrobial application

In some embodiments, the antimicrobial agent is ineffective for treating the antimicrobial resistant biological contammate in the absence of the therapeutic light In some embodiments, the antimicrobial agent is ineffective for treating the biological contaminate in the absence of the therapeutic light In some embodiments, the biological contaminate mcludes an aberrant microbial colonization, and further including includes using the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to reduce the level of colonization at the treatment site hi some embodiments, the aberrant microbial colonization, pnor to application of the treatment light, has a drug resistant phenotype with respect to the antimicrobial agent In some embodiments, controlling the dosage of the therapeutic light includes controlling the illumination of the portion of the treatment site at a power density of about 0.2 W/cm² to about 1 W/cm² and an energy density from about 100 J/cm²to about 400 J/cm² at the illuminated region. Exemplary antimicrobial agents that are appropriate for use in conjunction with optical photodamage to reduce bacterial counts include common antibiotics and pharmacologically acceptable salt thereof, including /3-lactams, glycopeptides, cyclic polypeptides, macrolides, ketolides, anilinouracils, lincosamides, chloramphenicols, tetracyclines, aminoglycosides, bacitracins, cefazolins, cephalosporins, mupirocins, nitroimidazoles, quinolones and fluoroquinolones, novobiocins, polymixins, cationic detergent antibiotics, oxazolidinones or other heterocyclic organic compounds, glycylcyclines, lipopeptides, cyclic lipopeptides, pleuromutilins, and gramicidins, daptomycins, linezolids, ansamycins, carbacephems, carbapenems, monobactams, platensimycins, streptogramins and tinidazoles. Various embodiments may include any of the features of techniques described above, either alone, or in any combination.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention may more fully be understood from the following description when read together with the accompanying drawings, which are to be regarded as illustrative in nature, and not limiting. The drawings are not necessarily to scale, emphasis instead being placed on the principles of the invention. In the drawings:

Figure 1 shows a typical phospholipid bilayer;

Figure 2 shows the chemical structure of a phospholipid;

Figure 3 shows dipole effects in phospholipid bilayer membranes ("^d);

Figure 4A shows a phospholipid bilayer in bacterial plasma membrane, mammalian mitochondrial membrane, or fugal mitochondrial membrane with a steady-state transmembrane potential prior to NIMELS irradiation. Figure 4B shows a transient-state plasma membrane potential in bacterial plasma membrane, mammalian mitochondrial membrane, or fugal mitochondrial membrane after NlMELS irradiation;

Figure 5 shows a phospholipid bilayer with trans-membrane proteins embedded therein; Figure 6 shows a general depiction of electron transport and proton pump;

Figure 7 shows the effects of NlMELS irradiation (at a single dosimetry) on MRSA trans-membrane potential which is measured by green fluorescence emission intensities in control and lased samples as a function of time in minutes post-lasing;

Figure 8 shows the effects of NIMELS irradiation (at a single dosimetry) on mitochondrial membrane potential of human embryonic kidney cells, which is measured by red fluorescence emission intensities in control and lased samples; and the effects of NIMELS irradiation (at a single dosimetry) on mitochondrial membrane potential of human embryonic kidney cells, which is measured as ratio of red to green fluorescence in control and lased samples;

Figure 9 shows the reduction in total glutathione concentration in MRSA as it correlates with reactive oxygen species (ROS) generation in these cells as the result of NIMELS irradiation (at several dosimetries); the decrease in glutathione concentration in lased samples is shown as percentage relative to the control;

Figure 10 shows the reduction in total glutathione concentration in human embryonic kidney cells as it correlates with reactive oxygen species (ROS) generation in these cells as the result of NIMELS irradiation (at two different dosimetries); the decrease in glutathione concentration in lased samples is shown as percentage relative to the control;

Figure 11 shows the synergistic effects of NIMELS and methicillin in growth inhibition of MRSA colonies; data show methicillin is being potentiated by sub-lethal NIMELS dosimetry; and

Figure 12 shows the synergistic effects of NIMELS and bacitracin in growth inhibition of MRSA colonies; arrows indicate the growth or a lack thereof of MRSA colonies in the two samples shown; images show that bacitracin is being potentiated by sub- lethal NIMELS dosimetry.

Figure 13 shows a bar chart depicting the synergistic effects, as indicated by experimental data, of NIMELS with methicillin, penicillin and erythromycin in growth inhibition of MRSA colonies.

Figure 14 illustrates the detection of decreased membrane potential in E. coli with sub-lethal NIMELS irradiation. Figure 15 illustrates the detection of increased glutathione in E. coli with sub-lethal NMELS irradiation.

Figure 16a illustrates five subjects initially culturing positive for erythromycin resistant MSSA, all showing positive responses to phototherapy. Figure 16b illustrates three subjects initially culturing positive for erythromycin resistant MSRA, all showing positive responses to phototherapy.

Fig. 17 illustrates an exemplary NEVIELS treatment system.

Figs. 18a-18d illustrate the delivery of treatment light from a NIMELS treatment system.

Fig. 19 is a perspective illustration of a diffusive fiber tip device.

Fig. 20 is a cross sectional representation of a diffusive fiber tip device that shows how the light emitted from a fiber optic initially interacts with a diffusion tube without the mirrors that form the reflective cavity.

Fig. 21 is a perspective illustration of the operation of the diffusing tube of Fig. 20 with the addition of the cavity mirrors.

Fig. 22 is a cross sectional illustration of a curved mirror embodiment of a diffusive fiber tip assembly.

Fig. 23 shows intensity profiles observed from the operation of the diffusive tip assembly.

Fig. 24 is a cross sectional illustration of an exemplary construction of a curved mirror embodiment of the diffusive tip assembly.

Fig. 25 is a cross sectional illustration of an exemplary construction of the diffusive reflector embodiment of the diffusive fiber tip assembly.

Fig. 26 is a drawing of a reusable diffusion tip encased in a disposable polypropylene outer jacket.

Fig 27 illustrates several exemplary embodiments of diffusion tips.

Fig 28 is an intensity CCD camera scan of a diffusion tip. Fig 29 is an illustration of a diffusion tip

Fig 30 is an illustration of a kit including a diffusion tip

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms "a", "an" and "the" also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise For example, reference to "a NIMELS wavelength" includes any wavelength within the ranges of the NTMELS wavelengths descπbed, as well as combinations of such wavelengths

The present invention is directed to methods and systems for enhancing bacterial succeptability to antimicrobial agents thereby reducmg the minimum inhibitory concentration (MIC) of the antimicrobial agent necessary to attenuate or eliminate microbial related pathology and/or enabling therapeutic use of antimicrobial agents that would otherwise be ineffective due to bactenal resistance According to methods and systems of the present invention, near mfrared optical radiation in selected energies and dosimetries (herein known as NIMELS, standing for "near mfrared microbial elimination system") are used to cause a depolarization of membranes withm the irradiated field, that will alter the absolute value of the membrane potential (ΔΨ) of the irradiated cells

This altered ΔΨ will cause an affiliated weakening of the proton motive force Δp, and the bioenergetics of all affected membranes Accordingly, the effects of NIMELS irradiation (NTMELS effect) can potentiate existing antimicrobial agents against microbes infecting and causing harm to human or animal hosts These NTMELS effects will affect many cellular anabolic reactions (e g , cell wall formation) and drug-resistance mechanisms (e g , efflux pumps) that require chemiosmotic electrochemical energy to function Hence, any membrane bound cellular resistance mechanisms or anabolic reactions that makes use of the membrane potential ΔΨ, proton motive force Δp, or the phosphorylation potential ΔGp for their functional energy needs, will be affected by the NTMELS effects, and accordingly provide therapeutic targets for the methods and systems of the present invention

The methods and systems of the present invention utilize optical radiation to sensitize undesirable microbial cells (e g , MRSA infection m skm) without substantial thermal or chemical damage to host tissues

In exemplary embodiments, the applied optical radiation used m accordance with methods and systems of the present invention includes one or more, and preferably two independent wavelengths ranging from about 850 nm to about 900 nm, at a NTMELS dosimetry, as described herein In one aspect, wavelengths from about 865 nm to about 875 nm are utilized In another aspect, such applied radiation has a wavelength from about 905 nm to about 945 nm at a NIMELS dosimetry In one aspect, such applied optical radiation has a wavelength from about 925 nm to about 935 nm In a particular aspect, a wavelength of (or narrow wavelength range including) 930 nm can be employed In some aspects of the present invention, multiple wavelength ranges include 870 and 930 nm, respectively

In one embodiment, the methods and systems of the present invention are used m treating, reducing and/or eliminating the infectious entities known to cause cutaneous or wound infections such as staphyloccocci and enterococci Staphyloccoccal and enterococcal infections can involve almost any skm surface on the body, and is known to cause numerous skm conditions such as boils, carbuncles, bullous impetigo and scalded skm syndrome Accordingly, one objective of the invention is to prevent or treat staphyloccoccal and enterococcal infections of the host skm, thereby treating the aforementioned conditions S aureus is also the cause of staphylococcal food poisoning, enteπtis, osteomilitis, toxic shock syndrome, endocarditis, meningitis, pneumonia, cystitis, septicemia and post- operative wound infections Accordingly, another objective of the invention is to prevent or treat such infections S aureus can be acquired while a patient is m a hospital or long- term care facility, and yet another object of the invention is to prevent or treat nosocomial infections m the host

The widespread use of antibiotics have led to the development of antibiotic-resistant strains of S aureus These strains are called methicillrn resistant staphylococcus aureus (MRSA) Infections caused by MRSA are frequently resistant to a wide vaπety of antibiotics (especially jS-lactams) and are associated with significantly higher rates of morbidity and mortality, higher costs, and longer hospital stays than mfections caused by non-MRSA microorganisms Risk factors for MRSA infection in the hospital include colonization of the nares, surgery, prior antibiotic therapy, admission to intensive care, exposure to a MRSA-colonized patient or health care worker, being m the hospital more than 48 hours, and having an indwelling catheter or other medical device that goes through the skm Thus, a further object of the invention is to prevent or treat drug resistant bacteπal infections of the host, preferably but not limited to MRSA mfections of the host The term "NIMELS dosimetry" denotes the power density (W/cm²) and the energy density (J/cm²) (where 1 Watt = 1 Joule per second) values at which a subject wavelength according to the invention is capable of generating a reactive oxygen species ("ROS") and thereby reduce the level of a biological contaminant m a target site The term also includes irradiating a cell to increase the sensitivity of the biological contaminant through the loweπng of ΔΨ with the concomitant generation of ROS of an antimicrobial or antineoplastic agent, wherein the contaminant is resistant to the agent otherwise This method can be effected without intolerable risks and/or intolerable side effects on the host subject's tissue other than the biological contaminant

By "potentiation" of an antibacterial agent, it is meant that the methods and systems of this invention counteract the resistance mechanisms m the microbe sufficiently for the agent to inhibit the growth and/or proliferation of said microbe at a lower concentration than in the absence of the present methods and systems In cases where resistance is essentially complete, i e , the agent has no apparent bacteπostatic or bacteriocidal effect on the microbial cells, potentiation means that the agent will inhibit the growth and/or proliferation of pathogenic cells at a therapeutically acceptable dosage, thereby treating the disease state

As used herein, the term "Membrane Dipole Potential Ψά" (m contrast to the Transmembrane Potential ΔΨ) refers to the potential formed between the highly hydrated lipid heads (hydrophihc) at the membrane surface and the low polar interior of the bilayer (hydrophobic) Lipid bilayers intrinsically possess a substantial Membrane Dipole Potential Ψd arising from the structural organization of dipolar groups and molecules, primarily the ester linkages of the phospholipids and water

Ψd does not depend upon the ions at the membrane surface and will be used herein to describe five different dipole potentials 1) Mammalian Plasma Membrane Dipole Potential Ψd-plas-mam, 2) Mammalian Mitochondrial Membrane Dipole Potential Ψd-mito-mam,

3) Fungal Plasma Membrane Dipole Potential Ϋd-plas-fungi,

4) Fungal Mitochondrial Membrane Dipole Potential Ψd-mito-fungi, and

5) Bacteπal Plasma Membrane Dipole Potential Ψd-plas-bact

As used herein, the term "Trans-Membrane Potential" refers to the electπcal potential difference between the aqueous phases separated by a membrane (dimensions mV) and will be given by the symbol (ΔΨ) ΔΨ does depend upon the ions at the membrane surface and will be used herein to describe three different plasma trans-membrane potentials

1) Mammalian Plasma Trans-Membrane Potential ΔΨ-plas-mam 2) Fungal Plasma Trans-Membrane Potential ΔΨ-plas-fungi

3) Bacterial Plasma Trans-Membrane Potential ΔΨ-plas-bact

As used herein, the term "Mitochondrial Trans-Membrane Potential" refers to the electπcal potential difference between the compartments separated by the mitochondrial inner membrane (dimensions mV) and will be used herein to describe two different mitochondrial trans-membrane potentials 1) Mammalian Mitochondrial Trans-Membrane Potential ΔΨ-mito-mam

2) Fungal Mitochondrial Trans-Membrane Potential ΔΨ-mito-fungi

As used herem, the term "bacterial plasma trans-membrane potential (ΔΨ-plas- bact)" refers to the electπcal potential difference m the bacteπal cell plasma membrane The bacterial plasma membrane potential is generated by the steady-state flow

(translocation) of electrons and protons (H⁺) across the bacteπal plasma membrane that occurs with normal electron transport and oxidative phosphorylation, within the bacteπal plasma membrane A common feature of all electron transport chains is the presence of a proton pump to create a transmembrane proton gradient Although bacteria lack mitochondπa, aerobic bacteria carry out oxidative phosphorylation (ATP production) by essentially the same process that occurs in eukaryotic mitochondπa

As used herem, the term "P-class ion pump" refers to a trans-membrane active transport protem assembly which contains an ATP-bmding site (ι e , it needs ATP to function) Dunng the transport process, one of the protem subunits is phosphorylated, and the transported ions are thought to move through the phosphorylated subunit This class of ion pumps includes the Na⁺/K⁺-ATPase pump m the mammalian plasma membrane, which maintains the Na⁺ and K⁺ electrochemical potential (ΔNa⁺/K⁺) and the pH gradients typical of ammal cells Another important member of the P-class ion pumps, transports protons (H⁺ ions) out of and K⁺ ions in to the cell As used herem, the term "steady-state plasma trans-membrane potential (ΔΨ- steady)" refers to the quantitative Plasma Membrane Potential of a mammalian, fungal or bactenal cell before irradiation in accordance with the methods and systems of the present invention that would continue into the future in the absence of such irradiation For example, the steady-state flow of electrons and protons across a bacteπal cell membiane that occurs during normal electron transport and oxidative phosphorylation would be in a steady- state due to a constant flow of conventional redox reactions occurπng across the membrane Conversely any modification of this redox state would cause a transient-state membrane potential ΔΨ-steady will be used herein to descnbe three (3) different steady-state plasma trans-membrane potentials, based on species 1) Steady-state mammalian plasma trans-membrane potential ΔΨ-steady-mam

2) Steady-state fungal plasma trans-membrane potential ΔΨ-steady-fungi

3) Steady-state bacterial plasma trans-membiane potential ΔΨ-steady-bact

As used herein, the term "Transient-state plasma membrane potential (ΔΨ-tran)" refers to the Plasma Membrane Potential of a mammalian, fungal or bacteπal cell after irradiation m accordance with the methods and systems of the present invention whereby the irradiation has changed the bioenergetics of the plasma membrane In a bacteria, ΔΨ-tran will also change the redox state of the cell, as the plasma membrane is where the ETS and cytochromes reside ΔΨ-tran is a state that would not occur without irradiation using methods of the present invention ΔΨ-tran will be used herein to descπbe three (3) different Transient-state plasma trans-membrane potentials based on species

1 ) Transient-state mammalian plasma trans-membrane potential ΔΨ-tran-mam

2) Transient-state fungal plasma trans-membrane potential ΔΨ-tran-fungi

3) Transient -state bacteπal plasma trans-membrane potential ΔΨ-tran-bact

As used herem, the term "steady-state mitochondrial membrane potential (ΔΨ- steady-mito)" refers to the quantitative Mitochondrial Membrane Potential of mammalian or fungal mitochondria before irradiation in accordance with the methods and systems of the present invention that would continue into the future, in the absence of such irradiation For example, the steady-state flow of electrons and protons across mitochondπal inner membrane that occurs during normal electron transport and oxidative phosphorylation would be in a steady-state because of a constant flow of conventional redox reactions occurring across the membrane Any modification of this redox state would cause a transient-state mitochondπal membrane potential ΔΨ-steady-mito will be used herein to descπbe two (2) different steady-state mitochondrial membrane potentials based on species 1) Steady-state mitochondrial mammalian potential ΔΨ-steady-mito-mam 2) Steady-state mitochondrial fungal potential ΔΨ-steady-mito-fungi

As used herein, the term "transient-state mitochondrial membrane potential (ΔΨ- tran-mito-mam oi ΔΨ-tran-mito-fungi)" refers to the membrane potential of a mammalian or fungal cell after irradiation m accordance with the methods and systems of the present invention whereby the irradiation has changed the bioenergetics of the mitochondrial inner membrane In mammalian and fungal cells, ΔΨ-tran-mito will also change the redox state of the cell, as the inner mitochondrial membrane is where the electron transport system (ETS) and cytochromes reside ΔΨ-tran-mito could also drastically affect (the Proton- motive force) Δp-mito-mam and Δp-mito-fungi, as these mitochondrial (H⁺) gradients are generated in the mitochondria, to produce adequate ATP for a myriad of cellular functions ΔΨ-tran-mito is a state that would not occur without irradiation m accordance with methods and systems of the present invention ΔΨ-tran-mito will be used herein to descπbe two (2) different transient-state mitochondrial membrane potentials based on species

1) Transient-state mitochondrial mammalian potential ΔΨ-tran-mito-mam

2) Transient-state mitochondrial fungal potential ΔΨ-tran-mito-fungi As used herein, the term "proton electrochemical gradient" (ΔμHT) (dimensions kJ mol-1) refers to the electncal and chemical properties across a membrane, particularly proton gradients, and represents a type of cellular potential energy available for work in a cell This proton electrochemical potential difference between the two sides of a membrane that engage m active transport involving proton pumps, is at times also called a chemiosmotic potential or proton motive force When Δμl-T is reduced by any means, it is a given that cellular anabolic pathways and resistance mechanisms m the affected cells are inhibited This can be accomplished by combining λn and Tn to irradiate a target site alone, or can be further enhanced with the simultaneous or sequential administration of a pharmacological agent configured and arranged for delivery to the target site (i e , the co- targetmg of an anabolic pathway with (λn and Tn) + (pharmacological molecule or molecules))

As used herein, the term "Ion Electrochemical Gradient (Δμx+)" refers to the electncal and chemical properties across a membrane caused by the concentration gradient of an ion (other than H⁺) and represents a type of cellular potential energy available for work m a cell In mammalian cells, the Na⁺ ion electrochemical gradient is maintained across the plasma membrane by active transport OfNa⁺ out of the cell This is a different gradient than the proton electrochemical potential, yet is generated from an ATP coupled pump, said ATP produced duπng oxidative phosphorylation from the mammalian mitochondπal proton-motive force (Δp-mito-marri) When Δμx⁺ is reduced by any means, it is a given that cellular anabolic pathways and resistance mechanisms in the affected cells are inhibited This can be accomplished by combining λn and Tn to irradiate a target site alone, or can be further enhanced with the simultaneous or sequential administration of a pharmacological agent configured and arranged for delivery to the target site (i e , the co- targeting of an anabolic pathway with (λn and Tn) + (pharmacological molecule or molecules)

As used herein, the term "co-targeting of a bacteπal anabolic pathway" refers to (the λn and Tn loweπng of (Δμl-T) and/oi (Δμx⁺) of cells at the target site to affect an anabolic pathway) + (a pharmacological molecule or molecules to affect the same bacteπal anabolic pathway) and can refer to any of the following bacteπal anabolic pathways that are capable of being inhibited with pharmacological molecules wherein the targeted anabolic pathway is peptidoglycan biosynthesis that is co-targeted by a pharmacological agent that binds at the active site of the bacteπal transpeptidase enzymes (penicillin binding proteins) which cross-links peptidoglycan in the bacteπal cell wall Inhibition of these enzymes ultimately cause cell lysis and death, wherein the targeted bacterial anabolic pathway is peptidoglycan biosynthesis that is co-targeted by a pharmacological agent that binds to acyl-D-alanyl-D- alanine groups in cell wall intermediates and hence prevents incorporation of N- acetylmuramic acid (NAM)- and N-acetylglucosamme (NAG)-peptide subunits into the peptidoglycan matrix (effectively inhibiting peptidoglycan biosynthesis by acting on transglycosylation and/or transpeptidation) thereby preventing the proper formation of peptidoglycan, in gram-positive bacteπa, wherein the targeted bacteπal anabolic pathway is peptidoglycan biosynthesis that is co-targeted by a pharmacological agent that binds with C₅₅-isoprenyl pyrophosphate and prevents pyrophosphatase from interacting with C₅₅- isoprenyl pyrophosphate thus reducing the amount of C₅₅-isoprenyl pyrophosphate that is available for carrying the building blocks peptidoglycan outside of the inner membrane, wherein the targeted anabolic pathway is bacteπal protein biosynthesis that is co-targeted by a pharmacological agent that binds to the 23S iRNA molecule m the subunit 5OS subumt of the bacterial πbosome, causing the accumulation of peptidyl-tRNA in the cell, hence depleting the free tRNA necessary for activation of α-ammo acids, and inhibiting transpeptidation by causmg premature dissociation of peptidyl tRNA from the πbosome, wherein the co-targeted pharmacological agent binds simultaneously to two domains of 23 S RNA of the 50 S bacteπal πbosomal subunit, and can thereby inhibit the formation of the bacteπal πbosomal subunits 5OS and 3OS (πbosomal subunit assembly), wherein the co- targeted pharmacological agent is chlorinated to increases its lipophilicity to penetrate into bacteπal cells, and binds to the 23S portion of the 5OS subunit of bacteπal πbosomes and prevents the translocation of the peptidyl -tRNA from the Ammoacyl site (A-site) to the Peptidyl site (P-site) thereby inhibiting the transpeptidase reaction, which results in an incomplete peptide being released from the πbosome, wherein the targeted anabolic pathway is bacteπal protem biosynthesis that is co-targeted by pharmacological agent that binds to the 3OS bacteπal πbosomal subunit and blocks the attachment of the ammo-acyl tRNA from binding to the acceptor site (A-site) of the πbosome, thereby inhibiting the codon-anticodon interaction and the elongation phase of protein synthesis, wherein the co- targeted pharmacological agent binds more avidly to the bacterial πbosomes, and in a different oπentation from the classical subclass of polyketide antimicrobials having an octahydrotetracene-2-carboxamide skeleton, so that they are active against strains of S aureus with a tet(M) πbosome and tet(K) efflux genetic determinant, wherein the targeted anabolic pathway is bacteπal protein biosynthesis that is co-targeted by a pharmacological agent that bmds to a specific aminoacyl-tRNA synthetase to prevent the esteπfϊcation of a specific ammo acid or its precursor to one of its compatible tRNA's, thus preventing formation of an ammoacyl-tRNA and hence halting the incorporation of a necessary ammo acid into bacterial proteins, wherein the targeted anabolic pathway is bacteπal protein biosynthesis that is co-targeted by a pharmacological agent that inhibits bacteπal protein synthesis before the initiation phase, by binding the 5OS rRNA through domain V of the 23S rRNA, along with interacting with the 16S rRNA of the 3OS πbosomal subunit, thus preventing binding of the initator of protein synthesis formyl -methionine (f-Met-tRNA), and the 30S nbosomal subunit, wherein the targeted anabolic pathway is bacterial protein biosynthesis that is co-targeted by a pharmacological agent that interacts with the 5OS subunit of bacterial πbosomes at protem L3 in the region of the 23 S rRNA P site near the peptidyl transferase center and hence inhibits peptidyl transferase activity and peptidyl transfer, blocks P-site interactions, and prevents the normal formation of active 50S nbosomal subumts, wherein the targeted anabolic pathway is DNA replication and transcription that is co-targeted by a pharmacological agent that inhibits Topoisomerase II (DNA gyrase) and/or Topoisomerase IV, wherem the targeted anabolic pathway is DNA replication and translation that is co-targeted by a pharmacological agent that inhibits DNA polymerase IHC, the enzyme required for the replication of chromosomal DNA in gram- positive bacteπa, but not present in gram-negative bacteπa, wherein the targeted anabolic pathway is DNA replication and transcπption that is co-targeted by a pharmacological hybird compound that inhibits Topoisomerase II (DNA gyrase) and/or Topoisomerase FV and/or DNA polymerase IIIC, wherein the targeted anabolic pathway is bacterial phospholipid biosynthesis that is co-targeted by a topical pharmacological agent that acts on phosphatidylethanolamme-nch cytoplasmic membranes and works well m combination with other topical synergistic agents, wherein the targeted anabolic pathway is bacterial fatty acid biosynthesis that is co-targeted by a pharmacological agent that inhibits bacteπal fatty acid biosynthesis through the selective targeting of /3-ketoacyl-(acyl-carπer-protem (ACP)) synthase MI (FabF/B), an essential enzymes m type II fatty acid synthesis, wherein the targeted anabolic pathway is maintenance of bacteπal plasma trans-membrane potential ΔΨ-plas-bact and the co-targeting pharmacological agent disrupts multiple aspects of bacteπal cell membrane function on its own, by binding pπmaπly to gram positive cytoplasmic membranes , not penetrating into the cells, and causing depolaπzation and loss of membrane potential that leads to inhibition of protem, DNA and RNA synthesis, wherein the co-targeting pharmacological agent increases the permeability of the bacterial cell wall, and hence allows inorganic cations to travel through the wall m an unrestπcted manner thereby destroying the ion gradient between the cytoplasm and extracellular environment, wherein the targeted anabolic pathway is maintenance of bacterial membrane selective permeability and bacterial plasma trans-membrane potential ΔΨ-plas-bact, and the co- targeting pharmacological agent is a cationic antibacteπal peptide that is selective for the negatively charged surface of bacteπal membranes relative to the neutral membrane surface of eukaryotic cells and leads to prokaryotic membrane permeabhzation and ultimate perforation and/or disintegration of bacteπal cell membranes, thereby promoting leakage of bacteπal cell contents and a breakdown of the transmembrane potential, wherein the co- targeting pharmacological agent inhibits bacteπa protease Peptide Deformylase, that catalyzes the removal of formyl groups from the N-termim of newly synthesized bacteπal polypeptides, and wherem the co-targeting pharmacological agent inhibits two-component regulatory systems m bacteria, such as the ability to respond to their environment through signal transduction across bacterial plasma membranes, these signal transduction processes being absent m mammalian membranes

As used herein, the term "proton-motive force (Ap)" refers to the stoπng of energy (acting like a kind of battery), as a combination of a proton and voltage gradient across a membrane The two components of Ap are ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺) Stated another way, Ap consists of the H⁺ transmembrane potential ΔΨ (negative (acidic) outside) and a transmembrane pH gradient ΔpH (alkaline mside) This potential energy stored m the form of an electrochemical gradient, is generated by the pumping of hydrogen ions across biological membranes (mitochondrial inner membranes or bacteπal and fungal plasma membranes) during chemiosmosis The Ap can be used for chemical, osmotic, or mechanical work m the cells The proton gradient is generally used in oxidative phosphorylation to dπve ATP synthesis and can be used to drive efflux pumps in bacteπa, fungi, or mammalian cells including cancerous cells Ap will be used herein to describe four (4) different proton motive forces m membranes, based on species, and is mathematically defined as (ΔP = ΔΨ + ΔpH) 1) Mammalian Mitochondπal Proton-motive force (Ap-mito-mam)

2) Fungal Mitochondπal Proton-motive force (Ap-mιto-Fungι)

3) Fungal Plasma Membrane Proton-motive force (Ap-plas-Fungι)

4) Bacteπal Plasma Membrane Proton-motive force (Ap-plas-Bact)

As used herein, the term "Bacteπal Plasma Membrane Proton-motive force (Δp- plas-Bact)" refers to the potential energy stored in the form of an electrochemical gradient (H⁺), across a bactenal plasma membrane, and is generated by the pumping of hydrogen ions across the plasma membrane duπng chemiosmosis Δp-plas-Bact is used m oxidative phosphorylation to dπve ATP synthesis m the bacteπal plasma membrane and can be used to dπve efflux pumps m bacterial cells As used herein, the term "phosphorylation potential (ΔGp)" refeis to the ΔG for ATP synthesis at any given ATP, ADP and Pi concentrations (dimensions kJ mol ')

As used herem the term "CCCP" refers to carbonyl cyanide m- chlorophenylhydrazone, a highly toxic ionophore and uncoupler of the respiratory chain CCCP increases the conductance of protons through membranes and acts as a classical uncoupler by uncoupling ATP synthesis from the Aμtt and dissipating both the ΔΨ and ΔpH

As used herein, the term "Reactive Oxygen Species", includes one of the following categoπes a) The Superoxide ion radical (O₂ ) b) Hydrogen Peroxide (non-radical) (H₂O₂) c) Hydroxyl radical (*OH) d) Hydroxy ion (OH )

These ROS generally occur through the reaction chain O₂ ^ O₂ + 2H+ ^ H₂O₂ ^ OH + *0H ^ OH (e-) (e-) (e-) (e-)

As used herein, the term "singlet oxygen" refers to ("1O₂") and is formed via an interaction with tπplet-excited molecules Smglet oxygen is a non-radical species with its electrons m anti-parallel spins Because smglet oxygen 10₂ does not have spin restriction of its electrons, it has a very high oxidizing power and is easily able to attack membranes (e g , via polyunsaturated fatty acids, or PUFAs) ammo acid residues, protein and DNA

As used herein, the term "NIMELS effect" refers to the modification of the bioenergetic "state" of irradiated cells at the level of the cell's plasma and mitochondrial membranes from ΔΨ-steady to ΔΨ-trans with the present invention Specifically, the NTMELS effect can weaken cellular anabolic pathways or antimicrobial and/or cancer resistance mechanisms that make use of the proton motive force or the chemiosmotic potential for their energy needs

As used herein, the term "peπplasmic space or periplasm" refers to the space between the plasma membrane and the outer membrane in gram-negative bacteπa and the space between the plasma membrane and the cell wall in gram-positive bacteπa and fungi such as the Candida and Trichophyton species This peπplasmic space is involved m various biochemical pathways including nutπent acquisition, synthesis of peptidoglycan, electron transport, and alteration of substances toxic to the cell In gram-positive bacteria like MRSA, the peπplasmic space is of significant clinical importance as it is where β- lactamase enzymes inactivate penicillin based antibiotics As used herein, the term "efflux pump" refers to an active transport protein assembly which exports molecules from the cytoplasm or peπplasm of a cell (such as antibiotics, antifungals, or poisons) for their removal from the cells to the external environment in an energy dependent fashion As used herein, the term "efflux pump inhibitor" refers to a compound or electromagnetic radiation delivery system and method which mterferes with the ability of an efflux pump to export molecules from a cell In particular, the efflux pump inhibitor of this invention is a form of electromagnetic radiation that will interfere with a pump's ability to excrete therapeutic antibiotics, anti-fungal agents, antineoplastic agents and poisons from cells via a modification of the ΔΨ-steady-mam , ΔΨ-steady-fungi or, ΔΨ-steady-bact

By a cell that "utilizes an efflux pump resistance mechanism," it is meant that the bacteπal cell exports anti-bacterial agents from their cytoplasm or peπplasm to the external environment of the cell and thereby reduce the concentration of these agents m the cell to a concentration below what is necessary to inhibit the growth and/or proliferation of the bacteπal cells

As used herein, the term "anti-bacterial molecule (or agent)" refers to a chemical or compound that is bacteπacidal or bacteπastatic Another pπncipal efficacy resides m the present invention's ability to potentiate anti-bacteπal molecules by inhibiting efflux pump activity in resistant bactenal strains, or inhibiting anabolic reactions and/or resistance mechanisms that require the proton motive force or chemiosmotic potential for energy

As used herein, a "sub-inhibitory concentration" of an antibacteπal agent refers to a concentration that is less than that required to inhibit a majoπty of the target cells m the population Generally, a sub-inhibitory concentration refers to a concentration that is less than the Minimum Inhibitory Concentration (MIC) As used herein, the term "Minimal Inhibitory Concentration" or MIC is defined as the lowest effective or therapeutic concentration that results m inhibition of growth of the microorganism The minimum inhibitory concentration (MIC) of an antibacteπal agent is therefore the maximum dilution of the agent that will still inhibit the growth of a test microorganism The minimum bactericidal concentration (MBCs) of an antibacteπal agent is the lowest concentration of the antimicrobial agent that will prevent the growth of an organism after subculture on to antibiotic-free media The minimum lethal concentration (MLC) of an antibacteπal agent is the maximum dilution of the product that will kill the test organism MIC/MLC values can be determined by a number of standard test procedures The most commonly employed methods are the tube dilution method and agar dilution methods Serial dilutions are made of the products m bacteπal growth media The test organisms are then added to the dilutions of the products, incubated, and scored for growth This procedure is a standard assay for antimicrobials The procedure incorporates the content and intent of the American Society for Microbiology (ASM) recommended methodology As used herein, the term "therapeutically effective amount" of an antibacterial agent refers to a concentration of an agent that will partially or completely relieve one or more of the symptoms caused by the target (pathogenic) cells In particular, a therapeutically effective amount refers to the amount of an agent that (1) reduces, if not eliminates, the population of target microbial cells m the patient's body, (2) inhibits (i e , slows, if not stops) proliferation of the target microbial cells m the patients body, (3) inhibits (i e , slows, if not stops) spread of the infection (4) relieves (if not, eliminates) symptoms associated with the infection The NIMELS effect lowers the therapeutic threshold by sensitizing the microbial targets to the antibiotic agent

As used herein, the term "Interaction coefficient" is defined as a numeπcal representation of the magnitude of the bacteπastatic/bacteπacidal interaction between the NIMELS laser and/or the antimicrobial molecule, with the target cells

Thermodynamics of Energy Transduction m Biological Membranes

The present mvention is directed to perturbing cell membrane biological thermodynamics (bioenergetics) and the consequent diminished capacity of the irradiated cells to adequately undergo normal energy transduction and energy transformation

The methods and systems of the present invention optically alter and modify Ψά- plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito-fungi and Ψd-plas-bact to set m motion further alterations of ΔΨ and Δp in the same membranes This is caused by the targeted near infrared irradiation of the C-H covalent bonds m the long chain fatty acids of lipid bilayers, causing a vaπation in the dipole potential Ψd

To aid with an understanding of the process of this bioenergetic modification, the following descnption of the application of thermodynamics to membrane bioenergetics and energy transduction m biological membranes is presented To begin, membranes (lipid bilayers, see, Figure 1) possess a significant dipole potential Ψd arising from the structural association of dipolar groups and molecules, primarily the ester linkages of the phospholipids (Figure 2) and water These dipolar groups are oriented such that the hydrocarbon phase is positive with respect to the outer membrane regions (Figure 3) The degree of the dipole potential is usually large, typically several hundreds of millivolts The second major potential, a separation of charge across the membrane, gives πse to the trans- membrane potential ΔΨ The trans-membrane potential is defined as the electric potential difference between the bulk aqueous phases at the two sides of the membrane and results from the selective transport of charged molecules across the membrane As a rule, the potential at the cytoplasm side of cell membranes is negative relative to the extracellular physiological solution (Figure 4A)

The dipole potential Ψd constitutes a large and functionally important part of the electrostatic potential of all plasma and mitochondπal membranes Ψd modifies the electric field mside the membrane, producing a virtual positive charge in the apolar bilayer center As a result of this "positive charge", lipid membranes exhibit a substantial (e g , up to six orders of magnitude) difference m the penetration rates between positively and negatively charged hydrophobic ions Ψd also plays an important role in the membrane permeability for lipophilic ions

Numerous cellular processes, such as binding and insertion of protems (enzymes), lateral diffusion of proteins, ligand-receptor recognition, and certain steps m membrane fusion to endogenous and exogenous molecules, critically depend on the physical properties ^d of the membrane bilayer Studies in model membrane systems have illustrated the ability of mono- and multivalent ions to cause isothermal phase transitions in pure lipids, different phase separations, and a distinct clusteπng of individual components m mixtures In membranes, changes such as these can exert physical influences on the conformational dynamics of membrane-embedded proteins (Figures 4B and 5), and more specifically, on proteins that go through large conformational rearrangements in their transmembrane domains duπng their operating cycles Most importantly, changes in Ψd is believed to modulate membrane enzyme activities

Energy Transduction

The energy transduction in biological membranes generally involves three interrelated mechanisms

1 ) The transduction of redox energy to "free energy" stored in a trans-membrane ionic electrochemical potential also called the membrane proton electrochemical gradient ΔμHT This proton electrochemical potential difference between the two sides of a membrane that engage m active transport involving proton pumps is at times also called a chemiosmotic potential or proton motive force

2) In mammalian cells, the (Na⁺) ion electrochemical gradient Δμx⁺ is maintained across the plasma membrane by active transport of (Na⁺) out of the cell This is a different gradient than the proton electrochemical potential, yet is generated from a (pump) via the ATP produced during oxidative phosphorylation from the Mammalian Mitochondrial Proton- motive force Δp-mito-mam.

3) The use of this "free energy" to create ATP (energy transformation) to impel active transport across membranes with the concomitant buildup of required solutes and metabolites in the cell is termed the phosphorylation potential ΔGp. In other words, ΔGp is the ΔG for ATP synthesis at any given set of ATP, ADP and P₁ concentrations.

Steady-state trans-membrane potential (ΔΨ-steady)

The state of a membrane "system" is in equilibrium when the values of its chemical potential gradient Aμ¥t and E (energy) are temporally independent and there is no flux of energy across the margins of the system. If the membrane system variables of ΔμU^{1^} and E are constant, but there is a net flux of energy moving across the_system, then this membrane system is in a steady-state and is temporally dependent.

It is this temporally dependent steady-state trans-membrane and/or mitochondrial potential (ΔΨ-steady) of a cell (a respiring, growing and dividing cell) that is of focus. This "steady-state" of the flow of electrons and protons, or Na⁺/K⁺ ions across a mitochondrial or plasma membrane during normal electron transport and oxidative phosphorylation, would most likely continue into the future, if unimpeded by an endogenous or exogenous event. Any exogenous modification of the membrane thermodynamics, would bring about a transient-state trans-membrane and/or mitochondrial potential ΔΨ-trans, and this change from ΔΨ-steady to ΔΨ-trans is an object of the present invention.

Mathematical relationships between the state variables ΔΨ-steady and ΔΨ-trans are called equations of state. In thermodynamics, a state function (state quantity), is a property or a system that depends only on the current state of the system. It does not depend on the way in which the system attained its particular state. The present invention facilitates a transition of state in a trans-membrane and/or mitochondrial potential ΔΨ, in a temporally dependent manner, to move the bioenergetics of a membrane from a thermodynamic steady- state condition ΔΨ-steady to one of energy stress and/or redox stress in a transition state ΔΨ-trans. This can occur in ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact-ΔΨ-steady- mito-mam and ΔΨ-steady-mito-fungi. Not wishing to be bound by theory, it is believed that this transition is caused by the targeted near infrared irradiation of the C-H covalent bonds in the long chain fatty acids of lipid bilayers (with 930 nm wavelength), causing a variation in the dipole potential Ψd, and the targeted near infrared irradiation of cytochrome chains (with λ of 870 nm), that will concurrently alter ΔΨ-steady and the redox potential of the membranes

The First Law of Thermodynamics and Membranes An elemental aspect of the First Law of Thermodynamics (which holds true for membrane systems) is that the energy of an insulated system is conserved and that heat and work are both considered as equivalent forms of energy Hence, the energy level of a membrane system (Ψd and ΔΨ ) can be altered by an mcrease or decrease of mechanical work exerted by a force or pressure actmg, respectively, over a given distance or within an element of volume, and/or non-destructive heat transmitted through a temperature gradient m the membrane

This law (the law of conservation of energy), posits that the total energy of a system insulated from its surroundings does not change Thus, addition of any amounts of (energy) heat and work to a system must be reflected in a change of the energy of the system

Absorption of infrared radiation

The individual photons of infrared radiation do not contam sufficient energy (e g , as measured m electron-volts) to induce electronic transitions (in molecules) as is seen with photons of ultraviolet radiation Because of this, absorption of infrared radiation is limited to compounds with small energy differences m the possible vibrational and rotational states of the molecular bonds

By definition, for a membrane bilayer to absorb infrared radiation, the vibrations or rotations within the lipid bilayer' s molecular bonds that absorb the infrared photons, must cause a net change m the dipole potential of the membrane If the frequency (wavelength) of the infrared radiation matches the vibrational frequency of the absorbing molecule (i e , C-H covalent bonds in long cham fatty acids) then radiation will be absorbed causmg a change in Ψd This can happen m Ψd-plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito- fungi and Ψd-plas-bact In other words, there can be a direct and targeted change m the enthalpy and entropy (ΔH and ΔS) of all cellular lipid bilayers with the methods and systems descπbed herein

The present mvention is based upon a combination of insights that have been introduced above and are deπved m part from empiπcal data, which mclude the following It has been appreciated that unique, single infrared wavelengths (about 870 nm and about 930 nm) are each capable of killing bacterial cells (prokaryotes) such as E cob and (eukaryotes) such as Chinese Hampster Ovary (CHO) cells, as a result of the generation and interaction of ROS and/or toxic singlet oxygen reactions. The present invention employs these infrared wavelengths, preferably in combination, but at 5 log less power density than is typically found in a confocal laser microscope such as that used in optical traps (~ to 500,000 w/cm² less power) to advantageously exploit the use of such wavelengths for therapeutic laser systems, to cause a bacteriostatic or bacteriocidal effect at an infection site, without causing thermal damage to the hosts tissues.

This is done for the expressed purpose of alteration, manipulation and depolarization of the ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito- mam and ΔΨ-steady-mito-fungi of all cells within the irradiation field. This is accomplished in the present invention by the targeted near infrared irradiation of the C-H covalent bonds in the long chain fatty acids of lipid bilayers (with 930 nm energy), resulting in a variation in the dipole potentials Ψd-plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito- fungi and Ψd-plas-bact of all biological membranes within the irradiation field. Secondly, the near infrared irradiation of cytochrome chains (with 870 nm), will additionally alter ΔΨ- steady and the redox potential of the membranes that have cytochromes (i.e., bacterial plasma membranes, and fungal and mammalian mitochondria).

Serving as direct chromophores (cytochromes and C-H bonds in long chain fatty acids), there will be a direct enthalpy and entropy change in the molecular dynamics of membrane lipids and cytochromes for all cellular lipid bilayers in the irradiation path of the present invention. This will alter each membrane dipole potential Ψd, and concurrently alter the absolute value of the membrane potential ΔΨ, of all membranes in the irradiated cells.

These changes occur through significantly increased molecular motions (viz. ΔS) of the lipids and metallo-protein reaction centers of the cytochromes, as they absorb energy from the NIMELS system in a linear one-photon process. As even a small thermodynamic shift in either the lipid bilayer and/or the cytochromes would be enough to change the dipole potential Ψd, the molecular shape (and hence the enzymatic reactivity) of an attached electron transport protein, or trans-membrane protein would be rendered less functional. This will directly affect and modify the ΔΨ in all membranes in the irradiated cells.

The NTMELS effect occurs in accordance with methods and systems described herein, importantly, without thermal or ablative mechanical damage to the cell membranes. This combined and targeted low dose irradiation approach is a distinct variation and improvement from existing methods that would otherwise cause actual thermal or mechanical damage to all membranes within the path of a beam of energy. Membrane Entropy and the Second Law of Thermodynamics

The conversion of heat mto other forms of energy is never perfect, and (according to the Second Law of Thermodynamics) must always be accompanied by an mcrease m entropy Entropy (m a membrane) is a state function whose change m a reaction descπbes the direction of a reaction due to changes m (energy) heat input or output and the associated molecular rearrangements

Even though heat and mechanical energy are equivalent in their fundamental nature (as forms of energy), there are limitations on the ability to convert heat energy into work i e , too much heat can permanently damage the membrane architecture and prevent work or beneficial energy changes in either direction

The NIMELS effect will modify the entropy "state" of irradiated cells at the level of the lipid bilayer in a temporally dependent manner This mcrease in entropy will alter the Ψd of all irradiated membranes (mitochondπal and plasma) and hence, thermodynamically alter the "steady-state" flow of electrons and protons across a cell membrane (Figures 6 and 7) This will in turn change the steady-state trans-membrane potential ΔΨ-steady to a transient-state membrane potential (ΔΨ-tran) This phenomenon will occur in

1) Mammalian Plasma Trans-membrane Potential ΔΨ-plas-mam,

2) Fungal Plasma Trans-membrane Potential ΔΨ-plas-fungi,

3) Bacteπal Plasma Trans-membrane Potential ΔΨ-plas-bact, 4) Mammalian Mitochondπal Trans-membiane Potential ΔΨ-mito-mam, and 5) Fungal Mitochondrial Trans-membiane Potential ΔΨ-mito-fungi

This is a direct result of the targeted enthalpy change at the level of the C-H bonds of the long chain fatty acids m the fluid mosaic membrane, causing a measure of dynamic disorder (m the membrane) which can alter the membranes corporeal properties This fluid mosaic increases in entropy and can disrupt the tertiary and quaternary properties of electron transport protems, cause redox stress, energy stress and subsequent generation of ROS, that will further damage membranes and additionally alter the bioenergetics

Since a pπme function of the electron transport system of respiring cells is to transduce energy under steady-state conditions, techniques according to the present invention are utilized to temporarily, mechano-optically uncouple many of the relevant thermodynamic interactions on that transduction process This can be done with the express intent of alteπng the absolute quantitative value of the proton electrochemical gradient ΔμFT^" and proton-motive force and Δp of the membranes This phenomenon can occur, inter aha, in 1) Mammalian Mitochondrial Proton-motive force (Δp-mito-mam),

2) Fungal Mitochondrial Proton-motive force (Δp-mito-Fungi),

3) Fungal Plasma Membrane Proton-motive force (Δp-plas-Fungij, and

4) Bacteπal Plasma Membrane Proton-motive force (Δp-plas-Bact) Such phenomena can in turn decrease the Gibbs free energy value AG available for the phosphorylation and synthesis of ATP (AGp) The present invention carries out these phenomena m order to inhibit the necessary energy dependent anabolic reactions, potentiating pharmacological therapies, and/or lowenng cellular resistance mechanisms (to antimicrobial, antifungal and antineoplastic molecules) as many of these resistance mechanisms make use of the proton motive force or the chemiosmotic potential for their energy needs, to resist and/or efflux these molecules Free Radical Generation m consequence of modifications of ΔΨ-steady

The action of chemical uncouplers for oxidative phosphorylation and other bioenergetic work is believed to depend on the energized state of the membrane (plasma or mitochondrial) Further, it is believed that the energized state of the bacteπal membrane or eukaryotic mitochondrial inner membrane, is an electrochemical proton gradient ΔμET that is established by primary proton translocation events occurring during cellular respiration and electron transport

Agents that directly dissipate (depolarize) the ΔμFf", (e g , by peπneabihzing the coupling membrane to the movement of protons or compensatory ions) short-circuits energy coupling, and inhibit bioenergetic work, by inducing a reduction in the membrane potential ΔΨ-steady This will occur while respiration (primary proton translocation) continues apace For example, the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induces a reduction m membrane potential ΔΨ-steady and induces a concomitant generation of ROS, as respiration continues These agents (uncouplers) generally cannot be used as antimicrobials, antifungals, or antineoplastics, because their effects are correspondingly toxic to all bacterial, fungal and mammalian cells

However, it has been shown that in many target cells that are resistant to antimicrobials a Δp uncoupler (like CCCP) will collapse the energy gradient required for an efflux pump and hence induce a strong increase in the intracellular accumulation of these drugs These results clearly indicate that some resistance mechanisms (such as drug efflux pumps) are driven by the proton motive force The scientific findings and experimental data of the present invention show that as a membrane is depolaπzed optically, the generation of ROS further potentiates the depolarization of affected cells, and further potentiate the antibacteπal effects of the present invention Free radical and RQS generation by irradiation with the NIMELS laser

By mechano-optically modifying many of the relevant thermodynamic interactions of the membrane energy transduction process, along with altermg ΔΨ-steady, the present invention can act as an optical uncoupler by lowering the ΔμHT and Ap of the following irradiated membranes 1) Mammalian Mitochondπal Proton-motive force (Δp-mito-mamj

2) Fungal Mitochondrial Proton-motive force (Δp-mito-Fungij

3) Fungal Plasma Membrane Proton-motive foice (Δp-plas-Fungij

4) Bacterial Plasma Membrane Proton-motive force (Δp-plas-Bactj

This lowered Δp will cause a senes of free radicals and radical oxygen species to be generated because of the altered redox state The generation of free radicals and reactive oxygen species has been proven experimentally and described herein with the alteration of ΔΨ-steady to ΔΨ-trans in the following

1) ΔΨ-steady-mam + (NIMELS Treatment) ->-> ΔΨ-trans-mam

2) ΔΨ-steady-fungi + (NIMELS Treatment) -> -> ΔΨ-trans-fungi 3) ΔΨ-steady-bact + (NIMELS Treatment) -> ^■-> ΔΨ-trans-bact

4) ΔΨ-mito-fungi + (NIMELS Treatment) ->-> ΔΨ-trans-rmto-fungi

5) ΔΨ-mito-mam + (NIMELS Treatment) -^ -> ΔΨ-trans-mito-mam

The altered redox state and generation of free radicals and ROS because of the ΔΨ- steady + (NIMELS Treatment) -^ -^ ΔΨ- trans phenomenon, can cause seπous further damage to biological membranes such as lipid peroxidation

Lipid peroxidation

Lipid peroxidation is a prevalent cause of biological cell injury and death m both the microbial and mammalian world In this process, strong oxidents cause the breakdown of membrane phospholipids that contain polyunsaturated fatty acids (PUFA's) The severity of the membrane damage can cause local reductions in membrane fluidity and full disruption of bilayer integrity

Peroxidation of mitochondrial membranes (mamalhan cells and fungi) will have detπmental consequences on the respiratory chams resulting m madequate production of ATP and collapse of the cellular energy cycle Peroxidation of the plasma membrane (bacteria) can affect membrane permeability, disfunction of membrane proteins such as poπns and efflux pumps, inhibition of signal transduction and improper cellular respiration and ATP formation (i e , the respiratory chains m prokaryotes are housed in the plasma membranes as prokaryotes do not have mitochondria) Free radical

A free radical is defined as an atom or molecule that contains an unpaired electron An example of the damage that a free radical can do in a biological environment is the one- electron (via an existing or generated free radical) removal from bis-allylic C-H bonds of polyunsaturated fatty acids (PUFAs) that will yield a carbon centered free radical R* + (PUFA)-CH(bis-allyhc C-H bond) ^ (PUFA)-C* + RH

This reaction can initiate lipid peroxidation damage of biological membranes A free radical can also add to a nonradical molecule, producing a free radical product (A* + B -> A-B*) or a nonradical product (A*+B -> A-B) An example of this would be the hydroxylation of an aromatic compound by *OH Reactive Oxygen Species (ROS)

Oxygen gas is actually a free radical species However, because it contains two unpaired electrons m different 7T-anti-bonding orbitals that have parallel spin m the ground state, the (spin restπction) rule generally prevents O₂ from receiving a parr of electrons with parallel spms without a catalyst Consequently O₂ must receive one electron at a time There are many significant donors m a cell (prokaryotic and eukaryotic) that are able to stimulate the one-electron reduction of oxygen, that will create an additional radical species

These are generally categoπzed as

The Superoxide ion radical (O₂ ) Hydrogen Peroxide (non-radical) (H₂O₂)

Hydroxyl radical (^*OH)

Hydroxy ion (OH ) The Reaction Chain is O₂ -> O₂ + 2H⁺ -> H₂O₂ -> OH + *0H -» OH (e-) (e-) (e-) (e-)

Superoxide

The danger of these molecules to cells is well categorized in the literature Superoxide, for example, can either act as an oxidizing or a reducing agent NADH -> NAD⁺ Of higher importance to an organism's metabolism, superoxide can reduce cytochrome C It is generally believed that the reaction rates of superoxide (O₂ ) with lipids (i e , membranes) protems, and DNA are too slow to have biological significance The protonated form of superoxide hydroperoxyl radical (HOO*) has a lower reduction potential than (O₂ ), yet is able to remove hydrogen atoms from PUFA' s Also of note, the pKa value of (HOO*) is 4 8 and the (acid) microenvironment near biologiocal membranes will favor the formation of hydroperoxyl radicals Furthermore, the reaction of superoxide (O₂ ) with any free F₆ ⁺³ will produce a "perferryl" intermediate which can also react with PUFA' s and induce lipid (membrane) peroxidation Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) is not a good oxidizing agent (by itself) and cannot remove hydrogen from PUFA' s It can, however, cross biological membranes (rather easily) to exert dangerous and harmful effects m other areas of cells For example, (H₂O₂) is highly reactive with transition metals mside microcellular environments, (such as Fe⁺² and Cu⁺) that can then create hydroxyl radicals (*0H) (known as the Fenton Reaction) An hydroxyl radical is one of the most reactive species known in biology Hydroxyl Radical

Hydroxyl radicals (*0H) will react with almost all kinds of biological molecules It has a very fast reaction rate that is essentially controlled by the hydroxyl radical (*0H) diffusion rate and the presence (or absence) of a molecule to react near the site of (*0H) creation In fact, the standard reduction potential (EO') for hydroxyl radical (*0H) is (+2 31 V) a value that is 7_X greater than (H₂O₂), and is categoπzed as the most reactive among the biologically relevant free radicals Hydroxyl radicals will initiate lipid peroxidation in biological membranes, in addition to damaging proteins and DNA Reactive Oxygen Species Created from the Peroxidation of PUFAs

Furthermore, the development of lipid peroxidation (from any source) will result in the genesis of three other reactive oxygen intermediate molecules from PUFA's

(a) alkyl hydroperoxides (ROOH),

Like H₂O₂, alkyl hydroperoxides are not technically radical species but are unstable in the presence of transition metals such as such as Fe⁺² and Cu⁺

(b) alkyl peroxyl radicles (ROO*), and

(c) alkoxyl radicles (RO*)

Alkyl peroxyl radicles and alkoxyl radicles are extremely reactive oxygen species and also contribute to the process of propagation of further lipid peroxidation The altered redox state of irradiated cells and generation of free radicals and ROS because of the ΔΨ- steady + (NIMELS Treatment) ->-> ΔΨ- trans phenomenon is another object of the present invention This is an additive effect to further alter cellular bioenergetics and inhibit necessary energy dependent anabolic reactions, potentiate pharmacological therapies, and/or lower cellular resistance mechanisms to antimicrobial, antifungal and antineoplastic molecules

ROS overproduction can damage cellular macromolecules, above all lipids Lipid oxidation has been shown to modify both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization and altered a packing density dependent reoπentation of the component of the dipole moment Ψά Oxidative damage of the acyl chains (in lipids) causes loss of double bonds, chain shortening, and the introduction of hydroperoxy groups Hence, these changes are believed to affect the structural characteπstics and dynamics of lipid bilayers and the dipole potential Ψά Antimicrobial Resistance

Antimicrobial resistance is defined as the ability of a microorganism to survive the effects of an antimicrobial drug or molecule Antimicrobial resistance can evolve naturally via natural selection, through a random mutation, or through genetic engineering Also, microbes can transfer resistance genes between one another via mechanisms such as plasmid exchange If a microorganism carries several resistance genes, it is called multi-drug resistant or, colloquially, a "superbug " Multi-drug resistance in pathogenic bacteria and fungi are a serious problem in the treatment of patients infected with such orgamsms At present, it is tremendously expensive and difficult to create or discover new antimicrobial drugs that are safe for human use Also, there have been resistant mutant orgamsms that have evolved challenging all known antimicrobial classes and mechanisms Hence, few antimicrobials have been able to maintain their long-term effectiveness Most of the mechanisms of antimicrobial drug resistance are known

The four mam mechanisms by which micro-organisms exhibit resistance to antimicrobials are a) Drug mactivation or modification, b) Alteration of target site, c) Alteration of metabolic pathway, and d) Reduced drug accumulation by decreasing drug permeability and/or increasing active efflux on the cell surface Resistant Microbes

Staphylococcus aureus (S aureus) is a good example of one of the major resistant bacteπal pathogens currently plaguing humanity This gram positive bacterium is primarily found on the mucous membranes and skm of close to half of the adult world-wide population S aureus is extremely adaptable to pressure from all known classes of antibiotics S aureus was the first bacteπum m which resistance to penicillin was found in 1947 Since then, almost complete resistance has been found to methicillm and oxacillin The "superbug" MRSA (methicillm resistant Staphylococcus aureus) was first detected m 1961, and is now ubiquitous m hospitals and communities worldwide Today, more than half of all S aureus infections m the United States are resistant to penicillin, methicillm, tetracycline and erythromycin Recently, m what were the new classes of antibiotics (antimicrobials of last resort) glycopeptides and oxazohdmones, there have been reports of significant resistance (Vancomycin since 1996 and Zyvox since 2003)

A new vaπant CA-MRSA, (community acquired MRSA) has also recently emerged as an epidemic, and is responsible for a group of rapidly progressive, fatal diseases including necrotizmg pneumonia, severe sepsis and necrotizing fasciitis Outbreaks of community-associated (CA)-MRSA infections are reported daily m correctional facilities, athletic teams, military recruits, in newborn nurseπes, and among active homosexual men CA-MRSA infections now appear to be almost endemic m many urban regions and cause most CA-S aureus infections

The scientific and medical community has been attempting to find potentiators of existmg antimicrobial drugs and inhibitors of drug resistance systems m bacteπa and fungi Such potentiators and/or inhibitors, if not toxic to humans, would be very valuable for the treatment of patients infected with pathogenic and drug-resistant microbes In the United States, as many as 80% of individuals are colonized with S aureus at some point Most are colonized only intermittently, 20-30% are persistently colomzed Healthcare workers, persons with diabetes, and patients on dialysis all have higher rates of colonization The anterior nares are the predominant site of colonization in adults, other potential sites of colonization include the axilla, rectum, and perineum

Selective Pharmacological Alteration of ΔΨ-Steady State m Bactena

There is a relatively new class of bactericidal antibiotics called the lipopeptides of which daptomycm is the first FDA approved member This antibiotic has demonstrated (in vitro and in vivo) that it can rapidly kill virtually all clinically relevant gram-positive bacteπa (such as MRSA) via a mechanism of action distmct from those of other antibiotics on the market at present

Daptomycm's mechanism of action involves a calcium-dependent incorporation of the lipopeptide compound mto the cytoplasmic membrane of bacteria On a molecular level, it is calcium binding between two aspartate residues (in the daptomycm molecule) that decreases its net negative charge and permits it to act better with the negatively charged phospholipids that are typically found m the cytoplasmic membrane of gram-positive bacteπa There is generally no interaction with fungi or mammalian cells at therapeutic levels, so it is a very selective molecule The effects of daptomycm have been proposed to result from this calcium- dependent action on the bacteπal cytoplasmic membrane that dissipates the transmembrane membrane electπcal potential gradient ΔμtT This is in effect, a selective chemical depolarization of only bacterial membranes It is well known that the maintenance of a correctly energized cytoplasmic membrane is essential to the survival and growth of bacteπal cells, nevertheless depolaπzation (m this manner) is not m and of itself a bacteπally lethal action For example, the antibiotic valmomycm, which causes depolaπzation in the presence of potassium ions, is bacteπostatic but not bactericidal as would be the case with CCCP

Conversely, m the absence of a proton motive force Δp, the mam component of which is the transmembrane electπcal potential gradient ΔμHT, cells cannot make ATP or take up necessary nutπents needed for growth and reproduction The collapse of ΔμFT^" explains the dissimilar (detπmental) effects produced by daptomycm (e g , inhibition of protein, RNA, DNA, peptidoglycan, lipoteichoic acid, and lipid biosynthesis)

Further research into the medical literature concerning the drug daptomycm, suggests that the addition of gentamicm or minocycline (to daptomycm) results in the enhancement of its bactericidal activity against MRSA As both gentamicm and minocycline can be effluxed out of MRSA cells through energy dependent pumps, and are inhibitors of protein synthesis (an anabolic function) at the level of the 3OS bacteπal πbosome, this indicates that dissipation of the transmembrane electπcal potential gradient ΔμtT by daptomycm can potentiate certain antimicrobial drugs This should occur as a result of resistance mechanisms that are rendered less useful by a reduction m the membrane potential ΔΨ and the fact that ATP is not available (i e , the concomitant lowered Δ Gp) for the anabolic function of protem synthesis Based on the above, it would be possible to optically inhibit the activity of drug efflux pumps and/or anabolic reactions m target cells by safely reducing the membrane potential ΔΨ (ΔΨ-steady + (NIMELS Treatment) ->-> ΔΨ- trans) of the cells in a given target area Methods according to the present invention accomplish this and other tasks with the use of selected infrared wavelengths, e g , about 870 nm and about 930 nm, independent of any exogenous chemical membrane-actmg agents such as daptomycm

Multidrug resistance efflux pumps

Multidrug resistance efflux pumps are now known to be present in gram-positive bacteπa, gram-negative bacteπa, and other eukaryotic cells Efflux pumps generally have a poly-specificity of transporters that confers a broad-spectrum of resistance mechanisms These can strengthen the effects of other mechamsms of antimicrobial resistance such as mutations of the antimicrobial targets or enzymatic modification of the antimicrobial molecules Active efflux for antimicrobials can be clinically relevant for /3-lactam antimicrobials, macrolides, fluoroquinolones, tetracyclines and other important antibiotic famihess

With efflux pump-based resistance, a microbe has the capacity to seize an antimicrobial agent or toxic compound and expel it to the exterior (environment) of the cell, thereby reducing the intracellular accumulation of the agent It is generally considered that the over-expression of one or more of these efflux pumps prevents the intracellular accumulation of the agent to thresholds necessary for their biological activity Universally m microbes, the efflux of drugs is coupled to the proton motive force that creates electrochemical potentials and/or the energy necessary (ATP) for the needs of these protein pumps This mcludes 1) Mammalian mitochondrial proton-motive force (Δp-mito-mam),

2) Fungal mitochondrial proton -motive force (Δp-mito-fungi),

3) Fungal plasma membrane proton-motive force (Δp-plas-fungi), and

4) Bacteπal plasma membrane proton-motive force (Δp-plas-bact)

Phylogenetically, bacteπal antibiotic efflux pumps belong to five superfamilies

(i) ABC (ATP -binding cassette), which are primary active transporters energized by ATP hydrolysis,

(n) SMR [small multidrug resistance subfamily of the DMT (drug/metabolite transporters) superfamily], (in) MATE [multi-antimicrobial extrusion subfamily of the MOP (multidrug/oligosacchaπdyl-lipid/polysacchaπde flippases) superfamily] , (iv) MFS (major facilitator superfamily), and

(v) RND (resistance/nodulation/division superfamily), which are all secondary active transporters driven by ion gradients

The approach of the current mvention to inhibit efflux pumps is a general modification (optical depolarization) of the membranes ΔΨ withm the irradiated area, leading to lower electrochemical gradients that will lower the phosphorylation potential ΔGp and energy available for the pumps functional energy needs It is also the object of the present invention to have the same photobiological mechanism inhibit the many different anabolic and energy dnven mechanisms of the target cells, including absorption of nutπents for normal growth

Reduction of efflux pump energy Targeting the driving force of the mechanism Today, there are no drugs that belong to the "energy-blocker" family of molecules that have been developed for clinical use as efflux pump inhibitors There are a couple of molecules that have been found to be "general" inhibitors of efflux pumps Two such molecules are reserpme and verapamil These molecules were originally recognized as inhibitors of vesicular monoamine transporters and blockers of transmembrane calcium entry (or calcium ion antagonists), respectively Verapamil is known as an inhibitor of MDR pumps m cancer cells and certain parasites and also improves the activity of tobramycin

Reserpme inhibits the activity of Bmr and NorA, two gram-positive efflux pumps, by altering the generation of the membrane proton-motive force Δp required for the function of MDR efflux pumps Although these molecules are able to inhibit the ABC transporters involved m the extrusion of antibiotics (ι e , tetracycline), the concentrations necessary to block bacterial efflux are neurotoxic m humans

Bacterial Plasma Trans-membrane Potential ΔΨ-plas-bact and cell wall synthesis

Dunng normal cellular metabolism, protons are extruded through the cytoplasmic membrane to form ΔΨ-plas-bact This function also acidifies (lower pH) the narrow region near the bacteπal plasma membrane It has been shown m the gram positive bacteπum Bacillus subtihs, that the activities of peptidoglycan autolysms are increased (i e , no longer inhibited) when the electron transport system was blocked by adding proton conductors This suggests that ΔΨ-plas-bact and ΔμtT (independent of storing energy for cellular enzymatic functions) potentially has a profound and exploitable influence on cell wall anabolic functions and physiology

In addition, it has been shown that ΔΨ-plas-bact uncouplers inhibit peptidoglycan formation with the accumulation of the nucleotide precursors involved in peptidoglycan synthesis, and the inhibition of transport of N-acetylglucosamme (GIcNAc), one of the major biopolymers in peptidoglycan

Also, there is reference to an antimicrobial compound called tachyplesm that decreases ΔΨ-plas-bact m gram positive and gram negative pathogens (Antimicrobial compositions and pharmaceutical preparations thereof United States Patent 5,610,139, the entire teaching of which is incorporated herein by reference ) This compound was shown at sub-lethal concentrations to have the ability to potentiate the cell wall synthesis inhibitor /3-lactam antibiotic ampicillm m MRSA It is desirable to couple the multiple influences of an optically lowered ΔΨ-plas-bact (i e , increased cell wall autolysis, inhibited cell wall synthesis, and cell wall antimicrobial potentiation) to any other relevant antimicrobial therapy that targets bacterial cell walls This is especially relevant m gram positive bacteπa such as MRSA that do not have efflux pumps as resistance mechanisms for cell wall inhibitory antimicrobial compounds

Cell wall inhibitory compounds do not need to gam entry through a membrane m gram positive bacteria, as is necessary with gram negative bacteria, to exhibit effects against the cell wall Experimental evidence has proven that the NIMELS laser and its concomitant optical ΔΨ-plas-bact lowering phenomenon is synergistic with cell wall inhibitory antimicrobials in MRSA This must function via the inhibition of anabolic (peπplasmic) ATP coupled functions, as MRSA does not have efflux pumps that function on peptidoglycan inhibitory antimicrobials, as they do not need to enter the cell to be effective In one aspect, the invention provides a method of modifying the dipole potential Ψd of all membranes within the path of a NIMELS beam (Ψd-plas-mam, Ψd-mito-mam, Ψd- plas-fungi, Ψd-rmto-fungi, and Ψd-plas-bact) to set m motion the cascade of further alterations of ΔΨ and Δp in the same membranes

The bioenergetic steady-state membrane potentials ΔΨ-steady of all irradiated cells (ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam and ΔΨ-steady- mito-fungi) are altered to ΔΨ-trans values (ΔΨ-trans-mam, ΔΨ-trans-fungi, ΔΨ-trans-Bact, ΔΨ-trans-mito-mam and ΔΨ-trans-mito-fungi) This results m a concomitant depolarization and quantifiable alteration in the absolute value of the Δp for all irradiated cells (Δp-mito- mam, Δp-mito-Fungi, Δp-plas-Fungi and Δp-plas-Bact) These phenomena occur without intolerable risks and/or intolerable adverse effects to biological subjects (e g , a mammalian tissue, cell or certain biochemical preparations such as a protein preparation) in/at the given target site other than the targeted biological contaminants (bacteria and fungi), by irradiating the target site with optical radiation of desired wavelength(s), power density level(s), and/or energy density level(s)

In certain embodiments, such applied optical radiation may have a wavelength from about 850 nm to about 900 nm, at a NIMELS dosimetry, as described herein In exemplary embodiments, wavelengths from about 865 nm to about 875 nm are utilized In further embodiments, such applied radiation may have a wavelength from about 905 nm to about 945 nm at a NIMELS dosimetry In certain embodiments, such applied optical radiation may have a wavelength from about 925 nm to about 935 nm In representative non-limiting embodiments exemplified hereinafter, the wavelength employed is 930 nm

Bioenergetic steady-state membrane potentials may be modified, in exemplary embodiments, as noted below, and may employ multiple wavelength ranges including ranges bracketing 870 and 930 nm, respectively

The NIMELS Potentiation Magnitude Scale (NPMS)

As discussed in more detail supra, NIMELS parameters include the average single or additive output power of the laser diodes and the wavelengths (870 nm and 930 nm) of the diodes This information, combined with the area of the laser beam or beams (cm²) at the target site, the power output of the laser system and the time of irradiation, provide the set of information which may be used to calculate effective and safe irradiation protocols according to the invention

Based on these novel resistance leversal and antimicrobial potentiation interactions available with the NIMELS laser, there needs to be a quantitative value for the "potentiation effect" that will hold true for each unique antimicrobial and laser dosimetry

A new set of parameters are defined that will take mto account the implementation of any different dosimetric value for the NTMELS laser and any MIC value for a given antimicrobial being examined This can be simply tailored to the NIMELS laser system and methods by creating only a set of variables that quantify CFU's of pathogenic organisms withm any given expeπmental or treatment parameter with the NIMELS system

These parameters create a scale called the NIMELS Potentiation Magnitude Scale (NPMS) and exploits the NEVIELS lasers inherent phenomenon of reversing resistance and/or potentiating the MIC of antimicrobial drugs, while also producing a measure of safety against burning and injuring adjacent tissues, with power, and/or treatment time The NPMS scale measures the NIMELS effect number (Ne) between 1 to 10, where the goal is to gam a Ne of >4 m reduction of CFU count of a pathogen, at any safe combination of antimicrobial concentration and NIMELS dosimetry Although CFU count is used here for quantifying pathogenic organism, other means of quantification such as, for example, dye detection methods or polymerase cham reaction (PCR) methods can also be used to obtain values for A, B, and Np parameters

The NIMELS effect number Ne is an interaction coefficient indicating to what extent the combined mhibitory/bacteπostatic effect of an antimicrobial drug is synergistic with the NIMELS laser against a pathogen target without significant harm to healthy tissue at the site of pathogen infection

The NIMELS potentiation number (Np) is a value indicating whether the antimicrobial at a given concentration is synergistic, or antagonistic, to the pathogen target without harm to healthy tissue Hence, within any given set of standard experimental or treatment parameters - A = CFU Count of pathogen with NPMELS alone,

• B = CFU Count of pathogen with antimicrobial alone,

• Np = CFU Count of pathogen with (NPMELS + Antimicrobial), and

• Ne = (A+B) / 2Np,

Interpretation of NTMELS effect number Ne where

If 2N^> < A + B then the (given) antimicrobial has been successfully potentiated with the

NTMELS laser at the employed concentrations and dosimetries then

IfNe = 1 then there is no potentiation effect IfNe > 1 then there is a potentiation effect If Ne ≥2 then there is at least a 50% potentiation effect on the antimicrobial If Ne >4 then there is at least a 75% potentiation effect on the antimicrobial IfNe >10 then there is at least a 90% potentiation effect on the antimicrobial

Sample calculation 1

• A = 110 CFU - B = 120 CFU

• Np = 75 CFU

• Ne = (1 10 CFU + 120 CFU) / 2(75) = 1 5 Sample calculation 2

- A = 150 CFU • B = 90 CFU • N/? = 30 CFU

• Ne = (150 CFU + 90 CFU) / 2(30) = 4

In general, it can be advantageous to use a lower dose of antimicrobials when treating microbial infections, as the antimicrobials are expensive and by and large associated with undesirable side effects that can include systemic kidney and/or liver damage Therefore, it is desirable to devise methods to lower and or potentiate the MIC of antimicrobials The present invention provides systems and methods to reduce the MIC of antimicrobial molecules when the area being treated is concomitantly treated with the NEVIELS laser system If the MIC of an antimicrobial is reduced for a localized and resistant local mfection

(e g , skin, diabetic foot, bedsore), the therapeutic efficacy of many of the older, cheaper and safer antimicrobials to treat these infections will be restored Therefore, decreasmg the MIC of an antimicrobial, by the addition of the NIMELS laser (e g , generating a value of Ne that is m one aspect > 1 and in another aspect >4 and yet in another aspect >10), represents a positive step forward in restoring the once lost therapeutic efficacy of antibiotics

Therefore, m one aspect, this invention provides methods and systems that will reduced the MIC of antimicrobial molecules necessary to eradicate or at least attenuate microbial pathogens via a depolarization of membranes within the irradiated field which will decrease the membrane potential ΔΨ of the irradiated cells This weakened ΔΨ will cause an affiliated weakening of the proton motive force Δp, and the associated bioenergetics of all affected membranes It is a further object of the present invention that this "NBVIELS effect" potentiate existing antimicrobial molecules against microbes infecting and causing harm to human hosts

In certain embodiments, such applied optical radiation has a wavelength from about 850 run to about 900 nm, at a NTMELS dosimetry, as descπbed herem In exemplary embodiments, wavelengths from about 865 nm to about 875 nm are utilized In further embodiments, such applied radiation has a wavelength from about 905 nm to about 945 nm at a NIMELS dosimetry In certain embodiments, such applied optical radiation has a wavelength from about 925 nm to about 935 nm In one aspect, the wavelength employed is 930 nm

Microbial pathogens that have their bioenergetic systems affected by the NIMELS laser system according to the present invention include microorganisms such as, for example, bacteria, fungi, molds, mycoplasmas, protozoa, and parasites Exemplary embodiments, as noted below may employ multiple wavelength ranges including ranges bracketing 870 and 930 nm, respectively In the methods according to one aspect of the invention, irradiation by the wavelength ranges contemplated are performed independently, in sequence, in a blended ratio, or essentially concurrently (all of which can utilize pulsed and/or continuous-wave, CW, operation). Irradiation with NIMELS energy at NIMELS dosimetry to the biological contaminant is applied prior to, subsequent to, or concomitant with the administration of an antimicrobial agent. However, said NIMELS energy at NIMELS dosimetry can be administered after antimicrobial agent has reached a "peak plasma level" in the infected individual or other mammal. It should be noted that the co-administered antimicrobial agent ought to have antimicrobial activity against any naturally sensitive variants of the resistant target contaminant.

The wavelengths irradiated according to the present methods and systems increase the sensitivity of a contaminant to the level of a similar non-resistant contaminant strain at a concentration of the antimicrobial agent of about 0.5 M or less, about 0.1 M or less, or about 0.01 M or less, about 0.005 M or less or about 0.005 M or less.

The methods of the invention slow or eliminate the progression of microbial contaminants in a target site, improve at least some symptoms or asymptomatic pathologic conditions associated with the contaminants, and/or increase the sensitivity of the contaminants to an antimicrobial agent. For example, the methods of the invention result in a reduction in the levels of microbial contaminants in a target site and/or potentiate the activity of antimicrobial compounds by increasing the sensitivity of a biological contaminant to an antimicrobial agent to which the biological contaminant has evolved or acquired resistance, without an adverse effect on a biological subject. The reduction in the levels of microbial contaminants can be, for example, at least 10%, 20%, 30%, 50%, 70%, 100% or more as compared to pretreatment levels. It is preferred that the bacterial reduction be approximately a 2 or 3 log reduction. With regard to sensitivity of a biological contaminant to an antimicrobial agent, the sensitivity is potentiated by at least 10% and preferably by several orders of magnitude.

In another aspect, the invention provides a system to implement the methods according to other aspects of the invention. Such a system includes a laser oscillator for generating the radiation, a controller for calculating and controlling the dosage of the radiation, and a delivery assembly (system) for transmitting the radiation to the treatment site through an application region. Suitable delivery assemblies/systems include hollow waveguides, fiber optics, and/or free space/beam optical transmission components. Suitable free space/beam optical transmission components include collimating lenses and/or aperture stops

In one form, the system utilizes two or more solid state diode lasers to function as a dual wavelength near-mfrared optical source The two or more diode lasers may be located in a single housing with a unified control The two wavelengths can include emission m two ranges from about 850 nm to about 900 nm and from about 905 nm to about 945 nm The laser oscillator of the present invention is used to emit a single wavelength (or a peak value, e g , central wavelength) in one of the ranges disclosed herein In certain embodiments, such a laser is used to emit radiation substantially within the about 865-875 nm and the about 925-935 nm ranges

Systems according to the present invention can include a suitable optical source for each individual wavelength range desired to be produced For example, a suitable solid stated laser diode, a vaπable ultra-short pulse laser oscillator, or an ion-doped (e g , with a suitable rare earth element) optical fiber or fiber laser is used In one form, a suitable neai infrared laser includes titanium-doped sapphire Other suitable laser sources including those with other types of solid state, liquid, or gas gam (active) media may be used within the scope of the present mvention

Accordmg to one embodiment of the present invention, a therapeutic system includes an optical radiation generation system adapted to generate optical radiation substantially m a first wavelength range from about 850 nm to about 900 nm, a delivery assembly for causing the optical radiation to be transmitted through an application region, and a controller operatively connected to the optical radiation generation device foi controlling the dosage of the radiation transmitted through the application region, such that the time integral of the power density and energy density of the transmitted radiation per unit area is below a predetermined threshold Also within this embodiment, are therapeutic systems especially adapted to generate optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm

According to further embodiments, a therapeutic system includes an optical radiation generation device that is configured to generate optical radiation substantially in a second wavelength range from about 905 nm to about 945 nm, m certain embodiments the noted first wavelength range is simultaneously or concurrently/sequentially produced by the optical radiation generation device Also withm the scope of this embodiment, are therapeutic systems especially adapted to generate optical radiation substantially m a first wavelength range from about 925 nm to about 935 nm The therapeutic system can further include a delivery assembly (system) for transmitting the optical radiation in the second wavelength range (and where applicable, the first wavelength range) through an application region, and a controller operatively for controlling the optical radiation generation device to selectively generate radiation substantially in the first wavelength range or substantially in the second wavelength range or any combinations thereof.

According to one embodiment, the delivery assembly comprises one or more optical fibers having an end configured and arranged for insertion in patient tissue at a location within an optical transmission range of the medical device, wherein the radiation is delivered at a NIMELS dosimetry to the tissue surrounding the medical device. The delivery assembly may further comprise a free beam optical system.

According to a further embodiment, the controller of the therapeutic system includes a power limiter to control the dosage of the radiation. The controller may further include memory for storing a patient's profile and dosimetry calculator for calculating the dosage needed for a particular target site based on the information input by an operator. In one aspect, the memory may also be used to store information about different types of diseases and the treatment profile, for example, the pattern of the radiation and the dosage of the radiation, associated with a particular application.

The optical radiation can be delivered from the therapeutic system to the application site in different patterns. The radiation can be produced and delivered as a continuous wave (CW), or pulsed, or a combination of each. For example, in a single wavelength pattern or in a multi-wavelength (e.g., dual-wavelength) pattern. For example, two wavelengths of radiation can be multiplexed (optically combined) or transmitted simultaneously to the same treatment site. Suitable optical combination techniques can be used, including, but not limited to, the use of polarizing beam splitters (combiners), and/or overlapping of focused outputs from suitable mirrors and/or lenses, or other suitable multiplexing/combining techniques. Alternatively, the radiation can be delivered in an alternating pattern, in which the radiation in two wavelengths are alternatively delivered to the same treatment site. An interval between two or more pulses may be selected as desired according to NIMELS techniques of the present invention. Each treatment may combine any of these modes of transmission. The intensity distributions of the delivered optical radiation can be selected as desired. Exemplary embodiments include top-hat or substantially top-hat (e.g., trapezoidal, etc.) intensity distributions. Other intensity distributions, such as Gaussian may be used. One of skill m the art will appreciate that the methods and systems of the invention may be used in conjunction with a variety of biological contaminants generally known to those skilled in the art The following lists are provided solely for the purpose of illustrating the broad scope of microorganisms which may be targeted according to the methods and devices of the present invention and are not intended to limit the scope of the invention

Accordingly, illustrative non-limitmg examples of biological contaminants (pathogens) include, but are not limited to, any bacteπa, such as, for example, Escherichia, Enter ob acter , Bacillus, Campylobacter, Corynebacterium, Klebsiella, Listeria, Mycobacterium, Neiseria, Pseudomonas, Salmonella, Streptococcus, Staphylococcus, Treponema, Vibrio and Yersinia

It will be understood that the target site to be irradiated need not be already infected with a biological contaminant Indeed, the methods of the present invention may be used "prophylactically," prior to infection Further embodiments include use on medical devices such as catheters, (e g , IV catheter, central venous line, arterial catheter, peripheral catheter, dialysis catheter, peπtoneal dialysis catheter, epidural catheter), artificial joints, stents, external fixator pms, chest tubes, gastronomy feeding tubes, etc

In certain instances, irradiation may be palliative as well as prophylactic Hence, the methods of the invention are used to irradiate a tissue or tissues for a therapeutically effective amount of time for treating or alleviating the symptoms of an infection The expression "treating or alleviating" means reducing, preventing, and/or reversing the symptoms of the individual treated according to the invention, as compared to the symptoms of an individual receiving no such treatment

One of skill m the art will appreciate that the invention is useful in conjunction with a variety of diseases caused by or otherwise associated with any microbial, fungal, and viral infection (see, Harrison's, Principles of Internal Medicine, 13^th Ed , McGraw Hill, New York (1994), the entire teaching of which is incorporated herem by reference) In certain embodiments, the methods and the systems according to the invention are used m concomitance with traditional therapeutic approaches available m the art (see, e g , Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th ed, 1990, Pergmon Press, the entire teaching of which is incorporated herein by reference ) to treat an infection by the administration of known antimicrobial agent compositions The terms "antimicrobial composition", "antimicrobial agent" refer to compounds and combinations thereof that are administered to an animal, including human, and which inhibit the proliferation of a microbial infection (e g , antibacterial, antifungal, and antiviral) The wide breath of applications contemplated include, for example, a variety of dermatological, podiatric, pediatric, and general medicine to mention but a few. The interaction between a target site being treated and the energy imparted is defined by a number of parameters including: the wavelength(s); the chemical and physical properties of the target site; the power density or irradiance of beam; whether a continuous wave (CW) or pulsed irradiation is being used; the laser beam spot size; the exposure time, energy density, and any change in the physical properties of the target site as a result of laser irradiation with any of these parameters. In addition, the physical properties (e.g., absorption and scattering coefficients, scattering anisotropy, thermal conductivity, heat capacity, and mechanical strength) of the target site may also affect the overall effects and outcomes.

The NTMELS dosimetry denotes the power density (W/cm²) and the energy density (J/cm²; where 1 Watt = 1 Joule/second) values at which a subject wavelength is capable of generating ROS and thereby reducing the level of a biological contaminant in a target site, and/or irradiating the contaminant to increase the sensitivity of the biological contaminant through the lowering of ΔΨ with concomitant generation of ROS to an antimicrobial agent that said contaminant is resistant to without intolerable risks and/or intolerable side effects on a biological moiety (e.g., a mammalian cell, tissue, or organ) other than the biological contaminant.

As discussed in Boulnois 1986, (Lasers Med. Sci. 1:47-66 (1986), the entire teaching of which is incorporated herein by reference), at low power densities (also referred to as irradiances) and/or energies, the laser-tissue interactions can be described as purely optical (photochemical), whereas at higher power densities photo-thermal interactions ensue. In certain embodiments, exemplified hereinafter, NIMELS dosimetry parameters lie between known photochemical and photo-thermal parameters in an area traditionally used for photodynamic therapy in conjunction with exogenous drugs, dyes, and/or chromophores, yet can function in the realm of photodynamic therapy without the need of exogenous drugs, dyes, and/or chromophores.

The energy density — also expressible as fluence, or the product (or integral) of particle or radiation flux and time — for medical laser applications in the art typically varies between about 1 J/cm² to about 10,000 J/cm² (five orders of magnitude), whereas the power density (irradiance) varies from about IxIO^"3 W/cm² to over about 10¹² W/cm² (15 orders of magnitude). Upon taking the reciprocal correlation between the power density and the irradiation exposure time, it can be observed that approximately the same energy density is required for any intended specific laser-tissue interaction. As a result, laser exposure duration (irradiation time) is the primary parameter that determines the nature and safety of laser-tissue interactions For example, if one were mathematically looking for thermal vapoπzation of tissue in vivo (non-ablative) (based on Boulnois 1986), it can be seen that to produce an energy density of 1000 J/cm² (see, Table 1) one could use any of the following dosimetry parameters

Table 1 Example of Values Deπved on the Basis of the Boulnois Table

POWER TIME ENERGY

DENSITY DENSIT

Y IxI O⁵ 0 01 1000

W/c sec J/cm

_2

IxIO⁴ 0 10 1000

W/c sec J/cm² m²

IxIO³ 1 00 1000

W/c sec J/cm² m^"

This progression descπbes a suitable method or basic algorithm that can be used for a NIMELS interaction agamst a biological contaminant m a tissue In other words, this mathematical relation is a reciprocal correlation to achieve a laser-tissue interaction phenomena This ratiomale can be used as a basis for dosimetry calculations for the observed antimicrobial phenomenon imparted by NIMELS energies with insertion of NIMELS expeπmental data m the energy density and time and power parameters

On the basis of the particular mteractions at the target site being irradiated (such as the chemical and physical properties of the target site, whether contmuous wave (CW) or pulsed irradiation is being used, the laser beam spot size, and any change in the physical properties of the target site, e g , absorption and scattering coefficients, scattering anisotropy, thermal conductivity, heat capacity, and mechamcal strength, as a result of laser irradiation with any of these parameters), a practitioner is able to adjust the power density and time to obtain the desired energy density

The examples provided herein show such relationships m the context of both in vitro and in vivo treatments Hence, m the context of treating subjects, for spot sizes having a diameter of 1 -4 cm, power density values were varied from about 0 2 W/cm² to about 5 W/cm² and preferably 0 3 W/cm² to about 0 7 W/cm² to stay withm safe and non- damaging/minimally damaging thermal laser-tissue interactions well below the level of "denatuπzation" and "tissue overheating" Other suitable spot sizes may be used With this reciprocal correlation, the threshold energy density needed for a NIMELS interaction with these wavelengths can be maintained independent of the spot-size so long as the desired energies are delivered. In exemplary embodiments, the optical energy is delivered through a uniform geometric distribution to the tissues (e.g., a fiat-top, or top-hat progression). With such a technique, a suitable NIMELS dosimetry sufficient to generate

ROS (a NEVIELS effect) can be calculated to reach the threshold energy densities required to reduce the level of a biological contaminant and/or to increase the sensitivity of the biological contaminant to an antimicrobial agent that said contaminant is resistant to, but below the level of "denarurization" and "tissue overheating".

NEVIELS dosimetries exemplified herein to target microbes in vivo, were from about 125 J/cm² to about 700 J/cm² and preferably 150 J/cm² to about 400 J/cm² for approximately 100 to 700 seconds. These power values do not approach power values associated with photoablative or photothermal (laser/tissue) interactions.

The intensity distribution of a collimated laser beam is given by the power density of the beam, and is defined as the ratio of laser output power to the area of the circle in

(cm²) and the spatial distribution pattern of the energy. Hence, the illumination pattern of a 1.5 cm irradiation spot with an incident Gaussian beam pattern of the area 1.77 cm² can produce at least six different power density values within the 1.77 cm² irradiation area.

These varying power densities increase in intensity (or concentration of power) over the surface area of the spot from 1 (on the outer periphery) to 6 at the center point. In certain embodiments of the invention, a beam pattern is provided which overcomes this inherent error associated with traditional laser beam emissions. NEvIELS parameters may be calculated as a function of treatment time (Tn) as follows: Tn = Energy Density/Power

Density.

In certain embodiments (see, e.g., the in vitro experiments hereinbelow), Tn is from about 50 to about 300 seconds; in other embodiments, Tn is from about 75 to about 200 seconds; in yet other embodiments, Tn is from about 100 to about 150 seconds. In in vivo embodiments, Tn is from about 100 to about 1200 seconds.

Utilizing the above relationships and desired optical intensity distributions, e.g., flat-top illumination geometries as described herein, a series of in vivo energy parameters have been experimentally proven as effective for NEVIELS microbial decontamination therapy in vitro. A key parameter for a given target site has thus been shown to be the energy density required for NIMELS therapy at a variety of different spot sizes and power densities.

"NEVIELS dosimetry" encompasses ranges of power density and/or energy density from a first threshold point at which a subject wavelength according to the invention is capable of optically reducing ΔΨ in a target site to a second end-point and/or to increase the sensitivity of the biological contaminant to an antimicrobial agent that said contaminant is resistant to via generation of ROS, immediately before those values at which an intolerable adverse risk or effect is detected (e g , thermal damage such as poration) on a biological moiety One of skill in the art will appreciate that under certain circumstances adverse effects and/or πsks at a target site (e g , a mammalian cell, tissues, or organ) may be tolerated in view of the inherent benefits accruing from the methods of the mvention Accordingly, the stopping point contemplated are those at which the adverse effects are considerable and, thus, undesired (e g , cell death, protein denaturation, DNA damage, morbidity, or mortality) In certain embodiments, e g , for in vivo applications, the power density range contemplated herein is from about 0 25 to about 40 W/cm² In other embodiments, the power density range is from about 0 5 W/cm² to about 25 W/cm² Currently preferred embodiments for decolonizing a microbial site on a subject utilize a power density range from about 0 3 W/cm² to about 0 7 W/cm² when antibacterial coumpounds are coadministered Currently preferred embodiments for decolonizing a microbial site on a subject utilize an energy density range from about 125 J/cm² to about 400 J/cm² when antibacterial coumpounds are coadministered

In further embodiments, power density ranges can encompass values from about 0 5 W/cm² to about 10 W/cm² Power densities exemplified herein are from about 0 5 W/cm² to about 5 W/cm² Power densities in vivo from about 1 5 to about 2 5 W/cm² have been shown to be effective for vaπous microbes with or without coadministration of antibiotics Empirical data appears to indicate that higher power density values are generally used when targeting a biological contaminant m an in vitro setting (e g , plates) rather than in vivo (e g , toe nail) In certain embodiments (see, in vitro examples below), the energy density range contemplated herein is greater than 50 J/cm⁹ but less than about 25,000 J/cm⁷ In other embodiments, the energy density range is from about 750 J/cm² to about 7,000 J/cm² In yet other embodiments, the energy density range is from about 1,500 J/cm² to about 6,000 J/cm² depending on whether the biological contaminant is to be targeted m an in vitro setting (e g , plates) or in vivo (e g , toe nail or surrounding a medical device) In certain embodiments (see, in vivo examples below), the energy density is from about 100 J/cm² to about 500 J/cm² In yet other in vivo embodiments, the energy density is from about 175 J/cm² to about 300 J/cm² In yet other embodiments, the energy density is from about 200 J/cm² to about 250 J/cm² In some embodiments, the energy density is from about 300 J/cm² to about 700 J/cm^~ In some other embodiments, the energy density is from about 300 J/cm² to about 500 J/cm² In yet others, the energy density is from about 300 J/cm² to about 450 J/cm²

Power densities empirically tested for various in vitro treatment of microbial species were from about 1 W/cm² to about 10 W/cm² One of skill m the art will appreciate that the identification of particularly suitable

NIMELS dosimetry values within the power density and energy density ranges contemplated herein for a given circumstance may be empirically done via routine expeπmentation Practitioners (e g , dentists) using near infrared energies m conjunction with peπodontal treatment routinely adjust power density and energy density based on the exigencies associated with each given patient (e g , adjust the parameters as a function of tissue color, tissue architecture, and depth of pathogen invasion) As an example, laser treatment of a peπodontal infection m a light-colored tissue (e g , a melanme deficient patient) will have greater thermal safety parameters than darker tissue, because the darker tissue will absorb near-infrared energy more efficiently, and hence transform these near- infrared energies to heat m the tissues faster Hence, the obvious need for the ability of a practitioner to identify multiple different NDVIELS dosimetry values for different therapy protocols

As illustrated infra, it has been found that antibiotic resistant bacteria may be effectively treated according to the methods of the present invention In addition, it has been found that the methods of this invention may be used to augment traditional approaches, to be used m combination with, m lieu of tradition therapy, or even serially as an effective therapeutic approach Accordmgly, the invention may be combined with antibiotic treatment The term "antibiotic" includes, but is not limited to, /3-lactams, penicillins, and cephalosporins, vancomycins, bacitracins, macrolides (erythromycins), ketolides (telithxomycm), lmcosamides (clindamycin), chloramphenicols, tetracyclines, aminoglycosides (gentamicms), amphoteπcns, amlinouracils, cefazolms, clindamycins, mupirocins, sulfonamides and trimethoprim, πfampicins, metronidazoles, qumolones, novobiocins, polymixms, oxazohdmone class (e g , lmezolid), glycylcyclmes (e g , tigecycline), cyclic hpopeptides (e g , daptomycm), pleuromutilms (e g , retapamulm) and gramicidins and the like and any salts or vaπants thereof It also understood that it is within the scope of the present invention that the tetracyclines include, but are not limited to, immunocyclme, chlortetracyclme, oxytetracychne, demeclocyclme, methacyclme, doxycyclme and minocycline and the like It is also further understood that it is withm the scope of the present invention that aminoglycoside antibiotics include, but are not limited to, gentamicm, amikacin and neomycin, and the like A common tenet m the search for inhibitors of drug resistance systems m bacteria, or a potentiator of antimicrobial agents has always been that such agents are preferably nontoxic to the mammalian tissues that are infected, in order to have any intrinsic value To accomplish this, most antimicrobials affect bacterial cellular processes that are not common to the mammalian host, and, hence, are less disruptive to host metabolic processes If antimicrobials, potentiators, and/or resistance reversal entities were to also affect the mammalian cells m the same manner as they damage the pathogens, over similar concentrations, they could not be used safely as therapeutic agents

In the current invention, the expeπmental data provided herein supports a universal alteration of ΔΨ and Δp among all cell types, and hence leads to the notion that not only the electro-mechanical, but also the electro-dynamical aspects of all cell membranes, have no differing properties that can adequately be separated This indicates that all cells in the path of the beam are affected with depolarization, not only the pathogenic (non-desired) cells By reaffirming what the photobiology and cellular energetics data of the NIMELS system has already illuminated (i e , that all of membrane energetics are affected m the same way across prokaryotic and eukaryotic species), techniques according to the present invention utilize this universal optical depolarizing effect to be independently exploited in non-desired cells, by adding antimicrobial agents to a therapeutic regimen, and potentiating such molecules m (only) non-desired cells Such a targeted therapeutic outcome can exploit the NIMELS laser's effect of universal depolarization, with the targeted toxicity of microbial antibiotics, the combination being somewhat transient to the metabolism of the host cells but highly disruptive and preferably lethal to the bacteπa

The examples below provide experimental evidence proving the concept of universal optical membrane depolarization coupled to our current understanding of photobiology and cellular energetics and the conservation of thermodynamics as applied to cellular processes

EXAMPLES

The following examples are included to demonstrate exemplary embodiments of the present invention and are not intended to limit the scope of the invention Those of skill in the art, will appreciate that many changes can be made in the specific embodiments and still obtain a like or similar result without departing from the spirit and scope of the present invention EXAMPLE I

Table 2: MIC values for Susceptible, Intermediate and Resistant S. aureus

Minimum Inhibitory Concentration (MIC) Interpretive Standards (μg/ml) for Staphylococcus sp.

Antimicrobial Agent Susceptible Intermediate Resistant

Penicillin <0.12 >0.25

Methicillin <s - >16

Aminoglycosides

Gentamicin <4 8 >16

Kanamycin <16 32 I >64

Macrolides

Erythromycin <0.5 1-4 >s

Tetracycline

Tetracycline <4 Q o I >16

Fluoroquinolone

Ciprofloxacin <1 2 >4

Folate Pathway Inhibitors

Trimethoprim <8 - >16

Ansamycins

Rifampin <1 2 >4

EXAMPLE II: BACTERIAL METHODS: NIMELS TREATMENT PARAMETERS FOR IN VITRO

MRSA EXPERIMENTS The following illustrates the general antibacterial methods according to the invention, using a MRSA model for the in vitro Experiments V and VIII-XII. A. Experiment Materials and Methods for MRSA:

Table 3 Method for CFU counts

Similar cell culture and kinetic protocols were performed with E coli for all NIMELS irradiation experiments A standardized suspension was ahquoted into selected wells in a 24-well tissue culture plate Following laser treatments, 100 μL was removed from each well and serially diluted to 1 1000 resulting m a final dilution of 1 5x10⁶ of initial culture An aliquot of each final dilution were spread onto separate plates The plates were then incubated at 37⁰C for approximately 16-20 hours Manual colony counts were performed and recorded

Table 4 Method for ΔΨ and ROS Assays

Again, similar cell culture and kinetic protocols were performed for all NIMELS irradiation with E coli A standardized suspension was ahquoted into selected wells in a 24 -well tissue culture plate Following laser treatments each lased and control sample were treated as per directions of individual assay

EXAMPLE III MAMMALIAN CELL METHODS NIMELS TREATMENT PARAMETERS FOR IN VITRO HEK293 EXPERIMENTS The following parameters illustrate the general methods according to the invention as applied to HEK293 cells for the in vitro expeπments

A Experiment Materials and Methods for HEK293 cells HEK293 cells were seeded into appropπate wells of a 24-well plate at a density of 1 x 10⁵ cells/ml (0 7ml total volume) in Freestyle medium (Invitrogen) Cells were incubated in a humidified incubator at 37 ⁰C m 8% CO₂ for approximately 48 hours prior to the expeπment Cells were approximately 90% confluent at the time of the expeπment equating to roughly 3 x 10⁵ total cells Immediately pπor to treatment, cells were washed m pre- warmed phosphate buffer saline (PBS) and overlaid with 2 ml of PBS during treatment

After laser treatment, cells were mechanically dislodged from the wells and transferred to 1 5 ml centrifuge tubes Mitochondrial membrane potential and total glutathione was determined

EXAMPLE FV NTMELS IN VITRO TESTS FOR CRT+ (YELLO W)AND CRT - (WHITE) S

A UREus EXPERIMENTS

We conducted experiments with crt- (white) mutants of S aureus that were genetically engineered with the crt gene (yellow carotenoid pigment) removed, and these mutants were subjected to previously determined non-lethal doses of NIMELS laser against wild type (yellow) S aureus The purpose of this expeπment was to test for the phenomenon of Radical Oxygen Species (ROS) generation and/or singlet oxygen generation with the NTMELS laser In the scientific literature, Lm et al had previously used a similar model, to test the antioxidant protection activity of the yellow S aureus *caratenoid) pigment against neutrophils (Lm et al , Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity, VoI 202, No 2, July 18, 2005 209-215, the entire teaching of which is incorporated herein by reference ) It has previously been determined that the golden color m S aureus is imparted by carotenoid (antioxidant) pigments capable of protecting the organism from oxygen damage, and when a mutant is isolated (crt ) that does not produce such carotenoid pigments, the mutant colomes are "white" in appearance and more susceptible to oxidative killing, and have impaired neutrophil survival

It was found that non-lethal dosimetπes of the NIMELS laser (to wild type S aureus) consistently killed up to 90% of the mutant "white" cells and did not kill the normal S aureus The only genetic difference m the two strains of S aureus is the lack of an antioxidant pigment in the mutant This experimental data strongly suggests that it is the endogenous generation of radical oxygen species and/or smglet oxygen that are killing the "white" S aureus

Table 5 Data

Dl - D4 Yellow Wild Type S aureus D5 - D6 White "crt " Mutant S Aureus

Table 6.

Samples Dl- D4 Yellow Wild Type S. aureus. Sam les D5 - D6 White "crt-" Mutant S. aureus.

EXAMPLE V NIMELS IN VITRO TESTS FOR ΔΨ ALTERATION IN MRSA₁ AND E COLI

There are selected fluorescent dyes that can be taken up by mtact cells and accumulate within the intact cells within 15 to 30 minutes without appreciable staining of other protoplasmic constituents These dye indicators of membrane potential have been available for many years and have been employed to study cell physiology The fluorescence mtensity of these dyes can be easily monitored, as their spectral fluorescent properties are responsive to changes in the value of the trans-membrane potentials ΔΨ- steady

These dyes generally operate by a potential-dependent partitioning between the extracellular medium and either the membrane or the cytoplasm of membranes This occurs by redistribution of the dye via interaction of the voltage potential with an ionic charge on the dye This fluorescence can be eliminated in about 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that maintenance of dye concentration is dependent on the mside-negative transmembrane potential maintained by functional ETS and Δp

Hypothesis Testing

The null hypothesis is μi - μ₂ = 0 μi is fluorescence intensity in a control cell culture (no laser) subjected to carbocyanme dye μ₂ is fluorescence intensity in the same cell culture pre-irradiated with sub-lethal dosimetry from the NTMELS laser The data mdicates that the fluorescence of cells is dissipated (less than control of unirradiated or "unlased" cells) by pre-treatment (of the cells) with the NTJVIELS laser system, indicating that the NTMELS laser interacted with respiratory processes and oxidative phosphorylation of the cells via the plasma membranes 0 Will uphold that the addition sub-lethal NTMEL irradiation on the cell culture has no effect on ΔΨ-steady 0

Will uphold that the addition sub-lethal NTMEL irradiation on the cell culture has a dissipation or depolarization effect on ΔΨ-steady Materials and Methods

BacLight™ Bacterial Membrane Potential Kit (B34950, Invitrogen U S ) The iføcLight™ Bacterial Membrane Potential Kit provides of carbocyanine dye DiOC2(3)

(3,3'-diethyloxacarbocyanine iodide, Component A) and CCCP (carbonyl cyanide 3- chlorophenylhydrazone, Component B), both in DMSO, and a 1 x PBS solution

(Component C).

DiOC2(3) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts toward red emission as the dye molecules self associate at the higher cytosolic concentrations caused by larger membrane potentials. Proton ionophores such as CCCP destroy membrane potential by eliminating the proton gradient, hence causing higher green fluorescence. 0 Detection of membrane potential ΔΨ in MRSA

Green fluorescence emission was calculated using population mean fluorescence intensities for control and lased samples at sub-lethal dosimetry:

Table 7.

5 The data shows that μi - μ₂ > 0 as the lased cells had less "Green fluorescence" as seen in Figure 8. These MRSA samples showed clear alteration and lowering of ΔΨ-steady- bact to one of ΔΨ-trans-bact with sub-lethal NIMELS dosimetry.

Detection of membrane potential ΔΨ in E. coli 0 Red/green ratios were calculated using population mean fluorescence intensities for control and lased samples at sub-lethal dosimetry:

The data shows that μi - μ₂ > 0 as the lased cells had less "Green fluorescence" as seen in Figure 19. These E. coli samples showed clear alteration and lowering of ΔΨ - steady-bact to one of ΔΨ -trans-bact with sublethal MMELS dosimetry. 5

EXAMPLE VI: NIMELS IN VITRO TESTS FOR ΔΨ-mito HUMAN EMBRYONIC KIDNEY CELLS WITH SUB-LETHAL LASER DOSIMETRY

Hypothesis Testing: 0 The null hypothesis is μ, - μ₂ = 0: a) μ] is fluorescence intensity m a mammalian control cell culture mitochondπa (no laser) subjected to a Mitochondπal Membrane Potential Detection Kit b) μ₂ is fluorescence intensity m the same mammalian cell culture pre-irradiated with sublethal dosimetry from the NIMELS laser and subjected to a Mitochondrial

Membrane Potential Detection Kit

The data shows that the fluorescence of mitochondπa is dissipated (less than control unlased cells) by pre-treatment (of the cells) with the NIMELS laser system, the results indicate that the NIMELS laser interacted with respiratory processes and oxidative phosphorylation of the cells m mitochondπa of mammalian cells

Will uphold that the addition sub-lethal NIMEL irradiation on the mammalian cell culture mitochondπa has no effect on ΔΨ-steady-mito-mam μ, - μ₂ > 0 Will uphold that the addition sub-lethal NDVIEL irradiation on the mammalian cell culture has a dissipation or depolaπzation effect on ΔΨ-steady-mito-mam

Mateπals and Methods

Mitochondπal Membrane Potential Detection Kit (APO LOGIX JC-I) (Cell Technology Inc , 950 Rengstorff Ave, Suite D, Mountain View CA 94043)

The loss of mitochondπal membrane potential (ΔΨ) is a hallmark for apoptosis The APO LOGIX JC-I Assay Kit measures the mitochondrial membrane potential in cells In non-apoptotic cells, JC-I (5,5\6,6'-tetrachloro-l,l ',3,3'-tetraethylbenz- lmidazolylcarbocyanme iodide) exists as a monomer in the cytosol (green) and also accumulates as aggregates m the mitochondπa which stam red Whereas, m apoptotic and necrotic cells, JC-I exists in monomeπc form and stams the cytosol green

Table 8 Mamalhan Cell Dosimetries

HEK-293 (Human Embryonic Kidney Cells) ΔΨ-mito tests:

The (APO LOGIX JC-I) kit measures membrane potential by conversion of green fluorescence to red fluorescence. The appearance of red color has been measured and plotted, which should only occur in cells with intact membranes, and the ratio of green to red is calculated for both control and lased samples.

Clearly in this test, the red fluorescence is reduced in the lased sample while the ratio of green to red increases, indicating depolarization. These results show that μ] - μ₂> O and that sub-lethal NIMELS irradiation on the mammalian cell mitochondria has a dissipation or depolarization effect on ΔΨ-steady-mito-mam, indicating a clear reduction in mammalian ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam.

EXAMPLE VII: NMELS IN VITRO TESTS FOR REACTIVE OXYGEN SPECIES (ROS)

These in vitro tests for generation of reactive oxygen species (ROS) were carried on after laser alteration of bacterial trans-membrane ΔΨ-steady-bact to ΔΨ-trans-bact, ΔΨ- steady-mito-fungi to ΔΨ-trans-mito-fungi, and ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam with sub-lethal laser dosimetry comparable to those used in ΔΨ tests above in previous examples.

Materials and Methods:

Total Glutathione Quantification Kit (Dojindo Laboratories; Kumamoto Techno Research Park, 2025-5 Tabaru, Mashiki-machi, Kamimashiki-gun; Kumamoto 861-2202, JAPAN) Glutathione (GSH) is the most abundant thiol (SH) compound in animal tissues, plant tissues, bacteria and yeast. GSH plays many different roles such as protection against reactive oxygen species and maintenance of protein SH groups. During these reactions,

GSH is converted into glutathione disulfide (GSSG: oxidized form of GSH). Since GSSG is enzymatically reduced by glutathione reductase, GSH is the dominant form in organisms. DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)), known as Ellman's Reagent, was developed for the detection of thiol compounds. In 1985, it was suggested that the glutathione recycling system by DTNB and glutathione reductase created a highly sensitive glutathione detection method. DTNB and glutathione (GSH) react to generate 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-nitro-5- thiobenzoic acid is a yellow colored product, GSH concentration in a sample solution can be determined by the measurement at 412 nm absorbance. GSH is generated from GSSG by glutathione reductase, and reacts with DTNB again to produce 2-nitro-5 -thiobenzoic acid. Therefore, this recycling reaction improves the sensitivity of total glutathione detection. At significant concentrations ROS will react rapidly and specifically with the target at a rate exceeding the rate of its reduction by the components of the glutathione antioxidant system (catalases, peroxidases, GSH).

Detection of Glutathione in MRSA at sub-lethal NIMELS dosimetry that alters ΔΨ-steady- bact to one of ΔΨ-trans-bact

A reduction in total glutathione in MRSA at sub-lethal NIMELS dosimetry that alters that alters ΔΨ-steady-bact to one of _.ΔΨ-trans-bact, is proof of generation of ROS with sub-lethal alteration of Trans-membrane ΔΨ-steady-bact to one of ΔΨ-trans-bact.

Detection of Glutathione in E.coli at sub-lethal NIMELS dosimetry that alters Transmembrane ΔΨ-steady to one of ΔΨ-trans

A reduction in total glutathione in E. coli at sub-lethal NIMELS dosimetry that alters ΔΨ-steady-bact to one of ΔΨ -trans-bact, is evidence of generation of ROS with sub- lethal alteration of Trans-membrane ΔΨ-steady-bact to one of ΔΨ -trans-bact.

Detection of glutathione in C. albicans at sub-lethal NIMELS that alters ΔΨ-steady- mito-fungi to ΔΨ-trans-mito-fungi and subsequently ΔΨ-steady-fungi to one of ΔΨ-trans- fungi.

Detection of Glutathione in C. albicans at sub-lethal NIMELS dosimetry that alters ΔΨ- steady-mito-fungi to ΔΨ-trans-mito-fungi and subsequently ΔΨ-steady-fungi to one of ΔΨ- trans-fungi

A reduction in total glutathione in C. albicans at sub-lethal NIMELS dosimetry that alters ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi and subsequently ΔΨ-steady-fungi to one of ΔΨ-trans-fungi, is proof of generation of ROS with sub-lethal alteration of Transmembrane ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi and subsequently ΔΨ-steady-fungi to one of ΔΨ-trans-fungi.

Detection of Glutathione in HEK-293 (Human Embryonic Kidney Cells) at sub-lethal NIMELS dosimetry that alters ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam

A reduction in total Glutathione in HEK-293 (Human Embryonic Kidney Cells) with sub-lethal NIMELS dosimetry that alters ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam, is proof of generation of ROS with NTMELS-mediated sub-lethal alteration of Transmembrane ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam. EXAMPLE Vπi Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with Erythromycin and Trimethoprim

In this example, it was determined whether a sub-lethal dose of the NIMEL laser will potentiate the effect of the antibiotic erythromycin more than the antibiotic trimethoprim in MRSA Efflux pumps play a major factor in erythromycin resistance There are no reported trimethoprim efflux pump resistance mechanisms in the gram positive S aureus

Background Erythromycin is a macrolide antibiotic that has an antibacteπal spectrum of action very similar to that of the /3-lactam penicillin In the past, it has been effective in the treatment of a wide range of gram-positive bacteπal infections effecting the skm and respiratory tract, and has been considered one of the safest antibiotics to use In the past, erythromycin has been used for people with allergies to penicillins Erythromycin's mechamsm of action is to prevent growth and replication of bacteria by obstructing bacteπal protem synthesis This is accomplished because erythromycin binds to the 23 S rRNA molecule in the 5OS of the bacteπal πbosome, thereby blocking the exit of the growing peptide chain thus inhibiting the translocation of peptides Erythromycin resistance (as with other marcohdes) is rampant, wide spread, and is accomplished via two significant resistance systems A) modification of the 23 S rRNA in the 5OS πbosomal subumt to msensitivity B) efflux of the drug out of cells

Tπmethopπm is an antibiotic that has histoπcally been used m the treatment of uπnary tract infections It is a member of the class of antimicrobials known as dihydro folate reductase inhibitors Tπmethopπm's mechamsm of action is to interfere with the system of bactenal dihydrofolate reductase (DHFR), because it is an analog of dihydro folic acid This causes competitive inhibition of DHFR due to a 1000 fold higher affinity for the enzyme than the natural substrate

Thus, tπmethopπm inhibits synthesis of the molecule tetrahydrofolic acid Tetrahydrofolic acid is an essential precursor m the de novo synthesis of the DNA nucleotide thymidylate Bacteπa are incapable of taking up folic acid from the environment (i e , the infection host) and are thus dependent on their own de novo synthesis of tetrahydrofolic acid Inhibition of the enzyme ultimately prevents DNA replication

Tπmethopπm resistance generally results from the overproduction of the normal chromosomal DHFR, or drug resistant DHFR enzymes Reports of tπmethopπm resistance W

S. aureus have indicated that the resistance is chromosomally of the mediated type or is encoded on large plasmids. Some strains have been reported to exhibit both chromosomal and plasmid-mediated trimethoprim resistance.

In the gram positive pathogen S. aureus, resistance to trimethoprim is due to genetic 5 mutation, and there have been no reports that trimethoprim is actively effluxed out of cells.

Efflux Pumps in Bacteria

A major route of drug resistance in bacteria and fungi is the active export (efflux) of antibiotics out of the cells such that a therapeutic concentration in not obtained in the 10 cytoplasm of the cell.

Active efflux of antibiotics (and other deleterious molecules) is mediated by a series of transmembrane proteins in the cytoplasmic membrane of gram positive bacteria and the outer membranes of gram negative bacteria.

Clinically, antibiotic resistance that is mediated via efflux pumps, is most relevant 15 in gram positive bacteria for marcolides, tetracyclines and fluoroquinolones. In gram negative bacteria, β-lactam efflux mediated resistance is also of high clinical relevance.

Hypothesis Testing

The null hypothesis is μ_t - μ₂ = 0 and μi - μ₃= 0 where: 20 a) μ] is sub-lethal dosimetry from the NDVIEL laser system on MRSA as a control and; b) μ₂ is the same sub-lethal dosimetry from the NIMEL laser system on MRSA with the addition of trimethoprim at resistant MIC just below effectiveness level and; c) μ₃ is the same sub-lethal dosimetry from the NIMEL laser system on MRSA with the addition of erythromycin at resistant MIC just below effectiveness level.

25 The data shows that the addition of the antibiotic trimethoprim or erythromycin, after sub-lethal irradiation, results in the reduction in growth of these MRSA colonies, as follows: 0

Will uphold that the addition of trimethoprim produces no deleterious effect after sub-lethal 30 NIMEL irradiation, on normal growth of MRSA colonies.

Will uphold that the addition of trimethoprim produces a deleterious effect after sub-lethal

NIMEL irradiation, on normal growth of MRSA colonies. μ, - μ₃= 0 Will uphold that the addition of erythromycin produces no deleterious effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies. μ, - μ₃ > 0

Will uphold that the addition of erythromycin produces a deleterious effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies.

Table 9.

Results:

This experiment clearly showed that under sub-lethal laser parameters with the NIMELS system, μ] - μ₂ = 0 and μι - μ₃ >= 0. This indicates that an efflux pump is being inhibited, and resistance to erythromycin being reversed by the NIMELS effect on ΔΨ- steady-bact of the MRSA.

EXAMPLE IX: Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with Tetracycline and Rifampin

The purpose of this experiment was to observe if a sub-lethal dose of the NDVIEL laser will potentiate the effect of the antibiotic tetracycline more than the antibiotic rifampin in MRSA. Efflux pumps are well researched, and play a major factor in tetracycline resistance. However, there are no reported rifampin efflux pump resistance mechanisms in the gram positive S. aureus.

This experiment was also previously run with erythromycin and trimethoprim, with data indicating that the NIMELS effect is able to damage efflux pump resistance mechanisms in erythromycin.

Tetracycline:

Tetracycline is considered a bacteriostatic antibiotic, meaning that it hampers the growth of bacteria by inhibiting protein synthesis. Tetracycline accomplishes this by inhibiting action of the bacterial 30S ribosome through the binding of the enzyme aminoacyl-tRNA. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines, or for a protein that protects bacterial πbosomes from the action of tetracyclines

Rifampin

Rifampin is a bacterial RNA polymerase inhibitor, and functions by directly blocking the elongation of RNA Rifampicin is typically used to treat mycobacteπal infections, but also plays a role m the treatment of methicillm-resistant Staphylococcus aureus (MRSA) m combmation with fusidic acid, a bacteπostatic protein synthesis inhibitor There are no reports of rifampin resistance via efflux pumps m MRSA

Hypothesis

The null hypothesis is μj - μ₂ = 0 and μi - μ₃= 0 where a) μi is sub-lethal dosimetry from the NEMEL laser system on MRSA as a control and, b) μ₂ is the same sub-lethal dosimetry from the NIMEL laser system on MRSA with the addition of tetracycline at resistant MIC just below effectiveness level and, c) μ₃ is the same sub-lethal dosimetry from the NIMEL laser system on MRSA with the addition of rifampin at resistant MIC just below effectiveness level

The data shows that the addition of the antibiotic tetracycline or rifampin, after sublethal irradiation, results m the reduction m growth of these MRSA colonies, as follows μ, - μ₂ = 0 Will uphold that the addition of tetracycline produces no deleterious effect after sub-lethal

NIMEL irradiation, on normal growth of MRSA colonies μ, - μ₂ > 0

Will uphold that the addition of tetracycline produces a deleterious effect after sub-lethal

NEMEL irradiation, on normal growth of MRSA colonies μ] - μ₃= 0

Will uphold that the addition of rifampin produces no deleteπous effect after sub-lethal

NIMEL irradiation, on normal growth of MRSA colonies μi - μs > 0

Will uphold that the addition of rifampin produces a deleteπous effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies Table 10.

Results:

This experiment clearly showed that under sub-lethal laser parameters with the NIMELS system, μi - μ₂ = 0 and μ] - μ₃ >= 0. This indicates that an efflux pump is being inhibited, and resistance to tetracycline is being reversed by the NTMELS effect on ΔΨ- steady-bact of the MRSA.

EXAMPLE X: Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with Methicillin and ΔΨ-plas-bact inhibition of cell wall synthesis Methicillm:

Methicillin is a /3-lactam that was previously used to treat infections caused by gram-positive bacteria, particularly /3-lactamase-producing organisms such as S. aureus that would otherwise be resistant to most penicillins, but is no longer clinically used. The term methicillin-resistant S. aureus (MRSA) continues to be used to describe S. aureus strains resistant to all penicillins. Mechanism of action

Like other /3-lactam antibiotics, methicillin acts by inhibiting the synthesis of peptidoglycan (bacterial cell walls). It has been shown in the gram positive bacterium Bacillus subtilis, that the activities of peptidoglycan autolysins are increased (i.e., no longer inhibited) when the ETS was blocked by adding proton conductors. This suggests that ΔΨ-plas-bact and ΔμHT (independent of storing energy for cellular enzymatic functions) potentially has a profound and exploitable influence on cell wall anabolic functions and physiology.

In addition, it has been reported that ΔΨ-plas-bact uncouplers inhibit peptidoglycan formation with the accumulation of the nucleotide precursors involved in peptidoglycan synthesis, and the inhibition of transport of N-acetylglucosamine (GIcNAc), one of the major biopolymers in peptidoglycan.

Hypothesis Testing:

Bacitracin will potentiate the multiple influences of an optically lowered ΔΨ-plas- bact on a growing cell wall (i.e., increased cell wall autolysis, inhibited cell wall synthesis). This is especially relevant in gram positive bacteria such as MRSA, that do not have efflux pumps as resistance mechanisms for cell wall inhibitory antimicrobial compounds. The null hypothesis is μi - μ₂ = 0 and μi - μ₃= 0 where: a) μi is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control and; b) μ₂ is the same sub-lethal dosimetry from the NTMEL laser system on MRSA with the addition of methicillin at resistant MIC just below effectiveness level and; μ_i - μ₂ = 0

Will uphold that the addition of methicillin produces no deleterious effect after sub-lethal

NMEL irradiation, on normal growth of MRSA colonies. μ_ι - μ₂ > 0

Will uphold that the addition of methicillin produces a deleterious effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies.

Results:

As shown in Figure 15, this experiment clearly showed that under sub-lethal laser parameters with the NIMELS system, μi - μ₂ >= 0, meaning that the addition of methicillin produces a deleterious effect after sub-lethal NIMEL irradiation on normal growth of

MRSA colonies as shown by CFU count. This suggest that methicillin (independent of an efflux pump) is being potentiated by the NMELS effect on ΔΨ-steady-bact of the MRSA. Hence, the NIMELS laser and its concomitant optical ΔΨ-plas-bact lowering phenomenon is synergistic with cell wall inhibitory antimicrobials in MRSA. Without wishing to be bound by theory, this must function via the inhibition of anabolic (periplasmic) ATP coupled functions, as MRSA does not have efflux pumps for methicillin.

EXAMPLE XI: Assessment of the impact of Sub-lethal doses of NMELS Laser on MRSA with Bacitracin and ΔΨ-plas-bact inhibition of cell wall synthesis

Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtilis. As a toxic and difficult-to-use antibiotic, bacitracin cannot generally be used orally, but is used topically.

Mechanism of action:

Bacitracin interferes with the dephosphorylation of the C₅₅-isoprenyl pyrophosphate, a molecule which carries the building blocks of the peptidoglycan bacterial cell wall outside of the inner membrane in gram negative organisms and the plasma membrane in gram positive organism.

It has been shown in the gram positive bacterium Bacillus subtilis, that the activities of peptidoglycan autolysins are increased (i.e., no longer inhibited) when the ETS was blocked by adding proton conductors. This indicates that ΔΨ-plas-bact and AμirC (independent of storing energy for cellular enzymatic functions) potentially has a profound and exploitable influence on cell wall anabolic functions and physiology.

Hypothesis Testing:

Bacitracin potentiates the multiple influences of an optically lowered ΔΨ-plas-bact on a growing cell wall (i.e., increased cell wall autolysis, inhibited cell wall synthesis). This is especially relevant in gram positive bacteria such as MRSA, that do not have efflux pumps as resistance mechanisms for cell wall inhibitory antimicrobial compounds. The null hypothesis is μi - μ₂ = 0 and μ, - μ₃ ⁼ 0 where: a) μi is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control and; b) μ₂ is the same sub-lethal dosimetry from the NIMEL laser system on MRSA with the addition of bacitracin at resistant MIC just below effectiveness level and; μ, - μ₂ = 0 Will uphold that the addition of bacitracin produces no deleterious effect after sub-lethal

NIMEL irradiation, on normal growth of MRSA colonies. μ, - μ₂ > 0

Will uphold that the addition of bacitracin produces a deleterious effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies.

Results:

As shown in Figure 16, this experiment clearly showed that under sub-lethal laser parameters with the NIMELS system, μj - μ₂ >= 0, meaning that the addition of bacitracin produces a deleterious effect after sub-lethal NIMEL irradiation, on normal growth of MRSA colonies. In Figure 16, arrows point to MRSA growth or a lack thereof in the two samples shown. This indicates that bacitracin (independent of an efflux pump) is being potentiated by the NIMELS effect on ΔΨ-steady-bact of the MRSA.

Hence, the NIMELS laser and its concomitant optical ΔΨ-plas-bact lowering phenomenon is synergistic with cell wall inhibitory antimicrobials in MRSA. Without wishing to be bound by theory, this most likely functions via the inhibition of anabolic (periplasmic) ATP coupled functions as MRSA does not have efflux pumps for bacitracin.

EXAMPLE XII: NIMELS Dosimetry Calculations The examples that follow describe selected experiments depicting the ability of the

NIMELS approach to impact upon the viability of various commonly found microorganisms at the wavelengths described herein. The microorganisms exemplified include E. coli K- 12, multi-drug resistant E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Candida albicans, and Trichophyton rubrum. As discussed in more details supra, NIMELS parameters include the average single or additive output power of the laser diodes, and the wavelengths (870 nm and 930 nm) of the diodes. This information, combined with the area of the laser beam or beams (cm²) at the target site, provide the initial set of information which may be used to calculate effective and safe irradiation protocols according to the invention. The power density of a given laser measures the potential effect of NIMELS at the target site. Power density is a function of any given laser output power and beam area, and may be calculated with the following equations: For a single wavelength:

1) Power Density (W/cm²) = Laser Output Power Beam Diameter (cm²) For dual wavelength treatments:

2) Power Density (W/cm²) = Laser (1) Output Power + Laser (2) Output Power

Beam Diameter (cm²) Beam Diameter (cm²) Beam area can be calculated by either:

3) Beam Area (cm²) = Diameter (cm)² * 0.7854 or Beam Area (cm²) = Pi * Radius (cm)²

The total photonic energy delivered into the tissue by one NIMELS laser diode system operating at a particular output power over a certain period is measured in Joules, and is calculated as follows:

4) Total Energy (Joules) = Laser Output Power (Watts) * Time (Sees.)

The total photonic energy delivered into the tissue by both NJMELS laser diode systems (both wavelengths) at the same time, at particular output powers over a certain period, is measured in Joules, and is calculated as follows:

5) Total Energy (Joules) = [Laser(l) Output Power (Watts) * Time (Sees)] + [Laser (2) Output Power (Watts) * Time(Secs)]

In practice, it is useful (but not necessary) to know the distribution and allocation of the total energy over the irradiation treatment area, in order to correctly measure dosage for maximal NIMELS beneficial response. Total energy distribution may be measured as energy density (Joules/cm²). As discussed infra, for a given wavelength of light, energy density is the most important factor in determining the tissue reaction. Energy density for one NIMELS wavelength may be derived as follows: 6) Energy Density (Joules/ cm²) = Laser Output power (Watts) * Time (sees)

Beam Area (cm²)

7) Energy Density (Joule/cm²) = Power Density (W/cm²) * Time (sees) When two NJJVIELS wavelengths are being used, the energy density may be derived as follows:

8) Energy Density (Joules/ cm²) = Laser (l)Output power (Watts) * Time (sees) Beam Area (cm²)

+ Laser (2) Output power (Watts) * Time (sees)

Beam Area (cm²) or, 9) Energy Density (Joule/cm2) = Power Density (1) (W/cm²) * Time (Sees)

+ Power Density (2) (W/cm²) * Time (Sees) To calculate the treatment time for a particular dosage, a practitioner may use either the energy density (J/cm²) or energy (J), as well as the output power (W), and beam area (cm²) using either one of the following equations:

10) Treatment Time (seconds) = Energy Density

(Joules/cm²)

Output power Density (W/cm²)

11) Treatment Time (seconds) = Energy (Joules)

Laser Output Power (Watts)

Because dosimetry calculations such as those exemplified in this Example can become burdensome, the therapeutic system may also include a computer database storing all researched treatment possibilities and dosimetries. The computer (a dosimetry and parameter calculator) in the controller is preprogrammed with algorithms based on the above-described formulas, so that any operator can easily retrieve the data and parameters on the screen, and input additional necessary data (such as: spot size, total energy desired, time and pulse width of each wavelength, tissue being irradiated, bacteria being irradiated) along with any other necessary information, so that any and all algorithms and calculations necessary for favorable treatment outcomes can be generated by the dosimetry and parameter calculator and hence run the laser.

In the examples that follow, in summary, when the bacterial cultures were exposed to the NJJVIELS laser, the bacterial kill rate (as measured by counting Colony Forming Units or CFU on post-treatment culture plates) ranged from 93.7% (multi-drug resistant E. coli) to 100% (all other bacteria and fungi).

EXAMPLE XIJJ: BACTERIAL METHODS: NMELS TREATMENT PARAMETERS FOR IN VITRO £. COLI TARGETING

The following parameters illustrate the methods according to the invention as applied to E. coli, at final temperatures well below those associated in the literature with thermal damage.

A. Experiment Materials and Methods for E. coli K-12: E. coli Kl 2 liquid cultures were grown in Luria Bertani (LB) medium (25 g/L).

Plates contained 35 mL of LB plate medium (25 g/L LB, 15 g/L bacteriological agar). Culture dilutions were performed using PBS. All protocols and manipulations were performed using sterile techniques. B Growth Kinetics

Drawing from a seed culture, multiple 50 mL LB cultures were inoculated and grown at 37 ⁰C overnight The next morning, the healthiest culture was chosen and used to inoculate 5% into 50 mL LB at 37 ⁰C and the O D ₆oo was monitored over time taking measurements every 30 to 45 minutes until the culture was in stationary phase C Master Stock Production

Starting with a culture in log phase (O D _6Oo approximately 0 75), 10 mL were placed at 4⁰C 10 mL of 50% glycerol were added and was ahquoted into 20 cryovials and snap frozen in liquid nitrogen The cryovials were then stored at -8O⁰C D Liquid Cultures

Liquid cultures of E coli Kl 2 were set up as descπbed previously An aliquot of 100 μL was removed from the subculture and serially diluted to 1 1200 m PBS This dilution was allowed to incubate at room temperature approximately 2 hours or until no further increase m O D _6Oo was observed m order to ensure that the cells in the PBS suspension would reach a static state (growth) with no significant doubling and a relatively consistent number of cells could be ahquoted further for testing

Once it was determined that the Kl 2 dilution was in a static state, 2 mL of this suspension were ahquoted mto selected wells of 24-well tissue culture plates for selected NIMELS expeπments at given dosimetry parameters The plates were incubated at room temperature until ready for use (approximately 2 hrs)

Following laser treatments, 100 μl was removed from each well and serially diluted to 1 1000 resulting in a final dilution of 1 12x10⁵ of initial Kl 2 culture Ahquots of 3 x 200 L of each final dilution were spread onto separate plates m triplicate The plates were then incubated at 37 ⁰C for approximately 16 hours Manual colony counts were performed and recorded A digital photograph of each plate was also taken

Similar cell culture and kinetic protocols were performed for all NIMELS irradiation tests with S aureus and C albicans in vitro tests For example, C albicans ATCC 14053 liquid cultures were grown in YM medium (21g/L, Difco) medium at 37°C A standardized suspension was ahquoted into selected wells m a 24-well tissue culture plate Following laser treatments, lOOμL was removed from each well and serially diluted to 1 1000 resultmg m a final dilution of 1 5x10⁵ of initial culture 3x100 μL of each final dilution were spread onto separate plates The plates were then incubated at 37⁰C for approximately 16-20 hours Manual colony counts were performed and recorded A digital photograph of each plate was also taken T rubrum ATCC 52022 liquid cultures were grown m peptone-dextrose (PD) medium at 37 ⁰C A standardized suspension was aliquoted into selected wells in a 24 -well tissue culture plate Following laser treatments, ahquots were removed from each well and spread onto separate plates The plates were then incubated at 37 ⁰C for approximately 91 hours Manual colony counts were performed and recorded after 66 hours and 91 hours of incubation While control wells all grew the organism, 100% of laser-treated wells as described herein had no growth A digital photograph of each plate was also taken

Thermal tests performed on PBS solution, starting from room temperature Ten (10) Watts of NIMELS laser energy were available for use m a 12 mmute lasmg cycle, before the

10 temperature of the system is raised close to the critical threshold of 44°C

Table 11 Time & Tem erature measurements for In Vitro NIMELS Dosimetries

15 EXAMPLE XIV DOSIMETRY VALUES FOR NIMELS LASER WAVELENGTH 930 NM FOR E

COLIIN VITRO TARGETING

The instant experiment demonstrates that the NTMELS single wavelength λ = 930 nm is associated with quantitatable antibacteπal efficacy against E coli in vitro withm safe 20 thermal parameters for mammalian tissues Experimental data in vitro demonstrates that if the threshold of total energy into the system with 930 nm alone of 5400 J and an energy density of 3056 J/cm is met in 25% less time, 100% antibacterial efficacy is still achievable.

Table 12. Sub-thermal NIMELS (X= 930) Dosimetry for In Vitro E. coli Targeting

Experimental data in vitro also demonstrates that treatments using a single energy with λ = 930 nm has antibacterial in vitro efficacy against the bacterial species S. aureus within safe thermal parameters for mammalian tissues.

It is also believed that if the threshold of total energy into the system of 5400 J and an energy density of 3056 J/cm² is met in 25% less time with S. aureus and other bacterial species, that 100% antibacterial efficacy will still be achieved.

Table 13. Sub-thermal NMELS (X= 930) Dosimetry for In Vitro S. aureus Targeting

Experimental in vitro data also showed that the NTMELS single wavelength of X = 930 nm has anti-fungal efficacy against in vitro C. albicans at ranges within safe thermal parameters for mammalian tissues.

It is also believed that if the threshold of total energy into the system of 5400 J and an energy density of 3056 J/cm² is met in 25% less time, that 100% antifungal efficacy will still be achieved.

Table 14. Sub-thermal NTMELS (X= 930) Dosimetry for In Vitro C. albicans Targeting

EXAMPLE XV: DOSIMETRY VALUES FOR NMELS LASER WAVELENGTH 870 NM IN VITRO

Experimental in vitro data also demonstrates that no significant kill is achieved up to a total energy of 7200 J, and energy density of 4074 J/cm² and a power density of 5.66 0 W/cm² with the wavelength of 870 nm alone against E. coli.

Table 15. E. coli Studies- Single wavelength X = 870 nm

10 Comparable results using radiation having X = 870 nm alone were also observed with S. aureus.

EXAMPLE XVI: NIMELS UNIQUE ALTERNATING SYNERGISTIC EFFECT BETWEEN 870 NM

AND 930 NM OPTICAL ENERGIES

15

Experimental in vitro data also demonstrates that there is a greater than additive effect between the two NIMELS wavelengths (X= 870 nm and 930 nm) when they are alternated (870 nm before 930 nm). The presence of the 870 nm NMELS wavelength as a first irradiance has been found to enhance the effect of the antibacterial efficacy of the

20 second 930 nm NMELS wavelength irradiance.

Experimental in vitro data demonstrates that this synergistic effect (combining the 870 nm wavelength to the 930 nm wavelength) allows for the 930 nm optical energy to be reduced. As shown herein, the optical energy was reduced to approximately 1/3 of the total energy and energy density required for NMELS 100 % E. coli antibacterial efficacy, when

25 the (870 nm before 930 nm) wavelengths are combined in an alternating manner.

Experimental in vitro data also demonstrates that this synergistic mechanism can allow for the 930 nm optical energy (total energy and energy density) to be reduced to approximately 1/2 of the total energy density necessary for NMELS 100% E. coli antibacterial efficacy if equal amounts of 870 nm optical energy are added to the system

30 before the 930 nm energy at 20% higher power densities. Table 16. E. coli data from Alternating NIMELS Wavelengths

This synergistic ability is significant to human tissue safety, as the 930 nm optical energy heats up tissues at a greater rate than the 870 nm optical energy, and it is beneficial to a mammalian system to produce the least amount of heat possible during treatment.

It is also believed that if the NIMELS optical energies (870 nm and 930 nm) are alternated in the above manner with other bacterial species, that the 100% antibacterial effect will be essentially the same.

EXAMPLE XVII: NTMELS UNIQUE SIMULTANEOUS SYNERGISTIC EFFECT BETWEEN λ= 870

NM AND λ= 930 NM OPTICAL ENERGIES

Experimental in vitro data also demonstrates that there is a greater than additive effect between the two NIMELS wavelengths (870 nm and 930 nm) when they are used simultaneously (870 nm combined with 930 nm). The presence of the 870 nm NIMELS wavelength and the 930 nm NTMELS wavelength as a simultaneous irradiance absolutely enhances the effect of the antibacterial efficacy of the NEVIELS system.

In vitro experimental data (see, for example, Tables LX and X below) demonstrated that by combining λ = 870 nm and λ = 930 nm (in this example used simultaneously) effectively reduces the 930 nm optical energy and density by about half of the total energy and energy density required when using a single treatment according to the invention.

Table 17. E. coli data from Combined NEvIEL Wavelengths

This simultaneous synergistic ability is significant to human tissue safety, as the 930 nm optical energy, heats up a system at a greater rate than the 870 nm optical energy, and it is beneficial to a mammalian system to produce the least amount of heat possible during treatment

Thus, NIMELS wavelengths (λ= 870 nm and 930 nm) may be used to achieve antibacterial and anti-fungal efficacy in an alternating mode or simultaneously or in any combination of such modes thereby reducing the exposure at the λ = 930 associated with temperature increases which are minimized

Expeπmental in vitro data also demonstrates that when E coli is irradiated alone with a (control) wavelength of λ = 830 nm, at the following parameters, the control 830 nm laser produced zero antibactenal efficacy for 12 minutes irradiation cycles, at identical parameters to the minimum NIMELS dosimetry associated with 100% antibactenal and anti-fungal efficacy with radiation of λ = 930 nm

Table 18 E coli Single Wavelength λ = 830 nm

Expeπmental in vitro data also demonstrates that when applied at safe thermal dosimetries, there is little additive effect when using radiance of λ = 830 nm in combination with λ = 930 nm The presence of the 830 nm control wavelength as a first irradiance is far inferior to the enhancement effect of the 870 nm NTMELS wavelength in producing synergistic antibacterial efficacy with the second 930 nm NIMELS wavelength

Table 19 E coli data from Substituted alternating 830 nm control Wavelength

Experimental in vitro data also demonstrates that when applied at safe thermal dosimetπes, there is less additive effect with the 830 nm wavelength, and the NIMELS 930 ran wavelength when they are used simultaneously In fact, expeπmental in vitro data demonstrates that 17% less total energy, 17% less energy density, and 17% less power density is required to achieve 100 % E coli antibacteπal efficacy when 870 nm is combined simultaneously with 930 nm vs the commercially available 830 nm This, again, substantially reduces heat and harm to an in vivo system being treated with the NIMELS wavelengths

Amount of Bacteπa Killed

In vitro data also showed that the NIMELS laser system in vitro is effective (within thermal tolerances) against solutions of bacteπa containing 2,000,000 (2 x 10⁶) Colony Forming Units (CFU's) of E coli and S aureus This is a 2x increase over what is typically seen in a 1 gm sample of infected human ulcer tissue Brown et al reported that miciobial cells in 75% of the diabetic patients tested were all at least 100,000 CFU/gm, and m 37 5% of the patients, quantities of microbial cells were greater than 1,000,000 (lxlO⁶)CFU (see Brown et al , Ostomy Wound Management, 401 47, issue 10, (2001), the entire teaching of which is incorporated herein by reference)

Thermal Parameters

Expeπmental in vitro data also demonstrates that the NDVIELS laser system can accomplish 100% antibacteπal and anti-fungal efficacy withm safe thermal tolerances for human tissues

EXAMPLE XVIII THE EFFECTS OF LOWER TEMPERATURES ON NEVIELS Cooling of Bacteπal species Expeπmental in vitro data also demonstrated that by substantially loweπng the starting temperature of bacteπal samples to 4°C for two hours m PBS before lasing cycle, that optical antibacterial efficacy was not achieved at any currently reproducible antibacteπal energies with the NIMELS laser system

EXAMPLE XIX MRSA/ ANTIMICROBIAL POTENTIATION

This example shows the use of NIMELS wavelengths (λ = 830 nm and 930nm) in m vitro targeting of MRSA to increase antimicrobial sensitivity to methicillin Four separate expeπments have been performed The data sets for these four experiments are presented in the tables that follow See, also, Figure 17, which shows (a) the synergistic effects of NIMELS with methicillin, penicillin and erythromycin in growth inhibition of MRSA colonies, data show that penicillin and methicillin is being potentiated by sub-lethal NEVIELS dosimetry by inhibiting the Bacteπal Plasma Membrane Proton-motive force (Ap- plas-Bact) thereby inhibiting peptidoglycan synthesis anabolic processes that are co- targeted with the drug, and (b) that erythromycin is potentiated to a greater extent, because the Nimels effect is inhibiting the Bacteπal Plasma Membrane Proton-motive foice (Ap- plas-Bact) that supplies the energy for protein synthesis anabolic processes and erythromycin resistance efflux pumps General Methods for CFU counts

Table 21

Table 22. MRSA Dosimetry Progression 11-06-06 Experiment #1

First lasing procedure : Both 870 and 930

Second lasin rocedure 930 alone

Experiment 1 - design:

Eight different laser dosages were used to treat a saline-suspension of logarithmically growing MRSA, labeled Al to Hl .

10 The treated and a control untreated suspension were diluted and plated in triplicate on trypic soy agar with or without 30 μg/ml methicillin. After 24hrs of growth at 37 ⁰C colonies were counted.

CFU (colony forming units) were compared between the plates with and without methicillin for both control (untreated) and treated MRSA.

Experiment 1 - results:

Conditions Dl through Hl showed a similar reduction in CFU on the methicillin plates in treated and untreated samples.

Conditions Al, Bl and Cl showed 30%, 33%, or 67% fewer CFU in the laser treated samples compared to the untreated controls, respectively.

This indicates that the treatments performed on sample Al, Bl and Cl sensitized the MRSA to the effects of methicillin.

Table 23. MRSA Data Progression

Table 24. MRSA Dosimetry Progression

MRSA Dosimetry

Progression

11-07-06

First lasmg procedure Both

870 and 930

Second lasmg procedure 930 alone

Expeπment 2 - des₁gn

Eight different laser dosages based on an effective dose established in expeπment 1 and previously were used to treat a salme-suspension of logarithmically growing MRSA, labeled A2 to H2

The treated and a control untreated suspension were diluted and plated m triplicate on trypic soy agar with or without 30 μg/ml methicillm After 24hrs of growth at 37 ⁰C colonies were counted

Expeπment 2 — results

Compaπson of CFU on plates with and without methicillm showed a significant increase m the effectiveness of methicillm m all laser treated samples with the exception of A2 and B2 This data is summarized m tabular form below

Table 25

Grouping Fold increase in methicillin sensitivity

A2 0 84

B2 0 91

C2 3 20

D2 2 44

E2 4 33

F2 2 13

G2 3 47

H2 1 62

Table 26 MRSA Data Progression

Table 27 Outlined Protocol for MRSA Study

Time _ ,

. . Task

(hrs)

Inoculate overnight culture

50 ml directly from glycerol stock 1 -18

Set up starter cultures

Three dilutions 1 50, 1 125, 1 250

T -4

Monitor ODrøo of starter cultures

Preparation of plating culture

At 10 00am, the culture which is at OD₆₀₀ = 1 0 is diluted 1 300 in PBS (50 mis final volume) and stored at RT for 1 hour T 0 (Room temp should be -25 °C)

Seeding of 24-well plates (8 plates in total) T +1 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR (8 24-well plates total) _ _ Dilution of treated samples

„ After laser treatment, 100 μl from each well is diluted serially to a final dilution of 1 1000 m PBS

Plating of treated samples

100 μl of final dilution is plated m qumtuphcate (5X) on TSB agar with and without 30 μg/ml methicillin (10 TSB plates per well)

Plates are incubated at 37 ⁰C 18-24hrs T +24 Colonies are counted on each plate (160 plates total)

Table 28 MRSA Dosimetry Progression

MRSA Dosimetry Progression

11-09-06

First lasing procedure Both 870 and 930

Second lasmg procedure 930 alone

Expeπment 3 - design

Eight different laser dosages based on an effective dose established in expeπments 1 and 2 and previously were used to treat a salme-suspension of logarithmically growing MRSA, labeled A3 to H3

The treated and a control untreated suspension were diluted and plated in pentuplicate on trypic soy agar with or without 30 μg/ml methicillin

10 After 24hrs of growth at 37 ⁰C colonies were counted Experiment 3 - results:

Comparison of CFU on plates with and without methicillin showed a significant increase in the effectiveness of methicillin in all laser treated samples. This data is summarized in tabular form below.

Table 29.

Grouping Fold increase in methicillin sensitivity

A3 1.98

B3 1.62

C3 1.91

D3 2.59

E3 2.09

F3 2.08

G3 3.16

H3 2.97

Table 30. MRSA Data Progression

Table 31 Outlined Protocol

Table 32 MRSA Dosimetry Progression

MRSA Dosimetry Progression First lasing procedure Both 870 and 930 Second lasin rocedure 930 alone

Independent Report for MRSA studies Expeπment 4 - design

Two different laser dosages based on an effective dose established in previous expeπments were used to treat a salme-suspension of logarithmically growing MRSA, labeled A4 to F4

10 The treated and a contiol untreated suspension were diluted and plated in pentuplicate on trypic soy agar with or without 30 μg/ml methicillin (Groups A4 and B4), 0 5 μg/ml penicillin G (Groups C4 and D4) or 4 μg/ml erythromycin (Groups E4 and F4)

After 24hrs of growth at 37 ⁰C colonies were counted Expeπment 4 - results

Laser treatment increases sensitivity of MRSA to each antibiotic tested by several fold This data is summarized below

Table 33

Series Drug

A4 Methicillin

B4 Methicillin

C4 Penicillin

D4 Penicillin

E4 Erythromycin

F4 Erythromycin

Table 34

Grouping Fold increase in antibiotic sensitivity

A4 2 19 B4 2 63

C4 2 21 D4 3 45

E4 50 50 F4 9 67

EXAMPLE XX: Non-thermal NIMELS Interaction Evidence for non-thermal NIMELS interaction:

It was demonstrated through experimentation (in vitro water bath studies), that the temperatures reached in the in vitro NIMELS experimentation, were not high enough in and of themselves to neutralize the pathogens.

In the chart that follows, it can clearly be seen that when simple E. coli Bacteria were challenged at 47.5 C continuously for 8 minutes in a test tube in a water bath, they achieved 91% growth of colonies. Therefore, it was demonstrated essentially that the NIMELS reaction is indeed photo-chemical in nature, and occurs in the absence of exogenous drugs and/or dyes.

Table 36.

Example XXI: Laser Treatment for Microbial Reduction and Elimination of Nasal Colonization of MRSA

The Nomir Near Infrared Microbial Elimination Laser System (NOVEON™ Model

1120 dual-wavelength diode laser was employed for this study. The laser operates in continuous wave format at two wavelengths, 870 ran (+/- 5 nm) and 930 ran (+/- 5 nm). This device is a class II non-significant risk laser device. The laser sources of this device are semiconductor laser arrays that are optically coupled to form a single fiber laser output. The delivery system consists of a single flexible optical fiber. The device delivers continuous wave laser light only.

The device is designed specifically to effect microbial cell optical destruction, while preserving and without substantial damage optically or thermally to the human tissue at the infection site being irradiated. In that regard, the NOVEON™ system was designed to harness the known photo-lethal characteristics of these precise energies to kill pathogenic microorganism at far lower energy levels and heat deposition than is generally necessary to kill pathogens using laser-based thermal sterilization means. Using exposure to the dual wavelength infrared NOVEON™ laser, at temperature levels inherently not lethal to the organism, we had accomplished in vitro successful reversal of MRSA resistance to Methicillin, Penicillin, Erythromycin and Tetracycline. It has also been shown in vitro, that MRSA that has been exposed to a sublethal dose by the NOVEON™ laser will become sensitive to antibiotics to which it was previously resistant.

Currently, topical intra-nasal antimicrobial agents are recognized as the preferred method for preventing (distal-site) infections because of their demonstrated effectiveness and widespread desire to minimize the use of systemic antimicrobials.

Thus, the design of this protocol includes a number of important factors have been considered. Foremost is the need to assure that the amount of energy used in the Nares is safe for the nasal and nares tissues. Furthermore, significant human and histological tests have been done with the Noveon laser in the areas that the study is treating

HUMAN STUDIES

Initial studies were performed to chart and ensure the thermal safety of laser energies on human dermal tissues. Exposure of dermal surfaces to both 870 nm and 930 nm simultaneously with a combined Power Density of 1.70 W/cm² for up to 233 seconds, results in a skin surface temperature of 100⁰F as measured with a laser infrared thermometer.

Exposure of dermal surfaces to 930 nm alone at a Power Density of 1.70 W/cm² for up to 142 seconds, results in a skin surface temperature of 97°F. At or above these doses to dermal infection sites, pain can result. It is therefore desirable from a standpoint of patient comfort not to exceed these doses. Table 37. Dosimetry Simultaneously Using 870 and 930 Nanometers

Additional testing of the device on the epithelial tissue of humans was conducted using a specially prepared dispersion tip designed to be inserted in the nares. Using a dispersion tip, laser energy was delivered to the nostrils circumferentially by an optical fiber (connected to the NOVEON™ laser) that terminates in a central diffusing tip. This was placed within the inner lumen of the nostril (nares).

A cylindrical diffusing optical fiber tip for near infrared light delivery was fabricated specifically for uniform illumination of a length of 1.5 cm, to then be placed in a transparent catheter (of given width) to prevent placement too far anteriorly in the nostril, and guarantee a uniform power density at all tissues proximal to the catheter within the nostril.

The tip included an optically transmissive, light diffusing, fiber tip assembly having an entrance aperture through a proximal reflector, a radiation-scattering, transmissive material (e.g. a poly-tetrafluoroethylene tube) surrounding an enclosed cavity (e.g. a cylindrical void filled with air or another substantially non-scattering, transparent medium), and a distal reflective surface. As radiation propagates through the fiber tip assembly, a portion of the radiation is scattered in a cylindrical (or partly cylindrical) pattern along the distal portion of the fiber tip. Radiation, which is not scattered during this initial pass through the tip, is reflected by at least one surface of the assembly and returned through the tip. During this second pass, the remaining radiation, (or a portion of the returning radiation), is scattered and emitted from the proximal portion of the tube. Multiple additional reflections off of the proximal and distal reflectors provide further homogenization of the intensity profile. Preferably the scattering medium has a prescribed inner diameter. This inner diameter of the scattering material is designed such that the interaction with this material and the multiple reflections off of the cavity reflectors interact to provide a substantially proscribed axial distribution of laser radiation over the length of the tip apparatus. Suitable choices of tip dimensions provide control over the emitted axial and azimuthal energy distributions.

To first document safety with the instrumentation, samples of turkey muscle (shown to be a suitable model for nasal mucosa, were irradiated with the above described dispersion tip. The maximum temperature attained during this experiment was 33.9 degrees Centigrade. Further, no specimen showed any burning or necrosis, despite use of exposure times that were double than any anticipated for use in human subjects.

Table 39. Dosimetry Simultaneously Using 870 and 930 Nanometers

Table 40. Dosimetry at 930 Nanometers

Studies have shown that there are five factors to consider regarding energy absorption and heat generation by the "y" emissions of near infrared diode lasers. These factors are: wavelength and optical penetration depth of the laser; absorption characteristics of exposed tissue; temporal mode (pulsed or continuous; exposure time; and power density of the laser beam.

Diode lasers in the near infrared range have a very low absorption coefficient in water; hence, they achieve relatively deep optical penetration in tissues that contain 80% water (such as the dermis, the oral mucosa, bone and the gingiva. With conventional near infrared diode soft tissue lasers, the depth of penetration (before photon absorption) of the greatest amount of the incident energy is about 1.5 cm. This allows the near infrared laser energy to pass through water with minimal absorption, producing thermal effects deeper in the tissue and the photons are absorbed by the deeper tissue pigments. This photobiology allows for controlled, deeper soft-tissue irradiation and decontamination, as the photons that emerge from the dispersion tip in a uniform dosimetry from the diffusing tip absorbed by blood and other tissue pigments.

Approached from other known dosimetry perspectives, if the conventional Power Equation is applied to in vivo NIMELS dosimetries [Power (Watts) = Work/ Time], the following examples illustrate the Power differences between current therapies: Photoablative dosimetry = 1000 J/cm² in l/l,000^Λ of a second; Thermal vaporizing dosimetry = 1000 J/cm² in 1 second; and NIMELS decontamination dosimetry = 500 J/cm² in 360 seconds.

This investigational protocol was designed to demonstrate that the Noveon Laser treatment is able to produce reduction in Nasal carriage of MRSA in patients with previously "culture positive" history. This investigational protocol was an open-label study of subjects who are colonized with MRSA in the nares (nostril). The study was done in two parts.

PART ONE Subjects

In this human study, three arms were produced. Subjects with a previous "culture positive" history who were found to be positive for MRSA colonization in the nares were randomized to one of three treatment groups: Arm # 1 : 1/3 of the subjects were treated with laser alone; Arm # 2: 1/3 of the subjects were be treated with topical H₂O₂ and then the laser after two minutes. This was done on day 1 and again on day three; and Arm #3: 1/3 of the subjects were treated with the laser and then a topical antibiotic three times a day for five days. Prior to enrollment in this study, prospective subjects met all of the following criteria: age >18 years and ≤IO years of age; previous positive MRSA culture; negative urine pregnancy test or post-menopausal for one year; willing to comply with study requirements, including return visits and self-application of topical antibiotics; and willing to provide informed consent to participate. Prospective subjects were excluded from this study if any of the following criteria were met: pregnancy; patients who are severely immunocompromised (such as may occur in AIDS, renal transplant regimens, immunosuppressed states consequent to malignancy or agents used in rendering oncologic care, or who suffer from end stage renal disease); diabetic patients; allergy to antimicrobials being used in the study (group 3). The exclusion of such groups in this instance was solely for purposes of performing a controlled clinical study, and it is particularly noted that the above exclusion groups are actually considered good candidates for the phototherapeutic treatments described herein, wherein such patients would actually benefit from therapeutic bacteπal photodamage in that reduced systemic doses of antibiotics could be given and infection sites could be better cleared Study Procedures: All participants underwent an initial quantitative assessment for nasal carriage of S aureus, during their first visit Each participant had the antenor nares (each nostπl) sampled for culture with a circular movement (three rotations on each side) of a sterile wood applicator plain Rayon® tipped swab m each nostπl and placed in a labled tube 2 ml of room temp phosphate buffered salme was placed m the tube after the removal of the swab (to completely cover the swab m the tube) Each swab was then placed back in the tube and the tube was then vortexed for 15 seconds to disperse isolates of MRSA and/or MSSA into the PBS solution Ahquots of PBS from the tube were plated m the following manner 100 μl from each tube was lawn plated m triplicate (3X) on selective Chromogemc MRSA And MSSA agar Plates were placed in incubator within 30 minutes of the plating procedure, and colonies were counted manually, and recorded 18 hrs after plating

On day one of the study, all subjects underwent this exact procedure in arms 1 and 2 of the study two minutes before the laser procedure They again were sampled in the same manner 2 minutes after the laser procedure In the third arm of the study, they were swabbed two minutes before the procedure and the first antibiotic administration was completed after the laser therapy The post/laser swab sample was taken for this arm 20 mm later

On day three of the study, all subjects from arms one two and three underwent the exact same procedures as day 1 of the study

On day five all subjects from all arms underwent just one swabbing per nostril with the exact same sampling procedure one time

Application of H202 3% OTC hydrogen peroxide was applied to a cotton pledget for application to the subject pπor to irradiation This was inserted m the nose for 120 seconds and then removed The subjects were then given doses of phototherapeutic near infrared radiation as descπbed Application of generic topical Antimicrobial The subjects were first given doses of phototherapeutic near infrared radiation as descπbed Subsequently, 2% erythromycin paste was applied to a cotton tipped swab for application to the subject following irradiation The swab was inserted approximately 1 cm in to the antenor nares and rotated 360 degrees several times and removed Patients were instructed to perform the exact application procedure 3 times a day for the remaining 5 days Treatment Description

The NOVEON™ laser was used for two (2) six-minute treatments in each nostril on day (1) and day (3) of the study. The dosimetries used are shown in the Table TT, below. The laser was calibrated before the first treatment of the day. Intermittent temperature testing of the treatment site was performed on each subject using a noncontact infrared thermometer (Raytek Minitemp), 30-60 second intervals. If a temperature of 1 10 F degrees was reached, or the patient complained of pain, the laser treatment was interrupted and only resumed when the patient was comfortable. Inturruption only occurred once in 40 treatments (20 nostrils x 2 treatments over three days), and was resumed 30 seconds later to completion.

Table 41 power

Laser density Length Diameter Area Trans set power nm W/cm2 mm mm cm2 percent W

930 0.46 10 12 3.77 80 2.17

870 0.185 10 12 3.77 80 0.87

930 0.277 10 12 3.77 80 1.30

80

930 0.405 10 12 3.77 80 1.91

870 0.16 10 12 3.77 80 0.75

930 0.243 10 12 3.77 80 1.14 both 0.54 10 12 3.77 80 2.54 both 0.46 10 12 3.77 70 2.48 Quantitative Assessments to measure Change in MRSA and MSSA colonies

The following Tables 42-44 represent the mean values of the triplicate CFU counts and plating of each swab from each nostril, pre and post laser therapy (for this data set the mean is the sum of the observed and counted CFU 's per plate, divided by the number of counted plates). Table 42

Table 43

Table 44

Third Analysis

Swab left right s. aureus MRSA s. aureus MRSA

Patient Average Average Average Average

Laser Alone

01-003 193 0 359 0

01-006 387 0 645 5

01-010 22 0 387 0

Laser w/ Peroxide

01-002 1 0 1 0

01-004 868 827 586 563

01-007 28 0 52 0

01-009 0 0 3493 3648

Laser w/ Erythromycin

01-001 0 0 0 0

01-005 0 0 0 0

01-008 0 0 0 0

Results We treated performed 36 treatments of 10 patients (20 infection sites) with zero negative sequelae from the laser m identified MRSA earners based on a physician's evaluation of all the patients 2 days following the second laser therapy

Patients 1 and 8 (m the laser plus antibiotic arm) were not treated a second time, as there was no growth of S aureus or MRSA colonies present on the pre-test swabs These patients were dismissed from the study by the pπncipal investigator The Laser alone arm was showed mconsequential colony reduction in MRSA and MSSA colomes in the nares The Laser plus H₂O₂ arm may have had some transient benefit in some of the patients, but no obvious long-term efficacy The remaining patient (01-005) in the Laser/erythromycm that began the study with culturable S aureus and MRSA showed a remarkable reduction in culturable bacterial from the colonization site as the treatments progressed, to the point of MRSA and MSSA eradication m both nostπls In this patient, the combination of near infrared bactenal photodamage and topical antibiotics eradicated the MRSA infection The heavily colonized nostπl showed at least a 3 log reduction of bioburden, and resulted m no culturable bacteπa, and the moderately colonized nostπl showed at least a 2 log reduction of bioburden, and resulted in no culturable bacteπa Notably, the MRSA colony in that patient was not sensitive to erythromycin pπor to phototherapy with the NOVEON™ laser system PART TWO

A second human study was conducted, to further evaluate the therapeutic potential of the NOVEON™ laser system, including its ability to reverse drug resistance m bacteπa The study was conducted in a similar manner as Part One, above Outcome measures assessed included both laboratory study and clinical observations

Positive anterior nares cultures were obtained in six patients (12 nostπls) having nasal colonization of MRSA or MSSA, before initiating bacterial photodamage through

10 doses of phototherapeutic near infrared radiation One patient had MRSA only, 3 had MSSA only, and 2 had both MRSA and MSSA All MRSA and MSSA were cultured and verified to be resistant to erythromycin

Application of Topical Antimicrobial Antimicrobial paste (generic 2% erythromycin) was 15 placed on a cotton tipped swab for application after phototherapeutic near mfrared radiation The swab was inserted approximately 1 cm in to the anteπor nares of the subject, rotated 360 degrees several times and removed The application of erythromycin was maintained for 3 times a day for the remainder of the study

The laser was calibrated before the first treatment of the day and between each 20 patient The NOVEON™ laser was used for four six-minute treatments of the nares at the following sets of dosimetries (Tables 45), which were evaluated for safety in previous studies Utilizing a 10 cm flat-top diffuser, each patient underwent exposure with the Noveon for 7 minutes (energy density - 207 J/cm2) to each anterior nostril on Day 1 and on Day 3 The treatment was divided into two parts, an approximately 3 -minute exposure using 25 a combination of 870 nm and 930 nm and an approximately 3-mmute exposure of 930 run alone Temperatures of the nares were recorded every 30 seconds with an IR temperature thermometer

Table 45

power set set IRB

Laser density Length Diameter Area Trans power Laser power Power nm W/cm2 mm mm cm2 percent W Amp W ratio

930 0 46 10 12 3 77 80 2 17 5 95 2 17 1 00

870 0 185 10 12 3 77 80 0 87 4 65 0 87 0 40

930 0 277 10 12 3 77 80 1 30 4 85 1 30 0 60 Bacteriology

Quantitative cultures from each nostril were obtained and plated in triplicate on chromogenic agar before and 20 minutes after exposure on day 1 and day 3. A final culture was taken on day 5. Anterior nares specimens were collected on rayon-tipped swabs, and stored in Amies liquid transport medium. The nasal swab was plated on Columbia colistin- nalidixic agar (CNA) with 5% sheep blood, then incubated 18 to 48 hours at 35⁰C in 5% CO2. S. aureus was identified by colony morphology and Staphaurex™ latex agglutination test (Murex Biotech Limited, Dartford, Kent, UK). Samples were frozen and stored at - 20⁰C. Results:

The Erythromycin resistant MRSA was completely cleared by culture in all 3 carriers, as was the E-mycin resistant MSSA in four of the five (5) carriers after the second laser treatment on day 3 and remained clear on day 5. In one patient the E-mycin resistant MSSA (baseline count > 1000 CFU's) showed a 3-log reduction in MSSA on the day 5 culture. No sequelae or adverse events were observed. The average maximum temperature of the nares reached in all patients was 99F.

Conclusions:

NOVEON™ laser exposure at a non-damaging energy density and approximately physiologic temperatures, re-sensitized erythromycin resistant MRSA and MSSA to 2% generic erythromycin paste. Photodamage to the organism results in sensitivity to antibiotics in otherwise drug resistant strains. The NOVEON™ laser system provides for local reduction of drug resistant microbes and a concomitant reduction of bio-burden in: e.g., wounds, mucosal or cutaneous tissues, and other colonized or infected areas such as surgical sites and tissue/medical device interfaces, which are prone to contamination particularly by nosocomial strains of microbes frequently having multidrug resistance phenotypes.

Exemplary NIMELS Systems

Figure 17 illustrates a schematic diagram of a therapeutic radiation treatment device according one embodiment of the present disclosure. The therapeutic system 110 includes an optical radiation generation device 112, a delivery assembly 114, an application region

116, and a controller 118. According one aspect of the present disclosure, the optical radiation generation device (source) includes one or more suitable lasers, Ll and L2 A suitable laser may be selected based on a degree of coherence In exemplary embodiments, a therapeutic system can include at least one diode laser configured and arranged to produce an output m the near infrared region Suitable diode lasers can include a semiconductor mateπals for producing radiation in desired wavelength ranges, e g , 850nm-900nm and 905nm-945nm Suitable diode laser configurations can include cleave-coupled, distπbuted feedback, distπbuted Bragg reflector, vertical cavity surface emitting lasers (VCSELS), etc

With contmued reference to Figure 17, in certain embodiments the delivery assembly 114 can generate a "flat-top" energy profile for uniform distribution of energy over large areas For example, a diffuser tip 10, may be included which diffuses treatment light with a uniform cylindrical energy profile in an application region 116 (e g a nasal cavity as descπbed in the example above) As noted, the optical radiation generation device 1 12 can include one or more lasers, e g , laser oscillators Ll and L2 In exemplary embodiments, one laser oscillator can be configured to emit optical radiation m a first wavelength range of 850 nm to 900 nm, and the other laser oscillator can be configured to emit radiation m a second wavelength range of 905 nm to 945 nm In certain embodiments, one laser oscillator is configured to emit radiation m a first wavelength range of 865 nm to 875 nm, and the other laser oscillator 28 is configured to emit radiation m a second wavelength range of 925 nm to 935 nm The geometry or configuration of the individual laser oscillators may be selected as desired, and the selection may be based on the intensity distributions produced by a particular oscillator geometry or configuration

With continued reference to Figure 17, m certain embodiments, the delivery assembly 114 includes an elongated flexible optical fiber 118 adapted for delivery of the dual wavelength radiation from the oscillators 26 and 28 to diffuser tip 10 to illuminate the application region 116 The delivery assembly 14 may have different formats (e g , including safety features to prevent thermal damage) based on the application requirements For example, in one form, the delivery assembly 114 or a portion thereof (e g tip 10) may be constructed with a size and with a shape for inserting into a patient's body In alternate forms, the delivery assembly 114 may be constructed with a corneal shape for emitting radiation in a divergmg-conical manner to apply the radiation to a relatively large area Hollow waveguides may be used for the delivery assembly 114 m certain embodiments Other size and shapes of the delivery assembly 14 may also be employed based on the requirements of the application site Lti exemplary embodiments, the delivery assembly 114 can be configured for free space or free beam application of the optical radiation, e g , making use of available transmission through tissue at NEVIELS wavelengths descπbed herein For example, at 930nm (and to a similar degree, 870nm), the applied optical radiation can penetrate patient tissue by up to 1 cm or more Such embodiments may be particularly well suited for use with in vivo medical devices as descnbed herein

In an exemplary embodiment, the controller 118 includes a power lirmter 124 connected to the laser oscillators Ll and L2 for controlling the dosage of the radiation transmitted through the application region 116, such that the time integral of the power density of the transmitted radiation per unit area is below a predetermined threshold, which is set up to prevent damages to the healthy tissue at the application site The controller 1 18 may further mclude a memory 126 for storing treatment information of patients The stored information of a particular patient may include, but not limited to, dosage of radiation, (for example, including which wavelength, power density, treatment tune, skin pigmentation parameters, etc ) and application site information (for example, including type of treatment site (lesion, cancer, etc ), size, depth, etc )

In an exemplary embodiment, the memory 126 may also be used to store information of different types of diseases and the treatment profile, for example, the pattern of the radiation and the dosage of the radiation, associated with a particular type of disease The controller 118 may further include a dosimetry calculator 128 to calculate the dosage needed for a particular patient based on the application type and other application site information input mto the controller by a physician In one form, the controller 118 further mcludes an imaging system for imaging the application site The imaging system gathers application site information based on the images of the application site and transfers the gathered information to the dosimetry calculator 128 for dosage calculation A physician also can manually calculate and input information gathered from the images to the controller 118

As shown in Figure 17, the controller may further include a control panel 130 through which, a physician can control the therapeutic system manually The therapeutic system 10 also can be controlled by a computer, which has a control platform, for example, a WINDOWS ™ based platform The parameters such as pulse intensity, pulse width, pulse repetition rate of the optical radiation can be controlled through both the computer and the control panel 30

Figures 18a-l 8d show different temporal patterns of the optical radiation that can be delivered from the therapeutic system to the application site The optical radiation can be delivered m one wavelength range only, for example, in the first wavelength range of 850 nm to 900 run, or m the range of 865 nm to 875 nm, or m the second wavelength range of 905 nm to 945 nm, or m the range of 925 nm to 935 nm, as shown m Figure 18a The radiation in the first wavelength range and the radiation m the second wavelength range also can be multiplexed by a multiplex system installed in the optical radiation generation device 112 and delivered to the application site m a multiplexed form, as shown in Figure 18b In an alternative form, the radiation in the first wavelength range and the radiation m the second wavelength range can be applied to the application site simultaneously without passing through a multiplex system Figure 18c shows that the optical radiation can be delivered in an intermission-alternating manner, for example, a first pulse m the first wavelength range, a second pulse in the second wavelength range, a third pulse in the first wavelength range agam, and a fourth pulse in the second wavelength range again, and so on The interval can be CW (Continuous Wave), one pulse as shown m Figure 18c, or two or more pulses (not shown) Figure 18d shows another pattern m which the application site is first treated by radiation m one of the two wavelength ranges, for example, the first wavelength range, and then treated by radiation in the other wavelength range The treatment pattern can be determined by the physician based on the type, and other information of the application site

Fiber diffusion Tip

As noted above, in some embodiments, treatment system 110 employs a diffusion tip 10 to diffuse therapeutic treatment light delivered from a therapeutic light source by optical fiber 118 The tip operates to provide a desired illumination profile (i e emitted intensity profile) at the application region 116 For example, as descπbed above, m embodiments where treatment light is applied to the nares, a substantially uniform cylindrical illumination profile is desirable Other embodiments of tip 10 may be used to direct treatment light to other areas such as tissue spaces (e g the peπodontal pocket or within a joint e g m an orthopedic surgical procedure), interfaces between body tissue and

- I l l - other surfaces (e g the surface of an implantable medical device), over a wide area such as a dermal surface, etc

The following describes several embodiments of diffuser tips suitable for use with the treatment techniques and devices descπbed herein

In one embodiment, diffuser tip 10 is an optically transmissive, light diffusing, fiber tip assembly having an entrance aperture through a proximal reflector, a radiation- scattering, transmissive mateπal surrounding an enclosed void (e g a cylindrical cavity), and a distal reflective surface is disclosed As radiation propagates through the fiber tip, a portion of the radiation is scattered in a cylindrical (or partly cylindrical) pattern along the distal portion of the fiber tip Radiation, which is not scattered during this initial pass through the tip, is reflected by at least one surface of the assembly and returned through the tip During this second pass, the remaining radiation, (or a portion of the returning radiation), is scattered and emitted from the proximal portion of the tube Multiple additional reflections off of the proximal and distal reflectors provide further homogemzation of the intensity profile Preferably the scattering medium has a prescribed inner diameter This inner diameter of the scatteπng material is designed such that the interaction with this mateπal and the multiple reflections off of the cavity reflectors mteract to provide a substantially proscπbed axial distribution of laser radiation over the length of the tip apparatus Various embodiments provide a diffusing tip with control over the emitted axial and azimuthal energy distributions

The diffusion techniques and devices disclosed herein are generally applicable for diffusing radiation from an optical fiber to provide a larger exposure area for photo- illummation Some embodiments are particularly useful as part of a fiber-optic based medical laser system in which a lower aspect ratio of length to diameter than typical diffuser designs is desirable Suitable laser systems include those descπbed herein along with those described in U S Patent Application No 11/930,941 filed 31 October 2007 and U S Patent Application Seπal No 11/981,486 filed 31 October 2007, the entire contents of both of which applications are incorporated herein by reference

Some embodiments provide substantially uniform energy distπbution to a major portion of the exposure area Some embodiments provide for constructing and implementing circumferential and/or sideways emitting diffusing tip assemblies for optical fibers to direct laser radiation in a radially outward pattern relative to the fiber's longitudinal axis. As used herein the term "optical fiber" is intended to encompass optically transmissive waveguides of various shapes and sizes.

Some diffusing tip designs are intended for a higher aspect ratio of length to diameter. Typical aspect ratios for prior art diffusing tip technologies are usually from 20 to 1 and higher, (e.g. 1 mm diameter and 20 mm length). Some embodiments of the diffusion tips described herein allow for producing diffusing tip assemblies with aspect ratios of about 10 or less, about 1 or less, or about .1 or less. For example, one embodiment may be used to produce uniform emission from a diffusing tip with 10mm diameter and 10 mm length. The aspect ratio of this diffusing tip would be 1.

In one embodiment, a diffusive tip assembly is disclosed for diffusing radiation from an optical fiber. The tip assembly includes a light transmissive, tubular housing, alignable with, and adapted to receive, the distal end of the fiber and serve as a diffusive scattering medium for light that has been emitted by the optical fiber. The assembly further includes a reflective cavity formed by reflectors on each side of the diffusive tube, such that the light is scattered by the tube on it's first pass through the tube, and is emitted outward to the illumination site. The un-scattered portion of the illumination is reflected back to further interact with the scattering tube. This second pass illumination is then scattered outward by the scattering tube to complement the light emitted on the first pass to produce the desired illumination profile. Additional 2nd, 3rd and 4th reflections with subsequent scattering from the diffusing tube can be added to produce additional homogeneity of the emitted axial energy profiles.

The reflective surfaces of the apparatus can also be modified to effect non-planar forms. Reflective structures are disclosed which control the spatial distribution of the light emitted from the tip. These techniques and structures permit, for example, an evenly distributed orthogonal projection of the radiation.

In another aspect, the diameter of the tubular scattering material and/ or the length of the diffusing tip can be controlled such that the diffusion of the radiation during the initial and reflected paths are complementary. By proper choice of such parameters, the cumulative energy profile, or fluence, along at least a portion of the fiber tip can be rendered uniform. The term "substantially uniform" is commonly used in the field of phototherapy to describe light diffusers that possess a uniformity of about +/-15% or less of the average intensity of light emitted from the diffusive tip assembly Thus embodiments of tip 10 provide a mechanism for substantially uniform cylindrical illumination of biological structures and other illumination applications

In some embodiments, the diffuser tips descπbed herem may be used to apply therapeutic light at NIMELS dosimetry and wavelengths (e g as descπbed above) without exhibiting heating to temperatures which are unwanted or intolerable at the treatment site (i e temperatures that would cause substantial thermal damage at the site, or discomfort to a patient undergoing treatment) For example, in some embodiments, the diffuser tip may absorb about 20% or less of the therapeutic light delivered from a therapeutic source at NIMELS dosimetry and wavelengths hi some embodiments, the diffuser tip may be operated to deliver therapeutic light at NIMELS dosimetry and wavelengths for treatment times on the order tens of seconds or on the order of minutes or more while remaining at an operatmg temperature of 110 F degrees or less, or IOOF degrees or less

In Fig 19 an optical fiber diffusive tip assembly 10 is shown including and optical fiber 12 having a light transmissive core 14, a cladding 16, a proximal first mirror 18, a diffusing tube 20, and a distal second mirror 22 The end face of fiber 12 is inserted through an aperture 24 m the first mirror 18

In Fig 20, the operation of diffuser tip 10 is shown where the radiation 28 from the fiber 12 expanding at angle defined by numerical aperture NA intersects the diffusing tube 20 As indicated, a distal portion 32 of diffusing tube 20 is illuminated by light which intersects the tub and is scattered radially outward

Fig 21 shows the operation of diffuser tip 10 where the radiation 28 from the fiber 12 which does not interact with the diffusing tube 20 reflects back into the void 30 from the distal mirror 22 The diffusing tube may be constructed from many mateπals with suitable optical and scatteπng properties, but preferably from those mateπals with low absorption (e g less than 20% of the incident intensity) and high scatteπng properties, of which expanded poly-tetrafluoroethylene (PTFE) is an example Radiation which propagates into the diffusing mateπal 20 is efficiently scattered m region 32 with a portion escaping the mateπal 20 and emergmg into external space A portion of the radiation 28 is returned into the void 30 where it is propagated into another region of the diffusing tube 20 or contmues to be reflected by first or second mirrors 18, 22 The radiation scattered out of tube 20 sums to a cumulative illumination pattern which depends on properties of tip 10 (e g the distance between the reflectors, the size of the inteπor void, the curvature of the reflectors, etc )

For example, Fig 22 shows the diffusive tip assembly 10 Shown are the optical fiber 12, a proximal first mirror 18, a diffusing tube 20, and a distal second mirror 22 having a curved reflective face Fig 23 shows a graph of the tip's light intensity (ordmate) as a function of axial position (ordmate) for a planar distal mirror 22 (left graph) and for a curved distal second mirror 22 (πght graph) For this configuration the void in the diffusion tube 20 was larger than preferred to provide a uniform axial intensity distribution In this design regime a properly curved second mirror 22 can mcrease the uniformity of the tip's axial mtensity profile

Fig 24 shows a method of construction of the curved second mirror 22 where a small form or sphere 34 is placed between the flexible reflective films 36 and the distal backing of the second mirror assembly 22 An example of a flexible reflective film is 3M Vikuity enhanced specular reflective film

Fig 25 shows a cross section of the fiber tip 10 having a proximal first mirror 18 with diffuse reflective surface 40 An example of this kind of film is white Backlight reflector sheet made of a polyester film such as that produced by Kimoto This alternative construction enables an improved radiant uniformity when factors including, but not limited to, cost and simplicity of construction are considered In some embodiments, the distal mirror 36 may be specular or diffuse, preferably specular The proximal mirror may be specular or diffuse, preferably diffuse

Fig 26 shows an embodiment of diffuser tip 10 where the diffusion tube is enclosed by a disposable, sterile, test tube sized appropriately for the diffusion tip assembly A preferred disposable tube is made from Polypropylene, due to its high transmission of visible and near infrared light, non shattering nature and ability to withstand high temperatures Alternate mateπals could include polycarbonate or Pyrex glass In some embodiments diffuser tip 10 is autoclavable and reusable In other embodiments, diffuser tip 20 is detachbly connected to fiber 12, and is disposable Fig 27 shows how the size of void 30 may be adjusted relative to the length of tip 10 m order to maintain a substantially uniform axial intensity distribution In general, as the void size is increased, the length of the tip that can be used to provide uniform illumination is increased up to the the point at which the wall thickness becomes extremely thm Above that pomt, other properties of tip 10 (e g the curvature of one or more of the reflectors) must be adjusted to maintain uniform illumination

Fig 28 shows an intensity profile of a diffusion tip 10 Axial intensity distribution uniformity of +/- 8% is achieved using the techniques taught herein Note also the lack of any light outside the cavity region This indicates that the cumulative illumination pattern for this tip is a substantially uniform cylindrical pattern characteπzed by uniform axial intensity distribution directed outward m the radial direction along diffusion tube 20 and substantially proscπbed illumination in the axial direction

Referring to Fig 29, m one embodiment, the piefeπed void size or preferred length for a given void size may be calculated as follows Light from fiber 12 propagates radial outward in a cone from aperture 24 as defined by the numerical aperture NA and the length L of tube 20 The radius of this cone at the distal end of tube 20 is defined as R The radius of void 30 is defined at R_VOi_d In one embodiment, for uniform axial illumination, the area of the void is chosen to be chosen to be 40% of the area of the cone at the distal end of tube 20 such that

R_V0ld = 4 ττR^Λ2 = ( 4 NA L)^Λ(l/2) = 0 63 NA L

Thus, for an embodiment where L= 10mm and NA=O 3, R_V0ld is about 1 89mm In an embodiment employing a standard 5/16 inch tube having a 3/16 inch void, L is equal to about one half inch Thus, uniform intensity profiles may be obtained using easily obtainable components for diffusion tips have aspect rations on the order of 1 to 10

In some embodiments, the fiber assembly elements are held together m a transparent tube, such as a heat shrink tubmg In one embodiment, the heat shrink tubing is a clear thm walled polyester tubmg In some embodiments, aluminum disks make convenient backing plates for both mirror assemblies Referring to Fig 30, a kit 300 includes a diffuser tip 10, e g of the types descπbed herein, suitable for use with a treatment system of the type descπbed above (e g system 110) to provide a desired distribution of treatment light at a target site Tip 10 may be may be detachably connected to the treatment system and may be configured to apply therapeutic light at NIMELS dosimetry and wavelengths without becoming subject to unwanted levels of heating at treatment site

Kit 300 also includes antimicrobial (e g antibiotic or antifungal) application 301 which is potentiated by treatment light delivered from the diffuser tip As described m detail above, antimicrobial application 301 may be potentiated by the treatment light to treat a resistant biological contaminate at a treatment site even though the antimicrobial application alone would be ineffective in treating the contaminate For example, in one embodiment, antimicrobial application 302 may be erythromycin potentiated by NIMELS treatment light to treat antibiotic resistant bacteπa such as MRSA or MSSA In various embodiments antimicrobial application 301 may be a topical application (e g a paste) or may be administered m other ways known m the art (e g ingested orally, administered intravenously, etc )

Kit 300 may also include instructions 302 instructing the use of antimicrobial application 301 in conjunction with potentiating treatment light diffused by tip 10 The instructions may provide guidance as to the types of contaminates which may be treated using the kit, along with information regarding suitable dosimetry for the treatment light (e g NIMELS dosimetry) The instructions may be provided m any media including pπnt or electronic formats

The above descπbed elements of Kit 300 may be contained in suitable packaging 303, e g a box or pouch In some embodiments the packaging may include steπle packaging For example, m some embodiments tip 10 may be steπhzed and packed in a sterile container

In various embodiments, diffusion tip 10 may include any number of sensors, e g , temperature sensors which may communicate (by wire or wirelessly) with therapeutic system 110 Information provided by these sensors may be used to control applied dosages of therapeutic light, e g for safety purposes Tip 10 may also include one or more cooling devices (e.g. a thermoelectric cooler), or attachments suitable for engagement with external cooling devices (e.g. tubular plumbing for circulation of cooling fluids).

It is to be understood that, as used herein, the phrases "light", "optical", etc. are not limited to the visible spectrum, but may refer to electromagnetic radiation at any wavelength including, e.g., the infrared.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A diffuser tip assembly adapted to receive the distal end of optical fiber, the assembly comprising: a reflective cavity comprising: a first reflector positioned proximal the distal end of the received fiber and comprising an aperture adapted to admit light emitted from the fiber into the cavity; and a second reflector positioned distal the first reflector; and a diffuser tube positioned between the first and second reflectors about a cavity axis extending from the first reflector to the second reflector, the diffuser tube comprising an inner void surrounded by an outer portion comprising a diffusive scattering material; wherein the cavity and diffuser tube are arranged such that at least a portion of light admitted into the cavity is scattered by the diffusive scattering material out of the tip assembly through the outer portion in a direction transverse to the cavity axis.

2. The diffuser tip assembly of claim 1 , wherein the cavity and diffuser tube are configured such that: light admitted into the cavity is directed from the aperture towards the second reflector; a portion of the light directed towards the second reflector impinges upon the diffusive scattering material and is scattered out of the tip assembly in a direction transverse to the axis; at least a portion of unscattered light impinges upon the second reflector and is reflected back towards the first reflector; and a portion of the light directed back towards the first reflector impinges upon the diffusive scattering material and is scattered out of the tip assembly in a direction transverse to the axis.

3 The diffuser tip assembly of any preceding claim, wherein the cavity and diffuser tube are configured such that light admitted into the cavity travels multiple passes between the first and second reflectors, and on each pass, at least a portion of the light is scattered by the diffusive scattering material out of the tip assembly m a direction transverse to the axis

4 The diffuser tip assembly of claim 3, wherein the light scattered out of the tip assembly on each pass combine to produce a cumulative illumination pattern

5 The diffuser tip assembly of claim 4, wherein the cumulative illumination pattern is characteπzed by substantially uniform axial intensity profile along at least a portion of the diffuser tube

6 The diffuser tip assembly of claim 5, wherein the cumulative illumination pattern is characteπzed by a substantially uniform azimuthal illumination profile

7 The diffuser tip assembly of any of claims 4-6, wherein the cumulative illumination pattern is characterized by substantially proscπbed illumination in the direction parallel to the axis

8 The diffuser tip assembly of any of claims 4-7, wherem the cumulative illumination pattern is a substantially uniform cylindrical illumination pattern emitted radially from the outer surface of the diffuser tube

9 The diffuser tip assembly of any of claims 4-8, wherein the cumulative illumination pattern is determined by at least on at least one chosen from the list consisting of a length of the diffuser tube, the diameter of the inner void of the diffuser tube, a numerical aperture associate with the aperture m the first reflector

10 The diffuser tip assembly of any preceding claim, wherein the inner void is filled with a substantially transparent non-scatteπng material

11 The diffuser tip assembly of any preceding claim, wherein the at least one of the first and second reflectors comprises a curved reflector

12. The diffuser tip assembly of any preceding claim, wherein the first reflector is a diffuse reflector and the second reflector is a specular reflector.

13. The diffuser tip assembly of any preceding claim, wherein the ratio of the distance between the first reflector and the second reflector along the cavity axis to the outer diameter of the diffusion tube is about 10 or less.

14. The diffuser tip assembly of any preceding claim, wherein the ratio of the distance between the first reflector and the second reflector along the cavity axis to the outer diameter of the diffusion tube is about 1 or less.

15. The diffuser tip assembly of any preceding claim, wherein the ratio of the distance between the first reflector and the second reflector along the cavity axis to the outer diameter of the diffusion tube is about 0.1 or less.

16. The diffuser tip assembly of any preceding claim, wherein the diffusive scattering material comprises at least one selected from the list consisting of: a plastic, a glass, a polymer, and a fluid.

17. The diffuser tip assembly of any preceding claim, wherein the diffusive scattering material comprises PTFE.

18. The diffuser tip assembly of any preceding claim, wherein the reflective cavity and the diffuser tube are autoclavable.

19. The diffuser tip assembly of any preceding claim, further comprising a substantially transparent outer jacket adapted to contain the reflective cavity and the diffuser tube.

20. The diffuser tip assembly of any preceding claim, wherein the outer jacket is detachable from the reflective cavity and the diffuser tube.

21. The diffuser tip assembly of any preceding claim, wherein the diffusive scattering material is adapted to scatter light in the near infrared.

22 The diffuser tip assembly of any of the preceding claims, wherein the tip assembly is adapted to scatter about 80% or more of the light delivered from the fiber while absorbing about 20% or less of the light delivered from the fiber

23 A kit for treating an antimicrobial resistant biological contaminate at a treatment site comprising a diffuser tip adapted to receive near infrared therapeutic light from a light delivery system and diffuse the light to illuminate at least a portion of the treatment site, a quantity of an antimicrobial, agent, instructions to use the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to treat the biological contaminate, and suitable packagmg

24 The kit of claim 23, wherein the diffuser tip is the diffuser tip assembly of any of claims 1 through 22

25 The kit of any preceding claim, wherein the therapeutic light comprises optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges

26 The kit of any preceding claim, wherein the treatment light compnses optical radiation at both wavelength ranges

27 The kit of any preceding claim, wherein the diffuser tip assembly is adapted to provide substantially uniform illumination of the illuminated portion of the treatment site

28 The kit of any preceding claim, wherein the diffuser tip assembly is adapted to illuminate the illuminated portion of the treatment site at a power density and an energy density which potentiates the antimicrobial agent at the treatment site without causing substantial photothermal or photomechanical damage to the treatment site

29. The kit of any preceding claim , wherein the diffuser tip assembly is adapted to illuminate the illuminated portion of the treatment site at a power density of about 0.2 W/cm² to about 1 W/cm² and an energy density from about 100 J/cm² to about 400 J/cm² at the illuminated target region.

30. The kit of any preceding claim , wherein the diffuser tip is adapted to operate for about 30 seconds or more at an operating temperature of 110° F or less.

31. The kit of any preceding claim, wherein the diffuser tip assembly is adapted for detachable connection to a distal end of an optical fiber which transmits the therapeutic light from a light source to the distal end of the fiber.

32. The kit of any preceding claim, wherein the antimicrobial agent is ineffective for treating the antimicrobial resistant biological contaminate in the absence of the therapeutic light.

33. The kit of any preceding claim, wherein the quantity of antimicrobial agent comprises a topical paste.

34. The kit of any preceding claim, wherein the antimicrobial agent comprises an antibiotic or a pharmacologically acceptable salt thereof, selected from the group consisting of: /3-lactams, glycopeptides, cyclic polypeptides, macrolides, ketolides, anilinouracils, lincosamides, chloramphenicols, tetracyclines, aminoglycosides, bacitracins, cefazolins, cephalosporins, mupirocins, nitroimidazoles, quinolones and fluoroquinolones, novobiocins, polymixins, cationic detergent antibiotics, oxazolidinones or other heterocyclic organic compounds, glycylcyclines, lipopeptides, cyclic lipopeptides, pleuromutilins, and gramicidins, daptomycins, linezolids, ansamycins, carbacephems, carbapenems, monobactams, platensimycins, streptogramins and tinidazoles.

35. The kit of any preceding claim, wherein the resistant biological contaminate comprises MRSA or MSSA.

36 The kit of any preceding claim, wherein the biological contaminate comprises an aberrant microbial colonization, and wherein the instructions comprise instruction to use the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to reduce the level of colonization at the treatment site

37 The kit of claim 36, wherem the an aberrant microbial colonization, prior to application of the treatment light, has a drug resistant phenotype with respect to the antimicrobial agent

38 The kit of any preceding claim, wherein the diffuser tip is sterilized

39 A therapeutic system for treatment of a biological contaminant at a treatment site compnsmg an optical radiation generation device configured and arranged to generate near infrared therapeutic light, a controller operatively connected to the optical radiation generation device for controlling dosage of the therapeutic light transmitted to the treatment site at a dosimetry sufficient to produce photodamage m the biological contaminant without causing substantial photothermal or photomechanical damage to biological tissue at the treatment site, a delivery assembly including an optical fiber which directs the therapeutic light to be transmitted to the treatment site, and a diffuser tip adapted to receive the therapeutic light from the delivery assembly and diffuse the therapeutic light to illuminate at least a portion of the treatment site with a prescπbed illumination pattern

40 The system of claim 39, wherein the diffuser tip is the diffuser tip assembly of any of claims 1 through 22

41 The system of any preceding claim, wherem the therapeutic light comprises optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges

42 The system of claim 41, wherem the treatment light comprises optical radiation at both wavelength ranges

43 The system of any preceding claim, wherem the diffuser tip assembly is adapted to provide substantially uniform illumination of at least a portion of the illuminated portion of the treatment site

44 The system of any preceding claim, wherem the diffuser tip assembly is adapted to illuminate the portion of the treatment site at a power density and an energy density which potentiates an antimicrobial application at the treatment site

45 The system of any preceding claim , wherein the diffuser tip assembly is adapted to illuminate the portion of the treatment site at a power density of about 0 3 W/cm² to about 7

W/cm² and an energy density from about 100 J/cm² to about 400 J/cm² at the illuminated portion of the treatment site

46 The system of any precedmg claim, wherem the diffuser tip is adapted to operate for about 30 seconds or more at an operating temperature of 110° F or less

47 The system of any preceding claim, wherem the diffuser tip assembly is adapted for detachable connection to a distal end of the optical fiber

48 A method treatment of a biological contaminant at a treatment site comprising generatmg near mfrared therapeutic light, controlling dosage of the therapeutic light transmitted to the treatment site at a dosimetry sufficient to produce photodamage in the biological contaminant without causing substantial photothermal or photomechanical damage to biological tissue at the treatment site, directing the therapeutic light to be transmitted to the treatment site, and using a diffuser tip, diffusing the therapeutic light to illuminate at least a portion of the treatment site with a prescπbed illumination pattern

49 The method of claim 48, wherem the diffuser tip is the diffuser tip assembly of any of claims 1 through 22

50 The method of any preceding claim, wherem the therapeutic light comprises optical radiation substantially in a first wavelength range from about 865 nm to about 875 nm or a second wavelength range having a wavelength from about 925 nm to about 935 nm, or both wavelength ranges

51. The method of claim 50, wherein the treatment light comprises optical radiation at both wavelength ranges.

52. The method of any preceding claim, wherein the diffusing the therapeutic light comprises providing substantially uniform illumination of at least a portion of the illuminated portion of the treatment site.

53. The method of any preceding claim, further comprising: applying a quantity of antimicrobial agent the treatment site; and illuminating the portion of the treatment site at a power density and an energy density which potentiates the antimicrobial application.

54. The method of any preceding claim, wherein the antimicrobial agent is ineffective for treating the antimicrobial resistant biological contaminate in the absence of the therapeutic light.

55. The method of claim 54, wherein the antimicrobial agent is ineffective for treating the biological contaminate in the absence of the therapeutic light.

56. The method of any of claims 53-55, wherein the biological contaminate comprises an aberrant microbial colonization, and further comprising comprises using the antimicrobial agent in conjunction with the therapeutic light to potentiate the antimicrobial agent to reduce the level of colonization at the treatment site.

57. The method of claim 56, wherein the aberrant microbial colonization, prior to application of the treatment light, has a drug resistant phenotype with respect to the antimicrobial agent.

58. The method of any preceding claim , wherein controlling the dosage of the therapeutic light comprises controlling the illumination of the portion of the treatment site at a power density of about 0.2 W/cm² to about 1 W/cm² and an energy density from about 100 J/cm² to about 400 J/cm² at the illuminated region.