# Patent

## Title
Inactivation of gram-positive bacteria

## Abstract

A method for inactivating medically important Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species, comprising exposure to visible light, and in particular light within the wavelength range 400-500 nm.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 11/997,227, filed Jul. 3, 2008, which is a U.S. National Stage of International Application No. PCT/GB2006/002841, filed Jul. 28, 2006, which claims the benefit of Great Britain Application No. 0515550.2, filed Jul. 29, 2005, the contents of which are hereby incorporated herein in their entirety by reference.


The present invention relates to a method for inactivating medically important Gram-positive bacteria including Staphylococcus aureus and methicillin (multi)-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species.
BACKGROUND OF THE INVENTION
Methicillin-resistant Staphylococcus aureus (MRSA) is becoming an increasingly problematic micro-organism, with infection rates rising and effective methods of control becoming more and more limited. In addition to the resistance of MRSA to antibiotics, there is a significant problem due to the availability of few effective sterilisation methods for environmental decontamination; for example in air and on contact surfaces. Public and media interest in the transmission and control of MRSA is escalating and it is becoming one of the most significant problems within the healthcare industry. Hospitals and nursing homes are the worst affected areas. Furthermore, community-acquired MRSA is also now being recognised as an increasing problem, with transmission occurring in public and social areas such as public gyms and sports centres.
As well as MRSA, other Gram-positive bacteria are known to cause health problems, particularly in the hospital environment. For example, Staphylococcus epidermidis, which is a Coagulase-Negative Staphylococcus (CONS), can cause infection, particularly in infants and in hospitalised patients who have received prosthetic implant surgery. Streptococcus pyogenes is a Gram-positive coccus commonly associated with infections such as pharyngitis, pyoderma, scarlet fever, erysipelas, cellulitis, streptococcal toxic-shock syndrome, rheumatic fever, glomerulonephritis, bacteraemia and necrotizing fasciitis, often referred to as “flesh-eating bacteria”. Enterococcus faecalis (another Gram-positive coccus) is a common cause of urinary tract and wound infections, as well as other infections including bacteraemia, endocarditis and meningitis in severely ill hospitalised patients. Multi-antibiotic resistance is also becoming a well-documented problem with enterococcal infections. Clostridium species, in particular C. difficile, have been associated with high mortality in elderly patients due to diarrohea-associated dehydration, medically known as antibiotic-associated pseudomembranous colitis.
Many techniques have been proposed for destroying harmful bacteria, such as MRSA. For example, U.S. Pat. No. 6,251,127 describes a photodynamic process for the inactivation of bacteria and fungal wound infections using methylene blue or toluidene blue. Light energy in combination with photosensitising agents is used to treat or detect pathologies of living tissue, including cancer and microbiological pathogens. The light used has wavelengths ranging from about 450 nm to about 850 nm. Tests demonstrate the efficacy of the light treatment in combination with the photosensitising agents for the destruction of Staphylococcus aureus in in-vivo infected wounds; and for in-vitro destruction of antibiotic-resistant Staphylococcus, Streptococcus, Enterococcus, E. coli, Pseudomonas, Haemophilus influenza and Candida albicans. In addition, wavelength spectra of activation of methylene blue and toluidene blue in the presence of various concentrations of the above bacteria and Candida have been provided.
Whilst in some environments, the methodology of U.S. Pat. No. 6,251,127 may be useful, it nevertheless suffers from the significant practical disadvantage that photosensitising agents must be applied to the bacteria that are to be inactivated. A similar problem arises with US2005/0049228, which also requires the combined use of a photosensitiser and light; in this case, in the range of 500 nm to 580 nm. The need for photosensitising agents is a significant limitation of these techniques.
An objective of the present invention is to provide a simple and effective technique for inactivating selected bacteria, in particular MRSA, and more generally the Staphylococcus, Streptococcus, Enterococcus and Clostridium species.
SUMMARY OF THE INVENTION
A method for inactivating one or more pathogenic gram-positive bacterial comprising exposure of the bacteria to visible light without using a photosensitiser.
Preferably said bacteria are selected from Staphylococcus, in particular MRSA, CONS, Streptococcus, Enterococcus and Clostridium species.
It is understood that the term pathogenic is used in the context of gram-positive bacterial species and/or strains, which are capable of causing disease or infection in a human or animal subject. It is also understood that some bacteria are often commensal in that they are able to colonise and/or live on/within a healthy host and not become pathogenic unless or until the host becomes immunocompromised and/or unhealthy due to some other form of disease or injury, such as a wound. Such “potentially” pathogenic bacteria are encompassed by the invention also.
Moreover, the term inactivation is understood to mean that said bacteria are killed, or damaged so as to reduce or inhibit bacterial replication. The methods and systems taught herein can therefore be considered as bactericidal and/or bacteriostatic and this may depend on the species/strain of bacteria, wavelength of light, dose, etc.
Exposing these bacteria to blue light, or white light containing blue light, has been found to stimulate an inactivation process. An advantage of using light in the visible-wavelength region is that there is no detrimental effect on human or animal health. Consequently, the method can be used for an extensive range of applications, such as air disinfection, contact-surface and materials disinfection and, most noteworthy, wound protection and tissue disinfection.
According to another aspect of the invention, there is provided a method for inactivating pathogenic gram positive bacteria including at least one of Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species comprising exposure of the bacteria to visible light having a wavelength in the range 400-500 nm. The visible light may have a wavelength in the range 400-450 nm. The light may have a wavelength in the range 400-420 nm. The light may have a wavelength of 405 nm.
According to yet another aspect of the invention, there is provided a system for inactivating pathogenic Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species, comprising the means for exposing them to visible light having a wavelength in the range of 400-500 nm. The wavelength of the light used is preferably in the range 400-500 nm. The wavelength may be in the range 400-450 nm, and more specifically in the range 400-420 nm, with optimal inactivation at 405 nm.
According to still another aspect of the invention, there is provided use of visible light having a wavelength in the range of 400-500 nm, especially 400-420 nm for inactivating pathogenic gram positive bacteria including at least one of Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species.



BRIEF DESCRIPTION OF THE DRAWINGS
Various aspects of the present invention will now be described by way of example only and with reference to the accompanying drawings, of which:
FIG. 1 shows the total emission spectrum of a Hamamatsu Xenon lamp;
FIG. 2 shows in greater detail the ultra-violet emission spectrum of the Xenon lamp of FIG. 1;
FIG. 3 is a plot of bacterial count of a methicillin-resistant S. aureus strain as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 4 is a plot of bacterial count of a second methicillin-resistant S. aureus strain as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 5 is a plot of bacterial count of S. aureus NCTC 4135 as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 6 is a plot of bacterial count of S. epidermidis NCTC 7944 as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 7 is a plot of bacterial count of Streptococcus pyogenes NCTC 8198 as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 8 is a plot of bacterial count of Enterococcus faecalis as a function of time of exposure to light of wavelength greater than 400 nm;
FIG. 9 is plots of bacterial count in a suspension of S. aureus NCTC 4135 as a function of time of exposure to light for different wavelength ranges;
FIG. 10 is a plot of bacterial log reduction as a function of wavelength (400-500 nm) for S. aureus NCTC 4135;
FIG. 11 is plots of bacterial count in a suspension of S. aureus NCTC 4135 as a function of time of exposure to light of wavelength greater than 400 nm for different light intensities;
FIG. 12 is a visual indication of the surface inactivation of S. aureus NCTC 4135 through exposure to light of wavelengths greater than 400 nm. Surface inactivation is evidenced by inhibition of S. aureus growth on the areas exposed to this light;
FIG. 13 is a plot of bacterial count of S. aureus NCTC 4135 as a function of time of exposure to light of 405 nm;
FIG. 14 is a plot of bacterial count of a methicillin-resistant S. aureus strain as a function of time of exposure to light of 405 nm;
FIG. 15 is a plot of bacterial count of Streptococcus pyogenes NCTC 8198 as a function of time of exposure to light of 405 nm, and
FIG. 16 is a plot of bacterial count of Clostridium perfringens 13124 as a function of time of exposure to light of 405 nm.



DETAILED DESCRIPTION OF THE DRAWINGS
Exposing MRSA to blue light has been found to cause significant inactivation. This narrow range of wavelength is part of the white-light spectrum. For all white-light sources, only a small fraction of the light output is in this range, typically one or two percent. Hence, to provide a sufficient amount of light and demonstrate the effectiveness of this technique, the source used was a Xenon lamp (Hamamatsu Photonics UK Limited). Emission spectra of the lamp are shown in FIGS. 1 and 2. The lamp was used in combination with an optical-fibre light guide and a selection of optical filters in order to allow exposure of the Staphylococcus aureus suspensions to specified wavelengths of visible light. The output of the light guide was maintained at a distance of 5 cm from the sample during all experiments.
To demonstrate the effectiveness of the technique, various studies have been carried out. The bacteria used were as follows: Staphylococcus aureus NCTC 4135; methicillin-resistant Staphylococcus aureus LMG 15975; methicillin-resistant Staphylococcus aureus 16a (clinical isolate), Staphylococcus epidermidis NCTC 7944, Streptococcus pyogenes NCTC 8198 Enterococcus faecali and Clostridium perfringens 13124. Each sample was serially diluted to the appropriate concentration using phosphate-buffered saline (PBS), plated out using nutrient agar (NA) and then incubated at 37° C. for 24 hours.
Suspensions of methicillin-resistant Staphylococcus aureus LMG 15975 and clinical isolate 16a were prepared and exposed to visible light. The light was transmitted through a 400 nm long-wave pass filter (50% cut-off in transmission at 400 nm) before impacting on the bacterial suspension. This allowed only wavelengths of 400 nm and above (visible light) to illuminate the sample. The results of these experiments are shown in FIGS. 3 and 4. From these, it can be seen that the light treatment causes significant reduction in the counts of both the culture collection MRSA (LMG 15975) and the highly resistant clinical isolate (16a). The control data refer to samples that were untreated over the same time interval.
Suspensions of Staphylococcus aureus NCTC 4135 were also exposed to visible-light treatment. Again, the light beam was transmitted through a 400 nm long-wave pass filter before impacting on the bacterial suspension, allowing only the transmission of wavelengths of 400 nm and above. From FIG. 5 it can be seen that the Xenon light source caused significant reduction in the Staphylococcus aureus count even with a high starting bacterial population of approximately 107 colony-forming units per milliliter (cfu/ml). Similar experiments were carried out using Staphylococcus epidermidis NCTC 7944, Streptococcus pyogenes NCTC 8198 and Enterococcus faecalis. The associated reductions in the bacterial population are shown in FIGS. 6, 7 and 8. In each of these a significant reduction in the bacterial count is observed.
Exposure tests using a range of filters were carried out. Bacterial suspensions were exposed to the following wavelength ranges for times up to 90 minutes: greater than 550 nm (using a 550 nm long-wave pass filter); greater than 500 nm (using a 500 nm long-wave pass filter), less than 500 nm (using a 500 nm short-wave pass filter); 400-500 nm (using a 400 nm long-wave pass filter and a 500 nm short-wave pass filter in combination); 450-500 nm (using a 450 nm long-wave pass filter and a 500 nm short-wave pass filter in combination); greater than 450 nm (using a 450 nm long-wave pass filter), and greater than 400 nm (using a 400 nm long-wave pass filter). The resultant inactivation curves in FIG. 9 allow only qualitative comparisons to be made since the filters do not have sharp cut-off wavelengths and the light intensities falling on the suspensions were different for the different curves. The results do however indicate that the wavelength region between 400 nm and 500 nm does provide a high rate of S. aureus inactivation.
Experiments were also carried out using bandpass filters each with a 10 nm FWHM (full-width, half-maximum). Suspensions of methicillin-resistant S. aureus LMG 15975 (approximately 105 cfu/ml population) were exposed to visible light transmitted through the following bandpass filters: 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 430 nm, 440 nm, and 450 nm. The intensity of the lamp was altered for each filter to ensure that the light power at the suspension was the same for each measurement, thus allowing direct comparison of results. The results of these experiments showed that samples exposed using the 400 nm, 405 nm and 415 nm bandpass filters have a reduced colony-forming-unit count/ml; that is, light of wavelengths within these narrow bandwidths had an inactivating effect on the S. aureus strains.
A more detailed analysis of wavelength sensitivity was performed using suspensions of S. aureus NCTC 4135, and this is shown in FIG. 10. The results show that samples exposed using the 400 nm, 405 nm, 410 nm, 415 nm and 420 nm bandpass filters have a reduced colony-forming-unit count/ml; that is, light of wavelengths within these narrow bandwidths had an inactivating effect on the S. aureus strains. From these results it can be deduced that visible-light exposure over the wavelength range 400-450 nm is the major inducing factor for Staphylococcal inactivation, with increased inactivation occurring over the range 400-420 nm and optimum inactivation occurring at 405 nm. Moreover, it has been observed that a lower dose is required at this wavelength and typically the dose is less than 200 J/cm2, such as less than 100 J/cm2.
In further experiments, Staphylococcus aureus NCTC 4135 suspensions were exposed to different intensities of visible-light treatment. These measurements were made using the 400 nm long-wave pass filter, that is, for wavelengths greater than 400 nm. FIG. 11 shows the results of these experiments. It can be seen that as the intensity of the light decreases, so to does the inactivation rate. The specific doses required for complete inactivation of Staphylococcal, Streptococcal and Enterococcal species using different filters and light intensities were determined. Sample results are shown in the Table below:














WAVELENGTH
DOSE
J/cm2/log


ORGANISM
RANGE
(J/cm2)
reduction


















S. aureus 4135
 >400 nm (100% intensity)
630
126


S. aureus 4135
>400 nm (75% intensity)
729
145.8


S. aureus 4135
>400 nm (50% intensity)
648
144


S. aureus 4135
<500 nm
189.6
37.92


S. aureus 4135
400-500 nm  
290.8
58.2


MRSA 15975
>400 nm
1260
252


MRSA 16a
>400 nm
945
189


S. epidermidis
>400 nm
840
168


NCTC 7944


Strep. pyogenes
>400 nm
1440
288


NCTC 8198


E. faecalis
>400 nm
2880
1440









The effect of visible-light exposure for surface decontamination was also examined. This was done by exposing S. aureus cells, which were plated onto nutrient agar, to the light treatment (through a 400 nm long-wave pass filter) prior to incubation. Examples of results are shown as the areas of growth inhibition on the culture plates in FIG. 12.
A similar treatment system to that used with the Xenon lamp was assembled using a 405 nm LED array as a light source. Experiments were carried out using Staphylococcus aureus NCTC 4135, MRSA 16a, Streptococcus pyogenes NCTC 8198 and Clostridium perfringens 13124. The associated reductions in the bacterial population are shown in FIGS. 13, 14, 15 and 16, respectively. The specific doses required for complete inactivation of Staphylococcus, Streptococcus and Clostridium species using the 405 nm LED array were determined. Sample results are shown in the Table below:















DOSE
J/cm2/log


ORGANISM
WAVELENGTH
(J/cm2)
reduction


















S. aureus 4135
405 nm
36
7.2


MRSA 16a
405 nm
45
9


Streptococcus pyogenes
405 nm
54
10.8


NCTC 8198


Clostridium perfringens 13124
405 nm
45
10.2









A comparison of the doses required for bacterial inactivation (5-log reduction) using light of wavelengths greater than 400 nm from the Xenon lamp and a 405 nm LED array is shown in the Table below:














DOSE (J/cm2)
J/cm2/log reduction











ORGANISM
>400 nm
405 nm
>400 nm
405 nm














S. aureus NCTC 4135
630
36
126
7.2


MRSA 16a
945
45
189
9


Cl. perfringens 13124
1440
54
288
10.8









The use of 400-500 nm, in particular 400-450 nm, wavelengths of visible light (blue light) has proved to be an effective means of inactivation of Staphylococcus strains, including MRSA, as well as CONS, Streptococcus, Enterococcus and Clostridium, with increased inhibition rates in the 400-420 nm range and in particular, around 405 nm. This demonstrates that a light source (continuous source, flashlamp, laser etc.) with output at wavelengths in these regions could potentially be used in clinical environments for the reduction in levels of methicillin-resistant Staphylococcus aureus, and other medically important Gram-positive species present in the air and on contact surfaces and materials, and most importantly, could be used for wound protection and tissue treatment. The exact parameters required would depend on the bacterial strain, the wavelength of the light being used and the light intensity. These can be readily determined experimentally.
Variations of the disclosed arrangements are possible without departing from the invention. For example, although both a Xenon lamp with a variety of different filters and a 405 nm LED array have been used as the inactivation source, it will be appreciated that any suitable light source can be used. Equally, although a particular experimental arrangement has been described here, it will be readily apparent that the light source used could be included in, for example, a hand-held device or could be designed to operate in or around areas that have to be kept free of MRSA. Accordingly the above description of the specific embodiment is made by way of example only and not for the purposes of limitation. It is clear that minor modifications may be made without significant changes to the operation described.

## Claims

1. A method for disinfecting air, contact surfaces or materials by inactivating one or more pathogenic Gram-positive bacteria in the air, on the contact surfaces or on the materials, said method comprising exposing the one or more pathogenic Gram-positive bacteria to visible light without using a photosensitizer, wherein the one or more pathogenic Gram-positive bacteria are selected from the group consisting of Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus, and Clostridium species, and wherein a portion of the visible light that inactivates the one or more pathogenic Gram-positive bacteria consists of wavelengths in the range 400-420 nm, and wherein the method is performed outside of the human body and the contact surfaces or the materials are non-living.

2. The method according to claim 1 wherein the visible light that inactivates has a wavelength of 405 nm.

3. A system for disinfecting air, contact surfaces or materials by inactivating one or more pathogenic Gram-positive bacteria in the air, on the contact surfaces or on the materials, the system comprising means for exposing said bacteria to visible light without using a photosensitizer, wherein the one or more pathogenic Gram-positive bacteria are selected from the group consisting of Methicillin-resistant Staphylococcus aureus (MRSA), Coagulase-Negative Staphylococcus (CONS), Streptococcus, Enterococcus and Clostridium species, and wherein a portion of the visible light that inactivates the one or more pathogenic Gram-positive bacteria consists of wavelengths in the range 400-420 nm, and wherein the system is for use outside of the human body and the contact surfaces or the materials are non-living.

4. The system according to claim 3 wherein the portion of the visible light that inactivates has a wavelength of 405 nm.