Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.
ABSTRACT In a previous study, we showed that 405-nm light photo-destroys methicillin-resistant Staphylococcus aureus (MRSA). The 390-420 nm spectral width of the 405-nm superluminous diode (SLD) source may raise safety concerns in clinical practice, because of the trace of ultraviolet (UV) light within the spectrum.
Here we report the effect of a different wavelength of blue light, one that has no trace of UV, on two strains of MRSA--the US-300 strain of CA-MRSA and the IS-853 strain of HA-MRSA--in vitro.
We cultured and plated each strain, and then irradiated each plate with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60 J/cm2 of energy a single time, using a 470-nm SLD phototherapy device. The irradiated specimens were then incubated at 35 degrees C for 24 h. Subsequently, digital images were made and quantified to obtain colony counts and the aggregate area occupied by bacteria.
Photo-irradiation produced a statistically significant dose-dependent reduction in both the number and the aggregate area of colonies formed by each strain (p < 0.001). The higher the dose the more bacteria were killed, but the effect was not linear, and was more impressive at lower doses than at higher doses. Nearly 30% of both strains was killed with as little as 3 J/cm2 of energy. As much as 90.4% of the US-300 and the IS-853 colonies, respectively, were killed with an energy density of 55 J/cm2. This same dose eradicated 91.7% and 94.8% of the aggregate area of the US-300 and the IS-853 strains, respectively.
At practical dose ranges, 470-nm blue light kills HA-MRSA and CA-MRSA in vitro, suggesting that a similar bactericidal effect may be attained in human cases of cutaneous and subcutaneous MRSA infections.
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ABSTRACT: This study was conducted to evaluate the disinfection effects of Staphylococcus aureus, and Escherichia coli treated with 461-nm LED and pH 5 at for 10 h. S. aureus strains were decreased by about 4 log CFU/mL after 461-nm LED irradiation treatment alone for 10 h. E. coli strains were inactivated by irradiation. However, when microorganisms were subjected to a combined treatment of 461-nm LED and pH 5, both strains were inactivated by irradiation for 7 h. The highest D-value was 5.05 h for S. aureus ATCC 27664 and the lowest D-value was 1.39 h for E.coli O157: H7 ATCC 35150 (pKorean Journal of Food Science and Technology. 08/2013; 45(4).
- Advances in Laserology - Selected Papers of Laser Florence 2011: a Window on the Laser Medicine World. 01/2012; 1486:113-118.
Photomedicine and Laser Surgery
Volume 27, Number 2, 2009
© Mary Ann Liebert, Inc.
Blue 470-nm Light Kills Methicillin-Resistant
Staphylococcus aureus (MRSA) in Vitro
Chukuka S. Enwemeka, Ph.D., FACSM,1Deborah Williams, M.D., Ph.D.,1,2Sombiri K. Enwemeka,1
Steve Hollosi, D.O.,2and David Yens, Ph.D.2
Background Data: In a previous study, we showed that 405-nm light photo-destroys methicillin-resistant Staphy-
lococcus aureus (MRSA). The 390–420 nm spectral width of the 405-nm superluminous diode (SLD) source may
raise safety concerns in clinical practice, because of the trace of ultraviolet (UV) light within the spectrum. Ob-
jective: Here we report the effect of a different wavelength of blue light, one that has no trace of UV, on two
strains of MRSA—the US-300 strain of CA-MRSA and the IS-853 strain of HA-MRSA—in vitro. Materials and
Methods: We cultured and plated each strain, and then irradiated each plate with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 25, 30, 35, 40, 45, 50, 55, or 60 J/cm2of energy a single time, using a 470-nm SLD phototherapy device. The
irradiated specimens were then incubated at 35°C for 24 h. Subsequently, digital images were made and quan-
tified to obtain colony counts and the aggregate area occupied by bacteria. Results: Photo-irradiation produced
a statistically significant dose-dependent reduction in both the number and the aggregate area of colonies formed
by each strain (p ? 0.001). The higher the dose the more bacteria were killed, but the effect was not linear, and
was more impressive at lower doses than at higher doses. Nearly 30% of both strains was killed with as little
as 3 J/cm2of energy. As much as 90.4% of the US-300 and the IS-853 colonies, respectively, were killed with
an energy density of 55 J/cm2. This same dose eradicated 91.7% and 94.8% of the aggregate area of the US-300
and the IS-853 strains, respectively. Conclusion: At practical dose ranges, 470-nm blue light kills HA-MRSA
and CA-MRSA in vitro, suggesting that a similar bactericidal effect may be attained in human cases of cuta-
neous and subcutaneous MRSA infections.
motile, aerobic, and facultatively anaerobic;1moreover, they
are capable of prolonged survival on a variety of environ-
mental surfaces. Staphylococcus aureus (S. aureus) is the most
virulent of the many staphylococcal species and is responsi-
ble for infections ranging from superficial skin and soft tis-
sue infections to those that are systemic and life-threaten-
ing.1S. aureus is part of the normal human flora, colonizing
the anterior nares, skin, vagina, axilla, perineum, and
oropharynx. These sites of colonization act as reservoirs for
future S. aureus infections.1
In the last few decades, S. aureus strains resistant to semi-
synthetic penicillins, such as methicillin, have emerged in
both nosocomial and community environments.2,3Two
grape-like clusters upon Gram’s staining. They are non-
ARE GRAM-POSITIVEBACTERIA that form
prominent strains of methicillin-resistant S. aureus (MRSA)
have been well studied: hospital-acquired methicillin-resis-
tant S. aureus (HA-MRSA) and community-acquired S. au-
reus (CA-MRSA).2–8Unlike HA-MRSA, which seems to be
limited to clinical settings, CA-MRSA has been found in
more common environments, such as computer keyboards,9
that were never before believed to harbor such deadly bac-
teria. Moreover, outbreaks of CA-MRSA have been reported
in schools, locker rooms, sporting arenas, and other locations
in which sports enthusiasts and athletes congregate.4,10–13
The CA-MRSA strain is clearly distinct from the HA-MRSA
strain, and infections with CA-MRSA have been reported in
rural and urban settings in individuals with no previous ex-
posure to medical environments. Moreover, the median age
for CA-MRSA infection is 23 y,3and the corresponding age
for HA-MRSA is 68 y.2,3,8,11While most individuals who de-
velop S. aureus infections do so with their own colonizing
1School of Health Professions, Behavioral, and Life Sciences, and 2New York College of Osteopathic Medicine, New York Institute of
Technology, Old Westbury, New York.
strains, reports have shown that MRSA may be acquired
from other people and fomites.2,12
Treatment of S. aureus infections has become increasingly
difficult, as available medications have had limited success
in combating the disease and stemming outbreaks of infec-
tion. Efforts have been made to develop new drugs, but even
the newest antibiotics have had limited success in control-
ling the spread of MRSA. Estimates indicate that two billion
people carry some strain of S. aureus worldwide, and of these
53 million have MRSA, usually in their nasal cavities.2,6,7
Currently fewer than 5% of Staphylococcusstrains remain sus-
ceptible to penicillin. In response to these increasing viru-
lence factors, new pharmaceutical targets within the bacterial
genome have been studied, resulting in the use of semisyn-
thetic penicillinase-resistant penicillins, such as methicillin,
in the treatment of these penicillin-resistant isolates. Even
then, 40–50% of S. aureus isolates remain resistant to methi-
cillin,1underscoring the need to find new ways to prevent
increased resistance and limit outbreaks of disease.
Blue light phototherapy appears to be a promising alter-
native approach to eradicating MRSA, given the responses
of other bacteria to blue light.14–17Papageorgiou et al.14
have shown that Propionibacterium acne, the bacteria that
causes acne, responds to blue light. They reported signifi-
cant improvements in patients with acne vulgaris follow-
ing treatment with combined blue (415 nm) and red (660
nm) light, and attributed their findings to the potential an-
tibacterial and anti-inflammatory effects of the blue and the
red light sources, respectively. In a recent report, Guffey
and Wilborn15examined the effects of 405-nm and 470-nm
light on two common aerobes, Staphylococcus aureus and
Pseudomonas aeruginosa, and the anaerobe P. acnes, in vitro.
They found both wavelengths to be bactericidal, but the kill
rate was higher with the 405-nm light source than the 470-
nm light source, which had a 90% kill rate for S. aureus and
a 95.1% kill rate for P. aeruginosa. However, neither wave-
length was effective on the anaerobe P. acne.14More re-
cently, Lipovsky et al.18demonstrated that high-intensity
broad-spectrum polychromatic light with wavelengths in
the range of 400–1000 nm kills bacteria in infected diabetic
In our pioneering studies,19,20we showed that 405-nm
light kills both HA-MRSA and CA-MRSA in vitro. The effect
was dose-dependent, with maximum eradication rates of
92–94% of each type of bacteria occurring within 8–10 min
of irradiation. Our light source had a spectral width of
390–420 nm, with 405-nm peak emission. Therefore it had a
trace of ultraviolet (UV) light, to which some of its bacteri-
cidal action could be attributed. This amount of UV light may
raise safety concerns in certain types of patient care situa-
tions, even though it is small, low in intensity, and less than
the amount received from several minutes of exposure to
sunlight. The UV light can be filtered from the 405-nm light
source; however, doing so removes some of the advantages
of using commercially available superluminous diodes
(SLDs), namely their ubiquity, ease of use, and relatively low
cost. To overcome this concern, we sought to assess the po-
tential bactericidal effect of a 470-nm SLD light source on
HA-MRSA and CA-MRSA in vitro. With a spectral width of
455–485 nm, the energy emitted by this SLD contains no UV
Materials and Methods
We cultured two strains of MRSA. The IS-853 strain was
obtained from Winthrop University Medical Center, Mine-
ola, New York, and the US-300 strain was obtained from the
New York Medical College, Valhalla, New York. These
strains represent HA-MRSA and CA-MRSA, respectively,
and were identified by standard identification procedures,
including Gram’s staining, hemolytic patterns seen on blood
agar, and catalase and coagulase production. As detailed in
our previous reports,19,20each strain was separately diluted
to a cell count of 5 ? 106/mL in 0.9% normal saline. Then
they were volumetrically streaked onto round 35-mm plates
of tryptic soy agar before being irradiated with the light
A Dynatron Solaris®708 device (Dynatronics Corp., Salt
Lake City, Utah, USA) fitted with a 470-nm light probe was
used to irradiate the bacteria. The 5.0-cm2applicator with
its cluster of 32 SLDs emits blue light with a peak at 470
nm (spectral width ? 455–485 nm), 150 mW average
power, and 30 mW cm–2irradiance. To minimize thermal
radiation, the applicator is cooled by an built-in fan posi-
tioned to dissipate any heat produced by the diodes. In
preliminary studies, we ascertained that the device did not
generate any measurable temperature rise within the range
of fluences used in this study. To ensure even irradiation
of each plate, we used 5.0-cm2culture plates, which were
the same size as the surface area of the applicator, which
was clamped at a distance of 1–2 mm perpendicularly
above each open plate. As each dose was selected, the treat-
ment time was automatically computed by the Solaris de-
vice to ensure that the bacteria were irradiated with 0, 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60
J/cm2of energy fluence. For example, to attain a fluence
of 3 J/cm2, the Solaris device calculated that 100 sec of ir-
radiation was needed, and the device automatically shut
down after 100 sec had elapsed. Each culture was irradi-
ated only once. Afterward, the bacteria were incubated at
35°C for 24 h.
Quantification of bacterial colonies and data analysis
Standard digital images of plates with bacteria colonies
were taken, scanned into the computer, and then the
colonies were quantified with Sigma Scan Pro 5 software
(Systat Software, Inc., Point Richmond, CA, USA). The
colony counts and the aggregate area occupied by the
colonies were then automatically computed and subjected
to statistical analysis. The experiment was repeated sev-
eral times; four times with the US-300 strain and five times
with the IS-853 strain, to ensure accurate results. Descrip-
tive data were generated, then analysis of variance
(ANOVA) was performed with SPSS Version 14 statistical
software (SPSS Inc., Chicago, IL, USA) to test the null hy-
potheses, namely: (1) there were no differences in the
colony counts and aggregate area of bacteria at the fluences
tested, and (2) the effect of irradiation did not differ be-
tween the two strains.
ENWEMEKA ET AL.222
Photo-irradiation with 470-nm light produced a statisti-
cally significant dose-dependent reduction in both the num-
ber and the aggregate area of colonies formed by each strain
of bacteria (p ? 0.001; Figs. 1, 2, and 3). Higher dosages re-
sulted in progressively greater eradication of each strain.
However, the effect occurred as a non-linear exponentially
decreasing curve, with greater dose-to-dose improvement at
lower dose ranges than at higher dose ranges. Whereas 50%
of each strain was killed with approximately 12 J/cm2flu-
ence, up to 35 J/cm2fluence was needed to eradicate about
80% of the bacteria. Moreover, 60 J/cm2did not kill 100% of
the bacteria, as might have been expected from the impres-
sive effects of the lower doses (Table 1 and Figs. 1–3).
On average, 90.4% of the US-300 and the IS-853 colonies,
respectively, were killed with a dose of 55 J/cm2, which ap-
peared to be the most optimal dose of all doses tested. This
same dose eradicated 91.7% and 94.8% of the aggregate area
of the US-300 and the IS-853 strains, respectively. There was
no statistically significant difference in the effect of 470-nm
light on the two strains of bacteria at any dose.
Despite the development of stronger antibiotics to com-
bat MRSA infections, outbreaks of the disease have been
on the rise, as effective remedies for common strains re-
main elusive.21–23Of great concern is the frequent outbreaks
of CA-MRSA, which accounts for an ever-increasing pro-
portion of MRSA cases seen in the United States and other
countries.2,21–23CA-MRSA differs from HA-MRSA in its ge-
netic and antibiotic susceptibility profile.24Moreover, it is
strongly linked with the virulence factor Panton-Valentin
leukocidin, a toxin that is associated with an increased risk
of invasive disease, as well as skin and subcutaneous tissue
infections. Its staphylococcal cassette chromosome mec type
encodes for penicillin-binding protein (PBP2a), which is not
inhibited by ?-lactam antibiotics.24As a result, medications
such as the oral cephalosporins, fluoroquinolones, tetracy-
clines, trimethoprim-sulfamethoxazole, and erythromycin,
as well as the semisynthetic forms of penicillin, such as naf-
cillin, have been minimally effective in combating CA-
MRSA infections. Even vancomycin, our last well-proven
antibiotic line of defense against the bacterium, is now meet-
ing resistance. MRSA continues to evolve genetically,25
making it difficult to find an effective pharmacological rem-
edy. Thus an effective treatment must not just eradicate cur-
rent strains of the bacteria, but future genetic variants as
well. Th fact that 470-nm blue light eradicates the two ge-
netically different strains of MRSA we tested in a single
treatment session indicates that phototherapy may be a vi-
able alternative to drug treatment , and that it also has the
potential to kill future variant strains of MRSA. Our statis-
tical analysis showed that both strains were killed with
equal efficacy, suggesting a common mechanism for the ef-
fects seen on the two strains.
The precise mechanism behind the photo-eradication of
MRSA is beyond the scope of this study. However, it is note-
worthy that as early as 1930, Gates26showed that at in-
creasingly higher fluences, light with wavelengths longer
than 400 nm can kill bacteria as does UV light. Irradiation
with UV photo-destroys bacteria and other pathogens be-
cause the light energy is absorbed by the pyrimidine bases
of DNA such as thymidine and cytosine. The absorbed en-
ergy opens the bond, allowing the UV-modified base to re-
act with nearby bases, thereby altering the structural con-
formation of the bases. The photo-irradiated cell dies when
the resulting rate of DNA damage exceeds the rate of re-
pair.26,27It is possible that blue light photo-damages DNA,
resulting in its bactericidal effect on MRSA and other bacte-
BLUE LIGHT KILLS MRSA223
gate colony area of the US-300 strain of MRSA.
Effect of 470-nm light on colony count and aggre-
gate colony area of the IS-853 strain of MRSA.
Effect of 470-nm light on colony count and aggre-
ria, such as Propionibacterium acne,14,17,28and Pseudomonas
aeruginosa,15even though the peaks of absorption of pyrim-
idine bases are known to lie outside the blue spectrum; thus
other mechanisms may be involved. For example, photody-
namic inactivation of the bacteria through excitation of in-
tracellular porphyrins cannot be ruled out, given a recent re-
port that showed that bacterial eradication diminishes with
oxygen depletion.29Further studies are needed to uncover
the precise mechanisms involved in the effects of blue light
As shown in the table, about one-third of the US-300 strain
and nearly the same amount of the IS-853 strain was killed
with as little as 3 J/cm2of energy (i.e., 100 sec of irradiation).
Similarly, more than 40% and 60% of the respective strains
were photo-destroyed at 7 J/cm2, with the eradication of
over 80% of each strain occurring at 35 J/cm2. These signif-
icant levels of photo-destruction at low dosages indicate that
irradiation with 470-nm LED light energy may be a practi-
cal, inexpensive alternative to treatment with pharmacolog-
ical agents, particularly in cases involving cutaneous and
ENWEMEKA ET AL. 224
TABLE 1.THE BACTERICIDAL EFFECTS OF A SAMPLE OF DOSES ON THE TWO STRAINS OF MRSA TESTED
(J/cm2) Colony count Aggregate areaColony countAggregate area
34.1 ? 6.71
48.0 ? 4.26
61.2 ? 4.17
80.7 ? 7.82
90.4 ? 5.60
90.6 ? 5.66
29.5 ? 6.82
40.9 ? 9.85
49.2 ? 9.39
80.1 ? 8.64
91.7 ? 2.72
89.2 ? 7.98
27.6 ? 12.51
67.3 ? 4.790
56.4 ? 6.530
84.5 ? 4.380
90.4 ? 3.240
88.5 ? 5.910
29.1 ? 7.78
63.2 ? 3.54
58.9 ? 5.37
87.8 ? 4.35
94.8 ? 1.96
88.3 ? 7.35
Only doses relevant to the discussion in this paper are shown in this table. The effects of the other doses are shown in Figs. 1 and 2.
US-300 MRSA killed ? SEM (%)
IS-853 MRSA killed ? SEM (%)
number assigned to each photo represents the irradiation dose (J/cm2). Panel 0 shows a control plate, that received no ir-
radiation, and the one in the bottom row shows the results of the highest dose tested (60 J/cm2).
Representative photographs showing colonies of HA-MRSA (IS-853 strain) irradiated with 470-nm blue light. The
subcutaneous MRSA infections that are susceptible to non-
invasive types of irradiation.
This study and our previous reports19,20on the effects of
405-nm blue light on MRSA were done sequentially and not
in parallel. Consequently, our ability to compare findings
with 405 and 470 nm wavelengths is limited, but it is note-
worthy that both wavelengths killed the two genetically dif-
ferent strains of MRSA in the same non-linear fashion, with
impressive eradication of bacteria occurring at lower dose
ranges, and higher doses resulting in progressively greater
eradication of each strain. But the 470-nm light does so with-
out the attendant dangers of UV irradiation. As noted in the
introduction, Guffey and Wilborn15showed that 405-nm and
470-nm light photo-destroy two common aerobes, Staphylo-
coccus aureus and Pseudomonas aeruginosa, in vitro, but not
anaerobic P. acnes. By measuring colony counts alone, they
showed that the kill rate was slightly higher with 405-nm
wavelength than the 470-nm wavelength. Our results are
consistent with this and other reports,14,17,19,20which indi-
cate that blue light is bactericidal.
We conclude that 470-nm light kills HA-MRSA and CA-
MRSA in vitro, suggesting that a similar effect may be
achieved in vivo in human cases of MRSA infection, partic-
ularly in cutaneous and subcutaneous cases of MRSA infec-
tions that are susceptible to non-invasive types of irradia-
We thank Dr. Charles Pavia for helping us procure the
MRSA samples used in this study, and we acknowledge with
thanks the grant support provided by Dynatronics Corpo-
ration, Salt Lake City, Utah.
No conflicting financial interests exist.
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Address reprint requests to:
Dr. Chukuka S. Enwemeka, Ph.D., F.A.C.S.M.
School of Health Professions
New York Institute of Technology
Old Westbury, NY 11568-8000
ENWEMEKA ET AL.226