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. Author manuscript; available in PMC: 2016 Apr 16.
Published in final edited form as: Lasers Surg Med. 2015 Jan 12;47(3):266–272. doi: 10.1002/lsm.22327

Optimization of the Antimicrobial Effect of Blue Light on Methicillin-Resistant Staphylococcus aureus (MRSA) In Vitro

Violet V Bumah 1, Daniela S Masson-Meyers 1, Susan Cashin 1, Chukuka S Enwemeka 1,2,*
PMCID: PMC4834034  NIHMSID: NIHMS775151  PMID: 25639752

Abstract

Background and Objective

In previous studies, we showed that irradiation with 405 nm or 470 nm light suppresses up to 92% methicillin-resistant Staphylococcus aureus (MRSA) growth in vitro and that the remaining bacteria re-colonize. In this study, the aim was to develop a protocol that yields 100% MRSA growth suppression.

Materials and Methods

We cultured 3 × 106 and 5 × 106 CFU/ml USA300 strain of MRSA and then irradiated each plate with varying fluences of 1–60 J/cm2 of 405 nm or 470 nm light, either once or twice at 6 hours intervals. Next, we plated 7 × 106 CFU/ml and irradiated it with 45, 50, 55, or 60 J/cm2 fluence, once, twice, or thrice at the same 6 hours intervals. In a third experiment, the same culture density was irradiated with 0, 165, 180, 220, or 240 J/cm2, either once, twice, or thrice.

Results

Irradiation with either wavelength significantly reduced the bacterial colonies regardless of bacterial density (P < 0.05). At 3 × 106 CFU/ml density, nearly 40% and 50% growth of MRSA were suppressed with as little as 3 J/cm2 of 405 nm and 470 nm wavelengths, respectively. Moreover, 100% of the colonies were suppressed with a single exposure to 55 or 60 J/cm2 of 470 nm light or double treatment with 50, 55, or 60 J/cm2 of 405 nm wavelength. At 5 × 106 CFU/ml density, irradiating twice with 50, 55, or 60 J/cm2 of either wavelength suppressed bacterial growth completely, lower fluences did not. The denser 7 × 106 CFU/ml culture required higher doses to achieve 100% suppression, either one shot with 220 J/cm2 of 470 nm light or two shots of the same dose using 405 nm.

Conclusion

The bactericidal effect of blue light can be optimized to yield 100% bacterial growth suppression, but with relatively high fluences for dense bacterial cultures, such as 7 × 106 CFU/ml.

Keywords: antimicrobial blue light, bacteria suppression, methicillin-resistant Staphylococcus aureus, phototherapy

INTRODUCTION

Since 1993 when the first case of community associated MRSA (CA-MRSA) was reported from a remote part of Western Australia with no hospital facility [1], pandemic strains of CA-MRSA have been found on beaches, computer keyboards, locker rooms, schools, athletic fields, and other locations involving human congregations [2,3]. Estimates suggest that MRSA infection now accounts for 44% of all hospital associated infections in the United States [4], and CA-MRSA has been reported in as many as 92% of persons hospitalized for MRSA infection. With the prevalence rate higher in 2010 compared to 2006, a majority of health care facilities in the United States have now heightened surveillance testing [5]. While the proportion of patients with hospital associated MRSA (HA-MRSA) has decreased, clinical cases of CA-MRSA have been on the rise [5–7], with pulsed-field type USA300 strain often isolated during community outbreaks [8,9].

The main form of treatment to date remains antibiotic therapy, even though 40–50% of Staphylococcus aureus isolates are now resistant to methicillin and less than 5% of staphylococci strains are known to be susceptible to penicillin. Moreover, uncontrolled use of antibiotics continues to worsen the situation [10,11], prompting the exploration of alternative treatment approaches. Other therapies currently under investigation include: the use of antibacterial clay [12], combination of honey and antibiotics [6], hyperbaric oxygen [13,14], photodynamic therapy (PDT) [15], and blue light phototherapy [16–20].

It has been reported that mineral leachates, such as ions of copper, iron, cobalt, nickel, and zinc, from certain varieties of clay have antibacterial properties against Escherichia coli and MRSA [12]. These findings support the time tested use of clay for wound care referred to in the 5,000-year-old ancient tablets of Nippur [21] and in the Ebers Papyrus written circa 1,600 BCE where clay was reportedly used as a therapeutic modality for wounds and abscesses as well as gastrointestinal diseases [22]. The ubiquitous and inexpensive nature of clay makes clay therapy a promising alternative to antibiotic treatment for certain cases of MRSA infection; however, the mechanism involved remains unknown and corroborative studies are sparse. Another form of treatment, photodynamic therapy (PDT) has been used beneficially to treat dermatological and ophthalmologic disorders [18,23]. Even though the excitation light used in PDT is itself harmless, particularly in the near infrared (NIR) and visible ranges [23–25], interest in PDT as an antimicrobial agent has waned because available photosensitizers are often non-targeted and known to cause serious side effects.

After our experiments in which we successfully eradicated two strains of MRSA with 405 nm and 470 nm light in vitro [16,17], we proposed a paradigm shift in favor of light as antimicrobial therapy for topical cases of MRSA infection. Since then, our group and others have shown that 405 nm, 415 nm, and 470 nm blue light suppress growth in cultures of Staphylococcus aureus (both MRSA and Methicillin-sensitive Staphylococcus aureus [MSSA]), E. coli, and other bacterial pathogens [16,17,19,26–31]. In further studies, we showed that the bactericidal effects of both 405 nm and 470 nm light on MRSA are commensurate, suppressing as much as 92% of standard 5 × 106 CFU/ml cultures [17]. More recently, we found that the remaining bacteria not cleared by 405 nm or 470 nm light can re-colonize and develop into larger viable colonies, and that a larger percentage of the bacteria is not eradicated when denser cultures—intended to mimic severe bacterial infection—i.e., 7 × 106 CFU/ml to 12 × 106 CFU/ml, are plated [20]. The latter finding suggests a need to explore the development of a protocol that could yield 100% bacterial suppression.

Thus, this study was conducted as an attempt to optimize the antimicrobial effect of both 405 nm and 470 nm light by exploring the use of a variety of irradiation protocols in order to achieve 100% suppression of MRSA growth in vitro. Developing a laboratory-based protocol that results in 100% bacterial suppression could be of immense benefit in the ongoing effort to advance the use of commonly available blue Light emitting diodes (LEDs) to eradicate MRSA infection.

MATERIALS AND METHODS

Bacterial Culture

USA300 strain of CA-MRSA, obtained from ATCC (ATCC® BAA-1680), was used in our experiments because it is the most common isolate in community outbreaks of MRSA [8,9]. The strain was identified by standard identification procedures, including Gram’s staining, hemolytic patterns seen on blood agar, catalase and coagulase production, and PCR. As detailed in our previous reports [16,17,20], bacteria cultures were separately diluted to concentrations of 3 × 106, 5 × 106, and 7 × 106 CFU/ml, in 0.9% normal saline, to reflect increasing bacterial loads. Then bacteria were volumetrically streaked onto round 35-mm Petri dishes containing tryptic soy agar (TSA) before irradiation with the 405 nm or 470 nm blue light. Control plates were not exposed to blue light.

Photo-irradiation

Plated cultures of bacteria were photo-irradiated with a Dynatron Solaris® 708 device (Dynatronics Corp., Salt Lake City, Utah, USA) fitted either with a 405 nm or 470 nm light probe. The 5.0 cm2 applicator of the 405 nm probe, with its cluster of 36 LEDs, emits violet-blue light with a peak at 405 nm (spectral width of 390–420 nm), 500 mW average power, and 100 mW cm− 2 irradiance. The power emitted by this probe at a distance of 0.3–0.5 mm from the Petri dishes was 180 mW. We have determined that this average power is attenuated to 81 mW, as measured at the bottom of the petri dish, i.e., after light has passed through the culture medium; thus, indicating that a total of 99 mW is absorbed within the culture. The interchangeable 5.0 cm2 470 nm applicator has a cluster of 32 LEDs, and emits blue light with a spectral width of 455–485 nm; 470 nm peak), 150 mW average power, and 30 mW cm− 2 irradiance. Similarly, at a distance of 0.3–0.5 mm from the culture, i.e., the surface of the culture medium, the average power emitted by the probe is 25 mW. Measurement of the average power at the bottom of the culture medium indicates that this average power is attenuated to 7 mW, suggesting that 18 mW is absorbed within the culture. In order to minimize thermal radiation, the applicator is cooled by a built-in fan positioned to dissipate any heat produced by the diodes. In previous studies, we ascertained that the device did not generate any measurable increase in temperature within the range of fluences studied [16,17] (Table 1).

TABLE 1.

Properties of Blue LEDs Used for the Experiments

Probe wavelength 405 nm 470 nm
Spectral width 390–420 nm 455–485 nm
LED clusters 36 LEDs 32 LEDs
Surface area of Applicator 5 cm2 5 cm2
Average power 500 mW 150 mW
Irradiance 100 mW/cm2 30 mW/cm2
Power emitted by probe at
  a distance of 0.3–0.5 cm		 180 mW 25 mW
Power absorbed by culture 99 mW 18 mW
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To ensure even irradiation of each plate, we used 5.0 cm2 Petri dishes, which were the same size as the surface area of the applicator. The applicator was clamped at a distance of 1–2 mm perpendicularly above each open plate. As each dose was selected, the treatment time was automatically computed by the Solaris device 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/cm2.

Research Design

Three series of experiments were conducted:

Series one experiments

We investigated one treatment protocol using 405 nm and 470 nm light (Fig. 1, Series 1).

Fig. 1.

Fig. 1

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Research Design.

Protocol 1

Cultures plated with 3 × 106 or 5 × 106 CFU/ml MRSA were irradiated with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60 J/cm2 once and incubated at 35 °C for 24 hours.

Protocol 2

Cultures plated with 3 × 106 or 5 × 106 CFU/ml MRSA were irradiated with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60 J/cm2 twice, with 6 hours of incubation at 35 °C between treatments. Then, plates were incubated for an additional 18 hours; total incubation time being 24 hours.

Series two experiments

One treatment protocol was investigated using 405 nm and 470 nm blue light (Fig. 1, Series 2).

Protocol 3

Plates with 7 × 106 CFU/ml of MRSA were irradiated with 0, 45, 50, 55, and 60 J/cm2 either once, twice, or thrice at intervals of 6 hours during which the plates were incubated at 35 °C. Following treatment, plates were incubated until 24 hour after the first irradiation. Thus, twice treated plates were incubated for 18 hours following the second irradiation, and thrice treated plates were incubated for 12 more hours after the third irradiation.

Series three experiments

Again, we studied one treatment protocol in this series of experiments (Fig. 1, Series 3).

Protocol 4

Here we examined the effect of 405 nm and 470 nm irradiation on 7 × 106 CFU/ml MRSA cultures exposed to 0, 165, 180, 220, or 240 J/cm2 fluences either twice or thrice at 6 hour intervals. Plates were incubated at 35 °C during the 6hours between treatments followed by further incubation to ensure 24 hours total incubation time.

Quantification of Bacterial Colonies and Data Analysis

Standard digital images of plates with bacterial colonies were taken, scanned into the computer, and colonies quantified with Sigma Scan Pro 5 software (Systat Software, Inc., Point Richmond, CA, USA). The number of colonies were then automatically computed and subjected to statistical analysis. Each experimental protocol was repeated three times to enhance data accuracy. Descriptive data were generated, and then analysis of variance (ANOVA) was performed with SPSS Version 18 statistical software (SPSS Inc., Chicago, IL, USA) to determine the effect of fluence on colony counts, and, in particular, to ascertain if any of the optimization protocols resulted in 100% bacterial growth suppression.

RESULTS

General Observation

Consistent with our previous reports, both wavelengths produced a statistically significant reduction in the number of bacterial colonies at each bacterial density tested, progressively with increases in fluence (P < 0.001). Moreover, the effect was non-linear even though higher fluences resulted in greater bacterial suppression [16,17,20]. In line with the aforementioned reports, more bacteria were cleared per unit fluence at the lower dose ranges; i.e., doses in the range of 1–11 J/cm2, with 5.0 J/cm2 dose clearing more than 50% of the colonies in the 3 × 106 CFU/ml and 5 × 106 CFU/ml plates.

Series One Experiments

Effect of single and double irradiation on 3 × 106 CFU/ml culture

Irradiation of the 3 × 106 CFU/ml culture with either 405 nm or 470 nm wavelength resulted in significant suppression of the MRSA colonies progressively as dose increased. In general, more bacterial growth was suppressed with double irradiation (Figs. 2 and 3). However, with either wavelength, the difference between single or double irradiation at each of the 18 fluences tested was minimal, except at 13, 15, 25, 55, and 60 J/cm2 dosages of 405 nm light (Fig. 2) and at 11, 15, 17, 19, and 45 J/cm2 dosages of 470 nm (Fig. 3) where bacterial growth suppression were significantly different (P < 0.05). Single irradiation with either 55 or 60 J/cm2 of 470 nm wavelength resulted in 100% bacterial suppression; 100% suppression was also achieved when the culture plates were twice exposed to either 50, 55, or 60 J/cm2 of 405 nm light, or 45, 50, 55, or 60 J/cm2 of 470 nm light (Figs. 2 and 3).

Fig. 2.

Fig. 2

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Comparative effect of single and double treatment of 405 nm blue light on MRSA USA300, 3 × 106 CFU/ml [*P < 0.05].

Fig. 3.

Fig. 3

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Comparative effect of single and double treatment of 470 nm blue light on MRSA USA300, 3 × 106 CFU/ml [*P < 0.05].

Effect of single and double irradiation on 5 × 106 CFU/ml culture

In general, irradiation of the denser 5 × 106 CFU/ml gave a similar result as treatment of the 3 × 106 CFU/ml culture, with double irradiation yielding minimally better results than single irradiation at most of the 18 fluences tested, and with progressively more bacterial growth suppression as fluence increased. However, double irradiation with either 3, 13, 17,19, 25, 30, or 55 J/cm2 of 405 nm light or 470 nm light at 50 or 55 J/cm2 fluence resulted in significantly more suppression than single treatment (P < 0.05; Figs. 4 and 5). Single irradiation with either wavelength did not result in 100% bacterial suppression at any of the fluences tested. However, double irradiation with 50, 55, or 60 J/cm2 of 405 nm light or 55 J/cm2 of 470 nm light gave 100% bacterial growth suppression (Figs. 4 and 5).

Fig. 4.

Fig. 4

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Comparative effect of single and double treatments of 405 nm blue light on MRSA USA300, 5 × 106 CFU/ml [*P < 0.05].

Fig. 5.

Fig. 5

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Comparative effect of single and double treatments of 470 nm blue light on MRSA USA300, 5 × 106 CFU/ml [*P < 0.05].

Series Two Experiments

Effect of single, double, or triple irradiation on 7 × 106 CFU/ml culture

Since 100% MRSA suppression was achieved mainly by exposing the bacteria twice to the higher fluences, we limited series two experiments to testing the effects of multiple irradiation of the bacteria with the high end doses, particularly as we explored the effect of blue light on the denser 7 × 106 CFU/ml culture. Each of the four fluences tested resulted in statistically significant reduction in MRSA colonies (P < 0.05). Progressively more bacteria were suppressed as irradiation was repeated. Triple irradiation gave the best result, particularly at 45 and 50 J/cm2 fluences at which there were significant differences between the three modes of treatment (Figs. 6 and 7). A comparison of the four fluences tested, showed no statistically significant differences in suppression of bacterial growth regardless of wavelength. Thus, the outcome of irradiation with 45 J/cm2 was as good as the outcome of irradiation at the higher 50, 55, or 60 J/cm2 dosages. Notwithstanding the number of irradiation, single, double, or triple, at none of the fluences tested was total bacterial growth suppressed.

Fig. 6.

Fig. 6

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Comparative effect of single, double, and triple treatments of 405 nm Blue light on MRSA USA300, 7 × 106 CFU/ml [*P < 0.05].

Fig. 7.

Fig. 7

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Comparative effect of single, double, and triple treatments of 470 nm Blue light on MRSA USA300, 7 × 106 CFU/ml.

Series Three Experiments

Effect of high dose single, double, or triple irradiation on 7 × 106 CFU/ml culture

Since we did not achieve 100% growth suppression of the denser 7 × 106 CFU/ml culture, regardless of the number of times the bacteria were irradiated, we repeated the last experiment with double the amount of each of four fluences used in protocol 3 of series two experiments. With either wavelength, three times irradiation of the bacteria using either 90, 100, 110, or 120 J/cm2 at 6 hours intervals did not fully suppress bacterial growth in this dense culture, 7 × 106 CFU/ml (Data not shown). However, single irradiation of the bacteria with 220 J/cm2 fluence of 470 nm light totally cleared the bacteria at this same culture density (Table 2), as did two time irradiation with 405 nm light at the same dose repeated at 6 hours interval (Tables 2 and 3). About the same suppression of bacterial growth was achieved using 240 J/cm2 (Tables 2 and 3).

TABLE 2.

Effect of High Fluences of 470 nm Blue Light on USA300 MRSA (7 × 106 CFU/ml)

Fluence
(J/cm2)		 Single
irradiation
(colony count,
%survival)		 Double
irradiation
(colony count,
%survival)		 Triple
irradiation
(colony count,
%survival)
0 100 100 100
220 0 0 0
240 1 0 0
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TABLE 3.

Effect of High Fluences of 405 nm Blue Light on MRSA USA300 (7 × 106 CFU/ml)

Fluence
(J/cm2)		 Single
irradiation
(colony count,
%survival)		 Double
irradiation
(colony count,
%survival)		 Triple
irradiation
(colony count,
%survival)
0 100 100 100
220 1.67 0 0
240 2.67 0 0
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DISCUSSION

Our results support previous findings that 405 nm and 470 nm light are bactericidal, and indeed, destroy MRSA particularly at higher doses [16,17,20,30,31]. Our finding that 100% bacterial growth suppression was achieved only with single irradiation of 3 × 106 CFU/ml culture but not the denser cultures, clearly shows that repeated irradiation is indeed necessary for effective bacterial growth suppression at relatively low fluences. This is evidenced by the fact that twice exposure to either 50, 55, and 60 J/cm2 of 405 nm light or 45, 50, 55, or 60 J/cm2 of the 470 nm wavelength completely suppressed the 3 × 106 CFU/ml culture. Moreover, double irradiation at similar doses, i.e., 50, 55, or 60 J/cm2 of 405 nm light or 55 J/cm2 of 470 nm light suppressed 100% of the bacterial colonies in the standard 5 × 106 CFU/ml culture. Thus, the overall outcome of the first series of experiments in this study is that 55 and 60 J/cm2 of 470 nm light completely suppressed the less dense 3 × 106 CFU/ml culture. While either 405 or 470 nm light has the potential to suppress 100% of the standard 5 × 106 CFU/ml culture in vitro, irradiation must be done twice to achieve this outcome.

The extent of infection—mild, moderate, or severe—is hard to predict at the beginning of treatment. Therefore, twice irradiation with 55 J/cm2 dose may not be a panacea for all MRSA infection, if infection is severe. And this is evidenced by the results of our series two and three experiments which clearly show that with the denser 7 × 106 CFU/ml culture this dose must be quadrupled, and in the case of 405 nm light, applied at least twice, in order to achieve 100% suppression of bacterial growth. Repeated double application of the 55 J/cm2 dose of either wavelength did not result in 100% suppression. Moreover, with either wavelength, even three times irradiation of the bacteria using either 90, 100, 110, or 120 J/cm2 at 6 hour intervals did not fully eradicate dense bacterial cultures (7 × 106 CFU/ml). Only when these fluences were quadrupled was a significant suppression of bacterial growth achieved. This finding is consistent with our previous study in which we showed that wavelength and bacterial density influence the bactericidal effect of blue light [20].

One remarkable observation is that double-treatment using 405 nm LED at the fluence of 13 J/cm2 (total fluence being 26 J/cm2) resulted resulted in about 85% suppression, and single treatment at 25 J/cm2 fluence resulted in about 75% MRSA suppression. Similarly, double-treatment using 470 nm LED at 13 J/cm2 fluence (total fluence being 26 J/cm2) resulted in about 89% killing of bacteria, whereas single treatment with 25 J/cm2 fluence gave about 75% killing. This finding may be explained by the fact that when treatment is done twice—in this case using 13 J/cm2—the first dose clears some of the bacteria during the time interval between the first and second dose. The second irradiation clears a further amount of bacteria since the underlying layer of MRSA colonies are now exposed to the second dose. Thus, the total amount of bacteria cleared is slightly higher when irradiation is done twice instead of once with a comparable total dose.

The overall outcome of this study is that under our experimental conditions and given our treatment protocol, 100% bacterial suppression is achievable with repeated irradiation using 55 J/cm2 for standard cultures or those that are less dense, or 220 J/cm2 fluence for those denser than the standard 5 × 106 CFU/ml, for example 7 × 106 CFU/ml. This finding indicates that MRSA growth suppression can be optimized.

Our experiment was designed to mimic practical clinical treatment protocols, such as patient medication which typically occurs at 4, 6, or 12 hour intervals. It is possible that the outcome of our experiment would have been different if a different irradiation regime was used to treat the bacterial colonies. Considering that it will be safer to achieve full MRSA suppression at lower fluences, efforts to explore the possibility of achieving 100% bacterial growth suppression at lower doses using other treatment protocols will be pursued.

In previous studies, we showed that 405 nm and 470 nm blue light suppresses the growth of both community associated (MRSA USA300) and hospital associated MRSA (IS853) strains [17,18], thus demonstrating the versatile nature of blue light to suppress multiple strains of MRSA. In recent works, we demonstrated that blue light suppresses the growth of Gram-negative Salmonella bacteria, again indicating the versatility of the antimicrobial effect of blue light irradiation (manuscript in preparation). These findings suggest that the mechanisms involved in the antibacterial effects of blue light broadly apply to different strains of bacteria.

Postulated mechanisms of action involve the absorption of this monochromatic light by endogenous chromophores within the bacteria which leads to the production of reactive oxygen species (ROS), and eventual destruction of bacteria membrane and cell death. At present, we are examining the effects of blue light on MRSA genome using DNA profiling research methodologies such as pulsed field electrophoresis, PCR, and whole genome sequencing to further determine the effects of blue light on the genome of MRSA.

CONCLUSION

Our results warrant the conclusion that: (1) 55 J/cm2 of either 405 or 470 nm light has the potential to suppress 100% of standard or less dense cultures of MRSA infection in vitro; however, irradiation must be done at least twice to achieve this outcome. (2) With denser cultures, such as 7 × 106 CFU/ml, this dose must be quadrupled and, in the case of 405 nm light, applied at least twice, in order to achieve 100% growth suppression. (3) Under our experimental conditions, 100% bacterial suppression is achievable with repeated irradiation using 55 J/cm2 for standard or less dense cultures, or 220 J/cm2 fluence for denser cultures.

Acknowledgments

We gratefully acknowledge the financial support provided by the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources and the National Center for Advancing Translational Sciences (Grant: UL1RR031973) and the financial support and infrastructure provided by the College of Health Sciences, University of Wisconsin-Milwaukee, USA. The authors thank Dynatronics Corporation for donating the Solaris® device used in this study.

Contract grant sponsor: National Center for Research Resources and the National Center for Advancing Translational Sciences; Contract grant number: UL1RR031973.

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