Inactivation of Mycobacterium smegmatis Following Exposure to 405-Nanometer Light from a Supraluminous Diode Array

Index: WOUNDS. 2013;25(5):131–135.

  Abstract: Objective. To determine the potential for blue light (405 nm) to produce a bactericidal effect on Mycobacterium smegmatis. Additionally, the study sought to evaluate a series of doses in terms of their respective bactericidal capabilities. Background Data. The effect of blue light on Staphylococcus aureus has been studied and it was found that a bactericidal outcome can be obtained with low doses of blue light.¹Methods. M. smegmatis was tested because of the recent appearance of the Mycobacterium family of organisms as a public health threat among persons receiving tattoos. The organism was treated in vitro with 405 nm light emitted from a supraluminous diode (SLD) array. Doses of 60 Jcm^-2, 90 Jcm^-2, 120 Jcm^-2, 150 Jcm^-2, 180 Jcm^-2, 215 Jcm^-2, and 250 Jcm^-2 were used. Colony counts were performed and compared to untreated controls using Student t tests and one-way ANOVA with Tukey post hoc analysis. Results. The results revealed statistically significant bactericidal effects of the blue light on M. smegmatis (F6, 28 = 50.518, P = 0.000). The treatment reduced the number of bacterial colonies at all doses, but 60 Jcm^-2 did not produce a statistically significant kill rate. All other doses produced a significant kill rate with 120 Jcm^-2, 150 Jcm^-2, and 215 Jcm^-2, demonstrating the most effective kill rates of 98.3%, 96.7%, and 100%, respectively. Conclusions. Appropriate doses of 405 nm light from an SLD array can kill M. smegmatis in vitro. A dose of at least 100 Jcm^-2 dose is needed for the most effective inactivation of the organism. The dose response for this organism to blue light is not linear. Some degree of effectiveness is lost at 180 Jcm^-2 and 250 Jcm^-2.

Introduction

  For several decades, antibiotics have been the treatment of choice for fighting infections. As effective as they have been, the development of antimicrobial resistance may bring an end to this era of antibiotic use.² New Delhi metallo-ß-lactamase-1(NDM-1), an enzyme that renders some bacteria resistant to almost all antibiotics, has recently been found in the United States.³ It is a common concern that bacteria associated with infectious diseases will become unresponsive to antibiotic treatment.² This concern has led to increased interest in light therapy as an alternative to antibiotic treatment.   Light therapy has the ability to provide equal killing effectiveness regardless of antibiotic resistance.² Commonly employed sources of light therapy include photodynamic therapy (PDT), ultraviolet C (UVC) light, and blue light. Currently, there is no evidence of microbial resistance to PDT, giving it great potential for fighting infections.⁴ However, PDT requires the addition of exogenous photosensitizers. Another source of light therapy, UVC light, is known to inhibit bacterial growth, but has produced detrimental effects such as burns, premature aging, suppression of the immune system, and even skin cancers on mammalian cells and host tissue.⁵ While all 3 are effective sources of light therapy, blue light can be administered without the addition of exogenous photosensitizers and is less harmful to human skin.⁶   Blue light has demonstrated a dose-dependent bactericidal effect on a number of organisms. For instance, 405 nm and 470 nm blue light achieved a bactericidal effect on both Staphylococcus aureus and Pseudomonas aeruginosa, in vitro.¹ In another study, 405 nm blue light resulted in inactivation of Shigella sonnei, Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Mycobacterium terrae.⁷ The mechanism of blue light inactivation is not completely understood. It is believed that bacterial inactivation results from the photo-excitation of porphyrins and the subsequent production of cytotoxic reactive oxygen species.^8-10   The genus Mycobacterium includes some very noteworthy pathogenic species that cause significant morbidity and mortality. The most well-known species, Mycobacterium tuberculosis and Mycobacterium leprae, cause tuberculosis and leprosy. Pathogens from the Mycobacterium tuberculosis complex such as M. bovis and M. tuberculosis remain among the most serious causes of infective disease worldwide.⁷ Recently, M. chelonae skin infections were reported in 14 New York residents who received tattoos between September 2011 and December 2011.¹¹ Blue light therapy has shown potential to be an effective treatment of Mycobacteria in vitro.⁷   Studies using PDT, UV, and blue light have demonstrated a bactericidal effect on Mycobacteria, but this outcome is not universal in all conditions. In a study on photodynamic therapy of M. smegmatis, it was discovered that photosensitizers must be cationic to achieve inactivation.¹² Ultraviolet light is commonly used for disinfection of food and water. Ultraviolet light has demonstrated a reduction in M. avium complex organisms when treating water, but the same is not the case when pasteurizing milk.^13-15 Obviously, research is necessary to determine the bactericidal effect of light therapy on Mycobacteria under various conditions and using specific techniques.   While research regarding blue light and Mycobacteria is developing, a bactericidal effect has recently been demonstrated. When a dose between 144 Jcm^-2 and 288 Jcm^-2 was administered, 405 nm blue light was shown to inactivate M. terrae by 4-5 log₁₀ (CFU mL^–1).⁷ To determine the most effective and least detrimental dose of blue light, a pattern of inactivation must be developed. Furthermore, to understand the impact blue light may have on fighting infection, microbial resistance must be determined. The purpose of this research was to evaluate, in vitro, the bactericidal effect of blue light on M. smegmatis in a similar fashion to the work by Murdoch et al.⁷

Methods

  The strain of the organism tested was M. smegmatis. This organism grows well in ambient air. The organism was obtained from a 20-hour-old culture. A suspension equivalent to a 0.5 McFarland Standard was prepared. Use of a 20-hour-old culture is standard microbiological practice and serves to minimize the lag time for new growth. The suspension was further diluted to 1/1000 using 100 microliter automatic pipettes for purposes of accuracy and reproducibility. All dilutions were made immediately before the treatment with blue light (405-nm). Ten microliter aliquots of the 1/1000 dilution of M. smegmatis were inoculated onto Middlebrook 7H10 agar plates (60 X 15 mm). Middlebrook 7H10 is a chemically defined medium formulated to supply the growth factors required for Mycobacterium species. Mature colonies are obtained sooner on Middlebrook 7H10 agar than on egg-based media such as Lowenstein-Jensen slants. The microorganism was applied to the surface of the agar plates in a star-streak pattern to enable colony counts to be performed.   The experimental treatment consisted of 7 subcomponents. The organism was prepared (n = 10 plates, 5 treatment and 5 control) and treated at 1 dose (Jcm^-2) on 1 day. The process was repeated a total of 7 times (7 different doses) on separate days. Controls for a given day’s subcomponent consisted of 5 plates handled under the same conditions of ambient light and temperature as the treated plates. Treated plates received exposures to blue light of 60 Jcm^-2, 90 Jcm^-2, 120 Jcm^-2, 150 Jcm^-2, 180 Jcm^-2, 215 Jcm^-2, and 250 Jcm^-2. Related quantitative data can be found in Table 1.   The treated and control plates were incubated at 35°C under aerobic conditions. M. smegmatis is a slow-growing, nonphotochromogenic Mycobacterium species and therefore required an extended incubation period as long as 3 days to 4 days for mature colonies to be observed. It can be readily distinguished from contaminating microbes due to the characteristic macroscopic morphology on the Middlebrook 7H10 agar plates. M. smegmatis forms rough, raised, nonpigmented, friable colonies, which some have likened to “bread crumbs.”¹⁶ Most contaminating organisms would form smooth, raised, circular colonies with a butyrous consistency.   Light exposures were achieved using the Dynatron® 701 Solaris™ (Dynatronics Corp, Salt Lake City, UT). This ultrasound and light therapy device is designed to accommodate a variety of light probes. For this experiment, the authors chose to illuminate the cultures using 405 nm wavelength produced by a SLD light probe that emitted a band of light focused around a primary wavelength. The probe consisted of a 5 cm² illuminating surface area and was composed of thirty-two 405 nm SLDs with an average power output of 160 mW. Dose was calculated in J/cm². Since power output for each probe was held constant, adjustment in time of irradiation provided the scale of doses (60 Jcm^-2, 90 Jcm^-2, 120 Jcm^-2, 150 Jcm^-2, 180 Jcm^-2, 215 Jcm^-2, 250 Jcm^-2). The ultrasound and light therapy device used in this study automatically calculates time of irradiation when desired dosage is selected.

Statistical Analysis

  Data were analyzed using a paired Student’s t test and a one-way ANOVA. Post hoc for the ANOVA was performed using Tukey’s honestly significant difference test. The software employed for the data analysis was SPSS 20.

Results

  Table 1 demonstrates the effectiveness of blue light in terms of its bactericidal impact on M. smegmatis. Each dose demonstrated a statistically significant kill rate (P  0.05). While each dose proved effective in terms of limiting the growth of the organism, differences in kill rate were observed. To evaluate these differences, a one-way ANOVA was performed. See Table 2 for the results of that analysis. One-way ANOVA revealed a significant dose effect (F6, 28 = 50.518, P  0.000). The associated post hoc test confirmed 120 Jcm^-2, 150 Jcm^-2, and 215 Jcm^-2 as the most effective doses (P  0.001). Figure 1 provides a graphical presentation of the kill rates by dose with standard error of each mean included.

Discussion

  Murdoch et al⁷ has shown that blue light could effectively inhibit the growth of M. smegmatis in vitro. The current study included additional dose levels to more completely examine any dose-specific effects. The Murdoch et al⁷ work further supported the intention of this study. Due to previous work,¹ the authors were already familiar with the effectiveness of blue light at inhibiting growth of other organisms.   The authors were initially encouraged to pursue this line of investigation by the work done by Maclean et al.¹⁷ As these authors pointed out, visible light, particularly 405 nm light, has the potential to serve as an effective and novel decontamination technique. The research agenda of the authors of the current study is precisely directed toward using light for antimicrobial applications in wound care.   It is clear that the dose level needed to effectively inhibit Mycobacterium species is significantly greater than that required with other organisms the authors have studied. Taking into account the research⁷ and the results from this study, an approximately 100 Jcm^-2 dose is needed to inactivate this organism. Also of interest is the fact that some of these higher doses (180 Jcm^-2) are not as effective as might be assumed (See Figure 1). The kill rates obtained in this work were not linear and the authors are not able to explain why this lack of predictable response occurred. The answer may lie in the unique nature of this organism. What sets the genus Mycobacterium apart from other bacteria is the composition of the cell wall that includes constituents such as peptidoglycan, arabinogalactan, and mycolic acids (waxy compounds).⁷ These compounds confer resistance to dessication, and are found in many commonly used disinfectants, antimicrobial agents, and phagocytosis.   Mycobacterium has become a more common threat to public health. In January 2012 there were 14 cases of skin infection attributed to Mycobacterium chelonae. The source of these skin infections was traced to the use of a nationally distributed, prediluted gray ink manufactured for the tattoo industry. The Centers for Disease Control, state and local health departments, along with the Food and Drug Administration, recently found nontuberculosis Mycobacterium contamination in tattoo inks used in 2 of 5 identified clusters. All infected persons were exposed to 1 of 4 different brands of ink.¹¹   Blue light is effective at limiting growth of M. smegmatis. The dose required to achieve this inhibition is greater than with other organisms commonly associated with human skin lesions. Still, the lack of negative effects on host tissue associated with blue light suggest this in vitro study may have potential application for clinical use.

Conclusions

  Blue light is an effective bactericidal agent to M. smegmatis in vitro. The dose level required to achieve this effect is high. At least a 100 Jcm^-2 dose is needed for effective inactivation of the organism. The dose response for this organism to blue light is not linear. Some degree of effectiveness is lost at 180 Jcm^-2 and 250 Jcm^-2.   The authors plan to continue examining the response of this organism to visible light. An additional study is planned to investigate the response obtained when other wavelengths are employed alone and in combination with blue light. The authors are also engaged in work to determine whether this organism can develop a resistance to dose levels that are initially effective.

References

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