Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5′-phosphate photolysis

https://doi.org/10.1016/j.jphotobiol.2017.07.009Get rights and content

Highlights

  • Violet light is a novel light source in the photolysis of FMN.

  • S. aureus inactivation under blue light illumination is lower than violet light.

  • Blue and violet light with FMN are optimized to inactivate S. aureus and MRSA.

  • FMN photolysis by violet light could inactivate S. aureus under low-energy dose.

Abstract

The light sensitive compound riboflavin-5′-phosphate (or flavin mononucleotide, FMN) generates reactive oxygen species (ROS) upon photo-irradiation. FMN is required by all flavoproteins because it is a cofactor of biological blue-light receptors. The photochemical effects of FMN after irradiation by blue or violet light on the inactivation of Staphylococcus aureus strains, including a methicillin-resistant strain (MRSA), were investigated in this study. Upon blue- or violet-light photo-treatment, FMN was shown to inactivate S. aureus due to the generated ROS. Effective bacterial inactivation can be achieved by FMN photolysis without an exogenous electron provider. Inactivation rates of 94.9 and 95.2% in S. aureus and MRSA, respectively, can be reached by blue light irradiation (2.0 mW/cm2) with 120 μM FMN for 120 min. A lower FMN concentration and a shorter time are required to reach similar effects by violet light irradiation. Inactivation rates of 96.3 and 97.0% in S. aureus and MRSA, respectively, can be reached by violet light irradiation (1.0 mW/cm2) with 30 μM FMN for 30 min. The sensitivity of the inherent photosensitizers is lower under blue-light irradiation. A long exposure photolytic treatment of FMN by blue light is required to inactivate S. aureus. Violet light was found to be more efficient in S. aureus inactivation at the same radiant intensity. FMN photolysis with blue or violet light irradiation enhanced the inactivation rates of S. aureus and MRSA. FMN photochemical treatment could be a supplemental technique in hygienic decontamination processes.

Introduction

Staphylococcus aureus is a common Gram-positive pathogenic colonizer found on the skin and in the nasal mucous membrane of humans and animals. It has been reported that S. aureus causes food poisoning and gastroenteritis as a result of food contamination [1]. S. aureus is a major human pathogen that produces a wide range of toxins accompanied with various disease symptoms [2]. It is one of the important nosocomial pathogens that cause wound infections associated with life-threatening diseases such as bacteraemia, pneumonia and endocarditis. S. aureus is a common indicator organism for the quality of hygienic standards.

The adaptability of S. aureus contributes to its prevalence in human populations. Many attempts to stop the spread of S. aureus have failed since the dissemination of its plasmids containing the penicillin-cleaving enzyme penicillinase. Consequently, stronger semisynthetic penicillinase-resistant penicillins have been synthesized against resistant isolates [3]. An organism exhibiting this type of resistance is referred to as methicillin (oxacillin)-resistant S. aureus (MRSA). This strain also builds resistance to most of the commonly used antimicrobial agents [4]. MRSA emerged in 1961, shortly after the introduction of this antibiotic [5]. MRSA is a virulent pathogen responsible for both health care-associated and community onset diseases [6]. It has been reported that approximately 20,000 hospitalized American patients died of MRSA infections in 2005 and the trend continues to increase [7]. Moreover, a new strain of MRSA was discovered in animals that can be transmitted to humans [8]. The development of new antibiotics for clinical use usually takes decades. Therefore, alternative therapies that can disinfect environmental, animal and human sources of MRSA are important.

Bacteria build resistance towards antibiotics efficiently during evolution. To solve this dilemma, different approaches have been proposed. One novel approach is to use antibacterial photodynamic inactivation of bacteria (aPDI) [9]. It has been reported that high-intensity ultraviolet light C (UVC) is an effective antimicrobial in both Gram-negative and Gram-positive strains [10]. However, UVC is the most hazardous type of UV irradiation due to its high energy. Visible light has been studied for the inactivation of microorganisms [11], [12], [13], [14]. High-intensity violet light has been shown to be effective in the inactivation of S. aureus. However, exposed Escherichia coli suspensions have shown negligible inactivation over a 30-min exposure time to high intensity violet light [15]. It has been proposed that violet light induces endogenous intracellular porphyrins to mimic the ability of the photosensitizer by formulating reactive singlet oxygen and other reactive oxygen species (ROS). Free radicals produced by such methods ultimately destroy the bacteria by disrupting its organelles and chromosomes [16]. It would be interesting to study whether adding a photosensitizer with visible light could enhance the efficacy of the inactivation of S. aureus.

The micronutrient riboflavin is required by the human body to undergo cellular processes, such as the TCA cycle, for energy production. It is also required by flavoproteins, such as the cofactors flavin adenine dinucleotide (FAD) and riboflavin-5′-phosphate (FMN), to metabolize fats, ketone bodies, carbohydrates and proteins [17]. The micronutrient FMN is an essential vitamin required for the functions of the human body.

Pathogenic microorganisms can be inactivated by irradiation by visible light under some specific conditions, such as appropriate photosensitizers [18]. Both riboflavin and FMN are sensitive to light [19]. They have been illuminated under UV [17], [19], [20], [21], [22] and blue light [23], [24] to reach a photo-excited state. After being light photo-energized, riboflavin is converted into triplet excited-state riboflavin. Superoxide anion (O2radical dot) or singlet oxygen is produced through the reaction of the triplet excited-state riboflavin [19], [25].

Blue light has been found to be the most efficient for the photo-decomposition of FMN in visible light [24]. Riboflavin and FMN photochemical treatments by blue light irradiation have been proven to be an effective technology for inactivation of E. coli with generated ROS [23], [24], [26]. Similar to DNA degradation, membrane peroxidation and destruction of endothelial cells, these harmful biochemical reactions create O2radical dot in cells [27]. E. coli is a Gram-negative bacterium. Our previous study showed 96% inactivation of E. coli after FMN incubation and blue light (462 nm) irradiation at 14.4 J/cm2 [23], [24]. It would be interesting to study whether FMN photochemical treatments could enhance the efficacy of inactivation of Gram-positive S. aureus, including methicillin-resistant strains.

The wavelength of blue light is longer than those of ultra-violet and violet light. Lights of shorter wavelengths, which have higher energy, might cause a higher degree of damage to cells. With an appropriate photosensitizer, irradiation by violet light might also inhibit pathogenic microorganisms under low-intensity specific conditions.

O2radical dot generated from light-excited riboflavin can be determined by nitro blue tetrazolium (NBT) [28]. The photochemically excited riboflavin from the NBT reduction method is first reduced by methionine into a semiquinone, which donates an electron to oxygen to form the O2radical dot source [29]. The O2radical dot level was higher in FMN than in riboflavin at the same level of blue light irradiation with NBT reduction [24].

FMN mediated photo-inactivation with violet or blue light is a potential disinfection method to eradicate environmental S. aureus and MRSA. The aim of the current study was to develop an effective antimicrobial method in vitro by applying either blue or violet light to FMN photochemical reactions under low-energy dose conditions. The study was to compare the effects of blue or violet light on the FMN photosensitization and inactivation of S. aureus by light intensity, irradiation time, and irradiation dosage. The cleavage of genomic DNA of S. aureus caused by the FMN photochemical treatment was also examined. Furthermore, the microbial viability of S. aureus and MRSA are the essential clue to estimating the efficiency of the novel hygienic process.

Section snippets

Chemicals

The chemicals used in this study, such as ethylenediaminetetraacetic acid (EDTA), FMN, isopropanol, l-methionine, mono‑potassium phosphate, potassium dihydrogen phosphate, sarcosine, sodium chloride, and urea, were all purchased from Sigma-Aldrich (St. Louis, MO, USA). NBT was purchased from Bio Basic, Inc. (Markham, Ontario, Canada). RNase was purchased from New England BioLabs, Inc. (Ipswich, MA). Tris-HCl was purchased from MDBio, Inc. (New Taipei, Taiwan). The reagent used for DNA staining

Effects of Blue and Violet Light Irradiation on FMN Spectra

The spectra (250–650 nm) of 120 μM FMN were measured during the course of blue and violet light irradiation of the photo-decomposition reactions. Fig. 3 shows the three peaks of FMN (260, 372, and 444 nm) in the spectrum of the dark control. The absorbance of FMN at 444 nm was decreased 50% for 10 min blue light irradiation and above 80% reduction for 20 and 30 min blue light irradiation, as shown in Fig. 3(A). The irradiation of violet light showed the highest efficiency in the photo-decomposition

Discussion

A photosensitizer can activate or catalyse the degradation of chemical compounds to generate ROS by irradiation. Pathogenic microorganisms can be inactivated by lights under appropriate photosensitizers. The present study showed the antibacterial photodynamic inactivation of S. aureus and MRSA by blue or violet light-induced ROS from FMN.

As shown in Fig. 3, the absorbance of FMN at 444 nm was decreased dramatically by irradiation with blue or violet light for 20 min. Such phenomena indicate that

Conclusions

The effects of blue and violet lights on the inactivation of pathogenic microorganisms with FMN photolysis were investigated. Violet light could be considered as a novel light source in FMN photo-decomposition.

Blue or violet light with FMN photolysis could be employed to inactivate microorganisms. The endogenous intracellular photosensitizers may be excited by blue and violet light, which donate an electron to excite FMN to form O2radical dot. At the same energy dose, the inactivation of pathogenic

Acknowledgments

The authors are grateful to Mr Chin-Hao Yu for his assistance with the experimental work.

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