High-intensity narrow-spectrum light inactivation and wavelength sensitivity of Staphylococcus aureus

Transcription

1 RESEARCH LETTER High-intensity narrow-spectrum light inactivation and wavelength sensitivity of Staphylococcus aureus Michelle Maclean, Scott J. MacGregor, John G. Anderson & Gerry Woolsey The Robertson Trust Laboratory for Electronic Sterilisation Technologies, Department of Electronic and Electrical Engineering, University of Strathclyde, Glasgow, Scotland Correspondence: Michelle Maclean, The Robertson Trust Laboratory for Electronic Sterilisation Technologies, Department of Electronic and Electrical Engineering, University of Strathclyde, Royal College Building, 24 George Street, Glasgow, Scotland, G1 1XW, UK. Tel.: ; fax: ; Received 19 December 27; accepted 15 May 28. First published online 16 June 28. DOI:1.1111/j x Editor: Mark Enright Keywords photodynamic inactivation; visible light; Staphylococcus aureus ; wavelength sensitivity. Introduction The increasing problem of microbial antibiotic resistance has generated interest in alternative methods of inactivating problematic organisms such as methicillin-resistant Staphylococcus aureus (MRSA). One area of interest involves the use of light-based treatment technologies. Much research has been carried out involving the photodynamic inactivation (PDI) of S. aureus and MRSA using light, particularly light of visible wavelengths, and exogenous photosensitisers such as phenothiazinium dyes, including toluidine blue O (Wilson & Yianni, 1995; Wainwright et al., 1998), methylene blue (Wainwright et al., 1998; Zeina et al., 21), and porphyrin derivatives such as hematoporphyrin (Bertoloni et al., 2). The induction of intracellular porphyrin production through pretreatment with d-aminolaevulinic acid followed by light exposure has also provided a successful method for staphylococcal PDI (Nitzan & Kauffman, 1999; Nitzan et al., 24). The observation that visible light alone has bactericidal properties has been previously documented for bacteria, Abstract This study was conducted to investigate the bactericidal effects of visible light on methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA), and subsequently identify the wavelength sensitivity of S. aureus, in order to establish the wavelengths inducing maximum inactivation. Staphylococcus aureus, including MRSA strains, were shown to be inactivated by exposure to high-intensity visible light, and, more specifically, through a series of studies using a xenon broadband white-light source in conjunction with a selection of optical filters, it was found that inactivation of S. aureus occurs upon exposure to blue light of wavelengths between 4 and 42 nm, with maximum inactivation occurring at 45 5 nm. This visible-light inactivation was achieved without the addition of exogenous photosensitisers. The significant safety benefit of these blue-light wavelengths over UV light, in addition to their ability to inactivate medically important microorganisms such as MRSA, emphasises the potential of exploiting these non-uv wavelengths for disinfection applications. most notably, the acne-associated bacterium Propionibacterium acnes. Research into P. acnes has found that irradiation of this organism with blue light leads to photosensitisation of intracellular porphyrins, stimulation of which leads to the production of reactive species, predominantly singlet delta oxygen ( 1 O 2 ), and consequently, cell death (Papageorgiou et al., 2; Ashkenazi et al., 23; Hamblin & Hasan, 24). Other bacteria that have been found to be susceptible to inactivation solely through visible-light exposure include Helicobacter pylori and some oral black-pigmented bacteria (Feuerstein et al., 24; Ganz et al., 25; Soukos et al., 25). Previous work on the use of pulsed UV-rich light (Mac- Gregor et al., 1998; Anderson et al., 2; Wang et al., 25) has now been expanded to investigate the bactericidal properties of high-intensity, narrow-band visible-light wavelengths against S. aureus and other bacterial pathogens (Anderson et al., 25; Maclean, 26). This, and the more recent work by Guffey & Wilborn (26a, b), which documented the inactivation of S. aureus using super luminous diodes, demonstrate that S. aureus can be inactivated

2 228 M. Maclean et al. optically using visible light without the use of exogenous photosensitisers or d-ala-induced porphyrins. The present study was carried out to investigate and fully characterise the bactericidal effect of visible light on S. aureus. Firstly, the bactericidal effect of high-intensity visible light (4 nm and longer) on S. aureus and MRSA was investigated. Subsequently, to identify the region of the visible spectrum that induces staphylococcal inactivation, the visible wavelength sensitivity of S. aureus was investigated through a series of optical filter studies. The results demonstrate that blue light between 4 and 42 nm, but optimally 45 nm ( 5 nm), has bactericidal effects against S. aureus. Materials and methods Microorganisms The bacteria used in this study were as follows: S. aureus NCTC 4135, Escherichia coli NCTC 91 (National Collection of Type Cultures, Collindale, UK), MRSA LMG (The Belgian Co-ordinated Collections of Micro-organisms, Gent, Belgium) and MRSA 16a (a clinical wound isolate obtained from the Royal Infirmary, Glasgow). Test strains were inoculated into 1 ml of nutrient broth (Oxoid, UK) and cultivated at 37 1C under rotary conditions (at 125 r.p.m.). After an 18-h incubation period, the broth was centrifuged at 3939 g for 1 min and the resultant pellet resuspended in 1 ml phosphate-buffered saline (PBS) (Oxoid, UK). This suspension was then diluted in PBS to give a population density of c CFU ml 1 for experimental use. Visible light source A xenon broadband white-light source (Hamamatsu Photonics UK Ltd), together with a 4 nm longwave pass (L-P) filter, was used for the visible-light exposure of bacterial Intensity (Arbitrary units) No Filter 4nm L-P Filter in place Wavelength (nm) Fig. 1. Emission spectrum of xenon lamp from 2 to 5 nm. Also shown is the emission spectrum when passed through a 4 nm longwave pass filter. suspensions and the relevant emission spectra are shown in Fig. 1. The use of this filter allowed transmission of wavelengths longer than 4 nm (i.e. visible light) and thus eliminated UV-light inactivation. High-intensity exposure experiments The experimental set-up was as follows: a 2 ml volume of bacterial suspension, with a population density of CFU ml 1, was transferred to one well of a 12-well multidish (Nunc, Denmark), which also contained a 7mm2mm magnetic follower. The 4 nm L-P filter was placed on top of the well. The dish was then positioned directly under the light source on a magnetic stirrer. This, in conjunction with the magnetic follower, permitted continuous mechanical agitation of the sample during light exposure. For the experiments detailed, all parameters were maintained constant: the sample volume used in each experiment was 2 ml and the irradiance of the xenon lamp through the 4 nm L-P filter was 35 4 mw cm 2 (measured using a radiant power meter and detector; L.O.T. -Oriel Ltd, UK). A separate sample well containing 2 ml bacterial suspension was used for each exposure time, and after each exposure, samples were plated onto nutrient agar (NA) (Oxoid, UK) (see Bacterial Plating and Enumeration ) and incubated at 37 1C for 24 h. Control samples were also set-up; these were subjected to identical conditions but were not exposed to high-intensity visible light, but left in normal laboratory lighting conditions. Filter study for identification of inactivation wavelengths The experimental set-up for the filter studies on S. aureus NCTC 4135 was identical to that described above for the high-intensity exposure experiments, with the 4 nm L-P filter being substituted with, firstly, a selection of commercially available L-P and shortwave pass (S-P) filters (L.O.T. -Oriel Ltd), which would identify the causative wavelength range, and secondly, a selection of narrow bandpass (B-P) filters ranging from 4 to 5 nm (L.O.T.-Oriel Ltd; Ealing Catalog. Inc.), which would identify the causative bandwidth to within 1 nm. For comparison of the germicidal efficiencies for each narrow bandwidth, the output intensity of the lamp was amended for each B-P filter so that the same irradiance (3.27 mw cm 2 ) was transmitted onto each bacterial sample for each filter. This 3.27 mw cm 2 irradiance was transmitted through each of the filters and illuminated each bacterial sample for an equal time period of 2 h. From these values, the absolute dose, also termed energy density, in Joules per square centimetre [irradiance (W cm 2 ) time (s)] for each 1 nm bandwidth being applied to the S. aureus

3 Light inactivation wavelength sensitivity of S. aureus 229 suspensions was calculated as 23.5 J cm 2 (3.27 mw cm 2 for 2 h). Bacterial plating and enumeration In order to obtain accurate viable cell counts, several standard plating methods were used in this study. The spiral and spread plate methods, using 5 and 1 ml sample volumes, respectively, were prepared on NA using a WASP 2 spiral plater (Don Whitley Scientific Ltd, UK). For samples with anticipated low CFU counts, pour plates, using a 1-mL sample volume, were prepared manually using molten NA. Spiral plates were enumerated using the supplied counting grid and tables. Spread and pour plates were counted manually with the aid of a colony counter (Stuart Scientific, UK). The resultant counts from each of these methods were then converted into viable CFU counts ml 1 of sample. Statistical analysis With regard to the replication and recording of experimental results, in the high-intensity exposure experiments each data point on the graphs represents the results from two independent experiments, with a minimum of triplicate samples being taken for each experiment. These results are documented as mean values with SDs being included. Significant differences in the light-treatment results were calculated at the 95% confidence interval using ANOVA (one way) with MINITAB software Release 15. Data on the germicidal efficiency of different wavelengths, as discussed later, were obtained from replicated CFU count data and calculated as mean log 1 reduction per unit dose. Results High-intensity exposure experiments Figure 2 shows the results for the exposure of S. aureus suspensions to high-intensity light of wavelengths 4 4 nm. It can be seen that the light had a significant bactericidal effect on S. aureus, with a 5-log 1 reduction being achieved after a dose of 63 J cm 1 (35 mw cm 2 3 min). The exposed E. coli suspensions demonstrated negligible inactivation over a 3-min exposure time, although exposures of 45 and 6 min did demonstrate significant differences compared with the associated control samples, indicating that a more prolonged exposure may induce further inactivation of the exposed E. coli population. In Fig. 3, it can be seen that when the MRSA strains were light-exposed, inactivation results that were similar to those obtained with S. aureus NCTC 4135 were observed (Fig. 2). The population densities of all control samples stayed constant throughout this series of experiments (Figs 3 and 4). Temperature was also monitored throughout the Bacterial count (Mean log 1 CFU ml 1 ) Exposure time (min) Fig. 2. Comparison of the effect of high-intensity visible light of wavelengths 4 4 nm on suspensions of Staphylococcus aureus NCTC 4135 and Escherichia coli NCTC 91. Data points for 15 min and above for S. aureus, and 45 min and above for E. coli, are significantly different from the respective controls. Bacterial count (Mean log 1 CFU ml 1 ) Exposure time (min) Fig. 3. Inactivation of MRSA strains in liquid suspension upon exposure to high-intensity visible light of wavelengths 4 4 nm. Data points for 15 min and above for MRSA LMG 15975, and 25 min and above for MRSA 16a, are significantly different from the respective controls. Bacterial count (Mean log 1 CFU ml 1 ) <5 nm >4 nm 1 >5 nm Control Exposure time (min) Fig. 4. Effect of different wavelength ranges on the inactivation rate of Staphylococcus aureus NCTC 4135 suspensions. Suspensions were exposed to high-intensity light of the following wavelength ranges: ( ) 5 nm and below; (m) 4 nm and above; () 5 nm and above. Data points for 1 min and above for o 5 nm exposure, and 15 min and above for 4 4 nm, are significantly different from the respective controls.

4 23 M. Maclean et al. Table 1. Log 1 reduction and germicidal efficiency (Z) values for the inactivation of Staphylococcus aureus NCTC 4135 following exposure to 1 nm bandwidths of light from 4 to 42 nm, each for a dose of 23.5 J cm 2 Bandwidth (nm) Initial Population, N (Log 1 CFU ml 1 ) Final Population, N (Log 1 CFU ml 1 ) Log 1 (N/N ) Reduction Z w (Log 1 (N/N )/J cm 2 ) 4 5nm 5.36(.2) 3.83 (.1) nm 5.29(.1) 2.89 (.7) nm 5.31(.3) 4.16 (.18) nm 5.2(.3) 4.65 (.7) nm 5.26(.1) 4.99 (.1) nm 5.35(.3) 5.25 (.5).1.4 Significant bacterial log1 reductions, calculated at a 95% confidence interval. (Data for 43 nm exposure shown for comparative purposes; data for 44 5 nm not shown). w Z, germicidal efficiency light-exposure experiments and was found to not rise above 37 1C, eliminating any thermal inactivation effects. Identification of inactivation wavelength range As a first step to identify the wavelength range within the visible spectrum inducing staphylococcal inactivation, different visible-wavelength ranges were selected using S-P and L-P filters using similar irradiances. Figure 4 shows the effects of the different wavelength ranges on the rate of inactivation of S. aureus NCTC 4135 cells in PBS suspensions. Exposure to wavelengths of 5 nm and below induced the most rapid inactivation rate, and this was likely the result of the inclusion of UV wavelengths, which are well known to have a strong germicidal effect. Wavelengths longer than 4 nm also caused total inactivation. When longer wavelengths of 5 nm and above were investigated, no inactivation was observed. This confirmed that the visible wavelengths inducing staphylococcal inactivation were in the wavelength region of 4 5 nm. Identification of the inactivation bandwidth In order to identify the narrow bandwidth of visible light between 4 and 5 nm inducing staphylococcal inactivation, B-P filters were used. The S. aureus suspensions exposed to each narrow 1 nm bandwidth between 4 and 5 nm received an absolute dose of 23.5 J cm 2, and significant log 1 reductions were achieved through exposure to 4 42 nm bandwidths, as shown in Table 1. Here it can be seen that the maximum log 1 reduction of S. aureus cells resulted from exposure to 45 5 nm wavelength light. Exposure to bandwidths of 43 5 nm did not cause significant inactivation of the bacteria. The inactivation capability at each wavelength can be quantified as the germicidal efficiency, defined as the log 1 reduction of a bacterial population by inactivation per unit dose in Joule per square centimetre (Wang et al., 25). Thus, germicidal efficiency, Z = log 1 (N/N )Jcm 2. Numerical data for the germicidal efficiencies achieved through light exposure to 1 nm bandwidths of light from 4 to 43 nm (with centre wavelength increments of 5 nm) are listed in Table 1. The results show that the germicidal efficiency peak of.12 log 1 Jcm 2 was at 45 nm, but the 4 nm light also demonstrated good germicidal activity for S. aureus, with a value of.64 log 1 Jcm 2. Bandwidths between 43 and 5 nm demonstrated no significant germicidal efficiency against S. aureus suspensions. Discussion Investigations using a high-intensity xenon lamp, in conjunction with a selection of commercially available L-P, S-P and B-P filters, have demonstrated the sensitivity of S. aureus to visible light, and also identified the bactericidal wavelengths inducing maximum visible-light inactivation to within a 1 nm bandwidth. The results have highlighted that inactivation is evident using 4 42-nm-wavelength blue light, with the most effective bactericidal activity at 45 5 nm. Wavelengths of longer than 43 nm were found to induce no effect on the viability of S. aureus cells. The occurrence of the peak at 45 nm suggests that an inactivation process is at its optimum within the S. aureus cells at this specific wavelength. The identification of this narrow band of inactivation wavelengths highlights that the vast majority of the illuminating wavelengths emitted by the broadband xenon lamp ( 4 4 nm) were superfluous to the inactivation process. Therefore, in Figs 2 and 3, although c. 63 J cm 2 was required for a 5-log 1 reduction of S. aureus and MRSA strains, this was the total irradiance, only a small fraction of which was responsible for the inactivation. Although this value is useful for comparison with other studies that have used broadband light sources, the experiments using the narrowband filters provide more meaningful values of absolute dose: 23.5 J cm 2 of 45 nm light resulting in a 2.4 log 1 reduction of S. aureus.

5 Light inactivation wavelength sensitivity of S. aureus 231 The use of these dose values can also be used to explain why throughout previous studies on the PDI of S. aureus using exogenous photosensitisers, inactivation in the absence of photosensitisers or d-ala-induced porphyrins has either been dismissed or not been discussed. Bertoloni et al. (2) and Zeina et al. (21) both used broadband light sources emitting visible light 4 7 nm (as used in this study; Figs 2 and 3) for illumination of the bacterial samples but only applied maximum doses of 3.6 and 2.16 J cm 2, respectively, compared with the much greater 63 J cm 2 dose applied in the present study (Figs 2 and 3). The finding that exposure to high-intensity visible light, at intensities that resulted in a 5 log 1 reduction in S. aureus and MRSA populations, had a negligible effect of E. coli reflects results found in previous PDI studies involving photosensitisers. In one study using pretreatment with d-ala, which used a white-light source for illumination (as in the present study), the inactivation of E. coli required at least 1-times higher doses to achieve similar activation levels to that for S. aureus (Nitzan & Kauffman, 1999). The sigmoidal shape of the inactivation curve for visiblelight inactivation reveals an initial period of low biocidal activity that may indicate a requirement for a build-up of energy, reactive molecules or cellular damage, which must occur before induction of bacterial inactivation is initiated. It is likely that the mechanism of inactivation is quite different to that of continuous UV-C exposure, which induces DNA damage, primarily as a result of UV absorption by the DNA bases in the wavelength region nm (Blatchley & Peel, 1991), or near-uv light, inactivation by which has been accredited to sublethal damage of DNA repair systems (Tyrrell & Peak, 1978). Visible-light inactivation, on the other hand, as established for other bacteria such as P. acnes, H. pylori and some black-pigmented bacteria (Ashkenazi et al., 23; Feuerstein et al., 24; Ganz et al., 25; Soukos et al., 25), has been accredited to the photo-stimulation of endogenous intracellular porphyrin molecules. These studies identified the stimulating wavelengths to be visible light in the wavelength region 4 5 nm, and more specifically 4 42 nm in the cases of P. acnes and H. pylori (Ashkenazi et al., 23; Elman et al., 23; Ganz et al., 25). Because of the similarity in causative wavelengths, it is hypothesised that the inactivation mechanism occurring with the S. aureus and MRSA strains in the present study is also the result of photostimulation of intracellular porphyrins, which results in the production of reactive species, predominantly singlet delta oxygen ( 1 O 2 ), and consequently, cell death. Papageorgiou et al. (2) investigated the effect of blue light on P. acnes and their findings are in general agreement with those established in this study. They found that the sensitivity of P. acnes to visible light was at a maximum in the blue region of 415 nm. Papageorgiou et al. (2) state that 415 nm corresponds to the absorption maximum of the specific porphyrin molecules produced by P. acnes. The maximum of 45 nm determined in the present study may indicate that porphyrin molecules that have different absorption maxima are present within S. aureus bacteria or are produced by them. The recent work of Guffey & Wilborn (26a, b), however, reports inactivation of S. aureus at both 45 and 47 nm. In their study, low populations of S. aureus plated onto agar surfaces were exposed to doses of magnitude similar to those used in the present study. For 45 nm inactivation, they measured an approximate single log 1 reduction in bacterial concentration for a dose of 15 J cm 2 compared with the reduction of 2.4 log 1 for a dose of 23 J cm 2 found in this work. At 47 nm, Guffey & Wilborn (26b) measured a log 1 reduction of around.4.5 for a dose of 15 J cm 2 ; our results, on the other hand, indicate that no inactivation of S. aureus occurs at 47 nm. The germicidal efficiency of the 45-nm-wavelength light is much lower than that for UV wavelengths; however, this disadvantage may be more than outweighed for some applications by the greater safety afforded at this wavelength. While it is well established that UV irradiation provides a method of inactivating pathogenic microorganisms, it is not apposite to expose humans to UV radiation because of the well-recognised risks of eye damage and skin cancer. Significantly, this study has shown that these blue wavelengths within the visible-light spectrum are capable of inactivating S. aureus, including MRSA, while posing a negligible threat to human health (ACGIH, 27). Acknowledgements The first author would like to thank The Engineering and Physical Sciences Research Council (EPSRC) for their support through a Doctoral Training Grant (awarded in 22/ 23). All authors would like to thank The Robertson Trust for their funding support. References American Conference of Governmental Industrial Hygienists (ACGIH) (27) Threshold Limit Values & Biological Exposure Indices. Signature Publications, Cincinnati, OH. Anderson JG, Rowan NJ, MacGregor SJ, Fouracre RA & Farish O (2) Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed light. IEEE Trans Plasma Sci 28: Anderson JG, Maclean M, Woolsey GA & MacGregor SJ. Inactivation of Gram-positive bacteria. International Patent WO 27/12875 A1. Patent Filing Date: July 25; Published February 27. Ashkenazi H, Malik Z, Harth Y & Nitzan Y (23) Eradication of Propionibacterium acnes by its endogenic porphyrins after

6 232 M. Maclean et al. illumination with high intensity blue light. FEMS Immunol Med Microbiol 35: Bertoloni G, Lauro FM, Cortella G & Merchant M (2) Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells. Biochim Biophys Acta 1475: Blatchley ER III & Peel MM (1991) Disinfection by ultraviolet irradiation. Disinfection, Sterilisation and Preservation, 4th edn (Block SS, ed), pp Lea & Febiger, Philadelphia, PA. Elman M, Slatkine M & Harth Y (23) The effective treatment of acne vulgaris by a high-intensity, narrow band nm light source. J Cosmet Laser Ther 5: Feuerstein O, Persman N & Weiss EI (24) Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: anin vitro study. Photochem Photobiol 8: Ganz RA, Viveiros J, Ahmad A, Ahmadi A, Khalil A, Tolkoff MJ, Nishioka NS & Hamblin MR (25) Helicobacter pylori in patients can be killed by visible light. Laser Surg Med 36: Guffey JS & Wilborn J (26a) Effects of combined 45-nm and 88-nm light on Staphylococcus aureus and Pseudomonas aeruginosa in vitro. Photomed Laser Surg 24: Guffey JS & Wilborn J (26b) In Vitro bactericidal effects of 45-nm and 47-nm blue light. Photomed Laser Surg 24: Hamblin MR & Hasan T (24) Photodynamic therapy: a new antimicrobial approach to infectious disease. Photochem Photobiol Sci 3: MacGregor SJ, Rowan NJ, McIlvaney L, Anderson JG, Fouracre RA & Farish O (1998) Light inactivation of food-related pathogenic bacteria using a pulsed power source. Lett Appl Microbiol 27: Maclean M (26) An investigation into the light inactivation of medically important microorganisms. PhD Thesis, University of Strathclyde, Glasgow, UK. Nitzan Y & Kauffman M (1999) Endogenous porphyrin production in bacteria by d-aminolaevulinic acid and subsequent bacterial photoeradication. Laser Med Sci 14: Nitzan Y, Salmon-Divon M, Shporen E & Malik Z (24) ALA induced photodynamic effects on Gram positive and negative bacteria. Photochem Photobiol Sci 3: Papageorgiou P, Katsambas A & Chu A (2) Phototherapy with blue (415 nm) and red (66 nm) light in the treatment of acne vulgaris. Br J Dermatol 142: Soukos NS, Som S, Abernethy AD, Ruggiero K, Dunham J, Lee C, Doukas AG & Goodson JM (25) Phototargeting oral blackpigmented bacteria. Antimicrob Agents Chemother 49: Tyrrell RM & Peak MJ (1978) Interactions between UV radiation of different energies in the inactivation of bacteria. J Bacteriol 136: Wainwright M, Phoenix DA, Laycock SL, Wareing DRA & Wright PA (1998) Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiol Lett 16: Wang T, MacGregor SJ, Anderson JG & Woolsey GA (25) Pulsed ultra-violet inactivation spectrum of Escherichia coli. Water Res 39: Wilson M & Yianni C (1995) Killing of methicillin-resistant Staphylococcus aureus by low-power laser light. J Med Microbiol 42: Zeina B, Greenman J, Purcell WM & Das B (21) Killing of cutaneous microbial species by photodynamic therapy. Br J Dermatol 144: