Background
There is global concern surrounding antibiotic resistant organisms, such as methicillin-resistant
Staphylococcus aureus (MRSA). These organisms negatively impact on healthcare systems by causing infections which are harder to treat due to reduced antibiotic choices, resulting in longer hospital stays and increased mortality of patients [
1]. To reduce transmission of these pathogenic organisms, new technologies are being developed to aid environmental decontamination and clinical treatment.
One such antimicrobial technology is 405 nm light. Violet-blue light in this region photo-excites intracellular porphyrins within microorganisms, producing a range of reactive oxygen species (ROS) which cause oxidative damage and cell death [
2‐
4]. Although less germicidal than ultraviolet (UV) light, 405 nm light has broad-spectrum antimicrobial action against Gram positive and negative bacteria, bacterial biofilms, endospores, yeasts, fungi and in some circumstances viruses [
5‐
11]. When used at appropriate irradiances, these wavelengths of visible light can also exert antimicrobial effects whilst being non-detrimental to mammalian cells [
12‐
14], giving it operational advantages over UV-light for applications such as continuous decontamination of occupied environments [
15‐
18] and wound decontamination [
12]. Additionally, it has recently been reported that 405 nm light can also have a synergistic antimicrobial effect with common chlorinated disinfectants, providing further support for its beneficial use for environmental decontamination [
19].
Although 405 nm light has extensive antimicrobial action and safety advantages, little is known about the potential for the development of bacterial resistance or tolerance to violet-blue 405 nm light inactivation. It is hypothesised that tolerance is unlikely due to the mechanism of inactivation [
13,
17,
20]. Similar to that of photodynamic inactivation (PDI), which makes use of an additional photosensitizer, the mechanism of inactivation is thought to be non-selective, as the ROS and
1O
2 produced cause unspecific damage to a wide spectrum of targets within bacterial cells [
13,
17,
20,
21]. Nevertheless there is little evidence available to support this hypothesis. Several studies have investigated tolerance formation following repeated PDI in a range of microorganisms including methicillin-sensitive and methicillin-resistant
S. aureus,
Escherichia coli,
Pseudomonas aeruginosa, Peptostreptococcus micros,
Actinobacillus actinomycetes and
Vibrio fischeri, in which none of the aforementioned species were found to become tolerant [
21‐
25]. However little is known about the potential for bacterial tolerance development to antimicrobial 405 nm light alone.
This study was carried out to determine if there is potential for tolerance development in exposed organisms, using the nosocomial pathogen
S. aureus as the model organism. The first stage of the study assessed whether pre-culture in low-level light-stress conditions would subsequently affect the susceptibility of the organism to 405 nm light inactivation and increase tolerance. Secondly, the study investigated the effect of repeated sub-lethal exposure to high-intensity 405 nm light on methicillin-sensitive and methicillin-resistant
S. aureus, with the number of sub-lethal exposures being extended past those carried out in previously published studies [
20,
26,
27]. To further investigate the likelihood of tolerance, the inactivation kinetics and antibiotic susceptibility of survivors after sub-lethal exposure were analysed and compared. The results provide novel fundamental evidence to support the hypothesis that tolerance following repeated exposure to 405 nm light is unlikely in both proliferating and non-proliferating bacteria.
Discussion
This study has addressed the question of the potential for S. aureus to become tolerant to antimicrobial 405 nm violet-blue light and has provided new information under carefully controlled experimental test conditions.
The results from the first stage of the study, demonstrated that MSSA cultured in 1 mW/cm
2 405 nm light appeared to exhibit a degree of tolerance to high-intensity 405 nm light, compared to when dark-cultured. This suggests that exposure to low-intensity 405 nm light during culture may have acted as a low-level stressor during growth, which could have increased the up-regulation of bacterial oxidative stress responses. Bacteria have developed several mechanisms to overcome oxidative stress, including enzymes to detoxify reactive oxygen species such as catalase, peroxidase and superoxide dismutase [
30]. To detect changes in the ability of MSSA to tolerate oxidative stress after culture in the presence of 1 mW/cm
2 405 nm light, compared to white light or complete darkness, a MIC assay using H
2O
2 was carried out, and carotenoid content was measured. Results determined that the average MIC of H
2O
2 was significantly higher for the organism when cultured in low-level (1 mW/cm
2) 405 nm light compared to white light or complete darkness. This demonstrates that during growth in low-irradiance 405 nm light there is likely an increase in the expression of protective enzymes e.g. an increase in catalase enzymes which would act as hydrogen peroxide scavengers. An increase in catalase enzyme, KatA, following photodynamic inactivation of
S. aureus (using blue light and exogenous porphyrins) was also demonstrated by Doselli et al. [
31], which further suggest this enzyme is up-regulated to try to help protect the bacteria against oxidative stress.
The presence of the carotenoid staphyloxanthin was also found to be significantly lower in MSSA cultured in low-level (1 mW/cm
2) 405 nm light compared to when grown in white light or darkness. As carotenoid pigments have anti-oxidative properties, in particular for protecting bacteria against
1O
2 stress [
32], you would expect MSSA cultivated in 1 mW/cm
2 405 nm light to be more sensitive to subsequent high-intensity 405 nm light exposure, due to the lower levels of staphyloxanthin present. However, as tolerance is increased upon exposure to high-intensity 405 nm light, it is likely there is an up-regulation of other oxidative stress responses within the bacteria and that the carotenoid pigment provided protection against ROS during growth in low-intensity 405 nm light. Further experiments investigating superoxide dismutase, catalase activity could help to confirm this.
Although the inactivation achieved at 216 J/cm
2 (Fig.
4) was less when the bacteria had been pre-cultured in low-irradiance (1 mW/cm
2) 405 nm light, it is important to note that when exposed to a higher dose of 270 J/cm
2 complete 5 log
10 inactivation was still able to be achieved (Fig.
5b). It is likely that complete inactivation is still able to occur due the level of ROS produced being greater than the level that the basal bacterial oxidative stress defence systems are able to scavenge [
30].
It is important to consider the possibility that bacteria may become less sensitive to high-irradiance 405 nm light after pre-culture in low-intensity, sub-lethal stress levels of 405 nm light, as this could have implications when violet-blue light is used to inactivate bacteria in nutritious environments where the bacteria are able to replicate, for example within wounds or surgical sites. In these cases it would be important to ensure a bactericidal dose was administered: if the dose administered or irradiance used is too low, the bacteria may not be completely inactivated and cause pathogenic organisms to potentially become more tolerant to subsequent applications of violet-blue light. Additionally, if the dose delivered is too low there may actually be an increase in population concentration, as it is thought visible light can encourage proliferation of microorganisms when used on nutrient rich areas such as wounds [
28].
Further investigations were carried out to investigate if growth in low-intensity 405 nm light was selective for MSSA which was able to adapt to a greater level of oxidative stress. Results demonstrated that this was not the case, with the sensitivity of MSSA returning to a similar level of that when cultivated in complete darkness alone. These results additionally suggest that the increased tolerance to high-intensity 405 nm light is caused by an up-regulation in bacterial stress response rather than selection for violet-blue light tolerant colonies due to growth conditions.
Further work should be carried out to investigate the oxidative stress response following growth in low-intensity 405 nm light fully, such as the levels of superoxide dismutase, which has been previously shown to be up-regulated in
S. aureus sensitive to PDI [
33]. Additionally it is known that there is a heat shock protein cascade after PDI [
30] and a study by St Denis et al. [
34] demonstrated that
E. coli exposed to external stress, before PDI showed a 2 log
10 less reduction in bacterial inactivation compared to normal PDI inactivation levels. Therefore it would be interesting to investigate if there was a heat shock protein cascade during growth in 1 mW/cm
2 violet-blue light, and if this up-regulation of proteins before high-intensity exposure may contribute to the stress tolerance seen. Additionally, investigations should explore why the increased level of tolerance is not seen after repeated growth in the low-level stress conditions. However as the bacteria can still be completely inactivated, and as results indicated repeated growth in low-irradiance light is unlikely to be a selective process, MSSA should continue to be susceptible to high-irradiance 405 nm light after growth in the presence of a low-level stressor.
It is also important to note that the changes in susceptibility observed here are in response to the organisms being pre-cultured in low-level 405 nm light. This is unlikely to occur in non-proliferating bacteria, such as is the case with environmental contamination [
17], as these organisms are stressed and not actively growing, thus up-regulation of stress responses is unlikely to occur, and actually when in a stationary stressed state, bacteria should become more susceptible to 405 nm light inactivation [
35].
The next stage of this study was to investigate the likelihood of tolerance development when non-proliferating bacteria were repeatedly exposed to a sub-lethal dose of high-intensity 405 nm light. MSSA and MRSA were subjected to 15 exposure-subculture-exposure cycles to 60 mW/cm2 405 nm light, resulting in a dose of 108 J/cm2 per sub-lethal exposure. Results demonstrated no significant change in the level of inactivation achieved, with 1.2 log10 and 1.4 log10 inactivation achieved for MSSA and MRSA after 15 sub-lethal exposures respectively, compared to an initial 1.3 log10 inactivation achieved for both.
The dose of 108 J/cm
2 was selected for exposure as it was shown to cause approximately 98% (1.6–1.8 log
10) inactivation of the organism used in this study (Figs.
3 and
4). When compared to doses used in other studies to achieve similar levels of inactivation, this dose is somewhat greater [
6,
7], however, the dose dependency will vary between studies, as it is specific to the light sources, the irradiances and the organisms used. LED arrays have variations in spectral output in terms of exact peak wavelength and bandwidth, and therefore this will affect the efficiency of bacterial inactivation. In addition, the overall doses used will vary depending on the output irradiance of the light source: the higher power the light source, the higher the irradiance, however, given that there will be a finite number of porphyrins per bacterial cell capable of photon absorption, there is likely to be a point at which the absorption of more photons would have little effect on the inactivation mechanism already in progress, therefore although more energy is being delivered, it may not translate into more effective kill.
Little other evidence has been documented regarding bacterial tolerance to 405 nm light. A short study by Guffey et al. [
26] demonstrated that there was potential for
S. aureus to become tolerant to a dose of 9 J/cm
2 405 nm light, as following 5 repeated exposures there was a decrease in the kill rate seen. The study demonstrated an initial increase in kill rate from 32.92 to 59.49% after the 1st to 4th sub-lethal exposures, however kill rate subsequently declined, with only 18.04% kill after the 7th repeated sub-lethal exposure, attributed to the development of resistance. These results are conflicting with those in this study, as our results indicate little change in bacterial response to 405 nm light inactivation following 15 sub-lethal exposures. In the present study, which used more than double the number of sub-lethal exposures and higher bacterial populations, natural variation in the level of inactivation occurred after repeated sub-lethal exposure, and this variation would be particularly apparent when using low population densities, as was the case in the study by Guffey et al. [
26], so potentially further repeated sub-lethal exposures may have shown a subsequent increase in kill rate.
Additionally, in the present study
S. aureus was repeatedly sub-lethally exposed whilst suspended in PBS. This allowed investigation of the sole effect that repeated antimicrobial violet-blue light exposure would have on bacterial cultures in a stationary, non-proliferating, state, representative of how bacteria would be found in the clinical environment. In the study by Guffey et al. [
26],
S. aureus was exposed to 405 nm light whilst seeded onto mannitol salt agar plates. Not only is mannitol known to be a ROS scavenger [
36], in this scenario the bacteria were likely to be in a metabolic state, tolerating the high salt conditions (7.5% NaCl) and fermenting mannitol, and therefore these processes may have affected the subsequent bacterial stress response to violet-blue light. Consequently the increased tolerance seen may be more relative to results in the earlier phase of this study, where low-level exposure to 405 nm light in nutritious conditions resulted in higher dose requirements for complete inactivation.
Conversely, two studies which exposed
Acinetobacter baumannii and
P. aeruginosa to 415 nm light, under exposure conditions similar to this study, demonstrated no evidence of tolerance formation [
20,
27]. Results are in keeping with study, with no tolerance to 415 nm light inactivation seen in 10
8 CFU/ml populations of
P. aeruginosa and
A. baumannii after 10 repeated sub-lethal exposures to a dose of 36 J/cm
2 and 70.2 J/cm
2 respectively [
20,
27]. These previous studies, along with the results in the present study, support the hypothesis that due to the unspecific mechanism of action and broad spectrum of intracellular targets, tolerance is not likely to occur.
Interestingly in the study by Zhang et al. [
27] a significant increase in the sensitivity of
A. baumannii was seen between the 1st exposure (4.52 log
10 reduction) and the 10th exposure (6.28 log
10 reduction) and inactivation curves revealed an increase in inactivation between 1, 6 and 9 sub-lethal exposures. These results were thought to indicate that a favourable mutation had occurred, increasing bacterial susceptibly to violet-blue light inactivation [
27]. To investigate if this phenomenon would also occur in
Staphylococcus aureus, the inactivation kinetics of surviving isolates after 5, 10 and 15 sub-lethal exposures were determined and all followed similar trends. Although these results do not indicate an increase in sensitivity of MSSA and MRSA to 405 nm light after repeated sub-lethal exposure, they do demonstrate consistent
Staphylococcal sensitivity to high-irradiance 405 nm light and further supports the hypothesis that tolerance to 405 nm light inactivation in unlikely. However, the potential for tolerance should be evaluated in other microorganisms normally susceptible to 405 nm light inactivation, including multidrug resistant organisms which are currently a great problem in healthcare settings [
37,
38].
Antibiotic susceptibility was also analysed to ensure that repeated 405 nm light exposure did not give rise to ‘stress hardening’, whereby as a result of continued exposure to this sub-lethal stress the bacteria would be able to adapt and develop protection mechanisms against other applied stresses [
39,
40]. Little significant variation was seen with the antibiotic susceptibility for both
S. aureus strains. Although a slight decrease in susceptibility of MSSA to mupirocin was noted after 5 sub-lethal exposures (an observation which was not observed following increased sub-lethal exposures to 10 or 15 cycles), it is worth noting that although the concentration of mupirocin antibiotic disc (5 μg) was below the recommended concentration used by EUCAST (200 μg), the zone of inhibition measured was still far greater (26 mm) than the EUCAST breakpoints for resistance (18 mm). Therefore, indicating that although significantly different to the initial non sub-lethally exposed MSSA, after 5 sub-lethal exposures MSSA is bacteria is still sensitive to mupirocin.
Furthermore, to the best of our knowledge these are the first results comparing repeated sub-lethal 405 nm light exposure and antibiotic susceptibility, and they indicate that sub-lethal exposure is unlikely to result in the development of antibiotic resistance. These findings are further supported by those of Pedigo et al. [
23], who demonstrated no antibiotic resistance occurred in MSSA after 25 repeat exposures to PDI, using a methylene blue photosensitizer and 670 nm light, compared to antibiotic resistance in MSSA after only 11 exposures to a 1 μg/ml oxacillin disk. Similarly, Grinholc et al. [
41] found no change in antibiotic susceptibility in MRSA before and after exposure to PDI (using protoporphyrin diarginate and 624 nm light) with the 26 different antibiotics tested.
Future studies should involve a greater range of commonly used antibiotics and broth microdilution studies should be carried out to enable the quantification of the MIC of each antibiotic before and after sub-lethal exposure. However, overall these results indicate repeated sub-lethal exposure to 405 nm light is unlikely to affect
S. aureus susceptibility to antibiotics, and supports the practical application of 405 nm light for decontamination applications within in the clinical environment [
42].
This study was designed to generate fundamental microbiological information with regards to the potential for the development of bacterial tolerance to 405 nm light. To investigate this,
S. aureus was exposed to high-irradiance light whilst in suspension, following either growth in low-level blue light stress conditions or previous sub-lethal exposures to 405 nm light. As discussed earlier, the 405 nm light levels used in the present study permitted establishment of the fundamental responses of the light-exposed bacteria. There is however, interest in utilising this antimicrobial technology for practical decontamination applications and it will therefore be important for future studies to progress investigations towards more clinically-relevant scenarios. This could include repeated exposure of bacteria on inert surfaces with low-irradiance 405 nm light more representative of levels which would be used for environmental decontamination (<1 mW/cm
2) [
15‐
18], or repeated exposure of organisms on tissue models using higher irradiance light, more suited for representation of wound treatments [
12]. It would also be important to investigate if there are any differences when bacteria are dried onto clinical surfaces compared to when suspended in biological fluids such as blood and secretions. A previous study by Murdoch et al. [
43], also demonstrated that bacteria can be more susceptible to inactivation when exposed whilst dried onto surfaces and in a desiccated state, and it is reasonable to consider that this increased susceptibility when on surfaces is likely to reduce the likelihood of persistent survival and tolerance/resistance. In addition, if the bacteria are present in biological fluids or embedded in a biological matrix then the inactivation effect could be enhanced by the excitation of photosensitive components present in the biological fluids or matrix (which accelerates the production of ROS) [
10,
11]. Conversely it must also be considered that inactivation efficacy of the 405 nm light could be hindered by the opaque transmission properties of the suspending fluid/matrix. Clearly much more research is required to understand not only the tolerance response of bacteria to 405 nm light but also the complex interplay between physico-chemical and environmental factors that can impact on the efficacy of the light induced inactivation in real clinical settings.
Finally, it would also be important to establish the influence of strain variance on the potential for tolerance. Although comparisons were made between methicillin sensitive and resistant strains of
S. aureus other studies have demonstrated that photodynamic inactivation and violet-light exposure can vary between exposed strains of the same organisms [
28,
44].