In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on Candida spp.

Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of Candida spp., Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm2 with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420–700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log10 reduction in the number of CFU/100 μL of Candida spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections.


Introduction
Candida spp. are commensal fungal species commonly colonizing human mucosal and skin surfaces, but they may become pathogenic in some particular scenarios such as treatment with antibiotics, immunocompromised patients, etc., producing in these cases infections that range from superficial to severe skin and mucosal lesions, to even systemic invasion at its worst degree [1]. For example, oral candidiasis is the most common opportunistic infection affecting the human oral cavity. It is caused by an overgrowth of Candida spp., being the most prevalent Candida albicans [2,3].
Due to the recurrence of Candida spp. infections, high systemic antifungal therapy have been widely used, thereby antifungal resistances are increasing. Moreover, patientdependent, interactions with other medical regimens and organ toxicity can happen [4].
Therefore, it is necessary to develop new treatments such as antimicrobial photodynamic therapy (aPDT). It is based on the use of photosensitizing molecules that are excited with visible light of the appropriate wavelength and reacts with the oxygen, generating reactive species of oxygen to destroy the target pathogen [5][6][7].
Superficial wound infections are potentially suitable for treatment by aPDT because of the ready accessibility of these wounds for both topical delivery of the photosensitizer and light, and because of the exposure to oxygen [6,8,9].
Future directions of aPDT include the combination with antimicrobials in order to enhance the microbial inactivation and prevent the regrowth when the light from aPDT is turned off and the photoinactivation ends. This original approach has already shown significant potential. It could help to implement the use of aPDT and reduce the amount of antimicrobials used and, thus, the multidrug resistance problem [14][15][16].
Chlorhexidine (CHX) is an antiseptic drug, mainly available in over-the-counter products as routine hand hygiene in healthcare personnel, to clean and prepare the skin before surgery, and before injections in order to help reduce the amount of microorganisms that potentially can cause skin infections [17][18][19][20]. CHX gluconate is also available as a prescription mouthwash to treat gingivitis and as a prescription oral chip to treat periodontal disease [21][22][23] and recently against COVID-19 in dentistry [24].
Here, we investigate the aPDT and the CHX uncombined or in combination against Candida spp. As a photosensitizing molecule, we use methylene blue (MB), the main member of the phenothiazine family, well known for its ability to produce singlet oxygen when it is irradiated by red light and react with molecular oxygen [6,8]. As a source of irradiation, we use a light-emitting diode (LED) lamp emitting in red or a white metal halide lamp (WMH) that emits at broad-spectrum white light which is comparable to the emission spectrum of daylight.
The aim is to compare the antimicrobial effect of MB-aPDT when a specific irradiation source or a non-specific broad-spectrum source is used to excite different concentrations of MB. Furthermore, the effects of the combination of aPDT with CHX are evaluated.

Materials and Methods
The procedure used tried to follow the materials and methods of our previous works and was adapted as follows [25][26][27] Figure S1 shows the lamps and their emission spectrums. Both were used at a fluence of 18 J/cm 2 . This fluence corresponds to a 42.86 min (≈43 min) irradiation time for the samples using the red-LED lamp and 3 min and 25 sec for the samples irradiated with WMH lamp.

In Vitro Photodynamic Treatment of Yeast Suspension
C. albicans, C. parapsilosis or C. krusei seeded on Sabouraud dextrose agar were cultured aerobically overnight at 35 • C. The inoculum was prepared in distilled water and adjusted to 5 ± 0.03 on the McFarland scale (concentrations in the range of >1 × 10 6 colony-forming units (CFU) per 100 µL and was deposited into 96-well microtiter plates. Two-fold serial dilutions concentrations from 640 µg/mL to 0.03 µg/mL of the MB were added, in absence or presence of 10 µg/mL of CHX (MB+/CHX−/light+) (MB+/CHX+/light+). The final volume in each well was 100 µL. Irradiation proceeded with no preincubation period; the suspensions were immediately subjected to irradiation with fluence of 18 J/cm 2 using the red-LED lamp or the broad spectrum-WMH lamp. Control samples were subjected to identical treatment, in the absence or presence of the photosensitizer, and were either kept in darkness or irradiated to evaluate the effect of each parameter: negative or initial control (MB−/CHX−/light−), irradiation control (MB−/CHX−/light+), control of photosensitizer in darkness (MB+/CHX−/light−) and antiseptic controls (MB−/CHX+/light−) (MB−/CHX+/light+). After completing the aPDT protocol, samples and controls were assessed in serial dilutions of each suspension and were cultured on blood agar and incubated overnight at 35 • C. The dilutions were made and aliquots were cultured to have blood agar plates with a number of CFUs in the range of 0 to 200 per plate in order to be able to count them reliably.

Efficacy
The efficacy of aPDT treatment was assessed by counting the number of CFU/100 µL using a Flash and Go automatic colony counter (IUL S.A., Barcelona, Spain). A reduction of 6 log 10 in the number of CFU/100 µL was considered indicative of fungicidal activity. The minimum concentration of MB that reduced yeast survival by 3 log 10 was also evaluated. All experiments were carried out at least five times. The results are expressed as mean and standard deviation.

Photoinactivation of Yeasts by MB-aPDT (MB+/CHX−/Light+)
MB-aPDT effectively inactivated Candida spp. achieving a reduction of 6 log 10 in the number of CFU/100 µL in all the studied strains ( Figure 1). The minimum concentration of MB required to achieve this effect was 320 µg/mL in all cases except in those irradiated with a WMH lamp in C. parapsilosis that required 80 µg/mL and in C. krusei between 320-640 µg/mL (Table 1). Analyzing in more detail the sensitivity of each strain to MB-aPDT, C. krusei is the most resistant and C. parapsilosis and C. albicans show a very similar ratio of response, although C. parapsilosis is slightly more sensitive to white light than C. albicans ( Figure 1 and Supplementary Material Figure S2I).

Fungicidal Effect of MB-aPDT Combined with CHX (MB+/CHX+/Light+)
The antimicrobial effect of MB-aPDT on Candida spp. was maintained in the presence of CHX, as evidenced by the 6 log 10 reduction in the number of CFU/100 µL in all experiments. Moreover, the combination of MB-aPDT using the WMH lamp + CHX achieves this degree of reduction on C. albicans decreasing 4-fold the required photosensitizer concentration (the necessary concentration is 1/4) ( Figure 1 and Table 1).
To achieve a 3 log 10 reduction in the number of CFU/100 µL when MB-aPDT is used in combination with CHX, the required concentration of MB needed is at least half compared to the concentration needed using MB-aPDT alone. The greatest reduction of the photosensitizer concentration (8-fold) is achieved against C. albicans using the red-LED lamp. On the other hand, the greatest reduction against C. parapsilosis occurs with the WMH irradiation (1/4-1/8 of the initial photosensitizer concentration) ( Figure 1 and Table 1). Samples with the different MB concentrations evaluated under the same conditions used in irradiation but keeping it in darkness (MB+/CHX−/light−) (dark MB in Figure 1) show significant reductions at the highest concentrations tested as follows: reductions of up to a maximum of 3.5 log 10 in C. albicans, 4 log 10 in C. parapsilosis and 4.5 log 10 in C. krusei were achieved by 640 µg/mL of MB. In all experiments, the effects of keeping the microbial suspension with the different MB concentrations in dark (light−) for 43 min or 3 min and 25 sec (using the time of the irradiation with the red-LED or the WMH lamp respectively) is similar, except for C. krusei (reduction of 4.5 log 10 after 43 min vs. 2.5 log 10 after 3 min and 25 sec) (Figure 1).

Control of Inoculum and Toxic
The irradiation with the red-LED lamp or with WMH lamp in the absence of photosensitizer and antimicrobial (MB−/CHX−/light+) did no significantly reduce the number of yeasts (reduction ≤0.3 log 10 , Figure 1).
In the absence of photosensitizer and irradiation, the tested concentration of CHX (10 µg/mL) (MB−/CHX+/light−) (dark MB-CHX with the value of 0 MB concentration in Figure 1) failed to effectively inactivate the yeast. A maximum reduction of 1 log 10 was observed against C. parapsilosis.
The cumulative effect of CHX and irradiation (MB−/CHX+/light+) ( Figure 1) reaches a maximum reduction in the number of CFU/100 µL of 1.3 log 10 against C. parapsilosis being the most sensitive strain to this effect.

Discussion
MB-aPDT is effective in eradicating Candida spp. (>6 log 10 reduction in the number of CFU/100 µL of C. albicans, C. parapsilosis or C. krusei) and the combination with CHX enhances the photoinactivation, i.e., the effect is achieved with lower aPDT-dose ( Figure 1 and Table 1).
Regarding the comparison of MB-aPDT results obtained with those reported by other authors, many variables should be considered. Table 2 summarizes different studies against Candida spp. in suspension, specifying the methodology and results. Daliri et al. reported a reduction of 3.43 log 10 of C. albicans using 200 µg/mL of MB which is notably lower than the one reached in the present study (MB concentration range of 80-160 µg/mL is able to inhibit 4 log 10 ). They used a bigger number of CFU in the inoculum and this could affect but the mismatch may be because they use a laser irradiation source [28]. Application times are usually short when lasers are used and it does not always guarantee adequate oxygenation [6]. Valkov Table 2).
Focusing on C. parapsilosis, Güzel Tunçcan et al. achieved a reduction of 4 log 10 with 25 µg/mL of MB. The comparison with our data and the possible explanation is very difficult because the methodology used is dissimilar [31].Černáková et al. demonstrated that using 9.6 µg/mL of MB inhibited between 1.13-1.27 log 10 of C. parapsilosis in suspension, similar results to this work (this concentration does not generate complete photoinactivation) [32]. Finally, Ahmed et al. used 100 µg/mL of MB and achieved reductions of 0.58-0.85 log 10 at fluences of 90-180 J/cm 2 respectively [33]. Again, the difference may be due to the fact that they used a laser irradiation source and therefore it would be less effective ( Table 2).
Against C. krusei, concentrations of 16 µg/mL [34] or150 µg/mL of MB [35] even at high fluences only achieves a maximum reduction of 0.65 log 10 . More similar result to ours was obtained by Souza et al. using 100 µg/mL with a reduction of 1.54 log 10 using a fluence of 28 J/cm 2 [36]. All MB-aPDT studies together lead us to conclude that C. krusei is the most resistant Candida spp. to MB-aPDT as well as it is more resistant to antifungals in general mainly due to the characteristics of its membrane [37] (Table 2).
Regarding the MB-aPDT combination with CHX, it stands out that >6 log 10 reduction in the number of CFU/100 µL of C. albicans was achieved reducing the concentration of photosensitizer needed from 320 to 80 µg/mL when WMH lamp was used ( Figure 1, Table 1 and Supplementary Figure S2). Furthermore, the addition of CHX halved the concentration of MB required to reach a reduction of 3 log 10 in C. albicans and C parapsilosis, and slightly less than half against C. krusei. Therefore, a synergistic effect is seen between MB at concentrations unable to achieve complete photoinactivation and CHX. These results are relevant because the presence of CHX could help to avoid the microbial regrowth of those microorganisms not completely destroyed when PDT is finished. This is one of the disadvantages of using aPDT for infections in the clinic, the risk of microbial regrowth after its application. The combination with antimicrobials could play a crucial role to overcome this limitation of aPDT in this context [14,15].
To our knowledge, there are not studies combining aPDT plus CHX in vivo against Candida spp. Recently, the effectiveness of MB-aPDT combined with CHX and zinc oxide ointment has been studied on wound healing process after rumenostomy. This study in cattle ratifies the use of aPDT and suggests that it could be performed for other surgical procedures as a complementary approach or an alternative for topical administration of antibiotics [38]. The combination of CHX plus aPDT has been tried against other microorganisms such as Porphyromonas gingivalis biofilm on a titanium surface in a dental framework. The application of CHX and subsequent aPDT using toluidine blue O was shown to be an efficient method to reduce P. gingivalis in titanium surfaces [39]. Regarding other studies of antimicrobials plus aPDT against Candida spp., Giroldo et al. demonstrated that yeasts, both in suspension and in biofilms, were much more susceptible to antifungal treatments after MB-aPDT, explained by the increase of membrane permeability caused by aPDT [40]. Regarding the in vitro combination of MB-aPDT with fluconazole against resistant strains of C. albicans, C. glabrata and C. krusei, a synergistic effect was found in fluconazole resistant strains of C. albicans and C. glabrata, but not against C. krusei. [34]. These results do not agree with those found by Snell et al. They showed that fluconazole did not increase the aPDT inactivation of C. albicans using MB or another photosensitizer of the protoporphyrin family. However, miconazole did enhance the fungicidal activity of aPDT [41]. Moreover, to our knowledge, there are no studies using aPDT in combination with antimicrobials that report antagonistic effects, which support the use of aPDT in combination with CXH due to the possible advantages [15].
Considering clinical practice, MB-aPDT (660 nm and 7.5 J/cm 2 ) has been tried in HIV patients diagnosed with oral candidiasis comparing it with an antifungal commonly used in candidiasis. After 30 days, the antimicrobial was effective, but there were recurrences except when 450 µg/mL of MB was used [42].
All together indicates that aPDT or antimicrobial alone may not be entirely effective against Candida spp. that is characterized for causing highly recurrent infection especially in predisposed or immunosuppressed patients. On the other hand, combined treatments such as aPDT plus antimicrobials may prevent recurrent infections and avoid resistance. In addition, the combination in terms of clinical application would decrease the intensity of blue staining caused when the MB is applied on the skin or mucous membranes, making the aPDT procedure more cosmetically appealing.
Regarding the concentration of 10 µg/mL CHX used for this study, it was chosen by taking into account other protocols for the combination of antimicrobials plus aPDT and considering that by itself produces no effect under experimental conditions [25,27],  [45].
Regarding the use of the red-LED lamp or the WHM lamp as a source of irradiation for aPDT, the second proved to be more effective in photoinactivating Candida spp. with the exception of against C. krusei (Figure 1, Table 1 and Supplementary Material Figure S2II). The use of LED lamp emitting in red matching the absorption spectra peak of the MB tends to be more efficient in the sense of not wasting irradiation energy and therefore red emission sources are usually the ones chosen for MB-aPDT studies (e.g., shown in Table 2). In addition, red LEDs lamps have added advantages at the time of transferring the use of aPDT to clinical application because they are available in all the PDT clinical units; in addition, these lamps stimulate cellular repair mechanisms in fibroblasts [46] and are already used to treat acne vulgaris, herpes simplex virus infection, shingles, or severe wound healing [6,47].
It is also worth noting the time factor to facilitate the use in the clinic since the WMH lamp needs 3 min and 25 seconds to photoinactivate Candida spp. compared to 43 min for the LED lamp, due to the greater irradiance of the former compare to the latter (90 mW/cm 2 vs. 7 mW/cm 2 respectively). Furthermore, the experiments were performed without preincubation of the photosensitizer MB with Candida spp. prior to irradiation. Andrade et al. and Soria-Lozano et al. demonstrated that a pre-incubation time did not produce greater inactivation of the microorganism, so it is not necessary to add more time to the aPDT procedure [12,48].
On the other hand, a broad-spectrum WMH lamp could be a model of daylight, i.e., which could be used as a source of irradiation for aPDT instead of this lamp. The ad-vantages are that the treatment could be carried out at home and it would require less equipment and personnel (cheaper). However, it also has disadvantages, such as the imprecision in the quantification of the dose of light or duration of exposure, considering that intensity of daylight depends on the season of the year, weather conditions, or geographic location [9,[49][50][51]. Another limitation for the use of daylight is the limitation to treat Candida infections not accessible for this light, such as the mouth or the genitalia, which otherwise are the most frequent. Nevertheless, the WMH lamp achieves better results than the LED lamp in this work, demonstrating its efficacy.  Overall, our study aims to open the way for the application of this alternative therapy, MB-aPDT alone or better in combination with CHX, either using lamps with a specific or broad-emission spectrum or even daylight as an irradiation source, to deal with cutaneous and mucosal candidiasis. However, it should be borne in mind that the present findings were obtained following in vitro irradiation of Candida spp., therefore clinical studies are required to confirm these results.