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. Author manuscript; available in PMC: 2020 Jul 10.
Published in final edited form as: Lasers Surg Med. 2019 Nov 19;52(6):569–575. doi: 10.1002/lsm.23180

Quinine Improves the Fungicidal Effects of Antimicrobial Blue Light: Implications for the Treatment of Cutaneous Candidiasis

Leon G Leanse 1,2, Xueping Sharon Goh 1,2, Tianhong Dai 1,2,*
PMCID: PMC7234921  NIHMSID: NIHMS1062860  PMID: 31746024

Abstract

Background and Objective:

Candida albicans is an opportunistic fungal pathogen of clinical importance and is the primary cause of fungal-associated wound infections, sepsis, or pneumonia in immunocompromised individuals. With the rise in antimicrobial resistance, it is becoming increasingly difficult to successfully treat fungal infections using traditional antifungals, signifying that alternative non-traditional approaches must be explored for their efficacy.

Study Design/Materials and Methods:

We investigated the combination of antimicrobial blue light (aBL) and quinine hydrochloride (Q-HCL) for improved inactivation of C. albicans, in vitro and in vivo, relative to either monotherapy. In addition, we evaluated the safety of this combination therapy in vivo using the TUNEL assay.

Results:

The combination of aBL (108 J/cm2) with Q-HCL (1 mg/mL) resulted in a significant improvement in the inactivation of C. albicans planktonic cells in vitro, where a 7.04 log10 colony forming units (CFU) reduction was achieved, compared with aBL alone that only inactivated 3.06 log10 CFU (P < 0.001) or Q-HCL alone which did not result in a loss of viability. aBL + Q-HCL was also effective at inactivating 48-hour biofilms, with an inactivation 1.73 log10 CFU at the dose of 108 J/cm2 aBL and 1 mg/mL Q-HCL, compared with only a 0.73 or 0.66 log10 CFU by aBL and Q-HCL alone, respectively (P < 0.001). Transmission electron microscopy revealed that aBL + Q-HCL induced morphological and ultrastructural changes consistent with cell wall and cytoplasmic damage. In addition, aBL + Q-HCL was effective at eliminating C. albicans within mouse abrasion wounds, with a 2.47 log10 relative luminescence unit (RLU) reduction at the dose of 324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL, compared with a 1.44 log10 RLU reduction by aBL alone. Q-HCL or nystatin alone did not significantly reduce the RLU. The TUNEL assay revealed some apoptotic cells before and 24 hours following treatment with aBL + Q-HCL.

Conclusion:

The combination of aBL + Q-HCL was effective at eliminating C. albicans both in vitro and in vivo. A comprehensive assessment of toxicity (cytotoxicity and genotoxicity) is required to fully determine the safety of aBL + Q-HCL therapy at different doses. In conclusion, the combination of aBL and Q-HCL may be a viable option for the treatment of cutaneous candidiasis.

Keywords: quinine, antimicrobial blue light, Candida albicans, wound infections

INTRODUCTION

Candida albicans is an important opportunistic fungal pathogen that is the cause of wound infection, sepsis, and pneumonia in immunocompromised patients [1,2]. The incidence of fungal infections is on the rise with an estimated 1 billion infections occurring every year, with Candida Spp. being the most important [3]. Furthermore, C. albicans is the most common etiological species of candidiasis, being responsible for up to 70% of all Candida related infections [1]. With the rise in antimicrobial resistance, traditional antifungals are becoming less effective and thus we need to look at alternative approaches to eliminate this clinically significant opportunistic pathogen [4–7]. Antimicrobial blue light (aBL; 405 nm) has been attracting a lot of attention in recent years, due to its inherent antimicrobial properties against a multitude of pathogenic agents [8–11]. In addition, previous studies have not found aBL to incur resistance development following repeated cycles of exposure [12,13]. The accepted mechanism of action of aBL is excitation of endogenous photosensitizing porphyrins that lead to the generation of reactive oxygen species, resulting in apoptosis [8]. The limited papers that have described the use of aBL against pathogenic fungi, have demonstrated that the relative antimicrobial efficacy of aBL against fungi is less than that of bacteria [14,15]. Therefore, it is imperative that we investigate innovative strategies to improve the fungicidal effects of aBL. Quinine is a potent antimalarial compound that has been shown to have inherent antibacterial and antifungal properties, albeit at high concentrations [16–18]. In a previous study, we found quinine hydrochloride (Q-HCL) to significantly enhance the effects of aBL in Gram-negative bacteria [19], Therefore, in this study we investigated the combined use of aBL and Q-HCL for the inactivation of C. albicans, both in vitro and in vivo.

MATERIALS AND METHODS

Blue Light Source

For all aBL exposures, a light emitting diode (LED; M405L3; Thorlabs, Newton, NJ) with a peak emission of 405 nm and a full width at half-maximum (FWHM) of 25 nm was used. The irradiance was regulated through varying the distance of the aperture of the LED from the target and measured using a PM100D power/energy meter (Thorlabs).

C. albicans Strain

For the purposes of the study, a bioluminescent strain of C. albicans CEC 749 was used for both in vitro and in vivo experiments, as described previously [14].

aBL + Q-HCL Inactivation of Planktonic C. albicans in vitro

C. albicans suspensions were cultured overnight to stationary phase in brain heart infusion (BHI) broth (BD) and adjusted to approximately 108 colony forming units (CFU)/mL prior to transferring the suspensions to 35 × 12 mm dishes. Prior to aBL exposure, Q-HCL (Sigma-Aldrich, St. Louis, MI; 1 mg/mL) was added to the 35 × 12 mm dishes containing the C. albicans suspensions. Untreated, aBL-treated alone and Q-HCL-treated alone samples were also included. aBL was irradiated at 60 mW/cm2, until radiant exposures of 27–108 J/cm2 were achieved (7.5–30 minutes; radiant exposure [J/cm2] = irradiance [W/cm2] × time [s]). During aBL irradiation, the bacterial suspension was stirred using a 12-mm magnetic bar (20 rpm) to prevent sedimentation. A total of four aliquots of aBL were delivered (27, 54, 81, and 108 J/cm2) and 30 μL of the suspension was withdrawn following each aliquot to determine the CFU as described previously [19]. Experiments were performed in triplicate.

Transmission Electron Microscopy (TEM) of C. albicans Treated With aBL + Q-HCL

C. albicans was irradiated with aBL (108 J/cm2) in the presence or absence of 1 mg/mL Q-HCL before fixing the cells in a 2.5% glutaraldehyde/ 2% paraformaldehyde solution (Sigma-Aldrich) and incubating overnight at 4°C. An untreated control and Q-HCL treated sample was also included. The following day, the pellets were washed with 0.1 M cacodylate buffer (Sigma-Aldrich; pH 7.2) in triplicate and re-suspended in the same buffer. Subsequently, hot agar (2% in distilled water) was added to the pellets where, once solidified, were processed for TEM. The pellets were fixed in osmium tetroxide in a sodium cacodylate buffer and then dehydrated using ethanol and embedded in Epon T812 (Tousimis, Rockville, MD). Sections were then cut on a Reichert-Jung Ultracut E microtome (Vienna, Austria), and collected on 200 mesh copper grids stained with lead citrate and uranyl acetate. The sections were then examined on a Philips CM-10 TEM (Eindhoven, The Netherlands). Multiple sections were analyzed microscopically with images that were most typical being presented in the study.

aBL + Q-HCL Inactivation of Fungal Biofilms

Biofilms are communities of microbes that are contained within a matrix of extracellular polymeric substances (EPS). Microbial biofilms are of importance to public health as they are associated with increased tolerance to routine antimicrobials [20]. Suspensions of C. albicans in BHI broth were initially incubated in 96-well microtiter plates (200 μL/well; approximately 105 CFU/mL) as described previously [21] for 48 hours to induce biofilm formation. The medium was changed daily to ensure a constant supply of nutrients. Following the incubation, the wells were carefully washed with phosphate-buffered saline (PBS) in triplicate, to remove the residual medium and planktonic cells. Prior to aBL irradiation, 200 μL of fresh PBS (in the presence or absence of 1 mg/mL Q-HCL) were added to wells and the biofilms were then exposed to aBL at an irradiance of 60 mW/cm2 until a radiant exposure of 108 J/cm2 was delivered. Subsequently, the cells were collected by thoroughly agitating the wells with a pipette tip and transferring the 200 μL cell/biofilm suspension to a 1.5-mL microcentrifuge tube. This was performed in triplicate, to ensure adequate removal of the biofilms. The cells were then placed in a water bath sonicator a (branson 2510 sonicator; Marshall Scientific, LLC, Hampton, NH), to ensure disruption of the biofilms for subsequent plating and CFU enumeration.

aBL + Q-HCL Inactivation of C. albicans Within Mouse Abrasion Wounds

Female BALB/c mice aged 6–8 weeks and weighing 17–19 g were purchased from Charles River Laboratories (Wilmington, MA). All animal procedures were approved by the institutional animal care and use committees of Massachusetts General Hospital (protocol number: 2015N000187) in accordance with the National Institute of Health guidelines. Mice were initially injected intraperitoneally with ketamine hydrochloride (Ketalar®; McKesson, Irving, TX)/xylazine hydrochloride (Rompun; Bayer HealthCare, LLC, Cambridge, MA) cocktail (20–100 mg/kg). The mice were then shaved and using a sterile #15 scalpel blade, and the tissue was carefully abraded within a defined 1.0 × 1.0-cm area, ensuring that the scraped area did not produce any blood. Subsequently (within a few minutes), a 100-μL C. albicans suspension containing approximately 108 CFU in PBS was inoculated directly into the wound and applied gently and uniformly with the use of a pipette tip. Three hours after inoculating the suspension (to produce an early onset infection), aBL was irradiated at 60 mW/cm2 in the presence or absence of 0.4 mg/cm2 Q-HCL (in a PBS suspension), until aliquots of 108, 216, or 324 J/cm2 were delivered. Bioluminescence imaging was carried out after each aliquot of aBL (108 J/cm2 aliquots) and immediately illuminated following imaging with a maximum interval of 3 minutes in between each aliquot of light. A vehicle control (PBS alone), Q-HCL control, and nystatin control (100-μL; 1 mg/mL) were also included. For each condition a group of 6 mice were used.

Bioluminescence imaging in vivo

The bioluminescence imaging of C. albicans in mouse abrasion wounds was performed by using an IVIS Lumina II In Vivo Imaging System (PerkinElmer, Inc, Hopkinton, MA). Prior to imaging (before and during treatment), 20 μL of coelenterazine (500 mg/mL; Gold Biotechnology, Inc., St. Louis, MO) was topically applied to the infected wounds, as described previously [14]. Following each aliquot of aBL, the wounds containing the bioluminescent C. albicans were then imaged for their relative luminescence as an indicator for cell viability, which was found previously to correlate with the CFU [14]. The system was operated using the Living Image software, which provides image acquisition tools including photon counting to permit real-time quantification of the relative luminescence units (RLU).

TUNEL Assay to Detect Apoptotic Cells in Healthy Mouse Skin Following aBL + Q-HCL Exposure

To evaluate the presence of apoptotic cells following aBL + Q-HCL therapy, healthy mouse skin was first simultaneously exposed to the 324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL (reflecting the maximum dose administered during the inactivation of C. albicans within mouse abrasion wounds). An untreated control was also included. Skin biopsy specimens were then isolated at 0 hours or 24 hours following treatment and fixed in a 10% phosphate-buffered formalin (Fisher Scientific, USA) for 48 hours as described previously [22]. Subsequently, tissue sections (4 μm thick) were analyzed with the use of the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) in accordance with the manufacturer’s instructions. Fluorescence images were captured using a NanoZoomer S60 Digital slide scanner with a fluorescein isothiocyanate as the fluor and DAPI as the nuclear counterstain. A positive control was included, which was treated with DNase I to induce DNA damage, with the use of an RQ1 RNasefree DNase (Promega), as per the manufacturer instructions.

Statistical Analyses

Data were presented as the mean ± standard error (SE). The differences between means being compared for significance, where aBL vs. aBL + Q-HCL groups were compared to evaluate potentiation using a paired t test. P-values of <0.05 were considered significant.

RESULTS

aBL + Q-HCL Effectively Eliminated C. albicans Planktonic Cells

Following irradiation with 108 J/cm2 aBL alone, 3.06 log10 CFU were inactivated. However, when in the presence of Q-HCL, the antimicrobial effects were significantly enhanced with a 7.04 log10 CFU inactivation, signifying that the effects were improved by approximately 104-fold (P < 0.0001; Fig. 1). Q-HCL at an equivalent dose did not affect the viability of C. albicans.

Fig. 1.

Fig. 1.

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Inactivation kinetic curves illustrating the log10 colony forming unit (CFU) reduction of Candida albicans planktonic cells at different aliquots of antimicrobial blue light (aBL) (0–108 J/cm2) in the presence or absence of 1 mg/mL quinine hydrochloride (Q-HCL). ***P < 0.001, **P < 0.01. Error bars: standard error of the mean.

TEM Revealed Morphological and Ultrastructural Changes Occurring as a Result of aBL + Q-HCL Exposure

Simultaneous exposure of aBL and Q-HCL resulted in significant changes in cell morphology (Fig. 2A). Cells became elongated (red arrow) with obvious detachment of the cytoplasm from the cell wall (black arrow). Furthermore, damage to the cytoplasm was evident with the presence of vacuole formation (yellow arrow). In the aBL alone treated cells, there was also evidence of changes in morphology with cells appearing more elongated (Fig. 2B). Furthermore, as with the combination-treated group, there was evidence of cytoplasmic detachment from the cell wall. The Q-HCL treated group did not have any obvious changes in morphology, except for what appeared to be swelling of the cell wall (Fig. 2C). The untreated control presented with characteristic C. albicans cellular morphology (Fig. 2D).

Fig. 2.

Fig. 2.

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Transmission Electron Microscopy images showing C. albicans cells following treatment with (A) antimicrobial blue light (aBL) + quinine hydrochloride (Q-HCL) (108 J/cm2 + 1 mg/mL), (B) aBL alone, (C) Q-HCL alone, and (D) untreated. Red arrow = elongated cell, yellow arrow = vacuole formation, and black arrow = cytoplasmic detachment from the cell wall. Bar: 500 nm.

aBL + Q-HCL Therapy was Effective at Eliminating C. albicans Biofilms

Exposure to aBL and Q-HCL simultaneously resulted in the inactivation of 1.73 log10 CFU in C. albicans biofilms, compared with aBL alone which only inactivated 0.73 log10 CFU. Interestingly, Q-HCL alone did have a significant effect on the viability of C. albicans, with an inactivation that was similar to that of aBL alone (0.66 log10 CFU; P > 0.05; Fig. 3).

Fig. 3.

Fig. 3.

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Bar graph illustrating the log10 colony forming unit (CFU) reduction of 48-hour C. albicans biofilms, following an aliquot of antimicrobial blue light (aBL) (108 J/cm2) in the presence or absence of 1 mg/mL quinine hydrochloride (Q-HCL). Untreated control and Q-HCL treated alone were also included. ***P < 0.001. Error bars: standard error of the mean.

aBL + Q-HCL Therapy was Effective at Inactivating C. albicans Within Mouse Abrasion Wounds

When C. albicans was treated with aliquots of 108, 216, and 324 J/cm2 aBL (30, 60, and 90 minutes, respectively) and 0.4 mg/cm2 Q-HCL simultaneously, it resulted in almost complete loss of the bioluminescence signal within the abrasion wound (Fig. 4E). By comparison, the aBL alone treated group still had an obvious bioluminescent signal following an equivalent dose (Fig. 4D). Quantitative analyses of RLU indicated that following 324 J/cm2 aBL simultaneously with 0.4 mg/cm2 Q-HCL, a 2.47 log10 RLU reduction was observed compared with aBL alone, at an equivalent dose of which only reduced the RLU by 1.44 log10 (indicating a 10-fold improvement in the inactivation; P < 0.0001; Fig. 4F). Furthermore, vehicle control (PBS) Q-HCL or nystatin alone did not influence the viability of C. albicans (Fig. 4A, B, C, and F).

Fig. 4.

Fig. 4.

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Representative images showing the bioluminescence of abrasion wounds infected with C. albicans that were (A) the vehicle control (PBS), (B) treated with 0.4 mg/cm2 quinine hydrochloride (Q-HCL) alone, (C) treated with nystatin 0.1 mg/cm2, (D) aBL alone, or (E) treated with aBL + Q-HCL. The pseudo-scale color bar semi-quantitatively indicates the relative luminescence unit (RLU). (F) Inactivation kinetic curves illustrating the log10 RLU reduction in mice following different aliquots of aBL with or without Q-HCL (n = 6). An vehicle control group, nystatin, and Q-HCL treated alone group were also included (n = 6). ****P < 0.0001. Error bars: standard error of the mean.

aBL + Q-HCL Therapy Resulted in Some Apoptosis in Healthy Mouse Skin

Fluorescence images were taken on representative mouse skin, at 0 or 24 hours following aBL + Q-HCL treatment (324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL). There was some increase in the number of apoptotic cells (white oval) following the simultaneous exposure of aBL and Q-HCL (Fig. 5A and B), when compared with the untreated control (Fig. 5C); which was determined qualitatively. This was evident both at 0 hours or at 24 hours following the therapy (Fig. 5A and B). The positive control that was treated with DNase I revealed a significant number of apoptotic cells (Fig. 5D).

Fig. 5.

Fig. 5.

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Representative TUNEL stained dorsal mouse skin sections to detect the presence of apoptotic cells resulting from (A) no treatment, (B) 0 hours post-treatment with aBL at 324 J/cm2 + 0.4 mg/cm2 Q-HCL (C) 24 hours’ post-treatment with aBL at 324 J/cm2 + 0.4 mg/cm2 Q-HCL. (D) a positive control treated with DNase I. Fluorescence of fluorescein and DAPI are represented by green and blue pseudo-color, respectively. DAPI is a nuclear counterstain. White ovals indicate apoptotic cells. Bar: 250 μm.

DISCUSSION

In the present study, we investigated the use of a novel combination therapy, aBL + Q-HCL, for the enhanced inactivation of C. albicans. While there have been several studies that have investigated the use of aBL for the inactivation of fungal organisms [14,15], this study represents the first of its kind, combining aBL and Q-HCL for the treatment of fungal infections. The results from this study demonstrated the enhanced antimicrobial effects (as high as 104-fold improvement) that were elicited when aBL was combined with Q-HCL on planktonic cells. A previous study demonstrated that chloroquine, the antimalarial drug, could be used successfully as an antifungal, when the cell was perturbed [15]. Furthermore, previous studies have demonstrated the inherent ability for aBL to damage the bacterial cell wall. Therefore, it is possible that perturbation of the cell wall occurring as a result of aBL irradiation may sensitize C. albicans to Q-HCL inactivation. In our previous study, we hypothesized that Q-HCL potentiated aBL activity through perturbation of the heme biosynthesis pathway, to result in porphyrin overproduction and thus enhanced photo-toxic effects [19]. However, further work would be required to corroborate this hypothesis. Looking at the TEM images, the aBL + Q-HCL exposure influenced the morphology, resulting in the elongation of cells. Typically, elongation can be a result of damage to the cell wall [23], which gives credence to potential sensitization of C. albicans to Q-HCL by aBL, specifically, as exposure to aBL alone also resulted in elongation of the cell.

Microbial biofilms are a tremendously important phenomenon with respect to public health, as they are extremely resistant to antimicrobial intervention [20]. Therefore, we investigated whether aBL + Q-HCL therapy was effective at eliminating mature C. albicans biofilms. We observed a significant potentiation in the antimicrobial effects of aBL + Q-HCL when compared with either monotherapy. An unexpected finding was that C. albicans biofilms were susceptible to Q-HCL alone to a similar extent to aBL. It is unclear why the C. albicans biofilms were susceptible to Q-HCL administered alone, and further studies would be required to investigate this.

The most important finding was the capacity for aBL + Q-HCL to significantly improve the fungicidal effects of aBL (and Q-HCL) within a mouse abrasion wound. We found the addition of Q-HCL improved the aBL activity by 10-fold with Q-HCL alone not eliciting any antimicrobial effects. In addition, the commonly used antifungal drug, nystatin, did not elicit any effects, which was mostly likely attributed to the fact that it was a single dose that was administered for only 90 minutes. Nystatin is commonly administered daily for up to 4 weeks for the treatment of cutaneous candidiasis [24], which could explain its ineffectiveness over the extremely limited treatment timeframe. Our findings strongly suggested that aBL + Q-HCL can effectively and rapidly treat early onset infections caused by C. albicans. It is important, however, to investigate whether this combination therapy is also efficacious for the treatment of more established infections. In addition, there were some apoptotic cells following aBL + Q-HCL therapy. In our previous study, we found there to be fewer apoptotic cells resulting from the aBL+Q-HCL treatment of naive mouse skin. However, lower radiant exposures of aBL were used (135 J/cm2 vs. 324 J/cm2) as well as lower concentrations of Q-HCL (0.2 mg/cm2 vs. 0.4 mg/cm2); which explains why fewer apoptotic cells were observed. Nevertheless, a more comprehensive assessment of safety to investigate potential cytotoxicity and genotoxicity, in vitro and in vivo, is required to fully assess toxicity associated with the therapy. It is reasonable to predict that higher therapeutic doses of aBL + Q-HCL may be required to treat more established infections, therefore, at these higher doses, we might observe some more significant toxicity.

CONCLUSION

In conclusion, we have investigated a novel combination antifungal therapy using aBL and Q-HCL for the inactivation of C. albicans. In this proof-of-principle study, we have demonstrated that the addition of Q-HCL significantly potentiated the effects of aBL. Therefore, the combined use of aBL and Q-HCL may offer an effective alternative strategy for the treatment of cutaneous candidiasis.

ACKNOWLEDGMENTS

We would like to Dr. Christophe d’Enfert (Unité Biologie et Pathogénicité Fongiques, F-75015 Paris, France) for providing the bioluminescent C. albicans strain.

Contract grant sponsor: NIH; Contract grant number: R01AI123312; Contract grant sponsor: DoD; Contract grant number: FA9550-17-1-0277; Contract grant sponsor: ASLMS; Contract grant number: BS.F04.18.

Footnotes

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

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