Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reduced virulence

Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reduced virulence

Journal Pre-proof Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reducing vir...

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Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reducing virulence Min Zhang , Haiying Yan , Mengjiao Lu , Decai Wang , Shujuan Sun PII: DOI: Reference:

S0924-8579(19)30251-1 https://doi.org/10.1016/j.ijantimicag.2019.09.008 ANTAGE 5804

To appear in:

International Journal of Antimicrobial Agents

Received date: Accepted date:

27 March 2019 11 September 2019

Please cite this article as: Min Zhang , Haiying Yan , Mengjiao Lu , Decai Wang , Shujuan Sun , Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reducing virulence, International Journal of Antimicrobial Agents (2019), doi: https://doi.org/10.1016/j.ijantimicag.2019.09.008

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Highlights ● Ribavirin displayed potent antifungal activity against Candida albicans in vitro. ● Ribavirin synergized with fluconazole against resistant Candida albicans in vitro. ● Antifungal effect of fluconazole was also enhanced by ribavirin in vivo. ● This drug combination inhibited the biofilm formation and hyphal growth. ● The combination also reduced the phospholipase activity of Candida albicans.

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Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reducing virulence

Min Zhang1, 2, Haiying Yan3, Mengjiao Lu4, Decai Wang1, Shujuan Sun3

1

School of Pharmaceutical Sciences, Shandong First Medical University, Taian, Shandong

Province, China; 2

Department of Pharmacy, Taian Municipal Hospital, Taian, Shandong Province, China;

3

Department of Pharmacy, Shandong Provincial Qianfoshan Hospital, the First Hospital

Affiliated with Shandong First Medical University, Jinan, Shandong Province, China; 4

Department of Pharmacy, Tianjin Baodi Hospital, Baodi Clinical College of Tianjin Medical

University, Tianjin, China;



Corresponding author

Tel: 86-531-89268365, Fax: 86-531-82961267 E-mail: s[email protected]

Abstract The incidence of fungal infections has increased continuously in recent years, and drug

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resistance, especially resistance to fluconazole (FLC), has emerged. To overcome this challenge, research on the antifungal activities of non-antifungal agents has gained more attention. In this study, we determined the anti-Candida activity of ribavirin (RBV), an antiviral drug commonly used in the clinic, and found that RBV displayed potent antifungal activity when used alone or in combination with FLC in vitro and in vivo. In vitro, the MIC80 values of RBV were 2-4 μg/mL for FLC-susceptible C. albicans and 8 μg/mL for FLC-resistant C. albicans. When RBV, at the dose of 1μg/mL, was combined with FLC, significant synergistic effects were exhibited against FLC-resistant C. albicans, and the MICs of FLC decreased from > 512 μg/mL to 0.25-1 μg/mL. Synergism was also exhibited against C. albicans biofilm. In vivo, RBV plus FLC significantly improved the survival of infected Galleria mellonella larvae when compared with the FLC-treated group over a 4-day period, and attenuated the damage of FLC-resistant C. albicans to G. melonella larvae tissue. Furthermore, mechanistic studies indicated that the antifungal effects of RBV used alone or in combination with FLC might be associated with the inhibition of biofilm formation, reducing extracellular phospholipase activity and inhibiting hyphal growth, but might not be related to the promotion of FLC uptake and the inhibition of FLC efflux. These results will provide a promising direction for overcoming drug resistance, and for expanding the clinical application of existing drugs. Keywords: FLC-resistant Candida albicans; Ribavirin; Fluconazole; Synergism; biofilms; Galleria mellonella model; Synergistic mechanisms

1. Introduction 3

In recent years, the incidence of invasive fungal infections has increased constantly, especially in patients with impaired immune systems or in hospitalized patients with severe underlying diseases [1]. Candida albicans is a common pathogenic fungus that can cause superficial fungal infections of the skin, mouth or mucous membranes, as well as life-threatening systemic infections [2]. The number of antifungal drugs available is increasing, although not enough. Azoles are considered to be the common therapeutic drugs for C. albicans infections due to their high efficiency and low toxicity, especially fluconazole (FLC). However, prolonged usage of FLC resulted in the emergence of resistant strains, introducing a great challenge to the prevention and treatment of C. albicans infection [3, 4]. At present, the main methods for overcoming fungal resistance include developing novel antifungal drugs and combining existing antifungal drugs. However, the development of novel antifungals is usually costly and time-consuming, while some existing antifungal drugs are expensive and have obvious side effects. Studying the antifungal effects of the existing clinical non-antifungal agents is one solution to overcome the current challenges. Inosine monophosphate dehydrogenase (IMPDH) is widely expressed in organisms, including humans, other mammals and microorganisms. Research on drugs targeting IMPDH has revealed that IMPDH inhibitors have good application prospects in immunosuppressive, antitumor, antiviral, antibacterial and antiprotozoal treatments [5-7]. Previous studies showed that IMPDH was associated with fungal virulence. Inhibition of IMPDH could attenuate the virulence of Cryptococcus neoformans and even kill it outright [8]. IMPDH inhibitors not only exhibit antifungal activity when used alone but also display synergistic antifungal effects when used in combination with antifungal agents against C. albicans and C. neoformans [8-10]. 4

Ribavirin (RBV), an IMPDH inhibitor, has been widely used for the prevention and treatment of viral diseases, but studies on its antifungal activity and the mechanism have not been reported thus far. RBV as an antiviral agent has long been used in the clinic and is inexpensive. Its toxicity is low, and the common adverse reactions include anemia and fatigue, among others. These symptoms disappear after stopping the drug. In this study, we determined the antifungal activities of RBV alone or in combination with FLC against planktonic cells and biofilms of C. albicans in vitro by the checkerboard microdilution method and XTT reduction assay, respectively. In addition, the in vivo antifungal effects of RBV combined with FLC against C. albicans were also investigated by the Galleria mellonella model, including survival analysis, and histological study. Furthermore, we explored the potential antifungal mechanisms by determining the extracellular phospholipase activity, observing the hyphal formation, and measuring the drug efflux pumps activity. These findings are very important for overcoming drug resistance, and targeting IMPDH might be an effective strategy for the development of new antifungals.

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2. Materials and methods 2.1 Strains and media Twelve C. albicans strains were obtained from the clinical laboratory of Shandong Qianfoshan Hospital in China. These strains have different sensitivities to FLC, and we distinguish their sensitivities according to CLSI document M27-S4, including six FLC-resistant and six FLC-susceptible C. albicans (Table 1). C. albicans (ATCC 10231), obtained from the pharmacological institute of Shandong University, was tested as a standard strain. All strains were stored at -80°C and subcultured at least twice on yeast-peptone-dextrose (YPD) solid medium at 35°C before each experiment.

2.2 Drugs Ribavirin (RBV) and fluconazole (FLC) were purchased from Dalian Meilun Biotech Co., Ltd. China. The stock solutions of RBV and FLC were prepared at a final concentration of 2560µg/mL in sterile deionized water and stored at 4°C.

2.3 Antifungal effects of RBV against C. albicans in vitro 2.3.1 Antifungal susceptibility testing Susceptibility testing of RBV and FLC alone and in combination was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines (CLSI, document M27-A3). For the checkerboard assays[11], the concentrations of FLC and RBV used were 0.125–64 µg/mL and 0.5–32 µg/mL against FLC-resistant C. albicans respectively, and 0.03125–16 µg/mL and 0.5–32 µg/mL against FLC-susceptible C. 6

albicans, respectively. Yeast suspensions (2 × 103 CFU/mL) were inoculated with RPMI-1640 medium in a 96-well plate. The medium was buffered with morpholinepropanesulfonic acid (MOPS), and the pH was adjusted to approximately 7.0 ± 0.1 by adding sodium hydroxide solution. A growth control containing C. albicans cell suspension and RPMI-1640 medium and a blank control containing only RPMI-1640 medium were used as negative controls. The plate was incubated at 35℃ for 24 h. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of drugs causing 80% growth inhibition compared to the growth control. The optical density was measured at 492 nm by microplate reader (BioTek Epoch). Both visual reading and optical density assessments were performed to determine the MIC80. All of the tests were performed in triplicate and were repeated at least three times. The interaction of FLC with RBV against C. albicans was evaluated by the fractional inhibitory concentration index (FICI) model. The expression of FICI was FICI=FICA+ FICB=CA/MICA+CB/MICB; FICI ≤ 0.5 represented synergy, FICI > 4.0 represented antagonism, and 0.5 < FICI ≤ 4 inferred no interaction [12].

2.3.2 Antibiofilm susceptibility testing Antibiofilm susceptibility testing of RBV combined with FLC against C. albicans was performed with preformed biofilms at different stages the method described by Ramage G et al. with slight modification [13]. Briefly, C. albicans cell suspensions (2 × 103 CFU/mL) were added to each of the wells of a 96-well plate except the blank control group. The biofilms were formed over four time intervals (4, 8, 12, and 24 h). At each time point, the wells containing biofilms were gently washed three times with sterile phosphate-buffered saline (PBS) to 7

remove the planktonic yeast. The drugs were then added at the final concentrations of 0.25-128 µg/mL for FLC and 1-64 µg/mL for RBV. The drug-free wells were set to control growth, and the plate was incubated at 35°C for 24 h. The biofilm formation was examined using XTT [2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2Htetrazolium-5-carboxanilide] reduction assay according to the protocol of Melo et al [14]. The absorbance was measured at 492nm by microplate reader (BioTek Epoch). The sessile minimum inhibitory concentration (sMIC) was defined as the lowest concentration of drugs causing 80% reduction in absorption compared to the growth control group. All results were performed in triplicate and were repeated three times.

2.4 Antifungal effects of RBV combined with FLC against C. albicans in vivo 2.4.1 Galleria mellonella survival assay A G. mellonella survival assay was performed as described previously with some modifications [15]. Eighty wax larvae were randomly divided into four groups: growth control group (PBS only), FLC-treated (160 µg/mL) group, RBV-treated (320 µg/mL) group, and FLC (160 µg/mL) plus RBV (320 µg/mL) group. All larvae were placed in a dark incubator at 35℃overnight before the experiment. FLC-resistant C. albicans (CA10) was dissolved in sterile PBS buffer containing cefazolin (20 µg/mL) to prevent bacterial contamination. Each larva was infected with 10 µL of yeast suspension (5 × 108 CFU/mL) through its last left proleg. After incubation at 35℃ for 2 h, each larva was injected with 10 µL of sterile PBS, FLC, RBV, or FLC plus RBV via the last right proleg. The larvae were incubated at 35℃ in the dark, and the numbers of G. mellonella that survived were recorded daily for a period of 4 8

days. The larvae that were black or did not respond to touch were considered dead. The survival curves were plotted by the Kaplan-Meier method using SPSS. Each experiment was performed in triplicate and was repeated at least three times.

2.4.2 Histological study of larvae tissue To evaluate the presence of FLC-resistant C.albicans in the tissues of G. mellonella, we performed histological studies on the above four groups and the blank control group. The experimental procedures were the same as before. Three larvae incubated for two days were randomly selected from each group and frozen at -20°C for two days. Tissue sections 20 μm thick were made in a freezing microtome (Leica CM1950) and allowed to dry overnight, after which the sections were stained with periodic acid Schiff (PAS). No fluorescent dye was added to the sample. The stained sections were observed under the bright-field function of a fluorescence microscope (Olympus FSX100) with a 4.2 × 10 objective.

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2.5 Antifungal mechanisms of RBV used alone or in combination with FLC against C. albicans 2.5.1 Phospholipase activity of C. albicans Phospholipase, one of the most important hydrolytic enzymes secreted by C. albicans, is an important virulence factor in C. albicans infection. Studies have demonstrated that the phospholipase activity of C. albicans is not only related to the resistance but also to a morphological switch [16, 17]. The phospholipase activity of C. albicans was determined using the egg yolk agar plate method. The egg yolk agar medium was prepared according to Padmavathi AR et al. [18] and contained 1% peptone, 3% glucose, 1.8% agar, 0.055% calcium chloride, 5.73% sodium chloride and 10% egg yolk emulsion. Yeast suspensions (1 × 107 CFU/mL) were incubated in RPMI-1640 with different drug groups, including the growth control (drug-free) group, the RBV (1 µg/mL) group, the FLC (0.25 µg/mL) group, and the RBV (1 µg/mL) plus FLC (0.25 µg/mL) group, for 24 h with shaking (200 rpm) at 35℃. The 10 μL suspensions were added to the sterilized egg yolk agar medium, and the plates were incubated at 35℃ for 3 days. Phospholipase activity was determined according to Mutlu Sariguzel F et al. [19]. The Pz was defined by the following equation: Phospholipase activity (Pz) = Diameter of the colony =/( Diameter of the colony plus the precipitation zone) The phospholipase activities were scored into five categories: very high (Pz ≤ 0.69), high (0.70 ≤ Pz ≤ 0.79), low (0.80 ≤ Pz ≤ 0.89), very low (0.90 ≤ Pz ≤ 0.99), and negative (Pz = 1) [20]. The experiment was repeated three times at different times.

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2.5.2 Morphogenesis analysis of C. albicans Filamentation analysis of C. albicans (CA4 and CA10) was performed according to the method of Pierce CG et al. with some modifications [21]. RPMI-1640 is a liquid medium that can induce hyphae formation [22, 23].Yeast suspensions (2 × 105 CFU/mL) were incubated in a 96-well plate with RPMI-1640 liquid medium and treated with different drug interventions, including a growth control (drug-free), RBV (8 μg/mL), FLC (2 μg/mL), and RBV (8 μg/mL) plus FLC (2 μg/mL). The plate was incubated at 35℃, with shaking for 4 h. The suspensions were then gently removed from each well of the 96-well plate with a micropipettor and were washed three times with sterile PBS to remove the planktonic yeast. The plate was then observed under the bright-field function of a fluorescence microscope to evaluate filamentation inhibition with a 40 × 10 objective by adjusting the phase contrast. No fluorescent dye was added to the sample. The experiment was repeated three times.

2.5.3 Rhodamine 6G uptake and efflux assay Rhodamine 6G (Rh6G) uptake and efflux assays were carried out as described previously [24]. Briefly, FLC-resistant C. albicans (1 × 105 CFU/mL) was incubated with YPD liquid medium at 35°C, and the cultures were shaken for approximately 18-19 h. The cells were then collected and washed three times with glucose-free PBS and adjusted to 5 × 107 CFU/mL. Then, the cell suspensions were de-energized for approximately 2 h in glucose-free PBS and then resuspended in the same buffer. For the Rh6G uptake assay, Rh6G and RBV were added to the suspensions at final concentration of 10 μM and 4 μg/mL, respectively. Cell suspensions without RBV treatment served as the control. For the Rh6G efflux assay, 10 μM Rh6G was added to the de-energized cell suspensions, which were then incubated at 35°C for 50 min. 11

Then, the suspensions were exposed to an ice water bath for 10 min to stop Rh6G uptake into cells. C. albicans cells were then harvested and washed three times with cold PBS buffer to remove excess extracellular Rh6G. RBV was added at a final concentration of 4 μg/mL, and the Rh6G alone group served as the control group. The fluorescence intensity of intracellular Rh6G was measured by flow cytometry (excitation at 488 nm, emission at 530 nm) every 10 and 30 min respectively, and the results were recorded until the intracellular fluorescence intensity values were essentially stable. The above experiment was repeated three times. The statistical significances were analyzed using Student’s t-test.

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3. Results 3.1 Antifungal effects of RBV against C. albicans in vitro 3.1.1 MIC80 determined by broth microdilution method The susceptibilities of twelve C. albicans strains were determined under planktonic states by the broth microdilution method, and the results are shown in Table 1. The MIC80 values of RBV were 2-4 μg/mL for FLC-susceptible C. albicans strains and 8 μg/mL for FLC-resistant C. albicans strains. When RBV was combined with FLC, significant synergistic effects were exhibited against FLC-resistant C. albicans, and the MIC80 of FLC decreased from > 512 μg/mL to 0.25-1 μg/mL when RBV was provided at a concentration of 1 μg/mL. We also tested the antifungal activities of RBA used alone or in combination with FLC against C. krusei and C. glabrata, but no antifungal activity was observed (data not shown). 3.1.2 sMIC80 determined by broth microdilution method The susceptibilities of FLC-susceptible C. albicans (CA4, CA8) and FLC-resistant C. albicans (CA10, CA16) were determined under biofilm states by XTT reduction assay, and the results are summarized in Table 2. RBV combined with FLC exhibited synergistic antifungal effects against FLC-susceptible C. albicans biofilms formed over 4, 8, 12, and 24 h (FICI < 0.5), while the synergism against FLC-resistant C. albicans biofilm was only observed when formed over 4, 8, and 12 h, indicating that RBV in combination with FLC has a synergistic antifungal effect at the early stage of FLC-resistant C. albicans biofilms, but not the mature biofilm. RBV used alone exhibited different anti-biofilm activities against C. albicans biofilms formed over different amounts of time. For biofilms formed over 4 h, the sMIC80 values were 8-16 μg/mL, while for biofilms formed over 8, 12 and 24 h, the sMIC80 values 13

were >64 μg/mL.

3.3 Synergistic effect of RBV combined with FLC against FLC-resistant C. albicans in vivo 3.3.1. Survival rate We evaluated the in vivo antifungal activities of FLC, RBV, and their combination in an infected G. mellonella model. Through repeated preliminary experimentation, we found that FLC (160 μg/mL) and RBV (320 μg/mL) was the optimal combination of drug concentrations, showing the strongest antifungal activity against FLC-resistant C. albicans in vivo, with the highest survival rate of G. mellonella. The concentrations of the drugs used in this experiment meet the requirements for safe human dosages. We used SPSS Statistics software to plot the survival curves, and the results are shown in Fig. 1. The survival assay showed that the FLC and RBV alone groups could not significantly improve the survival rate of the infected larvae when compared with the control group. However, the survival rate of larvae was significantly improved when RBV was provided in combination with FLC compared with the FLC-treated group over a 4-day period (P < 0.05). These results indicated that RBV in combination with FLC exhibited significant synergistic antifungal effect against C. albicans in vivo.

3.3.2. Histological study To better evaluate the infection of C. albicans in G. mellonella, we observed stained tissue sections. The effects of different drug interventions on G. mellonella are shown in Fig. 2. Infected tissues of G. mellonella were presented as melanized nodules after the PAS staining. In the blank control group, the tissue was relatively intact, and there were no melanized nodules observed, while melanized nodules were observed in all other groups. In the growth 14

control group and FLC-treated and RBV-treated groups, there were large areas of melanized nodules scattered all over the stained sections, and the tissues around the melanized nodules were loosely distributed. Compared with the growth control group and the drug alone group, the number of melanized nodules was significantly reduced in the RBV-FLC combination group, and the tissue was more complete. The results showed that compared with the FLC-treated group, the combination of RBV and FLC could significantly attenuate the tissue damage to G. melonella caused by FLC-resistant C. albicans.

3.4 Potential antifungal mechanisms studies 3.4.1 RBV combined with FLC reduced extracellular phospholipase activity of FLC-resistant C. albicans The phospholipase activity of C. albicans was determined using the egg yolk agar plate method. In this experiment, we diluted FLC and RBV to concentration ranges of 0.25-2 μg/mL and 1-8 μg/mL using two-fold dilution, respectively. We found that compared with other concentration combinations, FLC (0.25 μg/mL) plus RBV (1 μg/mL) significantly decreased the phospholipase activity of FLC-resistant C. albicans (Table 3). The Pz values of the control and the FLC and RBV alone groups were 0.64 ± 0.01, 0.65 ± 0.02 and 0.66 ± 0.01 respectively, showing very high phospholipase activity, while the Pz value of the drug combination group was 0.89 ± 0.03, which was significantly higher than that of the control group (P < 0.05). The combination of RBV and FLC had no significant effect on the phospholipase activity of FLC-susceptible C. albicans.This result indicated that the combination of FLC and RBV could significantly reduce the phospholipase activity of FLC-resistant C. albicans. 15

3.4.2 Effect of RBV and FLC on the hyphal formation of C. albicans In this experiment, the inhibitory effects of drugs on the hyphal formation of C. albicans (CA4, CA10) were tested at concentrations of 0.25-2 μg/mL for FLC and 1-8 μg/mL for RBV. The results showed that when 2 μg/mL FLC was provided in combination with 8 μg/mL RBV, the hyphal formation of C. albicans was significantly inhibited compared with other groups. As shown in Fig. 3, there were large areas of very long hyphae gathered together in the control group; in contrast, in the FLC or RBV alone group, there were still some hyphae gathered, but in much lesser quantities than in the control group. However, the hyphal length was significantly shorter, and there was almost no hypha aggregation in the combination group compared to the FLC- or RBV-treated group or the control group. From these results, we could see that RBV used alone or in combination with FLC could inhibit the hyphal formation of both FLC-resistant and FLC-susceptible C. albicans, and the inhibitory effect was more obvious when RBV combined with FLC, which might be related to reducing the virulence of C. albicans and reversing the fungal resistance.

3.4.3 Effect of RBV on the uptake and efflux of Rh6G in FLC-resistant C. albicans Both FLC and Rh6G are the substrates of a drug transporter pump on C. albicans cell membranes. In this study, we used Rh6G, which can emit fluorescence, as an alternative to FLC. Based on the result that 1μg/mL RBV significantly decreased the MICs of FLC against FLC-resistant C. albicans, we initially measured different concentrations (1, 2 and 4 μg/mL) of RBV to test whether RBV affects drug uptake or efflux. Through repeated preliminary 16

experimentation, we chose 4 μg/mL of RBV as the optimal drug concentration. As shown in Fig. 4, there was no significant difference in intracellular fluorescence intensity between the RBV group and control groups (P > 0.05), indicating that the synergistic antifungal effects of FLC in combination with RBV against FLC-resistant C. albicans were irrelevant to the uptake and efflux of FLC.

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4. Discussion In the past few decades, the incidence of candidiasis has been increasing worldwide [25]. In addition, it has been reported that C. albicans is the main pathogen causing bloodstream infections, which are associated with significant morbidity and mortality [26, 27]. In addition to high morbidity and mortality, the emergence of drug-resistant strains increases the pathogen’s refractoriness, which poses a substantial challenge for the prevention and treatment of clinical C. albicans infection. IMPDH, a key component of the de novo purine biosynthetic pathway, is essential for the growth and virulence of fungi and is a potential therapeutic target in C. albicans infections [28]. Previous studies have shown that IMPDH inhibitors not only have anti-Candida activity also induce synergism with antifungal agents against C. albicans and C. neoformans. For example, the immunosuppressant - mycophenolic acid (MPA) inhibited the growth of C. albicans, with an MIC50 of 0.25 μg/mL in vitro, and significantly increased the antifungal activity of amphotericin B against C. albicans, with the MIC of amphotericin B decreasing from 0.06 to 0.015 μg/mL [9, 10]. Mizoribine (MZP), another IMPDH inhibitor, was also found to possess antifungal activities against C. albicans in vitro, with an MIC < 0.4 μg/mL [9]. In this study, we first discovered that an antiviral agent, RBV, an IMPDH inhibitor, displayed potent antifungal activity when used alone or in combination with FLC against C. albicans. We found that the MIC80 values of RBV were 2-4 μg/mL for FLC-susceptible C. albicans and 8 μg/mL for FLC-resistant C. albicans. These concentrations meet the criteria for clinically safe drug use. RBV also exhibited significant synergistic effects when combined with FLC against all tested FLC-resistant C. albicans, with the MIC80 of FLC decreased from > 18

512 μg/mL to 0.25-1 μg/mL when the concentration of RBV was 1 μg/mL, and the FICIs were 0.125-0.127, much less than 0.5. However, for FLC-susceptible C. albicans, the FICIs were 0.5625-1.25, showing no interaction. RBV not only exhibits synergistic antifungal effects with FLC in vitro but also enhances the antifungal activity of FLC in vivo. In this paper, we studied the antifungal effect in vivo using G. mellonella infection model. G. mellonella is an ideal infection model of antifungal drug screening and evaluation in vivo because it is easy to operate and susceptible to infection [29, 30]. The survival assay showed that compared with the FLC-treated group, the survival rate of G. mellonella larvae was significantly improved when RBV was provided in combination with FLC over a 4-day period (P < 0.05). In addition, histopathological study exhibited that RBV significantly enhanced the efficacy of FLC in vivo and attenuate the damage of FLC-resistant C. albicans to G. melonella larvae tissue, demonstrating the synergistic antifungal effect of RBV and FLC in vivo. C. albicans biofilms are associated with infections caused by interventional therapies, including catheter-related bloodstream infections. The biofilms might also be involved in the persistence or deterioration of chronic inflammatory diseases and deep or systemic candidiasis [31, 32]. Biofilm formation is considered to be an important mechanism of antifungal resistance. Many studies have shown that C. albicans biofilms are resistant to most existing antifungal drugs, and the drug resistance caused by biofilms complicates the clinical application of FLC [33, 34]. Although most of the existing antifungal agents fail to eliminate Candida biofilms, there is still hope for some relatively new drug formulations, such as liposomal amphotericin B and amphotericin B lipid complex [35]. In addition, echinocandins 19

have been found to possess efficacy against C. albicans biofilms in a bioprosthetic model [36]. In this study, we found that RBV synergized with FLC against both FLC-susceptible and FLC-resistant C. albicans biofilms preformed for ≤12 h, with the FICI ranging from 0.032 to 0.254. A synergistic anti-biofilm effect might be able to overcome the resistance of C. albicans to FLC. C. albicans can participate in a yeast-hyphae transition in different environments [37], which has been described as a potential virulence factor for C. albicans and play an important role in the colonization and invasion of host tissues [38, 39]. The transition between the yeast and hyphal growth forms has been found to be important for the pathogenicity of C. albicans and can also effect the process of biofilm formation and its structure [39, 40]. Reports have revealed that the C. albicans strains that can form hyphae are more virulent [40, 41]. In this study, we found that RBV used alone or in combination with FLC could significantly inhibit the growth of hyphae, demonstrating that the inhibition of hyphal growth might be an antifungal mechanism of RBV or a synergistic mechanism of RBV combined with FLC against C. albicans. Phospholipase is one of the most important hydrolases of C. albicans and is considered to be another important virulence factor for C. albicans [42]. It is also considered to be a promising target for the discovery of novel antifungal agents [43, 44]. Here, we found that the FLC and RBV alone groups exhibited very high phospholipase activity, while the phospholipase activity of the combination group was significantly decreased compared with the other groups (P < 0.05). This finding suggested that the synergism against FLC-resistant C. albicans induced by the combination of FLC and RBV might be related to decreasing the 20

extracellular phospholipase activity. Drug efflux is one of the common mechanism of drug resistance [45, 46]. It has been confirmed that the FLC resistance of C. albicans is partially attributed to the high expression levels of multidrug transporters, which can lead to a decrease in the concentration of the drug, thereby weakening the effect of the drug and causing drug resistance [47, 48]. In this study, we determined the activities of drug uptake and efflux of FLC-resistant C. albicans by Rh6G assays and found that RBV could not promote Rh6G uptake or inhibit Rh6G efflux in FLC-resistant C. albicans. It is suggested that the synergistic antifungal effect of two agents might be not related to the uptake or efflux of FLC. Due to different requirements of different experiments, we used different concentrations of FLC and RBV in the different tests. For example, the survival rate of the larvae depended on the yeast concentration. Our previous research found that when larvae were infected with 5 × 108 CFU/mL of FLC-resistant C. albicans, the survival rates of larvae showed significant differences in the different groups [49], which could give a clear view of the effects of the agents in vivo. For antifungal susceptibility testing, we used a yeast suspension of 2 × 103 CFU/mL, which was consistent with the CLSI guidelines (document M27-A3). Therefore, depending on the different concentrations of yeast suspensions, we used different drug concentrations in different tests. The peak plasma concentration (Cmax) of RBV was approximately 1-2 mg/L. In conclusion, this study discovered a new pharmacological effect of the antiviral agent RBV by first finding that RBV not only possesses antifungal effects against both planktonic cells and preformed biofilms of C. albicans but also mediates significant synergistic effects on 21

the antifungal activities of FLC both in vitro and in vivo. This new pharmacological effect might be related to inhibition of biofilm, attenuation of phospholipase activity and inhibition of hyphal formation but is not related to drug uptake and efflux. A mammalian infected of model and more research on the mechanisms are needed in further studies. Although it is not easy to make a compound preparation of RBV and FLC due to their different T1/2 values, we believe these findings are very important for overcoming drug resistance, and targeting IMPDH might be a potential strategy for the development of new antifungal agents.

Declarations Funding: This work was supported by the Shandong Provincial Administration of Traditional Chinese Medicine of China (2017-166). Competing Interests: None declared. Ethical Approval: Not required.

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Figure 1 Figure 1. Survival curves of different treatments on infected G. mellonella over a 4-day period. Each group of G. mellonella larvae was injected with 10 μL of FLC-resistant C. albicans CA10 (5 × 108 CFU/mL) and was treated with PBS, FLC (160 μg/mL), RBV (320 μg/mL), and FLC (160 μg/mL) combined with RBV (320 μg/mL), respectively. * P < 0.05 when compared with the FLC-treated group.

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Figure 2 Figure 2. Histopathology of the G. mellonella larvae. G. mellonella were infected with 10 µL of yeast suspension (5 × 108 CFU/mL) and were treated with sterile PBS, FLC (160 μg/mL), RBV (320 μg/mL), or FLC (160 μg/mL) plus RBV (320 μg/mL). Tissue sections of infected larvae were stained with periodic acid Schiff reagent (PAS) and observed using a 4.2 × 10 objective. The melanized nodules are indicated by the arrows. The scale bar was 200 μm.

29

(a). CA4

(b). CA10 Figure 3 30

Figure 3. The effects of RBV and FLC on the hyphal formation of C. albicans. The hyphal formation of C. albicans was observed by fluorescence microscope. C.

albicans (2 × 105 CFU/mL) was incubated with RPMI-1640 medium and treated with different drugs, including a growth control (drug-free) group, an FLC (2 µg/mL) group, an RBV (8 µg/mL) group, and an FLC (2 µg/mL) combined with RBV (8 µg/mL) group, respectively. No fluorescent dye was added to the sample. The samples were observed under the bright-field function of a fluorescence microscope using a 40 × 10 objective. The scale bar was 50 μm. The experiment was repeated three times at different times.

(a)

(b) 31

Figure 4 Figure 4. The influence of tested RBV on Rh6G uptake/efflux. The uptake/efflux of Rh6G was determined by a flow cytometry in the absence and presence of RBV (4 μg/mL). MFIs represent the intracellular Rh6G in FLC-resistant C. albicans. Data are the means ± standard deviations of three replicates. Student’s t-test was used for statistical analyses. n.s., P > 0.05 when compared with the control group.

Table 1. The antifungal activities of RBV used alone or combined with FLC against C. albicans. MIC80 (μg/mL)

Strains

LA theory

MICFLC

MICRBV

CFLC

CRBV

FICI

IN

CA4

0.5

4

0.25

0.5

0.625

NI

CA5

0.25

4

0.03125

4

1.125

NI

CA8

0.5

4

0.03125

2

0.5625

NI

CA14

0.125

2

0.03125

2

1.25

NI

CA20

0.5

4

0.25

1

0.75

NI

CA129

0.25

2

0.125

0.5

0.75

NI

CA10

> 512

8

0.25

1

0.125

SYN

CA16

> 512

8

0.5

1

0.126

SYN

CA103

> 512

8

1

1

0.127

SYN

CA137

> 512

8

0.5

1

0.126

SYN

CA632

> 512

8

0.5

1

0.126

SYN

CA20003

> 512

8

0.5

1

0.126

SYN

MIC80 was defined the C. albicans was inhibited in 80%. CA, C. albicans; FLC, fluconazole; RBV, ribavirin; MICFLC, the MIC80 of FLC when used alone; MICRBV, the MIC80 of RBV when used alone; CFLC, the MIC80 of FLC when used in combination with RBV; CRBV, the MIC80 of RBV when used in combination with FLC; IN, interpretation; SYN, synergism; NI, no interaction. 32

Table 2. Antifungal effect of FLC combined with RBV against biofilm of C. albicans Strains

CA4

CA8

CA10

CA16

Time(h)

sMIC (μg/mL)

LA theory

MICFLC

MICRBV

CFLC

CRBV

FICI

IN

4

> 512

8

0.25

1

0.125

SYN

8

> 512

> 64

1

2

0.033

SYN

12

> 512

> 64

16

8

0.156

SYN

24

> 512

> 64

32

16

0.313

SYN

4

> 512

8

0.5

1

0.126

SYN

8

> 512

> 64

1

2

0.033

SYN

12

> 512

> 64

16

8

0.156

SYN

24

> 512

> 64

16

32

0.313

SYN

4

> 512

16

0.5

1

0.063

SYN

8

> 512

> 64

1

2

0.033

SYN

12

> 512

> 64

1

8

0.127

SYN

24

> 512

> 64

> 64

2

NI

4

> 512

8

0.5

1

0.126

SYN

8

> 512

> 64

1

2

0.033

SYN

12

> 512

> 64

1

8

0.127

SYN

24

> 512

> 64

> 64

2

NI

> 512

> 512

sMIC80 were read as the lowest concentrations that produced the 80% reduction in biofilms growth compared with that of the drug-free control. FLC, fluconazole; RBV,

33

ribavirin; MICFLC, the MIC80 of FLC when used alone; MICRBV, the MIC80 of RBV when used alone; CFLC, the MIC80 of FLC when used combined with RBV; CRBV, the MIC80 of RBV when used combined with FLC; IN, interpretation; SYN, synergism; NI, no interaction.

Table 3. Extracellular phospholipase activity of C. albicans treated with FLC and RBV. Strains

Groups

Pz value ± SD

Phospholipase activity

CA4

Control

0.78 ± 0.02

High

RBV

0.78 ± 0.02 n.s

High

FLC

0.79 ± 0.03 n.s

High

FLC + RBV

0.73 ± 0.02 n.s

High

Control

0.64 ± 0.01

Very high

RBV

0.66 ± 0.01 n.s

Very high

FLC

0.65 ± 0.02 n.s

Very high

FLC + RBV

0.89 ± 0.03 ***

Low

CA10

a Control, C. albicans alone; FLC, C. albicans

without drugs; RBV, C. albicans

treated RBV(1 μg/mL)

treated FLC (0.25 μg/mL) alone;

FLC + RBV, C. albicans

treated FLC (0.25 μg/mL) with RBV (1 μg/mL);

34

b Phospholipase activity of C. albicans was evaluated by Pz value, which was the averages of three independent experiments; SD, Standard deviation; c n.s, P > 0.05 when compared with the control group; compared with the control group.

35

***,

P < 0.05 when