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Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant Candida spp.
Reference: Tits Jana, Cools Freya, De Cremer Kaat, De Brucker Katrijn, Berman Judith, Verbruggen Kristof, Gevaert Bert, Cos Paul, Cammue Bruno P.A., Thevissen Karin.- Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant Candida spp. Antimicrobial agents and chemotherapy - ISSN 0066-4804 - 64:10(2020), e01296-20 Full text (Publisher's DOI): https://doi.org/10.1128/AAC.01296-20 To cite this reference: https://hdl.handle.net/10067/1705020151162165141
Institutional repository IRUA 1 Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant
2 Candida spp.
3 Jana Titsa, Freya Coolsb, Kaat De Cremera, Katrijn De Bruckera, Judith Bermanc, Kristof
4 Verbruggend, Bert Gevaertd, Paul Cosb, Bruno P.A. Cammuea, Karin Thevissena#
5
6 aCentre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium
7 bLaboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp,
8 Belgium
9 cDepartment of Molecular Microbiology & Biotechnology, School of Molecular Cell Biology and
10 Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
11 dPurna pharmaceuticals, Puurs, Belgium
12
13 Running Head: Fungicidal combination against resistant Candida spp.
14
15 #Address correspondence to Karin Thevissen, [email protected]
1
16 Abstract
17 The occurrence and recurrence of mucosal biofilm-related Candida infections, such as oral and
18 vulvovaginal candidiasis, is a serious clinical issue. Vaginal infections caused by Candida spp., for
19 example, affect 70‐75% of women at least once during their lives. Miconazole (MCZ) is the
20 preferred topical treatment against these fungal infections, yet it has only moderate antibiofilm
21 activity. Through screening of a drug repurposing library we identified the quaternary ammonium
22 compound domiphen bromide (DB) as a MCZ potentiator against Candida biofilms. DB displayed
23 synergistic C. albicans antibiofilm activity with MCZ, reducing viable biofilm cells 1000-fold. In
24 addition, the MCZ-DB combination also resulted in significant killing of biofilm cells of azole-
25 resistant C. albicans, C. glabrata, and C. auris isolates. In vivo, the MCZ-DB combination had
26 significantly improved activity in a vulvovaginal candidiasis rat model as compared to single
27 compound treatments. Data from an artificial evolution experiment indicated that resistance
28 development against the combination was not occurring, highlighting the potential of MCZ-DB
29 combination therapy to treat Candida biofilm-related infections.
30
31 Introduction
32 Various fungal species have the capacity to form biofilms, which are structured microbial
33 communities, surrounded by a self-produced extracellular polymer matrix and attached to biotic
34 or abiotic surfaces (1–3). Fungal biofilms are characterized by an increased tolerance to
35 commonly used antimycotics and are therefore difficult to eradicate (1, 3–6). The number of
36 people suffering from biofilm-related fungal infections is increasing, mainly due to rising numbers
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37 of immunocompromised individuals and increased use of indwelling medical devices, like
38 implants and catheters, on which biofilms can form (7–11).
39 Biofilm-related fungal infections can occur in the oral cavity, the respiratory -and urinary tract, on
40 reproductive organs, in wounds or on medical devices, and are dominated by the genus Candida,
41 of which Candida albicans is the most prevalent (12–17). However, the number of fungal
42 infections caused by non-albicans Candida species is rising with Candida glabrata infections being
43 first in frequency (18–24). C. glabrata is characterized by an innate resistance to azoles, which is
44 the preferred topical treatment against mucosal biofilm-related fungal infections. Hence, C.
45 glabrata mucosal biofilm-related infections are difficult to treat (24–26). Moreover, other
46 emerging Candida spp. present new challenges for antifungal drug development. Candida auris,
47 for instance, is known for its high levels of resistance to multiple antifungal drug classes and its
48 very efficient human-to-human transmissibility (27–31) .
49 Despite the increasing occurrence of fungal biofilm-related infections and their huge impact on
50 the healthcare system, only few novel antifungals with potent antibiofilm activity have been
51 developed during the last decades (12, 32–38). In addition to searches for novel types of
52 antimycotics a promising strategy to develop effective antibiofilm compounds is to increase the
53 activity of conventional antimycotics against fungal biofilms by combining them with a so-called
54 potentiator. This strategy may result in a fungicidal action of the combination against biofilms
55 (39–41). Potentiators can for example induce increased uptake of the antimycotic in the biofilm
56 cells or inhibit biofilm-specific tolerance pathways (42–47).
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57 The preferred antimycotics for topical treatment against mucosal biofilm-related fungal
58 infections are azoles. They include imidazoles (e.g. miconazole, MCZ) and triazoles (e.g.
59 fluconazole), and interfere with the biosynthesis of ergosterol by inhibiting the enzyme lanosterol
60 14‐alpha‐demethylase. Since ergosterol is a major constituent of the fungal membrane, its
61 depletion results in growth inhibition (48, 49). Additionally, MCZ causes an accumulation of
62 reactive oxygen species in planktonic fungal cultures and distinguishes itself from most other
63 fungistatic azoles by its fungicidal action (50–54). MCZ is a preferred topical treatment of mucosal
64 Candida infections, as intravenously administered MCZ is linked with liver toxicity and phlebitis
65 (55). MCZ at its normal therapeutic level is characterized by fungicidal activity against planktonic
66 Candida cultures, but only at very high doses (5 mM), it is fungicidal against Candida biofilm cells
67 (51, 56). Such high doses can only be achieved in antifungal lock therapy but not therapeutically
68 (57, 58). Hence, increasing miconazole’s fungicidal antibiofilm activity by combining it with a
69 potentiator is highly relevant. This study aimed at identifying compounds that increase the
70 fungicidal activity of MCZ against Candida biofilm cells.
71 We therefore screened 1,311 compounds of a drug repurposing library in combination with a sub-
72 inhibitory concentration of MCZ against mature C. albicans biofilms. The quaternary ammonium
73 compound domiphen bromide (DB) was selected as most promising MCZ potentiator and the
74 MCZ-DB combination was further characterized with regard to in vitro and in vivo activity.
75 Furthermore, as resistance to azoles is occurring (25, 59–62), resistance development against
76 MCZ-DB was studied by experimental evolution of C. glabrata cultures.
77
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78 Results
79 Screening for MCZ potentiators against C. albicans biofilms. Analogous to our previous study
80 (39), we screened another 1,311 off-patent drugs and bioactive agents (Pharmakon repositioning
81 library) to identify compounds that can enhance the antibiofilm activity of miconazole (MCZ)
82 against mature C. albicans biofilms, after which we assessed potential fungicidal activity of
83 synergistic antibiofilm MCZ-based combinations.
84 In this way, we identified five compounds that resulted in reduced viability of MCZ-treated C.
85 albicans biofilm cells and had primary applications other than as antimycotics (Table 1).
86 Checkerboard analyses and FICI determination confirmed that, out of these five potential hits,
87 only staurosporine, domiphen bromide (DB) and NSC-317926 act synergistically with MCZ to
88 reduce metabolic activity of C. albicans biofilms. These synergistic combinations were
89 characterized by FICI values of 0.49, 0.42 and 0.16, respectively. To determine whether these
90 synergistic MCZ-based combination treatments are fungicidal against C. albicans biofilm cells,
91 survival of biofilm cells after 24 h of drug exposure was assessed by determining CFUs. Maximal
92 concentrations of MCZ and the potentiators that did not result in significant killing of biofilm cells
93 upon single compound treatment were used in combination. These concentrations were
94 determined based on dose-response curves for fungicidal activity of the single compounds (as
95 assessed by CFU determination) and may vary slightly depending on the experimental setup, the
96 tested compounds and the organism in question. When potentiator concentrations are used that
97 are too low, potentiation will not occur, whereas the use of high, active potentiator
98 concentrations leaves little room for improvement, thereby complicating the analyses. DB was
99 most effective in potentiating MCZ toward fungicidal activity against biofilms, resulting in up to
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100 1000-fold reduction in viable biofilm cells (Fig. 1 and Fig. S1) relative to MCZ alone, and was
101 selected for further analysis.
102 Combination treatment of imidazoles and DB is fungicidal against C. albicans biofilms. To
103 investigate the compound specificity of this combination, the fungicidal activity of combinations
104 consisting of other azoles or other quaternary ammonium compounds against Candida biofilms
105 was investigated. Maximal concentrations of azoles and quaternary ammonium compounds that
106 did not result in significant killing of biofilm cells upon single compound treatment were used in
107 combination.
108 First, we assessed the fungicidal activity of combinations of MCZ with other quaternary
109 ammonium compounds (Fig. 1). A significant reduction in biofilm CFUs was observed upon
110 treatment of the biofilms with MCZ in combination with either DB or dequalinium chloride (DQ),
111 but not in combination with tetraethylammonium bromide (TB), benzethonium chloride (BTC) or
112 benzalkonium chloride (BKC). Treatment of biofilms with MCZ, combined with DQ or DB, resulted
113 in an approximately 100-fold and 1000-fold reduction, respectively, as compared to single
114 compound treatment. DB was selected as the most promising hit because of its most pronounced
115 MCZ potentiating effect.
116 We next tested whether DB also potentiates other imidazoles (e.g. ketoconazole and
117 clotrimazole) and triazoles (e.g. fluconazole and itraconazole) against C. albicans biofilms.
118 Imidazoles and triazoles are two subclasses of azoles, characterized by 2 -and 3 nitrogen atoms in
119 the azole ring, respectively (63). Significant reductions in viable biofilm CFUs, relative to single
120 treatments or DMSO were only observed for DB combinations with the other imidazoles tested
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121 (Fig. 1), but not with triazoles. These data indicate that the potentiating activity of DB toward
122 fungicidal antibiofilm activity is imidazole-specific.
123 Combination treatment of MCZ and DB is fungicidal against biofilms of azole-resistant Candida
124 spp. We aimed to gain insights into the combination’s antibiofilm activity spectrum as biofilm-
125 associated fungal infections and resistance occurrence are important problems. Therefore, we
126 further focused our research toward testing of the MCZ-DB combination against biofilms of
127 resistant Candida isolates, namely fluconazole-resistant C. albicans isolates, the intrinsically
128 azole-resistant C. glabrata and C. auris, reported to develop resistance to multiple antifungal drug
129 classes (25–27, 30). We used fluconazole-resistant isolates, characterized by fluconazole and
130 posaconazole MIC values of at least 64 mg/L and 8 mg/L, respectively. For each pathogen,
131 maximal concentrations of DB and MCZ that did not result in significant killing of biofilm cells
132 upon single compound treatment were selected and used in combination. The resulting
133 optimized MCZ-DB combinations were fungicidal against biofilms of all tested azole-resistant
134 Candida spp and resulted in significant CFU reductions as compared to single compound and
135 DMSO treatment (Fig. 2). Moreover, treatment of C. glabrata BG2 biofilms with a higher, active
136 DB concentration combined with MCZ resulted in a more than 100-fold reduction in viable biofilm
137 cells as compared to the DMSO only treatment (Fig. S2).
138 Combination treatment of MCZ and DB is fungicidal against planktonic cultures of C. albicans
139 and C. glabrata. To assess whether DB’s potentiating activity is biofilm-specific, planktonic
140 stationary phase cultures of the most prevalent Candida spp. C. albicans SC5314 and intrinsically
141 azole-resistant C. glabrata BG2 at a density and growth stage, similar to biofilms, were treated
142 with a concentration series of DB and MCZ alone or a combination of both, followed by CFU
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143 determination. Planktonic cell cultures in stationary phase share some characteristics with
144 biofilms as they have a lower metabolic activity as compared to exponential cultures and are
145 difficult to kill. Hence, stationary Candida cultures with a similar cell density to 48 h old biofilms
146 were used. For each pathogen, maximal concentrations of DB and MCZ that did not result in
147 significant killing of the cells upon single compound treatment were selected and used in
148 combination. The resulting optimized MCZ-DB combinations were fungicidal against planktonic
149 stationary phase cultures of both C. albicans SC5314 and C. glabrata BG2 and resulted in
150 significant CFU reductions as compared to single compound and DMSO treatments (Fig. 3). These
151 data indicate that DB’s potentiating effect on MCZ activity is not biofilm-specific.
152 Combination treatment of MCZ and DB does not result in resistance development. To determine
153 whether resistance against the MCZ-DB combination arises, an artificial evolution experiment was
154 performed. To this end, 3 independent cultures of C. glabrata BG2 (lineages) were consecutively
155 exposed to either MCZ alone (MCZ only-evolved cultures) or to MCZ combined with DB (MCZ-DB
156 evolved cultures). C. glabrata was used, since the growth inhibitory effect of the combination
157 treatment on planktonic C. glabrata cultures was more pronounced than on planktonic C.
158 albicans. The effect of the treatments on the different lineages was assessed after every evolution
159 cycle and doses for subsequent treatment of the evolved cultures were adapted if necessary,
160 thereby maintaining the selection pressure between the treatments and over time. During the
161 whole evolution experiment, we used a treatment that resulted in 35% of yeast growth inhibition
162 (IC35 according to (64)), representing a mild selection pressure that enabled direct inoculation for
163 consecutive rounds of evolution.
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164 After 61 cycles of evolution (representing more than 400 generations), sensitivity of the MCZ-DB
165 evolved cultures was compared to sensitivity of the starter cultures (cycle 0) toward the MCZ-DB
166 combination (Fig. 4). MCZ-DB treatment of starter cultures and MCZ-DB evolved cultures resulted
167 in a significant CFU reduction as compared to single compound treatments and DMSO only-
168 treatment. Furthermore, the MCZ-DB combination was at least as fungicidal on starter cultures
169 (cycle 0) as on the MCZ-DB evolved cultures (cycle 61). Note that for lineage 1, MCZ-DB evolved
170 cultures were significantly more sensitive to the MCZ-DB combination than the starter cultures.
171 Hence, resistance against the MCZ-DB combination was not observed after more than 60
172 consecutive experimental evolution cycles.
173 To check whether the mild selection pressure was high enough to induce experimental evolution
174 after 61 cycles, we assessed sensitivity of MCZ only-evolved cultures, MCZ-DB evolved cultures
175 and starter cultures to MCZ alone by determining MCZ inhibition halo’s. We observed that the
176 diameter of MCZ growth inhibition halo’s was reduced for all evolved cultures as compared to
177 those of starter cultures, pointing to MCZ resistance development in all cultures (Fig. S3). It could
178 therefore be concluded that the applied mild selection pressure was high enough to induce
179 experimental evolution in our setup. In conclusion, resistance against the MCZ-DB combination is
180 not occurring over time in our setup.
181 DB eliminates tolerance rather than altering the MIC of MCZ in both C. glabrata and C. albicans.
182 Azole tolerance is the appearance of cells that grow slowly at supraMIC concentrations of the
183 drug. For fluconazole in C. albicans, tolerance can be visualized with disk diffusion assays, where
184 susceptibility is measured as the size of the zone of inhibition (ZOI) and tolerance is quantified as
185 the fraction of growth (FoG) within the zone of inhibition (65). Furthermore, a number of adjuvant
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186 drugs were able to eliminate fluconazole tolerance when added throughout the agar plate with
187 fluconazole in the drug disk (66). Here, we used a modified plating assay, in which MCZ was
188 spotted at the center of plates which did or did not include 2 µM DB (Fig. S4). The size of the ZOI
189 did not change considerably, indicating that the minimal inhibitory concentration to MCZ was not
190 affected by the presence of DB. Rather, addition of DB to the plates cleared most of the tolerance
191 (partial growth within the ZOI) for both C. glabrata and C. albicans isolates. This implies that DB,
192 like inhibitors of Hsp90, calcineurin, PKC and the TOR pathway (66), inhibits tolerance rather than
193 resistance and that its effect on biofilms is likely an effect on drug tolerance as well.
194 DB enhances MCZ efficacy in vivo. To assess the potentiating effect of DB in vivo, a rat vaginitis
195 model was used. This model is based on infection with C. albicans strain B2630 as infection by
196 this strain results in high fungal burdens (67). This is in contrast to infection with C. albicans strain
197 SC5314 or C. glabrata strain BG2, which both are rapidly cleared by the animals (data not shown).
198 As a therapeutic dosage of 2% MCZ reduced the infection burden in this rat model already with
199 approx. 75% (Fig. S5), an additional potentiating effect by DB would not be notable. Hence, we
200 further used a formulation containing a suboptimal MCZ dose (1%). It is not unusual to use
201 suboptimal doses when studying antifungal combination treatments in vivo. For example, Lafleur
202 and colleagues (2013) showed that a suboptimal dose of fluconazole (0.005%) can be potentiated
203 by AC-17 in an in vivo guinea pig model of cutaneous candidiasis (68). Another study
204 demonstrated a synergistic effect of verapamil and a suboptimal itraconazole dose in vivo,
205 resulting in a reduced fungal lung burden in mice infected by Aspergillus fumigatus (69). To test
206 the MCZ-DB combination in vivo, a formulation containing 1% MCZ nitrate and 0.144 % DB was
207 used. The ratio of these concentrations was chosen based on the in vitro data, pointing to a ratio
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208 of 1:3-1:6 for optimal effects. As cytotoxicity of MCZ, assessed against the cell line MRC-5, was
209 only slightly affected by the presence of DB using this ratio (Table S1), we proceeded toward the
210 in vivo model. The infection burden over the test period is summarized using the “area under the
211 curve (AUC)” measurement (Fig. 5).
212 First, the efficacy of DB alone was evaluated. This treatment did not significantly change the AUC
213 compared to the vehicle-treated control. Secondly, the efficacy of treatment with MCZ or the
214 combination was assessed. While MCZ alone significantly reduced the AUC from 97.20 (95% CI,
215 91.25 to 103.2) Log(CFU)*h to 36.90 (95% CI, 17.88 to 55.92) Log(CFU)*h compared to the control
216 treatment (p < 0.001, linear mixed model), the combination with DB significantly further reduced
217 the AUC to 11.97 (95% CI, 0.3506 to 23.58) Log(CFU)*h (p < 0.001, linear mixed model).
218 Importantly, the combination proved to be statistically more efficient compared to treatment
219 with MCZ alone (p = 0.0217, linear mixed model).
220 A qualitative histological examination of vaginal tissue after H&E staining revealed changes in the
221 epithelial layer in all samples. Acanthosis (epidermal thickening) and hyperkeratosis were
222 observed in all samples, including in non-infected controls. These cornification effects were a
223 consequence of the administered hormone treatment. Inflammation grades were assigned based
224 on density and depth of inflammatory infiltration in the lamina propria (Fig. 6A-C). In the infected
225 control group, 75% (3/4) of the samples showed inflammation grades II or III in the epithelium. In
226 the MCZ treatment group, 70% (7/10) of all samples showed an inflammation grade II, while in
227 the combination therapy group, inflammation grades II or III were present in only 56% (5/9) of
228 the samples. PAS-D stained sections were then evaluated for presence of Candida (Fig. 6D). While
229 in the non-treated infected controls only 25% (1/4) of all samples stained positive for yeasts, after
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230 MCZ treatment 50% (5/10) of all samples showed signs of invasive Candida. For the combination
231 therapy, only 11% (1/9) showed signs of Candida infiltration.
232 Discussion
233 Fungal infections can currently be treated by 3 main classes of antimycotics, being azoles,
234 echinocandins and polyenes. However, fungal biofilms are characterized by an increased
235 tolerance to conventional antimycotics, only a few of which (echinocandins and liposomal
236 formulations of amphotericin B) can be used to treat biofilm-related infections (1, 3–6, 70–72).
237 Biofilms provide several different pathways that protect against the action of miconazole (MCZ),
238 a preferred treatment against mucosal Candida infections. We previously discovered that
239 treatment of C. albicans biofilms with MCZ induces genes classically considered important for
240 antifungal drug resistance: those involved in ergosterol biosynthesis and those encoding drug
241 efflux pumps (2016). Moreover, inhibitors of the electron transport chain act synergistically with
242 MCZ against C. albicans biofilms, but not against planktonic Candida cultures or in oxygen-
243 deprived conditions, suggesting a biofilm-specific oxygen-dependent tolerance mechanism (73).
244 This is consistent with the idea that antifungal tolerance is generally due to changes in stress
245 response pathways (reviewed in (74)).
246 In this study, we sought new combination treatments against biofilm-related Candida infections.
247 The number of patients suffering from such infections is growing. Vaginal infections caused by
248 Candida spp. for example already affect 70‐75% of women at least once during their lives and five
249 to eight percent of these patients subsequently develop recurrent vulvovaginal candidiasis (RVVC)
250 (7–11, 75). Azoles are the preferred treatment for mucosal Candida infections, yet failure to clear
251 the infections treated with azoles is increasingly problematic (25, 59–62). Most azoles are
12
252 fungistatic and not fungicidal, and hence, do not directly result in complete elimination of the
253 Candida cells. In most cases, clearance of (azole‐inhibited) Candida cells is performed by the
254 immune system (51, 76, 77).
255 Biofilm cells phenotypically exhibit an up to 1000-fold higher azole-resistance than planktonic
256 cultures and are less accessible to immune cells, and this contributes to the high recurrence rates
257 associated with fungal biofilm-related infections following antifungal treatment (3, 6, 78–80).
258 Biofilms acquire the ability to grow at higher drug concentrations only when they are in the
259 biofilm state, with growth rates that often differ from those of planktonic cells. This suggests that
260 biofilm drug responses are more akin to antifungal tolerance, which involves phenotypic
261 responses that can differ between isogenic cells as well as between different growth conditions
262 (74). Consistent with this idea, MCZ + DB inhibits MCZ tolerance and does not alter the resistance
263 level (MIC) of the strain. Importantly, the addition of DB to MCZ allows biofilm clearing, due to
264 the shift from the static effect of MCZ vs the cidal effect of the drug combination. Similar effects
265 on tolerance to fluconazole were seen with several different adjuvant drugs, albeit without
266 requiring the biofilm state (65, 66).
267 Thus, novel antibiofilm strategies that can increase the ability to clear fungal infections are
268 needed. In this study, we used an approach in which a potentiator enhances the activity of a
269 conventional antimycotic. Potentiation is often preferred over single compound treatment, since
270 it provides a broader drug activity spectrum, faster antifungal action, lowered dosing of toxic
271 drugs and a reduced risk for the emergence of fungal resistance (81). By screening a drug
272 repurposing library, composed of repurposed compounds that are characterized by a safe toxicity
273 profile and known dosing regimens, we aimed to provide a cost-effective and rapid solution to
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274 the paucity of available antifungal drugs. Such compounds do not require new clinical trials and
275 the costs of possible drug reformulation are much lower than the cost of developing an entirely
276 new drug (82, 83). In this context, 2‐adamantanamine, a derivative of amantadine (an anti‐
277 influenza A drug also used to treat some of the symptoms of Parkinson’s disease), was previously
278 found to potentiate azole antifungals against C. albicans biofilms (68). Other miconazole
279 potentiators against C. albicans biofilms have been discovered, more specifically antimalarial
280 artemisinins like artesunate and artemether, pyrvinium pamoate (an anthelmintic drug) or
281 hexachlorophene (a disinfectant) (39). However, these combinations appeared to be fungistatic
282 and not fungicidal.
283 In this study, domiphen bromide (DB) was selected as the most promising MCZ potentiator. The
284 MCZ-DB combination is fungicidal and is able to eradicate C. albicans biofilms by killing the cells
285 that compose them, potentially reducing the chance on recurrence. DB potentiates the activity of
286 MCZ and other imidazoles against C. albicans, but not of the tested triazoles, suggesting an
287 imidazole-specific potentiation and the possibility to develop multiple combination treatments
288 using the same compound. Furthermore, MCZ-DB is effective against biofilms of C. glabrata, the
289 second-most common cause of Candida infections after C. albicans, which is difficult to treat due
290 to its inherent azole resistance (18–26). Moreover, MCZ-DB is fungicidal against fluconazole-
291 resistant C. albicans isolates as well as against C. auris, an emerging pathogen characterized by
292 intrinsic resistance against fluconazole and also reported to develop resistance to the other
293 classes of antifungals (27, 30).
294 DB is a quaternary ammonium compound, that acts as a cationic surfactant. Such compounds are
295 commonly used as surface disinfectants (84, 85). For several decades, DB has been used at
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296 concentrations up to 0.1% in oral care products for daily use, like mouthwashes and toothpastes,
297 and is considered safe for mucosal applications (86). Furthermore, a double-blind placebo
298 controlled study has investigated tolerability and efficacy of DB in 31 patients suffering from acute
299 infectious dental diseases and showed that 0.5 mg/ 4-6 h of DB resulted in significantly less pain
300 and inflammation after 2 days. Apparently, treatment with DB led to a better prognosis, a good
301 response and fewer days of illness (87). Previous studies showed that DB has minimal cytotoxic
302 effects, which was confirmed by our cytotoxicity data (88).
303 We further demonstrated the DB-potentiating effect of MCZ in vivo. Indeed, using a rat
304 vulvovaginal candidiasis model, we showed superior activity of a combination treatment
305 consisting of MCZ and DB as compared to single compound treatment in curing the fungal
306 infection in vivo. Furthermore, histological data showed a similar grade of inflammation in animals
307 treated with MCZ-DB compared to MCZ alone. Importantly, histological data suggest a lower
308 grade of fungal infiltration in vaginal tissue after treatment with combination therapy.
309 Different quaternary ammonium compounds negatively affect cell membranes (89–91).
310 Understanding the mode of action of antifungal compounds or combinations is important, since
311 it may lead to a faster discovery of new antifungal therapies with a similar mode of action or to
312 the optimization of existing ones (92–94). Vacuolar sequestration of azoles is a recently
313 uncovered strategy of azole antifungal resistance, conserved in nonpathogenic and pathogenic
314 yeasts, that is proposed to reduce the effective azole concentration in the cells (95–97). Using
315 confocal microscopy and BCECF,AM staining, we found that DB’s action possibly increases the
316 permeability of the vacuolar membrane due to its surfactant properties, thereby releasing
317 sequestered azoles. However, additional research is required to confirm this hypothesis.
15
318 Finally, as shown for bacterial biofilms by Sass and colleagues, resistance to a single compound
319 and to combination treatment might already occur after 15 cycles of evolution (98). Here, our
320 evolution experiment, which was maintained for more than 60 consecutive testing cycles,
321 indicates the absence of resistance development in cultures that are grown in the presence of the
322 MCZ-DB combination; sensitivity toward the combination was unaffected after more than 400
323 generations under sublethal treatment with the combination. Thus, the MCZ-DB combination has
324 the potential to be developed into a next generation fungicidal and biofilm-active combination
325 therapy.
326
327 Materials and Methods
328 Strains and chemicals. C. albicans strain SC5314 (99), C. glabrata strain BG2 (100), fluconazole-
329 resistant C. albicans isolates Tansir_082 and Tansir_121B (clinical isolates, Prof Katrien Lagrou, UZ
330 Leuven, Belgium), a C. auris isolate (101) and C. albicans B2630 (67) were grown on YPD (1% yeast
331 extract, 2% bacteriological peptone (LabM, UK) and 2% glucose (Sigma-Aldrich, USA)) agar plates
332 at 30°C. MRC-5 cells (67) were used for cytotoxicity testing. Stock solutions of miconazole (MCZ)
333 (Sigma-Aldrich), ketoconazole (TCI Europe, Belgium), clotrimazole (Sigma-Aldrich), itraconazole
334 (TCI) and fluconazole (MP Biomedicals, France) were prepared in dimethyl sulfoxide (DMSO)
335 (VWR International, Belgium). RPMI 1640 medium (pH 7.0) with L-glutamine and without sodium
336 bicarbonate was purchased from Sigma-Aldrich and buffered with MOPS (Sigma-Aldrich). The
337 Pharmakon 1600 repositioning library (Microsource discovery systems, USA) was supplied by the
338 Centre of Drug Design and Discovery (Dr. Patrick Chaltin, KU Leuven, Belgium) and domiphen
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339 bromide and benzalkonium chloride were purchased from Chemische Fabrik Berg GmbH.
340 Benzethonium chloride and tetraethylammonium bromide were purchased from TCI Europe and
341 dequalinium chloride was purchased from Sigma-Aldrich USA.
342 Antibiofilm screening assay. The antibiofilm screening assay was performed as described
343 previously (39). Briefly, 24-h-old biofilms were treated with 10 µM of MCZ in combination with
344 25 µM of a compound from the Pharmakon 1600 library (2 mM stock solution in DMSO) in RPMI
345 (1,1% DMSO background). After 24 h at 37°C, biofilms were washed, quantified with Cell-Titre
346 Blue (CTB; Promega, USA) and percentage of metabolically active biofilm cells was expressed
347 relative to the control treatment (1,1% DMSO). A confirmation experiment for compounds,
348 resulting in less than 60% residual metabolic activity of C. albicans biofilms in combination with
349 10 µM MCZ compared to the control, was performed twice and compound-only controls were
350 included.
351 Biofilm checkerboard assay. To determine synergistic interactions between antifungal agents
352 and identified potentiators, a checkerboard assay was used, as previously described (39). Briefly,
353 a combination of antifungal compound (e.g. MCZ) and potentiator, two-fold diluted across rows
354 and columns of a microplate respectively, was added to C. albicans biofilms. After a 24 h
355 treatment at 37°C, biofilms were quantified with the CTB method. FICI (fractional inhibitory
356 concentration index) calculations were made to determine synergism (102). The FICI was
357 calculated by the formula FICI=[C(BEC-2A)/ BEC-2A]+[C(BEC-2B)/BEC-2B], in which C(BEC-2A) and
358 C(BEC-2B) are the BEC-2 values of the antifungal drugs in combination, and BEC-2A and BEC-2B are
359 the BEC-2 values of antifungal drugs A and B alone. The interaction was defined as synergistic for
360 a value of FICI≤0.5, indifferent for 0.5 17 361 Fungicidal activity assay. To assess whether a treatment is fungicidal against (i) biofilms or (ii) 362 planktonic cultures, a fungicidal activity assay was performed. (i) Biofilms of C. albicans, 363 fluconazole-resistant C. albicans strains, C. auris and C. glabrata were grown in RPMI and treated 364 with the azole (e.g. MCZ), the quaternary ammonium compound (e.g. DB) or a combination. 24 h 365 or 48 h (C. glabrata) after treatment, CFU determination was performed. To this end, biofilms 366 were washed with PBS, thoroughly scraped off from the bottom of the plate and dissolved in 100 367 µl triton (1%). Serial dilutions were plated on YPD agar plates, followed by an incubation period 368 of 24 h at 37°C and subsequent colony counting. (ii) Planktonic stationary cultures of C. albicans 369 SC5314 or C. glabrata BG2 at OD600nm=1 in RPMI were treated with MCZ, DB or a combination of 370 both. 2,5 h after treatment, shaking at 37°C, CFU determination was performed. To this end, 371 cultures were washed with and dissolved in PBS and serial dilutions were plated on YPD agar 372 plates, followed by an incubation period of 24 h at 37°C and subsequent colony counting. 373 Evolution experiment. The evolution experiment was performed in a volume of 500 µL RPMI 374 medium in a Masterblock® (Greiner bio-one, Germany). During the evolution experiment, we 375 used a treatment that resulted in 35% of growth inhibition on the cultures, representative for a 376 mild selection pressure. 3 independent cultures of C. glabrata BG2 (lineages) were exposed to 61 377 cycles of evolution. One cycle consisted of multiple steps. First, C. glabrata BG2 cells were grown 378 in RPMI medium in the presence of a concentration series of MCZ, either alone or combined with 379 a fixed concentration of 3.125 µM DB. 3.125 µM was the maximal DB concentration that did not 380 inhibit growth of C. glabrata BG2 cultures itself. Subsequently, optical density at 490 nm was 381 measured to determine the IC35 of MCZ. Finally, the culture grown in the IC35 of MCZ (OD600 of 382 approx. 1.0) was 100-fold diluted in RPMI and used to inoculate a subsequent evolution cycle. 18 383 Each cycle consisted of approx. 7 generations of C. glabrata, resulting in more than 400 384 generations over the entire experiment. 385 Halo assay and modified plating assay. A halo assay was performed to check whether the 386 selection pressure, applied in the evolution experiment, was high enough to induce experimental 387 evolution. To this end, overnight cultures of C. glabrata BG2 evolved and starter cultures were 388 diluted to an OD600nm=0.1 in YPD agar. 5 µL of a MCZ concentration series, dissolved in DMSO, 389 was spotted on the resulting YPD agar plates. After an incubation period of 24 h at 37°C, inhibitory 390 halos of MCZ were measured and pictures were taken. 391 To study the effect of DB on tolerance and MIC of MCZ, a modified plating assay was performed. 392 Here, C. albicans SC5314 and C. glabrata BG2 overnight cultures were diluted to an OD600nm=0.1 393 in YPD agar containing 2 µM DB (final DMSO concentration of 0.1%). 5 µL of 50 µM MCZ, dissolved 394 in DMSO, was spotted on the agar plates, followed by an incubation period of 24 h at 37°C. 395 Cytotoxicity assay. MRC-5 cells were grown into polystyrene 96-well plates at an initial 396 concentration of 1.5 * 105 cells/mL cells per well and incubated at 37°C and 5% CO2. In each well, 397 190 µL of cell suspension was added together with 10 µL of watery compound dilutions. Cell 398 growth was compared to untreated control wells (100% cell growth) and medium-control wells 399 (0% cell growth). After 3 days of incubation, cell viability was assessed using 10 µg/mL resazurin. 400 After an additional incubation of 4 hours, fluorescence was measured at λemission = 590 nm, λexcitation 401 = 550 nm using a spectrophotometer (Promega Discover) as described earlier. A compound is 402 classified non-toxic when the IC50 is greater than 20 µM. Tamoxifen was used as a positive 403 control. 19 404 Formulation for vaginitis rat model. A vaginal cream was developed for the application of MCZ, 405 DB or MCZ-DB. For all applications the viscosity of the corresponding cream was identical. As DB 406 affects viscosity of the cream, the formulation was slightly adapted as described below. First, the 407 placebo creams (Table 2) were prepared by heating the cetostearyl alcohol with the mineral oil, 408 the lauroyl polyoxyl-6 glycerides and the PEG-and glycol stearate to 70°C. Once the water had a 409 temperature of 70°C, it was mixed with the oil phase and cooled down under stirring. Once the 410 temperature was below 25 °C, water was added until final weight. After preparing the placebo 411 creams, MCZ nitrate and/or DB were added to obtain the final formulations. In this way the 412 following samples were prepared: 1% w/w MCZ nitrate cream, 0.144% w/w DB cream 413 (corresponding to 1/6 of the molar concentration of 1% w/w MCZ nitrate) and 1% w/w MCZ 414 nitrate with 0.144% w/w DB cream. After production, 0.2 mL of the different formulations was 415 filled into a 1 mL syringe (VWR) ready for application. Creams were prepared 3 days before 416 application and stored at 5°C until application. 417 Vaginitis rat model. All animal experiments were approved by the Ethical Commission of the 418 University of Antwerp (UA-ECD 2018-80) and carried out in strict accordance to all mandatory 419 guidelines (European Union directive 2010/63/EU on the protection of animals used for scientific 420 purposes and the Declaration of Helsinki). Female Wistar rats (± 180 g; Janvier Labs) were 421 ovariectomized 2 weeks prior to infection (67). To induce artificial oestrus, 17β-oestradiol 3- 422 benzoate (Sigma-Aldrich) and progesterone (Sigma-Aldrich) were administered 3 days prior to the 423 infection. Hormone treatment was repeated 2 and 7 days post-infection to maintain oestrus. At 424 the day of infection, 100 µL of an 1 x 108 CFU/mL C. albicans B2630 suspension was applied 425 vaginally. Vaginal swabs were taken at several days p.i. to assess the infection burden. 20 426 Simultaneously, oestrus was monitored by Giemsa staining of a fixated vaginal smear. Rats were 427 treated twice per day starting 24 hours after infection for 6 consecutive days. For the evaluation 428 of treatment with DB alone, 8 animals in total per group were used in 2 independent experiments. 429 Swabs were taken at 4, 9 and 14 days p.i.. To evaluate treatment with MCZ or the combination, 3 430 independent repeats with a total of 24 animals per treatment group were used. Vaginal swabs 431 were taken at 4, 7, 11 and 14 days p.i.. In all experiments, 4 vehicle-treated rats were used as 432 control animals after initial validation of the experimental setup of the controls. Group averages 433 of intravaginal burdens were used to plot graphs, and the area under the infection curve (AUC) 434 was calculated for each animal as a measure for infection burden. 435 Vaginal tissue collection and processing. Vaginal tissue was collected 14 days p.i.. Full-tissue 436 thickness samples were fixed in phosphate-buffered 4% paraformaldehyde for 4 hours and stored 437 in PBS at 4°C before processing. Tissue was subsequently dehydrated in graded ethanol solutions 438 and embedded in paraffin. Samples were cut into 4.5 µm sections and stained using routine 439 hematoxylin and eosin (H&E) method and periodic acid-Schiff-diastase (PAS-D) method to 440 observe tissue damage and candidial infiltration. Samples were observed by an independent 441 histopathologist (Histogenex, Belgium) and severity of inflammation was scored as grade I, II or 442 III based on density and depth of inflammatory infiltrations in the lamina propria as seen in the 443 H&E staining (Table 3). 444 Data analysis. GraphPad Prism 6 was used to analyze all in vitro data. Significant differences in 445 CFUs were determined on the mean log values with P < 0.05 considered as statistically significant. 446 Significant CFU reductions were determined by means of a two-way ANOVA, followed by the 447 appropriate multiple comparisons test. For the in vivo experiments, the area under curve (AUC) 21 448 was calculated for each individual rat based upon the natural logarithm of the burden plus 1, to 449 provide a summary value of the cumulative burden over all time points. Since batch effects cannot 450 be excluded, a linear mixed model was used, with AUC as dependent variable, treatment as fixed 451 effect and batch as random effect. This latter term corrects for the non-independence between 452 observations within the same batch. A posthoc analysis with Tukey correction for multiple testing 453 was used to assess pairwise differences between groups. Analysis of the in vivo data was carried 454 out using R, version 3.5.1 (R Core team (2018). R: A language and environment for statistical 455 computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R- 456 project.org/). 457 Acknowledgements 458 We want to thank Prof Katrien Lagrou, UZ Leuven, Belgium for providing the C. auris isolate and 459 the fluconazole-resistant C. albicans isolates. KT acknowledges receipt of an IOF (Industrial 460 Research Fund) mandate of the KU Leuven. Research was supported by VLAIO (grant 461 HBC.2018.0420) to BC, KT, PC, KV. The funders had no role in study design, data collection and 462 interpretation, or the decision to submit the work for publication. 463 References 464 1. Donlan RM. 2002. Biofilms : Microbial Life on Surfaces. Emerg Infect Dis 8:881–890. 465 2. Hall-stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural 466 environment to infectious diseases. Nat Rev Microbiol 2:95–108. 467 3. Ramage G, Rajendran R, Sherry L, Williams C. 2012. Fungal Biofilm Resistance. 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Biochem Biophys Res 774 Commun 135:397–402. 37 775 776 TABLE 1 Hits from MCZ potentiator screena Compound Application Mode of action PF-04981517 In vitro research tool CYP3A4-inhibitor (103) Domiphen Antiseptic Cell wall interactions; cell death (85, 104, bromide 105) QNZ (EVP4593) Possible therapeutic Inhibitor of NF-κB activation (106) agent to treat Huntington’s disease NSC-319726 Anticancer p53-mutant reactivator, metal ion chelator, redox modulator (107) Staurosporine Antibiotic, antifungal, Protein kinase inhibitor (108) antihypertensive, anticancer 777 aCompounds resulting in reduced viability of MCZ-treated C. albicans biofilm cells are presented 778 along with their primary application and mode of action. 779 38 780 TABLE 2 Quantitative formulation of the vaginal placebo creams Formulation of the vaginal placebo creams Chemical name Commercial name Function Supplier PEG-6, PEG-32 and glycol stearate Tefose 63 O/W Gattefossé emulsifier lauroyl polyoxyl-6 glycerides labrifil M 2130 CS Oil phase Gattefossé cetostearyl alcohol Lanette-D Stifner BASF Mineral oil Finavestan A 360 Oil-phase Total lubrifiants Water / Aqueous phase 781 782 39 783 TABLE 3 Scoring system for severity of inflammation based on H&E staining Grade Histopathological image 784 I Minimal inflammatory reaction with few chronic inflammatory 785 cells, especially lymphocytes in the lamina propria II Moderate inflammatory reaction with increasing extent and more 786 organization, with chronic inflammatory cells, eosinophils and vessels 787 III Severe inflammatory reaction with a dense organized granulomatous reaction with chronic inflammatory cells, 788 eosinophils, fibroblasts and vessels 789 790 40 791 FIG 1 Combining imidazole antifungals and specific quaternary ammonium compounds results 792 in fungicidal activity against C. albicans SC5314 biofilms. Biofilms of C. albicans SC5314 were 793 treated with combinations of imidazoles (either 150 µM miconazole (MCZ), 300 µM ketoconazole 794 (KTC) or 150 µM clotrimazole (CLT)) and a quaternary ammonium compound (QUAT). Maximal 795 concentrations of the quaternary ammonium compound and the imidazole antifungal that did 796 not result in significant killing of biofilm cells upon single compound treatment were selected and 797 used in combination. Following QUATs were used: 50 µM tetraethylammonium bromide (TB), 50 798 µM benzethonium chloride (BTC), 50 µM benzalkonium chloride (BKC), 12.5 µM dequalinium 799 chloride (DQ), or 37,5 µM domiphen bromide (DB). DMSO background was 1%. After 24 h of 800 treatment, the number of CFUs was determined. Mean log CFU values are shown for at least 5 801 biological replicates. Statistical analysis was performed to assign significant differences upon 802 combination treatment as compared to single compound and DMSO only treatment (‘control 803 treatments’). A 2-way ANOVA and either Sidak’s multiple comparisons test (MCZ) or Tukey’s 804 multiple comparisons test (KTC, CLT) was applied and significant differences (p<0.05) relative to 805 the control treatments are shown in orange. 806 FIG 2 Combining MCZ and DB results in fungicidal activity against biofilms of fluconazole- 807 resistant clinical isolates C. albicans Tansir_082 and C. albicans Tansir_121B, and of intrinsically 808 azole-resistant C. glabrata BG2 and C. auris. For each pathogen, maximal concentrations of DB 809 and MCZ that did not result in significant killing of biofilm cells upon single compound treatment 810 were selected and used in combination. Biofilms of fluconazole-resistant C. albicans isolates or C. 811 auris were treated with 150 µM miconazole (MCZ) combined with either 12.5 µM domiphen 812 bromide (DB) or 37.5 µM DB, respectively. Biofilms of C. glabrata BG2 were treated with 500 µM 41 813 MCZ combined with 25 µM DB. DMSO background was 1%. After 24 h (or 48 h for C. glabrata 814 BG2) of treatment, the number of CFUs was determined. Mean log CFU values are shown for at 815 least 4 biological replicates. Statistical analysis was performed to assign significant differences 816 upon combination treatment as compared to single compound or DMSO only treatment (‘control 817 treatments’). A 2-way ANOVA and Tukey’s multiple comparisons test was applied and significant 818 differences (p<0.05) relative to the control treatments are shown in orange. 819 FIG 3 Combining MCZ and DB results in fungicidal action against planktonic stationary phase 820 cultures of C. albicans SC5314 and C. glabrata BG2. For each pathogen, maximal concentrations 821 of DB and MCZ that did not result in significant killing of planktonic cells upon single compound 822 treatment were selected and used in combination. Planktonic stationary cultures of C. albicans 823 SC5314 or C. glabrata BG2 were treated with 25 µM domiphen bromide (DB), combined with 62.5 824 µM or 250 µM miconazole (MCZ), respectively. DMSO background was 1%. After 24 h of 825 treatment, the number of CFUs was determined. Mean log CFU values are shown for 4 biological 826 replicates. Statistical analysis was performed to assign significant differences upon combination 827 treatment as compared to single compound or DMSO only treatment (‘control treatments’). A 2- 828 way ANOVA and Tukey’s multiple comparisons test was applied and significant differences 829 (p<0.01) relative to the control treatments are shown in orange. 830 FIG 4 Resistance against the MCZ-DB combination is not occurring in MCZ-DB evolved C. 831 glabrata BG2 cultures. Sensitivity of three independent C. glabrata BG2 stationary phase cultures 832 (lineages), either evolved for 61 evolution cycles or not (cycle 0, starter culture), to 25 µM 833 domiphen bromide (DB), 250 µM miconazole (MCZ) or the combination was assessed by CFU 834 determination after 24 h incubation. DMSO background was 1%. Mean log CFU values are shown 42 835 for at least 4 biological replicates. Within each evolution cycle, a 2-way ANOVA and Tukey’s 836 multiple comparisons test was performed to assign significant differences upon combination 837 treatment as compared to single compound or DMSO only treatment. Significant differences 838 (p<0.001) are shown in orange. Between evolution cycles, significant differences upon 839 corresponding treatments were assessed by a 2-way ANOVA and Sidak’s multiple comparisons 840 test. ***, p < 0.001; ****, p < 0.0001. 841 FIG 5 Efficacy of treatment with 1% MCZ nitrate or 1% MCZ nitrate with 0.144% DB combination 842 in a C. albicans B2630 vaginitis rat model. First, 2 independent repeats were used to evaluate 843 the effect of DB alone (8 rats in total/group). Subsequently, to evaluate the effect of treatment 844 with MCZ or the combination, 3 independent repeats were used with a total of 24 rats per group 845 for treatments and 12 rats for control. The AUC representing the cumulative infection burden 846 over 14 days is shown. Differences in AUC between 1% MCZ + 0.144% DB and all other treatment 847 groups were tested using a linear mixed model followed by a posthoc analysis with Tukey 848 correction for multiple testing. Significant differences as compared to the control are shown in 849 orange. *, p < 0.05. 850 FIG 6 Histopathology of inflammation and infection stained with H&E (A-C) and PAS-D (D). A: 851 Grade I score showing minimal inflammation with few chronic inflammatory cells; B: Grade II 852 score showing moderate inflammation with chronic inflammatory cells; C: Grade III score showing 853 a severe and dense organized chronic granulomatous reaction; D: Candidial infiltration was 854 observed after PAS-D staining as a magenta colour (black arrows). Depth of inflammatory 855 infiltration in the lamina propria is indicated with a double black arrow (A-C). 43