Reversal of azole resistance in Candida albicans by sulfa antibacterial drugs

32 Pages·2017·2.37 MB·English

AAC Accepted Manuscript Posted Online 20 December 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00701-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Reversal of azole resistance in Candida albicans by sulfa
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antibacterial drugs
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4 Hassan E. Eldesouky1, Abdelrahman Mayhoub2,3, Tony R. Hazbun4,5 , and Mohamed N.
5 Seleem1,6*.
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6 1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, o
7 West Lafayette, IN 47906, USA w
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8 2Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University, lo
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9 Cairo, Egypt 11884 e
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10 3Biomedical Sciences, University of Science and Technology, Zewail City of Science and f
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11 Technology, Giza, Egypt o
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12 4Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, h
13 Purdue University, West Lafayette, Indiana 47906, USA ttp
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14 5Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47906, USA //a
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15 6Purdue Institute for Inflammation, Immunology, and Infectious Disease, Purdue University, c
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16 West Lafayette, IN 47907, USA a
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17 Running title: Reversal of azole resistance in Candida
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18 Key words: Candida albicans, antifungal, azole resistance, fluconazole resistance, sulfa /

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19 antibacterial, biofilm and Caenorhabditis elegans. n
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21 *Corresponding Author: ,
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22 Mohamed N. Seleem 9

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23 Department of Comparative Pathobiology
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24 Purdue University College of Veterinary Medicine s
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25 625 Harrison Street, Lynn 1298, West Lafayette, IN 47907-2027
26 Phone: [phone] Fax: [phone]
27 Email: [email protected]
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29 Abstract:
30 Invasive candidiasis presents an emerging global public health challenge due to the emergence of
31 resistance to the frontline treatment options, such as fluconazole. Hence, the identification of other
32 compounds capable of pairing with fluconazole and averting azole-resistance would potentially
33 prolong the clinical utility of this important group. In an effort to repurpose drugs in the field of
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34 antifungal drug discovery, we explored sulfa antibacterial drugs for the purpose of reverting azole w
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35 resistance in Candida. In this study, we assembled and investigated a library of 21 sulfa a
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36 antibacterial drugs for their ability to restore fluconazole sensitivity in Candida albicans.
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37 Surprisingly, the majority of assayed sulfa drugs (15) were found to exhibit synergistic relationships m

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38 with fluconazole by checkerboard assay with ΣFIC values ranging from < 0.0312 to 0.25. tp
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39 Remarkably, five sulfa drugs, were able to revert azole resistance in a clinically achievable range. a
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40 The structure-activity-relationships (SAR) of the amino benzene sulfonamide scaffold as antifungal s
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41 agents were studied. We also identified the possible mechanism of the synergistic interaction of or
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42 sulfa antibacterial drugs with azole antifungal drugs. Furthermore, the ability of sulfa antibacterial o
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43 drugs to inhibit Candida biofilm by 40% in vitro was confirmed. In addition, effect of sulfa- p
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44 fluconazole combination on Candida growth kinetics and efflux machinery was explored. Finally, ,

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45 using a Caenorhabditis elegans infection model, we demonstrated that the sulfa-fluconazole 1
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46 combination does possess potent antifungal activity in vivo reducing Candida in infected worms by y

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47 ~50% compared to the control. e
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51 1. Introduction:
52 Invasive candidiasis remains the single most important cause of fungal bloodstream infections
53 and the fourth leading cause of nosocomial bloodstream infections in the United States (1). The
54 mortality rate of invasive candidiasis can reach more than 40%, even with the introduction of
55 new antifungal therapies (2). Moreover, the development of new antifungal drugs is currently
56 unable to keep pace with the urgent demand for safe and effective treatment options.
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57 Collectively, this points to an urgent need for different strategies to develop antifungal drugs to w
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58 deal with this emerging scourge. lo
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59 The Infectious Diseases Society of America has adopted fluconazole as a primary drug of choice d

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60 for controlling and treating invasive candidiasis (3). Fluconazole has several advantages over m

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61 other antifungal drugs in terms of the cost, safety, oral bioavailability, and ability to cross the tt
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62 blood brain barrier (4, 5). However, the extensive use of fluconazole has increased the incidence a
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63 of resistance to the drug among different fungal strains, especially Candida albicans (6-8). a
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64 Unfortunately, fluconazole resistance has the potential to cross over to other azole drugs, such as .o
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65 itraconazole and voriconazole (9). o
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66 Several studies (10-13) have reported the ability of some drugs and compounds to revert azole
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67 resistance in C. albicans. Unfortunately, the concentrations required for the majority of these il 9
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68 drugs to suppress the azole resistance are generally above their clinically achievable 0
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69 concentration. In addition, some of these drugs can result in serious side effects, such as those b
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70 caused by tacrolimus and cyclosporin (14). u
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71 Interestingly, a few reports (15-18) have superficially outlined the ability of few sulfa
72 antibacterial drugs to act synergistically with different antifungals. For example,
73 sulfamethoxazole was found to exhibit synergistic activity with different azole antifungal drugs,
74 such as miconazole, ketoconazole, and clotrimazole, against C. albicans. Also, sulfamethoxazole
75 showed a synergistic interaction with caspofungin against different Aspergillus species, such as
76 Aspergillus fumigatus and Aspergillus niger.
77 Sulfa antibacterial drugs were the first systemic antimicrobial agents to be discovered and have
78 been used extensively for several decades in human and veterinary medicine to treat bacterial
79 infections (19). Sulfa drugs exert their antibacterial action by inhibiting the folate pathway
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80 through a competitive antagonism to the dihydropteroate synthase enzyme (DHPS), which is w
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81 required for the conversion of para-amino benzoic acid (PABA) into dihydrofolate (20). In fungi, lo
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82 sulfa drugs and dihydrofolate reductase (DHFR) inhibitors, such as methotrexate and d

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83 pyrimethamine, have been reported to inhibit the folate pathway by the same mechanism, albeit m

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84 very high concentrations are required to inhibit the growth (21). For instance, sulfamethoxazole tt
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85 and trimethoprim have been shown to have antimycotic activity against A. fumigatus through a
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86 inhibition of the folate pathway (22). Furthermore, interruption of the folate pathway in C. a
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87 albicans was found to inhibit ergosterol biosynthesis, which could explain the synergistic .o
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88 activity of folate inhibitors and azole antifungal agents (23, 24). o
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89 In an effort to repurpose drugs and explore new leads in the field of antifungal drug discovery,
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90 we explored sulfa antibacterial drugs for the purpose of reverting azole resistance in Candida. In il 9
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91 this study, we investigated a library of 21 sulfa antibacterial drugs for their ability to restore 0
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92 fluconazole sensitivity in C. albicans. b
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93 2- Materials and methods u
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94 2.1. Chemicals and reagents
95 RPMI1640 broth powder with glutamine, but without NaHCO3 was purchased from Thermo
96 Fisher Scientific (Waltham, MA), YPD broth medium and agar were obtained from BD
97 (Franklin Lakes, NJ). Fluconazole, rhodamine123, sulfaguanidine, and 4-aminophenyl sulfone
98 (dapsone) were obtained from Fisher Scientific (Pittsburgh, PA). Itraconazole, voriconazole, nile
99 red, sulfamonomethoxine, sulfathiazole, 5-fluorocytosine, sulfamethoxypyridazine,
100 sulfisomidine, sulfametopyrazine (sulfalene), sulfapyridine and succinylsulfathiazole were
101 obtained from TCI America (Portland, OR). Sulfabenzamide, sulfadiazine, sulfadimethoxine,
102 sulfamethazine, sulfanitran, sulfanilamide, sulfisoxazole (sulfafurazole), sulfametoxydiazine
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103 (sulfameter) and 3-(N-Morpholino) propanesulfonic acid (MOPS) were obtained from Sigma w
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104 Aldrich (St. Louis, MO). Sulfacetamide, sulfadoxine and sulfamerazine were obtained from Alfa lo
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105 Aesar (Tewksbury, MA). FK 506 and cyclosporin- A were obtained from (Biotang INC., d

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106 Lexington, MA). Sulfamethoxazole and amphotericin-B were obtained from Chem-Impex m

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107 International (Wood Dale, IL). Penicillin/streptomycin was obtained from Lonza (Walkersville, tt
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108 MD). Calcein-AM was obtained from BD Biosicences (San Jose, CA). a
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109 2.2. Clinical isolates and antifungal susceptibility testing. a
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110 A total of 53 Candida albicans clinical isolates (Table-1) were screened against fluconazole, .o
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111 itraconazole, voriconazole, 5-fluorocytosine and amphotericin-B following the Clinical and o
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112 Laboratory Standards Institute (CLSI) M27-A3 guidelines for yeast (25) to determine their
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113 minimum inhibitory concentrations (MICs). All experiments were carried out in triplicates and il 9
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114 repeated at least twice. 0
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115 2.3. Identification of azole-resistant Candida strains. b
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116 Resistance to fluconazole, itraconazole, and voriconazole were identified following the u
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117 guidelines of Clinical and Laboratory Standards Institute (25). Strains with MIC values of > 32
118 µg/ml fluconazole, ≥ 1 µg/ml itraconazole, and ≥ 1 µg/ml voriconazole, were considered azole
119 resistant (R) (26, 27). Candida albicans ATCC64124 was used as a reference strain for azole
120 resistance, due to their known genetic mutation in azole target (Erg11) (28).
121 2.4. Assembly of the sulfa antibacterial drugs and testing their antifungal activity.
122 A library of 21 FDA-approved sulfa antibacterial drugs (Table-2) was purchased from
123 commercial sources and was prepared as 10 mM stock solutions in dimethyl sulfoxide (DMSO).
124 The MICs of the sulfa antibacterial drugs were determined against azole-resistant C. albicans
125 strains according to the CLSI M27-A3 guidelines as described above (25).
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126 2.5. The interaction of sulfa antibacterial drugs with fluconazole against azole-resistant w
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127 Candida. lo
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128 The interaction between sulfa antibacterial drugs and azole antifungals against azole-resistant C. d

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129 albicans clinical isolates was investigated using the checkerboard assay and the fractional m

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130 inhibitory concentration index was calculated as described previously (29-33). An FIC index < tt
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131 0.5 is considered synergism (SYN); FIC index > 4 is considered antagonism; and a result > 0.5 - a
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132 4 is considered indifferent (IND). a
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133 2.6. Growth kinetic of sulfa antibacterial drugs r
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134 C. albicans strain NR 29448 was used in this experiment because it exhibited rapid resistance (~ n

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135 16 hours) to all azole antifungal drugs used in this study. All sulfa drugs that showed synergetic p
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136 activity with fluconazole were further studied in a growth kinetic curve to confirm their ability to ,

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137 revert azole resistance (34). Briefly, an overnight culture of C. albicans strain NR 29448 was 1
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138 adjusted to 2.5 x 103 CFU/ml in RPMI 1640 medium. Then each drug at its MICc (minimum y
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139 inhibitory concentration when combined with fluconazole) was incubated with C. albicans at 35º e
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140 C, either alone or in combination with fluconazole 1 µg/ml. The growth kinetic was monitored
141 at OD 595 at 0, 6, 10, 12, 24 and 48 hours after incubation.
142 2.7. Effect of sulfa drugs-fluconazole combinations on biofilm forming ability of C. albicans.
143 Biofilm forming C. albicans strains were used to study whether sulfa drugs-fluconazole
144 combinations can interfere with their abilities to form biofilms. Cells were prepared as
145 previously described (35-37). Briefly, an overnight cultures of 12 strains of C. albicans (Fig 2) in
146 YPD broth were diluted in RPMI 1640 medium to an inoculum size of 1 x 105 CFU/ml. Sulfa
147 drugs: sulfamethoxazole, sulfadiazine, sulfadoxine, sulfadimethoxine, and
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148 sulfamethoxypyridazine were added to the yeast cell suspension of C. albicans at concentrations w
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149 0.5, 0.25 and 0.125 x MIC in the presence of a fixed concentration of fluconazole (0.0625 lo
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150 µg/ml). Amphotericin-B, at concentrations 0.5, 0.25 and 0.125 x MIC, was used as a positive d

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151 control. Candida cells were then transferred to the wells of microtiter plates, and then incubated m

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152 at 35° C for 24 hours. The formed biofilms were rinsed twice with phosphate buffered saline tt
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153 (PBS) then left to air dry at room temperature. Air dried biofilms were stained for 10 min with a
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154 200 μl crystal violet (0.1%). Stained biofilms were rinsed three times with PBS and air dried for a
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155 one hour. The amount of crystal violet was quantitated by de-staining the biofilms for 10 min .o
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156 with 200 μl of absolute ethanol and then the absorbance of the crystal violet solution at OD 595 o
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157 nm was measured. All experiments were carried out in quadruplicates and repeated at least twice.
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158 2.8. Caenorhabditis elegans (C. elegans) infection study il 9
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159 To examine the efficacy of sulfa drugs in reverting azole resistant in vivo, we used the C. elegans 0
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160 animal model (38-41). The biofilm forming strain NR 29448 was found to colonize the worms b
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161 effectively, so we used it in this experiment. Briefly, L4-stage worms (strain AU37 genotype u
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162 (glp-4(bn2) I; sek-1(km4) X)) were infected with C. albicans NR 29448 for 3 hours at room
163 temperature. After infection, worms were washed five times with M9 buffer and transferred into
164 tubes (~20 worms per tube). Worms were treated for 24 hours (in triplicate) with a combination
165 of 10 x MICc sulfa drugs and 10 µg/ml fluconazole (SDZ was insoluble at 10 x MICc and was
166 excluded from this experiment). DMSO, 5-fluorocytosine at 10 x MIC (0.625 µg/ml) and
167 fluconazole (10 µg/ml) served as controls. Posttreatment, worms were examined microscopically
168 to evaluate morphological changes and ensure viability, then washed with M9 five times, then
169 disrupted using silicon carbide particles, and the resulting suspensions were serially diluted and
170 transferred to YPD agar plates containing penicillin (100 μg/ml), streptomycin (100 μg/ml).
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171 Plates were incubated for 48 hours at 35° C before the viable Candida colony forming unit w
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172 (CFU) per worm was determined. lo
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173 2.9. Effect of sulfa drug on efflux of rhodamine d

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174 To test if sulfa drugs have inhibitory effect on efflux pump in Candida, rhodamine accumulation m

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175 assay was performed (42). Briefly, C. albicans strain NR 29448 was grown overnight at 35° C in tt
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176 YPD broth, then transferred to a fresh YPD broth and incubated at 35° C for 4 hours until the a
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177 cells reached the mid log phase. Cells were harvested and adjusted to 1 x 106 CFU/ml in YPD .a
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178 broth. Candida cells were then incubated with sulfa drugs at sub-inhibitory concentrations (0.25 .o
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179 x MIC) for 1 hour. Then rhodamine 123 dye (Rh123) at a final concentration of 10 µM was o
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180 added to the cell suspension and incubated at 35° C in a reciprocating shaker for additional 30
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181 minutes. After incubation, 1 ml samples were taken and centrifuged at 5,000 g for 5 minutes. il 9
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182 Supernatants were discarded, and the pellets were washed three times with PBS to remove 0
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183 extracellular Rh123. Fluorescence at excitation and emission wavelengths of 485 and 538 nm b
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184 was recorded for 6 replicates for each drug using Spectramax-ix3 microplate reader. Rh123 u
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185 accumulations were expressed as arbitrary fluorescence per OD 595 (specific fluorescence).
186
187 2.10. Effect of para-amino benzoic acid (PABA) supplementation on synergistic activity of
188 sulfa antibacterial drugs and fluconazole
189 To study the effect of para-amino benzoic acid (PABA) on the synergistic relationship between
190 sulfa drugs and fluconazole, a checkerboard assay was performed as described above, in the
191 presence and absence of different concentrations of PABA (1, 4, 8, 16, 32, 64, 128 µg/ml). Then
192 FIC indices for each drug was calculated as described above.
193 2.11. Statistical Analysis
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194 Data are presented as means ± standard deviation. Statistical analyses were performed using w
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195 GraphPad Prism 6.0 (Graph Pad Software, La Jolla, CA, USA). P-values were calculated using lo
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196 one-way ANOVA. P-values < 0.05 were considered significant. d

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197 m

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198 3. Results: tt
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199 3.1. Antifungal susceptibility testing and identification of azole -resistant Candida a
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200 strains. s
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201 The MICs of fluconazole, itraconazole, voriconazole, amphotericin-B, and 5-fluorocytosine were r
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202 examined against a panel of 53 C. albicans strains. We identified ten strains as azole-resistant n

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203 (fluconazole, itraconazole, and voriconazole) (Table-1). These ten azole-resistant strains were p
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204 selected for further studies. ,

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205 3.2. Assembly of the sulfa antibacterial drugs and testing their antifungal activity. 1
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206 The library of 21 sulfa antibacterial drugs was initially screened at a single concentration of 1024 y

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207 µg/ml against C. albicans strains to identify any possible antifungal activity. Eight sulfa e
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208 antibacterial showed weak antifungal activity at high concentration of 512 µg/ml (Table-3). The
209 rest of the drugs did not show any antifungal activity up to 1024 µg/ml.
210
211 3.3. Sulfa drugs exert potent synergistic activity with fluconazole (checkerboard assay)
212 We assessed the activity of sulfa drugs in combination with fluconazole against azole resistant
213 Candida strains using the checkerboard assay (30, 42-44). As shown in (Table-3), 15 sulfa drugs
214 were found to exhibit synergistic relationships with fluconazole against seven of the ten Candida
215 strains tested with ΣFIC values ranging from < 0.0312 to < 0.25. Sulfamethoxazole and
216 sulfamonomethoxine were found to have the most potent synergistic activity. At 1/32 × MIC for
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217 sulfa, we observed more than 128-fold reduction in the MIC for fluconazole (when combined w
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218 with sulfamethoxazole or sulfamonomethoxine) for all seven clinical candida strains where lo
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219 synergy was detected (data not presented). Six drugs showed additive effect (indifference) with d

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220 FICI > 0.5 - 4. Interestingly, five drugs; sulfamethoxazole (SMX), sulfadiazine (SDZ), m

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221 sulfadoxine (SDX), sulfadimethoxine (SDM) and sulfamethoxypyridazine (SMP) reverted azole tt
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222 resistance in a clinically achievable range (MICc values were blew their achievable plasma a
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223 therapeutic concentrations). a
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224 3.4. Growth kinetic of sulfa antibacterial drugs. .o
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225 After confirming that the sulfa antibacterial drugs showed synergistic relationship with o
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226 fluconazole against azole-resistant Candida, we next assessed the growth kinetics of C. albicans
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227 strain NR 29448 when exposed to the sulfa drugs that showed synergistic activity with il 9
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228 fluconazole. As shown in (figure-1), the combination of sulfamethoxazole (16 µg/ml) and 0
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229 fluconazole (1 µg/ml) significantly inhibited the growth of C. albicans NR 29448. However, b
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230 neither sulfamethoxazole (256 µg/ml) nor fluconazole (32 µg/ml) was solely able to inhibit the u
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231 fungal growth such higher concentrations. A similar growth kinetics were observed for all other
232 active sulfa drugs (data not shown)
233
234 3.5. Sulfa drugs -fluconazole combinations do inhibit biofilm formation.