Clioquinol, an alternative antimicrobial agent against common pathogenic microbe

Clioquinol, an alternative antimicrobial agent against common pathogenic microbe

G Model MYCMED-788; No. of Pages 10 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx Available online at ScienceDirect www.sciencedirect.com Orig...

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MYCMED-788; No. of Pages 10 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx

Available online at

ScienceDirect www.sciencedirect.com

Original article/Article original

Clioquinol, an alternative antimicrobial agent against common pathogenic microbe Z. You a, X. Ran a, Y. Dai b, Y. Ran a,* a

Department of Dermatovenereology, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Wuhou District, 610041 Chengdu, Sichuan Province, China b Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 November 2017 Received in revised form 17 March 2018 Accepted 18 March 2018 Available online xxx

Skin and soft tissue infections (SSTIs) are very common in dermatology and the use of antimicrobial formulations are important in treating these diseases. With the increasing of drug-resistant strains, researchers need to find ways to enhance the effectiveness and/or reduce the drug resistance. Clioquinol was one of antiseptics that can inactivate microbes. It was lack of data of antimicrobial activity; meanwhile it was infrequently used in infection. In order to research the antimicrobial spectrum and activity of topical 3% clioquinol cream among common pathogenic microorganisms compared with other common topical pharmaceuticals, we used modified agar diffusion assay to judge drug susceptibility and compared with broth microdilution assay. Thirty strains of pathogenic fungi belonging to 14 species and 5 strains of pathogenic bacterium belonging to 4 species from clinic or standard strains were enrolled into the experiment. The inhibition zone around 3% clioquinol cream for all experiment isolates was observed. It could inhibit the growth of most fungal species with different strength, but the antibacterial activity was weak. For Candida tropicalis, Candida guilliermondii, Aspergillus terreus, Fusarium solani and Trichoderma harzianum, the inhibition zone was biggest among all the tested drugs. The antifungal activity for Dermatophytes and Candida albicans was moderate. Two assays had a degree of consistency. Based on results above, we identified the antifungal spectrum of 3% clioquinol cream was broad. The antimicrobial strength of 3% clioquinol cream depended on the species but it can act on most of the species.

C 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: 3% clioquinol Topical pharmaceuticals Modified agar diffusion assay Drug susceptibility

Introduction Skin and soft tissue infections (SSTIs) are very common in dermatology with severity ranging from mild, self-limited to lifethreatening infection. Among these diseases, bacterial infection with Staphylococcus aureus and b-hemolytic Streptococcias [1,2] the most common pathogens is most common. Besides Staphylococcus epidermidis and Propionibacterium acnes are associated with occurrence of acne vulgaris [3,4]. Dermatomycosis can be caused by yeast or filamentous fungi. For filamentous fungi, Dermatophytes are pathogens of tinea capitis, tinea pedis and etc. Aspergillus can lead to onychomycosis and otomycosis while Fusarium, Sporothrix complex often cause subcutaneous tissue infection. As for yeasts, Candida spp. especially C. albicans which can lead to oral candidiasis, candidal intertrigo etc. [5–8]. Lipophilic yeast Malassezia leads to superficial

* Corresponding author. E-mail address: [email protected] (Y. Ran).

skin mycosis such as pityriasis versicolor, Malassezia folliculitis and is related to acne, atopic dermatitis, psoriasis and eczema [9,10]. When treating these diseases, topical antimicrobials are often used and sometimes combined with oral formulations. Recently years, topical antifungal drugs including azoles and allylamines are widely used. As for topical antibiotics, mupirocin and fusidic acid both are highly effective. And azoles can also inhibit growth of some gram-positive bacterium [11,12]. Complex compounds like antimicrobials acting at different targets or antimicrobials plus glucocorticoids to reduce the adverse effects and/or enhance effect are commonly used. Accompanied by antibiotic abuse, drug-resistant strains such as methicillin-resistant S. aureus (MRSA), community-associated MRSA (CA-MRSA), macrolide-resistant Streptococci become more and more common [13–17]. Azoles or allylamines resistant fungi are also common though less than drug-resistant bacterias [18–22]. In this circumstance, lots of researchers want to develop new antimicrobials or use other ways to enhance the effectiveness and/ or reduce the drug resistance. Biocides are broad-spectrum chemical agents that inactivate microbes. When they are used

https://doi.org/10.1016/j.mycmed.2018.03.007 C 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc1156-5233/ nd/4.0/).

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on living tissue, they are usually referred to as antiseptics. Clioquinol is one of antiseptics that the exact chemical composition is 5-chloro-7-iodo-8-quinolinol. It can inhibit the growth of C. albicans and some species of bacteria based on study before [23,24]. And targets of clioquinol may be different from common formulations used now. When screening chemical monomers with antimicrobial activity, a series of pure products of different concentrations need to be tested for its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MFC) to determine its antimicrobial activity. However, marketed topical skin pharmaceuticals used in clinical work are mixture of bulk drug and matrix. Based on reasons above, the antimicrobial activity of marketed pharmaceutical products needs to be evaluated again. In this study, we aimed to use modified agar diffusion assay to investigate the antimicrobial properties of currently available antifungal, antibacterial and complex compounds marketed formulations. We investigated antifungal spectrum and activity of 3% clioquinol (CQ) compared with 1% terbinafine hydrochloride (TBF), 2% ketoconazole (KTZ), 1% bifonazole (BFZ), 0.1% triamcinolone acetonide and 1% econazole nitrate (TE), 1% naftifine and 0.25% ketoconazole (NK). Meanwhile, we compared antibacterial spectrum and activity of CQ with TE, 2% mupirocin (MP) and 2% fusidic acid (FA). One percent mometasone furoate (MF) without antimicrobial activity was used as negative control. Meantime, we used broth microdilution assay according to Clinical and Laboratory Standards Institute (CLSI) M38-A2 and M27-A3 to test antifungal activity for Candida spp. and Dermatophytes. Materials and methods Topical skin pharmaceuticals and antifungal agents We compared 3% clioquinol (Keailin1) (Jinyao, Tianjin, China) with other antimicrobial formulations. All the topical skin pharmaceuticals within the validity period were bought from state drugstores in Chengdu, China and stored at 4 8C until use. Allylamines: 1% terbinafine hydrochloride (Lamisil1) (Novartis, Nyon, Switzerland); azoles: 2% ketoconazole (Daktarin Gold1) (Xian Janssen, Xian, China), 1% bifonazole (Mycospor1) (Bayer, Beijing, China); antibacterial formulations: 2% mupirocin (Bactroban1) (SmithKline & French, Tianjin, China), 2% fusidic acid (Aoluo1) (Bright future, Hongkong, China); corticosteroid formulation: 1% mometasone furoate (Eloson1) (Schering-Plough, Shanghai, China); complex compounds: 0.1% triamcinolone

acetonide and 1% econazole nitrate (Pevisone1) (Xian Janssen, Xian, China), 1% naftifine and 0.25% ketoconazole (Biliang1) (Huapont, Chongqing, China). All the antifungal agents (clioquinol, terbinafine, ketoconazole, bifonazole, triamcinolone acetonide, econazole, natifine, fluconazole) were obtained as standard powders from Sigma-Aldrich, Germany. Complex compounds were mixed as the formulation. Microbes All the strains from clinical patients were identified by morphology and molecular biology method. Lab conserved strains and standard strains were stored in 80 8C. All the strains used in the study are listed in Tables 1 and 2. Candida parapsilosis ATCC22019 was used as quality control strain in broth microdilution assay. Culture media The preparation of Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), brain heart infusion (BHI), serum-rice flour agar, modified Lemming-Notmann agar (mLNA) were accorded to the article published by our team [25–28]. Blood plate, MuellerHinton agar (MH), Gifu anaerobic media (GAM) and MOPS were bought form Barebio, Hangzhou, China. RMPI 1640 was brought from Gibco, Carlsbad, USA. Modified agar diffusion assay [25–29] Strain activation Malassezia was cultivated in mLNA and Sporothrix globosa yeastform was cultivated in BHI at 35 8C for 7 days. Trichophyton rubrum was cultivated in PDA at 28 8C for 10 days. Other fungus species were cultivated in SDA at 28 8C for 7 days. S. Aureus, S. epidermidis and Escherichia coli were cultivated in blood plate at 37 8C for 3 days. P. acnes was cultivated in GAM at 37 8C for 5 days (using bio-bag (Mitsubishi, Japan) to create anaerobic environment). Culture media preparation Put 80 mL melt culture solution containing 2% agar into 150 mm-diameter culture dish to make SDA, mLNA, BHI, serumrice flour agar plates. Use 80 mL MH melt culture solution containing 4% agar to make MH plate. When making GAM plate, we used 90 mm-diameter culture dish with the same procedure as others.

Table 1 Fungus species and culture medium used in experiment. Isolates

Amount

Amount

Culture medium

Source

Culture medium

Candida spp.

C. albicans

3 strains

Candidal intertrigo, ATCC10231

Serum-rice flour agar Sabouraud dextrose agar (SDA)

Dimorphous fungus

C. tropicalis C. krusei C. guilliermondii M. furfur M. globosa M. sympodialis T. rubrum T. interdigitale M. canis S. globosa

3 3 1 2 2 1 3 3 3 2

Fusarium Aspergillus Trichoderma

F. solani A. terreus T. harzianum

1 strain 2 strains 1 strain

Mycelium Yeast Lab conserved strains, ATCC1369 Tinea pedis, ATCC6258 Candidal granuloma CBS1878, CBS1870 CBS9579, lab conserved strain CBS9593 Tinea pedis, ATCC28188 Tinea pedis, ATCC28185 Tinea capitis Mycelium Yeast Chronic ulcer Otomycosis Lymphoma with tinea pedis

Malassezia

Dermatophytes

strains strains strain strains strains strain strains strains strains strains

Modified Lemming-Notmann (mLNA)

Sabouraud dextrose agar (SDA)

Sporotrichosis Brain heart infusion (BHI) Sabouraud dextrose agar (SDA)

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Table 2 Bacteria species and culture medium used in experiment. Isolates

Amount

Source

Culture medium

S. aureus S. epidermidis E. coli P. acnes

2 1 1 1

ATCC25923, lab conserved strain CMCC26069 ATCC25922 Lab conserved strain

Mueller-Hinton agar (MH)

strains strain strain strain

Microbe suspension preparation Dissolve sesame-size colony in 1 mL sterilized normal saline, mix uniformity and adjust the concentration to appropriate range. Plate containing microorganism preparation Sterilized cotton swab that was dipped in microbe suspension wiped the suspension in the culture plates uniformity. The culture plate for Malassezia spp., S. globosa yeast-form, C. albicans mycelium-form was mLNA, BHI, serum-rice flour agar respectively. Other fungus species used SDA plates. As for bacteria, the plate used for P. acnes was GAM while other bacteria used MH plate. Punch and add sample Use 7 mm diameter-puncher to punch at the plates containing microorganism. Take the agar in hole out and add the topical cream sample including CQ, TBF, KTZ, BFZ, TE, NK, MF for fungus plate and CQ, MP, FA, TE, MF for bacteria plate. Each experiment used the newly opened drug tube. Make the sample and the edge of hole at the horizontal line and do not make the sample contaminate the plate outside the hole.

Gifu anaerobic medium (GAM)

Inoculum preparation The colonies were suspended in sterile saline water. A yeast stock suspension concentration was adjusted to (1  5)  106 cells per mL and working suspension concentration was (1  5)  103 per mL diluted by RMPI 1640 broth medium. As for Dermatophytes, numerical value was (1  5)  106 and (1  5)  104 per mL respectively. Antifungal assay Perform the test by using multiwall microdiulution plates (96-U shaped wells). Dispense the two times drug concentrations into the wells of Row 1 to 10 of the microdiultion plates in 100 mL volumes. Row 1 contains the highest drug concentration. Hundred and 200 mL RMPI 1640 broth medium was added into Row 11 and 12 respectively. Finally, 100 mL working suspension was added to each well from Row 1 to 11. Thus, the final solution concentration was 0.25 to 64 mg/mL and final suspension concentration was (0.5  2.5)  103 and (0.5  2.5)  104 cells per mL for yeast and Dermatophytes respectively. Microplates were incubated at 35 8C in humid incubator. Each strain tested three times.

Cultivation condition The temperature for Malassezia, S. globosa yeast-form, C. albicans mycelium-form and bacteria was 37 8C. Other fungus species were cultivated at 28 8C. P. acnes were cultivated in anaerobic environment using the bio-bag (Mitsubishi, Japan) while all the other isolated were aerobic microorganisms. The time for fungus and bacteria was 7 days and 3 days respectively.

Results reading Yeast was read after 48 h and Dermatophyptes were read after 96 h. MIC50 and MIC80 were used to evaluate the antifungal activity for yeast and Dermatophytes respectively.

Results observation Observe the inhibition zone around the hole and record the diameter (mm) or radius (mm) (when the edge of inhibition zone was exceeded the edge of plate). The final diameter was the average of maximum value and minimum value (when measuring radius, the final figure needed to multiply two to get the diameter). The experiment for the same strain was repeated twice.

Antimicrobial activity for Candida spp.

Statistical analysis Using Graphpad prism 5.0 to analyze the data. When comparing antimicrobial activity of different formulations for the same fungus species, ANOVA was used firstly and then Dunnett-t method was used to compare 3% clioquinol with other products. Statistical significance was set at P  0.05. Broth microdilution assay [30,31] Solution preparation A stock solution of antifungal agents 12.8 mg/mL was prepared in dimethyl sulfoxide (DMSO). The stock solutions were kept at room temperature for complete dissolving and then stored at 20 8C. The concentration of working solutions was 0.5  128 mg/ mL diluted by RMPI 1640 broth medium (with L-glutamine, without sodium bicarbonate). Strain activation Yeast was cultivated in 28 8C for 2 days and Dermatophytes was cultivated in 28 8C for 7  14 days.

Results

Modified agar diffusion assay In our study, the antifungal activity of 3% clioquinol to C. albicans yeast-form and Candia krusei was stronger than 1% terbinafine and 1% bifonazole and weaker than other drugs. When 3% clioquinol was compared with 1% terbinafine and 1% bifonazole, the differences were statistically significant. But when compared with other formulations, parts of differences were not statistically significant. Actually, the antifungal activity of formulations to C. krusei was weak except for 0.1% triamcinolone acetonide-1% econazole. As for C. tropicalis and C. guilliermondii, the activity of 3% clioquinol was strongest among all the drugs tested. Most of the differences were statistically significant except the difference between 3% clioquinol and 2% ketoconazole for C. tropicalis (Figs. 1 and 2). C. albicans has yeast and mycelium form. The mycelium form is the pathogenic form and yeast form can be transformed to it with appropriate conditions. So the inhibition zone of mycelium form may reflect the real antifungal activity better. In this form, 3% clioquinol was stronger than 1% terbinafine, 1% bifonazole and 2% ketoconazole and equal to the rest of drugs (Figs. 1 and 3). Broth microdilution assay The antifungal activity of clioquinol to C. albicans yeast-form was stronger than bifonazole and terbinafine and weaker than other drugs. Different from the results of modified agar diffusion assay, the antifungal activity of drugs to C. krusei was moderate to

Please cite this article in press as: You Z, et al. Clioquinol, an alternative antimicrobial agent against common pathogenic microbe. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.007

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Fig. 1. The inhibition zone of yeast to 3% clioquinol and other antifungal formulations. CQ: 3% clioquinol, TBF: 1% terbinafine, KTZ: 2% ketoconazole, BFZ: 1% bifonazole, TE: 0.1% triamcinolone acetonide-1% econazole, NK: 1% naftifine-0.25% ketoconazole, MP: 1% mometasone furoate.

strong except for terbinafine and bifonazole (fluconazole is naturally resistant to C. krusei). As for C. tropicalis and C. guilliermondii, the activity of clioquinol was similar or stronger than other antifungal agents (Table 3). Antimicrobial activity for Malassezia The inhibition zone and diameter of different formulations to Malassezia can be seen in Figs. 1 and 2 respectively. For Malassezia furfur, the antifungal activity between 3% clioquinol and 1% bifonazole, 0.1% triamcinolone acetonide-1% econazole respectively was no significant statistical difference. While comparing 3% clioquinol with 1% terbinafine, 2% ketoconazole, 1% naftifine-0.25% ketoconazole respectively, the antiM. furfur activity has significant statistical differences. But considering the absolute diameter of the inhibition zone, only the difference between 3% clioquinol cream and 2% ketoconazole was clinically significant. The anti-M. furfur activity of 2% ketoconazole was strongest. For Malassezia globosa, only the difference between 3% clioquinol and 0.1% triamcinolone acetonide-1% econazole was

no statistical significance. Still according to the absolute diameter, the difference between 3% clioquinol and 2% ketoconazole, 1% naftifine-0.25% ketoconazole respectively was meaningful in clinical work. For Malassezia sympodialis, the antifungal activity of 3% clioquinol cream was weaker than 2% ketoconazole cream, 1% naftifine-0.25% ketoconazole respectively and the difference was both statistically and clinically significant. While comparing with other formulations, the differences were no significance either in statistics or clinical work. Antimicrobial activity for Dermatophytes Modified agar diffusion assay For T. rubrum, 3% clioquinol was stronger than 2% ketoconazole and 1% bifonazole and weaker than 1% terbinafine and 1% naftifine0.25% ketoconazole, all the differences were significant in statistics. The antifungal activity was equal to 0.1% triamcinolone acetonide-1% econazole. As for Trichophyton interdigitale complex, 3% clioquinol was stronger or equal to other formulations except for 1% terbinafine and 1% naftifine-0.25% ketoconazole. Micro-

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Fig. 2. The diameter of inhibition zone (mm) for Candida spp., Malassezia, Dermatophytes, Fusarium, Aspergillus and Trichoderma to 3% clioquinol and other antifungal formulations.

sporum canis is often seen in tinea capitis. For this species, the antifungal activity of 3% clioquinol has no advantage over other formulations. It was just stronger than 1% bifonazole, equal to 2% ketoconazole and weaker than other formulations (Figs. 2 and 4). Actually, the strength of 1% terbinafine and 1% naftifine-0.25% ketoconazole to Dermatophytes were strongest among all the formulations tested. Three percent clioquinol had moderate antiDermatophytes activity. Broth microdilution assay For T. rubrum, clioquinol was stronger than ketoconazole and bifonazole and weaker than terbinafine, naftifine-ketoconazole, triamcinolone acetonide-econazole. As for T. interdigitale complex, clioquinol was equal or stronger to ketoconazole ketoconazole and bifonazole, but weaker than other formulations. The results of M. canis was similar to T. interdigitale complex (Table 3).

Antimicrobial activity for dimorphic fungus For mycelium form, the antifungal activity of 3% clioquinol was stronger than other formulations except for 1% naftifine-0.25% ketoconazole and the differences were statistically significant. Actually, the strength of 3% clioquinol, 1% terbinafine, 0.1% triamcinolone acetonide-1% econazole and 1% naftifine-0.25% ketoconazole were similar in clinical work considering the absolute diameter. For yeast form, the results were similar to the mycelium form (Figs. 3 and 4).

Antimicrobial activity for Aspergillus, Trichoderma, Fusarium The inhibition zone and diameter of different formulations to Aspergillus, Trichoderma, Fusarium can be seen in Figs. 3 and 4 respectively.

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Fig. 3. The diameter of inhibition zone (mm) for C. albicans and S. globosa to 3% clioquinol and other antifungal formulations. *: when compared with 3% clioquinol, the difference was statistically different (P < 0.05).

Table 3 Minimum inhibitory concentration (MIC) for Candida spp. and Dermatophytes. MIC range (mg/mL)

CQ

TBF

KTZ

BFZ

TE

NK

FLC

C. albicans C. tropicalis C. krusei C. guilliermondii T. rubrum T. interdigitale M. canis

2  2.4 0.8  1 24 0.75 1.3  4.8 1.3  3.3 2.8  4.3

> 64 > 64 8 > 64 1.6  2 0.67  1.6 0.5  1.5

12 0.25  4 0.25  1 0.75 48 2.25  4 3 $4

8  21.33 > 64 64 > 64  64  64 64

0.25 0.25  4 0.25 2 0.25  4 0.21  2.3 1.2  1.7

0.5  2 0.5  0.9 0.5  1 3 0.25  3 0.42  0.83 0.6  1.3

0.25  0.5 0.125  0.5 / 8 53.3  64 32  64 53.3

MIC: minimum inhibitory concentration, using MIC50 and MIC80 to evaluate antifungal activity of Candida spp. and Dermatophytes respectively. CQ: clioquinol; TBF: terbinafine; KTZ: ketoconazole; BFZ: bifonazole; TE: triamcinolone acetonide-econazole; NK: naftifine-ketoconazole; FLC: fluconazole.

For A. terreus, the antifungal activity between 3% clioquinol and 1% terbinafine, 0.1% triamcinolone acetonid-1% econazole, 1% naftifine-0.25% ketoconazole cream respectively was no significant statistical difference. All the antifungal activities of formulations above were strong. While comparing 3% clioquinol with 1% bifonazole or 2% ketoconazole, the anti-A. terreus activity has significant statistical differences. For F. solani, the antifungal activity of 3% clioquinol and 0.1% triamcinolone acetonid-1% econazole was strongest and the difference between 3% clioquinol and other formulations except for 0.1% triamcinolone acetonid-1% econazole was statistically significant. Trichoderma infection is rare; the strain was isolated from a female in immunosuppression state with lymphoma and tinea pedis. The difference between 3% clioquinol and all the other formulations was statistically significant. And the activity of 3% clioquinol was strongest among all the tested formulations. Antimicrobial activity for bacteria For S. aureus, the antibacterial activity of 3% clioquinol was weaker than 2% mupirocin and 2% fusidic acid respectively and the difference was statistically significant. The difference between 3% clioquinol and 0.1% triamcinolone acetonid-1% econazole was no statistical significance. For S. epidermidis, the antibacterial activity of 3% clioquinol was weaker than all the other formulations and the differences were statistically significant. For E. coli, the antibacterial activity of 3% clioquinol was equal to 0.1% triamcinolone acetonid-1% econazole and 2% fusidic acid. The differences were both statistically significant. The antibacterial activity of 3%

clioquinol was weaker than 2% mupirocin. As for P. acnes, the activity of 2% fusidic acid was strongest as we expected. The antibacterial activity of 3% clioquinol was as weak as 2% mupirocin. And 0.1% triamcinolone acetonid-1% econazole could not inhibit the growth of P. acnes (Figs. 5 and 6). Discussion Skin and soft tissue infections (SSTIs) are very common in dermatology. S. aureus and b-hemolytic Streptococci are most common species in bacteria infection while C. albicans, T. rubrum are most common species in fungus infection. With the increasing of drug-resistant strains, the need of new antimicrobials acting on different targets with common used drugs is urgent. Allylamines and azoles both are common used antifungal formulations. Antiseptics represent another group of topical antimicrobials that are used in modern healthcare and in consumer products. Antiseptics have a broader spectrum of activity than antibiotics and often act on multiple cellular targets. And clioquinol is one of it. It is worthy evaluating the antimicrobial activity of clioquinol among common pathogenic microorganisms. Clioquinol was firstly produced in 1934 as a topical antiseptic and marketed as an oral intestinal amebicide, being used to treat a wide range of intestinal diseases including lambliasis, shigellosis, chronic nonspecific diarrhea and traveller’s diarrhea. In the early 1970s, it was withdrawn from the market as an oral agent because of its association with subacute myelo-optic neuropathy (SMON). There is still controversy concerning the association between clioquinol and SMON, which was extremely rare outside Japan. The reason for the neurological side effect among Japanese is

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Fig. 4. The inhibition zone of filamentous fungi to 3% clioquinol and other antifungal formulations. CQ: 3% clioquinol; TBF: 1% terbinafine; KTZ: 2% ketoconazole; BFZ: 1% bifonazole; TE: 0.1% triamcinolone acetonide-1% Econazole; NK: 1% Naftifine-0.25% ketoconazole; MP: 1% mometasone furoate.

unknown, but it has been related to concomitant vitamin B deficiency [32–35]. In 2000, researchers found that clioquinol can be a method to treat Alzheimer’s disease as a metal chelator [35]. Then lots of experiments confirmed it can treat Alzheimer’s disease and inhibit tumors growth as metal chelator by inducing apoptosis, autophagy and cell cycle arrest [35–42]. In dermatological use topical clioquinol can cause skin irritation if concentration is high, but it is not a common contact allergen and has not been associated with the development of skin malignancies [24]. There were very few reports about the evaluation of antimicrobial activity. Alsterholm M et al. reported antimicrobial activity of combined corticosteroid and clioquinol preparation against C. Albicans, E. Coli, S. Aureus, S. epidermidis and Streptococcus pyogenes [24]. But there was no report about in vitro antimicrobial activity evaluation of clioquinol against common skin and soft tissue pathogenic microorganisms including yeast, filamentous fungi and bacteria. The standard method for drug susceptibility tests is MIC and MFC of chemical crude drugs made by CLSI. But it also has some

limitations. By now, drug susceptibility test for yeast just included Candida spp. and Cryptococcus neoformans. Meanwhile, breakpoint data from oropharyngeal candidiasis and invasive infection was not enough for all the antifungal drugs and all settings. For Dermatophytes, it may be hard to reach to spores concentration CLSI needed. Modified agar diffusion assay that is extensively used in our research laboratory and is carefully standardized [25–28] could be a supplement for broth microdilution methods. It can be used in both drug susceptibility test for antifungal powders and marketed pharmaceutical products even complex compounds. Lots of infectious dermatoses do not need oral formulations, using this assay to evaluate topical antimicrobial formulations is easy to achieve. Meanwhile, it can test the antifungal activity to some species of fungi like Malassezia spp. and dimorphic fungi yeastform that are not included in CLSI and antibacterial activity. Results reading is easier to achieve compared to broth microdilution methods. The agreement between different person and laboratory and data of different species need more study to confirm and achieve.

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Fig. 5. The inhibition zone of bacteria to 3% clioquinol and other antibiotics. CQ: 3% clioquinol, FA: 2% fusidic acid, MP: 2% mupirocin, TE: 0.1% triamcinolone acetonide-1% econazole, MP: 1% mometasone furoate.

Fig. 6. The diameter of inhibition zone (mm) for bacteria to 3% clioquinol and other antibiotics. *: when compared with 3% clioquinol, the difference was statistically different (P < 0.05).

In this study, we investigated in vitro antimicrobial activity of a number of topical skin pharmaceuticals widely used in dermatologic work and compared with 3% clioquinol cream using the modified agar diffusion assay. In our results based on modified agar diffusion assay, clioquinol can inhibit the growth of great majority fungi. For some species including C. albicans mycelium-form, C. tropicalis, C. guilliermondii, F. solani and T. harzianum, the antifungal activity of 3% clioquinol was strongest among all the topical skin pharmaceuticals. For S. globosa mycelium and yeast form, A. terreus, 3% clioquinol along with other formulations such as 1% natfitine-0.25% ketoconazole had the strongest antifungal activity. As for Dermatophytes and C. albicans yeast-form, 3% clioquinol had moderate strength against the fungus and was weaker than some common topical antifungal formulations such as 1% terbinafine and 1% natfitine-0.25% ketoconazole. The antifungal activity of 3% clioquinol against Malassezia was weak, though other topical skin pharmaceuticals except for 2% ketoconazole and 1% natfitine-0.25% ketoconazole cream were also weak. The strength of formulations tested for C. krusei was also weak except for 0.1% triamcinolone acetonid-1% econazole. As for the results based on broth microdilution assay,

most data were similar to the results above. Modified agar diffusion assay is used to test activity of marketed pharmaceutical products while broth microdilution assay is used to test antifungal powder. Based on our results, these two assays had a degree of consistency, which should do more experiment to confirm. Allylamines and azoles both can inhibit the synthesis of ergosterol through squalene epoxidase and 14a-sterol demethylase respectively. According the anti-tumor and anti-Alzheimer’s disease mechanism of clioquinol identified by other researchers, it may be related to the ability to chelate metal and then induce apoptosis, autophagy and cell cycle arrest [35–42]. The targets of clioquinol may be different with allylamines and azoles. Clioquinol was one of antiseptics, antiseptics have broad antimicrobial effects and can be suitable candidates for novel drugs. Researchers have previously explored the antimicrobial properties of pentane1, 5-diol (one of antiseptics) in vitro and in vivo with promising results [43–46]. Since antiseptics usually target several different cellular mechanisms they are less likely to promote resistance, although it is important to note that there are reports of resistance development [23]. Base on the results of our study and reasons above, clioquinol can be a promising formulation in topical use of superficial skin mycosis. It also can be combined with systematic antifungal formulations to reduce the occurrence of drugresistance and enhance the effect. It has broad antifungal spectrum and moderate to strong activity among common pathogenic fungus. As for other topical skin pharmaceuticals tested in the study, 1% terbinafine that belongs to allylamines had strong activity among Dermatophytes, A. terreus and weak activity among yeast. Unlike 1% terbinafine, 2% ketoconazole that belongs to azoles had strong activity among yeast especially Malassezia and weak or moderate activity among Dermatophytes. The antifungal activity of 1% bifonazole was weak among almost all the species, though it belongs to the same type of drugs as 2% ketoconazole. The antifungal spectrum and activity of 1% natfitine-0.25% ketoconazole was stronger than 1% terbinafine, 2% ketoconazole and 1% bifonazole. The result was consistent with the study before [25]. The effective constituent of 0.1% triamcinolone acetonide-1% econazole is econazole that belongs to azoles. But the antifungal activity was stronger than 2% ketoconazole especially against Dermatophytes and F. solani.

Please cite this article in press as: You Z, et al. Clioquinol, an alternative antimicrobial agent against common pathogenic microbe. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.007

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MYCMED-788; No. of Pages 10 Z. You et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx

Talking about bacteria, our results were very different with Alsterholm M et al. [24]. Among the species we investigated, the anti-bacterial activity of 3% clioquinol was very weak. But according to the results of Alsterholm M et al., the activity against S. Aureus, S. Epidermidis, E. coli was strong. As the results of our team and other researchers were opposite, the antibacterial activity of clioquinol needs more data to confirm. As for other antibacterial formulations, it is known that member of the azole class possesses antibacterial properties against some gram-positive bacteria. These bacteria contain P450 monooxygenases (P450s), some of which are homologues to 14a-sterol demethylase, the target enzyme of azoles. Our results showed 0.1% triamcinolone acetonid-1% econazole nitrate also inhibited growth of gram-negative E. coli though the activity was weak. Mupirocin has been used for longtime, the activity was strong among most species except for P. acnes. And fusidic acid could inhibit growth of all the species tested and had advantage over mupirocin among P. acnes. So mupirocin can be used in common bacterial skin infection while fusidic acid is always used in acne. In vitro study has to face a problem that the circumstance in skin can be influenced by factors like pH, temperature and salt concentrations. In this investigation, we used modified agar diffusion assay to try to simulate the circumstance of use of marketed topical skin pharmaceuticals in patients. Unlike the standard way of drug susceptibility test, we investigate the marketed pharmaceutical products, which are more close to the circumstances using in patients. This study obtained data of antimicrobial spectrum and activity of 3% clioquinol cream against common pathogenic microorganisms for the first time. The data can be important for the management of superficial skin infection and also highlights the need for new, topical, non-resistance promoting, antimicrobial treatment alternatives for skin infections. It also provides powerful evidences for use of 3% clioquinol cream in dermatology. Though this investigation has included most common pathogens in skin and soft tissue infection in dermatology, there are still species that are not tested. Meanwhile, some species were lack of the results of standard strains due to multiple reasons, more experiments need to complete to get more data. More strains need to be enrolled into experiments. Even though we used the marketed products to test, there is still gap between the results of in vitro study and in vivo use. And the targets of clioquinol to pathogenic microorganisms need more experiments to find and identify. With the fact that the increase of drug-resistance strains, development of new antimicrobial topical preparations is a significant topic for clinical doctors and researchers. This study provided evidences for the use of common skin topical pharmaceuticals. Clioquinol cream as antiseptic formulation could inhibit growth of most species of common pathogenic microorganisms. It still needs more data (clinical outcome, adverse effect and etc) to use 3% clioquinol in clinical work.

Author contributions Z.Y and X. Ran designed and performed experiment, analyzed data and contributed to writing the manuscript. Y.D performed experiment and contributed to the discussion. Y. Ran initiated original ideas, guided the design of experiment, data analysis and discussion and revised the manuscript.

Disclosure of interest The authors declare that they have no competing interest.

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Acknowledgements This study was supported by Tianjin Tianyao Pharmaceuticals Technology Co. Ltd, China.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://www.sciencedirect.com and https:// doi.org/10.1016/j.mycmed.2018.03.007.

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Please cite this article in press as: You Z, et al. Clioquinol, an alternative antimicrobial agent against common pathogenic microbe. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.007