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Keywords:

  • anti-Malassezia;
  • in vitro;
  • Malassezia furfur;
  • Malassezia pachydermatis;
  • xanthorrhizol

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  This study aimed at investigating the anti-Malassezia activity of xanthorrhizol (XTZ) isolated from Curcuma xanthorrhiza Roxb. against Malassezia furfur ATCC 14521 and Malassezia pachydermatis ATCC 14522.

Methods and Results:  The in vitro susceptibility tests for XTZ were carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), using broth microdilution method with endpoint after 48 h. Time-kill curves were determined at concentrations ranging from 0 to 25 μg ml−1. The MIC values of XTZ against M. furfur and M. pachydermatis were 1·25 and 0·25 μg ml−1, respectively. The MFC of XTZ was 5 μg ml−1 for M. furfur and 2·5 μg ml−1 for M. pachydermatis. Time-kill curves demonstrated that treatment with 25 μg ml−1 of XTZ for 5 h was able to kill 100% of M. furfur, while 20 μg ml−1 of XTZ for 15 min killed M. pachydermatis completely.

Conclusion:  XTZ shows potential as an anti-Malassezia agent for inhibiting the growth of M. furfur ATCC 14521 and M. pachydermatis ATCC 14522 in vitro.

Significance and Impact of the Study:  XTZ may be a useful alternative for treating Malassezia-associated diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Yeasts of the genus Malassezia are known to be the members of the skin microflora of human and other warm-blooded vertebrates, and they have received considerable attention in recent years from dermatologists, other clinicians, veterinarians and mycologists (Batra et al. 2005). Malassezia species may have a role in several cutaneous and systemic diseases, either as the causative agent of these conditions or associated with lesion (Ashbee 2006). Diseases with which Malassezia is associated include pityriasis versicolor, seborrhoeic dermatitis, seborrhoeic blepharitis, dandruff, folliculitis, atopic dermatitis, confluent reticulate papillomatosis and psoriasis (Kellett and MacDonald 1985; Bergbrant et al. 1991; Batra et al. 2005). These yeasts can also become opportunistic pathogens. Malassezia species have been reported as significant causes of fungal infections in patients with cancer and AIDS (Gabal 1998; Gupta et al. 2000).

Interest in the organism of Malasseziafurfur has increased considerably in recent years, as this yeast has been implicated as the primary cause of the scalp disease known as seborrhoeic dermatitis or dandruff (Bulmer and Bulmer 1999). More than 50% of the population in the world suffers from some level of dandruff and seborrhoeic dermatitis (Warner et al. 2001). It has also been recovered in blood cultures from neonate and adult patients undergoing lipid replacement therapy. Malasseziapachydermatis is a lipophilic yeast that has been shown to be a commensal inhabitant on the skin of normal dogs, cats and other animals. It can, however, become an opportunistic pathogen owing to the alterations on the skin surface environment and host defence (Rosales et al. 2005).

Plant extracts are promising sources for new natural antifungal drugs, even though they have relatively mild effect against human pathogenic fungi compared with commercial synthetic antifungal drugs (Hammer et al. 1999; Faleiro et al. 2003). Furthermore, a dramatic increase in fungal infections has been observed in the last decade, which may be attributed to the augmentation of the number of immunocompromised and immunosuppressed patients, more susceptible to opportunistic systemic and superficial mycoses (Rahalison et al. 1994). Fungal resistance to synthetic antibiotics in clinical use is rising, and it often develops rapidly (Metzger and Hoffmann 1997; Rocha et al. 2004). Given this problem, the potential enhancement of natural antifungal should be investigated.

In our previous reports (Hwang et al. 2000a,b; Rukayadi and Hwang 2006a,b; Rukayadi et al. 2006), xanthorrhizol (XTZ), a bioactive compound isolated from an edible medicinal plant, temulawak or java turmeric (Curcuma xanthorrhiza Roxb.), has been shown to possess bactericidal activity against oral bacteria, anticandidal activity, and also the ability to prevent and remove Streptococcus mutans biofilms. In this study, we determined the anti-Malassezia activity of XTZ against M. furfur and M. pachydermatis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Malassezia strains, growth conditions and inoculum preparation

Two strains of Malassezia, M. furfur ATCC 14521 and M. pachydermatis ATCC 14522 were obtained from the American Type Culture Collection (Rockville, MD, USA). These strains were grown on Sabouraud Dextrose Broth (SDB) or Sabouraud Dextrose Agar (SDA) (Difco, Sparks, MD, USA) supplemented with 1% (v/v) of pure olive oil (Yakuri Pure Chemicals, Kyoto, Japan) for M. furfur and SDB or SDA for M. pachydermatis, following incubation at 35°C during 2–7 days. Malassezia strains were maintained on the same medium described previously, at 4°C, with subcultures being carried out on a monthly basis. The same medium was used in all the experiments.

Inoculum suspensions were prepared by the method as described previously (Rukayadi et al. 2006). One millilitre of 48-h culture was centrifuged (3000 g at 4°C for 1 min), followed by washing the pellets twice with 1 ml of 50 mmol l−1, pH 7·0 phosphate buffered saline (PBS). Clusters of Malassezia cells were formed upon preparation of inoculum suspensions. The washing of these suspensions with PBS promotes single-cell status and more accurate turbidity measurements. A standard inoculum (a McFarland standard) for each isolate was 5 × 106 CFU ml−1.

Anti-Malassezia agents

XTZ was isolated in the pure form from the ethylacetate fraction on the methanol extract of C. xanthorrhiza Roxb., according to the method of Hwang et al. (2000b)). XTZ was dissolved in 10% dimethylsulfoxide (DMSO) to obtain stock solutions. It was found that DMSO at 10% did not kill the Malassezia. Zinc pyrithione (ZPT) (Dongsan Clean & Green, Seoul, Korea) was dissolved in sterile distillated water to obtain stock solution. The ranged concentration of individual antifungal agents was 1–100 μg ml−1. All the solutions prepared could be used freshly prior to experiments, or stored at −20 to −70°C until used.

In vitro susceptibility tests

In vitro susceptibility tests were performed to evaluate minimum inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) using broth microdilution method, as described in the guidelines of National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 (NCCLS 2002). However, the standardized RPMI-1640 medium does not support the growth of the Malassezia yeast cells, because it lacks the specific lipids they require (Gupta et al. 2000). SDB, SDA, SDBO (SDB supplemented with 1% olive oil) and SDAO (SDA supplemented with 1% olive oil) media were used for susceptibility testing in this study. In this study, we did not use fatty acid RPMI 1640 media (RPMI 1640, supplemented with fatty acids, bile salt lipids, oleic acid, Tween 20 and the n-octadecanoate fatty acid glycerol monostearate), a formulated media for testing the susceptibilities of Malassezia species (Velegraki et al. 2004). Sabouraud dextrose media is a rich media for the cultivation of yeasts, including Malassezia, and olive oil is a complex compound made of fatty acids, including oleic and linoleic acid, vitamins, volatile components, water-soluble components and microscopic bits of olive (Kaneko et al. 2005). Malasseziapachydermatis is nonlipid-dependent Malassezia and therefore, its growth can be supported by media with or without lipids. It is known that it can also grow in SDB or SDA while, M. furfur is the least lipid demanding Malassezia as far as lipid supplementation of media is concerned, and therefore, in most cases, growth can be supported in SDBO or SDAO. Briefly, MIC were determined for all species with the adjusted inoculum suspension of 5 × 106 CFU ml−1 (a McFarland standard) by diluting 1 : 10 with SDBO or SDB medium to make a final inoculum concentration of 5 × 105 CFU ml−1. Individual antifungal agents were diluted to 1 : 10 in SDBO or SDB medium containing 5 × 105-CFU ml−1 inoculum, yielding an initial inoculum of 4·5 × 105 CFU ml−1. The final range of concentration of individual antifungal agents was 0·1–10 μg ml−1. A 200-μl aliquot of each suspension was placed in 96-well round-bottom microtitration plates. The plates were incubated at 35°C, and endpoints were read visually after 48 h. The MIC was defined as the corresponding concentrations required for inhibiting the growth of the species relative to the controls grown in the absence of the antifungal agents. MFC were determined for each anti-Malassezia using the method described by Rukayadi et al. (2006).

Time-kill curves

Time-kill curves were constructed using the method as described previously (Rukayadi et al. 2006) in SDHO and SDH medium. The inoculum was adjusted spectrophotometrically to the density of a McFarland standard, 5 × 106 CFU ml−1. The final XTZ or ZPT concentrations were 0, 1, 5, 7·5, 10, 15, 20 and 25 μg ml−1. Predetermined time points were 0, 5, 15, 30, 45 min, 1, 2, 3, 4, 5, 6, 12, 16, 20 and 24 h. The experiment was repeated two times with two replicates per experiment.

Statistical analysis

Fisher's exact tests were applied to contingency tables (2 × 2) to compare the effectiveness of XTZ and ZPT in killing M. furfur and M. pachydermatis. A P value of less than 0·05 was taken as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

MIC and MFC of XTZ to M. furfur and M. pachydermatis in comparison with those of ZPT are summarized in Table 1. The killing activity of XTZ and ZPT for each species is shown in Fig. 1. Fisher's exact test was used to compare the effectiveness of XTZ and ZPT in 100% killing of M. furfur and M. pachydermatis (Table 2). To kill 100% of M. furfur, treatment with 25 μg ml−1 of XTZ for 5 h was more effective than that with 15 μg ml−1 for 12 h of ZPT (P < 0·043). For 100% killing of M. pachydermatis, treatment with 20 μg ml−1 of XTZ for 15 min was more effective than that with 20 μg ml−1 for 12 h of ZPT (P < 0·00001).

Table 1. In vitro antifungal activity of xanthorrhizol and zinc pyrithione against Malassezia furfur and Malassezia pachydermatis
YeastXanthorrhizol (μg ml−1)Zync pyrithione (μg ml−1)
MICMFCMICMFC
  1. MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration.

M. furfur ATCC 145211·2550·6255
M. pachydermatis ATCC 145220·252·50·252·5
image

Figure Figure.  Representative time-kill plots of Malassezia species following exposure to xanthorrhizol and zinc pyrithione at: 0 μg ml−1 (-bsl00046-); 1 μg ml−1 (-bsl00001-); 5 μg ml−1 (-bsl00066-); 7·5 μg ml−1 (-•-); 10 μg ml−1 (-bsl00067-); 15 μg ml−1 (-bsl00000-); 20 μg ml−1 (-bsl00084-); 25 μg ml−1 (-bsl00043-) after endpoint (24 h). (a) Xanthorrhizol against Malassezia furfur ATCC 14521; (b) Zinc pyrithione against M. furfur ATCC 14521; (c) Xanthorrhizol against Malassezia pachydermatis ATCC 14522; and (d) Zinc pyrithione against M. pachydermatis ATCC 14522.

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Table 2.   Contingency table for comparing the effectiveness of xanthorrhizol and zinc pyrithione treatment with Malassezia furfur and Malassezia pachydermatis
YeastConcentration (μg ml−1) and time (min or h) for 100% killingP value
XanthorrhizolZinc pyrithione
  1. P values were computed using the Fisher exact test. *Significantly different (P < 0·05, Fisher exact test).

M. furfur ATCC 1452125 μg ml−1 for 5 h15 μg ml−1 for 12 h0·043*
M. pachydermatis ATCC 1452225 μg ml−1 for 15 min20 μg ml−1 for 12 h0·00001*

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Recently, the potential antifungal effects of certain bioactive compounds from plants have attracted serious attention within the scientific community, largely as a result of the growing problem of multidrug resistance among pathogenic fungi (Cowan 1999; Tim Cushnie and Lamb 2005). In contrast, there are a few reports concerning the susceptibility of Malassezia to natural antifungal or anti-Malassezia agents. Rukayadi et al. (2006) have reported that XTZ has anticadidal activity. However, to our knowledge, there is no scientific information on anti-Malassezia properties of XTZ. This study emphasizes the importance of XTZ as an alternative anti-Malassezia agent against pathogenic fungi causing dandruff and other human and animal skin diseases, namely M. furfur and M. pachydermatis.

In an attempt to determine how effective XTZ may be in treating Malassezia-associated diseases, we studied the anti-Malassezia activity of XTZ against M. furfur and M. pachydermatis. ZPT was used as a positive control in comparison with anti-Malassezia activity of XTZ. ZPT has been used broadly for treating skin disease caused by Malassezia, including dandruff. Schmidt and Ruhl-Horster (1996) reported that MIC of ZPT against M. furfur was between 0·2 and 8 μg ml−1. Our result showed that MIC of ZPT against M. fufur was 0·625 μg ml−1, whereas, MIC of ZPT against M. pachydermatis was 0·25 μg ml−1 (Table 1). The susceptibility of ZPT against M. furfur and M. pachydermatis are in good agreement with previous report. The method used by Schmidt and Ruhl-Horster (1996) was different, and they used a microtitre plate assay with colorimetrically modified Leeming-Notman medium after incubation with alamarBlue. There was no previous report for anti-Malassezia of XTZ against M. furfur and M. pachydermatis. XTZ has been shown here to possess powerful in vitro activity against M. furfur and M. pachydermatis.

In vitro MFC of XTZ with endpoint after 48 h demonstrated that XTZ was able to kill the Melassezia strains with MFC of 5 μg ml−1 and 2·5 μg ml−1 for M. furfur and M. pachydermatis, repectively. These results were the same with the MFC of ZPT against those of the Malassezia strains (Table 1). Time-kill curves (Fig. 1) demonstrated that XTZ was able to kill 100% of M. furfur with 25 μg ml−1 for 5 h, whereas ZPT was able to kill 100% of M. furfur with 15 μg ml−1 for 12 h. To kill 100% of M. pachydermatis, it required 25 μg ml−1 for 5 min and 20 μg ml−1 for 12 h of XTZ and ZPT, respectively. Fisher's exact test analysis of these data showed that XTZ was more effective in killing of Malassezia strains than ZPT (Table 2).

Our findings demonstrate that XTZ has anti-Malassezia activity against M. furfur and M. pachydermatis in vitro. XTZ may be potentially valuable as a natural compound for treating Malassezia-associated diseases. However, the use of Malassezia ATCC strains mainly may not truly reflect the susceptibility of clinical isolates of Malassezia. Hence, future research is necessary to determine anti-Malassezia activity of XTZ against a range of clinical isolates.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was partly supported by the Yonsei Biomolecule Research Initiative of the BKZI project.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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