Otomycosis is defined as superficial fungal infection of the outer ear canal.1 Otomycosis has world-wide distribution, with higher prevalence in tropical and sub-tropical regions due to the hot, humid and dusty climate.2 Nevertheless studies pertaining prevalence of otomycosis remains scarce, despite its high occurrences particularly in tropical countries. Many species of fungi have been identified as causative agents of otomycosis. However, Aspergillus niger is the most commonly described agent in the literature, followed by Candida albicans. Antifungal medications vary widely, and no consensus has been derived as to which medications are most effective in otomycosis.3 Furthermore, in recent years, there are increasing antifungal drugs resistance. This drives the need to pursue for natural solution which is more effective and safe. Honey, a natural solution has been used as medicinal products since ancient times. Its antibacterial properties have been extensively documented.4 Nevertheless, studies on antifungal effects of local honeys are still limited.
In Malaysia, they are various types of local honeys available for consumptions. However, no extensive studies on their antifungal properties have been conducted.4-6 The three types of honey used in this study were selected based on their unique properties. Tualang honey is a multifloral honey, meanwhile Acacia honey is extraflorial honey, and Kelulut honey is derived from stingless bee. To the best of our knowledge, based on research and English literature review, this is the first study on the anti-fungal properties of different types of Malaysian local honey against two most common pathogenic fungi of otomycosis (A. niger and C. albicans).
MATERIALS AND METHODS
A. niger and C. albicans were obtained from the Mycology Laboratory, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia. These fungi have been isolated from patients with otomycosis. A. niger was identified based on microscopic features.7 Identification of C. albicans was based on the morphology on slide culture and biochemical characteristics by API 20C AUX (bioMerieux, France).
Tualang Honey was obtained from Federal Agriculture Marketing Authority (FAMA), Kedah, Malaysia. Meanwhile, Acacia honey and Kelulut honey were supplied by the USM-BJIM Project (Medicinal Trigonal Bee Rearing project). The honeys used in this study have been subjected to sterilization by 25kGy gamma-irradiation.
Preparation of conidial suspension
A. niger was subcultured on Sabouraud dextrose agar (SDA) (Oxoid, UK) and incubated at 30oC. Upon 48 h of incubation, the well grown sporulating colony was flooded with 5 ml of sterile water. The surface of the colony, which contained spores, was lightly scraped using sterile cotton swab. The mixture was filtered through sterile gauze to remove mycelia and debris. The filtrate containing conidia was then transferred to a sterile tube. Sterile water was added to the suspension until it reached turbidity of 0.5 MacFarland, which corresponded to 1-5 x 106 cfu/ml. C. albicans was sub cultured on SDA plate. Following 24-h incubation at 30oC, the colonies were scraped using sterile cotton swab and dipped into a sterile tube containing 3 ml of distilled water. The suspension turbidity was adjusted by adding sterile water to obtain turbidity of 0.5 MacFarland.T
Dilutions were made to produce conidial suspensions of 5x103 and 5x102 cfu/ml concentration. For each dilution, 100 ml of suspension was inoculated onto SDA for verification conidial concentration. The conidial suspension concentration of 5x103 cfu/ml was eventually used as standard inoculum concentration for this study.
Preparation of honey-incorporated culture media
Plain SDA agar plate was prepared in accordance to manufacturer’s instruction. Honey was added to SDA medium after sterilization prior to pouring into Petri dishes. The amount of honey incorporated into the medium was titrated to make up the following concentration; 5% (v/v), 10% (v/v), 15% (v/v) and 25% (v/v). Samples of prepared honey-SDA plates were incubated at 30oC for 24 h to check for contamination. All prepared plates were stored at 4oC prior to use.
Plate count procedure
Plate count procedure was performed based on previously reported study of similar purpose.8 All procedures were performed under aseptic condition. Freshly prepared conidial suspensions were vortexed to ensure homogenous suspension. One hundred microliter of conidial suspension was inoculated onto the prepared honey-SDA plates, in triplicates for each honey concentration. SDA plate without honey was used as control. The plates were incubated at 30oC. Colony count was performed after 24 and 48 h.
The fungal growth inhibition by Tualang, Acacia and Kelulut honeys are shown in Table 1 and Table 2. All three types of honeys showed antifungal activities, in which significant reduction of colony counts was seen in comparison to control plate (SDA without honey).
|Honey||Group||Colony count Mean(SD)||Mean difference (95% CI)||t-statisticsa (df)||p-value|
|Kelulut||Kelulut Control||15.30(1.83) 123.90(15.13)||-108.60(-118.89,-98.31)||-23.87(9)||<0.001|
|Honey||Group||Colony count Mean(SD)||Mean difference (95% CI)||t-statisticsa (df)||p-value|
|Acacia||Acacia Control||34.40(4.60) 127.10(16.52)||-92.70(-103.39, -82.015)||-19.63(9)||<0.001|
Figure 1 shows the effects of the three honeys on A. niger at four different honey concentrations (v/v). Tualang honey showed gradual reduction in A. niger colony count with increasing honey concentration from 5% (v/v) to 15% (v/v). No fungal colony seen at 25% (v/v). As for Acacia honey, at 5% (v/v), the colony counts of A. niger was rather similar to that of Tualang honey. At 10% (v/v) Acacia honey plate showed lesser colony count. No A. niger colony was seen at 15% (v/v) and 25% (v/v) Acacia honey. Kelulut honey 5% (v/v) plate showed much lesser A. niger colony count compared to those of Tualang and Acacia honeys. No A. niger growth seen on 10% (v/v), 15% (v/v) and 25% (v/v) Kelulut honey plates.
The effects of Tualang, Acacia and Kelulut honeys on C. albicans are shown in Figure 2. Tualang honey showed gradual reduction in C. albicans colony count. Total inhibition of C. albicans growth was seen at 25% (v/v) Tualang honey. Acacia honey, on the other hand, showed similar C. albicans colony count as Tualang honey at 5% (v/v) but scanty growth at 10% (v/v) and total growth inhibition at 15% (v/v) and 25% (v/v). Kelulut honey plates showed C. albicans colony count of less than 10 cfu at 5% (v/v), whereas at 10% (v/v), 15% (v/v) and 25% (v/v), total growth inhibition was noted. Therefore, comparing all types of honey, Kelulut honey seemed to have the strongest antifungal activity on both A. niger and C. albicans.
In the present study all three types of honey, Tualang, Acacia and Kelulut, demonstrated significant antifungal properties against A. niger and C. albicans. This finding is in line with previously reported studies that depicted antifungal efficacy of various honey on fungi. Nevertheless, some studies did report selected fungal resistance to selected honey.9-10
The in vitro antifungal activity of honey was reported by Estevinho et al. (2011), who observed that honey inhibited the growth of C. albicans, C. krusei and Cryptococcus neoformans.11 Obaseik-Ebor and Afonya (1984) compared the antifungal activity of honey distillate with some antimycotic preparations against C. albicans and found that all the strains resistant to conventional antimycotic agents were inhibited by active fraction of honey.12 Boukraa and Bouchegrane (2007) reports in vitro antifungal efficacy of Algeria honey on A. niger and C. albicans.13 Meanwhile Moussa et al. also using Algeria honey, demonstrated that it has in vitro antifungal activity against C. albicans, A. niger and Rhodotorula species.14-16
DeMera and Angert (2004) in Costa Rica reported antifungal effect of local honey towards C. albicans and a number of bacteria.17 Feas and Estevinho (2011) studied in vitro antifungal activity of Portugal organic honey.18 Meanwhile, Koc et al. (2009) reported that Turkey honey was able to inhibit forty strains of Candida spp. and Trichosporon spp.19 In addition, it also demonstrated antifungal activity towards fluconazole-resistant yeast strains.
In Australia, an in vitro study was conducted using four types of Australia honeys and their effects on Candida species.20 In an earlier study, six types of Australian honeys were tested against thirteen bacteria and C. albicans.10 Both studies used Medihoney® but their results were contradictory. Lusby et al. (2005) reported that none of the honeys used, including Medihoney, have antifungal effects against C. albicans.10 In addition, another report from the Republic of Slovenia showed that all six studied Slovenian honeys lacked antifungal effect on C. albicans, A. niger and Penicillium chrysogenum.10
In South Africa, an in vitro study using three types of South Africa honey; Wasbessie, Bluegum and Fynbos, showed only Wasbessie honey had inhibitory effect on C. albicans.21 In the present study, all three types of Malaysian honeys demonstrated inhibitory effect on both C. albicans and A. niger.
While most of the studies on antifungal effects of honey involved in vitro studies, in vivo studies have also been reported. Ngatu et al. (2011) have conducted an in vivo study in the Republic of Congo,22 in which efficacy of bee products such as Acacia honey and Brazilian green propolis extract on children with fungal infections (tinea capitis and tinea versicolor) has been assessed. They have proven that bee products showed beneficial effect in treatment of superficialmycosis.