ANTIFUNGAL ACTIVITY OF Cymbopogon nardus (L.) Rendle (CITRONELLA) AGAINST Microsporum canis FROM ANIMALS AND HOME ENVIRONMENT

Abstract

Dermatophytosis is a common zoonosis in urban centers. Dogs and cats have played an important role as its disseminators. Environmental decontamination is essential for the prevention of its propagation to humans and animals. However, sanitizers or disinfectants with antifungal activity, currently available, have high toxicity. The present study evaluated the in vitro effects of an extract of citronella (Cymbopogon nardus) on 31 Microsporum canis isolates from animals and home environments. Susceptibility tests were performed based on document M38-A2 (2008) of the Clinical and Laboratory Standards Institute with modifications for natural products. Although susceptibility variation was observed between the fungus tested, the concentrations that inhibited the growth of 50 and 90% of the microorganisms were low (19.5 and 78 µg/mL, respectively). Thus, this citronella extract showed potent fungistatic and fungicide activities against M. canis isolated from animals and home environments. Therefore, it could be an alternative for dermatophytosis prophylaxis in the home environment.

Dermatophytosis is a cutaneous mycosis caused by keratinophilic fungi, with high prevalence among adults and children that inhabit tropical regions. Domestic mammals, especially dogs and cats, clearly play a role as dissemination agents8. Several authors have linked animal dermatophytosis2 ^, 15 or surfaces contamination in houses to animal infections13.

The strategic treatment of dermatophytosis in animals should include environmental decontamination17. However, the products available present several limitations related to human health, such as toxicity and potential waste accumulation in the environment14 ^, 18. In addition, few studies have evaluated the activity of disinfectants against zoophilic fungi7.

In the search for alternative disinfectants, the present study evaluated the in vitro effects of an extract of Cymbopogon nardus (citronella) on dermatophytes isolated from domestic animals and the environment, in an effort to provide a new perspective in dermatophytosis prophylaxis.

Cymbopogon nardus (L.) Rendle, popularly known as citronella, is a plant from the Graminae family. Several authors have demonstrated the in vitro antifungal activity of essential oils of C. nardus, and other species of the genus Cymbopogon, on pathogenic fungi4 ^, 5 ^, 9. Hair and skin samples from dogs and cats with suspected dermatophytosis were collected by scraping the affected animals, in some veterinary clinics in Maringa (two from Clinica VeterináriadoUnicesumar, nine from Clínica Saúde Animal, nine from Clínica Ponto Cão and 11 from Zooloja), Parana, Brazil. Samples of domestic environments (e.g. floors and carpets) were collected by the carpet method3. The samples were grown on Mycosel Agar (Benton Dickinson, Sparks, MD, USA) for seven days at 25 °C. Of the 60 biological samples, including biotic and abiotic samples, 31 fungal isolates were obtained and identified as M. canis by micromorphological technique11. These samples, and the standard strain of Trichophyton rubrum (ATCC 28189), were tested against the hydroalcoholic extract of C. nardus to evaluate the in vitro antifungal activity. The leaves of C. nardus were collected in "Prof. Irenice Silva" medicinal plant garden of the State University of Maringa, PR, Brazil (lat: -24.35 long: -50.583333). Fresh leaves of C. nardus were cleaned with compressed air, cut into small pieces and submitted to turbo extraction for 15 minutes with 77 °GL (Gay Lussac) ethyl alcohol in a ratio of 20% (w/w) at room temperature. The extract was filtered, concentrated in a rotoevaporator and, subsequently, lyophilized. Then, the lyophilized extract of C. nardus was solubilized in dipropylene glycol at a rate of 10 mg/mL.

Firstly, the fungi were grown in potato dextrose agar for ten days at 25 °C. Afterward, the fungal structures were detached in sterile saline solution (0.85%). The inoculum concentration was adjusted to 1-5 ´ 104 colony forming units per millilitre (CFU/mL)12 ^, 20 in RPMI 1640 medium (Roswell Park Memorial Institute, Gibco) with L-glutamine without sodium bicarbonate, buffered with MOPS (3-[N-morpholino] propanesulfonic acid, 0.165 M, pH 7.2, Sigma) plus 2% glucose.

The susceptibility assay was performed using the microdilution broth method according to document M38-A2 (2008) of the Clinical and Laboratory Standards Institute1 with some modifications for natural products. Briefly, the extract was tested at concentrations that ranged from 9.7 µg/mL to 5,000 µg/mL. Terbinafine was used as a control (0.002 and 0.008 µg/mL). The reading was performed, by visual observation, after seven days of incubation at 25 °C. The minimum inhibitory concentration (MIC) was considered the lowest concentration that inhibited 100% of fungal growth compared with the control. For both the citronella extract and terbinafine, the MIC was calculated according to the inhibition of growth of 50% (MIC₅₀) and 90% (MIC₉₀) of the microorganisms.

The minimum fungicidal concentration (MFC) was determined by transferring the contents from MIC assay to plates with drug-free Mycosel Agar. The lowest concentration of the extract that inhibited fungi growth in complete medium was considered the MFC.

Ten houses were visited where 11 animals had confirmed cases of dermatophytosis. In eight, it was possible to isolate the pathogen from the environment. In three houses, asymptomatic animals were found with positive microbiological tests for M. canis. In four homes, the disease spread to humans or animals.

The MIC of citronella extract against 31 M. canis isolates and the control strain ranged from 9.75 to 625 µg/mL. However, in most of the studied samples, both the MIC₅₀ and MIC₉₀ were low (19.5 and 78 µg/mL, respectively). Only a low percentage of isolates (3.23%) demanded a high concentration of the citronella extract (625 µg/mL) to show fungicidal activity.

The citronella extract, according to the criteria established by SCORZONI et al. (2007)19, had moderate to strong antifungal activity (Table 1); it was strong for most of the M. canis samples tested (80.65%). The present data indicate that the fungistatic and fungicidal activities of the citronella extract were identical for most of the M. canis isolates tested. In only six isolates, the fungicidal concentration of the extract was slightly greater than the inhibitory concentration.

Table 1. - Activity in vitro of the citronella extract (CE) and terbinafine (TERB) on 31 isolates of Microsporum canis .

	Drugs	M. canis (31)
1 MIC range	CE	9.75-625
	TERB	0.001-1
MIC50	CE	19.50
	TERB	0.001
MIC90	CE	78.00
	TERB	0.001
2 MFC range	CE	9.75-625
MFC50	CE	19.50
MFC90	CE	78.00

¹Minimum inhibitory concentration - MIC (µg/mL); MIC₅₀ and MIC₉₀ for drug and extract: MIC capable of inhibiting 50% and 90% of the isolates, respectively. ²Minimum fungicidal concentration - MFC (µg/mL); The MFC₅₀ and MFC₉₀ are the MFCs capable of inhibiting 50% and 90% of the isolates, respectively. Trichophyton rubrum ATCC 28189 - MIC = 39 µg/mL, MFC = 39 µg/mL.

Our results demonstrated the in vitro efficiency of citronella extract on inhibiting M. canis obtained from animals and the home environment where these animals lived. Citronella essential oil has been used as an insect repellent and disinfectant10 ^, 16, but the high cost and manufacturing complexity limits its use. However, citronella extract use in controlling dermatophytosis is encouraged by its low cost, easy formula preparation, and accessibility. It is also rich in citronellal and geraniol10. The present study indicates a new option of low-cost disinfectants.

In the present study, the terbinafine MIC of 32 isolates ranged from 0.001 to 1 µg/mL, and the MIC₅₀ and MIC₉₀ were also low (0.001 µg/mL). These in vitro results suggest a homogeneous fungi population profile with regard to antifungal susceptibility. The MIC₉₀ for terbinafine was low, confirming the results reported by GUPTA et al. (2001)6. These data may suggest the use of terbinafine for the treatment of dermatophytosis caused by M. canis. Nevertheless, the high cost of this product makes its use prohibitive in environmental control.

In conclusion, the citronella extract showed strong antifungal activity, both fungistatic and fungicidal, against isolates of M. canis, suggesting its potential use in the control of zoonoses of fungal origin. Further studies should be conducted incorporating this extract in sanitizers for domestic environments where animals live, with the goal of use it in prophylaxis against dermatophytosis carried by pets.