Comparative study of the effect of Eucalyptus extract on Candida albicans and human pathogenic bacteria

Background & Aim: During recent decades, infections disease resulting from opportunistic fungi such as Candida albicans and resistance increasing of human pathogenic bacteria to current antibiotics has led to many problems for treating of these diseases. The aims of the present study was to investigate anti-fungi and anti-bacterial effects of Eucalyptus extract on Candida albicans species isolated from clinical samples and some standard human pathogenic bacteria. Experimental: Eucalyptus extract was provided using rotary apparatus and maceration method. Thirty isolates of Candida albicans were isolated from referred patients to gynecologist and then were purified and identified by valid keys. Finally, 30 isolated were used to growth inhibitory activity assay. Minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of eucalyptus extract against 5 standard bacteria inculding: Staphylococcus aureu, Shigella dysenteriae, Listeria monocytogenes, Vibrio cholera and Bacillus cereus were evaluated using micro broth dilution method. Results: Our results indicated that in compare to bacterial strains, fungus isolates showed more sensitivity to eucalyptus extract. The highest and lowest MIC of extract was recorded at 12.5 and 3.1 ppm for S. aureus and B. cereus, respectively. As MIC, the maximum MBC (20ppm) and minimum MBC (5ppm) of extract was recorded fro S. aureus and B. cereus respectively. Minimum inhibitory concentration of extract to fungal growth inhibitory was 50 ppm, whereas the maximum inhibiting concentration was 150 ppm. Recommended applications/industries: The positive and interesting results suggest the essential oil of E. globulus could be exploited antibiotic for the treatment of candidiasous disease caused by Candida albicans fungi, and some human pathogenic bacteria studied in this work. Type: Original Research Topic: Medicinal Plants Received July 24 2016 Accepted December 16 2016


Introduction
Although, fungal infections compared to bacterial and virus infections, was not much prevalent in the past, but they have been responsible for considerable increase of diseases during recent decades (Baker et al., 2012).Risk factors such as long and wide use of antibiotics, croton drugs, immuno suppressants medicament and basic disorders such as diabetes, AIDS and malignancies caused by fungal diseases, especially increased more than in the past (Goswami et al., 2000;Merenstein et al., 2013).Vaginal yeast infections known as candidiasis, is a disease caused by growth of more yeast around genital aerea of women (Consolaro et al., 2004;Giraldo et al., 2000).This is an important disease during the years in women who are sexually acitve, and it is increasingly spreading (Hay and Ashbee, 2010).Candida albicans species is the main causal agent that generates candida virginities disease with growth in two biological forms: yeast and mycelia forms in medium culture (De Vos et al., 2005).This dimorphic fungus growth mainly in the human body as digestive and genital systems, and culture of, and also is present on vaginal secretion of 10-25% of women without symptoms disease (Cates et al., 1990;Mathema et al., 2001).The sexual contact is not usually the main cause of fungus spread in candidiasis or yeast infection.Antibiotics, birth control pills, steroid consuming and also tight clothing, severe obesity and warm weather, cause increase numbers of yeast.
One of the most widespread methods of candidiasis controlling is the use of fungicides such as fluconazole, ketoconazole, itraconazole (Amber et al., 2010;Schuster et al., 2008).Resistant of C. albicans' isolates to normal treatment by topical medications such as miconazole and fluconazol antifungal cream has been reported (Hoehamer et al., 2010;Moosa et al., 2004).Bactreia are another pathogenic factor in human.Currently, human pathgenic bacteria are controlled using chemical drugs including different antibiotics.Regarding to broad-spectrum and fast-acting of antibiotics, they are the first choice of patients (Biyela et al., 2004;Levy, 1998;Neo, 2011).Continous and immethodical consumption of chemical drugs cause resistance to these combinations which result in weakened or neutralized effect of drugs and finally leads to raising the drug doze and tendency to apply new and more powerful formulae.Another disadvantage of using these drugs is their side effects which lead to emergence of diseases more dangerous than the primary ones (Pinto et al., 2009).During recent years, medical plants are being used as an appropriate alternative for chemical drugs without their negative side effects.Eucalyptus plant (Eucalyptus globules, Myrtaceae) is a flowering tree and shrub which have been adapted to hot and humid climate of Iran and is frequently used as medical plants to cure for the common cold.Antifungal and antibacterial effects of eucalyptus extract in human (Cermelli et al., 2008;Hammer et al., 1999;Saeeidi et al., 2014) and plant diseases (Ghorbani et al., 2014;Wilson et al., 1997) have been assessed, and it has been shown that in most cases in exprimental condition, eucalyptus has had good inhibiting effects on the growth of tested fungi and bacterial.Extract of Eucalyptuse has antiinflammatory, antiseptic, antibacterial and expectorating properties and has resulted to inhibit inflammation of nasal mucosa, pharyngeal mucosa, otitis, sinuses inflammation and vaginitis (Noumi et al., 2011).Analysis of chemical compound of eucalyptus extract content has shown that these compounds contain cineol, cytrol (Maciel et al., 2010), piperitone, α-Phellandrene and trepene (Gilles et al., 2010) .Because of biological and pharmacological properties of essential oil of Eukalyptus species and its applications as anesthetic, anodyne, antiseptic, astringent, deodorant, diaphoretic, in the international pharmacopeia and marke we were firstly interested, to investigate antifungal effects of eucalyptus extract on C. albicans isolates obtained from clinical samples and secondly to compare this with anticacterial effects of this extract against some human pathogenic standard bacteria.

Sample preparation
The plant used in this study was purchased from commercial source and based on morphological and botanical descriptions were identified as Eucalyptus globules by an expert botanist.The leaves of plant were dried in the shadow under the appropriate conditions, were grinded in a grinder and homogenized to a fine powder and then were maintain at room temperature for further studies.

Plant extraction
Plant extraction, was carried out using maceration method.For this propose, 10 gr dried powder of the eucalyptus leaf was put in 0.5 lit Erlenmeyer flask containing 100 ml ethanol (96%) and water.The flask content was mixed using a shaker in the room temperature with the speed of 130 rpm for 24 h, and then was filtered from Whatman No. 2. The solvent was separated and concentrated from the extract using a rotary evaporator (Heidolph, Germany), and was dried after being passed from a microbial filter with 0.45 micro meter pores in 60 C temperature for two days (Saeedi et al., 2012).The obtained extract was wieghted and solved in DSMO solvent and was kept in refrigrator in 4C until being used in anti-microbial tests.To determine the Maximum Inhibiting Concentration (MIC) of the extracts, was done using dilution method

Fungal isolates
Fungi isolates were isolated from patients' vaginas referred to the medical center using a sterile swab, by a gynecologist and then were quickly transferred to a sterile falcon tube containing sterile water for further culture and purification.Two solid culture medium including: Sabouraud Dextrose Agar and Sabouraud Dextrose Broth (Merck, Germany) were used for fungal culture and purification.One drop of fungi suspension was spread in a line across over the solid SDA medium in plates.All plates were transferred to an incubator at 37C and then were examined after 24 hrs incubation.Growth individual colonies were cultured again on the solid medium and fresh colonies were used for purification and identification.Each purified colony was identified according to the standard keys based on morphological critari.Isolates were kept for further studies in -20C (Otang et al., 2012).To prepare a suspension of fungal spores, a piece of C. albicans cultured on Sabouraud Dextrose Agar medium were transferred to liquid physiologic serum and kept in 37C.After fungi growth in liquid medium, the concentration of fungal suspension was measured using a spectrophotometer (Unico, USA) in 530 μm wavelength.Based on the obtained light absorption, a suspension with a concentration of 10 6 cells/mL was adjusted.

Bacterial strains
Standard bacteria strains including: Staphylococcus aureus ATCC1189, Shigella dysenteriae, ATCC1188, Listeria monocytogenes ATCC1298, Vibrio cholera ATCC1611, Bacillus cereus ATCC1015 were used to determine inhibiting effects of eucalyptus extract.After preliminary preparations, a colony of bacteria was cultured in 10 ml Tryptic Soy Broth (TSB, Merck, Germany) medium and was incubated in 37 C for 18 hrs; then, 100 ml of the fresh grown suspension of each bacterium was transferred to the solid TSB medium and was incubated in 37 C for 18 hours.

Preparing McFarland microbial suspension
To prepare a microbial suspension, one day before experiment, a piece of stock culture (solid medium) was transferred to nutrient agar medium (Merck, Germany).After growth of bacterial cells on the surface of culture medium, the surface was washed using a normal salt solution and concentrated microbial suspension was obtained.A drop of bacterial suspension was poured in leaded sterile tubes containing normal saline and its turbidity was measured using a spectrophotometer in 530 μm wavelength and was diluted with normal saline and then the turbidity of a bacterial suspension was equal to the turbidity of 0.5 McFarland suspension.The final concentration of the bacterial suspension was adjusted to 1.5 ×10 8 CFU.

The Minimum Inhibitory Concentration (MIC) Method
The minimum inhibitory concentration (MIC), is the lowest concentration of an antibiotic or plant extract that can inhibits the growth of a particular organism as bacteria or fungi.The MIC of eucalyptus extract was determined using broth micro-dilution method.For this proposes, one drop of each bacterial suspension (1.5 ×10 8 CFU) was added to well of microtiter plate containing Muller liquid Hinton nutrient medium (MHB).A serial dilution (ppm) of plant extract was done for each bacterial strain.Micro-titer plates were incubated at 37 o C and the MIC was recorded after 24 h.

The Minimum Bacterial Concentration (MBC)
The lowest concentration of plant extract which is able to reduce the viability of the initial bacterial inoculums by ≥99.9% is called MBC.The MBC is complementary to the MIC.MBC was determined by sub-culturing the test dilutions on to a fresh solid medium and incubated further for 18-24 h.The highest dilution that yielded no bacterial/fungal growth on solid medium was taken as MBC.

Statistical analysis
The mean values of studied parameters were taken from the measurements of three replicates and the "Standard Error" of the means was calculated.One-way ANOVA was applied to determine the significance of the results between different treatments and then Duncan multiple range tests (p < 0.05) were performed.All the statistical analyses were done using the Statistical Package for Social Sciences (SPSS) for Windows (version 18.0).

Results and discussion
The result of this study showed that various concentrations of eucalyptus extract can inhibit the growth rate of Candida albicans isolates in excremental condition.Increase of plant extract concentration resulted to high growth inhibitory effect on solid medium.Minimum inhibitory concentration was 50 ppm, whereas the maximum inhibiting concentration was 150 ppm (Fig. 1).
The results of investigating the effect of eucalyptus extract on bacteria indicated that eucalyptus extract was able to inhibit growth of all tested bacteria.The maximum inhibiting concentration (MIC) and maximum bacterial concentration (MBC) (MBC) was recorded for staphylococcus aureus strain and the minimum inhibitory concentration (MIC) minimum bacterial concentration (MBC) was recorded for Bacillus cereus strain, and (Fig. 1).Table 1.The results of minimum inhibiting concentration and minimum bacterial concentration of eucalyptus extract against human pathogenic bacteria.
The growth inhibition effect assay showed that the sensitivity of 30 tested isolates varied from 50-150 ppm of eucalyptus extract.The mean of inhibitory effect of eucalyptus extract was calculated to be 120 ppm.
Although aggressive and systemic fungal diseases are hardly prevalent (2% of the patients), the mortality rate resulting from these infections have been reported to be 26-56 percent.Regarding to, resistance of fungi including different species of Candida to current fungicides, many scientists have been interested to develop new treatment with the least side effects on humans to disease control (Patterson et al., 1996).In recent years, many human pathogenic fungi have been The present study, investigated the inhibitory effect of eucalyptus extract on Candida albicans isolates, as well as its comparative effect on inhibitory of some human pathogenic bacteria.The study of inhibitory effect of two essential oil of eucalyptus and lavender plant on the growth of clinical strains of Candida albicans isolated from animals infected by mucosal and cutaneous infections and comparing them with C. albicans resistant and sensitive to Itraconazole fungicide showed that the highest effect of Eucalyptus essential oil was at concentrations of 32 and 64 pm while, the highest effect for lavender essential oil was at concentrations of 8-16 ppm.Based on the obtained results of the present study and compared to effective concentrations, of plant extract, these values are lower than the ones for our data.This fact could be resulted from higher permeable nature of essential oil than extract.
Application of eucalyptus extract for male rats having diabetes led to reducing diabetes symptoms and indictors such as hyperglycemia and polyphagia as well as considerably reducing Candida albicans in the liquid around kidney and liver (Bokaeian et al., 2010).Uses of medical plants as synthetic medicines and determining their inhibitory effect has been investigated in various plants.The effect of plant extract of Myrtus communis as topical ointment application to inhibit the growth of C. albicans and comparing its effect with clotrimazole ointment shows that like clotrimazole, M. communis extract reduced itching in vaginal area of women having candidacies and this effect is similar to the effect of antibiotics (Genani et al., 2011).Antibacterial effect of essential oil related to 26 species of eucalyptus species on six strains of gram-positive bacteria showed that all species were able to inhibit the growth of bacteria in concentrations of 15-250 ppm under experimental condition, but none of them had any effects on gramnegative bacteria (Takahashi, 1966).In the present study, all gram-positive bacteria showed sensitivity to eucalyptus extract.Seed oil of eucalyptus has fungicide effect on Fusarium oxysporum species wich produce canceous toxin in human (Kottearachchi et al., 2012).These results confirm effectiveness of eucalyptus extract in inhibiting the growth of fungi.The results of our research showed that with increase of the extract, the inhibitory effect against fungi and bacteria was increased.The reason for this increase might be the change in effective material of plants due to alterations in plant culture condition and climate and this may consequently affect microorganisms differently.The inhibitory effect of essential oil of Eucalyptus plant on gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli has been proved (Bachir and Benali, 2012).The result of Saeidi et al's study showed that Eucalyptus extract has considerable MIC in 5 mg/ml against Staphylococcus aureus , and the highest amount of MBC is observed in 10 mg/ml concentration (Saeeidi et al., 2014).The minimum inhibitory concentrations of eucalyptus extract against:S.aureus, B. cereus, E. faecalis, Alicyclobacillus acidoterrestri and Propionibacterium acne were recorded as 3.9, 3.9, 7.8, 31, and 7.8 mg/ml, respectively (Takahashi et al., 2004) which is in accordance with the results of the present study.Findings of another study showed that inhibition zone diameters of ethanolic extract of eucalyptus at concentrations of 5, 7.5, 10 and 20 μl/ml was observed 1.4, 1.9, and 3.2 mm against staphylococcus aureus and 1.3, 1.5, 2, and 2.4 mm inhibitory zone against Escherichia coli (Ghalem and Mohamed, 2008).

Conclusion
It should be noted that during recent years, several reports about resistance of a series of C. albicans species-to chemical fungicide have been released.Based on the obtained data in this study, the results indicate that the extract of eucalyptus plays an important role in inhibiting the growth of C. albicans isolates and tested bacteria, and thus it can be used as an appropriate alternative for chemical anti-fungal medicine.Performing complementary tests with standard fungal isolates and various species of eucalyptus plants can be effective in introducing proper medicine in pharmaceutical industry.essential oils for antifungal activity against Botrytis cinerea.Plant Disease.81(2): 204-210.

Fig. 1 .
Fig. 1. the control sample of candida albicans in the medium without extract (A): isolate of candida albicans in 100 ppm concentration of eucalyptus essence, (B): isolate of candida albicans in 150 ppm of eucalyptus extract (C).
. So, many laboratories have attempted to find alternative drugs.Currently, medicinal plants are one the most appropriate alternative controlling factors.The provenance of medicinal plants using is traditional medicine that has resulted from experimental activities.So, based on these experiments, use of medicinal plants has open up new perspective in order to apply them in pharmaceutical industries for treatment of infectious disease