{"id":18295,"date":"2017-12-21T09:40:13","date_gmt":"2017-12-21T09:40:13","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=18295"},"modified":"2018-11-19T06:59:11","modified_gmt":"2018-11-19T06:59:11","slug":"a-comparative-study-between-specific-and-non-specific-antifungal-agents-to-treat-the-rhodotorula-mucilaginosa-athletes-foot","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol10no4\/a-comparative-study-between-specific-and-non-specific-antifungal-agents-to-treat-the-rhodotorula-mucilaginosa-athletes-foot\/","title":{"rendered":"A Comparative Study Between Specific and Non-Specific Antifungal Agents to Treat the Rhodotorula Mucilaginosa Athletes Foot"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

An\u00a0antifungal drug is a\u00a0pharmaceutical (fungicide\u00a0or\u00a0fungistatic)\u00a0used to treat and prevent\u00a0fungal infection as\u00a0athlete’s foot and\u00a0thrush (Baginski and Czub 2009) . Athlete’s foot, is a skin disease of the feet caused by fungus . Signs and symptoms often include itching, rough skin, and congestion .\u00a0In chronic cases the skin may\u00a0blister.\u00a0Athlete’s foot disease may infect any part of the foot, but most often grows between the toes (Bell-Syer et al<\/em>. 2012). Rhodotorula<\/em>\u00a0is an environmental yeast \u00a0that is found in air, soil, lakes, water, milk, and fruit juice.\u00a0Rhodotorula\u00a0species, part of the Basidiomycota phylum, colonies plants, humans, and other mammals (Larone 1995).\u00a0Rhodotorula\u00a0<\/em>produces pink to red colonies and blastoconidia that are single cell lacking pseudohyphae and hyphae. Several studies have isolated\u00a0Rhodotorula<\/em>\u00a0in different ecosystems and environments as well as described infections in Mammals.\u00a0Rhodotorula<\/em>\u00a0spp. have been recognized as emerging yeast pathogens in humans in the last two decades (Hagan et al<\/em>.1995).\u00a0Among the few sources to the pathogenicity of\u00a0Rhodotorula<\/em>\u00a0spp. in animals, there are several studies of an outbreak of skin infections in birds and sea animals and pulmonary infections and otomycosis in ruminants (Fernanda, 2012). This study aimed to select best antifungal agent to treat Rhodotorula mucilaginosa<\/em> athletes foot.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Antifungal Agents<\/strong><\/p>\n

In this study used two types of antifungal agents include Specific and non- specific antifungal agents. Specific antifungal agents were used to treat skin mycological infection between foot toes include \u00a0Ketoconazole, Clotrimazole ,Itraconazole, Fluconazole and Nystatin, While non-specific antifungal agents are using to treat skin mycological infection between foot toes include 1% Iodine, 3% Sodium bicarbonate, 3% H2<\/sub>O2<\/sub>, 2% Acetic acid and 10% Potassium permanganate. All non-specific antifungal agents were prepared from chemical agents stock solution (10% Iodine, 50% Sodium bicarbonate, 50% H2<\/sub>O2<\/sub>, 50% Potassium permanganate and 98% Acetic acid and use distilled water as a dilute of these substances}. The required concentration has been prepared by V1C1=V2C2 equation (Mary, 2005).<\/p>\n

Microbe’s Isolates<\/strong><\/p>\n

Rhodotorula mucilaginosa<\/em> \u00a0were isolate from toes skin (Hagan et al<\/em>.1995 ; Galan-Sanchez et al.<\/em>,1999) of sport man \u00a0by cut the small piece of infected skin between their toes figure (1) and kept in the cool container till trans to lab of Microbiology\/the Technical institute of Babylon than this small piece cultivated in the Sabouraud Dextrose Agar (SDA) media for 48 hours at 27\u00baC (Pawe\u0142 and Anna 2010).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: Infected skin between foot toes.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Lab Diagnosis of\u00a0 <\/strong>Rhodotorula Mucilaginosa<\/strong><\/em><\/p>\n

Macroscopic Appearance<\/strong><\/p>\n

Rhodotorula<\/em>\u00a0spp. are pigmented basidiomycetous yeasts in the family Sporidiobolaceae (Fell et al<\/em>. 2000) Rhodotorula<\/em>\u00a0spp. produce colonies that are pink to red in color but can also be orange to red on Sabouraud Dextrose agar due to the presence of carotenoid colours . Colony shape has been appeared as thin , smooth, and sometimes mucoid.\u00a0Rhodotorula<\/em>\u00a0spp. are nutritionally non-fastidious, grow easily on common media, and are characterized by a rapid growth rate(Larone 2002).<\/p>\n

Microscopic Appearance<\/strong><\/p>\n

A small portion of Rhodotorula mucilaginosa<\/em> colony cultivated in SDA media after incubation 48 hours at 27\u00baC taken by bacteriological loop and it place on the clean surface of class slide and mixed with one drop distilled water and\u00a0 one drop of Lactophenol-cotton blue dye. A cover slide was gradually applied with slowly pressure to expulsion air bubbles. The slide was then observed under microscope {Power zoom X10 , X40 , 100X objective lenses respectively} (Cumitech , 1980). Rhodotorula mucilaginosa<\/em> was appeared as Spherical to elongate cells and budding yeast cells . In some cases, rudimentary pseudomycelium can be observed (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts<\/em> 2008) figure (2).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2: <\/strong>Rhodotorula mucilaginosa<\/strong><\/em> cells are dyed by lactophenol cotton blue (<\/strong>100X<\/strong>)\u00a0\u039f\u00a0<\/strong>Refer to Budding yeast cells.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Biochemical Test<\/strong><\/p>\n

Rhodotorula mucilaginosa<\/em> production of urease; and inability to assimilate inositol or to ferment sugars (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts<\/em> 2008).<\/p>\n

Rhodotorula Mucilaginosa<\/strong><\/em> Cellular Counts<\/strong><\/p>\n

In this study the method of \u00a0Rhodotorula mucilaginosa<\/em> cells count same calculation method of \u00a0Candida albicans <\/em>cell count \u00a0include:<\/p>\n

Rhodotorula mucilaginosa<\/em> cells suspension was prepared by adding five ml of distilled water to fresh SDA media contains Rhodotorula mucilaginosa<\/em> (colonies aged 48 hour at 27\u00baC). Its turbidity was adjusted accordance to the absorbance of 0.08-0.10 at 625nm corresponding to 5 x 106<\/sup> CFU\/ml (Ricardo and Edeltrudes\u00a02013).<\/p>\n

Determination of Minimum Inhibitory Concentration (MIC)<\/strong><\/p>\n

MIC of the effective specific and non-specific antifungal agents were determined by tube dilution Method (Cruickshank, 1975)., Ten test tubes with 8 ml of Sabouraud Dextrose Broth (SDB) in each were taken and autoclaved. To the first tube, 2 ml of the each concentration (50% H2<\/sub>O2<\/sub>,10% Iodine and 98% Acetic acid) was added and serial double fold dilution was done up to the 10 tube and from the 10 tube, 2 ml of the mixture was discarded. To each tube 100\u03bcl of inoculums Rhodotorula mucilaginosa<\/em> suspension (5 x 106<\/sup> CFU\/ml) were added and mixed well . The tubes were incubated for 48 hours at 27\u00baC. The least concentration of each one specific and non-specific antifungal agents capable of inhibiting the\u00a0 Rhodotorula mucilaginosa<\/em>\u00a0 growth was considered MIC.<\/p>\n

Antifungal Susceptibility Testing<\/strong><\/p>\n

Specific Antifungal Susceptibility Testing<\/strong><\/p>\n

A standard antifungal disc diffusion susceptibility testing method (Clinical Laboratory Standards Institute 2009) were used by commercially available discs\u00a0 preloaded with Ketoconazole (10 \u00b5g) , Clotrimazole (50 \u00b5g), Nystatine (100 I.U.) , Itraconazole (25 \u00b5g) and Fluconazole (50 \u00b5g) were using to determine the inhibition zone against Rhodotorula mucilaginosa<\/em> in SDA media.<\/p>\n

Non-Specific Antifungal Susceptibility Testing<\/strong><\/p>\n

Ager well diffusion method (Magaldi et al<\/em>. 2004) were used to Non-Specific antifungal Susceptibility Testing.<\/p>\n

One hundred \u00b5l of Rhodotorula mucilaginosa<\/em><\/em> suspension were spread uniformly over SDA medium by using the class spreader, then left for one hour to dry of yeast cells on the media surface. By using cork borer, one central well (digs) was worked on the SDA media. One hundred microliter was taken from each non-specific antifungal agents (1% Iodine , 3% Sodium bicarbonate , 3% H2<\/sub>O2 <\/sub>, 2% Acetic acid and 10% Potassium permanganate) that has been prepared previously and put in these wells. Same previous steps were used again for distilled water which considered as control group. Number of petri-dish for each agent repeated five times.<\/p>\n

Inhibition activities of the non-specific antifungal agents were determined by measuring the zones inhibition formed around the well in millimeter. The plates were observed for presence of zones of inhibition around the well after 48 hours at 27\u00baC (Mohit 2013).<\/p>\n

Statistical Analysis<\/strong><\/p>\n

Data are presented as M\u00b1SE. For the statistical analysis, it was used one-way analysis of variance (ANOVA) using SPSS 13.0. Variances were considered significant if p < 0.05 (Joda 2008).<\/p>\n

Results and Discussion<\/strong><\/p>\n

The results of the present study showed a difference in the effectiveness of antifungal agents (Specific and Non-specific) against Rhodotorula mucilaginosa<\/em>.<\/strong><\/p>\n

Specific Antifungal Agents<\/strong><\/p>\n

In this study Rhodotorula mucilaginosa<\/em> isolates appeared more susceptible (sensitive) to Ketoconazole, Nystatin and Clotrimazole respectively whereas other azole antifungal drugs (Itraconazole and Fluconazole) did not show any effectiveness (Resistance), as figure (3) and table (1).<\/p>\n

The lowest MIC value (mg\/ml) of different specific antifungal agents used in this study against Rhodotorula mucilaginosa<\/em> showed in Ketoconazole in comparison with other agents used in this study, table (2).<\/p>\n

Table 1: Zone inhibition diameter of different specific antifungal agents against Rhodotorula mucilaginosa<\/em> in the SDA media in comparison with distilled water. The age of colonies 48 hours at 27\u00baC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Antifungal<\/strong><\/p>\n

\u00b5g or I.U.\/Disc<\/strong><\/td>\n

Inhibition Zone<\/strong><\/p>\n

M\u00b1SE <\/strong><\/td>\n<\/tr>\n

Ketoconazole (10 \u00b5g)<\/td>\n2.07\u00b10.04\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 A<\/td>\n<\/tr>\n
Clotrimazole (50 \u00b5g)<\/td>\n1.32\u00b10.04\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 B<\/td>\n<\/tr>\n
Nystatine (100 I.U.)<\/td>\n1.80\u00b10.05\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 A<\/td>\n<\/tr>\n
Itraconazole(25 \u00b5g)<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 C<\/td>\n<\/tr>\n
Fluconazole (50 \u00b5g)<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 C<\/td>\n<\/tr>\n
Control<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 C<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

M\u00b1SE =Mean \u00b1Stranded error<\/p>\n

Variant capital letters refer to significant values (P<0.05) between groups.<\/p>\n

Table 2:<\/strong> MIC value of different specific antifungal agents against Rhodotorula mucilaginosa<\/em> \u00a0in the SDA broth for 48 hour\u00a0 at 27\u00baC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Antifungal<\/strong><\/td>\nMIC value <\/strong><\/td>\n<\/tr>\n
Ketoconazole<\/td>\n0.190 \u00b5g\/ml<\/td>\n<\/tr>\n
Clotrimazole<\/td>\n39.06 \u00b5g\/ml<\/td>\n<\/tr>\n
Nystatine<\/td>\n79.65 I.U.\/ml<\/td>\n<\/tr>\n
Itraconazole<\/td>\n1.6 mg\/ml<\/td>\n<\/tr>\n
Fluconazole<\/td>\n2.4 mg\/ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 3: Inhibition Zone diameter (disc-diffusion method) of specific antifungal agents against\u00a0Rhodotorula mucilaginosa<\/em> in the SDA media for 48 hours at 27\u00b0C.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Ke= Ketoconazole (10 \u00b5g), Cl= Clotrimazole (50 \u00b5g), Ny= Nystatin (100 I.U.),<\/p>\n

Fl=Fluconazole (25 \u00b5g), It= Itraconazole (50 \u00b5g), C=Control group.<\/p>\n

The difference of antifungal drugs activity dependent on type\u00a0 and mechanism of action.<\/p>\n

The Mechanism of Action of azole antifungal group (Ketoconazole, Clotrimazole, Itraconazole and Fluconazole) act by inhibit CYP P450 14 \u03b1- demethylase in mould and yeast. CYP P450 14 \u03b1- demethylase enzyme is necessary to convert of lanosterol to ergosterol , While mechanism of action of Nystatin act by Linked to ergosterol in mycological \u00a0membrane causing membrane to become leaky (Myers 2006).<\/p>\n

The use of antifungal drugs in the therapy of fungal diseases can lead to the development of antifungal resistance.<\/p>\n

The resistance of Rhodotorula mucilaginosa<\/em> to Ketoconazole and\u00a0 Clotrimazole may be result from low intracellular antifungal concentration by stimulation of efflux pathway or decreased of antifungal penetration, modification of the specific active sites, up regulation of the specific enzyme and development of bypass pathways (Pem\u00e1n 2009).<\/p>\n

Non-Specific Antifungal Agents<\/strong><\/p>\n

In this study Rhodotorula mucilaginosa<\/em> isolates appeared more susceptible (Inhibition zone) to Acetic acid and H2<\/sub>O2<\/sub> respectively whereas other non-specific antifungal agents (Iodine ,Sodium bicarbonate and Potassium permanganate) did not show any effectiveness (Resistance)\u00a0 , as figure (4) and table (3).<\/p>\n

The lowest MIC value (mg\/ml) of different non-specific antifungal agents used in this study against Rhodotorula mucilaginosa<\/em> showed in acetic acid \u00a0in comparison with other agents, table (4).<\/p>\n

Table 3: Zone inhibition diameter of different non-specific antifungal agents against Rhodotorula mucilaginosa<\/em> in the SDA media in comparison with distilled water. The age of colonies 48 hours at 27\u00baC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Antifungal<\/strong><\/td>\nInhibition Zone<\/strong><\/p>\n

M\u00b1SE<\/strong><\/td>\n<\/tr>\n

Acetic acid (2%)<\/td>\n2.83\u00b10.11\u00a0\u00a0\u00a0\u00a0\u00a0 A<\/td>\n<\/tr>\n
H2<\/sub>O2<\/sub> (3%)<\/td>\n0.71\u00b10.03\u00a0\u00a0\u00a0\u00a0\u00a0 B<\/td>\n<\/tr>\n
Sodium bicarbonate (3%)<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0 C<\/td>\n<\/tr>\n
Iodine (1%)<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0 D<\/td>\n<\/tr>\n
Potassium permanganate (10%)<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0 D<\/td>\n<\/tr>\n
Control<\/td>\n0.00\u00b10.00\u00a0\u00a0\u00a0\u00a0\u00a0 D<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

M\u00b1SE =Mean \u00b1Stranded error<\/p>\n

Variant capital letters refer to significant values (P<0.05) between groups.<\/p>\n

Table 4:<\/strong> MIC value of different non- specific antifungal agents against Rhodotorula mucilaginosa\u00a0<\/em>in the SDA broth for 48 hour\u00a0 at 27\u00baC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Antifungal<\/strong><\/td>\nMIC value<\/strong><\/td>\n<\/tr>\n
Acetic acid<\/td>\n0.62 mg\/ml<\/td>\n<\/tr>\n
H2<\/sub>O2<\/sub><\/td>\n20 mg\/ml<\/td>\n<\/tr>\n
Sodium bicarbonate<\/td>\n120 mg\/ml<\/td>\n<\/tr>\n
Iodine<\/td>\n8 mg\/ml<\/td>\n<\/tr>\n
Potassium permanganate<\/td>\n40 mg\/ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 4: Inhibition Zone diameter (Agar well diffusion method) of specific antifungal agents against\u00a0Rhodotorula mucilaginosa<\/em> in the SDA media for 48 hours at 27\u00b0C.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

A=Acetic acid (2%) , N=Sodium bicarbonate (3%) , H=Hydrogen peroxide (3%) , P=Potassium permanganate (10%) , I=Iodine (1%) \u00a0C=Control.<\/p>\n

Some non-specific agents used in this study have antifungal effect but variety in cellular yeast damage depended on type, concentration and mechanism of action of antifungal agent used .<\/p>\n

CH3COOH has been mostly used in medical fields for more than 6000 years for the disinfection of wounds infections \u00a0and especially as an antiseptic agent in the treatment and prophylaxis of deferent microorganisms . The antimicrobial effect of acetic acid, even at concentrations as low as 5%, has been attributed to its ability to decrease pH both in intra- and extracellular conditions and therefore to altering the cell membrane’s transportation and integrity, as well as enzymatic activity, and even precipitating cytoplasmic proteins(Ryssel 2009) , Whereas antimicrobial activity of H2<\/sub>O2<\/sub>\u00a0acts as an oxidant by producing \u2022<\/sup>OH which linked with macromolecules of cell example lipids, proteins, and DNA. It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted (Block. and\u00a0 Peroxygen 1991).<\/p>\n

NaHCO3<\/sub> is used as an alkalinizing material for glutaraldehyde sterilization of medical apparatus . In watery solution, it ionizes to form sodium ions and bicarbonate ions ions. The dissociation of HCO\u2212<\/sup>3<\/sub> ions from NaHCO3<\/sub> increases the pH of a solution (Enfors\u00a0 and Molin 1975).<\/p>\n

I ions acts by lowering the O2<\/sub> concentration in aerobic microbes cell . I ions interacts with the respiratory chain of the microbes by blocking the transport of electrons through electrophilic reactions with the respiratory chain enzymes. I also interacts high affinity with the proteins of the cytoplasm membrane in a form with a positive (H2<\/sub>O +I) or neutral (I2<\/sub> or HOI) charge (Maris ,1995).<\/p>\n

KMnO4<\/sub> is a mild antiseptic with astringent properties. It is used in dermatology to treat weeping skin conditions (Anderson 2003). KMnO4<\/sub> oxidation of organic pollution also may induce biodegradability and\/or toxicity to microbes \u00a0(Bowers 1992), Also\u00a0 KMnO4<\/sub> act on the cell membrane phospholipids, containing unsaturated fatty acids, may be sensitive to MnO–<\/sup>4<\/sub> oxidation at C=C bonds that led to defect of cellular components (Bui and Cotton 2002).<\/p>\n

All non-specific antifungal agents which resistance to Rhodotorula mucilaginosa<\/em> may be resulted from:-<\/p>\n

First cause<\/strong><\/p>\n

Fungal resistance to chemical biocides is very limited. One commonly accepted theory about the mechanism of fungal resistance to biocides involves natural (intrinsic) resistance. A fungal cell may have an innate ability to present a permeability barrier to one or more biocides, or to inactivate a biocide due to the presence of existing enzymes (McDonnell and Russell 1999).<\/p>\n

Second cause<\/strong><\/p>\n

The defiance mechanism of\u00a0Rhodotorula mucilaginosa<\/em> to Potassium permanganate, Iodine and Sodium bicarbonate may be resulted from lowest concentrations were used to treatment of Rhodotorula mucilaginosa for that may be Increasing the concentration of these substances may increase their effectiveness against this yeast<\/em>.<\/em><\/p>\n

Conclusion<\/strong><\/p>\n

Specific antifungal drugs<\/strong><\/p>\n

Rhodotorula mucilaginosa<\/em> were highly sensitive to Clotrimazole and Ketoconazole and Nystatin and resistance to Fluconazole and Itraconazole.<\/p>\n

Non-Specific antifungal drugs<\/strong><\/p>\n

Rhodotorula mucilaginosa<\/em> were sensitive to Acetic acid and H2<\/sub>O2<\/sub> and resistance to Sodium bicarbonate, Iodine and Potassium permanganate.<\/p>\n

Recommendation<\/strong><\/p>\n

Molecular study of cellular defect of Rhodotorula mucilaginosa<\/em> after specific and non-specific antifungal agents treated.<\/p>\n

Studying Rhodotorula mucilaginosa<\/em> resistance to specific and non-specific antifungal agents in this study.<\/p>\n

Studying the direct use of antifungal agents (Clotrimazole, Fluconazole, Nystatin, 2% Acetic acid and 3% H2<\/sub>O2<\/sub>) in a sample of patients infected by Rhodotorula mucilaginosa athletes’ foot.<\/em><\/p>\n

Acknowledgments<\/strong><\/p>\n

The authors are <\/em>acknowledge to the <\/em>Al-Furat Al-Awsat Technical University \/Technical institute of Babylon in helping us to provide the providing the requirements of research.<\/em><\/p>\n

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