@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2018;24(1):1-6

Background
One of the most common opportunistic infections in the skin, mouth, digestive system, blood vessels and vaginal system is candidiasis which is caused by candida species [1, 2]. Candida albicans is the cause of most infections [3].
Previous Studies
… [4-6]. Among the existing biodiversity, bacteria are focused due to the diversity of population, as well as the ability to produce active metabolites with antimicrobial properties and have great value in biological control [7]. One of the most important bacteria that has this potentiality is bacillus that can prevent the growth and proliferation of fungi through the production of antifungal metabolites [8]. … [9-12]. According to studies conducted by the Canadian and US Healthcare Organizations in 2012, Bacillus amyloliquefaciens and its metabolites, apart from a transient inflammatory response, have no adverse effects or side effects on humans, vertebrates, invertebrates, plants and aquatic animals [14]. Therefore, recently, the use of these compounds as a biocontrol agent has become very important [15]. Iturins are a group of potent antifungal lipopeptides against a wide range of molds and pathogenic yeasts while they have little toxicity for mammals. … [16].
Aim(s)
According to studies conducted in databases, there is no domestic production for Iturins in Iran and Iran is dependent on this. The aim of this study was to extract and evaluate the antifungal effects of lipopeptide compounds by Isfahan native Bacillus amyloliquefaciens M13RW01 against Candida albicans, Candida glabrata, Candida Krusei, Candida parapsilosis and Candida tropicalis.
Research type
This study is experimental.
Research society, place and time
In this research, the antifungal activity of Bacillus amyloliquefaciens M13RW01 which was isolated by Naghdifar et al. in 2015 from Isfahan, was investigated [17].
Sampling method and number
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Used devices&materials
This strain of bacteria was cultivated in Nutrient agar medium (scharlau, Spain) and modified Peptone-Glucose-Yeast extract medium (peptone-glucose-yeast extract) (including 10gr/l peptone, 10 g/l glucose, 10 g/l yeast extract, 5g/l sodium chloride, 1.5 g/l dipotassium phosphate (K2HPO4), magnesium sulfate heptahydrate (MgSO4,7H2O), distilled water (PH=7) and heated in 37 ° C for 72 hours [9]. Standard samples of using Candida including Candia albicans CBS2747, Candida glabrata ATCC9003, Candida krusei CBS573, Candida tropicalis CBS94, and Candida parapsilosis ATCC90018 were prepared from Tehran University of Medical Sciences. Fungal strains were cultivated in plates containing Potato Dextrose Agar (PDA, Scharlau, Spain) and heated for 48 hours in 25 ° C. To extract the Iturin A from the Bacillus amyloliquefaciens M13RW01, 20 ml of PGY medium, prepared in a 100 ml flask was inoculated with 2 ml of suspension of the Bacillus amyloliquefaciens, equivalent to 0.5 McFarland. This medium was incubated in a shaker incubated at a temperature of 30 ° C and 150 ° rpm. After 18 hours, 12 ml of this medium was added to 100 ml of PGY medium by sterile pipettes and under laminar hood and incubated in incubator at 30 ° C and 150 rpm for 72 hours. Then, the medium was divided into falcon tubes under sterile conditions and centrifuged for 40 minutes in refrigerated centrifuges at 16000 x g in 5°C. The sedimentation was discarded and the supernatant was filtered by a 0.22 μm syringe filter. The PH of the achieved filtered substance was reached to 2 by adding 6 molar of hydrochloric acid. To this end, each time some acid was added to the filtered substance by sampler and after moving the container to unify the filtered substance, PH was being measured. The filtered substance was kept in the refrigerator for 24 hours at 5 ° C. Then the filtered subtance containing sediment was divided into falcon tubes and was centrifuged for 30 minutes by refrigerating centrifuge at 5 ° C and 16000 x g. This time, the supernatant was discarded. The obtained sediment was centrifuged and washed two times with distilled deionized sterilized water for 10 minutes at 5 ° C and at 16000xg. All of the above steps were carried out in sterile conditions underneath the laminar hood in the vicinity of dry ice and flame [4, 18-20]. A 0.5 mg extracted sediment containing antifungal metabolites was dissolved in a 1 ml solution of 50% methanol solution and centrifuged in a refrigerator for 10 minutes at 16000 x g at a temperature of 5 ° C and then the supernatant was filtered with a 0.22 μm syringe filter. The final filtered fluid was a one-milliliter methanolic extract containing the Iturin family [18]. In order to investigate the antagonism effect of extracted lipopeptide on five Candida species based on gel-diffusion technique, a suspension of yeast fungus equal to 0.5 Mcfarland was prepared and 20 μl of this suspension was added to the surface of the PDA medium by a sampler and each of yeast fungus were densely cultivated on PDA medium on the entire surface of the plate by sterile swabs. Then, by a sterile Pasteur pipette, in the center of PDA-containing plates, a 6 mm well was formed and the wells were filled with 50 μl of methanolic extract containing lipopeptide. Then these plates were incubated at a temperature of 30 ° C for 24 hours. This test was conducted for Candida albicans, Candida glabrata, Candida Krusei, Candida parapsilosis, and Candida tropicalis. As a positive control, 50 μl of IturinA (MO, St Louis, Sigma-Adrich; US) and as negative control, 50 μl methanol 50% were poured in two wells. Experiments were repeated three times [21]. In order to investigate the effect of extracted metabolite on the formation of germ tubes in Candida albicans, 5mm of human blood was prepared and poured into the test tube. Then, it took 5 to 10 minutes to drain the blood into the tubes. To isolate the serum, a tube containing blood was centrifuged for 10 minutes at 5000 rpm. Separately, 0.5 ml of serum was injected into three sterile tubes; the first tube contained serum and 50 μl of methanol solution 50% as negative control; the second tube contained serum and 50 μl of IturinA (Sigma-Aldrich, St Louis, MO, USA) as a positive control, and the third tube contained 50 μl extracted lipopeptide metabolite and serum. Then, the contents of each tube were well mixed with the shaker. Then, equal to a loop, cultivation of Candida albicans was entered into three tubes and the tubes were incubated for 2.5 hours at 37 ° C and 180 rpm. Finally, with the aid of a sampler, a drop of uniform content of the tubes was placed on a sterile lamina. After the lamination, the formation of the germ tube was examined under a microscope. The experimental was repeated three times [19]. In order to determine MIC (minimum inhibitory concentration) and MFC (maximum fungal concentration), Microdilution method was used [22].
Findings by Text
The extracted lipopeptide, similar to the Iturin A purchased from Sigma, had no antagonistic effects on Candida albicans, Candida tropicalis, Candida glabrata, Candida Krusei, and Candida parapsilosis (Figures 1 and 2).Candida albicans was able to form the germ tube after 2.5 hours of incubation in the blood serum without IturinA or without the bacterial-derived metabolite (Figure 3), but the extracted lipopeptide composition similar to that of IturinA inhibited the formation of the germ tube in the Candida albicans fungus (Figures 4 and 5). In addition, effects of surface degradation of Candida yeast cells were observed by the extracted compound (Figure 4).Despite the negative results of creating wells for yeast fungi, in order to control more, MIC and MFC were measured for Candida yeast fungi. The results showed the fungi growth in all tubes and dilution of the lipopeptide, so that in all the tubes (similar to the negative control tube without Iturin A), the sediment was observed which caused turbidity after shaking of the tubes. Based on the results obtained from the determination of MFC level, there was no evidence of inhibition of Candida yeast fungi growth in any dilution of extracted lipopeptide.
Main comparison to the similar studies
… [23-28]. According to studies by Magetdana et al. [23], Magetdana and Peypoux [24], and Timon et al. [25], it was shown that the lipopeptide compounds from the Iturin family, by forming the Iturin-phospholipid-ergosterol complex, increase lipid permeability of the fungi cell membranes by forming the ion pores and cause ions and essential micro molecular compounds of cells to leak out. It is possible that with the same mechanism, Iturin A prevents the production of germ tubes in Candida albicans. However, more research is needed to prove this. No study has ever been conducted on the prevention of germ tube in Candida albicans by Iturin. In only one study, the inhibition effect of Iturin A extracted from Bacillus subtilis YM10-20 has been shown on the germination of the spores of the penicillium roqueforti fungus [19]. ... [26, 29].
Suggestions
In order to know the antifungal activity of other lipopeptides compounds of the Iturin family, it is suggested that the effects of the compounds extracted from bacillus species and other strains be investigated on the yeast fungi. Also, antifungal activity of lipopeptides compounds of Bacillus amyloliquefaciens M13RW01 be investigated on other yeasts and fungi.
Limitations
One of the limitation of this study is that Iturin produced by used Bacillus amyloliquefaciens M13RW01 is probably not mycosubtilin type.
Conclusions
Lipopeptide metabolites extracted from amyloliquefaciens M13RW01, have no inhibitory or fetal effects on the studied species of Candida fungi, but inhibit the production of germ tubes by Candida albicans yeast.
Acknowledgments
Thanks to Ms. Fatemeh Khosrovanipour, for her sincere help in purchasing and providing standard Iturin ASigma-Aldrich, St Louis, MO) from Sigma Company in Germany.
Conflict of interest
Non-declared
Ethical Permissions

Funding Sources
This study was extracted from the master's thesis with the code 17230507931039 approved by the Islamic Azad University of Falavarjan.

TABLES and CHARTS

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