Journal de Mycologie Médicale (2011) 21, 188—197
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles ´ tabolites antimicrobiens actifs sur la membrane de micromyce ` tes Criblage de me ´ sicules chromatiques de phospholipide/polydiace ´ tyle ` ne du sol `a l’aide de ve M. Mehravar a,b, S. Sardari a,* a Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute, No. 69 Pasteur Ave., Tehran, 13164, Iran b Department of Microbiology, Faculty of Science, Shahed University, Tehran, Iran
Received 11 April 2011; accepted 8 July 2011
KEYWORDS Chromatic vesicles; Antimicrobial membrane activity; Soil microfungi; Iran
Summary Objective. — The focus of this study is screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles. Materials and methods. — In this work, soil samples were collected from desert, forest, farm, lake shore and mineral zones of Northern and Central parts of Iran. These parts were not studied for antimicrobial potential of the soil isolates producing metabolites with membrane activity in particular, from microfungi. In the primary screening that was performed to evaluate the antimicrobial activity, isolates were analyzed in terms of their general inhibition effects to indicator strains including Escherichia coli, Candida albicans, and Saccharomyces cerevisiae. In the secondary screening, isolates producing membrane-active metabolites were determined using a Rapid Chromatic Detection method. The chromatic technology is simple and this method provides a rapid and easy evaluation of interactions between antimicrobial membrane-active metabolites and lipid layers of vesicles as well. Results. — A total number of 59 species of fungi was isolated from the soil samples. It has been found that 20 isolates were effective against indicator strains. Based on color and fluorescence changes that are easily identified by the naked eye and fluorescent microscopy and can be recorded by UV-Vis spectrophotometery, one fungus showed antimicrobial membrane-activity effect against some of the indicator strains. This isolate was identified to the genus level that belonged to Aspergillus.
* Corresponding author. E-mail address: [email protected] (S. Sardari). 1156-5233/$ — see front matter # 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2011.07.005
Screening of antimicrobial membrane-active metabolites of soil microfungi
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Conclusion. — As resistance is barely developed against membrane-active antibiotics, in this paper, we demonstrated the application of the chromatic vesicle model for screening of antimicrobial membrane-active metabolites as potential new antibiotics from soil microfungi. # 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Vésicules chromatiques ; Activité membranaire antimicrobienne ; Micromycètes du sol ; Iran
Re ´sume ´ Objectif. — L’objectif de cette étude est de cribler des métabolites antimicrobiens de micromycètes du sol actifs sur la membrane en utilisant des vésicules de phospholipide/polydiacetylene (PDA). Mate´riels et me ´thodes. — Pour ce travail, des échantillons de sol ont été collectés dans des zones du nord et du centre de l’Iran : dans le désert, les forêts, les fermes, les bords de lacs et des zones minérales. Ces zones n’avaient pas été étudiées pour le potentiel antimicrobien des isolats du sol, particulièrement en ce qui concerne les micromycètes produisant des métabolites actifs sur les membranes. Dans un premier crible qui a été effectué pour évaluer l’activité antimicrobienne des isolats de micromycètes ont été analysés pour leur effet inhibiteur sur des souches tests d’Escherichia coli, de Candida albicans et de Saccharomyces cerevisae. Dans un second crible, les isolats produisant des métabolites actifs dans les membranes ont été testés à l’aide d’une méthode rapide de détection par la couleur. Cette technologie est simple et permet une évaluation facile et rapide des interactions entre les métabolites antimicrobiens actifs dans les membranes et ainsi que des couches lipidiques de vésicules. Re ´sultats. — Cinquante-neuf espèces de micromycètes ont été isolées à partir des échantillons du sol. Il a été trouvé que 20 isolats sont efficaces contre les souches tests. À partir des changements de couleur et de fluorescence qui sont facilement identifiables à l’œil nu et en microscopie à fluorescence, et enregistrés par spectrophotométrie, un isolat a montré qu’il possédait une activité membranaire antimicrobienne contre plusieurs souches tests. Cet isolat a été identifié comme appartenant au genre Aspergillus. Conclusion. — Comme la résistance contre des antibiotiques actifs sur les membranes est peu connue, nous avons démontré dans ce travail l’application efficace du modèle de vésicules chromatiques pour cribler des métabolites antimicrobiens actifs sur les membranes. Ces métabolites antimicrobiens sont donc des antibiotiques potentiels provenant des micromycètes du sol. # 2011 Elsevier Masson SAS. Tous droits réservés.
Introduction The repeated emergence of antibiotic-resistant microorganisms is a problem in public health and its economic impact has recently become a concern as well. Microorganisms usually possessed the ability to protect themselves from antibiotics [22]. Therefore, new antimicrobial molecules are necessary to confront this problem; specially anti-infective agents with fewer side effects, shorter lengths of treatment and in particular, drugs with new and fewer resistantprone targets to antimicrobial activity [3,32,33]. Membrane as a new and potential target is noteworthy for antibiotics’ action [2]. It has two significant advantages as a target for an antimicrobial agent: drugs that anchor the microorganism membrane show a fast and extensive antimicrobial effect because of allowing easy target access; another advantage is having a lower tendency to resistance because of difficulty in modifying their components in a way that is compatible with bacterial survival [2,35]. New ways to discover novel antibiotics have been pursued, such as genomics route, and combinatorial chemistry; but it has led to the introduction of a few marketed antibiotics [7]. Novel methods that are used to screen common microbes have been described [8,32]. Microorganisms are capable of producing natural products with widely divergent chemical structures [37]. Natural products have been the
main source of antibiotics and played a fundamental role in antibiotic drug discovery in the past [4,23]. Natural products are still the most important source for promising drug candidates [21]. Remarkably, 70 out of the 90-marketed antibiotics in the years 1982—2002 originated from natural products. Natural products versus synthetic molecules are still a worth promise [22] Antibiotics producing microorganisms have been isolated from the most diverse habitats [32]. Soil, in particular, is an ecological environment in which the great number of microorganisms has a constant interaction that partially determines physical, chemical and biological properties of this habitat [33]. Thus, this environment has a tremendous biodiversity that can be screened for antibiotic production [32]. Soil fungi such as Penicillium have had a high impact in producing clinical antibiotics [24,30]. Screening of antibiotics producing soil microfungi have been reported in several studies [1,10,25,27,32]. New microorganisms and products have been derived from uninhabited and undisturbed areas of the world such as China, Australia, Antarctica, India and Jordan [33]. Thakur et al. [33] and Takahashi et al. [32] studied antimicrobial activity of 110 actinomycete strains isolated from the soil of Indian protected areas and 200 fungal strains isolated from Brazilian soil respectively. In the mentioned surveys, antimicrobial activity of metabolites produced by the soil microorganisms has not been simultaneously studied with their membrane activity.
190 Various membrane-active compounds are released by microorganisms to their environments [31,34] that strongly interact with membrane components of the host cell [29]. The aim of this work is to evaluate antimicrobial membrane activity of 59 fungal isolates from Iranian soil samples by vesicle membrane model, phospholipid/polydiacetylene (lipid/PDA) vesicles, as a bioassay target. Lipid/PDA vesicles are aggregates that self-assembled in aqueous solutions and contain organized lipids and polymers. Vesicular particles can be useful biosensors due to their relative ease of preparation, stability, closely mimicking the cell membrane [15]. Previous studies indicated that the lipids and PDA most likely form interspersed microscopic phases within the vesicles and phospholipids incorporated within the PDA matrix have a bilayer structure, the dominant lipid organization within cellular membranes [19]. Particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids as colorimetric biosensors were shown to exhibit color changes following interactions with antimicrobial membrane-active metabolites. The color changes occur because of the structural perturbation of the lipids following their interactions with antimicrobial membrane-active compounds [17]. Previous studies have shown that biological processes leading to structural perturbations at the PDA vesicle interface, including ligand—receptor recognition [5], pH changes [6], and enzymatic catalysis [26] are responsible for the blue-red transitions in the lipid/ PDA vesicles. Here, we used lipid/PDA vesicles as a biosensing tool for detection of membrane-active metabolites produced by soil microfungi isolated from soil.
Materials and methods
M. Mehravar, S. Sardari evenly over the surface of Malt Extract agar (MEA, Merck, Germany) for fungi isolation that was complemented with 100 mg/ml chloramphenicol to inhibit bacterial contamination. Plates were incubated at 28 8C, and monitored after 48, 72, and 96 h for growing fungi. Fungi colonies were identified according to morphological characteristics such as color of colony and medium at its vicinity, surface texture, shape and size characters, aerial mycelium, and spore production. Repeated streaking on the same agar plates led to purified fungi colonies. All of these strains were isolated on the same media supplemented with chloramphenicol (100 mg/ml) to inhibit bacterial contamination. All isolates grew on MEA medium showed characteristic morphologies of fungi. Colony appearance, size and color were visible by the naked eye. Mycelia and spore production was studied by optical microscopy. Pure colonies of fungi were further studied by light microscopy using slide culture method [20]. Plates containing pure cultures were stored on test tubes containing MEA at 4 8C during two months until further examinations, and in a freezer at 70 8C in the presence of glycerol (50%, v/v) for a longer storage period.
Indicator strains Two yeasts, including Candida albicans ATCC 10231, Saccharomyces cerevisiae BY4743 and one bacterium, Escherichia coli ATCC 25922, were used to determine the antimicrobial activity of the isolated fungi strains. The above-mentioned yeasts were cultured in Sabouraud Dextrose broth (SDB) (Difco, USA) at 28 8C for 48 h and E. coli was cultured in Brain Heart Infusion broth (BHI) (Merck, Germany) at 37 8C for 24 h.
Source of fungi Screening of isolates for antimicrobial activity Soil samples were collected from Different areas in Iran. These habitats included desert, forest, farming, shore and mineral soils of Iran that respectively were collected from Ghaem Salt Mine road (35848.09900 N and 051825.9400 E), Garmsar, Semnan Province, pH: 7.2, non-rhizosphere soil; Asalem to Khalkhal road (37837.98500 N and 049802.14300 E), Guilan Province, pH: 6.9, rhizosphere soil; Kelibar cemetery (038852.13600 N and 047802.23000 E), East Azerbaijan Province, pH: 7, non-rhizosphere soil; Orumieh lake shore (37840.78800 N and 045802.74200 E), West Azerbaijan Province, pH: 8.7, non-rhizosphere soil; Tehran-Tabriz highway (38809.43500 N and 046845.30700 E), Tabriz, pH: 7.5, nonrhizosphere soil. The samples were taken up from a depth of 15 cm and three samples for each place were mixed. Samples were placed in sterile bags, closed tightly and stored in a refrigerator.
Isolation and storage of fungi For each collected sample, one gram of the soil was suspended in 10 mL of normal saline (NaCl 9 g/L) then incubated in an orbital shaker incubator at 28 8C with shaking at 200 rpm for 30 min. Mixtures were allowed to settle, and serial dilutions up to 104 were prepared using sterile normal saline and agitated with the vortex at the maximum speed. An aliquot of 0.1 ml of each dilution was taken and spread
Malt Extract agar plates were prepared and spot inoculated with fungi isolates in 4 points. After 4 days of incubation at 28 8C the plates were seeded with test organisms by overlay agar technique [33] in which, spot inoculated colonies were covered with a 0.75—0.8% agar layer of Sabouraud Maltose medium (for yeast) and BHI medium (for bacteria). The antimicrobial activity was observed after 48 h incubation at 28 8C for yeast and 24 h incubation at 37 8C for bacteria. Amphotericin B and chloramphenicol used as control antibiotic against the fungal and bacterial test, respectively. The antimicrobial activities were measured by the determination of the size of the inhibition zone.
Screening of isolates for detection of membrane activity Isolates which produced antimicrobial substances against target strains were studied for membrane activity by a Rapid Chromatic Detection method, using a Biomimetic Polymer Sensor, i.e., polydiacetylene (PDA) in conjunction with phospholipid as a membrane model vesicle [16,17,19,28,31]. The detection is based on the interaction of membrane-active metabolites secreted by isolates in broth culture extract, with phospholipid/polydiacetylene Vesicles. This detection platform generated dramatic visible color changes accom-
Screening of antimicrobial membrane-active metabolites of soil microfungi panied by intense fluorescence emission that are induced by membrane-active molecules secreted by isolates [31].
Construction of the vesicles To prepare polymerized vesicles, synthetic phospholipid, Dimyristoylphosphatidylcholine (DMPC), and The diacetylenic monomer, 10,12-tricocosadiynoic acid (TCDA), (both purchased from Sigma, USA) which had been separately dissolved in dichloromethane (one mg/ml) were mixed at the 2:3 molar ratio and prepared at a concentration of one mM. Lipids were dried together using vacuum to remove dichloromethane completely, then a white thin film of the lipids on the bottom of glass was yielded. Same amount of deionized water was added (typically to obtain one mM concentration). The suspension was sonicated at 70 8C for 8—9 min in a sonicating bath. The resulting vesicle solution was cooled to the room temperature and kept at 4 8C overnight. Prior to polymerization, the vesicles warmed to the room temperature and polymerized by irradiation at 254 nm for 40—60 s, resulting in intense blue color appearance due to polymerization of the diacetylene units [19].
Submerge culture and Organic extraction Isolates that showed antimicrobial activity against test organisms in the agar medium were grown in submerged culture in 500 ml flasks containing 100 ml of Malt Extract broth. A two-days-old broth culture grown on Malt Extract broth was used to inoculate the flasks. These cultures were grown in a rotary shaker (Adolf Kuhner AG, Switzerland) at 200 rpm, 28 8C, for 14 days. The resulting culture broths obtained following growth of each isolate in the culture media were separated from the biomass by centrifugation at 4000 rpm (Sigma, Germany) for 15 min. The supernatant was filtered through 0.45 mm membrane filter. Filtrate was used for extracellular antimicrobial activity by the agar well diffusion method against indicator strains [33]. Sterile cork borer was used for punching six mm diameter wells in appropriate agar medium plates previously seeded with one of the indicator strains. A volume of 100 mL of the supernatant of each isolate was dispensed in each well. Plates were kept at 4 8C for at least 2 h to allow the diffusion of produced antimicrobial metabolites. The diameter of an inhibition zone was determined after 24 h of incubation at 37 8C for bacteria, and after 48 h at 28 8C for yeasts. Antimicrobial compound was recovered from the culture of each active isolate by solvent extraction with ethyl acetate. Ethyl acetate was added to the centrifuged broth at the ratio 1:1 (v/v) along three times and shaken vigorously for five min in each time. The organic layers were collected and the organic solvent was evaporated to dryness in a rotary vacuum evaporator at 40 8C. The remainder was dissolved in 1 ml dimethylsulfoxid (DMSO). Resulting solution was studied for membrane activity.
Interaction of phospholipid/PDA vesicle solution with antimicrobial extracts for detection of membrane-active metabolites In order to determine the effect of the extract of each strain showing antimicrobial activity that was dissolved in
191
DMSO, phospholipid/PDA vesicles were used as the membrane model to detect of membrane-active metabolites. As mentioned previously [19] polydiacetylene polymers used in model vesicles have some important characteristics such as blue-red color transition under external perturbation that can be visible by the naked eye and fluorescence irradiation of red components in the emission range (500—550 nm) that can be recorded by fluorescence microscopy (Nikon, E200, Japan). Color transitions can be quantitatively recorded by UV-Vis measurements at a wavelength span between 300—700 nm and calculating CR% (colorimetric response). UV-Vis measurements: a quantitative value for the extent of blue-to-red color transition is given by the colorimetric response (%CR), which is calculated from the visible absorbance spectra acquired for the vesicle solutions. The colorimetric response is defined:
CR ¼
PB0 PB1 100 PB0
where PB ¼ A
Ablue blue þAred
A is the absorbance at either the ‘‘blue’’ component in the UV-Vis spectrum (640 nm) or ‘‘red’’ component (500 nm), PB0 is the red/blue ratio of the control sample (before induction of color change), and PBI is the value obtained for the vesicle solution after addition of the tested compound [15]. Test tubes containing 300—400 mL vesicle solutions and two mM Tris solution pH 8.5, reached the volume of 1000 mL by adding bioactive extracts of strains. The pH in the solutions was 8.5 in all experiments [18]. Tubes incubated at 28 8C for one hour. Amphotericin B and tetracycline were used as positive and negative control respectively.
Determination of the antibacterial spectrum of membrane active isolate In order to determinate the antimicrobial activity spectrum, according to the above-mentioned method the membrane active isolates were subjected to submerged culture to study antibacterial activity by agar well diffusion method against test bacteria including Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi PTCC 1609, S. pyogenes ATCC 8668, Bacillus subtilis PTCC 1365, Enterococcus faecalis ATCC 29212. Finally, membrane active isolates has been grown on sabouraud dextrose agar plate to study Morphologically and culturally such as colony size and texture, color, pigment production by the naked eye and optical microscopy [9]. Slide culture method followed by lactophenol cotton blue (Sigma, USA) staining was performed to observe aerial mycelia and conidiophores by light microscopy magnification 100 (Zeiss, Standard 20, Germany). In the present work, we screened fungi from north and central Iran’s unexplored habitats. These areas represent a diverse and unscreened ecosystem and the least investigated area for the isolation of membrane active metabolites-producing fungi. No scientific work has been carried out on the membrane active metabolites-producing fungi in various ecosystems of Iran.
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M. Mehravar, S. Sardari 20 mm inhibition zones. Eight isolates showed inhibition zones less than 10 mm against at least one test indicator.
Results Isolation of microfungi
Antimicrobial membrane activities of isolates Using the selective media and cultivation conditions described previously a total of 59 different microfungal isolates from several soil samples which, collected from desert, forest, farming, shore and mineral soil in north, west and central zones of Iran was obtained. The forest and desert soils give the higher number of microfungi isolates (22 and 13 isolates, respectively) with respect to other soils.
Antimicrobial activities of isolates All the isolates were tested for their ability to produce antimicrobial substances against three indicator strains included one Gram negative bacteria and two yeasts. We found 20 (33%) out of 59 fungi isolates to have in vitro antibacterial activity against at least one of the indicator strains. The results of the antimicrobial activity of active isolates are shown in Table 1. Six of them inhibited the growth of at least one indicator strains with more than
Artificial lipid/PDA vesicle solution has an intense blue color appearance. Microbial extracts separating from broth culture of active isolates before affecting on vesicle solution, were studied for their antimicrobial activity on indicator strains that one of them had membrane activity on lipid/PDA vesicles. By interacting the antimicrobial extracts with lipid/PDA vesicles and disrupting the vesicles, blue-red color transition was recognizable by the naked eye as shown in Fig. 1, as well as fluorescence emission can be recorded by fluorescence microscopy (Fig. 2). Published data further imply to the contribution of changes in fluidity within the lipid parts in inducing the blue-red transitions [19]. Using lipid/PDA vesicles for biosensing applications has been based on the observation that numerous membrane active compounds interacting with the lipid domains can cause to the blue-red transformations of
Table 1 Antimicrobial activities of soil microfungi isolates grown on Malt Extract agar medium. ´ antimicrobienne des isolats de micromyce ` tes du sol dans un milieu de croissance en ge ´ lose. Activite Isolate No.
Total strains isolated (%)
Number of active isolates (%)
The indicator strains and inhibition zone diameters (mm) on Malt Extract agar medium E. coli
Desert soil 08-1-1 08-1-2 08-1-3 08-1-4 08-1-5 Forest soil 08-20-1 08-20-2 08-20-3 08-20-4 08-20-5 Farming soil 08-29-1 08-29-2 08-29-3 08-29-4 Shore 08-35-1 08-35-2 08-35-3 Mineral soil 08-31-1 08-31-2 08-31-3 Control Chloramphenicol Amphotericin B Total
13 (22)
22 (37)
8 (14)
9 (15)
7 (12)
59
C. albicans
S. cerevisiae
5 (38) — 10 — 20 —
12 — 8 14 —
8 — — — 6
— — — 12 —
20 10 8 10 > 20
18 — — — 18
10 6 6 8
5 16 — —
— 20 — —
> 20 > 20 —
— 8 12
— 8 10
— — 8
— — —
6 6 —
23 —
— 18
— 22
5 (23)
4 (50)
3 (33)
3 (43)
20 (34)
Screening of antimicrobial membrane-active metabolites of soil microfungi [(Figure_1)TD$IG]
193
Figure 1 Color changes produced by interacting of vesicle solution with membrane active metabolites secreted by active microfungi. Results after adding: a: deionized water (control solution); b: antimicrobial extract of a non-membrane active microfungus 08-35-2 (according to Table 1); c: antimicrobial extract secreted by membrane active microfungus 08-29-2 (according to Table 1); d: antibiotic tetracycline as negative control; e: antibiotic amphotericin B as the positive control. ´ sicules avec les me ´ tabolites actifs dans les membranes et Changements de couleur produits par l’interaction de solution de ve ´ cre ´ te ´ s par les micromyce ` tes. Re ´ sultats apre ` s avoir ajoute ´ : a : l’eau distille ´ e (solution te ´ moin) ; b : des extraits antimicrobiens se ´ cre ´ te ´ s par des micromyce ` tes 08-35-2 non actifs dans les membranes (correspondant au Tableau 1) ; c : des extraits antimicrobiens se ´ cre ´ te ´ s par des micromyce ` te 08-29-2 actifs dans les membranes (correspondant au Tableau 1) ; d : l’antibiotique te ´ tracycline se ´ moin ne ´ gatif) ; e : l’antibiotique amphote ´ ricine B (te ´ moin positif). (te
the polymer that means PDA polymers in the mixed vesicles essentially work as the reporter module for membraneactive molecules [15]. Red components have fluorescence irradiation in 500— 550 nm; the intense disruption induces a stronger red color that is led to irradiate more fluorescence. Amphotericin B is an antifungal that its main target is the sterol presented in fungi membrane but amphotericin B in second preference targets lipids of membrane. As membrane-activity test results depicted in Fig. 2 color changes were only produced in tubes c and e, which belong to strain 08-29-2 and amphotericin B, respectively. Absorbance of blue and red components with calculation of Colorimetric response has been shown in Fig. 3. According to the results, strain 08-29-2 isolated from farming soil had membrane-activity effect. The amounts of colorimetric response for control, amphotericin B, tetracycline and strain 08-29-2 were calculated 0, 90, 5.5, 88%.
Determination of the antimicrobial spectrum The results of antimicrobial spectrum of strain 08-29-2 on agar medium based on the diameter of inhibition zones were against S. aureus: nine mm; S. pyogenes: 10 mm; B. subtilis: seven mm; S. typhi: six mm; E. faecalis: 5 mm; Ps. Aeruginosa: 6 mm; E. coli: 6 mm; C. albicans: 16 mm; S. cerevisae: 20 mm. Considering these results strain 08-29-2 has produced a larger inhibition zone (more than 10 mm) against yeasts. It shows that extract of strain 08-29-2 have rather dominant antifungal effect.
Characteristics of membrane active isolates Membrane active isolate 08-29-2 isolated from farming soil samples was studied on SDA medium. The colony of this strain was wide in size with a little bulge, globose shape, cottony, cream-white surface, yellowish-gold reverse, the
surface was smooth and this strain produced a yellow pigment on SDA. Aerial mycelia and conidiophores were observed by light microscopy. Considering the characteristics results and comparing them with other fungi [36], we identified the membrane active isolate as Aspergillus sp. Further study towards identification of the species is planned in our group.
Discussion The resistance of various pathogenic bacteria and fungi to existing bioactive secondary metabolites is an urgent focus of research, and new antifungal and antibacterial molecules are necessary to combat these pathogens. Furthermore, over the past 30 years a number of new infectious diseases have been discovered. Increasing number of duplications in screening for antimicrobial metabolites from natural products and the urgent demand for new leading structures in pharmacology have enforced the search for metabolites in microflora of undisturbed habitats and with potent targets in the mechanism of action [22]. Membrane is a potential target for antibiotic action, because there is a lower tendency that resistance can be developed against this target. Drugs that affect in the microorganism membrane seem to have maximum potential because they show a fast and extensive antimicrobial effect and are less prone to a resistance development [2,35]. In this study, we focused on fungal isolates. It is estimated that about 1.5 million fungi are present in the world, but 5% of these have yet to be discovered [11— 13]. Fungi are one of the major antibiotic-producing organisms, and one of the most diversified groups of organisms. The search for antibiotics from fungi is promising and six of the world’s 20 best selling drugs are indicated to be fungal derived [13]. In the present work, we screened fungi from north and central Iran’s unexplored habitats. These areas represent a diverse and unscreened ecosystem and the least investigated
194 [(Figure_2)TD$IG]
M. Mehravar, S. Sardari
Figure 2 Microscopy images of vesicles solution. a: fluorescence microscopy image of vesicles solution in emission 550 nm after adding antimicrobial extract of a membrane active microfungus 08-29-2 (according to Table 1); b: fluorescence microscopy image of control vesicles solution (vesicle solution after addition of deionized water); c: light microscopy of vesicle solution after adding antimicrobial extract of a membrane active microfungus 08-29-2; d: light microscopy of control vesicle solution (vesicle solution after addition of deionized water). ´ sicules. a : image en microscopie `a fluorescence d’une solution de ve ´ sicules Images en microscopie d’une solution contenant des ve ´ mission : 550 nm) apre ` s avoir ajoute ´ l’extrait antimicrobien provenant des micromyce ` te 08-29-2 actifs dans les membranes (e ´ sicules te ´ moin (solution de ve ´ sicules apre `s (correspondant au Tableau 1) ; b : image en microscopie `a fluorescence de la solution de ve ´ l’eau distille ´ e) ; c : image en microscopie optique de la solution de ve ´ sicules apre ` s avoir ajoute ´ l’extrait antimicrobien avoir ajoute ` te 08-29-2 actifs dans les membranes ; d : image en microscopie optique de la solution de ve ´ sicules te ´ moin provenant des micromyce ´ sicules apre ` s avoir ajoute ´ l’eau distille ´ e). (solution de ve
area for the isolation of membrane active metabolites-producing fungi. No scientific work has been carried out on the present subject, and the membrane active metabolites-producing fungi in various ecosystems of Iran. Considering the results in Table 1, a higher number (37%) of microfungi was isolated from forest soil. In accordance with previous reports this result is expected, since the similar studies have shown the importance of the moisture in growing fungi [14]. According to the results of the antimicrobial activity of isolates, a higher number of isolates (50%) from farming soil were active against test strains. Among active isolates, antifungal and antibacterial activities were observed in 10 isolates (50%) and five isolates (25%), respectively. Only five isolates (25%) of active isolates exhibited both antibacterial and antifungal activity. The detected activity regarding to nine active isolates (45%) that exhibited antimicrobial activity with more than 10 mm inhibition zone, can be considered very promising
considering that non-optimized cultures usually produce poor yields of active compounds and amounts of inhibition zones of crude extracts are, indeed, expected to be smaller than the control (a pure compound) inhibition zones. The results presented in Fig. 1 clearly demonstrate that the induced color transitions in the tubes c and e are directly related to interaction of membrane active compounds with the lipid bilayer in the vesicles. For example, antimicrobial extract of strain 08-29-2 gave rise to a blue-red transition like color changes induced by amphotericin B as the positive control, but antimicrobial extract of strain 08-35-2 did not induce colorimetric transitions. Considering the results of antimicrobial membrane activity of active isolates, strain 08-29-2 was the only one to produce color changes and irradiate fluorescence in vesicle solution that shows the membrane-activity of this strain. Despite the fact that strains 08-35-2, 08-1-4, 08-20-5 had potent antimicrobial activity against test bacteria and fungi; they did not disrupt vesicles and resulted in no color changes.
Screening of antimicrobial membrane-active metabolites of soil microfungi [(Figure_3)TD$IG]
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Figure 3 UV-Vis spectral response of color changes vesicle solutions under effect of membrane active compounds produced by active isolate. a: control: deionized water; b: amphotericin B (positive control); c: strain 08-29-2 (according to Table 1); d: Tetracycline (negative control). ´ ponse spectrale aux UV des changements de couleur des solutions de ve ´ sicules sous l’effet des compose ´ s actifs produits par les Re ´ moin : eau distille ´ e ; b : amphote ´ ricine (te ´ moin positif) ; c : Souche 08-29-2 (correspondant au Tableau 1) ; d : isolats. a : te ´ tracycline (te ´ moin ne ´ gatif). te
Furthermore considering the results in the study of Kolusheva et al. [17] addition of the membrane active peptides led to color changes in the vesicle solutions and Peptides that are not expected to bind cellular membranes did not induce detectable colorimetric transitions. In their study no colorimetric responses (negative controls) have been detected for various other soluble peptides and proteins that generally appear in physiological solutions, such as albumin. They found the different degrees of color changes induced by each peptide most likely depend upon their distinctive modes of interactions with the DMPC/PDA vesicle interface. According to the results of UV-Vis spectrophotometery in Fig. 3 to calculate colorimetric response, control sample and tetracycline as negative control have made the absorbance of blue component at 650 nm that improve to result in no color changes and absorbance of red component at 540 nm have been made by strain 08-29-2 and amphotericin B as positive control indicates that membrane active compounds interacted with lipid/PDA vesicles and led to blue-red color transitions.
The highest amount (90%) of colorimetric response represented by amphotericin B and strain 08-29-2 produced amounts of 88% in CR. DMSO as solvent control showed 55% in CR. DMSO has lonely a specific structure by which, cause to somewhat forcing effect on model vesicles that resulted in low color changes and colorimetric response of vesicle solution. Tetracycline as negative control gave 5.5% for the colorimetric response. Finally, we identified the active isolate 08-29-2 up to the genus level by macroscopic and microscopic methods. It belonged to Aspergillus sp. and attempt to identify and characterize selected isolate in species level and their membrane active metabolites will be continued in our group.
Conclusion The aim of the present study was screening of antimicrobial membrane-active metabolites from soil microfungi by using chromatic phospholipid/polydiacetylene vesicles. In our
196 laboratory a fungal isolate, belonged to Aspergillus sp., was isolated from farming soil of Kelibar cemetery, East Azerbaijan, Iran, (038852.13600 N and 047802.23000 E) exhibited promising antifungal activity. Also we found, the applied polydiacetylene polymers, as the membrane vesicle model to be very useful in screening of membrane active metabolites-producing fungi. The chromatic technology is simple and does not require knowledge of the composition of the membrane-active compounds secreted by microfungi. Exploitation of either visual color changes observed by the naked eye or fluorescence emission as a viable detection method is an important advantage of this system [31].
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The first author is grateful to the head of Drug Design and Bioinformatics Unit in Pasteur Institute of Iran for financial support to this study.
References [1] Ahmed Sheikh HM. Antimicrobial activity of certain bacteria and fungi isolated from soil mixed with human saliva against pathogenic microbes causing dermatological diseases. Saudi J Biol Sci 2010;17:331—9. [2] Bambeke FV, Mingeot-Leclercq MP, Struelens MJ, Tulkens PM. The bacterial envelope as a target for novel anti-MRSA antibiotics. Trends Pharmacol Sci 2008;29:124—34. [3] Brown ED, Wright GD. New targets and screening approaches in antimicrobial drug discovery. Chem Rev 2005;105:759—74. [4] Butler MS, Buss AD. Natural products — The future scaffolds for novel antibiotics? Biochem Pharmacol 2006;71:919—29. [5] Charych DH, Nagy JO, Spevak W, Bednarski MD. Direct colorimetric detection of a receptor—ligand interaction by a polymerized bilayer assembly. Science 1993;261:585—8. [6] Cheng Q, Stevens RC. Charge-induced chromatic transition of amino-acid derivatized polydiacetylene liposomes. Langmuir 1998;14:1974—6. [7] Coates ARM, Hu Y. Novel approaches to developing new antibiotics for bacterial infections. Brit J Pharmacol 2007;15:1147— 54. [8] Donadio S, Brandi L, Monciardini P, Sosio M, Gualerzi CO. Novel assays and novel strains — promising routes to new antibiotics? Expert Opin Drug Discov 2007;2:789—98. [9] Dugan FM. The identification of fungi-an illustrated introduction with keys, glossary and guide to literature. Minnesota: APS press; 2006. [10] Ganesh Kumar C, Mongolla P, Joseph J, Nageswar YVD, Kamal A. Antimicrobial activity from the extracts of fungal isolates of soil and dung samples from Kaziranga National Park, Assam. India J Mycol Med 2010;20:283—9. [11] Hawksworth DL. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol Res 1991;95:641— 55. [12] Hawksworth DL. Freshwater and marine lichen-forming fungi. Fungal Divers 2000;8:1—7.
M. Mehravar, S. Sardari [13] Ho WH, To PC, Hyde KD. Induction of antibiotic production of freshwater fungi using mix-culture fermentation. Fungal Divers 2003;12:45—51. [14] Indoor environmental quality: dampness and mold in buildings. Washington, D.C.; Centers for disease control and prevention (CDC), National Institute for Occupational Safety and Health.: http://www.cdc.gov/niosh/topics/indoorenv/mold.html [15] Jelinek R, Kolusheva S. Polymerized lipid vesicles as colorimetric biosensors for biotechnological applications. Biotechnol Adv 2001;19:109—18. [16] Ji EK, Ahn DJ, Kim JM. The Fluorescent Polydiacetylene Liposome. Bull Korean Chem Soc 2003;24:667—70. [17] Kolusheva S, Boyer L, Jelinek R. A colorimetric assay for rapid screening of antimicrobial peptides. Nat Biotechnol 2000;18: 225—7. [18] Kolusheva S, Shahal T, Jelinek R. Peptide-membrane interactions studied by a new phospholipid/polydiacetylene colorimetric vesicle assay. Biochem 2000;39:15851—9. [19] Kolusheva S, Wachtel E, Jelinek R. Biomimetic lipid/polymer colorimetric membranes: molecular and cooperative properties. Adv Lipid Res 2003;44:65—71. [20] Larone DH. Identification of fungi in culture. In: Larone DH, editor. Medically important fungi: a guide to identification. Washington: ASM Press; 2002. p. 82—175. [21] Luzhetskyy A, Pelzer S, Bechthold A. The future of natural products as a source of new antibiotics. Curr Opin Investig Drugs 2007;8:608—13. [22] McGowan JE. Economic impact of antimicrobial resistance. Emerg Infect Dis 2001;7:286—92. [23] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461—77. [24] Nicoletti R, Lopez-Gresa MP, Manzo E, Carella A, Ciavatta ML. Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens. Mycopathologia 2007; 163:295—301. [25] Nielsen MN, Sorensen J, Fels J, Pedersen HC. Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas fluorescens isolates from sugar beet rhizosphere. Appl Environ Microb 1998; 64:3563—9. [26] Okada SY, Jelinek R, Charych DH. Induced color change of conjugated polymeric vesicles by interfacial catalysis of phospholipase A2. Angew Chem Int Ed Engl 1999;38:655—9. [27] Petit P. Novel antimicrobial secondary metabolites from a Penicillium sp. isolated from Brazilian cerrado soil. Electron J Biotechn 2009;12:1—9. [28] Pevzner A, Kolusheva S, Orynbayeva Z, Jelinek R. Giant chromatic lipid/polydiacetylene vesicles for detection and visualization of membrane interactions. Adv Funct Mater 2008;18:242—7. [29] Prescott LM, Harley JP, Klein DA. Microbiology. New York: McGraw Hill; 2002. [30] Rancic A, Sokovic M, Karioti A, Vukojevic J, Skaltsa H. Isolation and structural elucidation of two secondary metabolites from the filamentous fungus Penicillium ochrochloron with antimicrobial activity. Environ Toxicol Phar 2006;22:80—4. [31] Silbert L, Shlush IB, Israel E, Porgador A, Kolusheva S, Jelinek R. Rapid chromatic detection of bacteria using a new biomimetic polymer sensor. Appl Environ Microbiol 2006;72:7339—44. [32] Takahashi JA, Monteiro de Castro MC, Souza GG, Lucas EM, Bracarense AP, Abreu LM. Isolation and screening of fungal species isolated from Brazilian cerrado soil for antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Streptococcus pyogenes and Listeria monocytogenes. J Mycol Med 2008;18:198—204. [33] Thakur D, Yadav A, Gogoi BK. Isolation and screening of Streptomces in soil of protected forest areas from the state of Assam and Tripura, India for antimicrobial metabolites. J Mycol Med 2007;17:242—9.
Screening of antimicrobial membrane-active metabolites of soil microfungi [34] Thanassi DG, Hultgren SJ. Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 2000;12:420—30. [35] Walsh CT, Kaback HR. Membrane transport as a potential target for antibiotic action. Ann NY Acad Sci 1974;235:519—41.
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[36] Watanabe T. Pictorial atlas of soil and seed fungi: morphologies of cultured fungi and key to species. Texas: CRC Press; 2002 . [37] Woodruff HB. Natural products from microorganisms. Science 1980;208:1225—9.
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles ´ tabolites antimicrobiens actifs sur la membrane de micromyce ` tes Criblage de me ´ sicules chromatiques de phospholipide/polydiace ´ tyle ` ne du sol `a l’aide de ve M. Mehravar a,b, S. Sardari a,* a Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute, No. 69 Pasteur Ave., Tehran, 13164, Iran b Department of Microbiology, Faculty of Science, Shahed University, Tehran, Iran
Received 11 April 2011; accepted 8 July 2011
KEYWORDS Chromatic vesicles; Antimicrobial membrane activity; Soil microfungi; Iran
Summary Objective. — The focus of this study is screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles. Materials and methods. — In this work, soil samples were collected from desert, forest, farm, lake shore and mineral zones of Northern and Central parts of Iran. These parts were not studied for antimicrobial potential of the soil isolates producing metabolites with membrane activity in particular, from microfungi. In the primary screening that was performed to evaluate the antimicrobial activity, isolates were analyzed in terms of their general inhibition effects to indicator strains including Escherichia coli, Candida albicans, and Saccharomyces cerevisiae. In the secondary screening, isolates producing membrane-active metabolites were determined using a Rapid Chromatic Detection method. The chromatic technology is simple and this method provides a rapid and easy evaluation of interactions between antimicrobial membrane-active metabolites and lipid layers of vesicles as well. Results. — A total number of 59 species of fungi was isolated from the soil samples. It has been found that 20 isolates were effective against indicator strains. Based on color and fluorescence changes that are easily identified by the naked eye and fluorescent microscopy and can be recorded by UV-Vis spectrophotometery, one fungus showed antimicrobial membrane-activity effect against some of the indicator strains. This isolate was identified to the genus level that belonged to Aspergillus.
* Corresponding author. E-mail address: [email protected] (S. Sardari). 1156-5233/$ — see front matter # 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2011.07.005
Screening of antimicrobial membrane-active metabolites of soil microfungi
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Conclusion. — As resistance is barely developed against membrane-active antibiotics, in this paper, we demonstrated the application of the chromatic vesicle model for screening of antimicrobial membrane-active metabolites as potential new antibiotics from soil microfungi. # 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Vésicules chromatiques ; Activité membranaire antimicrobienne ; Micromycètes du sol ; Iran
Re ´sume ´ Objectif. — L’objectif de cette étude est de cribler des métabolites antimicrobiens de micromycètes du sol actifs sur la membrane en utilisant des vésicules de phospholipide/polydiacetylene (PDA). Mate´riels et me ´thodes. — Pour ce travail, des échantillons de sol ont été collectés dans des zones du nord et du centre de l’Iran : dans le désert, les forêts, les fermes, les bords de lacs et des zones minérales. Ces zones n’avaient pas été étudiées pour le potentiel antimicrobien des isolats du sol, particulièrement en ce qui concerne les micromycètes produisant des métabolites actifs sur les membranes. Dans un premier crible qui a été effectué pour évaluer l’activité antimicrobienne des isolats de micromycètes ont été analysés pour leur effet inhibiteur sur des souches tests d’Escherichia coli, de Candida albicans et de Saccharomyces cerevisae. Dans un second crible, les isolats produisant des métabolites actifs dans les membranes ont été testés à l’aide d’une méthode rapide de détection par la couleur. Cette technologie est simple et permet une évaluation facile et rapide des interactions entre les métabolites antimicrobiens actifs dans les membranes et ainsi que des couches lipidiques de vésicules. Re ´sultats. — Cinquante-neuf espèces de micromycètes ont été isolées à partir des échantillons du sol. Il a été trouvé que 20 isolats sont efficaces contre les souches tests. À partir des changements de couleur et de fluorescence qui sont facilement identifiables à l’œil nu et en microscopie à fluorescence, et enregistrés par spectrophotométrie, un isolat a montré qu’il possédait une activité membranaire antimicrobienne contre plusieurs souches tests. Cet isolat a été identifié comme appartenant au genre Aspergillus. Conclusion. — Comme la résistance contre des antibiotiques actifs sur les membranes est peu connue, nous avons démontré dans ce travail l’application efficace du modèle de vésicules chromatiques pour cribler des métabolites antimicrobiens actifs sur les membranes. Ces métabolites antimicrobiens sont donc des antibiotiques potentiels provenant des micromycètes du sol. # 2011 Elsevier Masson SAS. Tous droits réservés.
Introduction The repeated emergence of antibiotic-resistant microorganisms is a problem in public health and its economic impact has recently become a concern as well. Microorganisms usually possessed the ability to protect themselves from antibiotics [22]. Therefore, new antimicrobial molecules are necessary to confront this problem; specially anti-infective agents with fewer side effects, shorter lengths of treatment and in particular, drugs with new and fewer resistantprone targets to antimicrobial activity [3,32,33]. Membrane as a new and potential target is noteworthy for antibiotics’ action [2]. It has two significant advantages as a target for an antimicrobial agent: drugs that anchor the microorganism membrane show a fast and extensive antimicrobial effect because of allowing easy target access; another advantage is having a lower tendency to resistance because of difficulty in modifying their components in a way that is compatible with bacterial survival [2,35]. New ways to discover novel antibiotics have been pursued, such as genomics route, and combinatorial chemistry; but it has led to the introduction of a few marketed antibiotics [7]. Novel methods that are used to screen common microbes have been described [8,32]. Microorganisms are capable of producing natural products with widely divergent chemical structures [37]. Natural products have been the
main source of antibiotics and played a fundamental role in antibiotic drug discovery in the past [4,23]. Natural products are still the most important source for promising drug candidates [21]. Remarkably, 70 out of the 90-marketed antibiotics in the years 1982—2002 originated from natural products. Natural products versus synthetic molecules are still a worth promise [22] Antibiotics producing microorganisms have been isolated from the most diverse habitats [32]. Soil, in particular, is an ecological environment in which the great number of microorganisms has a constant interaction that partially determines physical, chemical and biological properties of this habitat [33]. Thus, this environment has a tremendous biodiversity that can be screened for antibiotic production [32]. Soil fungi such as Penicillium have had a high impact in producing clinical antibiotics [24,30]. Screening of antibiotics producing soil microfungi have been reported in several studies [1,10,25,27,32]. New microorganisms and products have been derived from uninhabited and undisturbed areas of the world such as China, Australia, Antarctica, India and Jordan [33]. Thakur et al. [33] and Takahashi et al. [32] studied antimicrobial activity of 110 actinomycete strains isolated from the soil of Indian protected areas and 200 fungal strains isolated from Brazilian soil respectively. In the mentioned surveys, antimicrobial activity of metabolites produced by the soil microorganisms has not been simultaneously studied with their membrane activity.
190 Various membrane-active compounds are released by microorganisms to their environments [31,34] that strongly interact with membrane components of the host cell [29]. The aim of this work is to evaluate antimicrobial membrane activity of 59 fungal isolates from Iranian soil samples by vesicle membrane model, phospholipid/polydiacetylene (lipid/PDA) vesicles, as a bioassay target. Lipid/PDA vesicles are aggregates that self-assembled in aqueous solutions and contain organized lipids and polymers. Vesicular particles can be useful biosensors due to their relative ease of preparation, stability, closely mimicking the cell membrane [15]. Previous studies indicated that the lipids and PDA most likely form interspersed microscopic phases within the vesicles and phospholipids incorporated within the PDA matrix have a bilayer structure, the dominant lipid organization within cellular membranes [19]. Particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids as colorimetric biosensors were shown to exhibit color changes following interactions with antimicrobial membrane-active metabolites. The color changes occur because of the structural perturbation of the lipids following their interactions with antimicrobial membrane-active compounds [17]. Previous studies have shown that biological processes leading to structural perturbations at the PDA vesicle interface, including ligand—receptor recognition [5], pH changes [6], and enzymatic catalysis [26] are responsible for the blue-red transitions in the lipid/ PDA vesicles. Here, we used lipid/PDA vesicles as a biosensing tool for detection of membrane-active metabolites produced by soil microfungi isolated from soil.
Materials and methods
M. Mehravar, S. Sardari evenly over the surface of Malt Extract agar (MEA, Merck, Germany) for fungi isolation that was complemented with 100 mg/ml chloramphenicol to inhibit bacterial contamination. Plates were incubated at 28 8C, and monitored after 48, 72, and 96 h for growing fungi. Fungi colonies were identified according to morphological characteristics such as color of colony and medium at its vicinity, surface texture, shape and size characters, aerial mycelium, and spore production. Repeated streaking on the same agar plates led to purified fungi colonies. All of these strains were isolated on the same media supplemented with chloramphenicol (100 mg/ml) to inhibit bacterial contamination. All isolates grew on MEA medium showed characteristic morphologies of fungi. Colony appearance, size and color were visible by the naked eye. Mycelia and spore production was studied by optical microscopy. Pure colonies of fungi were further studied by light microscopy using slide culture method [20]. Plates containing pure cultures were stored on test tubes containing MEA at 4 8C during two months until further examinations, and in a freezer at 70 8C in the presence of glycerol (50%, v/v) for a longer storage period.
Indicator strains Two yeasts, including Candida albicans ATCC 10231, Saccharomyces cerevisiae BY4743 and one bacterium, Escherichia coli ATCC 25922, were used to determine the antimicrobial activity of the isolated fungi strains. The above-mentioned yeasts were cultured in Sabouraud Dextrose broth (SDB) (Difco, USA) at 28 8C for 48 h and E. coli was cultured in Brain Heart Infusion broth (BHI) (Merck, Germany) at 37 8C for 24 h.
Source of fungi Screening of isolates for antimicrobial activity Soil samples were collected from Different areas in Iran. These habitats included desert, forest, farming, shore and mineral soils of Iran that respectively were collected from Ghaem Salt Mine road (35848.09900 N and 051825.9400 E), Garmsar, Semnan Province, pH: 7.2, non-rhizosphere soil; Asalem to Khalkhal road (37837.98500 N and 049802.14300 E), Guilan Province, pH: 6.9, rhizosphere soil; Kelibar cemetery (038852.13600 N and 047802.23000 E), East Azerbaijan Province, pH: 7, non-rhizosphere soil; Orumieh lake shore (37840.78800 N and 045802.74200 E), West Azerbaijan Province, pH: 8.7, non-rhizosphere soil; Tehran-Tabriz highway (38809.43500 N and 046845.30700 E), Tabriz, pH: 7.5, nonrhizosphere soil. The samples were taken up from a depth of 15 cm and three samples for each place were mixed. Samples were placed in sterile bags, closed tightly and stored in a refrigerator.
Isolation and storage of fungi For each collected sample, one gram of the soil was suspended in 10 mL of normal saline (NaCl 9 g/L) then incubated in an orbital shaker incubator at 28 8C with shaking at 200 rpm for 30 min. Mixtures were allowed to settle, and serial dilutions up to 104 were prepared using sterile normal saline and agitated with the vortex at the maximum speed. An aliquot of 0.1 ml of each dilution was taken and spread
Malt Extract agar plates were prepared and spot inoculated with fungi isolates in 4 points. After 4 days of incubation at 28 8C the plates were seeded with test organisms by overlay agar technique [33] in which, spot inoculated colonies were covered with a 0.75—0.8% agar layer of Sabouraud Maltose medium (for yeast) and BHI medium (for bacteria). The antimicrobial activity was observed after 48 h incubation at 28 8C for yeast and 24 h incubation at 37 8C for bacteria. Amphotericin B and chloramphenicol used as control antibiotic against the fungal and bacterial test, respectively. The antimicrobial activities were measured by the determination of the size of the inhibition zone.
Screening of isolates for detection of membrane activity Isolates which produced antimicrobial substances against target strains were studied for membrane activity by a Rapid Chromatic Detection method, using a Biomimetic Polymer Sensor, i.e., polydiacetylene (PDA) in conjunction with phospholipid as a membrane model vesicle [16,17,19,28,31]. The detection is based on the interaction of membrane-active metabolites secreted by isolates in broth culture extract, with phospholipid/polydiacetylene Vesicles. This detection platform generated dramatic visible color changes accom-
Screening of antimicrobial membrane-active metabolites of soil microfungi panied by intense fluorescence emission that are induced by membrane-active molecules secreted by isolates [31].
Construction of the vesicles To prepare polymerized vesicles, synthetic phospholipid, Dimyristoylphosphatidylcholine (DMPC), and The diacetylenic monomer, 10,12-tricocosadiynoic acid (TCDA), (both purchased from Sigma, USA) which had been separately dissolved in dichloromethane (one mg/ml) were mixed at the 2:3 molar ratio and prepared at a concentration of one mM. Lipids were dried together using vacuum to remove dichloromethane completely, then a white thin film of the lipids on the bottom of glass was yielded. Same amount of deionized water was added (typically to obtain one mM concentration). The suspension was sonicated at 70 8C for 8—9 min in a sonicating bath. The resulting vesicle solution was cooled to the room temperature and kept at 4 8C overnight. Prior to polymerization, the vesicles warmed to the room temperature and polymerized by irradiation at 254 nm for 40—60 s, resulting in intense blue color appearance due to polymerization of the diacetylene units [19].
Submerge culture and Organic extraction Isolates that showed antimicrobial activity against test organisms in the agar medium were grown in submerged culture in 500 ml flasks containing 100 ml of Malt Extract broth. A two-days-old broth culture grown on Malt Extract broth was used to inoculate the flasks. These cultures were grown in a rotary shaker (Adolf Kuhner AG, Switzerland) at 200 rpm, 28 8C, for 14 days. The resulting culture broths obtained following growth of each isolate in the culture media were separated from the biomass by centrifugation at 4000 rpm (Sigma, Germany) for 15 min. The supernatant was filtered through 0.45 mm membrane filter. Filtrate was used for extracellular antimicrobial activity by the agar well diffusion method against indicator strains [33]. Sterile cork borer was used for punching six mm diameter wells in appropriate agar medium plates previously seeded with one of the indicator strains. A volume of 100 mL of the supernatant of each isolate was dispensed in each well. Plates were kept at 4 8C for at least 2 h to allow the diffusion of produced antimicrobial metabolites. The diameter of an inhibition zone was determined after 24 h of incubation at 37 8C for bacteria, and after 48 h at 28 8C for yeasts. Antimicrobial compound was recovered from the culture of each active isolate by solvent extraction with ethyl acetate. Ethyl acetate was added to the centrifuged broth at the ratio 1:1 (v/v) along three times and shaken vigorously for five min in each time. The organic layers were collected and the organic solvent was evaporated to dryness in a rotary vacuum evaporator at 40 8C. The remainder was dissolved in 1 ml dimethylsulfoxid (DMSO). Resulting solution was studied for membrane activity.
Interaction of phospholipid/PDA vesicle solution with antimicrobial extracts for detection of membrane-active metabolites In order to determine the effect of the extract of each strain showing antimicrobial activity that was dissolved in
191
DMSO, phospholipid/PDA vesicles were used as the membrane model to detect of membrane-active metabolites. As mentioned previously [19] polydiacetylene polymers used in model vesicles have some important characteristics such as blue-red color transition under external perturbation that can be visible by the naked eye and fluorescence irradiation of red components in the emission range (500—550 nm) that can be recorded by fluorescence microscopy (Nikon, E200, Japan). Color transitions can be quantitatively recorded by UV-Vis measurements at a wavelength span between 300—700 nm and calculating CR% (colorimetric response). UV-Vis measurements: a quantitative value for the extent of blue-to-red color transition is given by the colorimetric response (%CR), which is calculated from the visible absorbance spectra acquired for the vesicle solutions. The colorimetric response is defined:
CR ¼
PB0 PB1 100 PB0
where PB ¼ A
Ablue blue þAred
A is the absorbance at either the ‘‘blue’’ component in the UV-Vis spectrum (640 nm) or ‘‘red’’ component (500 nm), PB0 is the red/blue ratio of the control sample (before induction of color change), and PBI is the value obtained for the vesicle solution after addition of the tested compound [15]. Test tubes containing 300—400 mL vesicle solutions and two mM Tris solution pH 8.5, reached the volume of 1000 mL by adding bioactive extracts of strains. The pH in the solutions was 8.5 in all experiments [18]. Tubes incubated at 28 8C for one hour. Amphotericin B and tetracycline were used as positive and negative control respectively.
Determination of the antibacterial spectrum of membrane active isolate In order to determinate the antimicrobial activity spectrum, according to the above-mentioned method the membrane active isolates were subjected to submerged culture to study antibacterial activity by agar well diffusion method against test bacteria including Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi PTCC 1609, S. pyogenes ATCC 8668, Bacillus subtilis PTCC 1365, Enterococcus faecalis ATCC 29212. Finally, membrane active isolates has been grown on sabouraud dextrose agar plate to study Morphologically and culturally such as colony size and texture, color, pigment production by the naked eye and optical microscopy [9]. Slide culture method followed by lactophenol cotton blue (Sigma, USA) staining was performed to observe aerial mycelia and conidiophores by light microscopy magnification 100 (Zeiss, Standard 20, Germany). In the present work, we screened fungi from north and central Iran’s unexplored habitats. These areas represent a diverse and unscreened ecosystem and the least investigated area for the isolation of membrane active metabolites-producing fungi. No scientific work has been carried out on the membrane active metabolites-producing fungi in various ecosystems of Iran.
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M. Mehravar, S. Sardari 20 mm inhibition zones. Eight isolates showed inhibition zones less than 10 mm against at least one test indicator.
Results Isolation of microfungi
Antimicrobial membrane activities of isolates Using the selective media and cultivation conditions described previously a total of 59 different microfungal isolates from several soil samples which, collected from desert, forest, farming, shore and mineral soil in north, west and central zones of Iran was obtained. The forest and desert soils give the higher number of microfungi isolates (22 and 13 isolates, respectively) with respect to other soils.
Antimicrobial activities of isolates All the isolates were tested for their ability to produce antimicrobial substances against three indicator strains included one Gram negative bacteria and two yeasts. We found 20 (33%) out of 59 fungi isolates to have in vitro antibacterial activity against at least one of the indicator strains. The results of the antimicrobial activity of active isolates are shown in Table 1. Six of them inhibited the growth of at least one indicator strains with more than
Artificial lipid/PDA vesicle solution has an intense blue color appearance. Microbial extracts separating from broth culture of active isolates before affecting on vesicle solution, were studied for their antimicrobial activity on indicator strains that one of them had membrane activity on lipid/PDA vesicles. By interacting the antimicrobial extracts with lipid/PDA vesicles and disrupting the vesicles, blue-red color transition was recognizable by the naked eye as shown in Fig. 1, as well as fluorescence emission can be recorded by fluorescence microscopy (Fig. 2). Published data further imply to the contribution of changes in fluidity within the lipid parts in inducing the blue-red transitions [19]. Using lipid/PDA vesicles for biosensing applications has been based on the observation that numerous membrane active compounds interacting with the lipid domains can cause to the blue-red transformations of
Table 1 Antimicrobial activities of soil microfungi isolates grown on Malt Extract agar medium. ´ antimicrobienne des isolats de micromyce ` tes du sol dans un milieu de croissance en ge ´ lose. Activite Isolate No.
Total strains isolated (%)
Number of active isolates (%)
The indicator strains and inhibition zone diameters (mm) on Malt Extract agar medium E. coli
Desert soil 08-1-1 08-1-2 08-1-3 08-1-4 08-1-5 Forest soil 08-20-1 08-20-2 08-20-3 08-20-4 08-20-5 Farming soil 08-29-1 08-29-2 08-29-3 08-29-4 Shore 08-35-1 08-35-2 08-35-3 Mineral soil 08-31-1 08-31-2 08-31-3 Control Chloramphenicol Amphotericin B Total
13 (22)
22 (37)
8 (14)
9 (15)
7 (12)
59
C. albicans
S. cerevisiae
5 (38) — 10 — 20 —
12 — 8 14 —
8 — — — 6
— — — 12 —
20 10 8 10 > 20
18 — — — 18
10 6 6 8
5 16 — —
— 20 — —
> 20 > 20 —
— 8 12
— 8 10
— — 8
— — —
6 6 —
23 —
— 18
— 22
5 (23)
4 (50)
3 (33)
3 (43)
20 (34)
Screening of antimicrobial membrane-active metabolites of soil microfungi [(Figure_1)TD$IG]
193
Figure 1 Color changes produced by interacting of vesicle solution with membrane active metabolites secreted by active microfungi. Results after adding: a: deionized water (control solution); b: antimicrobial extract of a non-membrane active microfungus 08-35-2 (according to Table 1); c: antimicrobial extract secreted by membrane active microfungus 08-29-2 (according to Table 1); d: antibiotic tetracycline as negative control; e: antibiotic amphotericin B as the positive control. ´ sicules avec les me ´ tabolites actifs dans les membranes et Changements de couleur produits par l’interaction de solution de ve ´ cre ´ te ´ s par les micromyce ` tes. Re ´ sultats apre ` s avoir ajoute ´ : a : l’eau distille ´ e (solution te ´ moin) ; b : des extraits antimicrobiens se ´ cre ´ te ´ s par des micromyce ` tes 08-35-2 non actifs dans les membranes (correspondant au Tableau 1) ; c : des extraits antimicrobiens se ´ cre ´ te ´ s par des micromyce ` te 08-29-2 actifs dans les membranes (correspondant au Tableau 1) ; d : l’antibiotique te ´ tracycline se ´ moin ne ´ gatif) ; e : l’antibiotique amphote ´ ricine B (te ´ moin positif). (te
the polymer that means PDA polymers in the mixed vesicles essentially work as the reporter module for membraneactive molecules [15]. Red components have fluorescence irradiation in 500— 550 nm; the intense disruption induces a stronger red color that is led to irradiate more fluorescence. Amphotericin B is an antifungal that its main target is the sterol presented in fungi membrane but amphotericin B in second preference targets lipids of membrane. As membrane-activity test results depicted in Fig. 2 color changes were only produced in tubes c and e, which belong to strain 08-29-2 and amphotericin B, respectively. Absorbance of blue and red components with calculation of Colorimetric response has been shown in Fig. 3. According to the results, strain 08-29-2 isolated from farming soil had membrane-activity effect. The amounts of colorimetric response for control, amphotericin B, tetracycline and strain 08-29-2 were calculated 0, 90, 5.5, 88%.
Determination of the antimicrobial spectrum The results of antimicrobial spectrum of strain 08-29-2 on agar medium based on the diameter of inhibition zones were against S. aureus: nine mm; S. pyogenes: 10 mm; B. subtilis: seven mm; S. typhi: six mm; E. faecalis: 5 mm; Ps. Aeruginosa: 6 mm; E. coli: 6 mm; C. albicans: 16 mm; S. cerevisae: 20 mm. Considering these results strain 08-29-2 has produced a larger inhibition zone (more than 10 mm) against yeasts. It shows that extract of strain 08-29-2 have rather dominant antifungal effect.
Characteristics of membrane active isolates Membrane active isolate 08-29-2 isolated from farming soil samples was studied on SDA medium. The colony of this strain was wide in size with a little bulge, globose shape, cottony, cream-white surface, yellowish-gold reverse, the
surface was smooth and this strain produced a yellow pigment on SDA. Aerial mycelia and conidiophores were observed by light microscopy. Considering the characteristics results and comparing them with other fungi [36], we identified the membrane active isolate as Aspergillus sp. Further study towards identification of the species is planned in our group.
Discussion The resistance of various pathogenic bacteria and fungi to existing bioactive secondary metabolites is an urgent focus of research, and new antifungal and antibacterial molecules are necessary to combat these pathogens. Furthermore, over the past 30 years a number of new infectious diseases have been discovered. Increasing number of duplications in screening for antimicrobial metabolites from natural products and the urgent demand for new leading structures in pharmacology have enforced the search for metabolites in microflora of undisturbed habitats and with potent targets in the mechanism of action [22]. Membrane is a potential target for antibiotic action, because there is a lower tendency that resistance can be developed against this target. Drugs that affect in the microorganism membrane seem to have maximum potential because they show a fast and extensive antimicrobial effect and are less prone to a resistance development [2,35]. In this study, we focused on fungal isolates. It is estimated that about 1.5 million fungi are present in the world, but 5% of these have yet to be discovered [11— 13]. Fungi are one of the major antibiotic-producing organisms, and one of the most diversified groups of organisms. The search for antibiotics from fungi is promising and six of the world’s 20 best selling drugs are indicated to be fungal derived [13]. In the present work, we screened fungi from north and central Iran’s unexplored habitats. These areas represent a diverse and unscreened ecosystem and the least investigated
194 [(Figure_2)TD$IG]
M. Mehravar, S. Sardari
Figure 2 Microscopy images of vesicles solution. a: fluorescence microscopy image of vesicles solution in emission 550 nm after adding antimicrobial extract of a membrane active microfungus 08-29-2 (according to Table 1); b: fluorescence microscopy image of control vesicles solution (vesicle solution after addition of deionized water); c: light microscopy of vesicle solution after adding antimicrobial extract of a membrane active microfungus 08-29-2; d: light microscopy of control vesicle solution (vesicle solution after addition of deionized water). ´ sicules. a : image en microscopie `a fluorescence d’une solution de ve ´ sicules Images en microscopie d’une solution contenant des ve ´ mission : 550 nm) apre ` s avoir ajoute ´ l’extrait antimicrobien provenant des micromyce ` te 08-29-2 actifs dans les membranes (e ´ sicules te ´ moin (solution de ve ´ sicules apre `s (correspondant au Tableau 1) ; b : image en microscopie `a fluorescence de la solution de ve ´ l’eau distille ´ e) ; c : image en microscopie optique de la solution de ve ´ sicules apre ` s avoir ajoute ´ l’extrait antimicrobien avoir ajoute ` te 08-29-2 actifs dans les membranes ; d : image en microscopie optique de la solution de ve ´ sicules te ´ moin provenant des micromyce ´ sicules apre ` s avoir ajoute ´ l’eau distille ´ e). (solution de ve
area for the isolation of membrane active metabolites-producing fungi. No scientific work has been carried out on the present subject, and the membrane active metabolites-producing fungi in various ecosystems of Iran. Considering the results in Table 1, a higher number (37%) of microfungi was isolated from forest soil. In accordance with previous reports this result is expected, since the similar studies have shown the importance of the moisture in growing fungi [14]. According to the results of the antimicrobial activity of isolates, a higher number of isolates (50%) from farming soil were active against test strains. Among active isolates, antifungal and antibacterial activities were observed in 10 isolates (50%) and five isolates (25%), respectively. Only five isolates (25%) of active isolates exhibited both antibacterial and antifungal activity. The detected activity regarding to nine active isolates (45%) that exhibited antimicrobial activity with more than 10 mm inhibition zone, can be considered very promising
considering that non-optimized cultures usually produce poor yields of active compounds and amounts of inhibition zones of crude extracts are, indeed, expected to be smaller than the control (a pure compound) inhibition zones. The results presented in Fig. 1 clearly demonstrate that the induced color transitions in the tubes c and e are directly related to interaction of membrane active compounds with the lipid bilayer in the vesicles. For example, antimicrobial extract of strain 08-29-2 gave rise to a blue-red transition like color changes induced by amphotericin B as the positive control, but antimicrobial extract of strain 08-35-2 did not induce colorimetric transitions. Considering the results of antimicrobial membrane activity of active isolates, strain 08-29-2 was the only one to produce color changes and irradiate fluorescence in vesicle solution that shows the membrane-activity of this strain. Despite the fact that strains 08-35-2, 08-1-4, 08-20-5 had potent antimicrobial activity against test bacteria and fungi; they did not disrupt vesicles and resulted in no color changes.
Screening of antimicrobial membrane-active metabolites of soil microfungi [(Figure_3)TD$IG]
195
Figure 3 UV-Vis spectral response of color changes vesicle solutions under effect of membrane active compounds produced by active isolate. a: control: deionized water; b: amphotericin B (positive control); c: strain 08-29-2 (according to Table 1); d: Tetracycline (negative control). ´ ponse spectrale aux UV des changements de couleur des solutions de ve ´ sicules sous l’effet des compose ´ s actifs produits par les Re ´ moin : eau distille ´ e ; b : amphote ´ ricine (te ´ moin positif) ; c : Souche 08-29-2 (correspondant au Tableau 1) ; d : isolats. a : te ´ tracycline (te ´ moin ne ´ gatif). te
Furthermore considering the results in the study of Kolusheva et al. [17] addition of the membrane active peptides led to color changes in the vesicle solutions and Peptides that are not expected to bind cellular membranes did not induce detectable colorimetric transitions. In their study no colorimetric responses (negative controls) have been detected for various other soluble peptides and proteins that generally appear in physiological solutions, such as albumin. They found the different degrees of color changes induced by each peptide most likely depend upon their distinctive modes of interactions with the DMPC/PDA vesicle interface. According to the results of UV-Vis spectrophotometery in Fig. 3 to calculate colorimetric response, control sample and tetracycline as negative control have made the absorbance of blue component at 650 nm that improve to result in no color changes and absorbance of red component at 540 nm have been made by strain 08-29-2 and amphotericin B as positive control indicates that membrane active compounds interacted with lipid/PDA vesicles and led to blue-red color transitions.
The highest amount (90%) of colorimetric response represented by amphotericin B and strain 08-29-2 produced amounts of 88% in CR. DMSO as solvent control showed 55% in CR. DMSO has lonely a specific structure by which, cause to somewhat forcing effect on model vesicles that resulted in low color changes and colorimetric response of vesicle solution. Tetracycline as negative control gave 5.5% for the colorimetric response. Finally, we identified the active isolate 08-29-2 up to the genus level by macroscopic and microscopic methods. It belonged to Aspergillus sp. and attempt to identify and characterize selected isolate in species level and their membrane active metabolites will be continued in our group.
Conclusion The aim of the present study was screening of antimicrobial membrane-active metabolites from soil microfungi by using chromatic phospholipid/polydiacetylene vesicles. In our
196 laboratory a fungal isolate, belonged to Aspergillus sp., was isolated from farming soil of Kelibar cemetery, East Azerbaijan, Iran, (038852.13600 N and 047802.23000 E) exhibited promising antifungal activity. Also we found, the applied polydiacetylene polymers, as the membrane vesicle model to be very useful in screening of membrane active metabolites-producing fungi. The chromatic technology is simple and does not require knowledge of the composition of the membrane-active compounds secreted by microfungi. Exploitation of either visual color changes observed by the naked eye or fluorescence emission as a viable detection method is an important advantage of this system [31].
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The first author is grateful to the head of Drug Design and Bioinformatics Unit in Pasteur Institute of Iran for financial support to this study.
References [1] Ahmed Sheikh HM. Antimicrobial activity of certain bacteria and fungi isolated from soil mixed with human saliva against pathogenic microbes causing dermatological diseases. Saudi J Biol Sci 2010;17:331—9. [2] Bambeke FV, Mingeot-Leclercq MP, Struelens MJ, Tulkens PM. The bacterial envelope as a target for novel anti-MRSA antibiotics. Trends Pharmacol Sci 2008;29:124—34. [3] Brown ED, Wright GD. New targets and screening approaches in antimicrobial drug discovery. Chem Rev 2005;105:759—74. [4] Butler MS, Buss AD. Natural products — The future scaffolds for novel antibiotics? Biochem Pharmacol 2006;71:919—29. [5] Charych DH, Nagy JO, Spevak W, Bednarski MD. Direct colorimetric detection of a receptor—ligand interaction by a polymerized bilayer assembly. Science 1993;261:585—8. [6] Cheng Q, Stevens RC. Charge-induced chromatic transition of amino-acid derivatized polydiacetylene liposomes. Langmuir 1998;14:1974—6. [7] Coates ARM, Hu Y. Novel approaches to developing new antibiotics for bacterial infections. Brit J Pharmacol 2007;15:1147— 54. [8] Donadio S, Brandi L, Monciardini P, Sosio M, Gualerzi CO. Novel assays and novel strains — promising routes to new antibiotics? Expert Opin Drug Discov 2007;2:789—98. [9] Dugan FM. The identification of fungi-an illustrated introduction with keys, glossary and guide to literature. Minnesota: APS press; 2006. [10] Ganesh Kumar C, Mongolla P, Joseph J, Nageswar YVD, Kamal A. Antimicrobial activity from the extracts of fungal isolates of soil and dung samples from Kaziranga National Park, Assam. India J Mycol Med 2010;20:283—9. [11] Hawksworth DL. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol Res 1991;95:641— 55. [12] Hawksworth DL. Freshwater and marine lichen-forming fungi. Fungal Divers 2000;8:1—7.
M. Mehravar, S. Sardari [13] Ho WH, To PC, Hyde KD. Induction of antibiotic production of freshwater fungi using mix-culture fermentation. Fungal Divers 2003;12:45—51. [14] Indoor environmental quality: dampness and mold in buildings. Washington, D.C.; Centers for disease control and prevention (CDC), National Institute for Occupational Safety and Health.: http://www.cdc.gov/niosh/topics/indoorenv/mold.html [15] Jelinek R, Kolusheva S. Polymerized lipid vesicles as colorimetric biosensors for biotechnological applications. Biotechnol Adv 2001;19:109—18. [16] Ji EK, Ahn DJ, Kim JM. The Fluorescent Polydiacetylene Liposome. Bull Korean Chem Soc 2003;24:667—70. [17] Kolusheva S, Boyer L, Jelinek R. A colorimetric assay for rapid screening of antimicrobial peptides. Nat Biotechnol 2000;18: 225—7. [18] Kolusheva S, Shahal T, Jelinek R. Peptide-membrane interactions studied by a new phospholipid/polydiacetylene colorimetric vesicle assay. Biochem 2000;39:15851—9. [19] Kolusheva S, Wachtel E, Jelinek R. Biomimetic lipid/polymer colorimetric membranes: molecular and cooperative properties. Adv Lipid Res 2003;44:65—71. [20] Larone DH. Identification of fungi in culture. In: Larone DH, editor. Medically important fungi: a guide to identification. Washington: ASM Press; 2002. p. 82—175. [21] Luzhetskyy A, Pelzer S, Bechthold A. The future of natural products as a source of new antibiotics. Curr Opin Investig Drugs 2007;8:608—13. [22] McGowan JE. Economic impact of antimicrobial resistance. Emerg Infect Dis 2001;7:286—92. [23] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461—77. [24] Nicoletti R, Lopez-Gresa MP, Manzo E, Carella A, Ciavatta ML. Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens. Mycopathologia 2007; 163:295—301. [25] Nielsen MN, Sorensen J, Fels J, Pedersen HC. Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas fluorescens isolates from sugar beet rhizosphere. Appl Environ Microb 1998; 64:3563—9. [26] Okada SY, Jelinek R, Charych DH. Induced color change of conjugated polymeric vesicles by interfacial catalysis of phospholipase A2. Angew Chem Int Ed Engl 1999;38:655—9. [27] Petit P. Novel antimicrobial secondary metabolites from a Penicillium sp. isolated from Brazilian cerrado soil. Electron J Biotechn 2009;12:1—9. [28] Pevzner A, Kolusheva S, Orynbayeva Z, Jelinek R. Giant chromatic lipid/polydiacetylene vesicles for detection and visualization of membrane interactions. Adv Funct Mater 2008;18:242—7. [29] Prescott LM, Harley JP, Klein DA. Microbiology. New York: McGraw Hill; 2002. [30] Rancic A, Sokovic M, Karioti A, Vukojevic J, Skaltsa H. Isolation and structural elucidation of two secondary metabolites from the filamentous fungus Penicillium ochrochloron with antimicrobial activity. Environ Toxicol Phar 2006;22:80—4. [31] Silbert L, Shlush IB, Israel E, Porgador A, Kolusheva S, Jelinek R. Rapid chromatic detection of bacteria using a new biomimetic polymer sensor. Appl Environ Microbiol 2006;72:7339—44. [32] Takahashi JA, Monteiro de Castro MC, Souza GG, Lucas EM, Bracarense AP, Abreu LM. Isolation and screening of fungal species isolated from Brazilian cerrado soil for antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Streptococcus pyogenes and Listeria monocytogenes. J Mycol Med 2008;18:198—204. [33] Thakur D, Yadav A, Gogoi BK. Isolation and screening of Streptomces in soil of protected forest areas from the state of Assam and Tripura, India for antimicrobial metabolites. J Mycol Med 2007;17:242—9.
Screening of antimicrobial membrane-active metabolites of soil microfungi [34] Thanassi DG, Hultgren SJ. Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 2000;12:420—30. [35] Walsh CT, Kaback HR. Membrane transport as a potential target for antibiotic action. Ann NY Acad Sci 1974;235:519—41.
197
[36] Watanabe T. Pictorial atlas of soil and seed fungi: morphologies of cultured fungi and key to species. Texas: CRC Press; 2002 . [37] Woodruff HB. Natural products from microorganisms. Science 1980;208:1225—9.
