Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 7, Num. 1, 1996

Antifungal Activities Of Actinomycetes Isolated From

Moroccan Habitats

Y. OUHDOUCH, M. JANA, B. IMZILN,^ A. BOUSSAID and C. FINANCE*^

Universite Cadi Ayyad, Faculte des Sciences Semlalia, Departement de Biologie, Laboratoire de Microbiologie, ept. B.P. 15/S, Marrakech, Maroc and *Universite Henri Poincare, Nancy1, Faculte des Sciences Pharmaceutiques et Biologiques, GEVSM, Laboratoire de Microbiologie Moleculaire, B.P. 403, 54001 Nancy Cedex, France

Code Number: AC96003
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ABSTRACT. During a search for non-polyenic antifungal antibiotics ninety-six actinomycete strains were isolated from Moroccan soil and water samples. Twenty-six isolates showed strong activity against yeasts, moulds and bacteria. Four strains producing non-polyenic antifungal metabolites were investigated for their biological activity (spheroplast regeneration and ergosterol inhibition).

Although actinomycetes represent one of the richest sources of antibiotics, the chances of isolating new metabolites are slim unless adequate isolation strategies are adopted (Okami and Hotta, 1988).

Antibiotics used in human therapy are mostly antibacterial and show a wide range of chemical structures. As for antifungal agents the wall of mycetes represents a challenging target and compounds like nikkomycin and polyoxins, inhibiting chitin synthase (Nolan and Cross, 1988), are known. However many compounds, polyenes in particular, cannot be used because of their toxicity, while they are of interest in animal therapy, agriculture and industry.

The list of novel actinomycetes and products found in microbiologically poorly explored areas of the world stresses the advantage of investigating new habitats (Nolan and Cross, 1988), on the assumption that regions characterised by unique flora and fauna could also harbour less common actinomycetes (Okazaki and Naito, 1986).

Morocco presents many unexplored environments, such as rhizosphere of endemic plants, soils from the desert or from snowy peaks of Atlas, sea water from bays (e.g., Essaouira, where the temperature is around 20 C all year long). The present paper describes results obtained in a search for antifungal metabolites produced by actinomycetes isolated from such habitats, using different temperatures and culture media for isolation and growth (Iwai and Omura, 1982).

MATERIALS and METHODS

Samples and microbial strains. Samples were collected from various Moroccan ecosystems: rhizosphere of Phoenix dactylifera L. and Argania spinosa, Atlas mountain soil, Sahara sand, dung, sludge, well, river, lake and sea water, sediments and wastewater and analysed as quickly as possible (Pochon and Tardieux, 1962). Domestic wastewater samples were collected from the experimental stabilisation ponds of Marrakech before and after treatment.

The following organisms were used during the investigation: Bacillus subtilis, strain ATCC 9524, Candida albicans, strain ATCC 2091), Candida tropi- calis, strain R[2], CIP 203 (Collection of the Institut Pasteur, Paris), amphotericin B-nystatin resistant, Escherichia coli, strain ATCC 10536, Pythium irregu- lare, strain CIP 18, amphotericin B-nystatin resistant. Saccharomyces uvarum and Fusarium oxysporum f.sp. albedinis were obtained from the Phytopathology Laboratory of Cadi Ayyad University.

Isolation of actinomycetes. Samples (2g, wet weight) were diluted 10 times in sterile physiological water (NaCl, 9g/l), homogenised by vortexing and sonicated for 10-15min (Finance et al., 1985). Isolation was carried out on Olson agar medium (Olson, 1968) supplemented with 40ug/ml actidione. Plates were incubated at 25, 30, 37 and 42 C for 21dd. Isolates was maintained on Olson agar, incubated at 27 C for one week and then stored at +4 C for two months. Alternatively cultures were suspended in 20% sterile skim milk and frozen at - 80 C.

Antibiotic activity. Isolates were spot inoculated on Bennett, GBA (Shomurat et al., 1979) and Emerson agar. Plates were incubated at different temperatures (25, 30, 37 and 42 C) for 6dd then inverted for 40min over chloroform in a fume hood. Colonies were then covered with a 0.6 % agar layer of Sabouraud (for yeast), Bennett (for bacteria) or malt extract (for moulds) medium, previously seeded with test organisms. Incubation temperatures were 37 C for yeasts and bacteria and 25 C for moulds. Inhibition (20mm or more) was recorded after 24 and 48hrs.

Screening for antifungal non-polyenic metabolites. Active strain inocula were prepared from Olson medium according to Shirling and Gottlieb (1966). Isolates were grown in 20ml of Bennett medium for 48 hrs at 27 C and then for further 48hrs in 100ml of the same medium. Samples were collected regularly in order to estimate growth (absorbance at 620nm) and production of metabolites. Twenty-five ml of the culture were centrifuged at 4000rpm for 15min. The supernatant, sterilised by filtration, was used for biological assays. Antifungal activity was estimated by diffusion methods in Sabouraud agar medium (4% D-glucose, 1% Diagnostic Pasteur peptone and 1.5% agar), using double-layer activity, well or disc techniques and antibacterial activity by the agar cylinder method (Tortorano et al., 1979).

Spheroplast regeneration was estimated using S.uvarum (Ouhdouch, 1989) grown in 100ml flasks containing 20ml of Sabouraud medium (Difco) and incubated at 25 C under gentle shaking. Cells were harvested by centrifugation, the pellet was washed 3 times with sterile distilled water. One gram of cells was suspended in 4ml of a dithiotreitol 10^-2M and EDTA 25mM solution and incubated for 30min at 37 C, under gentle shaking. Cytohelicase (IBF Biotechnics) treatment was carried out on the pellets resuspended in 4ml of a NaHPO4 0.15M, citric acid 0.05M and KCl 0.14M buffer and incubated in presence of 22mg of cytohelicase at 37 C for 3hrs under gentle shaking. For regeneration washed spheroplasts (1ml) in isotonic buffer were introduced into flasks containing 19ml of Sabouraud medium, with sorbitol (osmotic stabiliser) and with or without sterile active extract. Light absorbance as well as the regeneration rate was estimated at 620nm and by counting the spheroplasts in a Malassez cell.

Ergosterol inhibition was tested by the agar cylinder and double layer method on Sabouraud medium with or without 50ug/ml ergosterol, in the presence of C.albicans.

UV spectra of extracts in methanol were recorded in the 220- 440nm range with a CECIL linear UV-spectrophotometer and compared with those of known polyenic antifungal antibiotics.

RESULTS and DISCUSSION

Isolation of actinomycetes. Actinomycetes were isolated from nearly all the environments explored.

The importance of the constituents of isolation media of growth temperature has already been stressed (Iwai and Omura, 1982). Olson medium, supplemented with actidione, appeared quite suitable for the isolation of actinomycetes from the different environments. In addition its transparency facilitates colony observation.

Although nutrient availability controls activity of soil actinomycetes, other factors, such as temperature may plays an important role (Goodfellow and Williams, 1983). The temperature of isolation of the 96 strains and the number of isolates with antifungal activity is shown in Tab. 1.

Antibiotic production. Twenty-six out of 96 strains show activity against the mycetes tested. Activity was tested on three different media and at four different temperatures (Table 2).

Active actinomycetes were isolated from a wide range of ecosystems, however 27% of active strains were obtained from the rhizosphere of P.dactylifera L. and A.spinosa. In Table 3 the antifungal activity of the 26 isolates at 30 C on Bennett medium, which appears the best for antibiotic production, is summarised.

No obvious relationships could be detected between isolation temperature and temperature of antibiotic production.

Non-polyenic antifungal activity. Nineteen out of the 26 isolates, showing antifungal activity, inhibited C.tropicalis R[2] or Pythium irregulare, amphotericin B-nystatin resistant strains (Table 4).

Four strains (SR10, SS2, D2 and LW1) appeared promising because of this character, activity against E.coli and B.subtilis (cell membrane without sterols) and no marked inhibition of antifungal activity by exogenous ergosterol (target of polyenic antifungal compounds). In addition the metabolites produced did not show an UV spectrum characteristic of the polyenic structure and inhibited Saccharomyces uvarum spheroplast regeneration.

Taxonomic characterisation of the four strains, as well as isolation, purification and structural elucidation of their metabolites, are in progress.

REFERENCES

Finance, C., Y.Ouhdouch, A.Jaques & R. Bonaly (1985). Nouvelles substances a action antifongique produites par des souches d'actinomycetales isolees a partir des milieux naturels. Ann.Biol.Clin., 43: 652

Goodfellow, M. & S.T.Williams (1983). Ecology of actinomycetes. Ann.Rev.Microbiol., 37: 189-216

Iwai, Y. & S.Omura (1982). Cultural conditions for screening of new antibiotics. J.Antibiot., 34: 123- 141

Nolan, R. & T.Cross (1988). Isolation and screening of actinomycetes. In: M.Goodfellow, S.T.Williams & M.Mordarski (eds.) Actinomycetes in Biotechnology. Academic Press, London, pp. 1-32

Okami, Y. & A.K.Hotta (1988). Search and discovery of new antibiotics. In: M.Goodfellow, S.T.Williams & M.Mordarski (eds.) Actinomycetes in Biotechnology. Academic Press, London, pp. 33-67

Okazaki, T. & A.Naito (1986). Studies on actinomycetes isolated from Australian soil. In: G.Szabo, S.Biro & M.Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akademiai Kiado, Budapest, pp. 739-741

Olson, E.H. (1968). Actinomycetes Isolation Agar. In Difco: Supplementary Literature. Difco Lab., Detroit, Michigan

Ouhdouch, Y.(1989). Bacteries actinomycetales rares productrices d'antifongiques: Criblage, selection et etude taxonomique d'une souche active, purification de l'antifongique elabore. Ph.D. Thesis, University Nancy 1, France.

Pochon, J. & P.Tardieux (1962). Technique d'Analyse en Microbiologie du Sol. Edition de la Tourelle, Saint-Mande

Shirling, E.. & D.Gottlieb (1966). Methods for characterization of Streptomyces species. Int.J.Syst.Bacteriol., 16: 313-334

Shomurat, T., J.Yoshida, S.Amano, M.Kojina & T.Niida (1979). Studies on actinomycetales producing antibiotics only in agar culture. I. Screening, taxonomy and morphology - productiv- ity relationship of Streptomyces halstedii, strain SF-1993. J.Antibiot., 32: 427-435

Tortorano, A.M., E.Cabrini & M.A.Vivani (1979). Sensibilite in vitro des levures a cinq antibiotiques. Comparaison de deux methodes C.M.I. en gelose et methode des disques. Bull.Soc.Fr.Myc.Med., 8: 69-74.

Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia

Table 1

Isolation
temperature    Number of   Strains with antifungal 
               isolates    activity
--------------------------------------------------
    25 C         20                   5
    30 C         29                   7
    37 C         31                   6
    42 C         16                   8
    Total        96                  26

Table 1. Total number of actinomycetes and of isolates with antifungal activity isolated at different temperatures.

                               Temperature

Medium           25 C         30 C         37 C        42 C
------------------------------------------------------------
Bennett           69          69           65          30
GBA               61          69           53          27
Emerson           30          39           19         
11

Table 2. Percentage of active strains on different media and at different temperatures.

Strain     Origin           Isolation   Activity against
                              temp.------------------------------
                                   C.alb- C.trop- F.oxy- P.irreg-
                                   icans  icalis  sporum ulare
-----------------------------------------------------------------
SR10 P.dactylifera rhizosphere 25 C    -       +     +      +
SA3  Atlas mountain soil       25 C    -       +     +      +
WW3  Domestic wastewater       25 C    -       -     -      -
WA4  River water               25 C    -       +     -      +
SS2  Desert sand               25 C    -       +     +      +
SR3  P.dactylifera rhizosphere 30 C    +       +     +      +
W3   Atlas lake water          30 C    -       +     -      +
IW5  Sahara well water         30 C    -       -     +      +
SS3  Desert sand               30 C    -       -     +      -
SRa  A.spinosa rhizosphere     30 C    +       +     +      -
D2   Dung                      30 C    +       -     +      +
AS7  Atlas mountain soil       30 C    +       -     +      -
SR13 P.dactylifera rhizosphere 37 C    +       +     +      +
AM3  Sludge                    37 C    -       -     -      -
SW3  Wastewater (outlet)       37 C    -       -     -      +
LW1  Essaouira bay water       37 C    +       +     +      +
S2   Sea sediment, Tangier     37 C    +       +     +      +
SS1  Desert sand               37 C    -       -     -      -
SR9  P.dactylifera rhizosphere 42 C    -       -     -      -
SR7  P.dactylifera rhizosphere 42 C    -       +     +      -
RIF1 Rif mountain soil         42 C    -       -     -      -
SA8  Atlas forest soil         42 C    -       -     -      -
IW3  Thermal water             42 C    -       +     +      +
AA1  Atlas mountain soil       42 C    -       -     -      -
SRA4 A.spinosa rhizosphere     42 C    +       +     +      +
SS6  Desert sand               42 C    -       -     -      -

Table 3. Origin and activity of the 26 actinomycete strains on Bennett agar at 30 C

Strain  Antibacterial Ergosterol UV(nm)abs.  Spheroplast       
         activity^a   effect^b    max        regeneration rate 
                      +     -                       (%)
----------------------------------------------------------------- 
SR10        +        14    15     none              1
SA3         -        10    20     291              90
SS2         +        17    18     none              2
SR3         -        05    16     340              80
W3          -        06    18     333              70
IW5         +        13    24     358              82
D2          +        17    20     none              2
AS7         +        05    16     318              75
SR13        -        07    15     362              85
AM3         -        08    17     340              92
LW1         +        16    15     none              3
S2          +        09    20     291              75
SR9         +        06    18     319              86
SR7         -        03    16     318              88
RIF1        -        12    18     291              78
SA8         -        15    21     333              90
IW3         -        16    25     305              75
AA1         -        10    22     379              95
SRA4        +        05    20     405              80
Reference^c -        06    13     362              70

Table 4. Results of the screening for non-polyenic antifungal producing actinomycetes (^a activity against E.coli and/or B.subtilis; ^b inhibition zone in mm in Sabouraud medium with (+) or without (-) 50ug/ml ergosterol; ^c amphotericin B)

Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia