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669

Screening of nonpolyenic antifungal metabolites


produced by clinical isolates of actinomycetes
S. Lemriss, F. Laurent, A. Couble, E. Casoli, J.M. Lancelin, D. Saintpierre-Bonaccio,
S. Rifai, A. Fassouane, and P. Boiron

Abstract: The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals.
This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic
antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three
yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging
to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that
clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances.
The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria:
antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all
three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further
investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.
Key words: actinomycetes, Streptomyces, clinical isolates, antifungal, non-polyene.
Résumé : L’objectif de notre travail a été la sélection de souches d’actinomycètes d’origine clinique productrices de
substances antifongiques de structure nonpolyénique. Ce choix a été fait afin d’éviter de redécouvrir les familles
d’antifongiques déjà connues, particulièrement celles de structure polyénique. Le criblage de cent dix souches a été effectué
contre trois souches de levures et trois souches de champignons filamenteux. Il a été réalisé par deux méthodes de
diffusion et sur deux milieux test. Parmi les cinquante quatre souches (49 %) montrant une activité antifongique, cinq
souches appartenant au genre Streptomyces sont actives contre tous les organismes test et se sont révélées prometteuses.
Ces résultats indiquent que les isolats d’origine clinique, comme ceux d’origine environnementale, sont une source
potentielle intéressante de substances antifongiques. La production de substances antifongiques de structure nonpolyénique
par ces cinq souches actives a été étudiée en utilisant trois critères : spectre antibactérien, inhibition par l’ergostérol et
analyse des spectres UV-visible des extraits actifs. Une seule souche répond aux trois critères de sélection et produit
des substances de structure nonpolyénique. Cette souche a été retenue pour des investigations complémentaires concernant
notamment la purification, la détermination de la structure et du mécanisme d’action du principe actif.
Mots clés : actinomycètes, Streptomyces, isolats cliniques, antifongique, non-polyène.

Lemriss et al. 674

Introduction thologies, such as the acquired immunodeficiency syndrome,


have been responsible for an ever-increasing incidence of
During the last decade, the development of immuno- fungal infections (Chabasse 1994).
suppressive therapy in medicine, the increase in iatrogenic The antifungal agents currently available for the treatment
factors and nosocomial diseases, and the advent of new pa- of systemic fungal infections are limited to polyenic anti-

Received 2 April 2003. Revision received 25 September 2003. Accepted 29 September 2003. Published on the NRC Research Press
Web site at http://cjm.nrc.ca on 16 December 2003.
S. Lemriss. UMR CNRS 5557 Ecologie Microbienne (Center for Microbial Ecology), Groupe de Recherche “Pathogènes
opportunistes et environnement”, Laboratoire de Mycologie Fondamentale et Appliquée aux Biotechnologies Industrielles, Institut
des Sciences Pharmaceutiques et Biologiques de Lyon, Université Claude Bernard Lyon 1, 8, avenue Rockefeller, 69373 Lyon
CEDEX 08, France; and Laboratoire de Biochimie Appliquée, Faculté des Sciences, Université Chouaib Doukkali, El jadida, Maroc.
F. Laurent, A. Couble, E. Casoli, D. Saintpierre-Bonaccio, and P. Boiron.1 UMR CNRS 5557 Ecologie Microbienne (Center for
Microbial Ecology), Groupe de Recherche “Pathogènes opportunistes et environnement”, Laboratoire de Mycologie Fondamentale et
Appliquée aux Biotechnologies Industrielles, Institut des Sciences Pharmaceutiques et Biologiques de Lyon, Université Claude
Bernard Lyon 1, 8, avenue Rockefeller, 69373 Lyon CEDEX 08, France.
S. Rifai and A. Fassouane. Laboratoire de Biochimie Appliquée, Faculté des Sciences, Université Chouaib Doukkali, El jadida,
Maroc.
J.M. Lancelin. Laboratoire de RMN Biomoléculaire associé au CNRS, Ecole Supérieure de Chimie Physique et Electronique de
Lyon, Université Claude Bernard Lyon 1, F-69622, Villeurbanne, France.
1
Corresponding author (e-mail: boiron@univ-lyon1.fr).

Can. J. Microbiol. 49: 669–674 (2003) doi: 10.1139/W03-088 © 2003 NRC Canada

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670 Can. J. Microbiol. Vol. 49, 2003

fungals of microbial origin, such as amphotericin B and its cans CIP 48.72, Candida albicans CIP 884.65, Candida
lipid formulations, and to synthetic products, such as azole tropicalis R2 CIP 1275.81 (a strain resistant to amphotericin
compounds, flucytosine, and other new antifungal agents. B and nystatin), Aspergillus fumigatus CIP 1082.74,
These synthetic products offer limited indication or are in Aspergillus fumigatus CIP 2279.94, and Trichophyton rubrum
the developmental stages. Amphotericin B remains the drug CIP 2043.92. All strains were maintained at 28 °C on
of choice for treating most fungal diseases because it has a Sabouraud’s agar.
broad spectrum and potent fungicidal activity, in spite of
well-known side effects (Andriole 1999; Lortholary et al.
Selection of actinomycete strains producing antifungal
1999). Although the azole antifungals are considered to be
metabolites
less toxic, their efficacies against deep-seated, life-threatening
mycoses are not fully satisfactory. In addition, it has been The selection of actinomycete strains was carried out by
reported that the frequency of multiazole-resistant strains be- agar diffusion methods in Casitone medium (9 g/L Bacto
longing to Candida species other than Candida albicans is casitone (Difco Laboratories, Sparks, U.S.A.), 5 g/L yeast
increasing (Hitchcok et al. 1993). In fact, there is a critical extract (Merck KGaA, Darmstadt, Germany), 10 g/L sodium
need for new fungicidal agents with original chemical struc- citrate (Prolabo, Paris, France), 20 g/L glucose (Merck),
ture (non-polyene), with a broad spectrum of activity, and 3.34 g/L di-sodium hydrogen phosphate (Merck), 0.54 g/L
that induce fewer side effects. potassium di-hydrogen phosphate (Merck), and 18 g/L agar
Actinomycetes have provided many important bioactive (Merck)) and in YMA medium (6.5 g/L yeast nitrogen base
compounds of high commercial value and continue to be (Difco), 1.5 g/L asparagine (Prolabo), 10 g/L glucose
routinely screened for new bioactive substances (Barakate et (Merck), and 20 g/L agar (Merck)) for both agar cylinders
al. 2002). It has been estimated that approximately two-thirds and well diffusion methods.
of naturally occurring antibiotics have been isolated from
these microorganisms (Okami and Hotta 1988). Environmen- Agar cylinders method (agar piece method)
tal actinomycetes, moreso that other groups of actinomycetes, Isolates were grown on Bennett’s agar (Jones 1949) plates
have been regarded as a major source of antibiotics (Saadoun for 4 days at 37 °C, then a calibrated cylinder (3 mm in di-
et al. 1999). Several reports show that some opportunistic ameter) was cut out and placed on the test media (Casitone
pathogenic actinomycetes, such as Nocardia brasiliensis, or YMA), which had previously been seeded with each fun-
Nocardia pseudobrasiliensis, and Nocardia otitidiscaviarum, gal test organism. Plates were kept first at 4 °C for at least
also produce novel bioactive substances (Tanaka et al. 1997; 4 h to allow the diffusion of any antifungal metabolites and
Komaki et al. 1999). For instance, a new antifungal macro- were then incubated at 28 °C (Barakate et al. 2002). Inhibi-
lide called brasilinolide B has recently been isolated a from tion diameters were determined after 24 h for yeasts, except
pathogenic Nocardia brasiliensis strain (Mikami et al. for C. tropicalis R2 (which requires 96 h in Casitone me-
2000). dium and 48 h in YMA medium), and after 48 h for filamen-
The aim of the present study was to screen the antifungal tous fungi.
nonpolyenic metabolites produced by clinical isolates from
an actinomycete collection. Well diffusion method
Isolates were grown in 10 mL of liquid Bennett’s medium
Materials and methods for 4 days at 37 °C. Twenty microlitres of this culture was
introduced into a calibrated well (3 mm in diameter) cut into
Strains and media each test medium, which had previously been seeded with
One hundred and ten actinomycete strains, recovered from each fungal test organism. Plates were incubated at 28 °C
clinical samples, were used in this study. They included 91 (Magaldi et al. 2001). The inhibition diameters were mea-
strains of Streptomyces spp., 11 Nocardia asteroides sensus sured after 24 h for yeasts, except for C. tropicalis R2 (96 h
stricto, 3 Nocardia farcinica, 3 Nocardia nova, 1 Nocardia in Casitone medium and 48 h in YMA medium), and after
brasiliensis, and 1 Nocardia otitidiscaviarum. Each strain 48 h for filamentous fungi.
was identified to the genus or species level by PCR methods
previously described, including enzymatic amplification of
Screening for actinomycete strains producing
16S rDNA with genus-specific primers or of a segment of
nonpolyenic antifungal metabolites
the 65-kDa heat shock protein gene combined with restriction
analysis of the amplimer (Laurent et al. 1999; Rodriguez et To select active actinomycete strains producing only non-
al. 2001; Steingrube et al. 1997). Four strains of actinomycetes polyenic antifungal agents, we carried out three experiments.
producing antifungal agents were obtained from Professor
D.P. Labeda (Peoria, Ill., U.S.A.) and were used as controls. Antibacterial activity
They included Streptomyces griseus (NRRL B-150), Strep- Antibacterial activity of selected strains was estimated by
tomyces noursei (NRRL B-1714), Streptomyces natalensis the agar cylinders method and the well diffusion method in
(NRRL B-5314), and Streptomyces nodosus (NRRL B-2371), Mueller–Hinton agar medium (Merck), against two Gram-
which produce cycloheximide, nystatin, pimaricin, and positive bacteria (Staphylococcus aureus ATCC 25923 and
amphotericin A and B, respectively. All isolates were main- Enterococcus faecalis ATCC 19433) and against four Gram-
tained at 4 °C on Bennett’s agar slants. negative bacteria (Enterobacter cloacae ATCC 13047, Pseu-
Six fungal species obtained from the fungi collection of domonas aeruginosa ATCC 10145, Proteus vulgaris ATCC
the Pasteur Institute were used as test strains: Candida albi- 13315, and Citrobacter freundii ATCC 8090).

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Lemriss et al. 671

actinomycete strains
Ergosterol inhibition

Total (%) of active


Ergosterol inhibition was tested by both diffusion methods
with or without ergosterol at 50 mg/mL (Fluka Chemika,
Buchs, Switzerland) in the presence of C. albicans CIP
884.65 (Ouhdouch et al. 2001). The results were compared

(41)
(47)
(49)
(40)
(45)
(48)
with those obtained with amphotericin B at 20 µg/well or
disc and with reference strains producing polyenic anti-

45
52
54
44
49
53
fungal agents.

UV-visible spectroscopic analysis

Trichophyton

CIP 2043.92
Production and extraction of active metabolites

(19)
(21)

(25)
(26)
rubrum

(3)

(5)
Isolates showing antifungal activity were precultured in a
250-mL flask containing 25 mL of liquid Bennett’s medium

3
21
23
5
28
29
and were incubated at 37 °C for 24 h. The preculture was
transferred into a 500-mL flask containing 225 mL of liquid

CIP 2279.94
Bennett’s medium. After 3 days of incubation at 37 °C, ei-

Note: CIP, Collection of Pasteur Institute, Paris, France; YMA, 6.5 g/L yeast nitrogen base, 1.5 g/L asparagine, 10 g/L glucose, and 20 g/L agar.
ther the whole culture was extracted twice with ethyl acetate

(11)

(11)
(10)

(10)
(1:1, v/v), or the pellet and the supernatant (obtained after

(5)

(6)
centrifugation at 4500 r/min (1 r = 2π rad) for 15 min) were

12
6
12
11
7
11
extracted with methanol (1:5, v/v) and twice with hexane
(1:1, v/v), respectively. The antifungal activity of the three
extracts for each active strain was determined by the well

CIP 1082.74
Aspergillus
diffusion and disc diffusion methods.

fumigatus
Table 1. Number of active actinomycetes strains, according to diffusion method and to test media (n = 110).

(12)

(12)
(10)

(10)
(6)

(6)
UV-visible spectra
The UV-visible spectra of the active extracts were re-

13
7
13
11
7
11
corded in the 200–500 nm range with a spectrophotometer
(Kontron Instruments, Uvikon 932) and compared with those Number (%) of active actinomycetes strains against:
spectra of known polyenic antifungals agents (Hacène et al.

CIP 884.65
1994; Ouhdouch et al. 2001).

(38)
(36)
(38)
(38)
(35)
(38)
42
40
42
42
39
42
Results and discussion
The antifungal activity of a collection of clinical actino-
mycetes was tested by using two diffusion methods and two
CIP 48.72
Candida
albicans

test media. Of 110 isolates studied, more than 49% produced


(38)
(38)
(38)
(36)
(35)
(36)
metabolites with antifungal activity against at least one out
42
42
42
40
39
of six test organisms (Table 1). However, this percentage is 40
higher than those reported by many authors (between 10%
and 34%) studying the activity of soil and aquatic actino-
tropicalis R2
CIP 1275.81

mycetes (Ouhdouch et al. 2001; Hacène et al. 1994; Hilali et


al. 2002). Therefore, clinical isolates of actinomycetes ap-
Candida

(10)
(19)
(27)
(29)
(5)
(8)

pear to be an especially interesting source of antifungal me-


tabolites, highlighting the interest to develop the screening
6
9
11
21
30
32

of these pathogenic microorganisms. Our data showed that


the test medium and the method of assay have a great influ-
ence on the results of the screening (Table 1). Thus, the sus-
Test medium

ceptibility of C. tropicalis R2 and T. rubrum was best


Casitone

Casitone

revealed by the well diffusion method in the YMA medium.


YMA

YMA
Total

Total

Conversely, activity against A. fumigatus was best revealed


by the agar piece method in the casitone medium. For
C. albicans, the results were similar, regardless of the
method or the test medium used. The need to select the most
Agar cylinders method

Well diffusion method

active strains provoked the interest in using different meth-


ods and different test media. These results indicate the value
Diffusion method

of using multiple methods and test media to detect active


strains.
The problem of rediscovery of well-known antifungal
families necessitates the development of tactics that are ca-
pable of discriminating potentially novel antifungal families.
The antifungal agents of microbial origin currently available

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672 Can. J. Microbiol. Vol. 49, 2003

Table 2. Antifungal activity of selected and reference strains.


Test strain†
Candida Candida Aspergillus Trichophyton
tropicalis R2 albicans fumigatus rubrum
Actinomycete strain and reference No. CIP 1275.81 CIP 48.72 CIP 884.65 CIP 1082.74 CIP 2279.94 CIP 2043.92
Streptomyces sp. 96.0333* 65 42 52 33 33 30
Streptomyces sp. 98.0395* 9 18 17 15 15 >30
Streptomyces sp. 98.0436* 14 20 19 15 13 >30
Streptomyces sp. 98.1259* 28 23 21 9 7.5 24
Streptomyces sp. 98.1549* 26 20 19 13 9 30
Streptomyces griseus NRRL B-150 3 3 3 3 3 3
Streptomyces noursei NRRL B-1714 3 25 21 22 20 14
Streptomyces nodosus NRRL B-2371 3 16 15 16 17 3
Streptomyces nalalensis NRRL B-5314 3 3 3 3 3 3
Note: CIP, Collection of Pasteur Institute, Paris, France; NRRL, Northern Regional Research Laboratory, Peoria, Illinois, U.S.A.
*Reference No. in the collection of actinomycetes of the Laboratoire de Mycologie Fondamentale et Appliquée aux Biotechnologies Industrielles,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon, Lyon, France.

Activity measured by diameter of inhibition zones (in mm).

for clinical use are primarily of polyenic structure, in partic- 98.0333 showed a reduction in the diameters of the inhibi-
ular amphotericin B and nystatin. Thus, we chose to screen tion zones (Table 3).
clinical actinomycetes for the production of nonpolyenic Finally, several authors have used spectroscopy to distin-
antifungal metabolites. First, we tested antifungal activity by guish polyenic and nonpolyenic substances (Hacène et al.
using a large number of species, including a strain of C. tropi- 1994; Ouhdouch et al. 2001; Barakate et al. 2002). The
calis R2 (a strain resistant to amphotericin B and nystatin). spectra of polyenes are characterized by a series of peaks be-
Among the 110 tested isolates, five (Streptomyces sp. 96.0333, tween 260 and 405 nm (Hamilton-Miller 1973). The UV-
Streptomyces sp. 98.0395, Streptomyces sp. 98.0436, Strep- visible spectroscopic analysis of the active extracts showed
tomyces sp. 98.1259, Streptomyces 98.1549) appeared prom- that those of strains 96.0333, 98.0395, and 98.0436 did not
ising because of the inhibition of C. tropicalis R2 and present such polyenic absorption bands (Fig. 1A). Con-
because of their broad spectrum activity against all of the versely, strains 98.1259 and 98.1549 did (heptaene)
other fungi (Table 2). (Fig. 1B).
These five strains were used in a second screening step for The results of the three assays showed that Streptomyces
the detection of potential nonpolyenic metabolites with sp. 98.1549 had no antibacterial activity and that Strepto-
antifungal activity. For such a screening procedure, previous myces sp. 98.1259 was only active against Staphylococcus
studies have used four different assays in different combina- aureus ATCC 25923. These two strains also showed an inhi-
tion: antibacterial activity, ergosterol inhibition, UV-visible bition of their reduced antifungal activity in the presence of
spectra of active extracts, and (or) inhibition of spheroplast exogenous ergosterol. Furthermore, the active extracts of
regeneration (Bastide et al. 1986; Hacène et al. 1994; these strains presented absorption bands characteristic of a
Ouhdouch et al. 2001; Hilali et al. 2002). In our study, we polyene (heptaene). These results indicate that these two
used only the first three assays, which are most currently strains produced polyenic compounds on the medium used
carried out. First of all, the ability of the five isolates to in- and the strains were therefore not retained.
hibit the growth of some bacteria was tested (Table 3). Evi- On the other hand, Streptomyces sp. 98.0395 and Strep-
dence of antibacterial activity against bacteria lacking sterols tomyces sp. 98.0436 showed activity against C. tropicalis R2
(target of polyenes) in their cell wall argues in favor of the and Staphylococcus aureus ATCC 25923, and their extracts
nonpolyenic nature of the active substance. Strain 98.1549 did not reveal polyene-type UV-visible spectra. However, the
showed no antibacterial activity, whereas strain 96.0333 re- inhibition zones were markedly reduced after the addition of
vealed a broad antibacterial spectrum against five of six test exogenous ergosterol. This can be explained by a co-
bacteria. The three other isolates (98.0395, 98.0436, and production of polyene and non-polyene products by the
98.1259) were only active against Staphylococcus aureus same strain. Therefore, these two strains were not retained in
ATCC 25923. the first times for the continuation of our work and will be
As sterols of yeast membranes are the targets of polyenes, subject to subsequent further investigations.
the addition of an exogenic sterol, like ergosterol, in growth Streptomyces sp. 96.0333, which was isolated from a ex-
medium interferes with the activity of the potential polyenic pectoration of a patient with a pneumopathy, was the only
substances produced by the isolates and reduces the inhibi- strain meeting all the selection criteria for the production of
tion diameters in the second screening assay. For non- potential nonpolyenic metabolite(s), i.e., it showed activity
polyenic metabolites, no interaction occurs and the diameter against C. tropicalis R2, it showed antibacterial activity
of the inhibition zones remains constant (Hamilton-Miller against five out of six test bacteria, and it showed no differ-
1974). This allowed us to easily detect the production of ence in activity before and after the addition of ergosterol.
polyenic metabolites. All isolates except Streptomyces sp. Moreover, the UV-visible spectroscopic analysis of the

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Lemriss et al. 673

Table 3. Results of three step screening method for nonpolyenic antifungal agents.
Antibacterial activity Ergosterol effect† UV-visible spectroscopic analysis
Gram-positive Gram-negative Polyene
Actinomycetes strain and reference No. bacteria bacteria Without With Active extract absorption (nm)
Streptomyces sp. 96.0333* ++ +++ 52 52 Methanolic extract Nondetected
Hexanic extract Nondetected
Streptomyces sp. 98.0395* + – 17 13 Methanolic extract Nondetected
Streptomyces sp. 98.0436* + – 19 10 Ethyl acetate extract Nondetected
Streptomyces sp. 98.1259* + – 21 12 Methanolic extract Heptaene (362,
381, 404)
Streptomyces sp. 98.1549* – – 19 8 Methanolic extract Heptaene (362,
382, 404)
Streptomyces noursei NRRL B-1714 – – 21 15 ND ND
Streptomyces nodosus NRRL B-2371 – – 15 8 ND ND
Reference ND ND 18.5‡ 8‡ Amphotericin B Heptaene (362,
381, 405)
Note: +, activity against Staphylococcus aureus ATCC 25923; ++, activity against S. aureus ATCC 25923 and Enterococcus faecalis ATCC 19433;
+++, activity against Enterobacter cloacae ATCC 13047, Proteus vulgaris ATCC 13315, and Citrobacter freundii ATCC 8090; –, no activity. NRRL,
Northern Regional Research Laboratory, Peoria, Ill., U.S.A.; ND, not done.
*Reference No. in the collection of actinomycetes of the Laboratoire de Mycologie Fondamentale et Appliquée aux Biotechnologies Industrielles,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon, Lyon, France.

Inhibition zones in mm with or without ergosterol at 50 mg/mL in Casitone medium.

Amphotericin at 20 µg/well or disc.

Fig. 1. UV-visible spectra of methanolic extracts of Streptomyces methanolic (pellet) and the hexanic (supernatant) extracts
sp. 96.0333 (A) and Streptomyces sp. 98.1259 (B). showed no absorption bands characteristic of polyene
antifungal metabolites. The presence of active substances in
the supernatant and in the mycelium suggests that one or
more potential nonpolyenic compounds are produced by this
strain, which explains that the activity could be due to their
cumulative effect. Isolation, purification, structural elucida-
tion, and mechanisms of action of these active products from
this strain are under investigation.

Acknowledgements
We thank Professor D.P. Labeda (USDA/Agricultural
Research Service, National Center for Agricultural Utilization
Research, Microbial Properties Research 1815 N. University
Street, Peoria, IL 61604, U.S.A.) for providing control strains
producing antifungal agents.

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