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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.

Rsum : Lobjectif de notre travail a t la slection de souches dactinomyctes dorigine clinique productrices de

substances antifongiques de structure nonpolynique. Ce choix a t fait afin dviter de redcouvrir les familles

dantifongiques dj connues, particulirement celles de structure polynique. Le criblage de cent dix souches a t effectu

contre trois souches de levures et trois souches de champignons filamenteux. Il a t ralis par deux mthodes de

diffusion et sur deux milieux test. Parmi les cinquante quatre souches (49 %) montrant une activit antifongique, cinq

souches appartenant au genre Streptomyces sont actives contre tous les organismes test et se sont rvles prometteuses.

Ces rsultats indiquent que les isolats dorigine clinique, comme ceux dorigine environnementale, sont une source

potentielle intressante de substances antifongiques. La production de substances antifongiques de structure nonpolynique

par ces cinq souches actives a t tudie en utilisant trois critres : spectre antibactrien, inhibition par lergostrol et

analyse des spectres UV-visible des extraits actifs. Une seule souche rpond aux trois critres de slection et produit

des substances de structure nonpolynique. Cette souche a t retenue pour des investigations complmentaires concernant

notamment la purification, la dtermination de la structure et du mcanisme daction du principe actif.

Mots cls : actinomyctes, Streptomyces, isolats cliniques, antifongique, non-polyne.

Lemriss et al. 674

Introduction

During the last decade, the development of immuno-suppressive therapy in medicine, the increase in iatrogenicfactors and nosocomial diseases, and the advent of new pa-

thologies, such as the acquired immunodeficiency syndrome,have been responsible for an ever-increasing incidence offungal infections (Chabasse 1994).

The antifungal agents currently available for the treatmentof systemic fungal infections are limited to polyenic anti-

Can. J. Microbiol. 49: 669674 (2003) doi: 10.1139/W03-088 2003 NRC Canada

669

Received 2 April 2003. Revision received 25 September 2003. Accepted 29 September 2003. Published on the NRC Research PressWeb site at http://cjm.nrc.ca on 16 December 2003.

S. Lemriss. UMR CNRS 5557 Ecologie Microbienne (Center for Microbial Ecology), Groupe de Recherche Pathognesopportunistes et environnement, Laboratoire de Mycologie Fondamentale et Applique aux Biotechnologies Industrielles, Institutdes Sciences Pharmaceutiques et Biologiques de Lyon, Universit Claude Bernard Lyon 1, 8, avenue Rockefeller, 69373 LyonCEDEX 08, France; and Laboratoire de Biochimie Applique, Facult des Sciences, Universit Chouaib Doukkali, El jadida, Maroc.F. Laurent, A. Couble, E. Casoli, D. Saintpierre-Bonaccio, and P. Boiron. 1 UMR CNRS 5557 Ecologie Microbienne (Center forMicrobial Ecology), Groupe de Recherche Pathognes opportunistes et environnement, Laboratoire de Mycologie Fondamentale etApplique aux Biotechnologies Industrielles, Institut des Sciences Pharmaceutiques et Biologiques de Lyon, Universit ClaudeBernard Lyon 1, 8, avenue Rockefeller, 69373 Lyon CEDEX 08, France.S. Rifai and A. Fassouane. Laboratoire de Biochimie Applique, Facult des Sciences, Universit Chouaib Doukkali, El jadida,Maroc.J.M. Lancelin. Laboratoire de RMN Biomolculaire associ au CNRS, Ecole Suprieure de Chimie Physique et Electronique deLyon, Universit Claude Bernard Lyon 1, F-69622, Villeurbanne, France.

1Corresponding author (e-mail: boiron@univ-lyon1.fr).

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fungals of microbial origin, such as amphotericin B and itslipid formulations, and to synthetic products, such as azolecompounds, flucytosine, and other new antifungal agents.These synthetic products offer limited indication or are inthe developmental stages. Amphotericin B remains the drugof choice for treating most fungal diseases because it has abroad spectrum and potent fungicidal activity, in spite of

well-known side effects (Andriole 1999; Lortholary et al.1999). Although the azole antifungals are considered to beless toxic, their efficacies against deep-seated, life-threateningmycoses are not fully satisfactory. In addition, it has beenreported that the frequency of multiazole-resistant strains be-longing to Candida species other than Candida albicans isincreasing (Hitchcok et al. 1993). In fact, there is a criticalneed for new fungicidal agents with original chemical struc-ture (non-polyene), with a broad spectrum of activity, andthat induce fewer side effects.

Actinomycetes have provided many important bioactivecompounds of high commercial value and continue to beroutinely screened for new bioactive substances (Barakate etal. 2002). It has been estimated that approximately two-thirds

of naturally occurring antibiotics have been isolated fromthese microorganisms (Okami and Hotta 1988). Environmen-tal actinomycetes, moreso that other groups of actinomycetes,have been regarded as a major source of antibiotics (Saadounet al. 1999). Several reports show that some opportunisticpathogenic actinomycetes, such as Nocardia brasiliensis, Nocardia pseudobrasiliensis, and Nocardia otitidiscaviarum,also produce novel bioactive substances (Tanaka et al. 1997;Komaki et al. 1999). For instance, a new antifungal macro-lide called brasilinolide B has recently been isolated a frompathogenic Nocardia brasiliensis strain (Mikami et al.2000).

The aim of the present study was to screen the antifungalnonpolyenic metabolites produced by clinical isolates from

an actinomycete collection.

Materials and methods

Strains and mediaOne hundred and ten actinomycete strains, recovered from

clinical samples, were used in this study. They included 91strains of Streptomyces spp., 11 Nocardia asteroides sensusstricto, 3 Nocardia farcinica, 3 Nocardia nova, 1 Nocardiabrasiliensis, a n d 1 Nocardia otitidiscaviarum. Each strainwas identified to the genus or species level by PCR methodspreviously described, including enzymatic amplification of16S rDNA with genus-specific primers or of a segment ofthe 65-kDa heat shock protein gene combined with restrictionanalysis of the amplimer (Laurent et al. 1999; Rodriguez etal. 2001; Steingrube et al. 1997). Four strains of actinomycetesproducing antifungal agents were obtained from ProfessorD.P. Labeda (Peoria, Ill., U.S.A.) and were used as controls.They included Streptomyces griseus (NRRL B-150), Strep-tomyces noursei (NRRL B-1714), Streptomyces natalensis(NRRL B-5314), and Streptomyces nodosus (NRRL B-2371),which produce cycloheximide, nystatin, pimaricin, andamphotericin A and B, respectively. All isolates were main-tained at 4 C on Bennetts agar slants.

Six fungal species obtained from the fungi collection ofthe Pasteur Institute were used as test strains: Candida albi-

cans CIP 48.72, Candida albicans CIP 884.65, Candidatropicalis R2 CIP 1275.81 (a strain resistant to amphotericinB and nystatin), Aspergillus fumigatus CIP 1082.74, Aspergillus fumigatus CIP 2279.94, and Trichophyton rubrumCIP 2043.92. All strains were maintained at 28 C onSabourauds agar.

Selection of actinomycete strains producing antifungalmetabolites

The selection of actinomycete strains was carried out byagar diffusion methods in Casitone medium (9 g/L Bactocasitone (Difco Laboratories, Sparks, U.S.A.), 5 g/L yeastextract (Merck KGaA, Darmstadt, Germany), 10 g/L sodiumcitrate (Prolabo, Paris, France), 20 g/L glucose (Merck),3.34 g/L di-sodium hydrogen phosphate (Merck), 0.54 g/Lpotassium di-hydrogen phosphate (Merck), and 18 g/L agar(Merck)) and in YMA medium (6.5 g/L yeast nitrogen base(Difco), 1.5 g/L asparagine (Prolabo), 10 g/L glucose(Merck), and 20 g/L agar (Merck)) for both agar cylindersand well diffusion methods.

Agar cylinders method (agar piece method)

Isolates were grown on Bennetts agar (Jones 1949) platesfor 4 days at 37 C, then a calibrated cylinder (3 mm in di-ameter) was cut out and placed on the test media (Casitoneor YMA), which had previously been seeded with each fun-gal test organism. Plates were kept first at 4 C for at least4 h to allow the diffusion of any antifungal metabolites andwere then incubated at 28 C (Barakate et al. 2002). Inhibi-tion diameters were determined after 24 h for yeasts, exceptfor C. tropicalis R2 (which requires 96 h in Casitone me-dium and 48 h in YMA medium), and after 48 h for filamen-tous fungi.

Well diffusion methodIsolates were grown in 10 mL of liquid Bennetts medium

for 4 days at 37 C. Twenty microlitres of this culture wasintroduced into a calibrated well (3 mm in diameter) cut intoeach test medium, which had previously been seeded witheach fungal test organism. Plates were incubated at 28 C(Magaldi et al. 2001). The inhibition diameters were mea-sured after 24 h for yeasts, except for C. tropicalis R2 (96 hin Casitone medium and 48 h in YMA medium), and after48 h for filamentous fungi.

Screening for actinomycete strains producingnonpolyenic antifungal metabolites

To select active actinomycete strains producing only non-polyenic antifungal agents, we carried out three experiments.

Antibacterial activity

Antibacterial activity of selected strains was estimated bythe agar cylinders method and the well diffusion method inMuellerHinton agar medium (Merck), against two Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 19433) and against four Gram-negative bacteria ( Enterobacter cloacae ATCC 13047, Pseu-domonas aeruginosa ATCC 10145, Proteus vulgaris ATCC13315, and Citrobacter freundii ATCC 8090).

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Ergosterol inhibitionErgosterol 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 CIP884.65 (Ouhdouch et al. 2001). The results were comparedwith those obtained with amphotericin B at 20 g/well ordisc and with reference strains producing polyenic anti-

fungal agents.

UV-visible spectroscopic analysis

Production and extraction of active metabolites

Isolates showing antifungal activity were precultured in a250-mL flask containing 25 mL of liquid Bennetts mediumand were incubated at 37 C for 24 h. The preculture wastransferred into a 500-mL flask containing 225 mL of liquidBennetts medium. After 3 days of incubation at 37 C, ei-ther the whole culture was extracted twice with ethyl acetate(1:1, v/v), or the pellet and the supernatant (obtained aftercentrifugation at 4500 r/min (1 r = 2 rad) for 15 min) wereextracted with methanol (1:5, v/v) and twice with hexane(1:1, v/v), respectively. The antifungal activity of the threeextracts for each active strain was determined by the welldiffusion and disc diffusion methods.

UV-visible spectra

The UV-visible spectra of the active extracts were re-corded in the 200500 nm range with a spectrophotometer(Kontron Instruments, Uvikon 932) and compared with thosespectra of known polyenic antifungals agents (Hacne et al.1994; Ouhdouch et al. 2001).

Results and discussion

The antifungal activity of a collection of clinical actino-mycetes was tested by using two diffusion methods and two

test media. Of 110 isolates studied, more than 49% producedmetabolites with antifungal activity against at least one outof six test organisms (Table 1). However, this percentage ishigher than those reported by many authors (between 10%and 34%) studying the activity of soil and aquatic actino-mycetes (Ouhdouch et al. 2001; Hacne et al. 1994; Hilali etal. 2002). Therefore, clinical isolates of actinomycetes ap-pear to be an especially interesting source of antifungal me-tabolites, highlighting the interest to develop the screeningof these pathogenic microorganisms. Our data showed thatthe test medium and the method of assay have a great influ-ence on the results of the screening (Table 1). Thus, the sus-ceptibility of C. tropicalis R2 and T. rubrum was bestrevealed by the well diffusion method in the YMA medium.Conversely, activity against A. fumigatus was best revealedby the agar piece method in the casitone medium. ForC. albicans, the results were similar, regardless of themethod or the test medium used. The need to select the mostactive strains provoked the interest in using different meth-ods and different test media. These results indicate the valueof using multiple methods and test media to detect activestrains.

The problem of rediscovery of well-known antifungalfamilies 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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Lemriss et al. 671

Diffusionmethod

Testmedium

Number(%)ofactiveactinomycetesstrainsagainst:

Total(%)ofactive

actinomycetestrains

Candida

tropicalisR2

Candida

albicans

Aspergillus

fumigatus

Trichophyton

rubrum

CIP1275.81

CIP48.72

CIP884.65

CIP1082.74

CIP2279.94

CIP2043.92

Agarcylindersmethod

Casito

ne

6(5)

42(38)

42(38)

13(12)

12(11)

3(3)

45(41)

YMA

9(8)

42(38)

40(36)

7(6)

6(5)

21(19)

52(47)

Total

11(10)

42(38)

42(38)

13(12)

12(11)

23(21)

54(49)

Welldiffusionmethod

Casito

ne

21(19)

40(36)

42(38)

11(10)

11(10)

5(5)

44(40)

YMA

30(27)

39(35)

39(35)

7(6)

7(6)

28(25)

49(45)

Total

32(29)

40(36)

42(38)

11(10)

11(10)

29(26)

53(48)

Note:CIP,CollectionofPasteurInstitute,Paris,France;YMA,6.5g/Lyeastnitrogenbase,1.5g/Lasparagine,10g/Lglucose,

and20g/Lagar.

Table1.Numberofactiveactinom

ycetesstrains,accordingtodiffusionmethodandtotestmedia(n=110).

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for clinical use are primarily of polyenic structure, in partic-

ular amphotericin B and nystatin. Thus, we chose to screenclinical actinomycetes for the production of nonpolyenicantifungal metabolites. First, we tested antifungal activity byusing a large number of species, including a strain of C. tropi-calis R2 (a strain resistant to amphotericin B and nystatin).Among the 110 tested isolates, five (Streptomyces sp. 96.0333,Streptomyces sp. 98.0395, Streptomyces sp. 98.0436, Strep-tomyces sp. 98.1259, Streptomyces 98.1549) appeared prom-ising because of the inhibition of C. tropicalis R2 andbecause of their broad spectrum activity against all of theother fungi (Table 2).

These five strains were used in a second screening step forthe detection of potential nonpolyenic metabolites withantifungal activity. For such a screening procedure, previousstudies have used four different assays in different combina-tion: antibacterial activity, ergosterol inhibition, UV-visiblespectra of active extracts, and (or) inhibition of spheroplastregeneration (Bastide et al. 1986; Hacne et al. 1994;Ouhdouch et al. 2001; Hilali et al. 2002). In our study, weused only the first three assays, which are most currentlycarried out. First of all, the ability of the five isolates to in-hibit the growth of some bacteria was tested (Table 3). Evi-dence of antibacterial activity against bacteria lacking sterols(target of polyenes) in their cell wall argues in favor of thenonpolyenic nature of the active substance. Strain 98.1549showed no antibacterial activity, whereas strain 96.0333 re-vealed a broad antibacterial spectrum against five of six test

bacteria. The three other isolates (98.0395, 98.0436, and98.1259) were only active against Staphylococcus aureusATCC 25923.

As sterols of yeast membranes are the targets of polyenes,the addition of an exogenic sterol, like ergosterol, in growthmedium interferes with the activity of the potential polyenicsubstances produced by the isolates and reduces the inhibi-tion diameters in the second screening assay. For non-polyenic metabolites, no interaction occurs and the diameterof the inhibition zones remains constant (Hamilton-Miller1974). This allowed us to easily detect the production ofpolyenic metabolites. All isolates except Streptomyces sp.

98.0333 showed a reduction in the diameters of the inhibi-

tion zones (Table 3).Finally, several authors have used spectroscopy to distin-

guish polyenic and nonpolyenic substances (Hacne et al.1994; Ouhdouch et al. 2001; Barakate et al. 2002). Thespectra of polyenes are characterized by a series of peaks be-tween 260 and 405 nm (Hamilton-Miller 1973). The UV-visible spectroscopic analysis of the active extracts showedthat those of strains 96.0333, 98.0395, and 98.0436 did notpresent such polyenic absorption bands (Fig. 1A). Con-versely, strains 98.1259 and 98.1549 did (heptaene)(Fig. 1B).

The results of the three assays showed that Streptomycessp. 98.1549 had no antibacterial activity and that Strepto-myces sp. 98.1259 was only active against Staphylococcusaureus ATCC 25923. These two strains also showed an inhi-bition of their reduced antifungal activity in the presence ofexogenous ergosterol. Furthermore, the active extracts ofthese strains presented absorption bands characteristic of apolyene (heptaene). These results indicate that these twostrains produced polyenic compounds on the medium usedand the strains were therefore not retained.

On the other hand, Streptomyces sp. 98.0395 and Strep-tomyces sp. 98.0436 showed activity against C. tropicalis R2and Staphylococcus aureus ATCC 25923, and their extractsdid not reveal polyene-type UV-visible spectra. However, theinhibition zones were markedly reduced after the addition ofexogenous ergosterol. This can be explained by a co-

production of polyene and non-polyene products by thesame strain. Therefore, these two strains were not retained inthe first times for the continuation of our work and will besubject to subsequent further investigations.

Streptomyces sp. 96.0333, which was isolated from a ex-pectoration of a patient with a pneumopathy, was the onlystrain meeting all the selection criteria for the production ofpotential nonpolyenic metabolite(s), i.e., it showed activityagainst C. tropicalis R2, it showed antibacterial activityagainst five out of six test bacteria, and it showed no differ-ence in activity before and after the addition of ergosterol.Moreover, the UV-visible spectroscopic analysis of the

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

Actinomycete strain and reference No.

Test strain

Candida

tropicalis R2

Candida

albicans

Aspergillus

fumigatus

Trichophyton

rubrum

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 Applique aux Biotechnologies Industrielles,

Institut des Sciences Pharmaceutiques et Biologiques de Lyon, Lyon, France.Activity measured by diameter of inhibition zones (in mm).

Table 2. Antifungal activity of selected and reference strains.

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methanolic (pellet) and the hexanic (supernatant) extractsshowed no absorption bands characteristic of polyeneantifungal metabolites. The presence of active substances inthe supernatant and in the mycelium suggests that one ormore potential nonpolyenic compounds are produced by thisstrain, which explains that the activity could be due to theircumulative effect. Isolation, purification, structural elucida-tion, and mechanisms of action of these active products fromthis strain are under investigation.

Acknowledgements

We thank Professor D.P. Labeda (USDA/AgriculturalResearch Service, National Center for Agricultural UtilizationResearch, Microbial Properties Research 1815 N. UniversityStreet, Peoria, IL 61604, U.S.A.) for providing control strainsproducing antifungal agents.

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