RAPAMYCIN (AY-22,989), A NEW ANTIFUNGAL ANTIBIOTIC
`
`I. TAXONOMY OF THE PRODUCING STREPTOMYCETE
`AND ISOLATION OF THE ACTIVE PRINCIPLE
`
`CLAUDE VEZINA, ALICIA KuDELSKI and S. N. SEHGAL
`
`Department of Microbiology, Ayerst Research Laboratories,
`Montreal, Quebec., Canada
`
`(Received for publication June 17, 1975)
`
`A streptomycete was isolated from an Easter Island soil sample and found to inhibit
`Candida albicans, Microsporum gypseum and Triclzophyton granulosum. The antibiotic(cid:173)
`producing microorganism was characterized and identified as Streptomyces hygroscopicus.
`The antifungal principle was extracted with organic solvent from the mycelium, isolated
`in crystalline form and named rapamycin. Rapamycin is mainly active against Candida
`albicans; minimum inhibitory concentration against ten strains ranged from 0.02 to 0.2
`µg/ml.
`Its apparent activity against Microsporum gypseum and Trichophyton granulosum
`is lower because of its instability in culture media on prolonged incubation required by
`these fungi. No activity was observed against gram-positive and gram-negative bacteria.
`Acute toxicity in mice is low.
`
`Cultures of a streptomycete isolated from an Easter Island (Rapa Nui) soil sample were
`found to inhibit the yeast, Candida albicans, and the dermatophytes, Microsporum gypseum and
`Trichophyton granulosum; antibacterial activity was only marginal and limited to some gram(cid:173)
`positive species, such as Sarcina /urea and Staphylococcus aureus; all gram-negative bacteria
`tested were resistant. The active principle was isolated from the mycelium of the streptomycete
`and the crystalline material obtained after purification was found to inhibit mainly Candida
`albicans; lesser activity was observed against the dermatophytes and no activity was demonstrated
`against any of the gram-positive and gram-negative bacteria tested. The antibiotic was named
`rapamycin [Etymol.: Rapa- (Rapa Nui=Easter Island), -mycin].
`Its structure is still unknown.
`This paper deals with the characterization of the producing streptomycete, the isolation and
`purification of the antibiotic and some of its biological properties.
`
`Identification of the Rapamycin-Producing Streptomycete
`
`Streptomycete strain AY B-994 was isolated from a soil sample collected in Easter Island
`(Rapa Nui): the soil was diluted in distilled water and the resulting suspensions were plated on
`yeast-starch agar according to the double-layer technique of PORTER et al. 11
`• After one week
`of incubation at 28°C the streptomycete colonies were purified by repeated streaking and the
`pure strains grown separately on yeast-starch agar plates to yield confluent growth. After 4-...
`10 days of incubation, discs (7 mm in diameter) were cut and transferred onto the surface of
`plates of Bacto-Blood Agar Base (Difeo Laboratories, Detroit, Mich.) inoculated with test
`bacteria and SABOURAUD dextrose agar inoculated with test yeast and dermatophytes. The
`Blood Agar Base plates were inoculated with a standardized bacterial inoculum grown at 37"C
`for S hours in Bacto-Nutrient broth; the SABOURAUD dextrose agar plates were inoculated with
`a standardized inoculum consisting of cells of yeast or spores of dermatophytes. Plates were
`
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`incubated at 37:ic for bacteria and yeast and at 28;)C for dermatophytes. Activity was read
`as diameter of inhibition zone after incubation of 18 hours, 48 hours and 5 days for bacteria,
`yeast and dermatophytes respectively. When tested under the above conditions strain A Y B-994
`showed the folJowing antimicrobial activity (diameter of inhibition zone in mm): Sarcina /urea,
`24; Staph.,vlococc11s aureus, 20; Escherichia coli, Enterobacter aerogenes, Proteus mirabilis and
`Pseudomonas aeruginosa, no zone of inhibition; Candida albicans, 24; Microsporum gypseum, 26;
`Tricl1ophyton granulosum, 27. Strain A Y B-994 was then maintained on tomato paste-oatmeal
`agar3
`' and preserved by Iyophilization 41
`The methods used for characterization \\.'ere those of the International Streptomyces Project
`published by SmRLING & GoTTLIEB51
`• Streptomycete strain AY B-994 was found to belong to ..
`the species Strepcomyces hygroscopicus in PRIDHAM & TRESNER's 21 key of classification; it was
`deposited in the ARS culture collection of USDA (Peoria, Ill.) and assigned the number NRRL
`5491.
`
`•
`
`1. Morphological Characteristics
`Streptomycete strain AY B-994 sporulates moderately to abundantly on most media studied.
`The morphology as revealed by light microscopy and electron microscopy is illustrated in Figs.
`1 and 2. Aerial mycelium is monopodially branched; sporophores are terminated by spore
`chains in the form of short, narrow, compact and closed coils (Spiral) of three or more turns;
`ten or more spores are present in each spiral. Spore color en masse is predominantly gray (gray
`series) turning gray-brown on prolonged incubation. Aerial mycelium is hygroscopic: on ab(cid:173)
`sorption of water, spores crowd in masses and a black pigment is produced, giving the appear(cid:173)
`ance of black, gelatinous spots on the surface of several solid media. Under the electron
`microscope spores are oval, smooth, 0.8 )< 1.6 p.
`
`Fig. I. Photomicrograph of Streptomyces hygro(cid:173)
`scopicus A Y B-994
`(ISP Medium 2, 14days at 25°C, x455)
`
`Fig. 2. Electron micrograph of Streptomyces hy(cid:173)
`g1-oscopicus A Y B-994
`(ISP Medium 2, 14 days at 25°C, x 2,500)
`
`,,:rx,t·:~:n: ·.
`;\ii!~.,.,.;:
`
`2. Cultural Characteristics
`The cultural characteristics of strain A Y B-994 are reported in Table l for International
`is by
`Streptomyces Project (ISP) media and various other media. Tomato paste-oatmeal agar:11
`far the best medium for growth, sporulation and development of black spots. After t\vo weeks
`of incubation at 28"C foJlowed by one week of storage at 4 "C the surface of this medium
`appears carbonaceus black; the preparation of homogenous spore suspensions from these black
`gelatinous masses is difficult. On this medium as well as on ISP Medium 1 (solidified) well
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`Table 1. Cultural characteristics of streptomycete strain A Y B-994
`
`Aerial mycelium
`
`Vegetative
`mycelium
`(growth and color) Development and color
`
`Rapid and
`aoundant;
`colorless to
`yellowish to
`grayish
`Rapid and
`at:>undant;
`yellowish
`
`Very abundant; grayish
`with many black spots
`
`Moderate to abundant;
`yellowish, with black
`spots
`
`Moderate to abundant;
`white to grayish to
`gray, with black spots
`
`Pigment
`
`I Spores/spiral; shape
`
`of sporophores**
`
`~ 10 spores/spiral;
`SCL >90?6
`
`> 10 spores/spiral
`
`Yellow
`
`> 10 spores/spiral;
`SCL 90?6
`
`Yellow
`
`Culture media*
`
`Tomato paste-
`oatmeal agara1
`
`Oatmeal agar
`(ISP Medium 3)
`
`Inorganic salts-
`starch agar
`(ISP Medium 4)
`
`Glycerol:
`asparagme agar
`(ISP Medium 5)
`
`Peptone-yeast
`extract-iron agar
`(ISP Medium 6)
`CzAPEK's solution
`agar (WAKSMAN'S
`Medium 1)
`
`BENNETT'S agar
`
`Tryptone-yeast
`extract agar
`(ISP Medium 1
`solidified)
`Yeast extract-malt Rapid and
`extract agar
`aoundant;
`yellow to dark
`(ISP Medium 2)
`yellowish
`Very abundant;
`pale yellowish
`to brownish
`'
`Slow, but
`eventually
`abundant;
`colorless to
`whitish; starch
`slowly hydrolyzed
`Slow, moderate
`after 14 days;
`colorless to
`pale yellowish
`to pinkish
`Poor; colorless to No sporulation (or
`pale yellowish;
`extremely poor)
`no H2S produced
`Slow, moderate
`after 14 days;
`colorless to
`pale yellowish
`Glucose-asparagine Rapid and
`agar (W AKSMAN's
`atiundant; pale
`yellowish to
`Medium 2)
`whitish, gray
`later
`Very abundant;
`pale to medium
`yellow to
`greenish
`Potato sucrose agar Moderate;
`I colorless to pale
`yellowish brown,
`! later yellow-gray
`SABOURAUD
`Rapid and
`dextrose agar
`at:>undant
`Rapid and
`SABOURAUD
`maltose agar
`aoundant
`I
`* For composition of ISP media refer to SmRLlNG & GoTTLIEa; 5' in this study Bacto dehydrated
`llledia \Vere used.
`** SCL=spirals, closed loops.
`
`Abundant; whitish to
`gray with black spots
`
`> 10 sporesjspiral;
`SCL 90?6
`
`Pale yellow
`
`Moderate; whitish to
`gray, with some black
`spots
`
`> 10 spores/spiral;
`SCL 9096
`
`None
`
`Very poor; grayish
`beige; no black spots
`
`-
`
`-
`
`Pale pink
`
`Yellow
`
`Poor, whitish to grayish
`
`ca 10 spores/spiral;
`SCL 70---8096
`
`None
`
`> IO spores/spiral;
`SCL 10 ..... 90?6
`
`Pale yellow
`
`> 10 spores/spiral;
`SCL 90?6
`
`Yellow
`
`Hyphae without
`spores; spirals
`irregular
`
`Yellow
`
`Moderate; whitish to
`yellowish, gray later,
`with black spots;
`surface brownish black
`after 28 days
`Moderate; whitish to
`gray to yellowish, with
`black spots
`
`Moderate; whitish to
`gray, with black spots
`
`Moderate to abundant
`
`Moderate
`
`I
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`isolated colonies are of two types. One type
`is powdery white, elevated and well sporulated;
`the other type is brownish, flat and much less
`sporulated. The white type is more productive.
`
`3. Physiological Characteristics
`The physiological characteristics of strain
`pH
`A Y B-994 are summarized in Table 2.
`and temperature ranges for growth and sporu(cid:173)
`lation are much wider than for production of
`rapamycin.
`From the observations reported above
`strain A Y B-994 appears to belong to the
`Its char(cid:173)
`species Streptomyces hygroscopicus.
`acteristics fit well within the range of charac(cid:173)
`teristics observed by TRESNER & BACK us61 for
`several strains which are recognized to belong
`to this species. Strain A Y B-994 also compares
`well to Streptomyces hygroscopicus, as recently
`determined by PRIDHAM & TRESNER21
`; it differs
`from the type strain listed by these authors
`in that it can also use raffinose and i-inositol.
`We feel these discrepancies do not justify the
`description of a new species for strain A Y
`B-994.
`
`Table 2. Physiological characteristics of strepto(cid:173)
`mycete strain A Y B-994
`I Reactions and other
`Parameters
`----------!-
`observations
`Slow, but eventually
`Hydrolysis of starch
`extensive
`(ISP Medium 4)
`Negative
`Decomposition of
`cellulose
`Production of hydrogen Negative
`sulfide (H2S)
`(ISP Medium 6)
`Production of tyrosinase Negative (melanin-
`(ISP Medium 7)
`negative)
`Very weakly positive
`Nitrate reduction
`I or negative
`(ISP Medium 8)
`Carbohydrate utilization Good growth on:
`(ISP Medium 9*)
`o-glucose, o-fructose,
`o-mannitol, i-inositol,
`starch. glycerol
`Moderate growth on:
`o-xylose, L-arabinose,
`L-rhamnosc, raffinose,
`lactose, o-maltose
`No growth on:
`sucrose, cellulose
`Growth at pH 5- > 8
`(optimum pH 6-8)
`Growth at 20.-w 37°C
`(optimum at25-27°C)
`no growth below
`18°C or above 47cc
`* Bacto-Carbon Utilization agar was used as
`the basal medium.
`
`Reaction to temperature
`
`Reaction to pH
`
`Fermentation of Rapamycio in Shake Flasks
`
`Strain AY B-994 was grown in several media at various pH and temperature values.
`Several sources of carbon and nitrogen were found to be suitable; optimal temperature and initial
`pH were 25°C and 6.0. Optimal composition of medium and agitation conditions have not
`been determined, but the following medium and conditions were found suitable for production
`of sufficient quantities of rapamycin for isolation and purification.
`A spore suspension was prepared by suspending the spores from a 14-day culture on tomato
`paste-oatmeal agar in distilled water; the suspension was diluted to read 70 ;'0 transmittance
`l 96 of the dilution used to
`(I-cm cuvette) in a Junior 6A Coleman spectrophotometer, and
`inoculate 500-ml Erlenmeyer flasks each containing 100 ml of the following medium (g-'liter):
`oatmeal, 50; "cerelose" (a pharmaceutical grade of glucose, Corn Products Corporation, New
`York, N.Y.), 20; N-Z Case (Sheffield Chemical, Norwich, N.Y.), 1; "blackstrap,, mo1asses, S;
`NaCl, 5 (in tap water; sterilization at 121°C for 20 minutes; pH after sterilization, 6.1). Flasks
`were incubated on a gyrotory shaker at 240 rev/min (2 11 -stroke), 25°C and 70 .90 R.H. Samples
`were taken daily for 7 day' and assayed microbiologically, using a standard solution of nystatin
`as a reference and Candida albicans as the test organism. When pure rapamycin became
`available, a standard curve could be drawn and unknowns· determined by
`interpolation.
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`A typical fermentation yielded the following results:
`
`Incubation
`
`pH
`Rapamycin (µg/ml)
`
`Oday I 3days
`6.0
`I
`-
`A two-stage fermentation could also be run by inoculating flasks as above for 24 hours and
`using 2 5'6 of this seed to inoculate production flasks prepared as above described; yields ob(cid:173)
`tained were essentially the same as in the single-stage fermentation.
`
`4days
`
`7. I
`45
`
`6.6
`40
`
`Sdays
`
`6days
`
`S.6
`100
`
`5.6
`100
`
`Isolation and Purification of Rapamycin
`
`After five days of incubation the fermentation broth was filtered through Celite and the
`mycelium, which contained most of the antibiotic, was harvested and washed with 1 ......... 2 volumes
`·of water. The washed mycelium was extracted three times with 5 volumes of methanol per
`weight of wet mycelium each time. The methanolic extracts were pooled and concentrated to
`the aqueous phase under reduced pressure. The concentrate was extracted three times with 1
`volume of methylene dichloride each time; the methylene dichloride extracts were combined,
`dehydrated with anhydrous sodium sulfate and evaporated to yield an oily residue.
`The oily residue was dissolved in 15 96 acetone in hexane and charged on a column of
`silica gel G (50 : l, w/v). The column was eluted with a solvent mixture of acetone and hexane
`containing gradually increasing concentrations of acetone. The elution of rapamycin was followed
`on TLC: an aliquot of each fraction was spotted
`on TLC silica gel G plates which were devel(cid:173)
`oped in acetone - hexane (40: 60). Rapamycin
`was eluted from the column when the develop(cid:173)
`ing mixture consisted ?f acetone and hexane
`(25 : 75). The fractions containing the antibio(cid:173)
`tic were combined and evaporated to dryness
`under vacuum. The product was crystallized
`from the residue with ether. Ten liters of
`fermentation broth generally yielded 300 mg
`·of pure, crstalline rapamycin.
`
`Table 3. Antimicrobial spectrum of rapamycin
`(A Y-22,989)
`
`Test organisms*
`
`Minimum
`inhibitory
`concentration
`(/lg/ml)
`
`Staphylococcus aure11s
`>100
`Staphylococcus aureus pen R
`> 100
`Streptococcus faeca/is
`>100
`Escherichia coli
`>100
`Enterobacter aerogenes
`> 100
`Klebsie/la pneumoniae
`>100
`Proteus mirabilis
`>100
`Proteus vulgaris
`>100
`Salmonella pullorum
`> 100
`Serralia 'fnarcescens
`> 100
`Pseudomonas aeruginosa
`> 100
`Candida alhicans (10 strains)
`<0.02-0.2
`Microsporum gypseum
`12.5
`Trichophyton granulosmn
`> 1,000
`* Medium for bacteria: Nutrient broth; medium
`for yeast and fungi: SABOURAUD dextrose broth.
`Incubation: 37°C for bacteria and yeast, 28°C
`for dermatophytes. Results read at 24 hours for
`bacteria, 48 hours for yeast, and 7 days for der(cid:173)
`matophytes.
`
`Biological Activity of Rapamycin
`
`The minimum inhibitory concentration of
`rapamycin for selected gram-positive and gram(cid:173)
`negative bacteria, yeast and dermatophytes is
`reported in Table 3. Rapamycin is very active
`against all strains of Candida albicans tested.
`It shows no activity against bacteria. The
`MIC's against Microsporum gypseum and Tri(cid:173)
`.chophyton granulosum are much higher than
`.anticipated from the inhibition zones observed
`
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`In the diffusion assay inhibition zones were largest for
`by the cylinder-plate diffusion method.
`T. granulosum and smallest for Candida albicans; inhibition of M. gypseum was intermediate.
`These contradictory results may be explained by the instability of rapamycin on prolonged incu(cid:173)
`bation in SABOURAUD Dextrose broth.
`Acute toxicity of rapamycin was studied in mice; LD50 (i.p.) is ca. 600 mg/kg; LDGo (p.o.)
`is >2,500 mg/kg.
`
`The able technical assistance of Mrs. TEODORA BLAZEKov1c-DESATI is gratefully acknowledged.
`
`Acknowledgments
`
`References
`
`1) PORTER, J. N.; J. J. WrLHEM & H. D. TRESNER: Method for the preferential isolation of actinomy(cid:173)
`cetes from soils. Appl. Microbiol. 8: 174-178, 1960
`2) PRIDHAM, T. G. & H. D. TRESNER: "Streptomyces WAKSMAN and HENRICI". in BERGEY's Manual
`of Determinative Bacteriology (Eds. R. E. BUCHANAN and N. E. GrneoNs), The Williams & Wilkins
`Company, Baltimore, pp. 748-829, 1974.
`3) PRIDHAM, T. G.; P. ANDERSON, C. FOLEY, L.A. LINDENFELSER, C. W. HESSELTINE & R. G. BENE(cid:173)
`"A selection of media for maintenance and taxonomic study of Streptomyces". bz Anti(cid:173)
`DICT:
`biotics Annual 1956/1957: 947-953, 1957
`4) RAPER, K. B. & D. F. ALEXANDER: Preservation of molds by the lyophil process. Mycologia 37:
`499,...,525, 1945
`5) SHIRLING, E. B. & D. GOTTLIEB: Methods for characterization of Streptomyces species.
`Bact. Nomen. Toxonomy 16: 313-340, 1966
`6) TRESNER, H. D. & E. J. BAcKus: A broadened concept of the characteristics of Streptomyces hy(cid:173)
`groscopicus. Appl. Microbiol. 4: 243 ...... 250, 1956
`
`Intl. Bull.
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