Spencer, 1942). The medium used most extensively for this purpose was that

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ASPERGILLUS CLAVATUS, AND THEIR ANTIBIOTIC SUBSTANCES1

SELMAN A. WAKSMAN, ELIZABETH S. HORNING AND ERNEST L. SPENCER NewJerseyAgriculturalExperiment Station,NewBrunswick, N.J.

Received for publication August 14,1942

It has been definitely established (Waksman, 1941; Waksman and Horning, 1942) that fungi capable ofproducing anti-bacterial substances are widely dis- tributed in nature and are not limited to any one or more taxonomic groups.

Suchfungi canbe readily isolated from soils, manures, composts, and dust by theuseof appropriate bacteria-enrichedagar media. Out of some 160 antagonistic fungi thusisolated, two organisms were selectedfor further study, because of their outstanding capacity to produce in culture media substances which inhibit in rather high dilutions the growth of various bacteria. These two organisms were

Aspergillus

fumigatus Fresenius, of which 16 strains wereisolated from different soils and composts, and Aspergillus clavatus Desmazieres, repre- sented by3 isolations. Themethods of testing the antibiotic action of the cul- turefiltrates ofthese organisms have beendescribed in detailelsewhere (Waks- man and Homing, 1942). This paper deals specifically with the formation, concentration, andisolation of the antibiotic substances produced by these two fungi under different conditions of culture and nutrition.

ASPERGILLUS FUMIGATUS

When grown in simple synthetic media A.

fumigatus

produces an antibiotic substance, which has beendesignatedas "fumigacin" (Waksman, Horningand Spencer, 1942). The medium used most

extensively

for this purposewas that ofCzapek-Dox oraglucose-nitrate solution, which consists of⁴⁰ g. glucose, 3 g. NaNO3, 1 g. KH2PO4, 0.5 g. KCl, 0.5 g. MgSO4 7H20, 0.01 g. FeSO4.7H20, and 1000ml. tap water.- The antibiotic substancewasfoundtobeproducedin thismediumduringthe earlystageofgrowthof theorganism:itspresencecould be demonstrated evenwithin3

days

incubation at

28°C;

it reachedamaximum in 7-8 days, and then gradually disappeared on further incubation. The re- duction inactivityis dueeither tothe destruction of theactive substanceorto its transformation into an inactive or at least ^a less active type of compound.

Inviewoftherecent announcement madebyOxford and Raistrick

(1942)

that fumigatin, a maroon-colored pigment produced by A.fumigatus, also has anti- bacterial properties, it was desirable to establish first the

possible

relation between the nature and formation of fumigatin and fumigacin, as well as their chemicalandbiologicaldifferences. Thiscanbedonebycomparingtheresults onfumigacinobtainedinthislaboratory,with those on

fumigatin reported

from

1Journal Series paper of the New Jersey Agricultural Experiment Station, Rutgers University, Departmentof SoilMicrobiology.

233

on May 14, 2021 by guesthttp://jb.asm.org/Downloaded from

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S. A. WAKSMAN, E. S. HORNING AND E. L. SPENCER

Raistrick's laboratory (AnslowandRaistrick, 1938; Oxford andRaistrick, 1942).

Thecomposition ofthe media used in the production of these two substances was different. Thesubstances are apparentlyproduced at different stages of growth ofthe fungus, fumigacinat anearly stage (3-10 days) and fumigatin at a late stage (35 days). The methods of isolation are also somewhat different, fumigacin being first adsorbedonnorit, then treated with ether and finally extracted inchloroform, whereas fumigatin is removed directly from the medium by chloroform. Because of these differences, the two substances can easily be differ-

TABLE 1

Distinguishingcharacteristics offumigatinandfumigacin-

CIARACTRMISTIC PROPERTIES YFUIGATIN* PUGACINt

Mediumproducedin Has been isolated from a Has been isolated from A.

culture of A.fumigatus in fumigatusculture inCzapek- Raulin-Thom medium Doxmedium

Stageof growth atwhich Late stage(30-35days) Early stage (3-10 days) substance is largely

formed

Methodof extraction from Direct extraction of medium Adsorption on norit, extrac-

medium with chloroform tion with chloroform, fol-

lowing ether treatment Crystallizationof From petroleum ether Fromalcohol, on cooling

substance

Appearance ofcrystals Maroon-coloredneedles Purewhiteneedles

Carbon content, percent 57.1 62.7

Presence ofnitrogen Absent Present (3.7percent)

Meltingpoint 1160 185°-1870

Chemicalnature Quinone Unknown

Unitsofantibiotic 12,000to 100,000 10,000-6,000,000 activity

Specific nature of anti- Little difference in activity Gram-negative bacteria (E.

biotic activity upongram-positive (spore- coli) are far less sensitive formers) and gram-nega- (10,000 units) than gram- tive(E. coli)bacteria positive bacteria (B.

mycoides-2,000,000, S. Lu- tea-6,000,000units).

BaseduponresultsobtainedinRaistrick's laboratory.

tBaseduponour ownresults.

entiatedfromeachother,asshown in table 1, whichsummarizestheir formation andproperties onthe basis of the results obtained in the two laboratories.

Fumigacin andfumigatin are thus shown to be distinctly different compounds, varying in the modeofformation, chemical nature, and antibacterial activity.

The capability ofa single organism to produce more than one substance which possesses antibiotic activity isno rare phenomenon among antagonistic microorganisms. Thishasbeen established,forexample, forPseudomonas

aeruginrosa,

whichproduces pyocyaninand pyocyanase; for Bacillus brevis which forms gram- icidin andtyrocidine;forActinomyces antibioticus, which produces the pigmented 2304

(3)

actinomycin A and the non-pigmented actinomycin B; and for Penicillium no- tatum, which forms the veryimportait antibiotic substance penicillin, the pig- mentchrysogenin, andasubstanceactive against certaingram-negative bacteria, such as Etcherichiacoli, which may bedesignated as the colon factor (Waksman and Homing, 1942).

TABLE 2

Production of antibiotic subotances by A.fumigatusin Czapek-Dox and Raulin-Thommedia

Activity of culture media*

S. lutea units.

B. mycoidesunits...

Reactionofmedia, pH...

Iodine reaction...

Yieldonchloroformextrac- tion(mg. perliter)...

Activity ofextractt S. lutea units...

B. mycoides units...

Yield byadsorptionon norit, ether-soluble (mg.

per liter).

Activity of ether-soluble material:

S. luteaunits...

B. mycoides units...

Yield by adsorptiononno- rit, chloroform soluble (mg. perliter)$...

Activityof chloroform-soluble material:

S. lutea units...

B. mycoidesunits...

NATuRt o0 MzDrum

Czapek-Dox

I

Raulin-Thom

Incubation, days 6

>1,000 750 7.4 0 0 522 100,000 10,000

54

10,000 1,000

156

>100,000 25,000

11

100 10 0 358

25

10

<10 7.8 0 336 1,000,000 20,000 200,000 20,000

46 68

100,000 120,000 20,000 40,000

226 128

300,000 120,000 100,000 50,000

6 1 11 25

10 70 3.5

+

166 3,000

<1,000

86

<1,000

104

<1,000

5

<5

236 20,000 10,000

116

<10

<10 5.0

+

470 10,000 10,000

287

<30,000 <3,000

152

<30,000

224

12,000 9,000

*Unitsofactivity=10/Amountofcultureormaterial necessary to inhibit thegrowth of thetestorganismin 10 ml.nutrient agar.

tOngrambasis ofdrymaterial isolated.

Adsorbed^onnorit,removedbyether,followed bychloroform.

One ofthe active strainsofA.fumigatus, namelyNo. 84, was used forade- tailed study of the production of the antibiotic substance fumigacin and the pigment fumigatin. The culture was grown on Czapek-Dox medium, found mostsuitableforthe formationoffumigacin,andontheRaulin-Thom

medium,

used by Raistrick and his associates

(Anslow

and Raistrick, 1938; Oxford and Raistrick,

1942)

forthe

production

of

fumigatin.

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2. A. WAKSMAN, E. 5. HORNING AND E. L. SPENCER

The course of formation, isolation, relative yields, and activity of the two substances are given in table 2. The substance produced in the Czapek-Dox medium had very high anti-bacterial properties, especially after6daysincuba- tion, whereas little antibiotic substance wasproduced intheRaulin-Thom medium. When the culture filtrate was extracted directly with chloroform, a fairly good yield of material possessing ahigh antibiotic activity was obtained from the 6- and 11-day old cultures on the Czapek-Dox medium and a more limited

yield

with a much lower activity from the Raulin-Thom medium. When the active substancewasabsorbedon norit, then removedbyetherandchloro- form,the Czapek-Dox mediumyielded a substance (fumigacin) withhigh antibiotic activity, whereas similar preparations obtained from the Raulin-Thom medium possessedlittle activity. The Czapek-Dox medium gave only aweak iodine test and a weak reactionwithconcentrated

sulfuric

acid(qualitativetests forquinones); however, the Raulin-Thom medium gavea strongreaction with these two reagents, thusconfirming the results of Anslow and Raistrick (1938) on theformation of the quinone fumigatinin this medium.

These results thus serve toillustrate further themarkeddifferenceinthefor- mation, chemical nature, and biological activity of fumigatin and fumigacin.

The latter substance may, therefore, beconsidered asthe true antibiotic agent produced by A.fumigatus. The pigment fumigatin also possesses some anti- bacterial properties; however, these appear to be those characteristic of qui- nones, to which group this substance belongs and which are known to act as bacteriostatic agents (Morganand Cooper, 1921, 1924;Oxford, 1942). It is of particular interesttonotethatwhereasfumigacinisactivechiefly againstgram- positive bacteria, especially cocci and spore-forming bacteria, and has comparatively little activity against various gram-negative bacteria, such as E.

coli and Aerobacter aerogenes, fumigatin, ina manner typical ofquinones, does notseemto exhibit suchmarkeddistinctions, ascouldbe seenfrom the results reportedbyOxfordand Raistrick (1942). Fumigatin was saidtodeteriorateon standing, inhibition of Staphylococcus aureus being reduced from 1:50,000 to 1:25,000 in 7

days.

Fumigacin, however, when kept inthe refrigerator for a month or more showed no deterioration, the inhibition of different strains of S. aureus by crude material varying from 1:200,000 to 1:500,000, and by the crvstalline material from 1:200,000 to 1:750,000.

ThegrowthofA.

fumigatus

onthe glucose-nitrate medium always results in analkaline reaction. This isbrought out

in,

table 3, where the results of an ex- perimentontheeffectofconcentrationofglucose andnitrate upon the production offumigacinarereported. Neither the amount of energy material nor the concentration of thenitrogen appeared tohave anysignificant effect upon the formation of this substance. The period of incubation seemed to be most important,theoptimumbeing6-9 daysat28°C. Thisperiodwas,therefore, used in allsubsequent experiments. It is particularly interesting to note the rapid disappearanceofthe active substance

after

9daysincubation. This maybe due toitsrapid

oxidation,

asbroughtoutintable4. The shallower cultures tended tolosetheiractivitymuchmorerapidlythan thedeepercultures. Theaddition 2306

(5)

of zinc, an element often found to play an important role in the metabolism of many fungi, had a somewhat depressive effect upon the active substance,

TABLE 3

Influence ofglucoseand nitrate concentration uponthe rate of production of fumigacin

ACTIVITYOFCULTIREFILTRATEIN

GLUCOSE NITRATERINONpH umNIstOF

INMIUM CONCENTRATION ATION

S.lhka B.mycoides B.subtiis percess perconS days

2 0.3 6 7.6 >300 300 100

2 0.3 9 7.9 1,000 200 100

2 0.3 12 8.0 30 15 < 10

2 0.6 6 7.6 >300 300 150

2 0.6 9 7.8 1,000 200 100

2 0.6 12 7.9 30 15 < 10

4 0.3 6 7.8 >300 250 100

4 0.3 9 7.7 1,000 100 75

4 0.3 12 6.6 100 50 20

4 0.6 6 7.5 >300 300 > 100

4 0.6 9 7.6 1,000 200 100

4 0.6 12 7.7 100 50 20

6 0.3 6 7.4 >300 200 100

6 0.3 9 3.9 1,000 100 100

6 0.3 12 3.5 100 50 50

6 0.6 6 7.4 >300 200 100

6 0.6 9 7.4 1,000 100 60

6 0.6 12 7.3 100 50 20

*Glucose present in allflasks;after12days,only thosereceiving 4 and 6 per cent still contained someglucose.

tSee first note1, table 2.

TABLE 4

Influenceofdepth ofculture andofzincsulfateuponthe formationoffumigacin

ACTIVITYt

VOLUIE OF MEDIUM ZnSO*7Hs0* 7 days 15 days

DiN1LITERFLASKC

S.hlta B. mycoides S.l,dea B.mycoides

150 - >1,000 200 100 20

250 - >1,000 300 250 50

350 - >1,000 150 1,000 60

500 - 700 100 1,000 100

150 + >1,000 200 30 20

350 + >1,000 200 60 20

* 10mg. perliter.

t On the basis of 1 ml. of the culture filtrate.

beyond the optimum

period

of incubation, and appeared to favor its rapid destruction.

(6)

The isolation of fumigacin could be most readily brought about by treating the culture filtrate with norit A(5g. perliter); complete adsorption took placein about 2-3hours, at37°C. The norit is then dried and treated with ether, followed by chloroform. Although the latter reagent is quite sufficient to remove virtually all the fumigacin from the norit, it was found that the preliminary treatment with ether facilitates considerably the subsequent crystallization of thefumigacin. The chloroform solution is evaporatedto dryness and the residue treated with ethylalcohol, in whichfumigacin isreadily soluble, especially on warming. On cooling the alcoholic solution in the refrigerator, fumigacin crystallizesoutintheformofwhite, needle-shapedcrystals. The mother liquor remainshighlyactive, thedegreeofactivity being similar,on adry weightbasis, to that ofthecrystalline fumigacin. This activity appeared tobe largely due to some ofthefumigacin remaining in solution.

It is sufficient to summarize the results of atypical experiment on the yield andactivity offumigacin. Six-dayoldcultures of A.fumigatuswereused. The filtratehada pH of 7.9 andanactivityof 1,000 Sarcina lutea, 450 Bacillusmy- coidesand300Bacillussubtilisunitsper1 ml. Tenliters of filtrateweretreated with norit. The following yields and activity were obtained;

ETER-SOLUBLEETHER-SOLUBEFRACTIONFRACTION(UMIGACI)|CHORtOFORM-SOLUBLEFRACTION

Yield

TESTORGANISM

69mg. 601mg.

Unitsofactivityongramweightbasi

Crude material Crude material

S. lutea.300,000 >2,000,000

B. mycoides.75,000 400,000

B.8ubtilib.75,000 200,000

E.coli. <100 200

Crystals

S. lutea 6,000,000

B.mycoides 3,600,000

B. subtilis 1, 0,000

The ether-solublefractionappearstocontainsome

fumigacin,

which isaccom-

panied by some non-active material, thus

reducing considerably

its

activity

on aunit weight basis.

Fumigacin not only possesses strong bacteriostatic

properties,

but is also actively bactericidal. This is

brought

out in table5. Six-hour old culturesof S. lutea were completely sterilized in 16 hours

by

the addition of 0.02 mg. of crude fumigacin to 1 ml. of culture.

Complete

sterilization of cultures of S.

aureus andofspore-formingbacteriawas

brought

about

only by

theadditionof 0.5 mg. ofactivesubstance per 1ml. ofculture. Sinceexcessive concentrations ofbactericidal agentsare

required

to sterilizeaculture

completely,

itiscustom- arytocalculate the disinfectant value of ^a substanceonthe basis of50per cent 238

(7)

destruction of cells. In a second experiment, older cultures of two bacteria were treated with varying amounts of fumigacin and, after allowing the substance to actupon the bacteria for 18 hours, plated out, in order to determine the numberofsurvivingcells. The results (table 6) show that theaddition of even aslittleas 4microgramsof crystalline fumigacin per 1 ml. of17-hour-oldcultures brought about more than 80 per cent destruction of S. lutea and 60 per cent destruction of B. mycoides. Furtherdata onthe antibiotic action of fumigacin against avarietyofbacteria aregiven in table 11.

TABLE 5

Bactericidal action offumigacin

Six-hour-oldbroth cultures of organismstreated with varying amounts of crude fumigacin, allowedto actfor16hoursat280^or37°C.

CONCEmNTRTON o0 IAc, MG. mE 1mr. OrcuLTR

T OR SM_

0 0.02 0.1 0.5

S.utea +++* 0 0

B.mycoides... +++ +++ ++ 0

B. subtilis... +++ ++ + 0

S.aureus...0+++ +++ +++

*+++ - goodgrowth, asdeterminedbystreakmethod; ++ - limitedgrowth;+ - traceofgrowth; 0 = no growth.

TABLE 6

Bactericidalactionofcrudecry8tallinefumigacin

Seventeen-hour-old culturestreatedwithfumigacinand incubated 18hours

BMCTERI LEFTPR1ML.

CONCENTUATIONofFUGACIN, MG. PER 13M.CULTUR.

S.nkaa B.mycoides

Crystallinepreparation

O 710,000 40,000

0.2 7,000 6,700

0.02 74,000 2,600

0.004 120,000 15,000

Crudepreparation

0.5 0 5,500

0.05 6,000 24,000

The action of fumigacin was also tested against fungi. The activity of the substanceagainsttheseorganismswasfoundtobeonlyvery limited. Growing cultures of A. fumigatus, however, appear ^to antagonize the growth of many fungi.

Aninterestingcorrelationwas foundto existbetweenthe growthofA.fumi- gatusatdifferenttemperatures and theproductionoffumigacin. Theorganism grows wellat 50°C, butit does not produce any active material. Asthe tem- perature ofincubation is reduced from 500 to 25°C., the amount of

fumigacin

(8)

produced gradually increases, without necessarily impairing the growth of the organism. Below 25°C., however, both the growth of the organism and the formationof the active substance arereduced.

Fumigacin has a certain degree of resistance to higher temperatures. On boiling the material in aqueous solution for 5-10 minutes, its activity was reduced, without being destroyed completely. Onheating at 80°C., for 15 nin- utes, theactivitywas reducedonlyslightly. Hence the substance may beconsidered as thermostable.

Fumigacin crystals are only sparingly soluble in water. When dissolved in alcohol and precipitated by addition of 9 volumes of water, the alcohol-water solution was foundto contain 0.25 mg. fumigacin per 1 ml.

Little is knownas yet concerning the activityof fumigacin in vivo. Itis to be recalled that Vaudremer (1913) reported that a group of patients suffering from tuberculosis were treated with extracts of A.fumigatus, with varying de- greesof success. The disease-producingorganism (Mycobacteriutm tuberculosis) wasrenderednon-pathogenic bysuch treatment.

Fumigacin appearedto befairly toxic to experimental animals, 1 mg. ofthe crystallinepreparationkillinga20gm.weight mousein24hours, wheninjected intraperitoneally. Whether thereis variation inthetoxicityoftheanti-bacterial substanceproduced by differentstrains of A.fumigatus, similartothe variation

in

theyield of active substance is stillto bedetermined.

ASPERGILLUS CLAVATUS

The fungus A. clavatus represents a group of organisms morphologically distinct from A. fumigatus. The type of antibiotic substance also proved to be very markedly different. Three cultures of this organism were isolated from stablemanure. They variedgreatlyinactivity, one (no. 129)beingmostactive and another (no. 130) possessing only limited activity. The mostactive strain was largely employed in these studies.

During the course of this work, there appeareda noteby WViesner (1942) re- porting resultsonthe formationofanantibiotic substance by A.clavatus. The activeagentappeared tobe similar tothat produced bythe strain used inthis study. Inviewof thefact, however, that onlypreliminarydatawerepresented, which appearto coveronly someof theproperties ofthe substance, the results of thefollowinginvestigationsarereportedwithoutfurthermodification.

A.clavatuswasgrown on avarietyofmedia, under differentculture conditions.

Theglucose-nitrate medium waschieflyemployed. The organism produced on thismediumasharp, pungent odor, similar to the putrefactive odor of many bacteria grown on protein media. The reaction of the media always became acid. The activesubstance was formed duringthe early stages of growth, even within 3days incubationat28°C. Theactivityofthe culture tended todimin- ish on further incubation (table 7). The highest activity was usually obtained in 6 days though the culture was still active even after 3 weeks. The culture filtrate showedtwomarkeddifferencesfromfumigacininanti-bacterial activity:

first, it was active against E. coli; and, second, it possessed greater activity 240

(9)

against B. subtilis than against B. mycoides. The active substance could be removed readily from the culture either by direct treatment with ether and chloroform or byadsorption onnorit and subsequent elution by ether and chloroform. It wasfar morereadilysoluble in ether than was fumigacin; it was also readily soluble in water. This substance appeared thus to be distinctly different from fumigacin in chemical properties and biological action. It possessed another important property, a high bactericidal action, which made it quite distinct from certain other antibiotic agents, such as penicillin.

Becauseof these specific chemical andbiological properties, this substance is recognizedasdistinctin natureand is designatedas clavacin.

The nature of the nitrogen source, its concentration, presence of certain growth-promoting substances,. neutralization of acid produced during growth, wereall foundto beof importance in theproductionofanti-bacterial agentsby certain fungi (Waksman andHorning, 1942). Inorder toestablishtheoptimum conditions for theproduction of clavacin, the influence of these factors upon the

TABLE 7

Production of clavacin in glucose nitrate mineral medium

ACTIVITYOF FILTRATE INCUBATIONAT pH

28tC.

S.lulta E.coli B.subtilis B.mycoides days

5 4.1 100 100 45

6 4.5 1,000 >30 300 150

7 300 100 150 100

11 4.9 250 100 250 100

growth of the organism and upon the production of the active substance was determined. The results of a typical experimentare presentedin table 8.

Theoriginalglucose-nitrate mediumappearedtobeasgoodfor theproduction of clavacin as was the modification of the medium by the replacement of the nitrate by another source of nitrogen, by an increase in concentration of the nitrate,orby neutralization of theacidproduced. Thelasttypeofmodification, in fact, actually tended to be injurious. Itwas found, on furtherstudy, however,thattheuseofrawsugar inplaceoftechnicalglucoseresultedinsomewhat greater activity. This suggests the possible function of some active agent or trace element present in the raw sugar.

In order to concentrate and isolate clavacin from the culture

medium,

the samemethodswereusedasinthe isolation of thefumigacin. The adsorptionof theactive substanceonthenorit,however,

proceeded

more

slowly

andwasless complete. The clavacincould be removed from the norit more

readily by

the use of ether than by chloroform. It could also be extracted directly from the culture filtratebymeansof thesetwo solvents; althoughsomewhat lower

yields

wereobtained, the activity was

high.

This is

brought

out intable 9.

The final procedure adopted for the extraction of clavacin consisted in the

(10)

treatment of the culture ifitrate with norit for 4-6 hours at37°C., and the re- moval of the active material by several treatments of the norit with ether, followed by chloroform. Since the reaction of the culture medium became acid

TABLE 8

Influenceofnitrogensourceandamendments upon the production of clavacin

ACTIVITY NITROGENSOIURCE INCUBATON pH

S.lida E.coli B.mycoides

days

NaNO3,3g./l... 7 4.5 250 100 80

NaNO8,6g./l.. 7. ....4.8 ....250 ....100 ...80

NaNOs, 3g./l.+CaCO3. 7 7.5 75 30 20

NaNOs, 3g./l. + corn

steep... 7 6.4 200 100 100

Peptone, 5g./... 7 3.6 200 100 100

(NH4)2S04, 3 g./l. +

CaCO... 7 6.7 75 10 10

NaNO,,3g./... 10 4.6 >100 100 100

NaNO3,6g./... 10 5.2 >100 100 100

NaNO3, 3g./l. + CaCO3 10 7.1 100 75 50

NaNOs, 3 g./l. + corn

steep... 10 6.2 >100 100 100

Peptone, 5g./lA.. 10.. ....3.1 ...>100 ....100 .100

(NH4)2804, 3 g./l. +

CaCO... 10 5.9 75 30 20

TABLE 9

Isolation of clavacinfromculture ;filtrate*

ACTIVITt

~~YIELD P ER UJT

S.hmca E.coli B.mycoides

mg.

Direct action with:

Ether... 286 200,000 200,000 100,000 Chloroform$... 204 300,000 200,000 60,000 Acidified, treatedwith norit:

Elutedwithether.892 60,000 20,000 20,000

Elutedwithchloroform$... 248 250,000 100,000 60,000 Treated directlywith norit:

Eluted withether.642 200,000 60,000 60,000

Elutedwith chloroform$. 30 20,000 150,000 150,000

*Seven-day-oldculture used.

tSee footnote1,table 2.

$Following extractionwithether.

during the growthof the organism, the culture filtrate wasusually neutralized with NaOH before the noritwasadded. Onevaporation of the ether andchloro- form, abrown gummy substance was produced. This was taken up in ethyl 242

(11)

alcohol, which appearedtoremove most of the activematerial. Aconsiderable gummy residue did not dissolve in the alcohol, but became soluble on subse- quenttreatment with NaOH solution. This alkalisolution also had some activity which was similar in nature to that of the alcohol-soluble fraction. It is impossibleto say atpresent whether more than one type of active substance was involved, or whether the substance was present in two different forms. The active substance appeared to be an acid, soluble in alcohol and in water; the sodium salt, however,wasinsolublein alcohol, but soluble in water. The latter appeared to be rather unstable, the activity disappearing on standing in the refrigerator.

Among the characteristic properties of clavacin, its high bactericidal action was particularly significant, as shown in table 10 and in figure 1. Different

TABLE 10

Bactericidal action of clavacin

Broth culture incubatedfor 6 and 18hoursandtreated with varyingamounts of clavacin Testmade 18hourslater

CONCENTRATION S. aures S.lukat B.subtilist

OFCLAVACIN IN

MICROGRASPER .

1ML.CULTURE 6hrs. 18hrs. 6 hrs. 18hrs. 6 hrs. 18hrs.

1,000 (t 0 0 0 0 0

300 0 0 0 0 0 0

100 0 + 0 0 0 0

30 +++ +++ 0 0 0 0

10 +++ +++ ++ + 0 0

3 +++ +++ ++ +

Control +++ +++ +++ +++ +++ +++

*Cultureincubatedat37°.

tCultureincubatedat28°C.

1 Seefootnote*,table 5.

bacteriavaried intheirreactionto thissubstance, S. aureusbeing lesssensitive than either S. lutea, thespore-forming B. subtilis andeven E. coli.

Older cultures of bacteriarequiredgreaterconcentrations of clavacinto

bring

about complete sterility. The action on S. lutea was similar to that on B.

subtilis. A 19-hour-old broth culture of S. lutea had fewer than 100,000 cells per 1 ml., as determined bythe plate method; after further incubation for 24 hours, thecontrol had3,500,000 cellsper 1ml. The addition of280 mg.ofclavacin gave complete sterility, 28 mg. reduced the number of bacteriato 150,000, and2.8mg.gave3,600,000bacteria,orthesamenumberasthe control.

Manybactericidalagents,whenusedin low concentrations, have been found toexert astimulating effectuponthenumbersofbacteria. Suchaneffect has also been obtainedfor clavacinactingupon E. coli, after 2.5and toa lesserex- tentafter 6 hours incubation. Thiseffect

disappeared

after 24hours.

Clavacinshowed also some fungistatic action,but to a rather limitedextent.

(12)

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(13)

Thefungi appearedto overcome the effect in a shorttime,either by inactivating the clavacinorby adjusting themselves to its action.

Clavacinis thermostable; heating at 100°C. for 10 minutes does not reduce its activity. It also appears to be fairly toxic to animals when injected into their tissues. Whether this toxicity is associated with strain specificity is still to be determined. Although Wiesner (1942) reported that he used his preparation for human treatment, no specific results were given.

TABLE 11

Bacteriostatic and bactericidal action offumigacinand clavacin

FJUMGACIN CrAVACIN,CRUDEMATERAL

TESTORGANISM* Bacteriostaticactiont Bactericidal Bacteriostatic Bactericidal Crystalsi Crudematerial¶ actiont actiont actiont A. aerogenes... <40,000 <40,000 >200 50,000 20 E. coli.... <40,000 40,000 >200 100,000 20 Salmonella schott-

muelleri. <40,000 <40,000 >200 60,000 20

Salmonellasp.

(Breslau type)... <40,000 <40,000 >200 75,000 20 Salmonella cholerae-

suis..<...<40,000 <40,000 >200 150,000 5 B. megatherium ... 1,250,000 1,000,000 20 100,000 5 B.cereus... 500,000 500,000 200 125,000 5 B. mycoides ... 1,250,000 500,000 200 200,000 2 B. subtilis ... 750,000 500,000 200 200,000 2

S.aureus3.... 750,000 750,000 200 100,000 2

S.aureus2. 500,000 250,000 >200 60,000 20

S. aureusH... 750,000 500,000 >200 75,000 20 S. aureusWl... 750,000 500,000 20011 200,000 5 S. aureusW2... 500,000 500,000 20011 200,000 2 S. lutea... 4,000,000 3,750,000 20 500,000 2

*Staphylococcus cultures,aswell as the gram-negative pathogens incubated at 370C.,

othersat280C.

tUnits ofactivity = dilution inplateortubeinhibiting growthcompletely.

$Microgramsrequiredto killbacteriain 1 ml. portionsof6-hour-old cultures.

§Watersaturated solution used.

¶Crude mother liquor,from which 2 lots of crystalswere removed onbasisof dried materialin solution.

11 Incomplete sterilization of cultureevenin 24 hours.

Comparative bacteriostatic and bactericidal

properties of fumigacin

and clavacin Asurvey wasmadeofboththebacteriostatic andthebactericidal properties of thetwosubstances,

fumigacin

and

clavacin,

upona

variety

of bacteria. These includedgram-negativeandgram-positive

organisms,

pathogenicandnon-pathogenic forms. Both agar and broth cultures were

employed

for measuring the bacteriostaticactionofthetwo

preparations.

Forbactericidal

studies,

six-hour- oldbroth cultures of thetest

organism

wereused. The resultsare

presented

in table 11.

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Fumigacin was found to have no importantbacteriostatic or bactericidal effect against thegram-negative bacteriaused in this test. Thatthe results were not absolutely negative isbrought out by thelimitedactivity againstE.coli. Fumi- gacin was most active against the gram-positive spore-forming bacteria and S.

lutea; it also possessed considerable activity against staphylococci. Clavacin, onthe otherhand, washighly active against gram-negative and gram-positive bacteria, therateof actionbeing similar. Clavacinshowedadegreeofbacterici- dalactivity comparable to its bacteriostatic capacity; fumigacin, however, had a rather wide ratio between these two properties. In many cases clavacin, when addedtothe culture before inoculation, showed bactericidal action upon 6-hour-old cultures of bacteria similar to its bacteriostatic action. On some organisms,suchasE. coli and S. aureus,strains2andH,thebacteridical action appearstobeevengreater thanthebacteriostaticeffect. Thisdiscrepancyisno doubt due to the fact that the first was measured in liquid cultures and the second on agar media. Eighteen-hour-old cultures, as well as mashed suspen- sions of bacteria, require larger amounts ofthe antibiotic agent for complete destructionofthe bacterial cells.

A comparison of the properties ofthese two antibiotic substances of fungus orgin with those of penicillin are presented below:

PENICL1IN PUMGACIN CLAVACIN

Solubility Water-soluble Sparinglysoluble in Water-soluble water, soluble in

alcohol

Activityinvitro Largely upon gram- Largelyupongram- Active upon gram- positive bacteria positive bacteria negative and some

gram-positive bacteria

Nature ofaction Largely Largely Highly

bacteriostatic bacteriostatic bactericidal Toxicity in vivo Lowtoxicity High toxicity Hightoxicity

SUMMARY

The results of a study ofthe antibiotic properties of two fungi, Aspergillus fumigatusand Aspergillus clavatus, isolated from soils, stable manure, and com-

posts are reported.

A. fumigatus grown in a glucose-nitrate mineral medium produces an anti- bacterial substance which was designated asfumigacin. This substance is dis- tinctinchemicalnatureandanti-bacterial activities from the pigment fumigatin also produced by this organism.

Fumigacinisformedintheculture medium duringthe early stages ofgrowth ofthe fungus, namelyin 5-9 days, and is then gradually destroyed on further incubation.

246

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Fumigacin can be concentrated and isolated by adding norit to the culture ifitrate, treating the norit with ether,andfinallyextracting the active substance inchloroform.

Fumigacin is readily soluble in alcohol and only sparingly soluble in water.

When the alcoholic solution is cooled, white, needle-shapedcrystalsoffumigacin areproduced.

Fumigacin is active against gram-positive bacteria and has comparatively little effect upon gram-negative organisms or fungi. It is both bacteriostatic and bactericidal, the ratio between thetwo properties being ratherwide.

A. clavatus also producesananti-bacterial substance in synthetic media. This substance, designated asclavacin, ismarkedlydifferent from fumigacin in both chemical nature and biological activity. It is nearly as active against gram- negative bacteria as against gram-positive organisms.

Clavacin is soluble inether,chloroform, alcohol andwater. Itappears to be anacid. In anacidstage,itismost soluble in alcohol. In the form of a salt, it is most soluble in water.

Clavacin possesses a high bactericidal action, almost comparable to its bacteriostatic properties. There is very little difference between the bactericidal action against gram-negative and gram-positive bacteria. This action is not instantaneous, but requiresacertain period of time. Older cultures of bacteria are more resistant than younger cultures.

Clavacin hasnot as yetbeen isolated in crystalline state.

Fumigacin andclavacinarefairly

toxnc

to animals.

Thetwosubstances described here, namely fumigacinand clavacin, are thus shown to possess selective bacteriostatic and bactericidal properties characteristic ofthe growing group ofantibiotic agents produced by microorganisms.

Fumigacin and clavacin differ in many respects from penicillin.

The authorsareindebtedto Mr. D. Hayman of the MicroanalyticalLabora- tory of Merck and Co. for the analysis ofthe crystalline fumigacin, Dr. H. J.

Metzger of the Department of Agricultural Biochemistry of the New Jersey Agricultural Experiment Station, and Mr. H.Robinson ofthe Merck Institute, formakingthetoxicitytests ofthetwo

preparations

described inthis paper.

REFERENCES

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LVII. Fumigatin (3-hydroxy4-methoxy-2:5-toluquinone) and spinulosin (3:6-dihy- droxy-4-methoxy-2:5-toluquinone), metabolic products respectively of Aspergillus fumigatusFreseniusand Penicillium spinulosumThom. Biochem.J.,32,687-696.

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248 8. A. WAKSMAN, E. S. HORNNG AND E. L. SPENCER

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