GROWTH CHARACTERISTICS AND ANTIBIOTIC PRODUCTION OFACTINOMYCETES ISOLATED FROM LITTORAL SEDIMENTS

AND MIATERIALS SUSPENDED IN SEA WATER'

A. GREIN2 AND S. P. MEYERSInstitute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey, and The MIarine

Laboratory, University of Miami, Miami, Florida

Received for publication April 11, 1958

The abundance of terrestrial actinomycetesand their role in numerous biological processesis well known. However, investigations dealingwith the marine counterparts of these organismsare extremely few and inconclusive. Microbio-logical studies, both bacterial and fungal, of thelittoral, benthic environment have largely ex-cluded the actinomycetes.

In his treatise on marine microbiology, ZoBell(1946) listed the occurrence of Mycobacterium,Actinomyces, Nocardia, and Micromonospora inthe sea, growing on dead marine algae, on sub-merged surfaces, and in bottom deposits. Speciesof Streptomyces were reported as being absentfrom the sea. Papers in which the presence ofmarine actinomycetes have been noted, havebeen mainly concerned with a tabulation andstudy of the other marine thallophytic florapresent. Consequently, the extent of the occur-rence of marine and halotolerant actinomycetesin the marine milieu can only be postulated.Only a few marine species of actinomycetes

are reported. Aronson (1926) described Myco-bacterium marinum, a marine pathogen causingspontaneous tuberculosis in salt water fish.ZoBell and Upham (1944) described 2 species ofActinomyces present in marine muds and alsoassociated with kelps. Humm and Shepard (1946)isolated 3 agar-digesting species of actinomycetesfrom marine material. The 2 species of Proactino-myces and 1 species of Actinomyces were collectedfrom intertidal marine sediments and beaches,

1 Contribution No. 194 from The Marine Labo-ratory, University of Miami. The development ofthis work at The Marine Laboratory, Universityof Miami, has been made possible by contractNonr 1811(00), Microbiology Branch, Office ofNaval Research.

2 Waksman-Farmitalia postdoctoral fellow.Present address: Laboratori di Microbiologia e

Chemoterapia Farmitalia (Milano), Italy.

as well as from seaweed, at Beaufort, NorthCarolina, and along the South Atlantic coast.All 3 species grew well in ordinary media and inthose prepared with as much as 6 per cent seasalt. The 7th edition of Bergey's iVianual recog-nizes the following marine species: NVocardiamarina (Krassilnikov) Waksman, (Proactino-myces flavus Humm and Shepard), Nocardiaatlantica (Humm and Shepard) Waksman (Pro-actinomyces atlanticus Humm and Shepard),Streptomyces marinus (Humm and Shepard)WVaksman (Actinomyces marinus Humm andShepard), and lMfycobacterium marinum Aronson.

Freitas and Bhat (1954) collected species ofNocardia and Streptomyces from deteriorating fishnets and cordage. Initial isolation of the organ-isms was made on nutrient media prepared withsea water or on media made isotonic with seawater by the addition of NaCl. Subsequenthalotolerant studies showed development ofcolonies on media of various salinities, from 0 to7 per cent NaCl.

Siebert and Schwartz (1956) isolated 1 strain ofNocardia and 6 strains of Streptomyces fromseaweed masses used in studies of sedimentationprocesses. Since the marine material was allowedto air dry before experimentation, it is possiblethat the actinomycetes collected were merely aircontaminants.

Chesters et al. (1956) collected approximately17 strains of alginate- and laminarin-decomposingactinomycetes from the decaying thalli of speciesof brown algae, from decaying masses of castseaweeds, from lagoon muds containing the re-mains of seaweed thalli, and from salt marsh soiland sand. Strains of Streptomyces were isolatedwhich could attack insoluble laminarin and uti-lize it as the sole carbon source.

Early in 1957, cooperative studies on marineactinomycetes vere begun concurrently at theInstitute of Microbiology, Rutgers University,

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and The Marine Laboratory, University ofMiami. The objectives of this work were toestablish the extent to which actinomycetes occurin the sea, the generic nature of this population,and selected aspects of their physiology, includingantibiosis. Preliminary results of this program arereported in this paper.

MATERIALS AND METHODS

Collections. Marine sediments, from variousinshore localities, constituted the primary mate-rial examined, although submerged wood andother substrates were also studied. Approximately100 separate bottom samples were tested, repre-senting 5 geographically different littoral habi-tats. The sediments were obtained in areas ofvarious depths, all completelv submerged at lowtide.The area, number of samples, and depth of

collection is given in table 1.Collections along the west coast of Florida

were made at Bokeelia, Ft. Myers, Marco Pass,Chokoloskee, Marco (Ten Thousand Islands),and Everglades City. The samples from Marco,Florida, were collected in the Gulf of Mexico,3 to 5 miles off shore. The New Jersey sampleswere taken near the mouth of the Shark River,adjacent to the Atlantic Ocean. Salinity readingsin the various brackish and marine sites werefrom 25 to 35 o/oo.The Florida samples were collected with a

coring device developed at The Marine Labora-tory. This apparatus consisted of a plastic coringtube (length, 11 in; diameter, 2 in) attached toa long pole. An adaptable plunger permitted theextrusion of the mud sample upward with itsremoval from the top end of the tube. Usually amud core, approximately 3 to 9 in long, dependingon the nature of the sediment, could be obtained

TABLE 1Collection data

Collection Area No. of Depth ofSamples Collection

Miami (Biscayne Bay), Flor-ida........................ 27 4-15 ft

Florida West Coast........... 58 3-19 ftLong Branch, New Jersey..... 8 6 ftBimini, The Bahama Islands 1 Shallow

waterGulf of St. Lawrence, Canada.. 2 33 ft

in this fashion. The topmost and lowermostportions of sediment were cut from the extrudedcore with a sterile spatula, and placed in sterile2 oz bottles. Several days elapsed before theFlorida west coast samples could be examined.The Biscayne Bay samples were usually studiedon the day of collection or on the following day.The New Jersey collections were made with anEckman dredge and transported in sterile quartjars for examination the following day.

All material was refrigerated at 4 to 8 C assoon as possible after its collection and transferto nutrient media. Standard soil plating meth-ods were used, with dilutions at 1:100, 1:1000,and 1:10,000.

Cultures. Species of terrestrial actinomycetesused in the salinity tolerance studies included,Streptomyces griseus 3475, 3570, S. coelicolor 3740,Nocardia corallina 3408, Mlifcromonospora sp.3452, and Mycobacterium smegmatis 607 ATCC.The following test bacteria were used in ourantibiosis work: Bacillus subtilis, B. cereus 8,Escherichia coli 52, Staphylococcus aureus 15,Klebsiella pneumoniae 133, and Pseudomonasaeruginosa 77.

Eight sea water isolates, belonging to the S.griseus and S. coelicolor groups, were selected forthe sea water tolerance experiment. These in-cluded: S. coelicolor group, (1) 266 GM, LongBranch, New Jersey, (2) 319 GM, Long Branch,New Jersey, (3) 379 GM, Long Branch, NewJersey, (4) 513 G, The Bahama Islands; S.griseus group, (1) 405 GM, Long Branch, NewJersey, (2) 450 GM, Everglades City, Florida,(3) 452 GM, Miami, Florida, (4) 512 G, TheBahama Islands. Isolate 266 GM was collectedfrom algae whereas 452 GM was isolated fromsubmerged banana stalk. All of the other isolateswere obtained from sediments.

Stock cultures of the marine isolates weremaintained on 1 per cent glucose, 1 per cent yeastextract (Difco) agar slants.

Representatives of terrestrial species were ob-tained from the culture collection of the Insti-tute of Microbiology, Rutgers University.

Media. Culture media were prepared withnatural sea water (SW), as well as with distilledwater. Sea water media is so designated through-out the paper, whereas media prepared withdistilled water will be listed without the "distilledwater" designation, or referred to as "ordinarymedia." The latter was used to ascertain whether

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autoclaving of the sea water agar together withvarious nutrients might inhibit the developmentof actinomycetes. Similarly, we wished to knowthe nature of the total actinomycete flora present,and its reaction to various salinities. The follow-ing media were used in our studies. The pH ofall media was adjusted to 7.0 to 7.5.

(1) Isolation media:-(a) Starch-casein agar:1 per cent soluble starch, 0.1 per cent technicalcasein (dissolved in NaOH), 1.5 per cent agar.

(b) Asparagine agar: 0.05 per cent asparagine,1.5 per cent agar.

(c) Glycerol-glycine agar (Lindenbein, 1952):2 per cent glycerin, 0.25 per cent glycine, 0.1per cent NaCl, 0.1 per cent K2HPO4, 0.01 percent FeSO4, 0.01 per cent MgSO4, 0.01 per centCaCO3, 1.5 per cent agar. This medium was alsoprepared without the added salts.

(2) Antibiotic media:-Antibiosis was deter-mined using the cross-streak method on glucoseyeast extract agar: 1.0 per cent yeast extract,1.0 per cent glucose, 1.5 per cent agar.

(3) Sea water tolerance media and experi-mental design:-Two basal liquid media were

prepared at various salinities, from 0 to 100 percent sea water.

(a) Medium A: 1 per cent glucose, 0.05 percent K2HPO4, and (b) Medium B: 1 per centglucose, 0.05 per cent K2HPO4, 0.05 per cent(NH4)2HPO4. A precipitate occurred in bothmedia, slightly greater in medium B.

In these experiments, the age of inoculum ofthe different isolates was kept constant. Theinoculum was prepared by washing each slantculture with 5 ml of sterile distilled water. Subse-quently, 0.1 ml of the resultant spore suspensionwas inoculated into 250-ml Erlenmeyer flasks,each containing 50 ml of the respective medium.Flasks containing medium A were then agitatedon a rotary shaker (280 rpm) for 10 days, whereasonly 5 days of incubation were allowed for me-dium B. The latter broth supported more rapidgrowth.

Following incubation, the broth culture of eachstrain was diluted to 10-8, except for the S. gri-seus group, where heavier growth necessitated a

dilution of 10-10. One ml of each final dilution wasthen plated out in duplicate on yeast extractagar and incubated at 28 C for 3 days. The totalnumber of colonies developing on each of 2 plateswas taken as an index of growth in the varioussea water broth concentrations. The inoculum

rate was checked by diluting the inoculum in thesame way as the samples.

RESULTS

Occurrence and growth characteristics. The gen-era Nocardia, Micromonospora, and Streptomyceswere represented in our collections. Species ofNocardia and Micromonospora constituted abouth of the total number of isolates, Nocardia beingmore prevalent. Streptomyces was the primarygenus collected, with isolates of the S. griseustype quite common. The 3 strains from theBahama Islands sample were all streptomycetes.The cultures of Streptomyces were separatedinto 8 general groups, based on the presence orabsence of soluble pigments and on the color ofthe vegetative and aerial mycelium. These groupsincluded types of S. coelicolor, S. chromogenes, andS. griseus, and groups of organisms characterizedby: (a) greenish-brown pigment and greenish-brown vegetative mycelium, (b) little solublepigment and yellow- to light yellow-brown vege-tative mycelium, (c) yellow brown soluble pig-ment, (d) no soluble pigment, pinkish vegetativemycelium, (e) light brown pigment and brownvegetative mycelium.No attempts were made to determine quanti-

tatively the number of actinomycetes in individ-ual samples, since, in many instances, the timeinterval between collection and subsequent exam-ination invalidated any quantitative measure-ments. Instead, a tabular account of the numberof isolates in the total samples from the generalarea studied is given in table 2.

Noticeable variations occurred in the totalnumber of organisms isolated at individual sta-tions within these localities. Preliminary observa-tions, using different selective media, suggestthat physiological dissimilarities may exist be-tween the organisms collected in New Jersey andthose collected in Florida.

Actinomycetes were found in sea water, al-though to a much lesser extent than that noted

TABLE 2Distribution of sediment collections and

isolates

Florida :lNew Bahamas Gulf of St.Jersey Lwec

No. of samples. 85 8 1 2No. of isolates... 87 172 3 -

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TABLE 3Antibiotic activity of marine actinomycetes

No. of IsolatesAntibiotic Spectrum Against

NewJersey Florida Total

Gram-positive andgram-negative bac-teria................ 46 24 70

Gram-positive bac-teria only........... 24 9 33

Inactive .............. 67 29 96Not tested 35 27 62

in the sediments examined. Previously sterilizedtrapping substrates, i. e., basswood panels, cellu-lose discs, banana stalk sections, submerged inthe marine area along the Florida coast, allshowed the development of colonies of strepto-mycetes on transfer of the exposed material tonutrient media.The starch casein agar was superior to the

other media tested for the initial isolation ofactinomycetes. An increase in the number ofcolonies occurred when distilled water was sub-stituted for sea water in the medium. The glyc-erol-glycine agar and asparagine agar were not as

effective as the starch-casein medium. Largenumbers of discrete myceloid colonies devel-oped on the asparagine medium and on mediaprepared with sea water. Further radial develop-ment of these colonies on the initial isolationmedium did not occur. Transfer of this growthto other media has been unsuccessful.

Antibiosis. The majority of the isolates were

tested for antibiotic activity against selectedgram-positive and gram-negative bacteria. Theresults of these tests are summarized in table 3.Approximately 50 per cent of the isolates exam-

ined exhibited various degrees of antibiosis.Selected isolates, found to have antibiotic

activity on glucose yeast extract agar, were sub-sequently tested on corresponding media pre-pared with sea water. Controls of the test bac-teria on this latter medium showed good growth,although slightly less than that on ordinarymedia. Strong inhibition of the test bacteria bythe actinomycete isolates on sea water media was

apparent. Although some of this inhibition maybe attributed to the slight decrease in bacterialgrowth on the sea water agar, most of the inhi-bition was due to antibiotic activity. It should

be noted that antibiosis was greater on themedium prepared with sea water.

Further studies are in progress to assesswhether the sea water isolates exhibit higheractivity in sea water broth, as contrasted toactivity in ordinary broth cultures, or broths madeisotonic with sea water by the addition of NaCl.

Spore germination on starch casein agar. Fourmarine isolates of the genus Streptomyces, and S.griseus 3570, were grown from spore suspensionson starch casein media, prepared both withdistilled water and with sea water. The sea waterisolates of Streptomyces used were collected ini-tially on media of different salinities, i. e., 241GM, 0 per cent sea water; 275 GM, 75 per centsea water; 254 GM, 100 per cent sea water;445 GM, 0 per cent sea water. Spore germinationoccurred on both media, with a slightly greaternumber of colonies on the sea water medium.Moreover, all of the isolates, with the exceptionof 241 GM, developed larger colonies on thestarch casein sea water medium. In addition, adiffusible pigment, noted in the colonies on thesea water plates, was absent in those on ordinarymedia. S. griseus 3570 also exhibited better de-velopment on the sea water agar, in size as wellas in number of colonies. A yellow pigmentationwas observed during the growth of this specieson sea water media.

Growth on nutrient media. The growth of 10different sea water isolates was examined on ordi-nary and sea water glucose yeast extract andstarch casein media. The average diametergrowth of 3 colonies was used for each measure-ment. After growth for 1 week, no appreciabledifference was noted in the amount of colonydevelopment between the two glucose yeast ex-tract media. However, after incubation for 2 to 3weeks, the average colony on the ordinary me-dium was consistently larger than that on thecorresponding sea water medium. Continuousenlargement of the colonies on the sea water basewas inhibited. Five out of a total of 10 strainsgrown on starch casein agar exhibited a similardevelopmental pattern. The remaining 5 strainsshowed approximately equal growth oa ordinarystarch casein media and on that prepared withsea water.The reasons for the failure of many of our sea

isolates to develop initially on pour plates ofstarch casein sea water media are not apparent.

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so --.-* *~'

-\0-

* 379 GM

0 *0 2 0 7 00 25 SO 75 100concentration of seo water

Figure 1. Growth of strains of Streptomycescoelicolor in various concentrations of sea water.Medium A.

The same isolates exhibited excellent growth onsubsequent transfer in pure culture to the samemedium. It was noted that inhibition of the de-velopment of actinomycetes also occurred on thesea water isolation plates, containing a mixedflora of bacteria and fungi.

Sea water tolerance. The results of these testsfor the S. coelicolor group are illustrated in figures1 (medium A) and 2 (medium B). The number ofcolonies are based on the average of data fromduplicate experiments. Maximum growth of theisolates, with the exception of 379 GM in me-dium B, occurred in broth with a sea waterconcentration of less than 50 per cent. Isolate379 GM does rather poorly in medium A, wherea nitrogen source is absent. Nitrogen is presentin medium B in the form of (NH4)2HPO4.

In general, there is a decrease in growth of thevarious isolates with an increase in the salinity ofthe medium. This may be due in part to theprecipitation of PO4= at the higher salinities. Inmedium A, 3 strains (S. coelicolor 3740, 266 GM,379 GM) show a decrease in the number of colo-

nies in the flasks of 0 per cent sea water. Inmedium B, only 319 GM and 379 GM exhibitedsimilar behavior. In both media, S. coelicolor3740 shows an increase in growth with salinity,up to 50 per cent sea water. Dissimilarities inpigmentation occurred in the broth cultures ofthe two media. Pigmentation was absent in thecultures of medium A, whereas in medium B, ablue violet color was apparent, decreasing inintensity with the concentration of sea water.At 100 per cent sea water, a slightly yellowishcolor was noted.

Considerable variability was observed amongthe strains of the S. griseus group. In medium A,maximum growth of isolates 405 GM, 512 G, andS. griseus 3475 occurred at a salinity concentra-tion of 75 per cent sea water. In medium B, wherea greater uniformity in development among thestrains occurred, maximum growth took placein broth cultures of 50 per cent sea water. Inmedium A, strains 450 GM, 452 GM, and 512 Gshowed maximum growth in broth of 100 percent sea water, whereas S. griseus 3475 and 405GM exhibited a decrease in growth at this

1501

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81

64

4'

24

0 25 50 75 100concentration of sea water

Figure 2. Growth of strains of Streptomycescoelicolor in various concentrations of sea water.Medium B.

. 266 GM

0 319 GM

*

513G /G/o / \

O _/ \ l

O \

E / / \ 6AO/' / \ s 1

9_/-~ \,0 X

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GREIN AND MEYERS

salinity. However, the latter isolate showed spec-tacular development in medium B, producing asmany as 900 colonies per ml in broth of 100 percent sea water.The results of these preliminary tests confirm

the observations noted in our earlier isolationwork, viz., a decrease in the concentration of seawater in the isolation medium generally permitsan increase in the number of developing actino-myeete colonies. Maximum development of theseorganisms occurred on media of salinities approx-imating that of 25 to 50 per cent sea water. TheS. griseus group shows a greater development onmedia of elevated salinities than do the strainsof S. coelicolor. This is borne out in part by ourisolation studies in which strains of S. griseuswere collected on media prepared in 100 per centsea water.

DISCUSSION

The regular isolation of the major genera ofactinomycetes from littoral sediments does notof course indicate that these organisms are in-digenous to the marine and brackish areasstudied. ZoBell and Upham (1944) noted thatmany common terrigenous bacteria, e. g., B.subtilis and E. coli, have been found in brackishor coastal waters. Similarly, Burke and Baird(1931) and Doudoroff (1940) conducted labora-tory experiments which indicated that manyfresh water and terrigenous bacteria can beadapted to tolerate salt concentrations which arehypertonic to sea water.

Conversely, the isolation of species, usuallyconsidered terrestrial, from marine localitiesdoes not necessarily imply that the organisms arecontaminants in the sea or even halotolerantterrestrial species. The occurrence of related oridentical species of bacteria and fungi in the seaand on the land has been noted previously(Wood, 1952; ZoBell, 1946; Ritchie, 1954; Aaron-son, 1956).The establishment of the marine affinity of

organisms isolated from the sea is far fromsimple. Initial growth on complex laboratorymedia prepared with sea water, or on media madeisotonic with sea water by the addition of NaCl,is not an absolute criterion of marine adaptation.MIany terrigenous species of fungi and bacteriadevelop readily on such media. A far better cri-terion, suggested by ZoBell (1946), is the isola-tion of the organism from the sea in areas remote

from possibilities of terrigenous contamination.But this is not practical for the investigatorworking primarily with organisms of the inshorearea. MacLeod and Onofrey (1956) propose that"true marine bacteria may well prove to be dis-tinguishable from land forms present as con-taminants in sea water, not by having a require-ment for sea water, but rather by having a readilydetectable need for Na+ in the medium forgrowth."Our preliminary studies suggest that the ae-

tinomyeetes collected, and especially the strepto-mycetes, do not represent an autochthonous ma-rine flora. More likely, they may be terrestrialforms that have become adapted to the salinityof sea water and sediments. Stapp (1953) reportedhalophilic tolerances within the streptomycetegroup. In our tests, various type cultures of ter-restrial actinomycetes, e. g., S. griseus 3475,3570, Nocardia corallina, a Jlicromonospora sp.,and Mycobacterium smegmatis, show wide salinitytolerances, with good growth on 100 per cent seawater nutrient media. No morphological differ-ences are apparent between our sea isolates andthe terrestrial-type species.Whether these marine tolerant or euryhaline

actinomycetes are merely survivors in littoralsediments or whether they participate in variousmarine metabolic processes can only be postu-lated. A large number of these isolates exhibitantibiotic activity in culture. Greenberg (1956)discusses the possible role of the production ofantibiotic substances by marine bacteria in thereduction of the number of enteric bacteria insea water.

This present investigation, as well as the gen-eral paucity of information on the ecology andphysiology of the specific microflora in littoralsediments, suggests the need for further exami-nation of marine tolerant and euryhaline actino-mycetes.

ACKNOWLEDGMENTS

The writers wish to express their sincereappreciation to Dr. Selman A. Waksman, Director of the Institute of Microbiology, for hisinitiation of this work and for his encouragementthroughout its development. The advice andassistance of various members of the Instituteof MVicrobiology is readily acknowledged. Otherindividuals, including Dr. D. Pramer, Depart-ment of Agricultural Microbiology, and Dr.

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H. H. Haskins, Department of Zoology, RutgersUniversity, and Dr. L. Provasoli, Haskins Lab-oratories, New York City, have contributedgenerously of their time and counsel. The tech-nical assistance of Mrs. C. Edith Marks isappreciated.

SUMMARY

A number of actinomycete species, includingstreptomycetes, were isolated from various ma-rine sediments. Certain preliminary observationsrelating to the physiology of these organismsare discussed, with special reference to the ef-fects of salinity in the medium and to the anti-biotic activity of the organisms. The ecologyof these species and their possible identity toterrestrial species is postulated.

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ARONSON, J. D. 1926 Spontaneous tuberculosisin salt water fish. J. Infectious Diseases,39, 315-320.

BURKE, V. AND BAIRD, L. A. 1931 Fate of freshwater bacteria in the sea. J. Bacteriol.,21, 287-298.

CHESTERS, C. G. C., APINIS, A., AND TURNER, M.1956 Studies of the decomposition of sea-weeds and seaweed products by microor-ganisms. Proc. Linnean Soc. London, 166,87-97.

DOUDOROFF, M. 1940 Experiments on the

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FREITAS, Y. M. AND BHAT, J. V. 1954 Micro-organisms associated with the deteriorationof fishnets and cordage. J. Univ. Bombay,23, 53-59.

GREENBERG, A. E. 1956 Survival of enteric or-ganisms in sea water. Public Health Repts.U. S., 71, 77-86.

HUMM, H. J. AND SHEPARD, K. S. 1946 Threenew agar-digesting actinomycetes. DukeUniv. Mar. Sta. Bull. No. 3, 76-80.

LINDENBEIN, W. 1952 Ueber einige chemischeinterresante Aktinomycetenstaemme und ihreKlassifizierung. Arch. Microbiol., 17, 361-383

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RITCHIE, D. 1954 A fungus flora of the sea.Science, 120, 578-579.

SIEBERT, G. AND SCHWARTZ, W. 1956 Unter-suchungen uber das Vorkommen von Mikro-organismen in entstehenden Sedimenten.Arch. Hydrobiol., 52, 321-366.

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WOOD, E. J. F. 1952 The micrococci in a marineenvironment. J. Gen. Microbiol., 6, 205-210.

ZoBELL, C. E. 1946 Marine microbiology.Chronica Botanica Co., Waltham, Massa-chusetts.

ZOBELL, C. E. AND UPHAM, H. C. 1944 A list ofmarine bacteria including descriptions ofsixty new species. Bull. Scripps Inst.Oceanog. Univ. Calif., 5, 239-292.

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