ANTIBIOTIC PRODUCTION BY MARINE MICROORGANISMS' WILLIAM D. ROSENFELD' AND CLAUDE E. ZoBELL Scripp8 Institution of Oceanography, University of California, La Jolla, California Received for publication June 17, 1947 The multiplication of most nonmarine bacteria is inhibited by sea water (Kofinek, 1927). ZoBell (1941) found that only from 4 to 15 per cent as many bacteria from soil, sewage, and other fresh-water or terrestrial sources formed colonies on nutrient agar prepared with sea water as on a similar medium prepared with distilled water. Sea water is also bactericidal for many nonmarine bacteria, gram-positive organisms being more sensitive than gram-negative forms (Beard and Meadowcroft, 1935; ZoBell, 1936). This was demonstrated by suspending pure or mixed cultures of bacteria from various fresh-water sources in sea water or other mineral solutions and determining the percentage of survival after different periods of time. Although gram-negative organisms display resistance to the lethal action of sea water, their viability in this medium varies widely among different species. In an investigation employing enteric bacteria, Trawinski (1929) reported survival periods in sea water ranging from 12 hours for Shigella dysenteiiae to 23 days for Salmonella enteritidis. Carpenter et at. (1938) noted that natural sea water killed 80 per cent of the organisms in sewage within half an hour. The bacteriostatic and bactericidal effects of sea water are greater than can be accounted for upon a basis of its salinity or osmotic pressure. Not only is natural sea water more bactericidal than synthetic sea water (ZoBell, 1946; Zo- Bell and Feltham, 1933), but it is also more bactericidal than heat-treated sea water (Kiribayashi and Aida, 1933; Waksman and Hotchkiss, 1937; ZoBell, 1936). De Giaxa (1889) observed that pathogens rapidly perish in raw sea water, although they may survive almost indefinitely in heat-treated sea water. Water from the Black Sea was found by Krassilnikov (1938) to be germicidal for terrestrial bacteria until it was boiled. He confirmed the observations of Beard and Meadowcroft (1935) and ZoBell (1936) that the bactericidal potency of sea water was decreased but not destroyed by passing it through Berkefeld, Chamberland, Coors, or similar filters. Lacking the properties of bacteriophage, the bactericidal property of sea water is attributed primarily to its content of antibiotic substances produced by microorganisms. Credence is lent to this view by the observation that the bactericidal principle occurs in greatest concentration in samples of sea water recently collected from zones of maximum bacterial population. The experimental results reported below demonstrate, the production of antibiotic substances by several species of microorganisms native to the sea. 1 Contributions from the Scripps Institution of Oceanography, New Series No. 327. This paper is a contribution from the American Petroleum Institute Research Project 43A. 2 Present address: California Research Corporation, La Habra, California. 393
394 W. D. ROSENFELD AND C. E. ZOBELL [VOL. 54 EXPERIMENTAL RESULTS Fifty-eight pure cultures of marine microorganisms were tested for their antimicrobial activities. The cultures employed as potential antagonists were taken from a collection previously described by ZoBell and Upham (1944) and were maintained in a medium of the following composition: Difco peptone... Difco yeast extract... (NH4)2SO4... FeSO4. (NH4)2S04.6H20... Aged sea water (75 per cent)... 5.0 g 1.0 g 1.0 g 0.1 g 1,000 ml The solution was adjusted to ph 7.4 to 7.6 before autoclaving. When a solid medium was required, 2.0 per cent of agar was added to this solution prior to sterilization. TABLE 1 Antimicrobial behavior of marine microorganism. SPEC]28 IIHST( FRSH- AJGONNSC GEUS SPE8 TE8TD WATER OR TERRESTRI ORGANISMS Bacillus... 9 4 Micrococus... 6 3 Actinomyces... 2 1 Serratia... 1 1 Pseudomonas... 19 0 Vibrio... 11 0 Flavobacterium...... 5 0 Achromobacter... 4 0 Sarcina...1 0 Totals. 58 9 All fresh-water or terrestrial species were tested to ensure their growth in this sea-water medium before subjecting them to the effects of marine antagonists. Both antagonistic and test inocula were taken from cultures grown for 48 hours at 27 C. Antimicrobial effects were surveyed in pour plates, each containing 20 ml of nutrient agar and seeded with 0.2 ml of a test culture. After the medium had solidified, the marine species under investigation were streaked over quadrants of the plates, which were then incubated at 27 C. Plates were examined at frequent intervals over a period of 17 days for growth suppression of the test organisms, as evidenced by clearing zones adjacent to the lines of growth of marine antagonists. Table 1 outlines the results noted and summarizes the distribution of antagonistic species among the various genera investigated. Although two antagonistic organisms were gram-negative, the majority of species exhibiting inhibitory powers were members of the gram-positive genera BaciUus and Micrococcus.
19471 ANTIBIOTICS FROM MARINE MICROORGANISMS 395 The susceptibility of the test organisms is described in tables 2, 3-, and 4. Of the 11 gram-positive cultures employed, only Staphylococcu aureu and Strepto- TABLE 2 Antimicrobial spectrum of marine Bacillus species TEST SPECIES DINDTD BY B. berberokoiles B. ebysos B. tha1sokels B. ubsnrixus Bacillus anthraci8... + + _ B. megatherium... B. mycoides... + + + + B. subtilis... Corynebacterium pseudodiphthericum.. + Micrococcus roseus... + + + + Mycobacterium lacticola... Proteus vulgaris... Salmonella typhimurium... Sarcina lutea... + + + Shigella paradysenteriae... Staphylococcus aureus... S. citrew... + + + Streptococcus faecalis... TABLE 3 Antimicrobial spectrum of marine Micrococcus species TEST SPECIES.na3IrE M. "Orioskm i. sed. M Bacillus anthracis... + * B. megatherium... * B. mycoides... B. subtilis... Corynebacterium pseudodiphthericum - _ * Micrococcus roseus... Mycobacterium lacticola... + * Proteus vulgaris... Salmonella typhimurium... Sareina lutea... + + + Shigella paradysenteriae... * Staphylococcus aureus... S. citreus... + * Streptococcus faecalis... This species not tested. coccu-sfaecalis failed to undergo inhibition by one or more maxine species. Three gram-negative species were tested, and none of these was susceptible. An attempt was made to demonstrate the presence of the inhibitory principle in cell-free solutions prepared by filtration of the antagonistic cultures. The BY
396 W. D. ROSENFELD AND C. E. ZOBELL[ [vol. 54 inhibitory organisms were grown in the broth described above for periods of 9 to 11 days, after which they were passed through Seitz or Mandler filters. The reaction of all filtrates approximated ph 8 and in each case was adjusted to ph 7.2 before employment of the sterile solutions as inhibitory agents. Pour plates of susceptible organisms were prepared in the manner already outlined. Sterile discs of filter paper were saturated with the culture filtrates and applied to the TABLE 4 Antimicrobial spectrum of marine microorganisms TEST SPECIES Actimomyces marinolimosus INIBITED 3Y S.rrsi.s waixerulira Bacillus anthracis... + B. megatherium... + + B. mycoides... + B. subtilis... + Corynebacterium pseudodiphthericum... Micrococcus roseus... + Mycobacterium lacticola... Proteus vulgaris... Salmonella typhimurium... Sarcina lutea... + Shigella paradysenteriae... - - Staphylococcus aureus... - - S. citreus...- - Streptococcus faecalis... - - TABLE 5 Decreases in antimicrobial activity resulting from filtration of antagonistic cultures ANTAONIST SUSCEP17L RE8ES Species Filter Tested Inhibited Actinomyces marinolimosus Seitz 4 1 Bacillus borborokoites Mandler 6 1 B. abysseus Seitz 5 1 B. thalas8okoites Seitz & Mandler 4 0 B. submarinus Seitz 4 1 Micrococcus maripuniceus Seitz 4 0 M. sedimenteus Mandler 1 0 Serratia marinorubra Mandler 3 0 agar surface. Such plates were incubated for 6 days, during which they were examined frequently for the presence of inhibition zones. The results are given in table 5. Little antagonistic activity was displayed by the cell-free preparations. Although such results may suggest that the inhibitory substance is intimately associated with its parent cell, there is the possibility that the active principle was removed by adsorption on the filter.
19471 1ANTIBIOTICS FROM MARINE MICROORGANISMS397 DISCUSSION Quantitative evidence of the effect of marine microorganss upon the inhibitory property of sea water has not been obtained. However, a presumptive interrelationship may be inferred from the incidence of antagonistic species. The behavior of antagonistic cultures when passed through germ-proof filters resembles that of sea water, the two undergoing similar decreases in antimicrobial potency. Further evidence for this correlation of activity is observed in a comparison of data reported herein with that testing the effect of sea water upon nonmarine bacteria. Of six organisms shown by Krassilnikov (1938) to be inhibited by unheated sea water and common to both investigations, only Staphylococcus aureus failed to demonsti ate a bacteriostatic response to marine bacteria. It is also significant that the response of test species to inhibition by marine organisms appears to parallel the giam reaction. This is in accordance with the general observation that gram-positive bacteria are more often inhibited by sea water than are gram-negative species. Isolations of specific antibiotics produced by marine bacteria have not been attempted, but it is evident that various species of microorganisms indigenous to the sea elaborate antimicrobial substances. The survey reported here, although very limited in scope, suggests that the marine environment should be considered as a potential source of antibiotics. SUMMARY Of 58 species of marine microorganisms tested, 9 have demonstrated antibiotic activity against nonmarine forms. The most actively antagonistic marine genera were Bacillus and Micrococcus. Similarities in the behavior of sea water and of antagonistic marine cultures indicate that the bacteriostatic or bactericidal activity of the former may be at least partially due to an autochthonous flora of antibiotic-producing organisms. It is suggested that the sea may represent a reservoir of microbial antagonists of possible importance. REFERENCES BEARD, P. J., AND MEADOWCROFT, N. F. 1935 Survival and rate of death of intestinal bacteria in sea water. Am. J. Pub. Health, 25, 1023-1026. CARPENTER, L. V., SETTR, L. R., AND WEINBERG, M. 1938 Chloramine treatment of sea water. Am. J. Pub. Health, 28, 929-934. GIAxA, D15 1889 Ueber das Verhalten einiger pathogener Mikroorganismen im Meer. wasser. Z. Hyg. Infektionskrankh., 6, 162-225. KIRBAYASI, S., AND AIDA, T. 1933 Fate of cholera vibrio in the sea water of Keelung Port, Formosa. J. Pub. Health Assoc. Japan, 9, 1-9. U. S. Pub. Health Eng. Abstracts, 14, W, 61 (1934). KofIINEK, J. 1927 Ein Beitrag zur Mikrobiologie des Meeres. Zentr. Bakt. Parasitenk., II, 71, 73-79. K8AssmIuNzov, N. A. 1938 The bactericidal action of sea water. Microbiology (U.S. S.R.), 7, 329-334. (Russian with English smmary.) TRAwINsKx, A. 1929 Etudes sur la vitalit6 des bacilles pathog%nes du groupe coli-typhique dans 1'eau de mer. Bull. inst. oc6anograph., no. 542, 1-3.
398 W. D. ROSENFELD AND C. E. ZOBELL [VOL. 54 WAKsmAN, S. A., AND HoTa ss, MARGARET 1937 Viability of bacteria in sea water. J. Bact., 33, 389-400. ZoBEzL, C. E. 1936 Bactericidal action of sea water. Proc. Soc. Exptl. Biol. Med., 34, 113-116. ZoBELL, C. E. 1941 Studies on marine bacteria. I. The cultural requirement of heterotrophic aerobes. J. Marine Research, 4, 42-75. ZOBELL, C. E. 1946 Marine microbiology. A monograph on hydrobacteriology. Chronica Botanica Co., Waltham, Mass. ZoBzLL, C. E., AND FELTHAM, C. B. 1933 Are there specific marine bacteria? Proc. Fifth Pac. Sci. Congr., 3, 2097-2100. ZOBELL, C. E., AND UPHAm, H. C. 1944 A list of marine bacteria including descriptions of sixty new species. Bull. Scripps Inst. Oceanogr., 5, 239-292.