subtilis, isolated from the air, readily lysed pneumococci, typhoid, CIDAL SUBSTANCES' (Hotchkiss and Dubos, 1940). BACTERIA WHICH PRODUCE BACTERI-

Transcription

1 THE ISOLATION FROM SOIL OF SPORE-FORMING BACTERIA WHICH PRODUCE BACTERI- CIDAL SUBSTANCES' Research Laboratory, Merck and Co., Inc., Rahway, N. J. Received for publication May 10, 1941 Within the past two years, the isolation of a number of crystalline bactericidal substances, from a soil bacillus, by Dubos and co-workers (1939, 1940) has aroused considerable interest. These agents were found to be bactericidal, in extremely small amounts, for many gram-positive bacteria and not for gramnegative bacteria. One of these substances, gramicidin, protected mice injected intraperitoneally with 10,000 or more lethal doses of pneumococci or streptococci. Subsequently, it was found that two of the substances, tyrothricin and tyrocidine hydrochloride, exhibited a marked degree of bactericidal activity, in vitro, against both gram-negative and gram-positive bacteria (Hotchkiss and Dubos, 1940). The ability of spore-forming bacteria to produce bactericidal substances has been recognized for many years, although Dubos was the first to isolate the active principles in a purified state. As early as 1907, Nicolle (1907) reported that a culture of Bacillus subtilis, isolated from the air, readily lysed pneumococci, typhoid, anthrax and other organisms in vitro. (Filtrates of cultures of B. subtilis likewise brought about complete or partial lysis of broth cultures of the same organisms). Other sporeforming bacteria such as Tyrothrix distortus, T. geniculatus and T. tenuis, isolated from cheese by Duclaux, were shown by Nicolle to produce similar bactericidal substances. Pringsheim (1920) isolated a spore-forming bacterium which appeared accidentally as a 1This paper was presented on December 28, 1940, before the Society of American Bacteriologists in St. Louis. 253

2 254 contaminant on plate cultures of Corynebacterium diphtheriae and formed zones in which the latter failed to develop. The bacillus considered to be of the Bacillus mesentericus type, also inhibited meningococci and other bacteria. Later, Much and Sartorius (1924), Rosenthal (1925), Frank and Ismet (1926), and more recently Cordon and Haenseler (1939), Hettche and Weber (1939) and Hoogerheide (1940) have clearly demonstrated that certain strains of spore-forming bacteria can produce agents which are bactericidal for a variety of pathogenic and saprophytic bacteria and for fungi. In the investigations mentioned, the bacilli, capable of destroying other bacteria, were obtained either accidentally or by indiscriminately isolating strains and testing each for anti-bacterial properties. Dubos obtained his strain by the enrichment culture technique, using gram-positive bacteria, staphylococci and streptococci, as the specific nutrient. Recently, Waksman and Woodruff (1940) isolated a number of microorganisms, antagonistic to pathogenic bacteria, by plating soils with washed agar media containing suspensions of various bacteria as the sole available nutrient. The present report describes a simple rapid method for isolating from soil, bacteria which produce bactericidal agents; their characteristics; and the bacteriostatic and bactericidal properties of the agents obtained from them. METHOD OF ISOLATING ANTAGONISTS The isolation method was to plate soil samples in low dilutions, 1-10,000 or less, on a suitable medium such as nutrient, tryptone or brainheart infusion agar in order to obtain a large number of microbial colonies on each plate. The resultant crowding of the colonies allows the potential antagonisms that may exist between the various microorganisms to express themselves as clear zones around the active antagonists. Occasionally, antagonistic action is evident when only a few colonies appear on the plates. The results obtained by this method are usually striking. After incubation of the plates at 28 C for 2 to 5 days, colonies with distinct zones can usually be seen on some of the plates. These

3 ISOLATION FROMX SOIL OF SPORE-FORMING BACTERIA 255 zones may vary in size from a narrow band around the colony to some whose width is many times the diameter of the colony (fig.1). In our experience, the antagonists have invariably been sporeforming bacteria and the microorganisms antagonized have generally been bacilli or short gram-negative rod-shaped bacteria and Downloaded from FIG. 1. SOIL PLATES SHOWN ING CLEAR ZONES SURROUNDING COLONIES OF ANTAGONISTIC AlICROORGANIS'MS occasionally fungi. Undoubtedly the type of antagonist obtained is influenced by the kind of medium used and the source of the mixed population. It seems reasonable to assume that by varying the plating medium and using different sources of microorganisms such as milk, water, sewage, etc., other types of aintagonists can probably be isolated. on March 17, 2019 by guest

4 256 It is recognized that the method is not specific in that it does not insure the isolation of antagonists for specific microorganisms, for example, for Brucella abortus or Hemophilus pertussis. However, this is of limited importance in view of the fact that most of the antagonistic microorganisms that have been isolated and the active agents obtained from them are effective not against a single species or genus but against a wide range of microorganisms. By. the method described, 24 strains of spore-forming bacteria were isolated from various soils. CHARACTERISTICS OF THE ISOLATED STRAINS The strains were purified by plating and tested further for antibacterial activity, in a number of ways, against gram-p,ositive and gram-negative bacteria, such as Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Eberthella typhosa and others. One method was to grow the isolated strains side by side with the test organism on solid media. Another method consisted in pouring melted nutrient agar, heavily seeded with the test organism, into sterile Petri dishes and then streaking the antagonist on the surface of the solidified agar. In these two methods, zones of inhibition were looked for after the plates had been incubated. It was found, however, that the best method was to add small amounts (0.1 ml to 1.0 ml) of a broth culture of the test organism to the supernatant fluid of centrifuged broth cultures of the antagonists. After incubating the mixture for 2 to 3 hours at 37 C. lysis was looked for and tests were made for bactericidal action by subculturing from the mixture onto fresh solid media. By this method, it was found that the supernatants of centrifuged 5-day old broth cultures of seven of the strains were bactericidal and bacteriolytic for S. aureus, B. subtilis, and Micrococcus sp. whereas E. coli and E. typhosa were not affected. The remaining strains had no effect on any of the test organisms. Of interest was the fact that 24 hr. broth cultures of the active strains were gram-negative whereas the inactive strains were gram-positive and remained so for 48 to 72 hrs. Biochemical differences between the two groups were also found as shown in table 1.

5 ISOLATION FROM SOIL OF SPORE-FORMING BACTERIA 257 The three inactive strains, A-15, 31 and 32, were picked at random from the 17 strains available. It can be seen that there is a greater tendency for the inactive strains to produce an acid reaction in glucose, lactose and sucrose broth. Indole is produced by only one strain A-27. The active strains form H2S in peptone media while the inactive strains do not. Practically all strains reduce nitrates and liquefy gelatin. The inactive strains hydrolyze starch but the active strains do not. There is little doubt, therefore, that there are relatively sharp differences between the two groups of spore-forming bacteria isolated. TABLE 1 Biochemical characteristics of "active" and "inactive" strain. NITRATE LIQUE- KYDROLY- STRAIN GLUCOSE LACTOSE SUCROSE INDOLE HIS RE EDUC- FACTION SAB of GAM TION OF GELA- STJC STAIN Active strains A-2 Alk Alk Alk _ - A-5 Alk Alk Alk A-21 Alk Alk Alk A-21 Alk Alk Alk _ A-23 Alk Alk Alk A-27 Alk Alk A _ A-34 Alk Alk Alk Inactive strains A-15 Alk Alk A _ - + _ + + A-31 Alk A A _ A-32 A A A ISOLATION AND PROPERTIES OF THE ACTIVE PRINCIPLES An attempt was made to isolate the bactericidal principle present in broth cultures of the gram-negative strains of bacilli. The procedure used was similar to that of Dubos (1939). Six of the seven strains were grown in shallow layers of 1 per cent tryptone broth at 370C. After 5 or 6 days, the cultures which were then alkaline, were adjusted to ph/4.6 with concentrated HCl; the precipitate that formed was allowed to settle overnight and removed by filtration. The filtrate was discarded and the filter paper contaning the precipitate was placed in 95 per cent alcohol.

6 258 After 24 hrs. the alcoholic extract of the precipitate was filtered through paper and 10 volumes of 1 per cent NaCl were added to it with constant, stirring. In every case, a white flocculent precipitate formed. The precipitate was separated by centrifugation, washed, dried and tested for bacteriostatic potency against a number of gram-positive and gram-negative bacteria. Cultures of the three inactive strains of bacilli whose characteristics are given in table 1 were similarly treated. No precipitate formed when these cultures were adjusted to ph 4.6. The cells, however, were centrifuged out and extracted with alcohol. Precipitates were not obtained when the alcoholic extracts were mixed with 1 % NaCl. BACTERIOSTATIC AND BACTERICIDAL ACTITY OF THE ISOLATED AGENTS The bacteriostatic potency of the isolated substances was determined by thoroughly mixing varying amounts of each suspended in water with 10 ml. of brain-heart infusion agar in Petri dishes. After the agar had set, the plates were streaked with a loopful of an 18- to 24-hour broth culture of each organism tested. The presence or absence of growth was recorded after the plates had been incubated for 24 hours. Since there were only small quantitative differences in the activities of the materials isolated from the six strains, the results obtained for only one of these materials, isolated from strain A-2, are presented in table 2. Twenty-five micrograms of material mixed with 10 ml. of agar completely inhibited the growth of Staphylococcus aureus and Staphylococcus albus, while as little as 5'y stopped the growth of the streptococci. Much larger quantities of the agent were necessary to inhibit the growth of the gram-negative bacteria. Four hundred micrograms reduced the growth of Neisseria catarrhalis and 6007 completely inhibited its growth. Hernophilus pertussis was not affected by 600,y, while the growth of E. coli was somewhat reduced by 1000y, an amount which was inhibitory for Salmonella paratyphi and E. typhosa. The agents in question are therefore highly bacteriostatic for gram-positive bacteria, while the gram-negative bacteria are considerably more resistant to

7 ISOLATION FROM SOIL OF SPORE-FORMING BACTERIA 259 their action; however, if sufficiently large quantities of the agent are used (and it should be noted that the quantities used are still small, i.e., 1 mgm. or less per milliliter of medium) the growth of members of the col-typhoid group can be inhibited. The bactericidal potency of the isolated compounds was de-- termined by adding different amounts of each, suspended in water, to tubes containing 0.1 ml of an 18- to 24-hr. broth culture of each test organism. The volume was adjusted to 2 ml. with sterile water and the mixture incubated at 37 C. After 2 hours a loopful of material from each tube was streaked on plates of TABLE 2 Bacteriostatic activity of agent isolated from cultures of strain A-S AMOUNT OF AGENT USED (MICROGRAMS PER ML.) TESTORGANIM_M i Control Staph. aureus Staph. albus Strep. hemolyticus Strep. viridans N. catarrhali l H. pertussis E. coli S. paratyphi E. typhosa = growth;- = no growth; ± = reduced growth. brain-heart infusion agar to determine whether any of the inoculated cells were alive. The results were recorded after the plates had been incubated for 24 hours. Since all of the isolated materials had similar activities, only the results obtained with the agent isolated from strain A-21 are given in table 3. The same sequence of results were obtained as in the bacteriostatic tests. The staphylococci and streptococci were killed by very small amounts of the agent, 507y or less. The streptococci were more sensitive than the staphylococci. As little as ly was sufficient to destroy Streptococcus hemolyticus, while 5 to 107 destroyed Streptococcus viridans. Larger amounts were required to kill E. coli, S. paratyphi, and E. typhosa. However, 200y brought about complete killing in all cases.

8 260 The cells of the active strains were entirely resistant to the bactericidal agents isolated from them. Through the courtesy of the Merck Institute for Therapeutic Research, the isolated materials were tested in mice against Type I pneumococcus infections. When both the active material and 10,000 lethal doses of pneumococci were injected intraperitoneally, it was found that 27y of the active material saved some of the mice whereas 87 gave complete or almost complete protection. These findings are similar to those of Dubos (1939). A study was made of the course of production of the active principle and its distribution in the culture medium and the cells of strain A-21. Two liter flasks containing 500 ml. of tryptose TABLE 3 Bactericidal activity of agent isolated from culturea of strain A-S1 TEST ORGAISM AMOUNTeOF!AGENT!USD (micbaoea&x Pza 2 ML. OF suspension) Staph. aureus Staph. albus Strep. hemolyticus Strep. viridans E. coli S. paratyphi E. typhosa = growth; - = no growth. broth were inoculated with 5 ml. of a 24 hr. broth culture. After 1, 2, 3, 4, 6, and 10 days at 37 C. the cells were removed by centrifugation. For rapid testing of the activity of the bactericidal agents, we have found a strain of micrococcus isolated from milk to be very useful since it grows readily and is rapidly lysed by very small amounts of active material. The supernatants were tested for activity by adding from 0.05 to 0.5 ml. to 1 ml. of a 5- hour culture of the micrococcus. The volume was raised to 2 ml. with sterile water and the tubes were incubated at 37 C. After 2 hours the degree of lysis was recorded and a loopful was streaked on solid media to determine the presence of living cells. The centrifuged cells were suspended in alcohol for 24 hours. The

9 ISOLATION FROM SOIL OF SPORE-FORMING BACTERIA 261 active material was precipitated with 1 per cent NaCl, collected by centrifugation, washed, dried and weighed. The data are presented in figure 2. We are able to give the values for the activity of the supernatants in milligrams because we have found in a number of experiments that about 4 micrograms will completely destroy 1 ml. of a 5-hour broth culture of the micrococcus, i.e., approximately 3 X 106 cells. It is evident from the graph that the amount of active material obtained by alcoholic extraction of the centrifuged DISTRIBUTION OF THE ACTIVE PRINCIPLE IN DEVELOPING CULTURES OF STRAIN A21 Z 150, hls 100 O 25- IN SUEDIMENT <50- IL 0 75 O IN SUPERNATANT 0.J INCUBATION PERIOD (DAYS) FIG. 2 sediment gradually increased with continued incubation until after 6 days a maximum yield of 137 mgm. was obtained. Later a decrease appeared to have occurred since only 94 mgm. were obtained from the 10-day-old culture. On the contrary, the amount of active material in the supernatant remained constant from day to day and represented only a small percentage of the total active material formed (13 per cent at the 6 day period). These results indicate, therefore, that only a small part of the total active material is present in solution in the culture, while most of the ma-

10 262 terial is in suspension or associated with the vegetative cells, spores and cellular debris and can be separated from the culture medium by centrifugation, without adjusting the medium to ph 4.6. SUMMARY A simple rapid method for isolating antagonistic microorganisms from soil is described, by which 24 strains of bacilli were obtained. These strains separate into two distinct groups: 1. An "active" group composed of strains whose broth cultures possessed readily demonstrable bactericidal properties. These strains formed H2S in peptone media, failed to hydrolyze starch and were gram-negative in 18- to 24-hour broth cultures. 2. An "inactive" group composed of strains whose broth cultures were not bactericidal. These strains did not form H2S in peptone media, hydrolyzed starch and were gram-positive in 24- to 48-hour broth cultures. The bactericidal agent of the active strains was readily isolated by extracting with ethanol the precipitate that formed on adjusting the cultures to ph 4.6 to 4.8 or by a similar extraction of the sediment obtained by centrifuging such cultures without adjustment of ph. The potent substances were finally obtained as grey or tan powders by precipitating them from the alcoholic extracts with a 1 per cent aqueous solution of NaCl. Attempts to obtain similar substances from cultures of some of the inactive strains were unsuccessful. The isolated agents were bactericidal in very small amounts ( mgm. per 2 ml. of suspension) for various grampositive bacteria; larger amounts were necessary to inhibit or destroy gram-negative bacteria, particularly those belonging to the colon-typhoid group. The agents obtained from the various active strains appeared to have similar anti-bacterial properties. The amount of bactericidal material present in a developing culture increased with continued incubation until a maximum was reached in about 6 days. Most of this material was present either in suspension or associated with the vegetative cells, spores and cellular debris of the culture and could be separated from the culture medium by centrifugation.

11 ISOLATION FROM SOIL OF SPORE-FORMING BACTERIA 263 It is evident from the data presented that antagonistic sporeforming bacteria are commonly present in soil and can be readily isolated. However, the ability to produce the bactericidal agents described is not a property of all spore-forming bacteria but is limited to a group of bacilli which are closely related in morphological and biochemical characteristics. REFERENCES CORDON, T. C., AND HAENSELER, C. M A bacterium antagonistic to Rhizoctonia solani. Soil Sci., 47: DuBos, R. J Studies on a bactericidal agent extracted from a soil bacillus. J. Exptl. Med., 70: 1-17, FRANEK, H., AND ISMET, A Ueber Cytolyse. Zentr. Bakt. Parasitenk. (I), 99: HETTCHE, H. O., AND WEBER, B Die Ursache der bakteriziden Wirkung von Mesentericus-filtraten. Arch. Hyg. Bakt., 123: HOOGERHEIDE, J. C An agent isolated from a soil bacillus, which inhibits encapsulation of Friedliinder's bacterium and is highly bactericidal for Gram-positive microorganisms. J. Franklin Inst., 229: HOuCHKISS, R. D., AND DuBos, R. J Fractionation of the bactericidal agent from cultures of a soil bacillus. J. Biol. Chem., 32: HOTCHKISS, R. D., AND DUBOS, R. J Bactericidal fractions from an aerobic sporulating bacillus. J. Biol. Chem., 136: MUCH, H., AND SARTORIUS, F Ueber die neuartigen Lysine des Mycoides "Much." Med. Klin., 20: NICOLLE, M Action du Bacillus subtiles sur diverses bacteries. Ann. inst. Pasteur, 21: PRINGSHEIM, E. G Ueber die gegenseitige Schadigung und Forderung von Bakterien. Zentr. Bakt. Parasitenk. (II), 51: ROSENTHAL, L Microbes bacterio-lytiques (Lysobacteries). Compt. rend., 92: WAKSMAN, S. A., AND WOODRUFF, H. B The soil as a source of microorganisms antagonistic to disease-producing bacteria. J. Bact., 40: