EFFECT OF STAPHYLOCOCCUS AUREUS EXTRACTS ON VARIOUS BACTERIA LEO G. NUTINI, SR. THOMAS AQUIN KELLY, AND SR. MARGARET ANN McDOWELL Laboratories of the Institutum Divi Thomae, Cincinnati, Ohio, and its associated unit at St. Mary of the Springs College, Columbus, Ohio Received for publication January 7, 1946 For some time past, reports dealing with the effect of protein-free alcoholic extracts of various animal tissues on the growth of certain bacteria in vitro and in vivo have come from these laboratories (Nutini and Kreke, 1942; Nutini, Kreke, and Schroeder, 1945; Nutini and Lynch, 1945). The effectiveness of such extracts suggested further research using extracts of specific bacteria instead of the animal tissues. These bacterial extracts, by the nature of their preparation, differ from the bacterial filtrates investigated by other workers. Observations on the manner in which bacterial filtrates affect the growth of microorganisms have been made since the time of Pasteur. Within relatively recent years research concerned with bacteriostatic agents, initiated by Dubos (1939), has been carried on by such workers as Hettche and Weber (1939), Sarnowiec (1939), Waksman and Woodruff (1940), and Hotchkiss and Dubos (1941). The influence of living cells of one bacterial strain on the life processes of another grown in its presence has also been studied extensively by a number of workers, among whom may be mentioned McLeod and Govenlock (1921), Dujardin- Beaumetz (1932), Duliscouet (1935), and Waksman and Woodruff (1940; 1942). The literature has been briefly reviewed by Stokes and Woodward (1942) and exhaustively studied by Waksman (1945). It was the purpose of the present investigation to test the effect on bacterial growth in vitro of (a) protein-free alcoholic extracts of Staphylococcus aureus cells alone, (b) protein-free alcoholic extracts of medium obtained from ultraviolet-irradiated and nonirradiated cultures of Staphylococcus aureus, and (c) filtered, sterilized, but unextracted medium from ultraviolet-irradiated and nonirradiated cultures of Staphylococcus aureus. METHODS Source of Staphylococcus aureus. The culture of Staphylococcus aureus was one isolated from an infected tonsil (Pathological Laboratory, Good Samaritan Hospital, Cincinnati, Ohio). It was the same strain as that used for the test organism in experiments employing animal tissue extracts in these laboratories (Nutini and Lynch, 1945). Cell extract. The cell extract was prepared as follows: Large Roux flasks containing 250 ml of nutrient agar were inoculated aseptically with 3 ml of a 24- hour-old broth culture of Staphylococcus aureus, plugged, and incubated for 48 hours at 37 C. At the end of that time the bacterial cells were washed from the 533
534 L. G. NUTINI, T. A. KELLY, AND M. A. McDOWELL agar surface with 20 ml normal saline. The washings were subjected to the procedures previously described for animal tissues (Nutini and Kreke, 1942). Extracts of medium from nonirradiated cultures. Erlenmeyer flasks (250 ml) containing 100 ml of sterile nutrient broth were inoculated with 0.1 ml of a 24- hour-old broth culture of Staphylococcus aureus and incubated at 37 C for 48 hours. The bacterial cells were then removed by centrifugation. An extract of the supematant broth was prepared exactly as for the cells with the exception of the omission of the freezing and thawing, which was considered uinnecessary in the absence of cellular material. In order to determine whether active substances were adsorbed by the filters, some extracts were sterilized by using Seitz and others by using Berkefeld filters. Extracts of medium from irradiated cultures. Large quantities of 48-hour-old broth cultures of Staphylococcus aureus were introduced into special large glass (Coming 9741, grade 3) tubes and exposed to nonfiltered ultraviolet radiation from a Burdick A.C. quartz mercury arc at a distance of 45 cm for 24- and 48- hour periods. The tubes were rotated from time to time. After centrifugation the supematant broth was divided into two parts and an alcoholic extract prepared from one part. Filtrates of irradiated cultures. The second portion of the irradiated broth was sterilized by Seitz filtration in order to compare the effectiveness of the irradiated, protein-free alcoholic extracts of medium with that of untreated, irradiated, sterilized filtrates. Filtrates of nonirradiated cultures. The nonirradiated broth medium of 48- hour-old cultures was centrifuged and the supematant broth sterilized. Measurement of activity. Bacterial growth in terms of increase or decrease in colony numbers was estimated by the pour plate method. To 15 ml of warm, sterile nutrient agar, volumes of the extract or filtrate to be tested were added aseptically to give concentrations of 0.1, 0.5, 1.0, and 5.0 per cent. The extract or filtrate was thoroughly mixed through the agar by rotating the tube. It was then poured into petri plates containing 0.1 ml of a 1:10,000 dilution of a 24-hour broth culture of the test organisms, Staphylococcus aureus, Escherichia coli, strain 4265, Aerobacter aerogenes, strain 211, and Shigella dysenteriae, strain 9665. (The last three organisms were from the American Type Culture Collection, but the strain of Staphylococcus aureus was the same as that used in making the extracts and filtrates.) The plates were incubated for 48 hours at 37 C, and growth was determined by making colony counts on a Wolfhuegel plate counter. Control organisms were cultured in plates containing agar only, or agar and a sterile nutrient broth in the same percentage of concentration as was used for the experimental plates. Colony counts for the control plates were taken as 100 per cent, and the difference between these counts and those obtained on the experimentals was calculated. Results with the pour plate method are subject to about 25 per cent experimental error, so that only extracts producing stimulatory or inhibitory effects in terms of colony numbers lying well beyond this range were considered significant. Triplicate experiments were conducted. In several experiments Hopkins
EFFECT OF STAPHYLOCOCCUS AUREUS EXTRACTS ON BACTERIA tubes were also used as checks, but the cell volume was too small for accurate reading. Investigations were also undertaken to determine whether changes had occurred in the biochemistry of the test organisms. These experiments consisted of the fermentation of lactose, sucrose, and glucose; coagulation of litmus milk; liquefaction of gelatin; indole formation; and nitrate reduction. Only Staphylococccus aureus cell extract and the Seitz-filtered broth extract were used in these experiments. 535 TABLE 1 Effect of extracts of Staphylococcus aureus cells and of irradiated and nonirradiated broth media on the growth of organisms TYPE OF EXTRACT USED 0 E. COLI A. AEROGENES S. AUREUS S.DYSE. RIAE Cell extract 0.1 No effect Inhibition Complete inh. Complete inh. 0.5 Slight inh. Complete inh. Complete inh. Complete inh. 1.0 Inhibition Inhibition Complete inh. Stimulation 5.0 No effect Inhibition Stimulation Stimulation Broth extract, Seitz 0.1 No effect Inhibition No effectt No effect filter 0.5 No effect* Slight inh. No effectt Inhibition 1.0 No effect* Slight inh. No effectt Inhibition 5.0 Stimulation Slight inh. No effect Inhibition Broth extract, Berke- 0.1 Slight stim. No effect No effect No effect feld filter 0.5 No effect No effect No effect No effect 1.0 No effect No effect No effect No effect 5.0 No effect No effect No effect Slight inh. Extract of 24-hr. irra- 0.1 Stimulation Inhibition Inhibition No effect diated broth, Seitz 0.5 Stimulation Slight inh. Inhibition Stimulation filter 1.0 Stimulation Slight inh. Inhibition Stimulation 5.0 Slight stim. Inhibition Inhibition Stimulation Control colonies, 100 per cent; slight stimulation, 125 to 175 per cent; slight inhibition, 75 to 25 per cent; inhibition, 25 to 0 per cent; stimulation, 175 to 500 per cent; no effect, 75 to 125 per cent. * Prevented coagulation of milk. t Coagulated milk. RESULTS The effects of several concentrations of Staphylococcus aureus extracts and filtrates on the growth of 4 test bacteria are given in tables 1 and 2. A survey of the data shows that the growth response varied with the species of the test organism, the material tested, and its concentration, as well as with the irradiation of the Staphylococcus aureus cultures from which the test materials were prepared. The mode of filtration, whether by Seitz or Berkefeld, produced no appreciable difference in the response of the organisms to the broth extracts and filtrates.
536 L. G. NUTINI, T. A. KELLY, AND M. A. McDOWELL Cell extract. Extracts of Staphylococcus aureus cells had a predominatingly inhibitory effect on the growth of the 4 test organisms. The exceptions were in the stimulating action of higher concentrations of the cell extracts on Staphylococcus aureus and Shigella dysenteriae. Broth extracts. The broth extracts, with the single exception of growth stimulation of Escherichia coli by a 5 per cent concentration, had no effect or inhibited growth of the test organisms. It is apparent from table 1 that extracts produced from broth media of 24-hour irradiated Staphylococcus aureus cultures differed in their action from nonirradiated preparations. Filtrates. The action of the simple filtrates, on the other hand, was essentially one of stimulation of growth. Filtrates of 24-hour irradiated cultures of Staph- TABLE 2 Effect of filtrates of irradiated and nonirradiated broth media in which Staphylococcus aureus was cultured TYP 01 PLTRATZ *. COLI A.A.ROGENES S. AUREUS S. DYSZNTEURSA Filtrate of nonirra- 0.1 Stimulation No effect No effect No effect diated broth 0.5 Stimulation No effect No effect No effect 1.0 Stimulation Slight inh. Stimulation Slight stim. 5 0 Slight stim. Slight inh. Stimulation Stimulation Filtrate of 24-hr. irra- 0.1 Slight inh. Inhibition No effect No effect diated broth 0.5 No effect Inhibition Slight stim. No effect 1.0 No effect Inhibition Slight stim. No effect 5.0 No effect Inhibition Stimulation Inhibition Filtrate of 48-hr. irra- 0.1 Stimulation No effect No effect No effect diated broth 0.5 Stimulation No effect Slight stim. Slight stim. 1.0 Stimulation No effect Stimulation No effect 5.0 Slight inh. No effect No effect No effect See footnote to table 1 for definition of terms of response. ylococcus aureus differed somewhat in their effect on the growth of the 4 test organisms from nonirradiated filtrates. After 48-hour irradiation of cultures of Staphylococcus aureus the action of broth filtrates on the growth of organisms was essentially the same as that of filtrates of nonirradiated cultures. Biochemical changes. Observations on biochemical tests showed that the presence of the cell or broth extracts did not interfere with the fermentation of sugars, indole formation, nitrate reduction, or the liquefaction of gelatin. Broth extract, as shown in table 1, prevented coagulation of milk by Escherichia coli. The coagulating system was also affected in Staphylococcus aureus, which, like other strains obtained from similar sources, does not coagulate milk. Coagulation was observed in the presence of the broth extract, as indicated in table 1. In vitro and in vivo investigations of extracts of Staphylococcus aureus, as well as those from other organisms, are under way in these laboratories.
EFFECT OF STAPHYLOCOCCUS AUREUS EXTRACTS ON BACTERIA 537 DISCUSSION As Waksman (1944) points out, such bacterial preparations as those described here may affect one or more of the metabolic functions of the bacterial cell which is submitted to investigation. Among the probable mechanisms involved are interference with cell division, modification of the respiratory and other enzymatic systems, and changes in the surface tension of the cell and in the osmotic pressure, with accompanying changes in the utilization of nutrient materials and growth-regulatory substances. The results, though inconclusive, add evidence to Waksman's (1944) observation that antibiotic substances are selective in their action on bacteria. This investigation also confirms the report of Loofbourow and Morgan (1940) that different concentrations of the same cellular extract can produce opposite effects on bacterial growth. SUMMARY Several protein-free alcoholic extracts of a virulent strain of Staphylococcus aureus cells and of filterable bacterial products in the media in which they were grown were prepared. The effects of these and simple filtrates on the growth of Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, and Aerobacter aeroegenes were investigated. Extracts of both cells and the media in which they were grown, while showing some slight differences, were predominantly inhibitory in their growth effects on the test organisms. Cell-free filtrates of media in which Staphylococcus aureus was grown were, in general, stimulatory in their action on the growth ofthe test organisms. Ultraviolet irradiation of Staphylococcus aureus cultures slightly modified the action of both the broth extract and the filtrates. There was a reversal of the growth response of the test organisms with higher concentrations of the cell extract (2 instances) and filtrates of the 48-hour ultraviolet irradiated cultures of Staphylococcus aureus (1 instance). Biochemical tests indicate that in some cases the alcoholic broth extract may interfere with the coagulase reactions of the bacteria in culture. REFERENCES DUBOS, R. J. 1939 Studies on a bactericidal agent extracted from a soil bacillus. J. Exptl. Med., 70, 1-7; 249-256. DUJARDIN-BEAUMETZ, E. 1932 Action antibiotique d'une vari6t6 de staphylocoque A l'egard des bacilles gram-positifs et acido-resistants. Compt. rend. soc. biol., 110, 1210-1213. DULISCOUET, R., AND BALLET, B. 1935 Curieuses proprietds des staphylocoque chez les porteurs de bacilles diphtheriques; indications prognostiques et applications therapeutiques. Presse med., 43, 1297-1300. HETTCHE, H. O., AND WEBER, B. 1939 Die Ursache der bakteriziden Wirkung von Mesentericusfiltraten. Arch. Hyg. Bakt., 123, 69-80. HOTCHKISS, R. D., AND DUBOS, R. J. 1941 The isolation of bacterial substances from cultures of Bacillus brevis. J. Biol. Chem., 141, 155-162.
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