Wright (1933) expressed the opinion that the beneficial effect of blood on. Bacteriology, The University of Texas, Austin, Texas

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1 AN ANTIBRUCELLA FACTOR IN PEPTONES V. T. SCHUHARDT, L. J. RODE, J. W. FOSTER, AND GLENDA OGLESBY The Brucellosi8 Research Laboratory of the Clayton Foundation and the Department of Bacteriology, The University of Texas, Austin, Texas Received for publication August 30, 1948 The inhibition of bacterial growth by liquid media containing hydrolytic products of proteins has been described by a number of workers. McLeod and his colleagues (McLeod and Wyon, 1921; Wyon, 1923; Wyon and McLeod, 1923; Gordon and McLeod, 1926; McLeod, Wheatley, and Phelon, 1927) showed that peptones and certain amino acids tend to inhibit the growth of many bacteria and that serum tends to overcome this inhibition. Oxidizing agents, either added to peptone media or produced in the media, have been shown to inhibit various bacteria selectively (Novy and Freer, 1902; Burnet, 1927; Dubos, 1929a, b,c, 1930; McLeod, 1930; Braun, 1931; Braun and Guggenheim, 1932; Wright, 1929, 1933). Most of these authors have attributed such inhibition to the establishment and maintenance of redox potentials unsuitable for growth of the test bacteria. Wright, however, is inclined to disagree with the redox potential hypothesis and to attribute inhibition of growth to the oxidation of certain constituents of the peptone that, in the oxidized form, are toxic to certain bacteria. He believes that blood, serum, and meat and yeast extracts exert their beneficial effect on the bacteriological media by virtue of their capacity to reduce toxic, oxidized constituents of peptones. He noted that thioglycolic acid and glucose exert stimulatory effects upon the growth of the pneumococcus. This enhancement of meat infusion media by glucose for the cultivation of pneumococci and streptococci was noted by Mueller (1922a,b) and others, but such enhancement was not explained in terms of the neutralization of toxic components of the medium. Wright (1933) expressed the opinion that the beneficial effect of blood on cultures of Hemophilus spp. might be due to the neutralization of inhibitory substances in the peptone rather than the provision of essential growth factors. This prediction seems to be substantiated by the work of Pollock (1947), who found that the growth-promoting power of whole blood for Hemophilus pertussis could be replaced by purified albumin, by activated charcoal, or by starch. Pollock is inclined to attribute the toxicity of the culture medium to fatty acids in the peptone. He found activated charcoal to be just as effective as whole blood in promoting the growth of H. pertussis when the former was incorporated in the medium during the growth of the organism. However, the charcoal was not capable of removing the toxic substance from the liquid medium when the "norit" was removed by centrifugation or filtration prior to inoculation. A number of authors have correlated the toxicity of peptones with the size of the test inoculum. This observation fits well into both the redox potential hypothesis and the oxidized substances concept of the nature of the inhibition 1

2 2 SCHUHARDT, RODE, FOSTER, AND OGLESBY [VOL. 57 of growth. The only mention of the inhibition of Brucella spp. by peptone broth is that of Wright (1933), whose observations were based on the size of the inocula tested. He found that Brucella melitensis would grow from an inoculum of 0.1 ml of a 10-1 dilution in a medium in which the peptone was added to a heart infusion before heating, whereas an inoculum of 0.1 ml of a 10C4 dilution failed to grow in the same medium when the peptone was added in the conventional manner after removal of the heat-coagulable proteins of the heart infusion. He attributed the growth in the former medium to the reduction of toxic substances in the peptone by the heart infusion proteins before and during heat coagulation. In the course of an experiment to determine the in vitro effect of sulfonamides on BruceUa abortus, using small inocula in Difco tryptose broth, we encountered failure to obtain growth in the control tubes. When a repetition of the experiment resulted in a similar failure of growth in the controls and dilution plates showed the presence of at least 400 to 400,000 viable organisms in the inocula used, we decided to attempt to determine the nature of the inadequacy of the tryptose broth for the culture of B. abortus. EXPERIMENTAL RESULTS Demonstration of the antibrucella factor in peptone. In our preliminary experiments, tubes containing 10 ml of a 2 per cent aqueous solution of tryptose, or other peptone, adjusted to ph 7.0 were inoculated with 0.1-ml amounts of undiluted and decimal dilutions (through 10-9) of a 48-hour broth culture of B. abortus. The tubes were incubated in appropriate C02 environments at 37 C and were examined for visible growth at 2, 3, and 5 days. Negative tubes frequently were incubated for 15 days or longer without developing visible growth. Table 1 gives a typical result with a toxic tryptose preparation, the same tryptose after the addition of 2 per cent agar, and a nontoxic tryptose broth. These results indicate an antibrucella property of the toxic tryptose capable of inhibiting growth of at least 100 million times the number of B. abottus cells that will grow in nontoxic tryptose broth. Also, the results show the capacity of 2 per cent agar to neutralize the antibrucella factor in toxic tryptose. Three of 10 lots of Difco tryptose so tested and 9 of 10 lots of Difco peptone were found to inhibit all but the undiluted or the 10-1 dilution inocula. Other peptones tested ranged from nontoxic to toxic in varying degrees as indicated by the size of the inocula inhibited. Table 2 gives the results of an experiment to determine the toxicity of the tryptose factor for various cultures of the three Brucella species. Thirteen cultures of B. abortus, including an avirulent, acclimated strain, virulent, acclimated strains, and virulent, C02-requiring strains, were inhibited in arl but the tubes receiving the one or two largest inocula. Two of four cultures of Brucella suis were equally inhibited, whereas all of four cultures of B. melitensis proved more resistant. Single cultures of Pseudomonas fluorescens, Serratia marcescens, Salmonella typhosa, Staphylococcus albus, and Streptococcus salivarius were not inhibited by the toxic tryptose at any level of the inocula tested. The anti-

3 1949] ANTIBRUCELLA FACTOR IN PEPTONES 3 brucella factor in the only lot of Difco peptone tested proved more uniformly toxic to B. suis and B. melitensis than did the tryptose factor. P To determine whether the inhibitory action of the tryptose is brucellacidal or brucellastatic, 0.5 ml of a 10- dilution of a 48-hour broth culture of B. abortu8 TABLE 1 Inhibitory action of toxic tryptose on B. abortus 1*57 and the effect of 2 per cent agar on the antibrucella factor INOCPLLA TOXIC TRYPOE BROTH TO=C TRYPToSE PLUS 2 NONTOXIC TRYPTOSE BROTH PHI CENT AGAR 0.1 ml of a 48-hour broth culture days days days Undil _ ' _ _ S _ _ ' e growth;- no growth. TABLE 2 The effect of the toxic tryptose factor on brucellae INOCU7A B. ANORTUS Acclimated C02-requiring 0.1 ml of a 48-hour broth culture o: G W w w_ B. SUIS B.-MELITENSIS Undil ' ' '- _ Growth results recorded for the 5-day reading only. was inoculated into flasks containing 50 ml each of (a) 2 per cent toxic tryptose, (b) 2 per cent nontoxic tryptose, and (c) physiological saline. The flasks were incubated at 37 C and plate counts were made on each flask immediately after inoculation and at intervals up to 80 hours. Table 3 gives the results of this

4 4 CIUEIARDT, RODE, FOSTER, AND OGLESBY test, which indicate that the antibrucella factor in tryptose is definitely brucellacidal. Neutralization of toxicity of peptone. Following the observation that Difco agar in concentrations above 0.2 per cent would reverse the antibrucella activity of the toxic peptones, we conducted a search for other neutralizing agents. Crude aqueous extracts, approximating 20 per cent wet weight, were prepared from all available fresh fruits and vegetables, agar, lean beef, and liver. After the ph was adjusted to 7.0, varying dilutions of these extracts were added to tubes of 2 per cent toxic tryptose broth. Additional tubes of the toxic broth received 10 per cent beef serum, 0.5 and 1.0 per cent starch, 0.5 and 1.0 psr cent* glucose, 2 per cent corn steep liquor, 0.5 per cent vitamin-free casein, 0.5 par cent "casamino acids," the ash equivalent of 2 per cent agar, and various mineral and B vitamin supplements. In every instance except the 10 per cent serum the toxic INCUBATION PERUOD TABLE 3 Brucellacidal action of toxic tryptose on B. abortus 1257 PIATE COUNTS PER ML Toxic tryptose Nontoxic tryptose Saline hours TNTC TNTC TNTC TNTC 32 1 TNTC TNTC 48 0 Confluent growth TNTC 56 0 Confluent growth TNTC 72 0 Confluent growth TNTC 80 0 Confluent growth TNTC TNTC = colonies too numerous to count. [VOL. 57 tryptose was autoclaved after the addition of the supplement. These tubes and controls of the toxic tryptose broth were each inoculated with 0.1 ml of a 10-2 dilution of a 48-hour broth culture of B. abortus. The controls showed no growth. No evidence of detoxification was observed in the tubes receiving the vitamins or mineral supplements, the casamino acids, the starch, or the agar ash. The toxicity was neutralized, at least to the extent tested, by serum, glucose, casein, corn steep liquor, liver extract, beef and agar extracts, and by a number of the fresh fruit and vegetable extracts. In subsequent experiments various toxic peptones (2 per cent) were neutralized by minimum glucose concentrations ranging from 0.05 per cent to 0.5 per cent. The glucose neutralization occurred only when the sugar was heat-sterilized in the medium, not when added aseptically or when filtered. The toxicity gradually returned when the glucose broth was allowed to stand after sterilization. Detoxification of the peptones

5 19491 ANTIBRUCELLA FACTOR IN PEPTONES 5 by various other reducing agents, including thio-acids, has been observed and will be reported in detail in a subsequent paper. Chemical properties and attempts to purify the toxic factor in peptone. Exposure of a toxic 2 per cent tryptose broth to a ph of 10.0 at room temperature for 4 to 7 hours resulted in a slight reduction of the toxicity; exposure for 48 hours resulted in a marked reduction in toxicity. Autoclaving such broth for 10 minutes at 121 C at ph 3.0 or 7.0 had no effect on the toxicity Autoclaving at ph 8.0 had a very slight effect, whereas autoclaving at ph 9.0 to 10.0 resulted in complete detoxification. Three aliquots of 2 per cent toxic tryptose were adjusted to ph 3.0, 7.0, and 10.0, and 1 per cent norit was added to each. After a 30-minute shaking at room temperature, the norit was removed by filtration and the adsorbed broths were adjusted to ph 7.0 and tested. for residual toxicity. Aliquots treated with charcoal at ph 3.0 and 10.0 were completely detoxified, whereas the aliquot treated at ph 7.0 was incompletely detoxified. Subsequently, 0.4 per cent norit was established as the minimum concentration capable of removing all toxicity from 2 per cent toxic tryptose at a ph of 2.5; however, some destruction of peptone toxicity was noted at ph levels below 3.0. Therefore, for maximum recovery of the toxic factor from 2 per cent peptone by norit adsorption, the use of 0.6 to 1.0 per cent concentrations of the charcoal at ph levels above 3.0 is desirable. After establishing the capacity of norit to adsorb the antibrucella factor from aqueous solutions of toxic peptones, we began a search for a suitable solvent to elute the factor from the charcoal. Buffered aqueous solutions at various ph levels, ethyl alcohol, methyl alcohol, ethyl ether, chloroform, and acetone at room temperature and at boiling temperatures failed to elute the toxic factor from norit. Since these failures could be due to destruction of the antibrucella factor rather than to failure to elute, we decided to determine whether or not these and other organic solvents would remove the factor from toxic tryptose. Samples of toxic tryptose powder were subjected to continuous hot extraction in a Soxhlet apparatus for 15 hours by ethyl ether, ethyl alcohol, methyl alcohol, chloroform, acetone, benzene, and pyridine. After extraction, the tryptose was freed of all residual solvent and was employed in a 2 per cent concentration for the preparation of tryptose broth. The seven extracted tryptose broths and an unextracted control were then tested for toxicity by the decimal dilution inocula method. Table 4 records the 5-day growth results of this experiment, which indicate that, although a trace of the toxic factor was removed by ethyl and methyl alcohols, only pyridine of the solvents tested removed all of the toxic factor from the tryptose. Subsequently, pyridine proved effective in eluting the antibrucella factor from norit, and the eluate from both tryptose and peptone, after elimination of the pyridine and solution of the concentrate in water, proved toxic when added in varying dilutions to nontoxic tryptose broth or to nontoxic "N-Z-case" (Sheffield Farms Company) broth. This successful concentration of the toxic tryptose and peptone factors neces-

6 6 5CHUHARDT, RODE, FOSTER, AND OGLESBY [VOL. 57 sitated the development of a quantitative assay technique that might be used to determine progress in studies directed at further concentration and possible purification of the factor. Consequently, we established an arbitrary ABF unit (antibrucella factor) as follows: The ABF unit is the number of milligrams of toxic material per ml of assay medium required to inhibit visible growth for 48 hours at 37 C from a 0.1-ml inoculum of a 10- dilution of a 48-hour broth culture of B. abortue The assay medium is 0.5 per cent N-Z-case broth at ph 7.0. By this technique, the single toxic tryptose tested gave an ABF unit value of 5 mg. Eight of the nine lots of toxic peptone ranged in ABF unit values from 4 mg to 11 mg. The ninth lot gave an ABF unit value greater than 20 mg. Since the latter peptone alone at 2 per cent concentrations had proved toxic for B. abortus, it became apparent that our assay medium was exerting some neutraliz- TABLE 4 Effect of Soxhlet extraction with various solvents upon the toxicity of tryptose for B. abortus INOCULA GIROWTH IN ROTHS PREPARED FROM TRYPTOSE ZXTRACTED WI VARIOUS SOLVENTS 0.1 ml of a 48-hour Unex- Ethyl Ethyl Methyl Chloro A B Py*d broth culture tracted ether alcohol alcohol form cetone enzene yride Undil ' S ' ' ' + Growth results recorded for 5-day reading only. ing tendency on the antibrucella factor of that peptone. This observation was confirmed by showing that increasing concentrations of N-Z-case would neutralize correspondingly larger amounts of the antibrucella factor. In spite of this ABF neutralization by some, as yet unknown, constituent of a nontoxic peptone, the quantitative assay has proved valuable in our concentration studies. To date, we have been able to concentrate the antibrucella factor from tryptose a maximum of 60-fold, from an ABF unit of 5 mg to an ABF unit of 0.08 mg. The peptone factor has been more resistant to concentration, and as yet we have succeeded in concentrating this factor only to an ABF unit of 0.33 mg. Efforts at concentration so far have utilized hydrochloric acid and phosphotungstic acid precipitations, selective precipitations by various volumes of acetone, amyl acetate, ethyl ether, ethyl alcohol, methyl alcohol, and n-butyl alcohol. Also, we have tried selective adsorptions with aluminum oxide and various synthetic

7 1949] ANTIBRUCELLA FACTOR IN PEPTONES 7 resins. The best results obtained so far utilize norit adsorption, pyridine elution, precipitation of the active factor from the pyridine with 10 volumes of ethyl ether, and the solution of the ether precipitate in water. General impressions gained from the purification studies of the antibrucella factor lead us to conclude that we are dealing with an amphoteric substance, either present in relatively large amounts in the peptone or present in association with neutralizing substances that accompany the toxic factor through the purification process. The evidence leads us to suspect oxidized polypeptides or amino acids as the toxic factors, and experiments designed to test this supposition are in progress and have yielded some confirmatory evidence. SUMMARY An antibrucella factor has been found in certain lots of Difco tryptose that proved to be uniformly brucellacidal to relatively large inocula of each of 13 cultures of Brucella abortus tested. Brucella suis and Brucella melitensis cultures exhibited varying degrees of susceptibility to the toxic tryptose factor. A similar, if not identical, factor has been found in most samples of Difco peptone tested. The antibrucella factor can be adsorbed from aqueous solutions of the peptones by "norit." It can also be extracted from the dry peptones or eluted from norit by pyridine. Ethyl ether will precipitate the factor from pyridine. A 60-fold concentration of the factor has been accomplished by these methods. The antibrucella factor appears to be an amphoteric compound either present in the peptones in large amounts or present in association with neutralizing substances that accompany the toxic factor through the purification process. Oxidized polypeptides or amino acids are suspected as the toxic agents. REFERENCES BRAUN, H Zur Assimilation und Dissimilation bei Bakterien. Zentr. Bakt. Parasitenk., I, Orig., 122, BRAUN, H., AND GUGGENHEIm, K Ueber Atmungstypen bei fakultativ aeroben pathogenen Bakterien. Zent. Bakt. Parsitenk., I, Orig., 127, BURNET, F. M The action of cyanides on bacteria. J. Path. Bact., 30, DUBOS, RENt 1929a The inhibition of growth of certain facultative anaerobes as related to oxidation-reduction processes in the medium. J. Exptl. Med., 49, DUBOS, RENh 1929b The relation of bacteriostatic action of certain dyes to oxidationreduction processes. J. Exptl. Med., 49, DUBOS, RENA 1929c The role of carbohydrates in biological oxidations and reductions. Experiments with the pneumococcus. J. Exptl. Med., 50, DuBos, RENA 1930 The bacteriostatic action of certain components of commercial peptones as affected by conditions of oxidation and reduction. J. Exptl. Med., 52, GORDON, J., AND MCLEOD, J. W Inhibition of bacterial growth by some amino acids and its bearing on the use of tryptic digests as culture media. J. Path. Bact., 29, McLEOD, J. W Bacterial respiration. In Med. Res. Council (Brit.), A system of bacteriology, 1, MCLEOD, J. W., WHEATLEY, B., AND PHELON, H. V On some of the unexplained difficulties met with in cultivating the gonococcus; the part played by amino acids. Brit. J. Exptl. Path., 8,

8 8 SCHUHARDT, RODE, FOSTER, AND OGLESBY [VOL. 57 MCLEOD, J. W., AND WYON, G. A The supposed importance of vitamins in promoting bacterial growth. J. Path. Bact., 24, MUIELLER, J. HOWARD 1922a Studies on cultural requirements of bacteria. I. J. Bact., 7, MUELLER, J. HOWARD 1922b Studies on cultural requirements of bacteria. II. J. Bact., 7, Novy, F. G., AND FREER, P. C Ueber das keimtotende Vermogen der organischen Peroxide. Zentr. Bakt. Parsitenk., I, Ref., 31, 299. POLLOCK, M. R The growth of Hemophilus pertu8sis on media without blood. Brit. J. Exptl. Path., 28, WRIGHT, HEDLEY D The effect of certain factors upon the growth of the pneumococcus. J. Path. Bact., 82, WRIGHT, HEDLEY D The importance of adequate reduction of peptone in the preparation of media for the pneumococcus and other organisms. J. Path. Bact., 37, WYON, G. A Vitamins and bacterial growth. J. Path. Bact., 26, WYON, G. A., AND MCLEOD, J. W Preliminary note on inhibition of bacterial growth by amino acids. J. Hyg., 21,