Lafayette, Indiana. Animals. Adult albino mice of the Purdue. Swiss and Rockland Swis strains were used. Toxin and antiserum. The toxin and antitoxin

Transcription

1 THE EFFECT OF CALCIUM BINDING AGENTS ON THE VIRULENCE OF CLOSTRIDIUM PERFRINGENS FOR THE WHITE MOUSE' ERNEST ALAN MEYER AND MERWIN MOSKOWITZ Division of Bacteriology, Department of Biological Sciences, Purdue University, Lafayette, Indiana Received for publication June 7, 1954 Clostridium perfringens type A produces a variety of toxins. It has been shown that the alpha toxin (lecithinase) produced by this organism apparently plays a major role in its virulence (Evans, 1947) using the guinea pig as the test animal. If lecithinase is necesary for virulence, the organisms should be avirulent when the lecithinase is inhibited. Calcium ion is necessary for lecithinase activity, and addition of appropriate amounts of calcium binding agents will effectively inhibit this activity in vitro. The present paper is concerned with the effect of simultaneously injecting calcium binding agents and C. perfringen organisms in the white mouse. Although frequently used in gas gangrene studies, the white mouse is considerably more resistant to infection with C. pefringens than is the guinea pig. Some method of producing preliminary tissue breakdown is generally employed for initiating C. perfringens infection in the mouse. Two of the more frequently encountered methods of causing tissue breakdown are the use of high concentrations of ionizable calcium salts (Bullock and Cramer, 1919) or toxic culture filtrates (de- Kruif and Bolhann, 1917) as suspending agents for the organisms. In the course of the present study, it has been observed that washed C. perfringen organisms injected subcutaneously in comparatively large numbers are capable of causing the death of the white mouse, in the absence of any added tissue damaging agent.,aterials AD METHODS Culture. The SR12 strain of C. perfringens type A was used throughout. It was obtained through the courtesy of the National Institutes of Health, Bethesda, Md. Organisms were transferred weekly in fluid thioglycolate medium and in egg meat medium. 1 This work is part of a thesis submitted in partial fulfilment for the Master of Science degree by Ernest Alan Meyer. Animals. Adult albino mice of the Purdue Swiss and Rockland Swis strains were used. Toxin and antiserum. The toxin and antitoxin studies were -carried out using a C. perfringens type A toxin and antiserum provided by the Lederle Co., Pearl River, New York. The toxin was labeled 400 MLD per ml (intravenous mouse units). The subcutaneous LD5o for mice, determined at the time of the present study, was equivalent to approximately 55 of the above units, or 0.14 ml of the undiluted toxin. The antitoxin was labeled 65 units per ml. A preliminary toxin-antitoxin titration established that 0.65 antitoxin unit was just sufficient to inhibit completely the in vitro lecithinase activity of 20 toxin units. Lecithovitellin. One hen's egg yolk was mixed in 250 ml of physiological saline solution, heated at 56 C for 30 minutes, and filtered through a Seitz filter with a no. 6 pad after cooling. This solution was stored at 1 C until used. Washed vegetative organisms. In the experiments in which large numbers of vegetative organisms were used, an identical procedure was employed for obtaining the cells, differing only in the amount of original medium used and the number of fractions made up from the original pellet.2 The technique was as follows: Immediately after sterilization, tubes of media (about 15 ml per tube) were cooled and inoculated with a loopful of a 12 hour SR12 culture of C. perfringene in egg meat medium. Incubation was at 37 C for 10 hours. Tubes of culture fluid were then pooled and centrifuged. The clear supernatant was discarded. The organisms were washed twice in 0.85 per cent saline. They were resuspended in saline and divided evenly into the number of portions, usually two or four, needed for the test. The organisms were again centrifuged, and the supernatant removed and discarded. A final wash- 2 No spores were observed when the organisms were grown under these conditions. 111

2 112 ERNEST A. MEYER AND MERWIN MOSKOWITZ [VOL. 69 ing was carried out in saline or 5 per cent calcium chloride. The packed organisms were then suspended in a small amount, usually 1.1 ml, of the same agent used in the final wash and inoculated subcutaneously in 0.1 ml amounts in mice. In order to count the number of viable organisms present, one-tenth ml of the above suspensions of organisms was added to a sterile 99.9 ml saline blank. Tenfold serial dilutions were made from 10-5 to One-tenth ml of each of these dilutions was added in quintuplicate to tubes of freshly prepared fluid thioglycolate medium. These were incubated at 37 C, and presence or absence of growth was noted in 24 hours. The Most Probable Number (MPN) of organisms was computed using the tables of Hoskins (1934). Lecithovitellin test. One ml of lecithovitellin prepared as described above was mixed with an equal volume of the diluted test material and incubated at 37 C. Readings were made at 24 hours, on the basis of the amount of lipoid material which had risen to the top of the tubes. Tubes were centrifuged before readings were made. Maximal lecithovitellin readings were reported at 3 +. Hemolysie tet. One ml of two per cent human red blood cells was mixed with an equal volume of test material and incubated at 37 C. Readings were made at 24 hours. Maximal hemolysis readings were reported as 3 +. TABLE 1 Relation of culture medium to mouse mortality caused by the washed SR1f organisms Culture Medium Suspending Agent A prox. Viable Cells per 0.1 ml Fluid thioglycolate with- NaCl 107 0/5 out added glucose CaCl /5 Fluid thioglycolate without added glucose or agar Brain heart infusion Brain heart infusion plus 1% added sodium thioglycolate NaCl CaCl2 NaCl CaCl2 NaCl CaCl EXPERIMENTAL RESULTS Early unsuccessful attempts at producing consistently high mouse mortality with C. petfringens included the inoculation of washed vegetative organisms in comparatively small numbers, and of washed vegetative organisms plus a sublethal dose of toxin. Further experiments demonstrated that under certain conditions large numbers of subcutaneously inoculated washed vegetative organisms which had been grown in brain heart infusion medium (Difco) caused a consistently high mouse mortality. Injluence of growth medium on the ability of C. perfringens to produce mouse mortality. The SR12 strain was grown on each of the following: (a) fluid thioglycolate medium without added glucose, (b) fluid thioglycolate medium without added glucose or agar, (a) brain heart infusion medium, and (d) brain heart infusion medium plus one per cent added sodium thioglycolate. The organisms from each of these media were harvested, washed, divided into two equal fractions, and suspended in physiological saline and five per cent calcium chloride, respectively. The organisms were inoculated subcutaneously into the abdominal region of mice. The number of viable organisms per mouse dose was determined as described. The results (table 1) show that a subcutaneous injection of washed C. pefringens of the order of 108 organisms which had been grown in brain heart infusion medium was sufficient to produce mortality in 36 out of 40 test mice. On the other hand, the same approximate number of organsms grown in fluid thioglycolate medium caused the death of but 4 out of 23 test nimals. Calcium chloride as a suspending agent for the injected organisms had no effect on mouse mortality when compared with the saline suspending agent. The basis for the use of calcium chloride here was for its effect on the tissues (Bullock and Cramer, 1919) and not for the activation of the lecithinase. In vitro lcithinase and hemolytic activity of toxin and washed organisms. The mechanism by which the mice were killed by the organisms grown in brain heart infusion medium is not known. Inasmuch as lecithinase has been shown to play a prominent role in experimental guinea pig infection by C. perfringens, a high lecithbinase activity of the washed organisms would seem to be a possible explanation for the establishment of the

3 1955] CALCIUM BINDING AGENTS ON VIRULENCE OF C. PERFRINGENS lethal infection in the mouse. In this experiment the in vitro lecithinase activity of a dose of washed organisms (prepared as described earlier) was compared with that of C. pefringens toxin (table 2). The organisms were grown on brain heart infusion medium; dilutions were made in physiological saline solution. The count of viable organisms indicates that the first tube in the series contained 1.4 X 10' organisms. The previous experiment showed that this number of brain heart infusion-grown organisms constitutes more than an LDI)o mouse dose; subsequent experiments have consistently confirmed this. The lecithovitellin activity of such a dose of organisms in saline, however, most closely resembled that of 1 toxin units or about 2 per cent of an LDso of toxin (55 units). In vitro effect of sodium citrate, sodium oxalate, and specific antisrum on the lecithinase and hemolytic activity of toxin and washed organism. This experiment was designed to determine if calcium binding agents could prevent in vitro lecithinase activity of the washed SR12 organisms and the approximate level of binding agent required. After the organisms were washed in saline, aliquots were washed and ultimately suspended in 0.27 M sodium citrate and 0.27 M sodium oxalate, as well as in antium. The level of antiserum used (6.5 units per ml) had been shown in a preliminar titration to just neutralize the lecithinase activity of 20 units of toxin. Onetenth ml of the above suspensons was used in the first tube in each series of dilutions. Counts of viable organisms were made as previously described. All dilutions of toxin and organisms were made up to 1.0 ml with physiological saline. One ml of 2 per cent red blood oells or lecithovitellin was added to each tube, the tubes shaken and incubated at 37 C. Final rdings were made at 24 hr. Control tubes containing red cells with no toxin or organisms added manifested no hemolysis. The results (table 2) indicate that the concentration of calcium binding agents used was effective in inhibiting the lecithovitellin reaction; at the same time, however, hemolysis is seen to persist. This remaining hemolysis is probably due to some hemolytic product of the organisms not dependent on calcium ion for activity. An example of such a product of C. perfringens is the theta hemolysin. The almost complete absence of he- TABLE 2 Comparison of lecithinase and hemolytic activity of toxin and washed vegetative cells, and the inhibition of this activity by sodium citrate, sodium oxalate, and antiserum. Units of Toxin ; 1V Dilution of SR12 Organisms Undiluted Toxin plhs Na Chloride (0.145 Y) J= SR12 plus Na Chloride (0.145 x) Toxin plus Na Citrate (0.27 x) 4- SR12 plus Na Citrate (0.27 x) =1 Toxin (lus Na Oxialate (0.27 m) SRI2 (lus Na Oxalate (0.27 x)i 4- Toxin plus Antiserum (6.5 units/ml) SR12 plus Antiserum (6.5 units/ml) 113 * The lecithovitellin titer of this tube most closely resembled the toxin tube containing 1H units of toxin. The viable cell counts of the organisms used above were: Suspending agent MPN organism*/o.i ml Sodium chloride X 109 Sodium citrate. 1.1 X 109 Sodium oxalate X 10l Antiserum X 10' molysis in the presence of antiserum, on the other hand, suggests that the hemolytic activity of the various toxic factors has been prevented as a result of the presence of specific antibody. In vivo effect of sodium citrate, sodium oalate, and antiserum used as surpending agents for the washw organisms. This experiment was designed to determine if calcium binding agents or specific antiserum, when used as suspending agents for the washed organisms, had any mked effect on the mouse mortality produced by the washed organisms (table 3). Two concentrations of sodium citrate (0.16 X and 0.50 m) and two concentrations of sodium oxalate (0.16 M and 0.27 M-saturated) were used.

4 114 ERNEST A. MEYER AND MERWIN MOSKOWITZ [VOL.. 69 TABLE 3 The effect on mouse mortality of calcium binding substances and anti8erum as suspending agents for the washed organisms MPN Mice Expt Suspending Agent (organisms/ (died/ 0.1 ml) total) % (0.145 M) NaCl 2.4 X 108 8/ M Na citrate 2.5 X 108 8/ M Na oxalate 2.4 X 109 9/ % (0.145 m) NaCl 2.4 X 10' 6/ M Na citrate 3.3 X 108 5/ M Na oxalate 9.2 X 108 9/ % (0.145 M) NaCI 4.9 X 108 6/ units antiserum 2.4 X 109 5/ units antiserum 2.4 X 10' 1/10 Injections of these salts alone had no apparent effect on the mice. The dilution of antiserum containing 0.65 units per 0.1 ml had previously been shown to be the amount required for the in vitro inhibition of the lecithinase activity of 20 toxin units. Effect of heating of brain heart infusion-grown organisms on their ability to produce mouse mortality. It was considered a possibility that the deaths observed were produced by a lethal dose TABLE 4 Comparison of mouse mortality produced by heated and unheated brain heart infusion-grown organisms MPN Mice Suspending Agent Heating (organisms/ (died/ 0.1 ml) total) 0.85% saline None 3.3 X 109 7/10 5% calcium chlo- None 4.9 X 10' 7/10 ride 0.85% saline 60 C, 30 min 240* 1/10 5% calcium chlo- 60 C, 30 min 130 0/10 ride 0.85% saline 100 C, 30 min 240 0/10 5% calcium chlo- 100 C, 30 min 240 0/10 ride * These numbers refer to the number of live bacteria after heating. The actual number of bacteria present-living and dead-are the same as in the unheated preparations. of a heat stable toxin associated with the washed organisms, present at the time of animal inoculation. The experiment which follows compares the mouse mortality produced by heated and unheated suspensions of this organism. The organisms were prepared as has been described. After the second washing in saline, the cells were divided evenly among six centrifuge tubes. Three tubes ultimately contained 0.85 per cent saline as a suspending agent, the other three containing 5 per cent CaCl2 solution. The cells in each tube were suspended to a volume of 1.1 ml. One pair of tubes (CaCl2 and saline) was not heated, one pair was heated to 60 C for 30 min and quickly cooled, and the other pair immersed in boiling water for 30 min and cooled. The results (table 4) indicate that the ability of these organisms to produce mouse death is not due to the presence within the cells of a lethal dose of heat stable toxin. DISCUSSION Calcium binding agents have been used as suspending agents for washed C. pefringens8 organisms injected into mice. These agents were used because: (a) the alpha toxin, lecithinase, is said to be an important factor in the virulence of C. perfringens, (b) calcium ions are required for the action of this lecithinase, and (c) it is possible to inhibit the in vitro lecithinase activity with calcium binding agents. The use of the white mouse in this study instead of the guinea pig presented an initial problem: how to produce mouse mortality upon injection of gas gangrene organisms in the absence of calcium salts. It has been shown here that comparatively large numbers of organisms grown in brain heart infusion medium, washed and suspended in saline and inoculated subcutaneously, are capable of consistently producing more than 50 per cent mortality in white mice. On the other hand, a very low mortality rate was observed in mice receiving comparable numbers of organisms grown in fluid thioglycolate medium. Two possible mechanisms are suggested for the mouse deaths observed in the above mentioned experiment: (a) some product associated with the brain heart infusion grown organism enabled them to initiate infection and ultimately cause the death of the mouse, or (b) death was brought about by a lethal dose of toxic substance present in the

5 1955] CALCIUM BINDING AGENTS ON VIRULENCE OF C. PERFRINGENS inoculated organisms, and no further proliferation need have taken place. Whichever may have been the case, large numbers of C. perfringens type A, grown on brain heart infusion medium, cause the death of the mouse. This does not occur with organisms grown on fluid thioglycolate medium, and the virulence of the organism when grown on the former medium is undoubtedly due to a substance(s) produced in that medium which is not produced in the fluid thioglycolate medium. Of the known toxis of this organism, the one whose production is most consistently related to virulence for the guinea pig is the alpha toxin, lecithinase. The role of the various toxins in the virulence of this organism for the mouse has not been so extensively investigated. If lecithinase does play a major role in virulence for the mouse, a high lecithinase titer in the washed organisms would be cause for speculation that the mice had been killed by the toxin associated with the washed organi. However, comparison of lecithinase titrations of washed vegetative cells and of an LD5o of toxin indicates that the lecithinase titer of a lethal dose of washed organisms amounts to but two per cent of that of the LDuo toxin dose. Additional experiments suggest that the part played by lecithins in the death of the mouse is an insignificant one. Amounts of calcium binding agents which successfully inhibited the in vitro lecithovitellin reaction of a typical dose of organisms were used to suspend the washed vegetative cells which were to be inoculated into mice. The mouse mortality was not markedly different when these agents were employed than when physiological saline was used as a suspending agent. Further, when the injected organisms were suspended in an amount of antiserum (which contains antibodies directed at many of the antigenic products of the organism) just sufficient to inhibit the in vitro lecithinase and hemolytic activity of an LDIo of toxin, mouse mortality was markedly reduced. It was considered possible that a heat stable toxin was involved in mouse death. An experiment was performed in which organisms were heated for 30 minutes at 60 C and 100 C, respectively, before inoculation into mice. The almost complete failure of the heated organisms to kill mice suggests that if a preformed lethal dose of toxin is involved in mouse mortality, it is destroyed by heating at 60 C for 30 minutes. The 115 marked decrease in the number of organi present after heating also suggests that large numbers of viable organisms may be necesary to set up the lethal infection. The possibility that lecithinase may be of minor importance in the virulence of this orga ism for the mouse is strengthened by the work of Kass et al. (1945). In a study directed toward elucidating the relation of lecithinase and hyaluronidase in C. perfringens to virulence for the mouse, they reported finding a number of strains of this organism which produced no lecithinase yet were virulent for the mouse via the intramuscular route. Further, they reported that they had isolated three strains of C. perfringens which produced neither lecithinase nor hyaluronidase yet possessed virulence for the mouse by this route. This work is part of a program in progress here on the role of specific toins on the initiation of gas gangrene infections. The rationale is selectively to inhibit the different toxins produced by C. perfringens and observe which ones are necessary for the initiation of the infection. The white mouse was used in these initial experiments for reasons of economy, inasmuch as we wished to screen procedures on large numbers of animals. This work is being continued using different species of animals, and when techniques are worked out, other toxins will be used. SUMMARY Vegetative cells of Clostridium peifringens type A, grown in brain heart infusion medium, washed and suspended in saline, have been shown capable of killing mice when large numbers are injected subcutaneously in the absence of any added necrotizing agent. The percentage of mice killed was markedly smaller when the organim was grown on fluid thioglycolate medium. Comparison of the lecithinase activity of a dose of washed vegetative cells with a toxin of C. perfringens type A showed that the organisms grown in brain heart infusion medium contained approximately one-fiftieth of the lecithinase activity of an LDI50 of toxin, and that the fluid thioglycolate-grown organisms exhibited no lecithina activity under the conditions used. A concentration of antisum which inhibited the in vitro lecithovitellin and hemolysin reactions of the washed organisms reduced mouse

6 116 ERNEST A. MEYER AND MERWIN MOSKOWITZ [VOL. 69 mortality markedly when used as a suspending agent for the washed organisms. This concentration of antiserum did not significantly reduce the viable organism count. Concentrations of sodium citrate and sodium oxalate which inhibited the in vitro lecithovitellin reaction of the washed organisms had no marked effect on mouse mortality when used as suspending agents for the washed organisms. REFERENCES BULLOCK, W. E., AND CRAMER, W On a new factor in the mechanism of bacterial infection. Proc. Roy. Soc. (London), B, 90, DEKRUIF, P. H., AND BOLLMANN, J. L The toxin on Bacillus welchii. II. The mechanism of infection with B. welchii. J. Infectious Diseases, 21, EVANS, D. G Anticollagenase in immunity to C. welchii type A infection. Brit. J. Exptl. Pathol., 28, HoSJlNS, J. K Most probable numbers for evaluation of coli-aerogenes test by fermentation tube method. Public Health Repts. (U. S.), 49, KASS, E. H., LICHSTEIN, H. C., AND WAISBREN, B. H Occurrence of hyaluronidase and lecithinase in relation to virulence in C. welchii. Proc. Soc. Exptl. Biol. Med., 68,