Pauline Stark

The Destruction of Botulinum Toxin by Intestinal Bacteria.

In our studies of the destruction of toxins by bacteria a number of interesting, and perhaps important, observations have been made. Among these the destruction of the toxin of Clostridium botulinum by certain intestinal types of bacteria may be worthy of particular note. Several investigators have shown that if large numbers of detoxified botulinum spores are fed to experimental animals death from botulism will ensue, indicating that, under some conditions, the organisms can grow and produce their toxin in the intestinal tract. Since it is well known that the organisms of botulism are frequently ingested with such foods as raw vegetables, fruits and milk, the question naturally arises as to why the disease is not commonly transmitted in this way. It may also be questioned whether some of the so-called autointoxications are in fact incipient botulism arising from the limited growth of these organisms in the intestinal tract, with the production of sublethal quantities of toxin. It occurred to us that the probable explanation was to be found in the action of the intestinal bacteria upon the toxins which might be formed in the alimentary tract. In Table I are given data which indicate the destruction of botulinum toxin by Bact. coli, Bact. communior, Bact. aerogenes and Proteus vulgaris. In these experiments botulinum toxin (A type) was added to beef infusion broth so that the resulting medium contained approximately 100 m.l.d per cc. This toxin-broth mixture was inoculated with the different test organisms and allowed to incubate at 20° C. for two weeks. Uninoculated tubes of the toxin-broth mixture were of course incubated at the same temperature over the same period of time and subjected to the same filtering process in order to serve as controls. The unit amounts of toxin given in Table I are in terms of approximate m.l.d. values as determined on the control tubes after incubation.

Destruction of Botulinum Toxin by Bacillus subtilis.

In previous papers 1 , 2 it has been shown that a variety of bacteria have the ability to destroy the toxin produced by Clostridium botulinum. This power, though definite, is relatively slight as compared with the ability of certain organisms to destroy the toxin of the diphtheria organism. 3 In our work on the destruction of the toxin of botulism there has not appeared to be any general relationship between the proteolytic activities of the organisms tested and their ability to destroy botulinum toxin. In fact, it has been found that such an active proteolytic organism as Clostridium sporogenes has little or no demonstrable capacity to destroy this toxin, under the conditions of our experiments. 4 On the other hand, Dack, 5 working with mice, showed a definite destruction of botulinum toxin by several strains of Clostridium sporogenes and also by a variety of other spore forming anaerobic organisms. The purpose of this note is to report the results obtained with Bacillus subtilis, which shows a more marked destruction of botulinum toxin than does any one of the other organisms with which we have worked. The toxin was made by growing Clostridium botulinum, type A, in a medium composed of: beef infusion, 4% peptone, 0.5% glucose, 0.7% di-basic potassium phosphate, and 0.3% mono-basic potassium phosphate. The culture was grown in this medium for 8 days at 37° C. It was then filtered and the sterility of the filtrate verified by cultural tests. Some of the filtrate was then inoculated with a laboratory culture of Bacillus subtilis and incubated for 14 days at 37° C. Other portions of the same filtrate uninoculated were similarly incubated as controls.

Extracellular Production of Toxin by Clostridium botulinum.

It is well known that bacterial free filtrates containing the toxin of Clostridium botulinum increase in toxicity when mixed with certain substances such as normal blood serum and non-specific antitoxins. The observed increases have, as a rule, been relatively small (2 to 10 fold) and the observations have been variously interpreted by the several investigators. The possibility that the toxin might be produced extracellularly by the action of the enzymes of the organism on a suitable substrate has not been seriously entertained by the more prominent workers in this field; on the contrary, the belief is generally held that the toxin is produced only intracellularly. From our own work with Clostridium botulinum (Type A) we have been led to the conclusion that the toxin is produced entirely intracellularly, or nearly so, in some media, while it may be produced both intracellularly and extracellularly in certain other media. If a bacterial free filtrate of the botulinum organism is mixed with sterile skimmed milk and the mixture incubated at 37° C. until proteolysis ensues, a material increase in toxicity takes place. This is illustrated by the example given in Table I. In this experiment 1 part of the culture filtrate was mixed with 4 parts of sterile skimmed milk and the mixture incubated at 37° C. for 4 days. As a control, some of the same filtrate was incubated alone at the same temperature for the same period of time. After incubation, series of guinea pigs were inoculated with, (1) the incubated filtrate mixed with 4 parts of sterile physiological salt solution, (2) the incubated filtrate mixed with 4 parts of sterile skimmed milk within an hour previous to inoculation, and (3) the incubated filtrate-milk mixture referred to previously.

Further Observations on Apparent Effect of Diphtheria Toxin on Growth of Bacteria.

In a previous report 1 we have noted the fact that diphtheria toxin when added to cultures of Bacillus cereus in a concentration of 30 m.l.d. per cc. leads to a slight but definite retardation of growth. While our data do not prove that the inhibitory factor for B. cereus is in fact the toxin rather than some other by-product of growth of the diphtheria organism, there is some basis for believing that this might be the case. Of particular interest is the fact that the addition of antitoxin appears to counteract this inhibitory action. In the present paper we wish to record that similar results have been obtained with Proteus vulgaris and Staphylococcus albus. The apparent inhibitory effect of the diphtheria toxin upon the growth of these organisms is of about the same magnitude as in the case of B. cereus. This is a preliminary report.