ON THE DEVELOPMENT OF STRAINS OF BACTERIA RESIS-

Transcription

1 214 ON THE DEVELOPMENT OF STRAINS OF BACTERIA RESIS- TANT TO LYSOZYME ACTION AND THE RELATION OF LYSOZYME ACTION TO INTRACELLULAR DIGESTION. ALEXANDER FLEMING, F.R.C.S., AND V. D. ALLISON, M.D. From the Laboratories of the Inoculation Department, St. Mary's Hospital, London. Received for publication April 15th, IN a previous communication (Fleming and Allison, 1922) it has been shown that when tears, sputum, egg-white or other lysozyme-containing substance is embedded in an agar plate and the surface of the plate is thickly planted with a microbe specially susceptible to lysozyme action (e. g. M. lysodeikticus), there appears a zone of complete inhibition of growth around the tissue or secretion. It has been shown also that after a week or two there may be developed in the inhibition zone a few colonies of the microbe, and if these colonies are subcultured they are found to be more resistant to the lytic action of the tissue or secretion than was the original culture. Not only are they more resistant to the lytic action of the tissue or secretion with which they have been grown in contact, but they are equally resistant to all other tissues or secretions; for instance, colonies which have developed in the neighbourhood of a piece of tonsil or cartilage have acquired as much resistance to tears or egg-white as they have to the tonsil or cartilage. Thus in one operation there has been developed a strain of the microbe relatively resistant to the bacteriolytic action of tissues. Is this Acquired Resistance to Lysis Temnporary or Permanent? When originally isolated, strains of M. lysodeikticus, which had by the method outlined above been made resistant to various tissues and secretions, were tested with egg-white, and their susceptibility to lysis by this substance was noted. Nine months afterwards the same strains, which in the meantime had been repeatedly subcultured at intervals of 1 or 2 weeks, were again tested with egg-white in the same way. The results obtained are set forth in Table I. It will be seen from this table that at the end of 9 months the "resistant" strains had not changed in character, but were just as resistant to lysozyme as they were when first isolated. Development of Highly Resistant Strains of Bacteria. Attempts to develop a highly resistant strain were made with M. lysodeikticus and also with a Streptococcus facalis. The method used was to plant the microbes thickly into broth containing that dilution of egg-white which just completely dissolved the microbes in 24 hours at 450C. (as judged by nakedeye observation). In the case of M. lysodeikticuts this was a 1 in 350,000 dilution and with streptococcus 1 in 4000 dilution of the egg-white. Although

2 LYSOZYME. 215 in these concentrations almost all the bacteria were dissolved, some of them survived and grew out, and these were again subcultured into stronger and stronger concentrations of egg-white in broth. In this way the cultures were made more and more resistant to the bacteriolytic action of egg-white, so that in the case of M. lysodeikticus, the culture which originally was completely dissolved in 3 hours by egg-white 1 in 64,000 was after 91 weeks only dissolved by egg-white 1 in 800, while the streptococcus which originally was completely dissolved by a 1 in 800 dilution of egg-white was after 67 weeks' treatment only partially dissolved by a 1 in 5 dilution. TABLE I.-Illustrating the Permanence of the Resistanzce to Bacteriolysis of.m. lysodeikticus Grown in the Neighbourhood of Tissues and Secretions. Tissue to which strain was made resistant. Lowest concentration of egg-white which in 3 hours at 450C. showed any lysis of a suspension of the bacteria. When first isolated. After 9 months. Tears. 1 in 243, in 243,000 Nasal mucus. 1,, 243,000. I,, 243,000 Liver 1,, 243,000. 1,, 243,000 Kidney 1,, 243,000. 1,, 243,000 Spleen. 1,, 81,000. 1,, 243,000 Stomach. 1,, 243,000. 1,, 243,000 Tonsil. 1,, 243,000. 1,, 243,000 Cartilage. 1,, 243,000 1,, 243,000 Control (non-resistant). 1,, 2,137,000. 1,, 2,137,000 Table II shows the gradual increase in the resistance to lysis by egg-white of cultures treated in the above manner: TABLE II.-Showing the Gradual Increase of Resistance to Lysis of Bacteria Grown in Increasing Concentrations of Egg- White. Number of Lysis shown in 3 hours at by egg-white in dilutions as undergrowth in e 1/00. 1/ 1wieb. ot / / / / /6400., 1/ /12, /16,000. 1/32,000. 1/64,000. M.Slyodeikticus Before +j ± + AC AC AC ± 91 +'. AC... ±. 1/6. 1/10. 1/20. 1/ , 1/180. 1/320. 1/640. 1/800. 1/ / '6400. Streptococcus Before ± AC AC ± ¾ AC ± AC&ACAC AC ACAC ± ± P P.. + = solution; AC = almost complete; * = slight; - = no solution.

3 216 A. FLEMING AND V. D. ALLISON. Bactericidal Power of Egg-White to "Resistant" and "NNon-Resistant" Strains of Streptococcus. It has been shown above that by growing streptococci in egg-white they acquire the power of resisting the bacteriolytic action of this substance. Experiments were done to see how far they had acquired a resistance to the bactericidal power of egg-white. A series of dilutions of egg-white were made, and to 10 c.mm. of each of these dilutions was added 10 c.mm. of a suitably diluted broth culture of the resistant and the non-resistant streptococci. These were incubated for 1 hour at 370 C. and then plated on agar. The results are shown on Table III: TABLE III.-Bactericidal Power of Egg-White to "Resistant" and "Non- Resistant" Strains of Streptococcus. Number of colonies of streptococci. Non-resistant strain. Resistant strain. 1 in in in in in Control (no egg-white) It will be seen from these figures that the resistant strain has acquired a definite well-marked increase of resistance to the bactericidal power of eggwhite. Does a Streptococcus which has been made Resistant to Egg-White offer anly Increased Resistance to the Bactericidal Power of Humant Blood? To settle this question bactericidal experiments were done with resistant and non-resistant strains in slide-cells. Serial dilutions of a broth-culture of both strains were miade, and 2 5 c.mm. of each of these dilutions were added to 50 c.mm. of human defibrinated blood and placed in slide-cells. The results obtained were as shown in Table IV: TABLE IV. Bactericidal Power of Human Blood on Streptococci which had been made Resistant to Egg-White. Number of colonies developing in blood planted with dilutions of broth-culture as under. 1/1. 1/10. 1/100. 1/1000. Streptococcuxs J: Resistant Non-resistant Streptococcus A: Resistant Non-resistant

4 LYSOZYME. 217 The figures given in Table IV show that there is a very definite diminution of the susceptibility to the bactericidal power of human blood of two strains of streptococci, which had been made resistant to the bacteriolytic power of egg-white. Is an Egg-White Resistant Strain of Streptococcus less susceptible to Intracellular Digestion by Leucocytes? It has been shown by many experiments that the bactericidal power of human blood to streptococci is largely a function of leucocytes, and in all probability is mainly dependent on phagocytosis and intracellular digestion of the microbes. Having shown that the strains of streptococci which had been made resistant to egg-white are less susceptible to the bactericidal power of Non-resistant. Egg-resistant. Typical leucocytes after incubation of streptococci with human blood for 3 hours at 370 C. FIG. 1.-Intracellular digestion of non-resistant and egg-resistant faecal streptococci. the blood, it was interesting to see whether they were also less susceptible to intracellular digestion than were the normal cocci. Bacterial suspensions of agar cultures of the normal and resistant strains were made. These were mixed with equal volumes of normal human defibrinated blood and incubated for 3 hours at 370C. in capillary tubes, after which films were made and stained, and the state of the bacteria inside the leucocytes was observed. There was found to be much more intracellular digestion of the normal strain than there was of the resistant strain. It is, of course, impossible to express this numerically, but whereas with the non-resistant strain there were only a few normally staining cocci inside the leucocytes, with the resistant strain there were inany well-stained cocci and comparatively few shadows. Fig. 1 shows typical examples of leucocytes filled with each type of coccus, and the difference in the appearance of the resistant and the non-resistant strains is obvious.

5 218 A. FLEMING AND V. D. ALLISON. This phagocytic experiment was made in 1924, and the cultures were put aside and not opened for two years, when subcultures were made and a similar phagocytic experiment was performed with the same resullt, showing that the streptococci which had been made resistant to the lysozyme of egg-white, had maintained their resistance to intra-cellular digestion by human leucocytes for two years. From these results it would appear that there may be some connection between the bacteriolytic power of egg-white and the power of leucocytes to digest microbes which have been phagocyted; in other words, the intracellular digestion of bacteria by leucocytes may be due to a ferment similar to that which we have described as occurring in many animal secretions and tissues, and which we have called lysozyme. DISCUSSION. The observations detailed above show that by growing bacteria in eggwhite, cultures can be obtained which are resistant not only to the bacteriolytic and bactericidal action of the egg-white, but also to the bactericidal power of human blood and to intracellular digestion by human leucocytes. It has been shown that bacteria can be made resistant to egg-white by growing them in contact with human tissues and secretions, and although the direct experiment has not been made, it is extremely likely that these bacteria grown in contact with human tissues are also resistant to the bactericidal power of the blood and to leucocytes. It is also very probable that the virulence of the microbes could be enhanced in this way, and it was hoped that by growing these bacteria in egg-white they would be rendered virulent, but even at the end of the experiments they were still non-virulent for animals. It must be remembered, however, that the two microbes chosen for the experiment were both very susceptible to the lytic action of the tissues, and were quite avirulent, and it is possible that had microbes been chosen which were slightly pathogenic for animals, similar treatment would have resulted in an increase in their virulence. Murray (1924) has shown that by growing meningococci in contact with leucocytes the virulence of the cocci is increased, and it is probable that this is due to an enhanced resistance to lysozyme as in the experiments detailed above. SUMMARY. Bacteria grown in contact with tissues or secretions are rendered comparatively resistant to their antibacterial action. This resistance to lysozyme action is not specific, in that bacteria made resistant to one tissue are equally resistant to all tissues and secretions. By repeated subculture into stronger and stronger dilutions of egg-white bacteria were made to acquire considerable resistance to the bacteriolytic action of this substance. At the same time, however, they acquired resistance to the bactericidal power of the blood and to intracellular digestion by leucocytes. REFERENCES. FLEMMING, A., AND ALLISON, V. D.-(1922) Proc. R. Soc., B, 94, 142. MURRAY, E. G. D.-(1924) J. Hyg., 22, 175.