A Comparison o f the Activities Antiseptics on Bacteria and on Leucocytes.

Transcription

1 171 A Comparison o f the Activities Antiseptics on Bacteria and on Leucocytes. By Alexander Fleming, F.R.C.S. (Communicated by Sir Almroth Wright, F.R.S. Received November 21, 1923.) (From the Laboratories of the Institute of Pathology, St. Mary s Hospital.) Many experiments have indicated that the chemical antiseptics in common use, in addition to combining with the bacterial protoplasm and so producing death of the bacterium, will also enter into combination with other albuminous matter and are, in fact, general protoplasmic poisons. The relative toxicities of these antiseptics to bacteria and to the cells of the body have to a certain extent been investigated, for instance, many experiments have been done on their inhibitory action on the phagocytosis of bacteria by leucocytes. It has been shown, however, that apart from phagocytosis the leucocytes have a powerful lethal action on bacteria, and little or no work has been published on the effect of these chemicals on the actual bactericidal power of leucocytes. In this communication, advantage is taken of new methods of technique to compare the toxicity of antiseptics on bacteria and on body cells as exemplified by the leucocytes of the blood, and the results will show clearly that in certain concentrations most of the antiseptics will favour the growth of bacteria in blood, owing to the fact that they destroy the antibacterial power of the leucocytes more readily than they inhibit the growth of bacteria. In the experiments to be described the staphylococcus was employed as the test micro-organism. This is a convenient test organism for several reasons : 1. Because it is a microbe pathogenic to man and is frequently found in superficial infections such as are usually treated with antiseptics. 2. Because it is a bacterium to which there is little or no bactericidal power in the blood fluids. 3. Because under certain conditions (in confined spaces) it will develop in a fluid medium in separate colonies. While the blood fluids have little or no bactericidal power on the staphylococcus it can easily be shown that the whole blood has a powerful destructive action on the cocci. That this bactericidal action is vested in the leucocytes has been shown in a number of ways of which the simplest was by causing VOL. xcvi. b. o

2 172 A. Fleming. A Comparison o f the the leucocytes to emigrate from blood on to the walls of a capillary tube and then testing their bactericidal action. Some blood was taken up into a capillary tube and allowed to stand in the incubator for an hour during which time the leucocytes emigrated from the clot and adhered to the walls of the capillary tube. The clot was then expelled and in its place there was drawn up a weak suspension of staphylococci in serum. The tube was sealed and incubated for 24 hours in a horizontal position, when it was found that there developed only one or two colonies of staphylococci, whereas in the control tube without leucocytes there might be 100 or more colonies.* This method of experimentation has been described by Sir Almroth Wright (1), and he has given to the tube containing leucocytes prepared in this way the name Lined Tube. This will be made use of later in the investigation of the action of antiseptics. The simplest and most dramatic method of demonstrating the antistaphylococcal action of blood is by means of another device of Sir Almroth Wright, which he has called a Slide Cell. This method has been fully described by its author (1), but a brief reference to the technique is necessary here, as the method will be much made use of in the later experiments. The culture cells in this method are made from two microscopic slides separated by means of five narrow strips of vaselined paper arranged at intervals transversely to the long axis of the slides. By means of these strips of paper the space between the two slides is divided into four very thin compartments or cells open at each end, and which will contain rather more than 50 c.mm. of blood. If staphylococci be added to human blood in such quantity that there are in every 50 c.mm. of blood about 100 cocci, and if this mixture be placed in slide cells in 50 c.mm. quantities, it will be found that after incubation only some two or three colonies will develop in each cell. If, however, before incubation, the bactericidal action of the leucocytes is destroyed, say by heating to 48 C., it will be found that all the cocci implanted will grow out. This method, then, furnishes us with a very convenient way of measuring the effect of solutions of chemicals on the bactericidal power of leucocytes. Having thus briefly described the methods to be employed, we can proceed to an investigation of the action of antiseptic solutions. * In these capillary tubes filled with fluid medium staphylococci, if not too thickly planted, will develop as separate, opaque, white, woolly colonies which are quite easy to enumerate.

3 Activities o f Antiseptics on Bacteria and on Leucocytes. 173 Effect of a Chemical Antiseptic on the Bactericidal Power of Leucocytes Isolated from Human Blood. Human serum was thinly implanted with staphylococci. Serial dilutions of Carbolic Acid were made in normal saline solution and to 25 c.mm. of each of these dilutions was added 25 c.mm. of the infected serum. These mixtures were immediately drawn up into Lined Tubes, prepared as described above, and the tubes were sealed and incubated for 24 hours. The colonies developing in the tubes were then counted. Controls were put up in which the antiseptic was replaced by normal salt solution and also in which the antiseptic-serumstaphylococcus mixture was taken up into ordinary capillary tubes instead of lined tubes. The results obtained w*ere as follows : Number of colonies of staphylococci which develop in- Control Serum containing Carbolic Acid in a concentration of no antiseptic. 1/ /6400 1/3200 1/1600 1/800 1/400 1/200 Unlined tube (control) Lined tube From this it will be seen that whereas only four colonies developed in the control Lined tube (where the leucocytes were acting in the absence of any antiseptic) 82 colonies developed in the tube containing carbolic acid in a concentration of 1 in 400. That this was due to the action of the carbolic acid on the leucocytes lining the tube, and not to a stimulating effect of the antiseptic on the growth of the cocci, was shown by the fact that in the unlined tubes the same number of staphylococci (within the limits of experimental error) developed in the tube containing carbolic acid 1 in 400 (132) and in the control without antiseptic (147). We see, therefore, that Carbolic Acid in a concentration of 1 in 400 will almost completely destroy the bactericidal power of human leucocytes, while, at the same time, it has practically no inhibitory action on the growth of staphylococci.

4 1/4 A. Fleming. A Comparison o f the Effect of a very short Exposure to an Antiseptic on the Bactericidal power of Human The preceding experiment showed the effect on the leucocytes of prolonged exposure to Carbolic Acid. The effect of an almost momentary exposure of the leucocytes to antiseptics was investigated in the manner to be described. Lined tubes were made as in the previous experiment; immediately after the clot had been removed from the capillary a volume of the antiseptic solution was sucked up over the leucocytes which were attached to the walls of the pipette, and the antiseptic was immediately followed by several volumes of serum (to wash away any of the antiseptic which might adhere to the walls), and, lastly, a measured volume of serum containing a small number of staphylococci. This measured volume was allowed to remain in the space originally occupied by the blood, that is in the Lined portion of the tube. The tubes were then sealed and incubated horizontally for 24 hours, when they were examined. The results obtained are shown in Table I. T a b l e I. Antiseptic used. Number of colonies developing. Unlined tube (no antiseptic) Innumerable (over 200). Lined tube (no antiseptic)... One. Lined tubes treated with Iodine, 1 in Innumerable. Carbolic Acid, 1 in Picric Acid, 1 in Mercuric Chloride, 1 in Eusol Alcohol... Ether Chloroform... Flavine, 1 in (not so many). These results show that even a momentary application of the antiseptics to the leucocytes is sufficient to destroy their bactericidal power. It might be argued that the washing of the tube with several volumes of serum had mechanically removed the leucocytes, but this could not wholly account for the results, because the control tube, which only grew one colony of staphylococci, had been subjected to the same amount of washing, and because in the case of some of the antiseptics (Iodine, Picric Acid, and Mercuric Chloride), duplicate tubes went through exactly the same procedure, and on microscopic examination were found to have a good lining of leucocytes.

5 Activities o f Antiseptics on Bacteria and on Leucocytes. 175 Effect of Antiseptics on the Antibacterial Lower of Blood. The procedure employed in the investigation of this question was as follows : A weak suspension of a 24-hour broth culture of staphylococci was made and 25 c.mm. of this was added to 1 c.c. of fresh defibrinated human blood. The concentration of the staphylococci aimed at was such that when the blood was thus infected it should contain rather less than 100 cocci in each 25 c.mm. Serial dilutions of the antiseptic were made in normal salt solution, and to 25 c.mm. of each of these dilutions was added 25 c.mm. of the infected blood. These were mixed and introduced into slide cells (described above) which were then sealed with paraffin and incubated at 37 C. After incubation for 1, 2 or more days the number of colonies which had developed in the cells were enumerated. The results obtained are set forth in Table II. One experiment may be cited in detail as explanatory of the general results obtained. Here the antiseptic used was Carbolic Acid and the results obtained are shown graphically in fig. 1. Control (no Carbolic Acid, 1 in antiseptic). 20,000 10,000 5,000 Carbolic Acid, 1 in 2,500 1, Fig. 1.Showing the effect of Carbolic Acid on the Growth of Staphylococci in Blood. (25 c.mm. of infected blood and 25 c.mm. of antiseptic in each cell. 25 c.mm. of infected blood planted on agar gave 56 colonies.) It will be seen that in the control cell (without antiseptic) out of 56 cocci implanted only one colony has developed. With concentrations of Carbolic Acid from 1 in 20,000 to 1 in 2,500 the results were not materially different from

6 Table II.Showing the effect of Antiseptics on the Antibacterial power of defibrinated human blood. Eusol i _ Alkaline hypochlorite Urea Alcohol Acetone Glycerine Hydrogen peroxide Chloroform f Saturated solu-^ ^ tion in y Ether ^normal saline J /80 1/160 1/320 1/640 1/1280 1/2560 1/5120 1/ / /40000 Chloramine T, Carbolic Acid Emetine A. Fler B crq <rs. Antiseptic. No. of cocci implanted into 25 c.mm. blood. Control no antiseptic. Percentage of the implanted cocci which survived and developed into colonies in dilutions of antiseptic in blood as under. 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024

7 1/200 1/400 1/800 1/1000 1/3200 1/6400 1/ / / /320 x 10:i 1/640 x 103 nties of Antiseptics on Bacteria and on c> o o Cb C* Formalin... Iodine... Picric Acid... Potassium Permanganate Zinc Permanganate Sodium Salicylate... Quinine hydrochloride <) l I / / /20000 I & i-h X i 1/80 x 10:i 1/160 x 10 * Novarseno billon Flavine Brilliant Green f t)

8 178 A. Fleming. A Comparison o f the the control. This range of dilution, where the antiseptic solution has no effect either on the bacteria or on the antibacterial power of the blood, might be called the Indifferent Zone A In the cell containing a concentration of the antiseptic of 1 in 1280, there is a great increase in the number of colonies and in the next cell, which contains 1 in 640 Carbolic Acid, it will be seen that every staphylococcus implanted has grown out. Here then we have an Zone, where the antiseptic, far from having any inhibitory action on the bacteria, increases the growth, owing to its interfering with, or completely destroying, the bactericidal power of the blood. Finally, in the cell containing 1 in 320 Carbolic Acid, no colonies develop, and here we may assume that, although the antibacterial power of the blood is destroyed, this concentration of antiseptic is sufficient to completely inhibit the growth of the bacteria. This might be called the Antiseptic Zone. A consideration of Table II will show that with nearly all the antiseptics tested an Antibactericidal Zone is manifest, in which there is a greater development of cocci than there is in the control cells. The width of this zone varies considerably, being very wide in the case of Picric Acid and Emetine and narrow and ill-marked in the case or Mercuric Chloride. Some of the results call for comment. Eusol.This is an antiseptic which has been recommended for intravenous injection in cases of septicaemia (2). It will be seen from the table that equal parts of this antiseptic and blood furnish a good culture medium for staphylococci and that practically all the staphylococci introduced grow out in this mixture. In view of this result, which has been confirmed by many experiments, it seems hardly possible that anything but harm could result from large injections of Eusol into the blood stream. The amount recommended for injection was 150 to 200 c.c., which when diluted with the total volume of blood, say 5,000 c.c., would make the concentration such that it would fall into the Indifferent Zone in which no effect would be produced either on the cocci or on the blood cells. Quinine Hydrochloride.This is another antiseptic which has been used as an intravenous injection with the object of destroying the bacteria in the blood in cases of septicaemia. Here again the amount used, about five grains, when diluted with the blood furnishes a concentration far below that which has any inhibitory action on the bacteria or even on the blood cells. The experiments show clearly, however, that were it possible to inject sufficient to make a concentration in the blood of something like 1 in 1600, the protective mechanism of the blood would be paralysed without the bacteria being inhibited,

9 Activities o f Antiseptics on Bacteria and on Leucocytes. 179 and quite apart from the direct poisoning effects of the quinine, it is difficult to see how there could be any other result than an exacerbation of the infection. Formalin.In the past this has been used as an intravenous injection in the treatment of pulmonary tubercle, but here again it can be seen that with a concentration of 1 in 6400 the bactericidal power of the blood is seriously interfered with, so that the bacteria grow out freely, while in lesser concentration it is without action. Flavine.With this antiseptic no Antibactericidal Zone could be demonstrated by the slide-cell method. Flavine, however, acts very slowly both on bacteria and on blood cells, and it has been shown by Sir Almroth Wright that the lethal action of the blood cells on the bacteria is a rapid one, so that it might well be that, when the mixtures are put up in this way, Flavine does not destroy the leucocytes before they have dealt with the cocci. Browning (3) showed that when the time of action of Flavine was not more than 20 minutes it required a concentration of 1 in 500 to materially reduce the phagocytic power of the leucocytes, but the author (4) subsequently showed that when Flavine was allowed to remain in contact with the leucocytes for 5 hours the phagocytic power of these was seriously interfered with by a concentration of 1 in 500,000. It might, however, be argued that, as a result of these experiments, Flavine is an antiseptic which might be used with advantage as an intravenous injection In septicaemia. A simple experiment will show that this is extremely unlikely. A rabbit weighing 2'5 kilos, was injected with 20 c.c. of 1 in 1,000 Flavine intravenously, and blood was taken before, and again at intervals after, the injection. The blood was defibrinated and to 200 c.mm. of the defibrinated blood was added 10 c.mm. of a weak suspension of staphylococci, after which the infected blood was placed in slide cells in 50 c.mm. quantities and incubated at 37 C. In this way, it could easily be determined whether or not the blood after injection had any inhibitory power on the growth of the cocci. It was found that the blood taken 1 minute after the injection had some inhibitory power on the growth of staphylococci, although the inhibition was by no means complete. Within 3 minutes, however, this inhibitory power was completely lost, so that whatever effect Flavine could have had on the bacteria could not have lasted as long as 3 minutes, and with a slow acting antiseptic such as this, it seems clear that the antibacterial effect of such an intravenous injection would be negligible. These experiments show that there is little hope that any of the antiseptics in common use could be successfully introduced into the blood stream to destroy the circulating bacteria in cases of septicaemia. They have, however, a much

10 180 Activities o f Antiseptics on Bacteria and on Leucocytes. wider bearing, and are directly applicable to tbe question of the use of antiseptics in septic wounds. The antiseptic would behave in the same way to the pus cells, which are merely leucocytes emigrated from the blood stream, as it would to the leucocytes in defibrinated blood or the leucocytes which have emigrated from a blood clot on to the walls of a capillary tube. It may be assumed that any of the commonly used antiseptics, in the strength in which they are used in septic wounds, are capable of destroying bacteria with which they come in contact. In the wound, however, the st antiseptic diminishes, with greater or less rapidity, from its entering into combination with albuminous and cellular matter in the wound, from its being absorbed to the dressings, and probably from many other causes. Sooner or later, therefore, a concentration would be arrived at which would no longer have any inhibitory action on the bacteria, although it would paralyse the activities of the leucocytes which form the main defence in the wound against the spread of infection. If this stage were of long duration it seems certain that definite harm would result, but fortunately, with most of the antiseptics, this stage is only a short one, so that the bacteria have little time to flourish before fresh leucocytes emigrate from the walls of the wound to take the place of those destroyed by the antiseptic. In this connection, it is noteworthy that in the later stages of the late War, when many different antiseptics had had very extensive trial in the treatment of septic wounds, those which enjoyed the greatest popularity were those which lost their potency most rapidly in the wound, namely, the hypochlorites. It was shown by the author (5) that if Eusol or Dakin s fluid were applied to a wound their potency was reduced in less than a quarter of an hour to such an extent that they ceased to have an inhibitory effect either on bacteria or on leucocytes. It is probable that this very rapid diminution in strength, with a corresponding shortening of the time during which the antiseptic was acting in an Antibactericidal concentration, had much to do with their popularity. REFERENCES. (1) Wright, Lancet, vol. I, pp. 365, 417, 473 (1923). (2) Lorrain Smith, Drennan, R ettie & Campbell, British Medical Journal, vol. I I, page 129 (1915). (3) Browning, Gulbransen, Kennaway & Thornton, *British Medical Journal, vol. I, page 73 (1917). (4) Fleming, Lancet, vol. II, page 341 (1917). (5) Fleming, British Journal of Surgery, vol. VII, page 99 (1919).