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On the antibacterial action of cultures of a Penicillium, with special reference to their use in the isolation of B. injluenzae 1929 l Alexander Fleming Fleming, Alexander. 1929. On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to Their Use in the Isolation of B. jnflrunzae. British ]ournal of Experimental Path- ology, Vol. 10, pages 22fS236. WHILE WORKING WITH STAPHYLOCOCCUS formed in the mould culture and variants a number of culture-plates which had diffused into the surround- were set aside on the laboratory bench ing medium. It was found that broth and examined from time to time. In in which the mould had been grown the examinations these plates were nec- at room temperature for one or two essarily exposed to the air and they weeks had acquired marked inhibitory, became contaminated with various mi- bactericidal and bacteriolytic proper- cro-organisms. It was noticed that ties to many of the more common around a large colony of a contam- pathogenic bacteria. inating mould the staphylococcus colonies became transparent and were CHARACTERS OF THE MOULD obviously undergoing lysis. Subcultures of this mould were The colony appears as a white made and experiments conducted with fluffy mass Lvhich rapidly increases a view to ascertaining something of in s&e and after a few davs sporulates, the properties of the bacteriolytic sub- the centre becoming dark green and stance which had evidently been later in old cultures darkens to almost 186 CHEMOTHERAPY black. In four or five days a bright yel- In the rest of this article allusion low colour is produced which diffuses will constantly be made to experiments into the medium. In certain condi- with filtrates of a broth culture of this tions a reddish colour can be observed mould, so for convenience and to in the growth. avoid the repetition of the rather cum- In broth the mould grows on the bersome phrase “mould broth filtrate,” surface as a white fluffy growth the name “penicillin” will be used. changing in a few days to a dark This will denote the filtrate of a broth green felted mass. The broth becomes culture of the particular penicillium bright yellow and this yellow pigment with which we are concerned. is not extracted by CHC&. The reac- tion of the broth becomes markedly METHODSOF EXAMINING CULTURES FOR alkaline, the PH varying from 8.5 to 9. ANTIBACTERIALSUBSTANCES Acid is produced m 3 or 4 days in glucose and saccharose broth. There The simplest method of examining is no acid production in 7 days in for inhibitory power is to cut a fur- lactose, mannite or dulcite broth. row in an agar plate (or a plate of Growth is slow at 37’C. and is most other suitable culture material), and rapid about 2O’C. No growth is ob- fill this in with a mixture of equal parts served under anaerobic conditions. of agar and the broth in which the In its morphology this organism is a mould has grown. When this has solid- penicillium and in all its characters it ified, cultures of various microbes can most closely resembles P. rubrum. be streaked at right angles from the Biourge (1923) states that he has never furrow to the edge of the plate. The found P. rubrum in nature and that it inhibitory substance diffuses very rap- is an “animal de laboratoire.” This idly in the agar, so that in the few penicillium is not uncommon in the hours before the microbes show visible air of the laboratory. growth it has spread out for a centi- metre or more in sufficient concentra- tion to inhibit growth of a sensitive IS THE ANTIBAcTERIAL BODY ELABORATED microbe. On further incubation it will IN CULTURE BY ALL MOULDS? be seen that the proximal portion of A number of other moulds were the culture for perhaps one centimetre grown in broth at room temperature becomes transparent, and on examina- and the culture fluids were tested for tion of this portion of the culture it antibacterial substances at various in- is found that practically all the mi- tervals up to one month. The species crobes are dissolved, indicating that examined were: Eidamia viridiscens, the anti-bacterial substance has con- Botrytis cineria, Aspergillus fumigatus, tinued to diffuse into the agar in suffi- Sporotricbum, Cladosporium, Penicil- cient concentration to induce dissolu- hum, 8 strains. Of these it was found tion of the bacteria. This simple that only one strain of penicillium method therefore suffices to demon- produced any inhibitory substance, strate the bacteria-inhibitory and and that one had exactly the same cul- bacteriolytic properties of the mould tural characters as the original one culture, and also by the extent of the from the contaminated plate. area of inhibition gives some measure It is clear, therefore, that the pro- of the sensitiveness of the particular duction of this antibacterial substance microbe tested. Figure 2 shows the is not common to all moulds or to all degree of inhibition obtained with types of penicillium. various microbes tested in this way. Fleming l The antibacterial action of cultures of a Penicillium 187 l%e inhibitory power can be accurately titrated by making serial dilutions of penicillin in fresh nutrient broth, and then implanting all the tubes with the same vol- ume of a bacterial suspension and incubating them. The in- hibition can then readily be seen by noting the opacity of the broth. For the estimation of the antibacterial power of a mould culture it is unnecessary to filter as the mould grows only slowly at 37’C., and in 24 hours, when the results are read, no growth of mould is perceptible. Staphylococcus is a very suitable microbe on which to test the broth as it is hard , lives well in culture, grows water and weak saline solutions. My rapi J ly, and is very sensitive to pen- colleague, Mr. Ridley, has found that icillin. if penicillin is evaporated at a low The bactericidal power can be temperature to a sticky mass the active tested in the same way except that at principle can be completely extracted intervals measured quantities are ex- by absolute alcohol. It is insoluble in planted so that the number of sur- ether or chloroform. viving microbes can be estimated. Rate of development of inhibitory substance in culture. A 500 cc. Erlen- PROPERTIES OF THE meyer flask containing ZOO cc. of ANTIBACTERIAL SUBSTANCE broth was planted with mould spores and incubated at room temperature Ejfect of heat. Heating for 1 hour (IO’ to 2O’C.). The inhibitory power at 56’ or 8O’C. has no effect on the of the broth to staphylococcus was antibacterial power of penicillin. Boil- tested at intervals, ing for a few minutes hardly affects it. Boiling for 1 hour reduces it to less After Complete inhibition in than one quarter its previous strength 5 days I in 20 dilution. if the fluid is alkaline, but if it is 6 ” 1 in 40 ” neutral or very slightly acid then the 7 99 1 in 200 ” reduction is much less. Autoclaving 8 9, 1 in 500 ” for 20 minutes at IlS’C. practically Grown at 2O’C. the development destroys it, of the active principle is more rapid Effect of filtration. Passage through and a good sample will completely in- a Seitz filter does not diminish the hibit staphylococci in a I in 500 or 1 antibacterial power. This is the best in 800 dilution in 6 or 7 days. As the method of obtaining sterile active culture ages the antibacterial power mould broth, falls and may in 14 days at 2O’C. have Volubility. It is freely soluble in almost disappeared. 188 CHEhtOTHERAPY The antibacterial power of penicil- and non-pathogenic, are inhibited by lin falls when it is kept at room tem- penicillin. The first table shows the in- perature. hibition by the agar plate method and If the reaction of penicillin is al- the second shows the inhibitory power tered from its original pH of 9 to a when diluted in nutrient broth. pH of 6.8 it is much more stable. Certain interesting facts emerge The small drops of bright ellow from these Tables. It is clear that pen- fluid which collect on the sur Y ace of icillin contains a bacteria-inhibitor the mould may have a high antibac- substance which is very active towar Bs terial titre. One specimen of such some microbes, while not affecting fluid completely inhibited the growth others. The members of the coli- of staphylococci in a dilution of 1 in typhoid group are unaffected as are 20,000 while the broth in which the other intestinal bacilli such as I3. mould was growing, tested at the same pyocyaneus [Pseudomonas], B. p-o- time, inhibited staphylococcal growth teus [Proteus] and V. cholerae in 1 in 800. [Vibrio]. Other bacteria which are in- If the mould is grown on solid sensitive to penicillin are the enteroc- medium and the felted mass picked occus, some of the Gram-negative off and extracted in normal salt solu- cocci of the mouth, Friedlander’s tion for 24 hours it is found that the pneumobacillus [ Klebsiella], and B. extract has bacteriolytic properties. infiuenzae (Pfeiff er) [ Hemophilus 1, If this extract is mixed with a thick while the action on B. dysenteriae suspension of staphylococcus suspen- (Flexner) [ Shigella], and B. pseudo- sion and incubated for 2 hours at tuberculosis rodentium is almost neg- 45’C. it will be found that the opacity ligible. The anthrax bacillus is com- of the suspension has markedly dimin- pletely inhibited in a 1 in IO dilution ished and after 24 hours the previously but in this case the inhibitory influence opaque suspension will have become is trifling when compared with the almost clear. effect on the pyogenic cocci. Influence of the medium on the anti- It is on the pyogenic cocci and on bacterial titre of the mould culture. bacilli of the diphtheria group that So far as has been ascertained nutrient the action is most manifest. broth is the most suitable medium for Staphylococci are very sensitive, and the production of penicillin. The ad- the inhibitory effect is practically the dition of glucose or saccharose, which same on all strains, whatever the colour are fermented by the mould with the or type of the staphylococcus. production of a&d, delays or prevents Streptococcus pyogenes is also very the appearance of the antibacterial sensitive. There were small differences substance. Dilution of the broth with in the titre with different strains, but it water delays the formation of the anti- may be said generally that it is slightly bacterial s&stance and diminishes the more sensitive than staphylococcus. concentration which is ultimately Pneumococci are equally sensitive reached. with Streptococcus pyogenes. The green streptococci vary very INHIBITORY POWER OF PENICILLIN considerably, a few strains being al- ON THE GROWTH OF BACTERIA most unaffected while others are as sensitive as S. pyogenes. Gonococci, Tables II and III show the extent meningococci, and some of the Gram- to which various microbes, pathogenic negative cocci found in nasal catar- Flewzing - The antibacterial action of cultures of a Penicillium 189 TALW IL-Znbibitory Power of Penicillin on Various Microbes (Agar Plate Method) Extent of inhibition in mm‘ from penicillin embedded in agar, serum agar, or Type of microbe blood agar plates Experiment 1: S~apbylococcus pyogenes 23 Smeptococcus pyogenes 17 Streptococcus viridans (mouth) 17 Diphtheroid bacillus 27 Sarcina 10 Micrococcus lysodiekhus 6 Micrococcus from air (1) 20 Micrococcus from air (2) 4 B. anhacis ~Bacillusl 0 B. ty@9orz0 [Salmonellal 0 Enterococcus [Streptococcus? 1 0 Experiment 2: Stapl9ylococcus pyogenes 24 Streptococcus pyogenes 30 Sweptococcus viridans (mouth) 25 Pneumococcus 30 Diphtheroid bacillus 35 B. pyocyaneus IPseudomonas 0 B. pneumoniae (Friedlander) [Klebsiellal 0 B. coli [Escherichial 0 B. paratyphosus A LSalmonella1 0 Experiment 3: Staphylococcus pyogenes 16 Gonococcus [Neisserial 16 Meningococcus [Neisserial 17 Experiment 4: Staphylococcus pyogenes 17 Staphylococcus epidermidis 18 Streptococcus pyogenes 15 Streptococcus viridans (faeces) 5 B. dipbtheriae (2 strains) [Corynebacteriuml 14 Diphtheroid bacillus 10 Gram-negative coccus from the mouth (1) 12 Gram-negative coccus from the mouth (2) 0 B. cofi [Escherichial 0 B. influenzae (Pfeiffer) 6 strains [Hemophilusl 0 TABLE III.-MGbitory Power of Penicillin on Difierent Bacteria.’ Dilution of penicillin in broth l/f. l/lo. 1/20. 1/46. 1/80. l/lOO. 1/200. 1/40 Staphylococcus aweus 0 0 0 0 0 0 0 0 ,, epidemidis 0 0 0 0 0 0 0 0 Pneumococcus 0 0 0 0 0 0 0 0 Streptococcus (haemolytic) 0 0 0 0 0 0 0 0 9, viridam (mouth) 0 0 0 0 0 0 9, faecalis ++ ++ ++ ++ ++ B. antbracis [Bacillus1 0 0 + E B. pseudo-tuberculosis rodentium :+ E B. pullorum [Salmonella] : : ++ z E B. dysenteriae [Shigella] ++ ++ B. coli [Ekcherichia] :+ z z B. typhosus [Salmonella] B. pyocyaneus [Pseudomonas] :: z z B. proteus [Proteus] g v. cbolerae [Vibrio] z z :z 1/60. 1/120. 1/300. 1/600 B. dipbthriae (3 strains) [Corynebacterium] 0 * ++ Streptococcus pyogenes (13 strains) 0 0 0 z ,9 0 0 2 ++ ,9 fae&s (i: ZZ) ++ 9, 0 viridans at random from faeces (1 strain) t *, 9, ?? (2 strains) 0 ,. 9, 99 $9 (1 strain) 0 ,, $9 9, M (I strain) ,, 9, 9, 9, (1 strain) :+ 9. ,, 0 at random from mouth (1 strain) 9, ,, ,* ,, (2 strains) 0 ,. 9. *, ,, (1 strain) 0 .LFxEND: 0 z no growth; k = trace of growth; + z poor growth; ++ = normal growth. Fleming l The antibacterial action of cultures of a Penicikm 191 rhal conditions are about as sensitive is necessary to inhibit completely the as are staphylococci. Many of the culture in broth. In the weaker con- Gram-negative cocci found in the centrations it will be seen that at first mouth and throat are, however, quite there is growth of the staphylococci insensitive. and only after some hours are the B. dipbtberiae [ Corynebacterium] cocci killed off. The same thing can is less affected than staphylococcus but be seen if a series of dilutions of pen- is yet completely inhibited by a lYO icillin in broth are heavily infected dilution of a fair sample of penicillin. with staphylococcus and incubated. If It may be noted here that penicillin, the cultures are examined after 4 hours which is strongly inhibitory to many it may be seen that growth has taken bacteria, does not inhibit the growth place apparently equally in all the of the original penicillium which was tubes but when examined after being used in its preparation. incubated overnight, the tubes con- taining penicillin in concentrations THE RATE OF KILLING OF greater than 1 in 300 or 1 in 400 are STAPHYLOCOCCI BY PENICILLIN perfectly clear while the control tube shows a heavy growth. This is a clear Some bactericidal agents like the illustration of the bacteriolytic action hypochlorites are extremely rapid in of penicillin. their action, others like flavine or no- varsenobillon are slow. Experiments TOXICITY OF PENICILLIN were made to find into which category penicillin fell. The toxicity to animals of power- To 1 cc. volumes of dilutions in fully antibacterial mould broth filtrates broth of penicillin were added 10 appears to be very low. Twenty cc. c.mm. volumes of a I in 1000 dilution injected intravenously into a rabbit of a staphylococcus broth culture. The were not more toxic than the same tubes were then incubated at 37’C. quantity of broth. Half a cc. injected and at intervals 10 c.mm. volumes intraperitoneally into a mouse weigh- were removed and plated with the ing about 20 gm. induced no toxic following result: symptoms. Constant irrigation of large infected surfaces in man was not ac- Number of colonies devel- companied by any toxic symptoms, oping after sojourn in while irrigation of the human con- pemcillin in concentrations as under: junctiva every hour for a day had no irritant effect. Control I/80 1/40 1/20 l/l0 In &TO pencillin which completely inhibits the growth of staphylococci Before 27 27 27 27 27 in a dilution of 1 in 600 does not inter- After 2 hours 116 73 Sl 48 23 fere with leucocytic function to a After 4% hours a 13 1 2 5 greater extent than does ordinary After 8 hours w 0 0 0 0 broth. After 12hours m 0 0 0 0 USE OF PENICILLIN IN THE ISOLATION It appears, therefore, that penicillin OF B. lNFLu#?Ni?AE (PFEIFFER) belongs to the group of slow acting AND OTHER ORGANISMS antiseptics, and the staphylococci are only completely killed after an inter- It sometimes happens that in the val of over 4% hours even in a con- human body a pathogenic microbe centration 30 or 40 times stronger than may be difficult to isolate because it 192 CHEMOTHERAPY occurs in association with others which favourable for the growth of B. in- grow more profusely and which mask fluenzae must be used, e.g. boiled it. If in such a case the first microbe blood agar, as by the repression of the is insensitive to penicillin and the ob- pneumococci and the staphylococci scuring microbes are sensitive, then by the symbiotic effect of these, so fa- the use of this substance these latter miliar in cultures of sputum on blood can be inhibited while the former are agar, is lost and if blood agar alone allowed to develop normally. Such an is used the colonies of B. influenzae example occurs in the body, certainly may be so minute as to be easily with I?. in@enzue (Pfeiffer) and prob- missed. ably with Bordet’s whooping-cough From a number of observations bacillus and other organisms. Pfeiffer’s which have been made on sputum bacillus, occurring as it does in the postnasal and throat swabs it seems respiratory tract, is usually associated likely that by the use of penicillin, with streptococci, pneumococci, sta- organisms of the B. influence group phylococci and Gram-negative cocci. will be isolated from a great variety All of these, with the exception of of pathological conditions as well as some of the Gram-negative cocci, are from individuals who are apparently highly sensitive to penicillin and by healthy. the addition of some of this to the medium the can be completely in- DISCUSSION hibited whi Y B. influenzae is unaf- e fected. A definite quantity of the pen- It has been demonstrated that a icillin may be incorporated with the species of penicillium produces in cul- molten culture medium before the ture a very powerful antibacterial sub- plates are made, but an easier and very stance which affects different bacteria satisfactory method is to spread the in different degrees. Speaking gener- infected material, sputum, nasal mucus, ally it may be said that the least sensi- and so forth, on the plate in the usual tive bacteria are the Gram-negative way and then over one half of the bacilli, and the most susceptible are plate spread 2 to 6 drops (according the pyogenic cocci. Inhibitory sub- to potency) of the penicillin. This stances have been described in old small amount of fluid soaks into the cultures of many organisms; generally agar and after cultivation for 24 hours the inhibition is more or less specific it will be found that the half of the to the microbe which has been used plate without the penicillin will show for the culture, and the inhibitory the normal growth while on the peni- substances are seldom strong enough cillin treated half there will be nothing to withstand even slight dilution with but B. influenzae with Gram-negative fresh nutrient material. Penicillin is not cocci and occasionally some other inhibitory to the original penicillium microbe. This makes it infinitely easier used in its preparation. to isolate these penicillin-insensitive Emmerich and other workers have organisms, and repeatedly B. influen- shown that old cultures of B. pyo- zae has been isolated in this way when cyanem acquire a marked bacteriolytic they have not been seen in films of power. The bacteriolytic agent, pvo- sputum and when it has not been cyanase, possesses properties similar possible to detect them in plates not to penicillin in that its heat resistance treated with penicillin. Of course if is the same and it exists in the filtrate this method is adopted then a medium of a fluid culture, It resembles peni- Fleming * The antibacterial action of cultures of a Penicillium 193 cillin also in that it acts only on certain in this paper, and to our mycologist, microbes. It differs however in being Mr. la Touche, for his suggestions as relatively extremely weak in its action to the identity of the penicillium. and in acting on quite different types of bacteria. The bacilli of anthrax, SUMMARY diphtheria, cholera and typhoid are those most sensitive to pyocyanase, 1. A certain type of penicillium while the pyogenic cocci are unaf- produces in culture a powerful anti- fected, but the percentages of pyo- bacterial substance. The antibacterial cyaneus filtrate necessary for the in- power of the culture reaches its maxi- hibition of these organisms was 40, 33, mum in about 7 days at 2O’C. and 40 and 60 respectively (Bocchia, after 10 days diminishes until it has 1909). This degree of inhibition is almost disappeared in 4 weeks. hardly comparable with 0.2% or 2. The best medium found for the less of penicillin which is necessary to production of the antibacterial sub- completely inhibit the pyogenic cocci stance has been ordinary nutrient or the 1o/O necessary for B. dipbtheriae. broth. Penicillin, in regard to infections 3. The active agent is readily fil- with sensitive microbes, appears to terable and the name “penicillin” has have some advantages over the well- been given to filtrates of broth cul- known chemical antiseptics. A good tures of the mould. sample will completely inhibit sta- 4. Penicillin loses most of its power phylococci, Streptococcus pyogenes after 10 to 14 days at room tempera- and pneumococcus in a dilution of 1 ture but can be preserved longer by in 800. It is therefore a more powerful neutralization. inhibitory agent than is carbolic acid 5. The active agent is not de- and it can be applied to an infected stroved by boiling for a few minutes surface undiluted as it is non-irritant but ‘in alkaline solution boiling for 1 and non-toxic. If applied, therefore, on hour markedly reduces the po\ver. a dressing, it will still be effective even Autoclaving for 20 minutes at 115’C. when diluted 800 times which is more practically destrois it. It is sohtblc in than can be said of the chemical an- alcohol but insoluble in ether or chlo- tiseptics in use. Experiments in con- roform. nection with its value in the treatment 6. The action is very marked on of pyogenic infections are in progress. the pyogenic cocci and the diphtheria In addition to its possible use in the group of bacilli. Many bacteria are treatment of bacterial infections pen- quite insensitive, e.g. the coli-tvphoid icillin is certainly useful to the bac- group, the influenza-bacillus -group, teriologist for its power of inhibiting and the cnterococcus. unwanted microbes in bacterial cul- 7. Penicillin is non-toxic to animals tures so that penicillin insensitive in enormous doses and is non-irritant. bacteria can readily be isolated. A It doses (sic) not interfere with leu- notable instance of this is the very cocytic function to a greater degree easy isolation of Pfeiffer’s bacillus of than does ordinary broth. influenza when penicillin is used. 8. It is suggested that it mav be an In conclusion my thanks are due to efficient antiseptic for application to, my colleagues, Mr. Ridley and Mr. or injection into, areas infected with Craddock, for their help in carrying penicillin-sensitive microbes. out some of the experiments described 9. The use of penicillin on culture 194 CHEMOTHERAPY plates renders obvious many bacterial IO. Its value as an aid to the isola- inhibitions which are not very evident tion of B. influenzae has been demon- in ordinary cultures. strated. Comment The history of the development of of penicillin or less, while current com- penicillin is now quite well known. This mercial production gives yields of at paper presents the first scientific obser- least 5000 pg./ml. But because of the high vations of this substance. activity of penicillin against certain bac- Many earlier workers had noted the teria, he was able to study the effects of effects of various microorganisms on these crude broths. He showed that not pathogenic bacteria. Fleming was the first all bacteria were affected equally by pen- to actually develop a clear conception of icillin, indicating a high degree of spe- what this action was due to. It is obvi- cificity in its action. He further described ous that he considers penicillin to be a the bactericidal and bacteriolytic action substance, and although he does not say of the antibiotic and showed that a period this in so many words, he considered it of growth was needed before either ac- to be a chemical compound with a defi- tion was manifest. We know now that nite structure. He presented sufficient this is because penicillin specifically in- basic information so that later workers hibits the synthesis of the cell walls of could develop penicillin into a practical sensitive bacteria. This means that as they substance. continue to grow without synthesizing He noted that this substance was pro- cell walls, they are killed or lysed be- duced only by a specific species of mold. cause they become osmotically fragile. This observation is important because it Fleming perceived only dimly the showed that the action of the mold broth practical applications of penicillin. He filtrate was not due to some nonspecific obviously was aware that such a sub- effect, such as a change of PH during stance would be highly useful if it would growth. kill pathogenic bacteria in humans, but He devised a crude assay for deter- such a hope seemed much in the future mining the potency of the substance. in view of the low broth potencies he With this assay he was able to study the was obtaining. It took the development stability and certain chemical properties of the sulfa drugs (see page 195) and of penicillin. He was able to follow its the impetus of World War II before production in culture. By current stand- the tremendous technological and medi- ards of penicillin production, Fleming’s cal problems could be adequately solved material was of very low potency. His which led to the development of peni- broths probably had around IO pg./ml. cillin as a practical chemotherapeutant.
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