BACTERIOPHAGESi THE ABSORPTION OF STAPHYLOCOCCUS

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1 THE ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGESi MORRIS L. RAKIETEN, TONY L. RAKIETEN AND SIMON DOFF Department of Immunology, Yale University School of Medicine and Department of Bacteriology, Long Island College of Medicine, Brooklyn, New York In a series of papers beginning in 1933 Levine and Frisch have affirmed that extracts derived from susceptible cultures of Salmonella block the lytic activity of bacteriophages active against that group of bacteria. These investigations have been confirmed by Burnet (1934), utilizing organisms and phages belonging to the same group as well as several strains of Shigella. These extracts apparently reflect quite accurately those surface antigenic components responsible for bacteriophage absorption on living and heat-killed susceptible cultures. In the course of our studies with staphylococcus bacteriophages we have been struck by the fact that these phages are exceedingly difficult to isolate from sources rich in phages for members of the enteric group. It may very well be that one of the factors contributing to this difficulty is the absorbing quality of such substances as molds, yeasts, phage-resistant staphylococci, and body exudates (Applebaum and MacNeal, 1931; Rakieten, 1932). Other bacteriophages, especially those attacking organisms of the enteric group are not affected at all by the same agents that have a capacity for what may be termed "non-specific absorption" of staphylococcus phages. The absorption of staphylococcus phages by organisms other than staphylococci (to be reported later) has revealed evidence of heterogenetic antigenic relations between staphylococci and otherwise totally unrelated organisms. This report deals with (1) the absorption and inactivation of staphylococcus bacteriophages by extracts prepared from susceptible and resistant strains 1 Presented in part before the Society of American Bacteriologists, New York, December, JOuRNAL OF BACTURIOLOGY, VOL. 32, NO. 5

2 506 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF of staphylococci, and (2) the absorption of similar phages by heatkilled susceptible and resistant cultures belonging to the same group of organisms. 1. STUDIES WITH BACTERIAL EXTRACTS The early reports of Levine and Frisch concerning the preparation of bacterial extracts resulted in our attempt to apply their technique to phage-susceptible strains of staphylococci. All such attempts resulted in preparations weak in phage-inhibiting power, or lacking this property. The method of Burnet (1934) yielded extracts whose phage-inactivating power closely paralleled the susceptibility of the culture to bacteriophagy. TECHNIQUE Bacteria were grown on 1.5 per cent Savita agar2 (Rakieten, 1932) in Roux bottles (150 to 200 cc. per bottle) for 48 hours at 350C. and then washed off with 10 cc. of sterile distilled water. The washings were placed in a 56 to 570C. water-bath for 48 hours. Bacteria which settled rapidly were resuspended by occasional shaking. After 48 hours the autolysates were centrifuged at 3000 r.p.m. for one hour and the clear to slightly opalescent supernatant fluid was removed. Only those extracts which on testing proved to be free of living bacteria were used. For demonstrating the effect of the extracts on several races of bacteriophage serial dilutions of the extracts were prepared, broth being used as the diluent, and to each dilution of extract an equal volume of bacteriophage was added. The bacteriophage was so diluted that at least 200 to 500 plaques could be counted in the final mixture, provided no inhibitory effect had occurred. After the extract-bacteriophage mixtures were incubated for 20 hours at 350C., 0.02 cc. of each mixture was layered over a segment of an agar plate previously seeded with a susceptible culture in such a manner that a homogeneous layer was provided for the demonstration of plaques. After a further incubation for 20 hours at 350C. the plates were counted for plaques. A method which reveals its effectiveness by a diminution in 2Unless otherwise noted media used in this study were Savita broth and agar.

3 ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES 507 plaque count is certainly a better criterion of inactivation than are standards of turbidity in a test-tube. In appearance, secondary cultures may often correspond to controls uninoculated with bacteriophage, and yet contain practically as much phage by plaque count as does a tube showing complete lysis. Such tubes would be read as absence of bacteriophagy. It has been demonstrated by d'herelle (1920), Eliava and Pozerski (1921), Asheshov (1933), and others, that it is possible to isolate from secondary cultures organisms which are completely susceptible. Only when the entire secondary culture is used is resistance to the bacteriophage, responsible for the appearance of the secondary culture, demonstrable consistently. Experimental A. To equal quantities of increasing dilutions of extracts of 8 strains of susceptible staphylococci, an equal and constant amount of bacteriophage was added, the final volume being 2 cc. After incubation for 20 hours at 350C., 0.02 cc. from each tube of each series was layered over homologous cultures previously streaked on segments of agar plates. Plaque counts were made after 20 hours at 350C. The results are summarized in table 1. These results corroborate those of Levine and Frisch and of Burnet: extracts of susceptible cultures possess phage-blocking power. The most potent extracts were derived from those strains which, on the whole, were the most susceptible. In this category we would place Staphylococcus H43, H741, Vnt., and PTR, since they are the ones that are lysed in the shortest period and do not give rise to secondary cultures. The remaining cultures (with the exception of Staph. v), although susceptible, give rise in most instances to secondary cultures after a period of 48 hours. Their phage-inactivating power, or rather the potency of their extracts in this direction is considerably less than is that of the other strains. Staph. v, the only Staphylococcu albums in this series is completely susceptible and never gives rise to secondary culture, even on prolonged incubation. It constitutes an exception in that its extract, which on the basis of strain susceptibility ought to be as powerful in blocking phage M15 as some of the other strains, is relatively weak.

4 508 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF B. All of the extracts in this series were obtained from cultures susceptible to our polyvalent phages, but with the exception of Staphylococcus v, these cultures are resistant to Bacteriophage V. Extract v is derived from strain v, which is susceptible to phage V and to all of our other staphylococcus phages. Hence, with the exception noted above, all of these extracts may be considered to have been derived from cultures resistant to Bacteriophage V. The technique used is that described above, except that the extract-phage mixtures were plated on Staph. v, it being the only EXTRACT (FINAL DILUTION) TABLE 1 Effect of extracts on bacteriophage M15 NUMBER OF PLAQUES IN 0.02 CC. OF BXTPWTPRACTPE MIXTURE, HOMOLOGOUS SUSCEPTIBLE CULTURES Staph. Staph. Staph. Staph.Staph.Staph. aphph-.staph. v Pg. Vnt. H43 H741 Arc. Sch. PTR. 1: : : : : : :160 NT :320 NT NT :640 NT NT NT NT NT 1:1280 NT NT 304 NT NT NT NT NT Bacteriophage control NT (not tested in this dilution). culture susceptible to Bacteriophage V. The results are indicated in table 2. On the basis of these results one may conclude that extracts derived from strains of staphylococci not susceptible to a phage lack on the whole, significant phage-inactivating power. The one susceptible culture, v, does provide an extract that in dilutions as high as 1:20 markedly reduces corpuscular count. Test-tube methods show that this extract, in a dilution of 1: 20, plus phage V and the susceptible culture prevents lysis. In higher dilutions of extract, under the same conditions, lysis in the test-tube takes

5 ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES 509 place. While a slight reduction in plaque count is noted in high concentrations of extracts of some of the non-susceptible cultures, test-tube experiments fail to show any inhibitory action of these extracts on phage V. The diminution may be due to a slight amount of susceptible substance in these cultures, not demonstrable except by experiments involving extracts. C. Extracts have been prepared from three completely resistant staphylococci, i.e., strains resistant to all of our races of polyvalent phages. As far as could be determined, extracts prepared from these three strains failed to inhibit the action of any of these phages when they were subsequently tested either for diminution of plaque count, or lysis in the test-tube. EXTRACT (lnwg DILUTION) TABLE 2 Effect of staphylococcus extracts on monovalent phase V NUMBER OF PLAQUES IN 0.02 CC. OF EXTRACT-PHAGE BMXTURE. SUSCEPTIBLE CULTURE V Staph. Vnt. Staph. Pg. Staph. Bch. Staph. V Staph. H741 1: : : : NT 144 NT 1:80 NT 241 NT 201 NT 1: : Bacteriophage V 236 control D. The results recorded have been obtained many times with these and with 12 other extracts prepared from susceptible cultures of staphylococci. Extracts from susceptible cultures always successfully inactivated bacteriophage, whereas extracts from resistant cultures, with one exception, lacked this property. This unusual culture is a Staphylococcus albus, which is not only completely resistant to all of our races of phages, but within a period of 3 to 8 hours is able to absorb completely the phage without showing lysis of any degree. Filtrates of this culture to which phage has been added and incubated for 8 hours are devoid of lytic action, whereas phage control filtrates contain millions of

6 510 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF corpuscles. An 18-hour culture heated at either 600C. for an hour, or autoclaved at 15 pounds pressure for 15 minutes will absorb phage completely in 24 hours at 350C. Extracts were prepared from this culture several times in 1935 and again recently; none has shown phage-inactivating power. Levine and Frisch (1933) have noted that in one case, at least, an extract from a phage-resistant strain possessed inactivating properties. Burnet (1934) also encountered the same sort of an exceptional strain, an R variant of a Y strain of Shigella paradysenteriae. According to Burnet the phage-inactivating principle of these extracts is precipitated by the addition of the homologous antibacterial serum, and our observations are in accord with this statement, the supernatant fluids in no way blocking the lytic action of bacteriophage. A 1: 20 dilution of rabbit anti-staphylo- TABLE 3 Removal of phage-inactivating power of extract v by anti-serum PLAQUE COUNT ON STAPH. V Serum 255 (1:40) plus extract v (1:2); plus Staph. Bp. V Extract v plus Staph. Bp. V... 1 Control Bp. V coccus v serum was diluted with an equal volume of staphylococcus extract v. The mixture and the controls were held in a 550C. bath for an hour and in the refrigerator overnight, whereupon the finely suspended aggregates were thrown down and the supernatant fluids removed. Equal quantities of staphylococcus phage V were added to the supernatants, incubated for 20 hours at 350C. and finally 0.02 cc. of each was layered over a segment of an agar plate previously treated with susceptible culture v. After incubation at 350C. for 18 hours plaque counts were made. The results are recorded in table 3. Similar results may be obtained with other extracts and other sera providing the latter contain agglutinins and precipitins for the organisms providing the extracts. Once the bacteriophage has been inactivated by an extract it

7 ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES 511 has not been possible by the dilution method to show any increase in corpuscular count. In this respect inactivation by extracts differs from neutralization by anti-phagic serum (Andrews and Elford, 1933). All of the extracts are deprived of phage-inactivating ability by filtration through Chamberland L3, and L5 candles, Berkefeld V, and Seitz filters, but potency is not diminished by heating for an hour at temperatures from 60 to 100'C. Activity is only slightly lessened when the extract is autoclaved at 15 pounds for 15 minutes. 2. ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES BY HEAT- KILLED CULTURES OF STAPHYLOCOCCI It has been demonstrated that heat-killed susceptible cultures will absorb bacteriophage, (Da Costa Cruz, 1922; Jaumain and Meuleman, 1922; Bail and Okuda, 1923; Prausnitz and Firle, 1924; Burnet, 1930; Levine and Frisch, 1934). It is also agreed that with few exceptions lyso-resistant strains (secondary cultures) do not bind homologous phage (d'herelle, 1921; Jaumain and Meuleman, 1922). Our interest in this problem aside from showing that staphylococcus phages behave as do those active with other bacterial species, centered on the question as to whether bacteriophage absorbed to dead cells may under suitable conditions be liberated and proliferate at the expense of living susceptible bacteria. If this could be demonstrated experimentally one might conclude that dead bacteria may serve as carriers of phage and that outside of the test-tube this union might, under certain conditions, be broken with the subsequent liberation of the active lytic agent. Experimental evidence regarding the absorption of staphylococcus phage by heat-killed susceptible cultures is easily obtainable especially if the tests are made at 350C. It appears that the most susceptible of our strains, when killed at 600C. may even absorb phage at ice-box temperature. Strains that are only partially susceptible under no circumstances completely absorb phage. The absorption is not entirely irreversible, for active corpuscles may be demonstrated if susceptible living cells are

8 512 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF added to the tubes containing heat-killed culture-absorbed phage mixtures. When the absorption is carried out at 200C. no plaques are demonstrable; yet complete lysis of susceptible cultures may occur when they are added, and with this there is a multiplication of phage corpuscles. EXPERIMENTAL A. Absorption of phage M16 by heat-killed cultures. Nine strains of staphylococci, which, with one exception (strain Gre.), are completely lysable by bacteriophage M16 were tested for their ability to absorb bacteriophage after being heated at 600, TABLE 4 Absorption of phase M16 by heat-killed (623C. for an hour) staphylococci INCUBATED ICE BOX CltJULTURE 35OC., 22 (4C., 22 PLAQUE COUNT LAQUE COUNT Staphylococcus aureus Sch Staphylococcus aureus Arc Staphylococcus aureus PTR Staphylococcus aureus Mtg Staphylococcus aureus Pg Staphylococcus aureus H Staphylococcus aureus H Staphylococcus albus v Staphylococcus albus Gre Control Bp. (Staph. aur. Vnt. used as susceptible strain) and 1000C. for an hour, and after being autoclaved at 15 pounds for 15 minutes. Eighteen-hour broth cultures, after being heated at 620C. for an hour were centrifuged, the supernatant fluids removed, and the sedimented bacteria resuspended in 5 cc. of fresh broth. The supernatants were devoid of phagebinding power. To 5 cc. of broth, 2 cc. of the bacterial suspension and 1 cc. of bacteriophage were added, the bacteriophage being so diluted that 0.02 cc. would yield approximately 500 plaques providing no absorption had taken place. The mixtures were placed at 350C. and in the ice box for 22 hours, when 0.02 cc. was layered over a segment of an agar plate previously seeded with susceptible

9 ABSORPTION OF STAPHYLOCOCCTUS BACTERIOPHAGES 513 culture. The results of such an experiment are indicated in table 4. It is clear that susceptible cultures of staphylococci heated for one hour at 620C. are capable of absorbing phage, especially when tests are made at incubator temperatures, although there is also a very marked degree of absorption to all of the totally susceptible cultures even at temperatures as low as 4VC. Staphylococcus Gre., an albus variety, is but incompletely lysed by phage M16, and shows no bacteriophage-binding power at ice-box temperature. Similar results were obtained with the susceptible cultures when they had been autoclaved and were tested for absorption. These results with our autoclaved cultures are not in accordance with those of Prausnitz and Firle (1924) who reported that phagesusceptible cultures lose their ability to absorb phage when heated to 1000C. or above. It is interesting to recall at this point that extracts made from some of the above strains also retained their phage-inactivating power, when autoclaved for the same period, and also to note, as described in a preceding section, that a living culture of Staphylococcus albus (Gec.) may absorb phage without undergoing lysis. This strain when autoclaved also absorbs phage, but an extract of this culture has no inactivating power. Having demonstrated that heat-killed susceptible cultures absorb phage, attempts were made to ascertain whether such absorbed phage could be freed and bring about bacteriophagy, as evidenced by increase in plaque count, or lysis in the test-tube. All experiments to free phage from heat-killed susceptible culture, by such means as plasmolysis, or plasmoptysis failed. However, it is interesting to note that bacteriophage controls were undiminished in titer for as long as 11 days in distilled water, or hypertonic or hypotonic salt solution. The presence of free lytic principle could be demonstrated by the addition of a small amount of living susceptible culture to the tubes containing the absorbed phage. This resulted not only in the appearance of plaques but also in some instances in complete lysis in the test-tube. Experiment. An 18-hour broth culture (10 cc.) of Staph. Vnt. was killed at 62 C. for one hour. After centrifugation the super-

10 514 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF natant was removed and the bacteria resuspended in one-half the original volume of fresh broth. Of this suspension, 0.25 cc. plus -an equal volume of bacteriophage were added to 5 cc. of broth and incubated for 20 hours at 350C. A test for absorption was then made by overlaying 0.02 cc. on a susceptible culture. To the absorbed phage-culture mixture was added 0.05 cc. of an 18-hour culture of Staph. Vnt. (susceptible). The tubes with controls were then incubated for 20 hours at 35WC., being observed at frequent intervals for macroscopic evidence of lysis. They were then filtered through L5 Chamberland candles and the fil- Test Staph. Vnt. (heat-killed) 0.25 cc. plus 0.25 cc. of Bp. M16. Incubated 350C., 20 hours. Plaque test reveals no plaques cc. of Staph. Vnt. added, incubated 350C., 20 hours. Test-tube shows no evidence of lysis. Filtrate (0.02 cc.) overlayed on Staph. Vnt. shows after incubation 400 plaques. Control 2 Staph. Vnt. (heat-killed) 0.25 cc. incubated 350C., 20 hours. Plaque test reveals no plaques. Staph. Vnt. added, incubated 350C., 20 hours. Test-tube shows no evidence of lysis. Plaque test negative TABLE 5 Control 1 Bp. M16, 0.25 cc. incubated 350C., 20 hours. Plaque test reveals in 0.02 cc. approximately 400 plaques. Staph. Vnt. added, incubated 350C., 20 hours. Test-tube shows complete lysis cc. of filtrate shows after incubation confluent plaques. Control cc. of Staph. Vnt. added to broth, incubated 350C., 20 hours. Filtrate shows no evidence of plaques. trates tested for ability to produce plaques. The method used and the results obtained are indicated in table 5. When experiments of similar nature were repeated at 20'C. absorption of phage could also be demonstrated as indicated above, but when living culture was added complete lysis generally occurred in the test tube. The results of these and many other experiments of like nature show that bacteriophage absorbed upon heat-killed susceptible culture may under the influence of added living cells be freed. However, it is also possible that a very few corpuscles may have escaped absorption, and on the

11 ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES 515 addition of living cells proliferate, the result being the demonstration of plaques or lysis in the test tube. Though we have not been able to demonstrate it, it appears that test-tube evidence of lysis, under these conditions, depends on the rate of cell growth. At 200C., the living staphylococci develop less rapidly than at 350C. and the balance between cell multiplication and bacteriophage invasion by the freed phage is such that complete lysis takes place. Whereas at 350C., while bacteriophagy does take place, as is evidenced by plaque demonstration, failure to clear in a broth tube culture is due to the higher rate of growth under these conditions. The bacteriophage development lags behind bacterial proliferation so that the end result is a tube that contains bacteriophage, although macroscopically it is similar to the bacterial control. Only plaque demonstration will show that bacteriophagy has occurred, under these conditions. It is also conceivable that at 350C. the bacteriophage freed from the heat-killed culture may lack some of the types originally present in the unabsorbed phage, the result being failure of the tube to clear completely. A plaque method allows any and all types of phages to manifest themselves. Furthermore, if one or more types of the original phage are lacking the secondary culture which develops is always susceptible to the original complete phage. This may well represent a source of error in interpreting what a secondary culture is in terms of susceptibility or resistance to lysis. Experiments were so conducted that although absorption of bacteriophage took place at 350C., living culture was added and the tube incubated at 200C. In these instances practically complete lysis took place in the test tube. Whereas if the absorption was carried out at 200C. and the tube with added living culture incubated at 350C., bacteriophagy was demonstrable only by the plaque method. DISCUSSION Working with staphylococci and staphylococcus bacteriophages one can prepare aqueous extracts of bacteria that will inhibit the action of bacteriophage. The ability of a given extract to inactivate a bacteriophage will depend largely on the susceptibility

12 516 M. L. RAKIETEN, T. L. RAKIETEN AND S. DOFF of the culture furnishing the extract. With the cultures that we have examined, the most potent extracts were derived from highly susceptible bacteria. Extracts which act only in high concentrations have originated from cultures which are not completely lysed, reflecting probably a surface antigen, which so far as bacteriophagy is concerned is not homogeneous. That the composition of the surface antigen largely determines susceptibility or resistance to bacteriophage has been expressed by Burnet (1929) and the results with these staphylococcus extracts strengthens this viewpoint. Viewed somewhat differently the relation between bacterial extracts and phage bears out the early claims of d'herelle that the first step in bacteriophagy is an absorption on the surface of the susceptible cell. It is probable that the susceptibility of other cells to other viruses has the same basis. Any change in the make-up of the surface antigen, one not necessarily as striking as the S -- R, will result in a change in susceptibility toward a bacteriophage, and this change is accurately reflected in the capacity of the extract to inactivate phage. A susceptible culture gives rise to an extract that neutralizes bacteriophage, while a secondary culture made by subjecting this susceptible strain to a bacteriophage and therefore resistant to that phage, gives rise to an extract devoid of inactivating power. However, phages capable of lysing that secondary culture are also inhibited by the extract of that culture. Strains of resistant bacteria can be isolated that can absorb phage in both the living and heat-killed states, without showing any evidence of bacteriophagy (such as strain Gec.), yet extracts prepared from this culture fail to inactivate phage. It may be that such uncommon strains of staphylococcus are composed of at least two surface components from the point of view of the bacteriophage, one susceptible, therefore capable of absorbing phage, the other resistant, the final result being a growth of the bacteria with the concomitant disappearance of the bacteriophage. We feel with d'herelle, Asheshov, Burnet and others that the best method for demonstrating bacteriophagy, or the effect of any agent on bacteriophage is one that entails plaque demonstration. The experience of several years with phages confirms the

13 ABSORPTION OF STAPHYLOCOCCUS BACTERIOPHAGES 517 belief that turbidity standards do not necessarily mean phage absorption or destruction, and that the demonstration of lysis against a few isolated colonies from such a culture is indicative that phage has not affected that culture. Asheshov (1933) has pointed out that in any phage-resistant culture (secondary) one can isolate colonies that are susceptible; it is only when the entire secondary culture is used that resistance to bacteriophage is consistently evident. Weak phages or a phage robbed of one or more of its types may never give rise to complete lysis in the test-tube, but with a plaque method macroscopic evidence of bacteriophagy is easily obtainable. Our results with heat-killed cultures lead us to believe that absorption of bacteriophages by such means is not completely irreversible; that while the temperature at which the absorption is made to occur is of little importance, evidence of disunion of bacteriophage is obtained when the living cells are added and the experiment carried out at 20'C. CONCLUSIONS 1. Susceptible cultures of staphylococci furnish extracts which inhibit bacteriophage. 2. There appears to be a relation between the susceptibility of a culture to phage and the ability of an extract of that culture to inactivate bacteriophage. 3. Extracts are heat-stable, being active after autoclaving. 4. Extracts lose their phage-inactivating capacity on filtering through L3, L5 Chamberland candles, Berkefeld and Seitz filters. 5. The phage-inactivating property of an extract is lost on precipitating with homologous anti-bacterial serum. 6. Heat-killed susceptible cultures of staphylococci absorb phage, as do the cultures after being autoclaved. 7. The union between bacteriophage and heat-killed culture under certain conditions appears to be reversible. REFERENCES ANDREWES, C. H., AND ELFORD, W. J Brit. Jour. Exp. Path., 14, 376. APPLEBAUM, M., AND MACNEAL, W. J Jour. Inf. Die., 49, 225. ASHESHOV, I. N., ASHESHOV, I., SARANJAN, K., LAHIRI, M. N., AND CHATTERJI, S. K Ind. Jour. Med. Research, 20, 1101.

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