INHIBITION OF A STAPHYLOCOCCAL BY A SOLUBLE SUBSTANCE PRODUCED BY A NONHEMOLYTIC MICROCOCCUS SPECIES PINGHUI LIU' Kitasato Institute for Infectious Diseases, Tokyo, Japan Received for publication June 2, 1954

Inhibition of the activity of a bacterial toxin usually grew into the line of micrococcus. The by antitoxic immune sera or by chemicals such as picture resembles those in an article published by formalin is commonly observed. The inhibition Elek and Levy (1950). The inhibition of caused by metabolic products of other types of in their cases, however, was caused by specific , however, has never been described. The antihemolytic sera. In order to avoid the confu- present communication describes a preliminary sion with specific antisera which are often called observation of one such phenomenon. "antihemolysin", the soluble substance produced by the micrococcus will be referred to as "hemol- EXPERIMENTAL METHODS AND RESULTS ysin inhibiting substance" (HIS). A colony of a yellow pigment forming, non- Identification of the hemolysin inhibited by hemolytic Micrococcus species (tentatively identi- hemolysin inhibiting substance. It is well known fied as Micrococcus luteus) was found on a human that staphylococci from various sources produce blood close to a hemolytic staphylo- several different types of (Glenny and coccal colony. It was also noted that the zone of Stevens, 1935; Smith and Price, 1938; Williams hemolysis of this (the term and Harper, 1947). It was necessary to identify Micrococcus is not used here for this organism to the type of hemolysin concerned in this phe- avoid confusion with the other) was slightly but nomenon. definitely indented on the side near the colony of The staphylococcus was isolated from an the micrococcus. This suggested that the hemol- urethral culture. It was negative, ysis was inhibited by some soluble substances mannitol negative, produced white pigment, and produced by the nonhemolytic micrococcus. appeared to be a Staphylococcus albus which is Observation of hemolysis on blood agar plate. commonly found on human skin. It produced a Since both organisms started growing at the same definite and wide zone of hemolysis on human, time on this plate, it was understandable that the rabbit, and sheep blood agar plate. When a 24 inhibition of hemolysis was not marked because hour old culture plate was placed in an ice chest once hemolysis occurs, the effect of antihemolysis (4 C), no phenomenon resembling so-called "hot- will never appear. It was decided to see the effect cold" lysis was observed. of this antihemolytic activity by allowing the The alpha-lysin is active against rabbit red cells micrococcus to grow for 24 hours in advance on but not against human cells (Parker, 1924; blood agar plate and then the hemolytic Glenny and Stevens, 1935). The hemolysin con- staphylococcus around it. Figure 1 shows one cerned here was, therefore, not an alpha-lysin. such culture. The horizontal line is the micrococ- It is not a beta-lysin because it did not show so- cus grown for 24 hours at 37 C in advance, and called "hot-cold" lysis (Smith and Price, 1938). the staphylococcus was then streaked vertically The probable identity of the third type of lysin, on both sides of the former. The plate was incu- the gamma-lysin described by Smith and Price bated for another 24 hours. It can be noted that (1938), with the fourth type, the delta-lysin de- the portions of both staphylococcal colonies near scribed by Williams and Harper (1947), was the line of the micrococcus do not exhibit any pointed out by Elek and Levy (195G). The hemolysis although the growth of the organism delta-lysin was studied in detail by Marks and itself was never inhibited. The staphylococcus Vaughan (1950) who stated that "complete lysis 1 Present address: Medical Laboratory, Mercy produced by staphylococcal cultures or extracts Hospital, Cedar Rapids, Iowa. in human blood agar is due to delta-lysin". The 718 19541 INHIBITION OF STAPHYLOCOCCAL HENIOLYSIN7719 hemolysin concerned here was identical with the delta-lysin studied by these workers in its activity on various types of red cells, optimal conditions for its production, the length of time in which the hemolysin production reached its peak, non- filtrability through Seitz filter, and inhibition of hemolytic activity by various types of normal animal sera. The amount of normal human serum necessary to inhibit one unit of this hemolysin was also equal to that stated by Marks and Vaughan (1950), i.e., 0.05 ml inhibiting one hemolytic unit. It was, therefore, identified as delta-lysin. Several strains of coagulase positive were also examined, and their hemolysis was inhibited by this micrococcus on human blood agar plate but not on rabbit blood agar. This observation further supports the Figure 1. Inhibition of the hemolysis of a conclusion that the hemolysin involved was a Staphylococcus albus on human blood agar plate delta-lysin because most of these strains produced by a nonhemolytic yellow pigment forming micro- both alpha and delta-lysins and the alpha-lysin is coccus species. The horizontal line is the micro- not active on human cells. The hemolysis on coccus grown for 24 hours at 37 C in advance, and human blood produced by these coagulase posi- the staphylococcus was then streaked vertically tive strains is, therefore, caused solely by the and separately on both sides of the micrococcus delta-lysin while that on rabbit blood agar was colony. The plate was incubated for another 24 caused by both types of hemolysins. hours. Note the absence of hemolysis of staphylo- Elek and Levy (1950) postulated the existence coccal colony near the line of the micrococcus. of another lysin designated as epsilon by them, but since these workers used only rabbit and of this technique to the old one has been con- sheep cells and not human cells, it was impossible firmed in the present study. It is to grow the to compare the results obtained here with their staphylococcus on the surface of a nutrient agar observations. plate covered by cellophane. The nutrition from Distribution of hemolysin inhibiting substance the agar will pass through the cellophane to among yellow pigment forming Micrococcus species. support the growth of the organism while the Five strains of similar yellow pigment forming hemolysin does not diffuse through this substance. Micrococcus species were isolated from clinical A high concentration of hemolysin can thus be materials and tested for hemolysin inhibiting obtained by washing off the growth with sterile substance activity. They all exhibited similar saline and centrifuging the suspension. No sig- inhibitory effect on delta-lysin on human blood nificant difference in the titer of hemolysin was agar plate. Twenty strains of coagulase negative observed by using various types of nutrient agar, Staphylococcus albus and ten strains of coagulase and the blood agar base (Difco) was used in most positive Staphylococcus aureus were tested and of these experiments. An ordinary wrapping cello- their hemolysis on human blood agar plate was phane was cut to fit the petri dishes, sterilized inhibited by the above mentioned strains of at 15 lb, for 10 minutes with moisture. These Micrococcus species. sterilized sheets were placed aseptically on The phenomenon described here, therefore, is freshly poured, undried agar plates and inocu- with 3 of 18 hour old broth culture of a common lated drops fairly occurrence. of the organism. The inoculum was distributed Production of delta hemolysin. It has been a over the whole surface of the plate by a sterile common practice since Burnet's work (1929) to cotton swab, and the plates were then incubated produce staphylococcal hemolysin in a semisolid with 10 per cent CO2 at 37 C. It is important that agar incubated with high concentration of CO2. the cellophane sheets be free from wrinkles be- A better technique, however, has been described cause this will reduce the area of attachment of by Marks and Vaughan (1950). The superiority the cellophane to the agar and also reduce con- 720 PINGHUI LIU [VOL. 68 siderably the amount of hemolysin harvested when they were mixed because the titer of hemol- from a plate. The hemolysin production usually ysin inhibiting substance was practically the reached the maximum between 24-30 hours of same when the red cell was added immediately incubation, and harvests were made at these time after these substances were mixed (i.e., without intervals. Three ml of saline were used for each 30 minutes' incubation of the mixture at 37 C). plate to wash off the growth, and the suspensions The mechanism of the hemolysin inhibition. were allowed to stand for 1 hour at 4 C before There can be two processes by which a hemolysis centrifugation. may be inhibited. The first is to neutralize the Marks and Vaughan (1950) defined one unit of hemolysin itself before it combines with red cells, delta-lysin as that amount which caused 50 per as is done by antihemolytic sera. The second is to cent lysis of 0.025 ml of 20 per cent human red block the receptors of the hemolysin on the sur- cell suspension. The same amount of red cells was face of red cells. Since the hemolysins are cheinical used, but for the sake of convenience, 0.5 ml of substances with definite structures, the receptors 1 per cent suspension was employed in most of on red cells must be specific for each hemolysin. these experiments. The reading was made after It is well known that various types of red cells one hour incubation at 37 C. Complete hemolysis vary considerably with regards to their suscepti- instead of 50 per cent lysis was taken as one bility to the action of various types of hemolysin. hemolytic unit because 50 per cent lysis was This variation in susceptibility exists even among difficult to evaluate in cases of hemolysin inhibit- different types of staphylococcal hemolysins as ing test by hemolysin inhibiting substance which mentioned above. Since the micrococcus is closely will be described later. The hemolytic unit used related to staphylococcus, it was thought more here was, therefore, twice as large as that em- likely that the inhibition of the hemolysis was due ployed by Marks and Vaughan (1950). The above to the latter process, i.e., the blocking of red cell mentioned technique usually yielded a hemolytic surface by a substance structurally resembling the extract with about 64 to 128 units per ml. No at- delta-lysin and thus preventing the access of the tempt was made to improve the medium to obtain hemolysin to the red cell. An example of the pro- a higher titer of hemolysin because it was not the cedure employed is given in table 1. As will be purpose of this study. seen in the table, the red cell never became resist- Production of "hemolysis inhibiting substance" ant to the action of hemolysin by combining with (HIS). The optimal condition for production of hemolysin inhibiting substance. When the hemol- hemolysin inhibiting substance by the micrococ- vsin was mixed with varying dilutions of hemoly- cus was found to be identical with that for hemo- sin inhibiting substance, its hemolytic activitv lysin production. The hemolysin inhibiting sub- was inhibited by a very high dilution of this sub- stance production was practically nil in broth stance. One ml of the extract produced with the culture throughout the entire period of examina- micrococcus by the above mentioned technique tion which lasted from 12 hours to 5 days. usually yielded from 512 to 1,012 hemolysin Semisolid agar-CO2 technique yielded some inhibiting substance units. An example of the hemolysin inhibiting substance, but the best experimental procedure is given in table 2. yield was obtained on the cellophane plate de- The nature of the hemolysin inhibiting substance. scribed for hemolysin production. The peak of The hemolysin inhibiting substance has many hemolysin inhibiting substance production also characteristics similar to the delta-lysin which it reached maximum around 24 hours of incubation. inhibits. As mentioned before the optimal con- The only difference was that the production of ditions for its production, the length of time in hemolysin inhibiting substance does not require which its production reached peak, nondialyza- a high concentration of CO2 which is essential for bility through cellophane, inhibition of its pro- hemolysin production. One unit of hemolysin duction by normal animal sera, etc., are the char- inhibiting substance was arbitrarily defined as acteristics they have in common. However, many that amount which completely inhibits one unit differences were also observed between the two of delta-hemolysin following 30 minutes' incuba- substances, and these differences are summarized tion of the mixture of both substances at 37 C. in table 3. The combination of the delta-lysin and hemolysin Aside from the hemolytic activity, the hemoly- inhibiting substance seemed to occur immediately sin inhibiting substance differs from the hemoly- 19541 INHIBITION OF STAPHYLOCOCCAL HEMOLYSIN 721 TABLE 1 The effect of the "hemolysin inhibiting substance" (HIS) on human red cells

Tube 1 2 3 4 6 7 8 Hemol- Red Cell dcl 06 5 0.5 C05l0 l Control Saline 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ml HIS 1.0 serially diluted twofold to tube 8 1% Red cell $||0ml0.5 |______O__. ___ _ _ Incubation at 37 C for 1 hour with occasional stirring Centrifuge with 3,000 rpm for 10 minutes Discard the supernatant fluid and wash red cells three times with sterile saline Saline 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.5 ml Shake to resuspend the red cells Hemolysin, lp/ml 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ml Incubation at 37 C for 1 hour with occasional stirring

Result A jA Af A A A Q Numerals in "Result" indicate degrees of hemolysis. A indicates complete hemolysis and Q indicates complete absence of hemolysis. TABLE 2 The effect of the "hemolysin inhibiting substance" (HIS) on the delta-hemolysin

Tube1Tube|1 1 2 3 ....@* @ * 8 | 9 10t° HemolysinControl |ControlRed Cell

Saline 0.5 0.5 0.5 .... 0.5 0.5 0.5 0.5 1.0 ml HIS | 0.5 | serially diluted twofold to tube 10 Hemolysin, 2 ;l/ml 0.5 j 0.5 j 0.5 j .... 0.5 0.5 0.5 j 0.5 j ml Incubation at 37 C for 30 minutes 1%redcell 05 |0.5 |0.5 .... 0.5 |0.5 |0.5 0.5 j0.5ml Incubation at 37 C for 1 hour with occasional stirring Result IQ|QI|I|Q||a|A| Numerals in "Result" indicate degrees of hemolysis. A indicates complete hemolysis and Q indicates complete absence of hemolysis. 1 and a indicate de- grees of hemolysis. sin in that its production does not require a high treatment (Marks and Vaughan, 1950). Marks concentration of C02, and the titer is not signifi- and Vaughan (1950) pointed out that delta-lysin cantly reduced by the Seitz filter which reduces has many characteristics similar to fatty acids. greatly the titer of delta-lysin. The hemolysin The hemolysin inhibiting substance resembles inhibiting activity of the hemolysin inhibiting more closely protein in that its activity is de- substance is completely lost by heating at 100 C stroyed by heat, and the extracts containing this for 10 minutes while the hemolytic activity of the substance exhibit strong biuret reactions. delta-lysin is not completely destroyed bv this It has already been pointed out by Marks and 722 PINGHUI LIU [VOL. 68 TABLE 3 dela-lysin on human red cells and the substance The differences between the delta-lysin and the in normal animal sera which inhibits the hemo- "hemolysin inhibiting substance" (HIS) lytic activity of the delta-lysin are similar in struc- ture with the hemolysin inhibiting substance pro- dela- HIS by the micrococcus. The data presented Hemolysin duced HI above, although limited, seemed to indicate that Hemolysis...... + the hemolysin inhibiting substance is a protein. CO2 required for production. .. Yes No A search was made to find this hemolysin in- Absorbed by Seitz filter ...... Yes No hibiting phenomenon among closely related Activity destroyed by heating organisms. The combinations of organisms em- 100 C 10 minutes...... No Yes ployed included groups A, C, and G hemolytic streptococci with a nonhemolytic , Vaughan (1950) that the hemolytic activity of the hemolytic varieties of fecal streptococci with delta-lysin is inhibited by various animal sera, nonhemolytic type, hemolytic varieties of Escher- and both of the albumin and globulin fractions of ichia coli with a nonhemolytic type. None of these a normal horse serum were capable of neutralizing combinations exhibited a phenomenon similar to this hemolysin. The hemolysin inhibiting sub- that described above on blood agar plate. How- stance may be similar to the protein fractions in ever, only a few strains were used for each of these the animal sera which neutralize the delta-lysin. combinations, and it is possible that the same phenomenon may be found with other combina- DISCUSSION tions of organisms. Observation of the above mentioned phenome- SUMMARY non has never been described, but it is not par- The delta-hemolysin of staphylococci was found ticularly surprising because the Staphylococcus to be inhibited by a soluble substance produced and the Micrococcus are closely related. Produc- by a nonhemolytic yellow pigment forming tions of bacterial exotoxins may be due to the Micrococcu8 species. Theproductionof thishemol- excretion of an incomplete product which is a part ysis inhibiting substance seemed to be quite of a nontoxic respiratory enzyme. In an unfavor- common because all of the five strains of yellow able condition, the organism may not be able to pigment forming Micrococcus species isolated complete the process of enzyme synthesis and the from clinical materials were found to produce this balf products; either the prosthetic group or the substance. protein carriers of the enzyme are excreted into The hemolysin inhibiting substance has no the surrounding medium. The best example is the effect on the human red cells, and its activity is mechanism for toxin production of Corynebacte- exhibited by combining directly with the delta- rium diphtheriae (Pappenheimer and Hendee, hemolysin. This substance seems to be a protein 1947). In the absence of iron which is necessary in nature, and a possible mode of its production for the synthesis of cytochrome b, the protein and activity is discussed. carrier of this enzyme is excreted into the sur- rounding medium. It was postulated by these REFERENCES workers that the toxicity of the protein carrier BURNET, F. M. 1929 The exotoxin of Staphylo- (the toxin) is due to its affinity to some respira- coccus pyogenees aureus. J. Pathol. Bac- tory enzyme in the tissue because the combina- teriol., 32, 717-734. tion of this substance may interfere with some EL3K, S. D., AND Luvy, E. 1950 Distribution normal enzyme synthesizing process. of hemolysins in pathogenic and non-patho- J. Pathol. a related such as genic staphylococci. Bacteriol., In pair of closely organisms 62, 541-554. some of their Staphylococcus and Micrococcu, GLENNY, A. T., AND STBVENS, M. F. 1936 Staph- enzymes may be similar in structure, and when ylococcus toxins and antitoxin. J. Pathol. their half products are mixed, it is possible that Bacteriol., 40, 201-210. they will combine readily. The combination thus MARKS, J., AND VAUGHAN, A. C. T. 1950 Staph- results in the inhibition of the hemolytic activity. ylococcal delta-hemolysin. J. Pathol. Bac- It is also possible that the receptors for the teriol., 62, 597-615. 1954] INHIBITION OF STAPHYLOCOCCAL HEMOLYSIN 723

PAPPENHEIMER, A. M., JR., AND HENDEE, E. D. SMITH, M. L., AND PRICE, S. A. 1938 I. Staphylo- 1947 Diphtherial toxin. IV. The iron en- coccus beta-hemolysin. II. Staphylococcus zymes of Corynebacterium diphtheriae and gamma-hemolysin. J. Pathol. Bacteriol., 47, their possible relation to diphtheria toxin. 361-393. J. Biol. Chem., 171, 701-713. WILLIAMS, R. E. O., AND HARPER, G. J. 1947 PARKER, J. T. 1924 The production of an exo- Staphylococcal hemolysin on sheep blood agar toxin by certain strains of Staphylococcus with evidence for a fourth hemolysin. J. aureus. J. Exptl. Med., 40, 761-772. Pathol. Bacteriol., 59, 69-78.