HEMOLYSIN-INHIBITING SUBSTANCE IN STAPHYLOCOCCUS AUREUS STRAINS

Transcription

1 HEMOLYSIN-INHIBITING SUBSTANCE IN STAPHYLOCOCCUS AUREUS STRAINS E. T. CETIN Departnment of lliicrobiology and Contagious Diseases, Facuilty of Medicine, Istanbul University, Istanbul, T'urkey Received for publication 25 March 1963 ABSTRACT QETIN, E. T. (Istanbul University, Istanbul, Turkey). Hemolysin-inhibiting substance in Staphylococcus aureus strains. J. Bacteriol. 86: Of 144 Staphylococcus aureus strains isolated from pathological specimens, 3.4% did not cause hemolysis on sheep blood agar; the remainder produced hemolytic and semihemolytic zones, most of which were surrounded by a dense red band. Most of the strains causing pronounced hemolysis and a large dense red band on sheep blood agar also produced a dense red band on human blood agar after incubation at 37 C for about 1 week. In the dense red band on human blood agar, circles of hemolysis were observed when petri dishes were kept at room temperature for approximately 1 more week. The dense red band inhibited delta hemolysis of some of the S. aureus strains growing nearby. Certain strains failing to produce the dense red band on human blood agar inhibited delta hemolysis of other strains grown near them. A hemolysis-inhibiting substance (HIS) was produced in broth and agar media, and could be extracted from agar cultures. HIS was stable for 1 hr at 56 C and for 15 min at 100 C. It lost 75% of its effect when heated for 30 min at 100 C and became ineffective when heated at the same temperature for 45 min. Conditioned hemolysis occurred when some saprophytic gram-positive bacteria grew near or within the dense red band. These organisms also produced conditioned hemolysis without the presence of a visible dense red band, when growing near the strains inhibiting delta hemolysis. When such strains were grown on human blood agar, a conditioned hemolysis also occurred after the border of the medium was flamed. During investigations (by measuring pigment production, plasmacoagulase, hemolysis, and mannitol fermentation activities) of different hemolysins of Staphylococcus aureus strains, it was observed that some strains formed a dense red band around colonies grown on human blood agar plates. Weld (1962) reported a similar observation. In earlier experiments here, some of the strains had been noted to produce a dense red region on sheep blood agar, and it had been reported that strains of S. aureus that produced beta hemolysin developed a curious appearance on sheep blood agar. But explanations of this phenomenon were not quite satisfactory, and the role of various factors was suggested by various authors. It was observed that the dense red band formed by some strains on human blood agar was able to inhibit hemolysis of certain S. aureus strains that lyse human blood. Liu (1954) found a substance in Micrococcus luteus strains that in a similar manner inhibited the delta hemolysin of S. aureus; he termed it HIS (hemolysin-inhibiting substance). In this article, some results of my investigations are presented. MATERIALS ANI) METHODS The hemolyzing potentialities of 144 S. aureus strains, isolated from pathological specimens, were tested on blood agar plates prepared with blood or washed erythrocytes of human, sheep, and rabbit. To prepare these plates, an agar medium containing 1, 1.5, or 2% Difco agar in beef infusion broth was dispensed in 16-ml volumes to tubes. These media, when needed, were melted at 100 C and transferred to a water bath at 52 C. Portions (0.75 ml) of defibrinated blood or washed erythrocytes of human, sheep, or rabbit were put into petri dishes (9 cm in diameter). The melted agar content of each tube was then added to the dish, which was moved circularly and linearly to mix the blood and agar homogeneously. For other tests, blood agar plates were incubated at 37 C, then put at room 407

2 ... ~~~~~~~~~~~~~~~~~~~~~~~~~~~...j..... temperature (24, 28 C) or in a refrigerator at 2 C, or both. Blood agar plates prepared with washed rabbit erythrocytes became completely hemolyzed if kept for more than 48 hr at 37 C. The color of the plates prepared with washed sheep or human erythrocytes turned to dark brown. As the color of those prepared with human and sheelp 1was blood not altered during long incubation periods, such )lates were used in most of the tests. Inoculations on blood agar plates were made from agar or broth cultures of strains grown for 24 to 48 hr. This inoculation was done with a loop, so as to form a round shape 0.5 cm in diameter. RESULTS Elffect of S. aureais strains on sheep blood agar. It was observed that, of 144 S. aureus strains, five did not produce hemolysis on sheep blood agar; in the remainder, completely hemolyzed or semihemolyzed zones of differing diameters were produced around the growth regions when the plates were incubated for 9 days at 37 C, or for 1 day at 37 C, and then for 8 days at 2 C in a refrigerator. MToreover, dense red bands were produced around the growth regions of some strains. From the strains that produced considerable zones of hemolysis or wide dense bands, 30 strains were selected and used in the following experiments. As no difference was observed in blood agar media containing 1, 1.5, or 2% agar, 2% agar medium was used in the experiments. 'B... f 22 ^ : :. 408 CETIN FIG. 1. Dense eled band around growth region of Staphylococcus aureus on human blood agar. J. BACTERIOL. Effect of 30 S. aureus strains on sheep, human, and rabbit blood agar. The 30 strains of S. aureus were inoculated in streaks with a platinum wire onto sheep, human, and rabbit blood agar plates. Inoculated human blood agar plates were kept for 9 days at 37 C, or for 4 days at 37 C and 8 days at room temperature or in a refrigerator. Since rabbit blood agar plates hemolyzed if kept for a long period, they were incubated for 2 days at 37 C and then kept at room temperature or in a refrigerator for 4 days. Sheep blood agar plates were incubated for 9 days at 37 C, or for 1 to 5 days at 37 C and then for 8 days at 2 C in a refrigerator. A dense red band was formed by 15 strains on sheep blood agar, and by 18 strains on human blood agar (Fig. 1). All but four of the strains producing the dense red band on sheep blood agar gave rise to the same phenomenon on human blood agar. The dense red band was obviously present on sheep blood agar even after l day, and it progressively increased in diameter. The band on human blood agar became visible after the third day and reached 5 to 15 mm in diameter on the twelfth day. Eight strains of S. aureus (strains 465 and 484, producing the widest dense red band on sheep blood agar; 376 and 436, producing a wide dense red band on human blood agar; 400 and 402, producing hemolysis on human blood agar; and 419 and 422, producing hemolysis in the form of concentric circles in the dense red band on sheep blood agar) were chosen for further experiments, described below. Table 1 shows the properties of these strains. Effects of S. aureus strains on sheep blood agar. S. aureus strains 465 and 484 produced a large dense red band on sheep blood agar. The boundary of the dense red band was quite obvious, and the band, on the whole, was deep red in color. The diameter increased progressively when plates were kept in an incubator for long periods (Table 2). When sheep blood agar plates inoculated with strains 465 and 484 were incubated for 1 day at 37 C and then kept in a refrigerator at 2 C, the results were as follows. A 5-mm dense red band formed on day 1, when the plates were being incubated at 37 C, and remained the same size, under refrigeration at 2 C, on days 2 and 3. On days 5, 7, and 9 (plates refrigerated at 2 C), there appeared 0.5 mm of hemolysis, a 4-mm dense red band, and 0.5 mm of semihemolysis.

3 VOL. 86, 1963 HEMOLYSIN INHIBITING SUBSTANCE IN S. AUREUS 409 TABLE 1. Properties of Staphylococcus aureus strains when incubated at 37 C for 2 days Property Results with strain Hemolysis of sheep blood Hemolysis of human blood _ Hemolysis of rabbit blood Plasmacoagulase ± + Pigment production Mannitol fermentation...-i TABLE 2. Effects of Staphylococcus aureus strains 465 and 484 on sheep blood agar incubated for 10 days at 37 C Day Diameter* mm * Of dense red band formed. On day 11 (plates incubated at 37 C), there appeared 0.5 mm of hemolysis, 4 mm of semihemolysis, three hemolytic circles in a dense red band 3 mm in diameter, and 0.5 mm of semihemolysis. Effects of S. aureus strains on human blood agar. S. aureus strain 436, when incubated for 1 week at 37 C, produced a dense red band 10 mm in diameter around the growth region. Nothing was observed, meanwhile, on a plate inoculated with strain 484. When the plate inoculated with strain 436 was kept for 1 more week at room temperature, three hemolytic circles were produced 5, 7, and 10 mm from the growth region (Fig. 2). Production of the substance causing the dense red band, from filtrates of cultures of S. aureus on human blood agar, nutrient agar, and broth. Human blood agar medium extracts of the S. aureus cultures were prepared as follows. A 24-hr agar culture of the strain was inoculated onto blood agar in a petri dish at six equidistant intervals (each 1.5 cm from the edge) and also exactly in the center of the plate, so as to form a circle 0.5 cm in diameter. When the plates were incubated for 4 days and then kept at room temperature, it was observed that the dense red bands around the growth were touching each other and that no void region remained. The bacterial growth regions in human blood agar were cut off with a sterile scalpel, and the remainder was cut into small pieces. The contents of five petri dishes thus prepared were collected in a flask, and 40 ml of distilled water were added. The flask was left overnight in a refrigerator; then the supernatant fluid was filtered through a Seitz pad. The same manipulations were performed on agar plates (without blood), and an extract was obtained and filtered in the same way. Human blood agar plates were cut off in the center in a circular shape 1 cm in diameter, and FIG. 2. Dense red band and hemolytic circles around growth region of Staphylococcus aureus on human blood agar. IM-

4 410 CETIN J. BACTERIOL. FIG. 3. Inhibition of delta hemolysis of Staphylococcus aureus by the dense red band on human blood agar. Colony a: S. aureus strain producing the dense red band; colonies b, c, d: three S. aureus strains causing delta hemolysis. FIG. 4. Inhibition of delta hemolysis by a Staphylococcus aureus strain not producing a dense red band. Colony a: S. aureus strain not producing a dense red band. Colony b: S. aureus strain capable of producing delta hemolysis but only at 2.5 cm from colony a. Colony c: saprophytic gram-positive coccus producing conditioned hemolysis in the region inhibiting delta hemolysis. the fractions of medium were taken off with a sterile scalpel. The filtrates were poured into these holes, and the plates were kept for 4 days at 37 C and for 4 more days at room temperature. At this time, a dense red band 3 mm in width was observed around each hole. After Seitz filtration of a 48-hr broth culture of the S. aureus strain, the same procedure was carried out with this filtrate. A dense red band 2 mm in width occurred on human blood agar. These experiments clearly showed that the substance was produced in blood agar as well as in agar and broth cultures, and that it was filterable through Seitz pads. As the filtrate derived from agar medium behaved in the same way as that from blood agar, the following experiments were carried out with the agar medium extract. Inhibiting effect (on human blood agar) of S. aureus strains on hemolysis of other S. aureus strains. It was observed that, after production of the dense red band around a S. aureus colony on human blood agar, an inhibiting effect on the hemolysis of other S. aureus strains (400 and 402) was present when these strains were inoculated on the same plate, near the dense red band. When the S. aureus strain was inoculated outside of but closely proximal to the dense red band, a hemolytic zone was produced around itself, but it stopped at the margin of the dense red band. If the S. aureus strain was inoculated within the dense red band, an arciform hemolytic area, the center of which was the strain itself, was observed outside the dense red band (Fig. 3). On the other hand, some S. aureus strains did not produce a dense red band when inoculated on human blood agar, incubated at 37 C for 6 days, and then kept at room temperature (Fig. 4, a). It was noted that a delta-hemolytic S. aureus strain (inoculated in a straight line 0.5 to 2 cm from the growth region of the first strain) could not produce delta hemolysin within 2.5 cm of the growth region of the first strain, but could produce a very narrow hemolytic zone at this distance (Fig. 4, b). Hemolysis-inhibiting substance in filtrates of agar culture extract and broth culture. Human blood agar plates were used for production of the dense red band by filtrates of agar culture extract and broth culture of S. aureus. A square-shaped surface area (1 cm2) of the medium was cut and taken off with a sterile scalpel. The filtrates (the

5 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... VOL. 86, 1963 HEMOLYSIN-INHIBITING SUBSTANCE IN S. A UREUS 411 production of which was detailed above) were dropped with a pipette into the square hole. After 6 days of incubation at 37 C and storage at I.. room temperature, dense red bands were produced around the square holes (Fig. 5). Delta lysin was then dropped, by a thin pipette, within and outside the band. The dense red band inhibited delta hemolysis in the way explained in 0 the preceding section (Fig. 5). This fact evidenced the presence of a substance, filterable and ; inhibiting delta hemolysis, around the growth region of the S. aureus. Conditioned hemolysis on human blood agar. After inoculation of S. aureus strains 376 and 419 in the center of separate I)etri dishes containing... human blood agar, the plates were incubated at.-o C for 6 days. A dense red band was produced (Fig. 6, a). A colony of saprophytic gram-positive _. bacteria (cocci, bacilli, and corynebacteria),. growing 7 cm away from the growth region of the S. aureus that produced a dense red band, gave rise to a narrow zone of hemolysis around FIG. 6. Conditioned hemolysis on human blood its colony and resulted in a conditioned hemoly- agar. Colony a: Staphylococcus aureus strain prosis in a crescent or lens shape within the dense ducing the dense red band. Colony b: saprophytic gram-positive coccus growing outside the dense red red band (Fig. 6,b).Inthenex5days,th band and causing a conditioned hemolysis in a lens shape. Colony c: saprophytic gram-positive coccus growing within the dense red band and causing a.:e: ::.,conditioned hemolysis. molysis increased in width. Another colony of _EOc_ saprophytic bacteria growing in the dense red band also formed conditioned hemolysis (Fig. 6, c). As explained above, a S. aureus strain that did not produce a dense red band (Fig. 4, a) inhibited delta hemolysis of another S. aureus strain in the region within 2.5 cm of its colony (Fig. 4, b). A saprop)hytic gram-positive coccus p)roduced a conditioned hemolysis in the region where delta - hemolysis was inhibited (Fig. 4, c). Human blood agar inoculated with S. aureus...strain 484 was incubated for 6 days at 37 C;.... neither a hemolytic circle nor a dense red band e;-..,....~,.,...~.., was observed. As a saprophytic colony was noted near the border of a petri dish, that part of the FIG5InhIbItIon deltalysnbyhispro blood agar was cut off; the boundary of the cut FIG. 5. Inhibition of lysin HIS. Production of a dense red band by the filtrate of nutrient region was flamed. On the following day, between agar culture of Staphylococcus aureus dropped into the growth region of strain 484 and the flamed a square hole on human blood agar. Inhibition of part, a hemolysis arch (0.5 mm in width) having delta lysin in three regions around the square dense its concavity toward the growth region was noted red band. 15 mm from the colony. The part included in the

6 412 CETIN concavity of the arch inhibited hemolysis of the other strains. Some properties of HIS. HIS was resistant for 1 hr at 56 C and for 15 min at 100 C. After heating for 30 min at 100 C, it lost 75% of its effect, and its effect was destroyed if heated at the same temperature for 45 min. No change was noted in respect to the HIS effect after 4 weeks of incubation at 37 C. HIS lost two-thirds of its effect when diluted 10-fold and became ineffective when diluted 100-fold. HIS was found not to be dialyzable through cellophane. DISCUSSION During experiments, it was observed that some S. aureus strains formed a dense red band on human blood agar. After incubation at 37 C for a given time, followed by keeping at room temperature, hemolysis circles occurred in that region. Some writers previously observed the dense red band and hemolysis circles on sheep blood agar and reported them as being a result of beta hemolysin (Bigger, Boland, and O'Meara, 1927; Kortenhaus, 1929; Glenny and Stevens, 1935; Flaum and Forssman, 1936; Roy, 1937; Flamm, 1957). It was also observed, during the present experiments, that the dense red band occurring on human blood agar inhibited hemolysis of other S. aureus strains. Moreover, some strains growing on human blood agar inhibited hemolysis of other strains, though they did not produce a visible dense red band. The invisible region underwent conditioned hemolysis under some circumstances, and a circular hemolysis was also produced. For instance, a saprophytic gram-positive bacterium caused conditioned hemolysis. Conditioned hemolysis also occurred if the border of blood agar, in the middle of which a S. aureus strain had been grown, was heated by a thin flame of gas. Some substance occurring during heating could be the cause of such conditioned hemolysis. In the dense red band on human blood agar, a conditioned hemolysis also occurred with some saprophytic gram-positive bacteria growing near that region. The conditioned hemolysis on human blood agar was similar to that occurring on sheep blood agar plates (called beta conditioned hemolysis). J. BACTERiOL. Is it possible to interpret both these phenomena in the same way? Christie and Graydon (1941) reported that all nonhemolytic S. aureus strains hydrolyzing butterfat produced hemolysis on sheep blood agar on which staphylococci producing beta hemolysin had been grown previously. Williams and Harper (1947) called this phenomenon ",B conditioned hemolysis," and concluded that this was the result of delta hemolysis. Beta lysin acts on erythrocytes of calf, sheep, and goat. Some writers reported that beta hemolysin might affect human erythrocytes. Christie and North (1941) claimed that, in the presence of alpha hemolysin, beta hemolysin might lyse human erythrocytes. In spite of these data, there is a unanimous belief that beta lysins do not lyse human erythrocytes. My experience led me to the conclusion that beta lysin by itself or together with alpha lysin could not lyse human erythrocytes. It was reported that delta hemolysin increased the beta conditioned hemolysis on sheep blood agar. It was observed that the region of conditioned hemolysis on human blood agar inhibited the hemolysin effect of S. aureus strains lysing human blood, which effect ought to be delta hemolysin. Thus, the state of things is quite different in human blood agar as compared with sheep blood agar. In the case of human blood agar, delta hemolysis is, on the contrary, inhibited. Munch-Peterson (1954) reported that some diphtheroid bacteria secrete a substance that protects sheep and calf erythrocytes from the effect of beta hemolysin. fiu (1954) showed that some strains of M. luteus inhibited the delta hemolysins of some strains of staphylococci. Furthermore, he reported that HIS was different from delta hemolysin, as it was produced without CO2, did not cause hemolysis, was not adsorbed to asbestos, and was destroyed in 10 min at 100 C. I have not found a paper dealing with a substance in S. aureus strains inhibiting hemolysis of other S. aureus strains. The properties of the substance here extracted from agar medium were quite similar to those of Liu's substance. The substance in question was not dialyzable through cellophane and was rather resistant to heat. On the other hand, this substance was, in some ways,

7 VOL. 86, 1963 HEMOLYSIN-INHIBITING SUBSTANCE IN S. AUREUS 413 similar to the so-called beta lysin obtained from S. aureus strains that produce a dense red band in sheep blood agar. The substance extracted from agar medium and filtered through Seitz pads produced a dense red band in sheep blood agar, the red density of which remained unchanged if kept in a refrigerator or at room temperature. This dense red band inhibited the activity of alpha hemolysin and the production of cyclohemolysis, but conditioned hemolysis resulted when some saprophytic strains were inoculated near it. Such an observation suggested the presence of an inhibiting factor, together with beta hemolysin, in the extract of agar. In fact, this idea has already been put forward by some investigators (Bigger, 1933; Seiffert, 1935; Levine, 1937; Smith and Price, 1938; Elek, 1959). As a result of the present experiments, it was concluded that, in human blood agar, some S. aureus strains produce a substance that inhibits hemolysis of other strains and diffuses into the agar. A dense red band occurs on human blood agar where some of the strains producing a hemolysin-inhibiting substance grow. Moreover, most of these strains caused beta hemolysis, showing a dense red band in sheep blood agar. In human blood agar, a conditioned hemolysis occurred in visible or invisible regions. After the production of that dense red band on human blood agar, circles of hemolysis were formed in that region if petri dishes were left at room temperature for some days. It is possible that failure of the effect of the interfering substance produced by staphylococei results in conditioned hemolysis and hemolytic cireles. ACKNOWLEDGMENT The author wishes to thank P. Rountree, Fairfax Institute of Pathology, Royal Prince Alfred Hospital, Sydney, Australia, for her interest and suggestions. LITERATURE CITED BIGGER, J. W The production of staphylococcal haemolysin with observations on its mode of action. J. Pathol. Bacteriol. 36: BIGGER, J. W., C. R. BOLAND, ANI) R. A. Q. O'MEARA A new method of preparing staphylococcal haemolysin. J. Pathol. Bacteriol. 30: CHRISTIE, R., AND J. J. GRAYDON Observations on staphylococcal haemolysins and staphylococcal lipase. Australian J. Exptl. Biol. Med. Sci. 19:9-16. CHRISTIE, R., AND E. A. NORTH Observations on staphylococcal beta toxin and its production by strains isolated from staphylococcal lesions in man. Australian J. Exptl. Biol. Med. Sci. 19: ELEK, S. D Staphylococcus pyogenes and its relation to disease. E. S. Livingstone Ltd., London. FLAMM, H Zonenhamolyse, Hamolysesteigerung, und Hamolysehemmung bei Staphylokokken. Schweiz. Z. Allgem. Pathol. Bakteriol. 20: FLAUM, A., AND J. FORSSMAN Studies in staphylococci. V. Experimental investigations of staphylolysin. Acta Pathol. Microbiol. Scand. 13: GLENNY, A. T., AND M. F. STEVENS Staphylococcus toxins and antitoxins. J. Pathol. Bacteriol. 40: KORTENHAUS, F Zonenzyklohamolyse auf Blutagar bei Staphylokokken sowie bei Viridans-Streptokokken und Pneumokokken. Zentr. Bakteriol. Parasitenk. Abt. I 113: LEVINE, B. S Studies of staphylococcus toxin. The phenomenon of hot-cold lysis by active staphylococcus filtrates. J. Infect. D)iseases 61: LIU, P Inhibition of a staphylococcal hemolysin by a soluble substance produced by a nonhemolytic Micrococcus species. J. Bacteriol. 68: MUNCH-PETERSON, E A corynebacterial agent which protects ruminant erythrocytes against staphylococcal E toxin. Australian J. Exptl. Biol. Med. Sci. 32: Roy, T. E The titration of alpha and beta haemolysins in staphylococcus toxin. J. linmunol. 33: SEIFFERT, W Das Staphylotoxin. Zentr. Bakteriol. Abt. I Orig. 135: SMITH, M. L., AND S. A. PRICE Staphylococcus -y haemolysin. J. Pathol. Bacteriol. 47 : WELD, J. T Production of dense red band around growth of Staphylococcus aureus on blood agar plates. Proc. Soc. Exptl. Biol. Med. 109: WILLIAMS, R. E. O., AND G. J. HARPER Staphylococcal haemolysins on sheep-blood agar with evidence for a fourth haemolysin. J. Pathol. Bacteriol. 59:69-78.