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Correlation Between Growth in Antibiotic-Medium and Hemolytic Activity of Group C and G Streptococci
Zbl. Bakt. 272, 283-289 (1990) © Gustav Fischer Verlag, StuttgartlNew York
Correlation Between Growth in Antibiotic-Medium and Hemolytic Activity of Group C and G Streptococci ANGELA C. D. DE CASTRO, BERNADETE T. FERREIRA, and LESLIE C. BENCHETRIT Streptococcal Reference Center, Department of Medical Microbiology, Institute of Microbiology, Federal University, 21944 Rio de Janeiro, RJ, Brazil
Received March 23, 1988 . Accepted in revised form September 20, 1989
Abstract The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin 0 (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.
Zusammenfassung Es wurde der EinfluS subinhibitorischer Penicillin-Konzentrationen auf die Bildung freier und gebundener Hamolysine durch Streptokokken unter Verwendung von Schaf-Erythrozyten untersucht. Ein deutlicher Abfall zellfreier und gebundener Aktivitaten wurde in Gegenwart von 113 der MIK des Penicillins bei Gruppe C-Streptokokken beobachtet, wahrend bei Gruppe G-Streptokokken ein Anstieg auftrat. Kaliumferricyanid und Antistreptolysin 0 (Gruppe A-Streptokokken) hemmten die freien Aktivitaen beider Stamme. Die zellgebundenen Aktivitaten wurden durch RNA-Zusatz wahrend des bakteriellen Wachstums in Kontrollmedien und in Gegenwart verschiedener Zusatze stimuliert.
Introduction Streptolysins 0 (SLO) and S (SLS) of group A streptococci have been well characterized (1). SLO is an immunogenic, oxygen-labile protein and can only be activated in a reduced atmosphere. The toxin is elaborated by most strains of group A streptococci
284
A. C. D. de Castro, B. T. Ferreira, and L. C. Benchetrit
and located between the cell membrane and the cell wall (5). Group G streptococci and human isolates of group C also produce an oxygen-labile hemolysin that is antigenically similar to that of group A (1). The SLS molecule is small, apparently not immunogenic and associated with various types of carriers. Almost all strains of group A, C and G streptococci have the capability of elaborating this hemolysin in vitro. Subminimal inhibitory concentrations of antimicrobial agents alter the expression of several products by microorganisms. Among drug-exposed gram-positive cocci, the expression of staphylococcal penicillinase (16), lipase (18), exfoliative toxin(19) and hemolysin (13, 20) as well as that of group A streptococcal serum-opacity factor (11) and enzymes hyaluronidase (2, 7), deoxyribonuclease (17), nicotinamide adenine dinucleotidase (11) and pneumococcal hemolysin (15) have been followed in vitro and shown to occur at levels different from those present in control cultures during bacterial growth. Previous studies at this laboratory have shown that aerobic growth of streptococci on sheep blood agar plates containing penicillin was accompanied by an increase in the surface hemolytic activity of a group G strain when compared to control cultures (6) and a decrease in that of a group C (unpublished data). In this context, we carried out a more precise examination of the elaboration of cellbound and free hemolytic activities by two strains of group C and group G streptococci which is described in the present report.
Material and Methods
Bacterial strains. The streptococci used in this study had been isolated from throat cultures as previously described (3). The strains were from the collection of this laboratory. Stock cultures were stored in a lyophilized state in sheep blood. Purity of the cultures was assessed by streaking the bacteria onto 5% sheep blood agar plates. Media. Todd-Hewitt broth (THB; Difco Laboratories) was used for bacterial growth in the presence or absence of penicillin (113 of the MIC). RNA (Sigma) was added as indicated at a concentration of 4mg/ml in broth (4). The drug and the nucleic acid were added at the time of inoculation. Culture supernatants and cells. One drop of an overnight 35°C THB culture was added to 7 ml of penicillin medium and incubated for 16 h at 35°C. The culture was then mixed and optical density readings of the bacterial suspension were carried out at 540 nm (7). Cultures were cleared from cells by low-speed centrifugation. The pellet was washed twice with 10 mM sodium phosphate buffer pH 6.8 and resuspended in 6.86 ml of the same buffer. The culture supernatants were passed through membrane filters (0.45 [.tm) before testing for hemolysin activity. Red blood cells. Sheep red blood cells (SRBC s) were harvested after three washings with phosphate buffer and resuspended in the same buffer at a 1% final concentration. Thereafter the suspension was distributed in 3.5 ml aliquots into glass tubes and centrifuged. The SRBC button was used for the hemolysin studies. Hemolysin assay. Cell-bound and free hemolysin activities were determined in the reaction systems described in Table 1. The reaction mixtures were incubated for 45 min at 35°C and then centrifuged at 500 g for 20 min. Absorbances of supernatants were measured at 540 nm. The control reactions are also shown in Table l. Inhibition studies. Inhibition reactions were performed using antistreptolysin 0 (ASLO; Difco), potassium ferricyanide, pronase or heat denaturation as follows: prior to addition of culture supernatant (reaction system IV; Table 1) to SRBCs, ASLO (group A streptococcus), the oxidizing agent or pronase were added to these supernatnat fluids and the mixtures were
Penicillin and Streptococcal Hemolysins
285
Table 1. Composition of reaction systems for determination of hemolytic activities present in cultures of streptococci a Reaction system b
I II III IV V VI VII VIII IX X XI XII a
b
Bacterial Culture Sodium suspensIOn supernatant phosphate buffer (ml) (ml) (m!) 6.86 5.6 6.86 5.6
0.14 6.86 7.0 1.4 1.26 1.4 6.72 6.86 1.26 1.12 1.26
Distilled water (ml)
ToddHewitt broth (ml)
Mercaptoethanol (ml)
0.14 0.14
5.6 5.6
0.14 0.14
5.6 5.6
0.14 0.14 0.14 0.14 0.14 0.14
An SRBC button was present in all systems. See text for details on preparation. Systems II, V, VIII and XI were used as positive controls, i.e. complete lysis for the systems I, IV, VII and X, respectively. Systems III, VI, IX and XII were used as negative controls, i.e. absence of lysis for the same systems.
incubated for 30 min at 35°C. Heat denaturation was carried out for 30 min in boiling water. The residual hemolytic cytolytic activities were then determined as described above and the results compared to those obtained in control tubes without any inhibitor.
Results Not all of the hemolytic activity present in the bacterial culture is associated with bacterial (sedimentable) cells. Table 2 presents the distribution of cell-associated and free hemolysis. A major portion of the activity is bound to the bacteria (group C, 66%; group G, 60%). The non-associated activity does not seem to arise from continual dissociation from cells, since subsequent washing of cells does not result in an increase of free hemolysin. The cell-bound (groups C and G) and free (group G) hemolytic activities were not stimulated by addition of 2-mercaptoethanol (Table 2). When the thiol reagent was added to the system containing the group C cell-free hemolysin, a marked increase of activity was determined (Table 2). Inactivation of the free hemolytic activities was also performed with pronase-treated or heat-denatured supernatants and also with the oxidizing agent, potassium ferricyani de. The inhibition reached nearly 100% of the initial activity for both streptococcal groups. Hemolysin neutralization was also performed with antibodies to group A streptolysin. The ASLO preparation had a total (100%) inhibitory effect on the free hemolysin of streptococci of groups C and G.
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A. C. D. de Castro, B. T. Ferreira, and L. C. Benchetrit
Table 2. Hemolytic activity in streptococcal cultures Serological group
Antibiotic level
Culture density
Mercaptoethanol
Hemolysin activity Cell-bound Free
C
0
0.84"
+h
1/3 MIC
0.26
+
0
0.83
+
1/3 MIC
0.58
+
1.2C 1.2 0.4 0.2 1.3 1.3 1.6 1.8
G
0.6 d 0.3 0.09 0.06 0.9 1.0 1.25 1.4
" Optical density readings (540 nm) of bacterial cultures grown in THB for 16 h at 35°C. When present, the reagent had been added to the reaction mixtures at a final concentration of 0.03 M. c Ratio: optical density readings (540 nm) of the supernatant fluid obtained after lysis of SRBC's with whole bacterial cellsloptical density readings of bacterial growth. d The ratio was calculated after using whole culture supernatants instead of whole cells to lyse SRBS's.
h
Table 3. Capability of streptococcal cells grown in the presence of RNA to lyse sheep red blood cells" Serological group
Antibiotic level
Culture density
Hemolytic activity
C
0 1/3 MIC 0 1/3 MIC
0.85 h 0.62 0.90 0.68
1.3 c,d 2.1
G
1.5
2.0
The nucleic acid was added to the culture medium at a final concentration of 4 mg/ml at inoculation time. h,c See Table 1. d Identical results were obtained when 2-mercaptoethanol was added to the reaction mixtures. a
The group C streptococcus was inoculated onto blood agar plates containing 113 of the MIC of penicillin. The colonies looked less hemolytic and indeed quantitative determination of the activity demonstrated that the drug had inhibited the production of both cell-bound and free hemolysins (TabeI2). However, growth of group G in the penicillin-supplemented medium was accompanied by an increase in the two forms of the hemolysin (Table 2). The fraction of activity associated with cells of the two streptococcal groups was also calculated and was found to be similar to that in control organisms. The preceding experiments were performed using cultures to which the drug had been added at the time of inoculation. Bacterial growth was also carried out after incorporation of RNA into culture media. Tables 2 and 3 show that production of cell-
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287
bound hemolysin by RNA-grown and control bacteria was not significantly different. In studies where the broth for bacterial growth contained the nucleic acid and the betalactam antibiotic, only growth of the group C was stimulated (Table 3). However, these hemolysin levels associated to cells increased in cultures of the strains of groups C and G. Discussion Hemolytic streptococci which are pathogenic for humans include Lancefield groups A, C and G. All three serological groups elaborate, in vivo, antigenically similar SLO's (1,21). The results of the present study are in accordance with earlier characterizations of the hemolysin (1, 12). The soluble hemolysins present in the supernatant of broth cultures of group C and group G strains were inactivated by group A ASLO antibodies. The group C substance was more active in the reduced form (Table 2). In contrast, the group G hemolysin was barely affected by the reducing agent although being more active or produced in higher amounts than that of group G organisms. Oxygen had an inactivating effect on the group G hemolysin since supernatants submitted to magnetic stirring at 4°C for 18 h had no measurable activity (not shown). These results also indicate that the microbial toxin appears in broth cultures that may have relatively high reducing properties. Indeed, supernatants to which resazurin had been added did not change the color of the medium. Thus, with regard to the group G extracellular hemolysin, there may be specific strain characteristics or physical and chemical properties of the lytic macromolecule to be accounted for. The cell-bound hemolytic activities occurred at similar levels in the two strains and none was affected by mercaptoethanol (Table 2). The activities were only slightly stimulated by the presence of RNA in the culture media (Tables 2 and 3). Since it was of interest to demonstrate to which extent lysis of SRBC could occur, erythrocytes were treated with distilled water and absorbancies were measured spectrophotometrically (Table 1). Indeed no significant differences between water-induced lysis and that caused by the cell-bound hemolysins were observed. In addition, the higher lytic capability of these substances was analogous to the activity which appeared to be tightly bound to group A streptococcal cells during growth in liquid media (12). Furthermore, the soluble hemolysin was thermolabile to a relatively high extent, much like the group A SLO (12). The effects of subMICs of antimicrobial agents on bacterial morphology and on extracellular products have been well documented (6, 9, 15). In a previous report, the hyaluronidase activity was shown to be increased for the same strain of group G when this microorganism was cultivated in the presence of penicillin (7), and strains of group A also presented the same behavior (2). The penicillinase production by Staphylococcus aureus can be increased or decreased when microorganisms are exposed to subMICs of erythromycin, clindamycin und pristinamycin (16). In respect of hemolytic activity, it was observed that group A streptococci exposed to penicillin showed an inhibition of SLS (9). We observed the same phenomenon for the group C strain. However, the hemolytic activity of group G streptococci seemed to be increased in the presence of penicillin. The increase of hemolytic activity expression by microorganisms grown in the presence of antibiotics was also reported by Hallander et al. (13). They verified an increase of the hemolysin liberated by S. aureus exposed to beta-Iactam antibiotics. Lorian et al. (15), observed that the pneumococcus, an alpha-hemolysis
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A. C. D. de Castro, B. T. Ferreira, and L. C. Benchetrit
producing microorganism, was capable of showing beta-hemolysis when exposed to subMlCs of several antibiotics that act on cell walls. This pneumolysin is also inactivated by oxydizing agents and active only in the reduced form (14). Because group C organisms grew less than those of group G in the presence of 113 of the MIC of penicillin, the question could be raised as to whether the low levels of free and cell-bound hemolytic activity measured in the cultures were due to the small number of bacterial cells. It was therefore decided to carry out the following experiment: a group C control culture was diluted until it presented the same absorbance as that of a culture in the drug-supplemented medium and the activity measured. Low but measurable levels of hemolytic activity were determined. There is a relative lack of knowledge about the biologic and toxic properties of hemolysins of groups C and G. More is known about the toxins of streptococci of groups A and B (1, 10), their possible contribution to the virulence of the microorganisms (both species) and relationship to the pathogenesis of either rheumatic fever or acute glomerulonephritis (group A). Group A SLO is produced in patients with streptococcal infection and there are experimental results showing that SLS can also be elaborated during bacterial growth in vivo (mouse animal model, 8). In addition, our results indicate that substances that may be considered as virulence factors of groups C and G streptococci can be altered by subMICs of penicillin, a drug used for treatment of streptococcal infections. The differences found in the hemolytic activities of the two distinct strains of streptococci suggest that the mechanism of action of low doses of the drug is not a uniform feature and may represent biochemical characteristics of the microorganisms tested.
Acknowledgments. This study was supported by grants from CNPq (Brasilia), CEPG (Federal University of Rio de Janeiro) and FINEPIPADCT (Rio de Janeiro). During the study Bernadete T. Ferreira was the recipient of a fellowship from CNPq. We thank FontouraWyeth for the gift of penicillin.
References 1. Alouf, J. E.: Streptococcal toxins (streptolysin 0, streptolysin S, erythrogenic toxin). Pharm. Ther. 11 (1980) 661-717 2. Benchetrit, L. c., C. C. Avelino, and C. M. Oliveira: Effect of subminimal inhibitory concentrations of penicillin on hyaluronidase production by group A streptococci. Zbl. Bakt. Hyg., I. Abt. Orig. A 251 (1981) 152-156 3. Benchetrit, L. c., A. A. Borges Neto, A. M. S. Figueiredo, and C. M. Oliveira: Occurrence of group A and nongroup A betahemolytic streptococci in human infections in Rio de Janeiro. Rev. Microbiol. (Sao Paulo) 11 (1980) 50-54 4. Bernheimer, A. W. and M. Rodbart: The effect of nucleic acids and of carbohydrates on the formation of streptolysin. J. Exp.Med. 88 (1948) 149-167 5. Calandra, G. B. and T. S. Theodore: Cellular location of streptolysin o. Infect. Immun. 12 (1975) 750-753 6. Castro, A. C. D., L. C. Benchetrit, and L. Barrucand: Efeito de concentra~oes subinibit6rias de penicilina sobre a morfologia de estreptococos do group G. Rev. Bras. Pat. Clin. 21 (1985) 123-129 7. Castro, A. C. D., L. C. Benchetrit, and C. M. Oliveira: Efeito de concentra~6es subinibit6rias de penicilina sobre antigenos de estreptococos do grupo G. Rev. Inst. Med. Trop. (Sao Paulo) 28 (1986) 234-238
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8. Duncan, j. L.: Streptococcal growth and toxin production in vivo. Infect. Immun. 40 (1983) 501-505
9. Ferne, M., S. B. Rabinowitz, Z. Duchan, and j. Michel: Morphologic and enzymatic
changes of beta-hemolytic group A streptococci due to subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococci and Streptococcal Diseases, pp. 148-150. Reedbooks, Chertsey (1982) 10. Ferrieri, P.: Characterization of a hemolysin isolated from group B streptococci. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococci and Streptococcal Diseases, pp. 142-143. Reedbooks, Chertsey (1982) 11. Gemmell, C. G. and M. K. Abdul-Amir: Effect of certain antibiotics on the formation of cellular antigens and extracellular products by group A streptococci. In: M. T. Parker (ed.), Pathogenic Streptococci, pp. 67-68. Reedbooks, Chertsey (1979) 12. Ginsburg, I.: Mechanisms of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae. J. Infect. Dis. 3 (1972) 294-340 13. Hallander, H. 0., G. Laurel, and G. Loftstrom: Stimulation of staphylococcal haemolysin production by low concentrations of penicillin. Acta path. microbiol. scand. 68 (1966) 142-148 14. Kanclerski, K. and R. Mollby: Production and purification of Streptococcus pneumoniae hemolysin (pneumolysin). J. Clin. Microbiol. 25 (1987) 222-225 15. Lorian, V., A. Waluschka, and B. Popoola: Pneumococcal beta-hemolysin produced under the effect of antibiotics. App. Microbiol. 25 (1973) 290-294 16. Michel, j. P. Stessman, and j. Stessman: Effects of subminimal inhibitory concentrations of erythromycin, clindamycin and pristinamycin on the penicillinase production of Staphylococcus aureus. Antimicrob. Agents Chemother. 17 (1980) 13-15 17. Michel, J., M. Ferne, R. Borinski, Z. Kornberg, S. Bergner-Rabinowitz, and I. Ginsburg: Effects of subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin on group A streptococci. Eur. J. Clin. Microbiol. 1 (1982) 375-380 18. Shibl, A. M.: Effect of antibiotics on production of enzymes and toxins by microorganisms. Rev. Infect. Dis. 5 (1983) 865-875 19. Shibl, A. M.: Influence of subinhibitory concentrations of antibiotics on virulence of staphylococci. Rev. Infect. Dis. 9 (1987) 704-712 20. Shibl, A. M. and I. A. Al-Sowaygh: Differential inhibition of bacterial growth and hemolysin production by lincosamide antibiotics. J. Bact. 137 (1979) 1022-1023 21. Wannamaker,L.: The extracellular products of group A streptococci. In: S. E. Read and j. B. Zabriskie (eds.), Streptococcal Diseases and the Immune Response, pp. 177-184. Academic Press, New York-London (1980)
Angela C. D. de Castro, Streptococcal Reference Center, Department of Medical Microbiology, Institute of Microbiology, Federal University, P. Box 68040, 21944 Rio de Janeiro, RJ, Brazil
19 Zbl. Bakt. 272/3
Correlation Between Growth in Antibiotic-Medium and Hemolytic Activity of Group C and G Streptococci ANGELA C. D. DE CASTRO, BERNADETE T. FERREIRA, and LESLIE C. BENCHETRIT Streptococcal Reference Center, Department of Medical Microbiology, Institute of Microbiology, Federal University, 21944 Rio de Janeiro, RJ, Brazil
Received March 23, 1988 . Accepted in revised form September 20, 1989
Abstract The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin 0 (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.
Zusammenfassung Es wurde der EinfluS subinhibitorischer Penicillin-Konzentrationen auf die Bildung freier und gebundener Hamolysine durch Streptokokken unter Verwendung von Schaf-Erythrozyten untersucht. Ein deutlicher Abfall zellfreier und gebundener Aktivitaten wurde in Gegenwart von 113 der MIK des Penicillins bei Gruppe C-Streptokokken beobachtet, wahrend bei Gruppe G-Streptokokken ein Anstieg auftrat. Kaliumferricyanid und Antistreptolysin 0 (Gruppe A-Streptokokken) hemmten die freien Aktivitaen beider Stamme. Die zellgebundenen Aktivitaten wurden durch RNA-Zusatz wahrend des bakteriellen Wachstums in Kontrollmedien und in Gegenwart verschiedener Zusatze stimuliert.
Introduction Streptolysins 0 (SLO) and S (SLS) of group A streptococci have been well characterized (1). SLO is an immunogenic, oxygen-labile protein and can only be activated in a reduced atmosphere. The toxin is elaborated by most strains of group A streptococci
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A. C. D. de Castro, B. T. Ferreira, and L. C. Benchetrit
and located between the cell membrane and the cell wall (5). Group G streptococci and human isolates of group C also produce an oxygen-labile hemolysin that is antigenically similar to that of group A (1). The SLS molecule is small, apparently not immunogenic and associated with various types of carriers. Almost all strains of group A, C and G streptococci have the capability of elaborating this hemolysin in vitro. Subminimal inhibitory concentrations of antimicrobial agents alter the expression of several products by microorganisms. Among drug-exposed gram-positive cocci, the expression of staphylococcal penicillinase (16), lipase (18), exfoliative toxin(19) and hemolysin (13, 20) as well as that of group A streptococcal serum-opacity factor (11) and enzymes hyaluronidase (2, 7), deoxyribonuclease (17), nicotinamide adenine dinucleotidase (11) and pneumococcal hemolysin (15) have been followed in vitro and shown to occur at levels different from those present in control cultures during bacterial growth. Previous studies at this laboratory have shown that aerobic growth of streptococci on sheep blood agar plates containing penicillin was accompanied by an increase in the surface hemolytic activity of a group G strain when compared to control cultures (6) and a decrease in that of a group C (unpublished data). In this context, we carried out a more precise examination of the elaboration of cellbound and free hemolytic activities by two strains of group C and group G streptococci which is described in the present report.
Material and Methods
Bacterial strains. The streptococci used in this study had been isolated from throat cultures as previously described (3). The strains were from the collection of this laboratory. Stock cultures were stored in a lyophilized state in sheep blood. Purity of the cultures was assessed by streaking the bacteria onto 5% sheep blood agar plates. Media. Todd-Hewitt broth (THB; Difco Laboratories) was used for bacterial growth in the presence or absence of penicillin (113 of the MIC). RNA (Sigma) was added as indicated at a concentration of 4mg/ml in broth (4). The drug and the nucleic acid were added at the time of inoculation. Culture supernatants and cells. One drop of an overnight 35°C THB culture was added to 7 ml of penicillin medium and incubated for 16 h at 35°C. The culture was then mixed and optical density readings of the bacterial suspension were carried out at 540 nm (7). Cultures were cleared from cells by low-speed centrifugation. The pellet was washed twice with 10 mM sodium phosphate buffer pH 6.8 and resuspended in 6.86 ml of the same buffer. The culture supernatants were passed through membrane filters (0.45 [.tm) before testing for hemolysin activity. Red blood cells. Sheep red blood cells (SRBC s) were harvested after three washings with phosphate buffer and resuspended in the same buffer at a 1% final concentration. Thereafter the suspension was distributed in 3.5 ml aliquots into glass tubes and centrifuged. The SRBC button was used for the hemolysin studies. Hemolysin assay. Cell-bound and free hemolysin activities were determined in the reaction systems described in Table 1. The reaction mixtures were incubated for 45 min at 35°C and then centrifuged at 500 g for 20 min. Absorbances of supernatants were measured at 540 nm. The control reactions are also shown in Table l. Inhibition studies. Inhibition reactions were performed using antistreptolysin 0 (ASLO; Difco), potassium ferricyanide, pronase or heat denaturation as follows: prior to addition of culture supernatant (reaction system IV; Table 1) to SRBCs, ASLO (group A streptococcus), the oxidizing agent or pronase were added to these supernatnat fluids and the mixtures were
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Table 1. Composition of reaction systems for determination of hemolytic activities present in cultures of streptococci a Reaction system b
I II III IV V VI VII VIII IX X XI XII a
b
Bacterial Culture Sodium suspensIOn supernatant phosphate buffer (ml) (ml) (m!) 6.86 5.6 6.86 5.6
0.14 6.86 7.0 1.4 1.26 1.4 6.72 6.86 1.26 1.12 1.26
Distilled water (ml)
ToddHewitt broth (ml)
Mercaptoethanol (ml)
0.14 0.14
5.6 5.6
0.14 0.14
5.6 5.6
0.14 0.14 0.14 0.14 0.14 0.14
An SRBC button was present in all systems. See text for details on preparation. Systems II, V, VIII and XI were used as positive controls, i.e. complete lysis for the systems I, IV, VII and X, respectively. Systems III, VI, IX and XII were used as negative controls, i.e. absence of lysis for the same systems.
incubated for 30 min at 35°C. Heat denaturation was carried out for 30 min in boiling water. The residual hemolytic cytolytic activities were then determined as described above and the results compared to those obtained in control tubes without any inhibitor.
Results Not all of the hemolytic activity present in the bacterial culture is associated with bacterial (sedimentable) cells. Table 2 presents the distribution of cell-associated and free hemolysis. A major portion of the activity is bound to the bacteria (group C, 66%; group G, 60%). The non-associated activity does not seem to arise from continual dissociation from cells, since subsequent washing of cells does not result in an increase of free hemolysin. The cell-bound (groups C and G) and free (group G) hemolytic activities were not stimulated by addition of 2-mercaptoethanol (Table 2). When the thiol reagent was added to the system containing the group C cell-free hemolysin, a marked increase of activity was determined (Table 2). Inactivation of the free hemolytic activities was also performed with pronase-treated or heat-denatured supernatants and also with the oxidizing agent, potassium ferricyani de. The inhibition reached nearly 100% of the initial activity for both streptococcal groups. Hemolysin neutralization was also performed with antibodies to group A streptolysin. The ASLO preparation had a total (100%) inhibitory effect on the free hemolysin of streptococci of groups C and G.
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A. C. D. de Castro, B. T. Ferreira, and L. C. Benchetrit
Table 2. Hemolytic activity in streptococcal cultures Serological group
Antibiotic level
Culture density
Mercaptoethanol
Hemolysin activity Cell-bound Free
C
0
0.84"
+h
1/3 MIC
0.26
+
0
0.83
+
1/3 MIC
0.58
+
1.2C 1.2 0.4 0.2 1.3 1.3 1.6 1.8
G
0.6 d 0.3 0.09 0.06 0.9 1.0 1.25 1.4
" Optical density readings (540 nm) of bacterial cultures grown in THB for 16 h at 35°C. When present, the reagent had been added to the reaction mixtures at a final concentration of 0.03 M. c Ratio: optical density readings (540 nm) of the supernatant fluid obtained after lysis of SRBC's with whole bacterial cellsloptical density readings of bacterial growth. d The ratio was calculated after using whole culture supernatants instead of whole cells to lyse SRBS's.
h
Table 3. Capability of streptococcal cells grown in the presence of RNA to lyse sheep red blood cells" Serological group
Antibiotic level
Culture density
Hemolytic activity
C
0 1/3 MIC 0 1/3 MIC
0.85 h 0.62 0.90 0.68
1.3 c,d 2.1
G
1.5
2.0
The nucleic acid was added to the culture medium at a final concentration of 4 mg/ml at inoculation time. h,c See Table 1. d Identical results were obtained when 2-mercaptoethanol was added to the reaction mixtures. a
The group C streptococcus was inoculated onto blood agar plates containing 113 of the MIC of penicillin. The colonies looked less hemolytic and indeed quantitative determination of the activity demonstrated that the drug had inhibited the production of both cell-bound and free hemolysins (TabeI2). However, growth of group G in the penicillin-supplemented medium was accompanied by an increase in the two forms of the hemolysin (Table 2). The fraction of activity associated with cells of the two streptococcal groups was also calculated and was found to be similar to that in control organisms. The preceding experiments were performed using cultures to which the drug had been added at the time of inoculation. Bacterial growth was also carried out after incorporation of RNA into culture media. Tables 2 and 3 show that production of cell-
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bound hemolysin by RNA-grown and control bacteria was not significantly different. In studies where the broth for bacterial growth contained the nucleic acid and the betalactam antibiotic, only growth of the group C was stimulated (Table 3). However, these hemolysin levels associated to cells increased in cultures of the strains of groups C and G. Discussion Hemolytic streptococci which are pathogenic for humans include Lancefield groups A, C and G. All three serological groups elaborate, in vivo, antigenically similar SLO's (1,21). The results of the present study are in accordance with earlier characterizations of the hemolysin (1, 12). The soluble hemolysins present in the supernatant of broth cultures of group C and group G strains were inactivated by group A ASLO antibodies. The group C substance was more active in the reduced form (Table 2). In contrast, the group G hemolysin was barely affected by the reducing agent although being more active or produced in higher amounts than that of group G organisms. Oxygen had an inactivating effect on the group G hemolysin since supernatants submitted to magnetic stirring at 4°C for 18 h had no measurable activity (not shown). These results also indicate that the microbial toxin appears in broth cultures that may have relatively high reducing properties. Indeed, supernatants to which resazurin had been added did not change the color of the medium. Thus, with regard to the group G extracellular hemolysin, there may be specific strain characteristics or physical and chemical properties of the lytic macromolecule to be accounted for. The cell-bound hemolytic activities occurred at similar levels in the two strains and none was affected by mercaptoethanol (Table 2). The activities were only slightly stimulated by the presence of RNA in the culture media (Tables 2 and 3). Since it was of interest to demonstrate to which extent lysis of SRBC could occur, erythrocytes were treated with distilled water and absorbancies were measured spectrophotometrically (Table 1). Indeed no significant differences between water-induced lysis and that caused by the cell-bound hemolysins were observed. In addition, the higher lytic capability of these substances was analogous to the activity which appeared to be tightly bound to group A streptococcal cells during growth in liquid media (12). Furthermore, the soluble hemolysin was thermolabile to a relatively high extent, much like the group A SLO (12). The effects of subMICs of antimicrobial agents on bacterial morphology and on extracellular products have been well documented (6, 9, 15). In a previous report, the hyaluronidase activity was shown to be increased for the same strain of group G when this microorganism was cultivated in the presence of penicillin (7), and strains of group A also presented the same behavior (2). The penicillinase production by Staphylococcus aureus can be increased or decreased when microorganisms are exposed to subMICs of erythromycin, clindamycin und pristinamycin (16). In respect of hemolytic activity, it was observed that group A streptococci exposed to penicillin showed an inhibition of SLS (9). We observed the same phenomenon for the group C strain. However, the hemolytic activity of group G streptococci seemed to be increased in the presence of penicillin. The increase of hemolytic activity expression by microorganisms grown in the presence of antibiotics was also reported by Hallander et al. (13). They verified an increase of the hemolysin liberated by S. aureus exposed to beta-Iactam antibiotics. Lorian et al. (15), observed that the pneumococcus, an alpha-hemolysis
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producing microorganism, was capable of showing beta-hemolysis when exposed to subMlCs of several antibiotics that act on cell walls. This pneumolysin is also inactivated by oxydizing agents and active only in the reduced form (14). Because group C organisms grew less than those of group G in the presence of 113 of the MIC of penicillin, the question could be raised as to whether the low levels of free and cell-bound hemolytic activity measured in the cultures were due to the small number of bacterial cells. It was therefore decided to carry out the following experiment: a group C control culture was diluted until it presented the same absorbance as that of a culture in the drug-supplemented medium and the activity measured. Low but measurable levels of hemolytic activity were determined. There is a relative lack of knowledge about the biologic and toxic properties of hemolysins of groups C and G. More is known about the toxins of streptococci of groups A and B (1, 10), their possible contribution to the virulence of the microorganisms (both species) and relationship to the pathogenesis of either rheumatic fever or acute glomerulonephritis (group A). Group A SLO is produced in patients with streptococcal infection and there are experimental results showing that SLS can also be elaborated during bacterial growth in vivo (mouse animal model, 8). In addition, our results indicate that substances that may be considered as virulence factors of groups C and G streptococci can be altered by subMICs of penicillin, a drug used for treatment of streptococcal infections. The differences found in the hemolytic activities of the two distinct strains of streptococci suggest that the mechanism of action of low doses of the drug is not a uniform feature and may represent biochemical characteristics of the microorganisms tested.
Acknowledgments. This study was supported by grants from CNPq (Brasilia), CEPG (Federal University of Rio de Janeiro) and FINEPIPADCT (Rio de Janeiro). During the study Bernadete T. Ferreira was the recipient of a fellowship from CNPq. We thank FontouraWyeth for the gift of penicillin.
References 1. Alouf, J. E.: Streptococcal toxins (streptolysin 0, streptolysin S, erythrogenic toxin). Pharm. Ther. 11 (1980) 661-717 2. Benchetrit, L. c., C. C. Avelino, and C. M. Oliveira: Effect of subminimal inhibitory concentrations of penicillin on hyaluronidase production by group A streptococci. Zbl. Bakt. Hyg., I. Abt. Orig. A 251 (1981) 152-156 3. Benchetrit, L. c., A. A. Borges Neto, A. M. S. Figueiredo, and C. M. Oliveira: Occurrence of group A and nongroup A betahemolytic streptococci in human infections in Rio de Janeiro. Rev. Microbiol. (Sao Paulo) 11 (1980) 50-54 4. Bernheimer, A. W. and M. Rodbart: The effect of nucleic acids and of carbohydrates on the formation of streptolysin. J. Exp.Med. 88 (1948) 149-167 5. Calandra, G. B. and T. S. Theodore: Cellular location of streptolysin o. Infect. Immun. 12 (1975) 750-753 6. Castro, A. C. D., L. C. Benchetrit, and L. Barrucand: Efeito de concentra~oes subinibit6rias de penicilina sobre a morfologia de estreptococos do group G. Rev. Bras. Pat. Clin. 21 (1985) 123-129 7. Castro, A. C. D., L. C. Benchetrit, and C. M. Oliveira: Efeito de concentra~6es subinibit6rias de penicilina sobre antigenos de estreptococos do grupo G. Rev. Inst. Med. Trop. (Sao Paulo) 28 (1986) 234-238
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8. Duncan, j. L.: Streptococcal growth and toxin production in vivo. Infect. Immun. 40 (1983) 501-505
9. Ferne, M., S. B. Rabinowitz, Z. Duchan, and j. Michel: Morphologic and enzymatic
changes of beta-hemolytic group A streptococci due to subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococci and Streptococcal Diseases, pp. 148-150. Reedbooks, Chertsey (1982) 10. Ferrieri, P.: Characterization of a hemolysin isolated from group B streptococci. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococci and Streptococcal Diseases, pp. 142-143. Reedbooks, Chertsey (1982) 11. Gemmell, C. G. and M. K. Abdul-Amir: Effect of certain antibiotics on the formation of cellular antigens and extracellular products by group A streptococci. In: M. T. Parker (ed.), Pathogenic Streptococci, pp. 67-68. Reedbooks, Chertsey (1979) 12. Ginsburg, I.: Mechanisms of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae. J. Infect. Dis. 3 (1972) 294-340 13. Hallander, H. 0., G. Laurel, and G. Loftstrom: Stimulation of staphylococcal haemolysin production by low concentrations of penicillin. Acta path. microbiol. scand. 68 (1966) 142-148 14. Kanclerski, K. and R. Mollby: Production and purification of Streptococcus pneumoniae hemolysin (pneumolysin). J. Clin. Microbiol. 25 (1987) 222-225 15. Lorian, V., A. Waluschka, and B. Popoola: Pneumococcal beta-hemolysin produced under the effect of antibiotics. App. Microbiol. 25 (1973) 290-294 16. Michel, j. P. Stessman, and j. Stessman: Effects of subminimal inhibitory concentrations of erythromycin, clindamycin and pristinamycin on the penicillinase production of Staphylococcus aureus. Antimicrob. Agents Chemother. 17 (1980) 13-15 17. Michel, J., M. Ferne, R. Borinski, Z. Kornberg, S. Bergner-Rabinowitz, and I. Ginsburg: Effects of subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin on group A streptococci. Eur. J. Clin. Microbiol. 1 (1982) 375-380 18. Shibl, A. M.: Effect of antibiotics on production of enzymes and toxins by microorganisms. Rev. Infect. Dis. 5 (1983) 865-875 19. Shibl, A. M.: Influence of subinhibitory concentrations of antibiotics on virulence of staphylococci. Rev. Infect. Dis. 9 (1987) 704-712 20. Shibl, A. M. and I. A. Al-Sowaygh: Differential inhibition of bacterial growth and hemolysin production by lincosamide antibiotics. J. Bact. 137 (1979) 1022-1023 21. Wannamaker,L.: The extracellular products of group A streptococci. In: S. E. Read and j. B. Zabriskie (eds.), Streptococcal Diseases and the Immune Response, pp. 177-184. Academic Press, New York-London (1980)
Angela C. D. de Castro, Streptococcal Reference Center, Department of Medical Microbiology, Institute of Microbiology, Federal University, P. Box 68040, 21944 Rio de Janeiro, RJ, Brazil
19 Zbl. Bakt. 272/3