INFrCTION AND ImUNmTY, Apr. 1977, p. 37-42 Copyright 0 1977 American Society for Microbiology
Vol. 16, No. 1
Printed in U.S.A.
Bacteriolytic Activity in Staphylococci GIUSEPPE SATfA, PIETRO E. VARALDO,* GRAZIA GRAZI, AND ROBERTA FONTANA Istituto di Microbiologia dell'Universitd di Genova, Genoa, Italy Received for publication 26 August 1976
The bacteriolytic activity ofStaphylococcus aureus and Staphylococcus albus strains was tested with various media. Whereas S. aureus strains were found to be active under all conditions, the percentage of active S. albus strains was significantly influenced by the composition of the medium. Ionic strength and concentration of the organic nitrogen source were found to be the main factors affecting the expression of bacteriolytic activity of staphylococci. Virtually all of 318 S. aureus and 603 S. albus strains were active on a medium containing 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, and 0.9% agar. The optimal conditions for the bacteriolytic activity of S. aureus strains were different from those of most S. albus strains. Within S. albus, optimal conditions differed also from strain to strain. It is suggested that further studies on this subject may prove useful for the identification and taxonomy of staphylococci. A possible relationship between the production of extracellular bacteriolytic enzymes and pathogenic properties of staphylococci is also considered. The production of bacteriolytic enzymes by a variety of bacteria is well established (8, 9, 24, 27, 36). Several of these enzymes have been studied in detail (10, 18, 32-35, 39). However, their role in nature and particularly in the physiology of cells, even though hypotheses on this subject have been advanced by several authors (12, 30), is largely unknown. This may also be due to the fact that little attention has been paid to whether these enzymes are produced just by sporadic strains or by the entire bacterial taxa. No bacterial family or genus in which all strains produce bacteriolytic activity has been reported so far. Moreover, different yet related bacteria might produce different bacteriolytic enzymes detectable under different testing conditions. Strains of Staphylococcus aureus are known to produce extracellular bacteriolytic enzymes (25, 26, 38). It is now well established that the production of such enzymes can be considered a characteristic common to all S. aureus strains; on the contrary, discordant findings have been reported with regard to the production of bacteriolytic enzymes by Staphylococcus albus strains (1, 2, 13-17, 19, 20, 22, 23). (In this report, the old name S. albus refers to all staphylococcal strains other than S. aureus. This name is preferred to both S. epidermidis and "coagulase-negative staphylococci," the former having recently been used to indicate only a particular group of staphylococci other than S. aureus [6, 7, 29] and the latter being confus-
ing because of the occurrence of coagulase-negative strains of S. aureus [4, 31].) In this study we have tested the bacteriolytic activity of several hundred staphylococcal strains under various conditions. The results obtained showed that a particularly rich growth medium and a relatively high sodium chloride concentration are required for detecting the bacteriolytic activity of many S. albus strains. Under ideal conditions, all S. aureus and S. albus strains were found to produce easily detectable bacteriolytic activity. However, the bacteriolytic activity of S. aureus strains appeared different, and easily distinguished, from that of S. albus strains. MATERIALS AND METHODS Bacterial strains. A total of 318 S. aureus and 603 S. albus strains were used in this study. All strains were of human origin. Most were isolated from clinical materials, including blood, urine, sputum, throat swabs, etc., that were examined in the Clinical Bacteriology Laboratory of the Institute of Microbiology of Genoa University. Some strains were also isolated from the skin, nose, and throat of healthy volunteers. All strains were gram-positive and catalase-positive cocci and were recognized as belonging to the genus Staphylococcus (5, 11, 28). Subsequently, S. aureus strains were identified according to current criteria (4, 6, 7). All strains not meeting such criteria were indicated as S. albus. Preparation of the substrate for bacteriolytic activity. Heat-killed cells of Micrococcus lysodeikticus ATCC 4698 were used as substrate for bacteriolytic 37
38
SATTA ET AL.
activity. Samples (0.1 ml) of a 48-h broth culture of this strain were uniformly distributed to the surface of several plates containing brain heart infusion agar (BHIA, Difco). Plates were incubated for 48 to 72 h at 30°C. The dense bacterial growth was then harvested and washed twice with distilled water. Cells were finally suspended in distilled water, and the concentration of the suspension was spectrophotometrically adjusted at 400 optical units/ml. This suspension was heated twice at 121°C for 20 min and stored at 4°C. Media. To study the variability of bacteriolytic activity of staphylococci, the following commercial and synthetic media were used: BHIA (Difco); tryptose phosphate broth (Difco) supplemented with 1.5% agar (TP1); nutrient agar (NA; Difco); ToddHewitt broth (Oxoid) supplemented with 1.5% agar (TH); D.S.T. agar (Oxoid); tryptic soy agar (TSA; Biolife); 1.2% peptone (Difco), 1% beef extract, 0.5% glucose, 0.25% disodium phosphate, and 1.5% agar, pH 6.9 (SG); 3% Peptonum Siccum (A. Costantino & C., Favria, Torino, Italy), 0.2% glucose, 0.5% sodium chloride, and 1.5% agar, pH 6.9 (M9). To study the relationship between the bacteriolytic activity of staphylococci and the composition of the growth medium, 27 different media were prepared by combining three different concentrations of peptone (1, 2.2, and 3.5%; Difco), three of glucose (0, 0.3, and 0.6%), and three of sodium chloride (0, 1.5, and 3%). These media were supplemented with 1.5% agar. The pH was adjusted at 6.9. For more detailed studies on the effects of sodium chloride, a base medium (1.2% Peptonum Siccum [Costantino], 0.3% glucose, and 1.5% agar, pH 6.9) was supplemented with NaCl to obtain eight different final concentrations: 0, 0.75, 1.5, 2.25, 3, 4, 6, and 8%. A rich medium containing 3% Peptonum Siccum (Costantino), 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, and 0.9% agar, pH 6.9, was used to test the bacteriolytic activity of all 921 strains studied. All media were sterilized by autoclaving and stored at 40C. A suspension of heat-killed M. lyso-
deikticus cells was added to the melted media to
obtain a final concentration of 2 optical units/ml. The media were then poured into petri dishes. Inoculation of the media. A multiple inoculating device manufactured in our laboratory was used in this study. This device contains 22 appropriately disposed needles, which were sterilized by flaming. Simultaneous inoculation of 22 isolates from a master plate into the medium contained inr a petri dish could be performed by this device. Master plates were made by marking the surfaces of BHIA plates with the sterile needles of the device and heavily inoculating the 22 respective sites with the strains studied. Twelve- to 24-h incubation produced dense bacterial growth, which could be picked up with a single impression of the corresponding needles and inoculated into the above-mentioned media. Examination of the bacteriolytic activity. The inoculated media were incubated for 24 h at 37°C and then maintained for 12 h at room temperature.
INFIECT. IMMUN. Zones of transparency occurring around the spots of bacterial growth indicated lysis of substrate by the growing strains. However, a certain variability in the diameters of the spots of bacterial growth could be observed, so that the distance between the edge of the spot and the edge of the zone of transparency, rather than the diameter of the entire zone of transparency, was considered more indicative of the degree of bacteriolytic activity. The results were scored according to the following scheme: +++, very large zone of transparency, exceeding the spot of bacterial growth by more than 6 mm; + +, large zone of transparency, exceeding the spot of bacterial growth by 3 to 6 mm; +, moderate to small zone of transparency, exceeding the spot of bacterial growth by less than 3 mm; -, no detectable zone of transparency.
RESULTS Bacteriolytic activity of S. aureus and S. albus strains on various commercial and synthetic media. Table 1 summarizes the incidence of bacteriolytic activity production by S. aureus and S. albus strains as reported to date. Authors who had found virtually 100% of S. aureus and contrasting percentages of S. albus strains able to produce bacteriolytic activity had generally been using different media. This would suggest that the bacteriolytic activity of S. albus strains may require particular and more critical conditions than S. aureus. Therefore, we tested the bacteriolytic activity of 132 S. aureus and 132 S. albus strains on various commercial and synthetic media, some approximating those used by other authors. On these media all S. aureus strains were active, whereas S. albus strains showed different activity from one medium to another (Table 2). Significance of the essential components of growth media. The same strains were then tested on a set of chemically defined media containing varying amounts of peptone as an organic nitrogen source, glucose as an organic carbon source, and NaCl as a salt, in order to confirm the dependence of bacteriolytic activity on the medium composition and to identify the components responsible for the variability ofS. albus activity. Results concerning S. aureus and S. albus strains are summarized in Tables 3 and 4, respectively. All S. aureus and S. albus strains were found to produce an easily detectable bacteriolytic activity on some of these media. The importance of the organic nitrogen concentration was evident: both the incidence of bacteriolytic activity production and the size of zones of transparency showed a general increase when the peptone concentration was raised from 1 to 3.5%. However, the ionic strength was the most important parameter affecting bacteriolytic activity. The lytic activ-
VOL. -16, 1977
BACTERIOLYTIC ACTIVITY OF STAPHYLOCOCCI
39
TABLz 1. Reported incidence of bacteriolytic activity production by staphylococcal strains S. aureus S. albus Author (reference)
Strains
tested 30 55 133 82 126 503 1,189 50 28 100
Active strains
Strains %
tested
Active strains
%
Welsch (37) 30 100 Kashiba et al. (20) 55 100 42 11 26.2 Omori et al. (23) 133 100 203 3 1.5 Goldbach and Haenel (13) 82 100 43 15 34.9 Jay (19) 120 95.2 49 4 8.2 Hawiger (15) 503 100 35 13 37.1 Grossgebauer et al. (14) 1,170 98.6 156 0 0 Arvidson et al. (2) 50 100 15 1 6.7 Afanasieva and Shevyakova (1) 28 100 14 5 35.7 Holt (17) 100 100 238a 33 13.9 Heczko (16) 478 62 13 Kloos and Schleifer (22) 46 46 100 886 422 47.6 a Including 62 strains reported as belonging to Baird-Parker's subgroups Ml to M4, which should now be considered Staphylococcus strains (6).
TABLE 2. Bacteriolytic activity of S. aureus and S. albus strains on various commercial and synthetic
chloride. The relationship between NaCl concentration of the medium and lytic activity exmediaa hibited by staphylococcal strains was examined in more detail, using a base medium to which % of strains showing zones of transparency NaCl was added in various concentrations. The results given in Table 5 confirm and expand on S. aureus S. albus diumb the data already shown in Tables 3 and 4. All S. + ++ +++ + ++ +++ aureus strains were active in the range of 0.75 BHIA 97 3 36 11 1 to 3% NaCl concentration. Only 2% of S. albus TP1 90 10 43 17 3 strains were inactive at the concentration of NA 97 3 37 1 2.25% NaCl. However, all strains examined, TH 95 5 44 8 both S. aureus and S. albus, did produce a zone DST 6 94 50 7 2 of transparency, at least at one of the NaCl TSA 93 7 20 1 SG 29 71 5 43 testing concentrations. The optimal NaCl conM9 92 8 66 13 3 centrations for bacteriolytic activity of S. aureus strains (0.75 and 1.5%) were found to be a One hundred and thirty-two S. aureus and 132 S. albus strains were tested. Symbols +, + +, and lower than those for most S. albus strains (2.25 + + + indicate different sizes of zones of transpar- and 3%). It was also confirmed that all S. aureus strains were similarly affected by the ionic ency (see text). b See Materials and Methods for abbreviations of strength of the medium with respect to their media. bacteriolytic activity. On the contrary, S. albus strains exhibited heterogeneous bacteriolytic ity of S. aureus strains varied uniformly with activity patterns. In fact, even though the madifferent concentrations of NaCl. S. albus jority ofthe latter strains showed optimal activstrains usually differed from S. aureus regard- ity at NaCl concentrations of 2.25 and 3%, some ing their lytic activity in response to the same were most active at lower concentrations (0.75 parameter; furthermore, they showed marked and 1.5%), or even in a medium without NaCl. differences among themselves. The effect of Moreover, the highest incidence of very large ionic strength appeared to be most pronounced zones of transparency (+++ +) was found at an under conditions of low peptone concentration. NaCl concentration of 4%, which inhibited the The carbon source concentration seemed to be activity of roughly one-third of these strains. of less importance: only under conditions of Finally, in media of optimal ionic strengths adequate peptone and NaCl concentrations did different S. albus strains exhibited marked the glucose concentration prove to be signifi- variability in the size and features of the zones cant. Perhaps the utilization of glucose by of transparency. Incidence of bacteriolytic activity among growing bacteria and the accompanying local reduction of pH interfered with expression of 921 staphylococci. As already stated elsewhere in this report, all strains tested were able to bacteriolytic activity. Further analysis of the effect of sodium produce a detectable bacoriolytic activity on
40
INFECT. IMMUN.
SATTA ET AL.
TABLE 3. Bacteriolytic activity of S. aureus strains on chemically defined media varying in peptone, glucose, and NaCI concentrationsa % of strains showing zones of transparency Medium composition +
++
3.5% peptone
2.2% peptone
1% peptone +++
+
++
No glucose, no NaCl 89 98 0.3% glucose, no NaCl 6 82 6 0.6% glucose, no NaCl 73 No glucose, 1.5% NaCl 91 9 0.3% glucose, 1.5% NaCl 2 11 87 0.6% glucose, 1.5% NaCl 84 16 No glucose, 3% NaCl 98 2 98 0.3% glucose, 3% NaCl 21 78 1 2 0.6% glucose, 3% NaCl 7 92 1 1 a One hundred and thirty-two strains were tested. Symbols +, zones of transparency (see text).
+++
1 74 28 64 2 94 83
+
++
69 23 60 26 72 36
98 1 1 + +, and + + + indicate
4 16
+++
31 68 9 38 77 23 11 89 13 87 2 93 6 95 4 different sizes of
TABLE 4. Bacteriolytic activity of S. albus strains on chemically defined media varying in peptone, glucose, and NaCl concentrations a % of strains showing zones of transparency
Medium composition _
1% peptone ++ ~~~~~+
3.5% peptone
2.2% peptone +++
+
++
+++
+
++
+++
No glucose, no NaCl 1 35 43 45 7 0.3% glucose, no NaCl 15 1 1 26 50 5 1 0.6% glucose, no NaCl 15 22 28 5 1 No glucose, 1.5% NaCl 57 3 72 75 5 16 2 0.3% glucose, 1.5% NaCl 53 5 1 71 23 42 56 2 0.6% glucose, 1.5% NaCl 48 1 65 17 46 47 3 No glucose, 3% NaCl 70 6 71 9 1 71 10 1 0.3% glucose, 3% NaCl 46 44 2 34 40 34 23 49 14 0.6% glucose, 3% NaCl 36 43 3 39 34 30 23 25 34 a +, One hundred and thirty-two strains were tested. Symbols + +, and + + + indicate different sizes of zones of transparency (see text).
one particular medium, even though the latter did not prove optimal for all strains. To confirm these results, a greater number of staphylococci (318 S. aureus and 603 S. albus strains) were tested on a similar medium of the following composition: 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, 0.9% agar, pH 6.9. The reduced agar content ensured an easier diffusion into the medium of substances produced by growing bacteria. On this medium, all S. aureus strains were active and gave similar zones of transparency; S. albus proved active in over 99% of cases, although producing variable zones of transparency, both for size and features, from strain to strain. These results are summarized in Table 6. S. aureus homogeneity and S. albus heterogeneity regarding bacteriolytic activity were thus confirmed. It was also proved that the activity of virtually all staphylococcal strains could be detected by using a rich medium with
a suitable ionic strength. The very few strains not producing any zone of transparency do not invalidate this statement. It is known, in fact,
that separation of staphylococci from micrococci is sometimes uncertain in the absence of data on deoxyribonucleic acid base composition (3, 21).
DISCUSSION In accordance with data in a preliminary report (31), the present results indicate that the production of bacteriolytic activity is a characteristic common to virtually all Staphylococcus strains. The variable and lower incidences obtained by previous authors, particularly in connection with S. albus, could be explained by the following facts: (i) S. albus strains often require a richer medium than S. aureus strains to show a constantly detectable bacteriolytic activity; (ii) the NaCl concentration capable of ensuring optimal bacteriolytic activity is most often dif-
VOL. 16, 1977
BACTERIOLYTIC ACTIVITY OF STAPHYLOCOCCI
TABLE 5. Bacteriolytic activity of S. aureus and S. albus strains in response to different concentrations of NaCla % of strains showing zone of transparency
+
0 0.75 1.5 2.25 3 4 6 8
S. albus
S. aureus
% NaCl
94 10 2 64 93 41 1
++
85 93 36 7
+++
+
++
+++
5 5
17 40 62 53
2 7 5 44
1 1
60 26
34 36
3
56 29
2
a One hundred and thirty-two S. aureus and 132 S. albus strain were tested. Symbols +, + +, and + + + indicate different sizes of zones of transparency (see text).
TABLE 6. Bacteriolytic activity of 318 S. aureus and 603 S. albus strains on a single appropriate medium Bacteriolytic
No. of strains
% of strains
251 67
79 21
4 327 256 16
Vol. 16, No. 1
Printed in U.S.A.
Bacteriolytic Activity in Staphylococci GIUSEPPE SATfA, PIETRO E. VARALDO,* GRAZIA GRAZI, AND ROBERTA FONTANA Istituto di Microbiologia dell'Universitd di Genova, Genoa, Italy Received for publication 26 August 1976
The bacteriolytic activity ofStaphylococcus aureus and Staphylococcus albus strains was tested with various media. Whereas S. aureus strains were found to be active under all conditions, the percentage of active S. albus strains was significantly influenced by the composition of the medium. Ionic strength and concentration of the organic nitrogen source were found to be the main factors affecting the expression of bacteriolytic activity of staphylococci. Virtually all of 318 S. aureus and 603 S. albus strains were active on a medium containing 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, and 0.9% agar. The optimal conditions for the bacteriolytic activity of S. aureus strains were different from those of most S. albus strains. Within S. albus, optimal conditions differed also from strain to strain. It is suggested that further studies on this subject may prove useful for the identification and taxonomy of staphylococci. A possible relationship between the production of extracellular bacteriolytic enzymes and pathogenic properties of staphylococci is also considered. The production of bacteriolytic enzymes by a variety of bacteria is well established (8, 9, 24, 27, 36). Several of these enzymes have been studied in detail (10, 18, 32-35, 39). However, their role in nature and particularly in the physiology of cells, even though hypotheses on this subject have been advanced by several authors (12, 30), is largely unknown. This may also be due to the fact that little attention has been paid to whether these enzymes are produced just by sporadic strains or by the entire bacterial taxa. No bacterial family or genus in which all strains produce bacteriolytic activity has been reported so far. Moreover, different yet related bacteria might produce different bacteriolytic enzymes detectable under different testing conditions. Strains of Staphylococcus aureus are known to produce extracellular bacteriolytic enzymes (25, 26, 38). It is now well established that the production of such enzymes can be considered a characteristic common to all S. aureus strains; on the contrary, discordant findings have been reported with regard to the production of bacteriolytic enzymes by Staphylococcus albus strains (1, 2, 13-17, 19, 20, 22, 23). (In this report, the old name S. albus refers to all staphylococcal strains other than S. aureus. This name is preferred to both S. epidermidis and "coagulase-negative staphylococci," the former having recently been used to indicate only a particular group of staphylococci other than S. aureus [6, 7, 29] and the latter being confus-
ing because of the occurrence of coagulase-negative strains of S. aureus [4, 31].) In this study we have tested the bacteriolytic activity of several hundred staphylococcal strains under various conditions. The results obtained showed that a particularly rich growth medium and a relatively high sodium chloride concentration are required for detecting the bacteriolytic activity of many S. albus strains. Under ideal conditions, all S. aureus and S. albus strains were found to produce easily detectable bacteriolytic activity. However, the bacteriolytic activity of S. aureus strains appeared different, and easily distinguished, from that of S. albus strains. MATERIALS AND METHODS Bacterial strains. A total of 318 S. aureus and 603 S. albus strains were used in this study. All strains were of human origin. Most were isolated from clinical materials, including blood, urine, sputum, throat swabs, etc., that were examined in the Clinical Bacteriology Laboratory of the Institute of Microbiology of Genoa University. Some strains were also isolated from the skin, nose, and throat of healthy volunteers. All strains were gram-positive and catalase-positive cocci and were recognized as belonging to the genus Staphylococcus (5, 11, 28). Subsequently, S. aureus strains were identified according to current criteria (4, 6, 7). All strains not meeting such criteria were indicated as S. albus. Preparation of the substrate for bacteriolytic activity. Heat-killed cells of Micrococcus lysodeikticus ATCC 4698 were used as substrate for bacteriolytic 37
38
SATTA ET AL.
activity. Samples (0.1 ml) of a 48-h broth culture of this strain were uniformly distributed to the surface of several plates containing brain heart infusion agar (BHIA, Difco). Plates were incubated for 48 to 72 h at 30°C. The dense bacterial growth was then harvested and washed twice with distilled water. Cells were finally suspended in distilled water, and the concentration of the suspension was spectrophotometrically adjusted at 400 optical units/ml. This suspension was heated twice at 121°C for 20 min and stored at 4°C. Media. To study the variability of bacteriolytic activity of staphylococci, the following commercial and synthetic media were used: BHIA (Difco); tryptose phosphate broth (Difco) supplemented with 1.5% agar (TP1); nutrient agar (NA; Difco); ToddHewitt broth (Oxoid) supplemented with 1.5% agar (TH); D.S.T. agar (Oxoid); tryptic soy agar (TSA; Biolife); 1.2% peptone (Difco), 1% beef extract, 0.5% glucose, 0.25% disodium phosphate, and 1.5% agar, pH 6.9 (SG); 3% Peptonum Siccum (A. Costantino & C., Favria, Torino, Italy), 0.2% glucose, 0.5% sodium chloride, and 1.5% agar, pH 6.9 (M9). To study the relationship between the bacteriolytic activity of staphylococci and the composition of the growth medium, 27 different media were prepared by combining three different concentrations of peptone (1, 2.2, and 3.5%; Difco), three of glucose (0, 0.3, and 0.6%), and three of sodium chloride (0, 1.5, and 3%). These media were supplemented with 1.5% agar. The pH was adjusted at 6.9. For more detailed studies on the effects of sodium chloride, a base medium (1.2% Peptonum Siccum [Costantino], 0.3% glucose, and 1.5% agar, pH 6.9) was supplemented with NaCl to obtain eight different final concentrations: 0, 0.75, 1.5, 2.25, 3, 4, 6, and 8%. A rich medium containing 3% Peptonum Siccum (Costantino), 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, and 0.9% agar, pH 6.9, was used to test the bacteriolytic activity of all 921 strains studied. All media were sterilized by autoclaving and stored at 40C. A suspension of heat-killed M. lyso-
deikticus cells was added to the melted media to
obtain a final concentration of 2 optical units/ml. The media were then poured into petri dishes. Inoculation of the media. A multiple inoculating device manufactured in our laboratory was used in this study. This device contains 22 appropriately disposed needles, which were sterilized by flaming. Simultaneous inoculation of 22 isolates from a master plate into the medium contained inr a petri dish could be performed by this device. Master plates were made by marking the surfaces of BHIA plates with the sterile needles of the device and heavily inoculating the 22 respective sites with the strains studied. Twelve- to 24-h incubation produced dense bacterial growth, which could be picked up with a single impression of the corresponding needles and inoculated into the above-mentioned media. Examination of the bacteriolytic activity. The inoculated media were incubated for 24 h at 37°C and then maintained for 12 h at room temperature.
INFIECT. IMMUN. Zones of transparency occurring around the spots of bacterial growth indicated lysis of substrate by the growing strains. However, a certain variability in the diameters of the spots of bacterial growth could be observed, so that the distance between the edge of the spot and the edge of the zone of transparency, rather than the diameter of the entire zone of transparency, was considered more indicative of the degree of bacteriolytic activity. The results were scored according to the following scheme: +++, very large zone of transparency, exceeding the spot of bacterial growth by more than 6 mm; + +, large zone of transparency, exceeding the spot of bacterial growth by 3 to 6 mm; +, moderate to small zone of transparency, exceeding the spot of bacterial growth by less than 3 mm; -, no detectable zone of transparency.
RESULTS Bacteriolytic activity of S. aureus and S. albus strains on various commercial and synthetic media. Table 1 summarizes the incidence of bacteriolytic activity production by S. aureus and S. albus strains as reported to date. Authors who had found virtually 100% of S. aureus and contrasting percentages of S. albus strains able to produce bacteriolytic activity had generally been using different media. This would suggest that the bacteriolytic activity of S. albus strains may require particular and more critical conditions than S. aureus. Therefore, we tested the bacteriolytic activity of 132 S. aureus and 132 S. albus strains on various commercial and synthetic media, some approximating those used by other authors. On these media all S. aureus strains were active, whereas S. albus strains showed different activity from one medium to another (Table 2). Significance of the essential components of growth media. The same strains were then tested on a set of chemically defined media containing varying amounts of peptone as an organic nitrogen source, glucose as an organic carbon source, and NaCl as a salt, in order to confirm the dependence of bacteriolytic activity on the medium composition and to identify the components responsible for the variability ofS. albus activity. Results concerning S. aureus and S. albus strains are summarized in Tables 3 and 4, respectively. All S. aureus and S. albus strains were found to produce an easily detectable bacteriolytic activity on some of these media. The importance of the organic nitrogen concentration was evident: both the incidence of bacteriolytic activity production and the size of zones of transparency showed a general increase when the peptone concentration was raised from 1 to 3.5%. However, the ionic strength was the most important parameter affecting bacteriolytic activity. The lytic activ-
VOL. -16, 1977
BACTERIOLYTIC ACTIVITY OF STAPHYLOCOCCI
39
TABLz 1. Reported incidence of bacteriolytic activity production by staphylococcal strains S. aureus S. albus Author (reference)
Strains
tested 30 55 133 82 126 503 1,189 50 28 100
Active strains
Strains %
tested
Active strains
%
Welsch (37) 30 100 Kashiba et al. (20) 55 100 42 11 26.2 Omori et al. (23) 133 100 203 3 1.5 Goldbach and Haenel (13) 82 100 43 15 34.9 Jay (19) 120 95.2 49 4 8.2 Hawiger (15) 503 100 35 13 37.1 Grossgebauer et al. (14) 1,170 98.6 156 0 0 Arvidson et al. (2) 50 100 15 1 6.7 Afanasieva and Shevyakova (1) 28 100 14 5 35.7 Holt (17) 100 100 238a 33 13.9 Heczko (16) 478 62 13 Kloos and Schleifer (22) 46 46 100 886 422 47.6 a Including 62 strains reported as belonging to Baird-Parker's subgroups Ml to M4, which should now be considered Staphylococcus strains (6).
TABLE 2. Bacteriolytic activity of S. aureus and S. albus strains on various commercial and synthetic
chloride. The relationship between NaCl concentration of the medium and lytic activity exmediaa hibited by staphylococcal strains was examined in more detail, using a base medium to which % of strains showing zones of transparency NaCl was added in various concentrations. The results given in Table 5 confirm and expand on S. aureus S. albus diumb the data already shown in Tables 3 and 4. All S. + ++ +++ + ++ +++ aureus strains were active in the range of 0.75 BHIA 97 3 36 11 1 to 3% NaCl concentration. Only 2% of S. albus TP1 90 10 43 17 3 strains were inactive at the concentration of NA 97 3 37 1 2.25% NaCl. However, all strains examined, TH 95 5 44 8 both S. aureus and S. albus, did produce a zone DST 6 94 50 7 2 of transparency, at least at one of the NaCl TSA 93 7 20 1 SG 29 71 5 43 testing concentrations. The optimal NaCl conM9 92 8 66 13 3 centrations for bacteriolytic activity of S. aureus strains (0.75 and 1.5%) were found to be a One hundred and thirty-two S. aureus and 132 S. albus strains were tested. Symbols +, + +, and lower than those for most S. albus strains (2.25 + + + indicate different sizes of zones of transpar- and 3%). It was also confirmed that all S. aureus strains were similarly affected by the ionic ency (see text). b See Materials and Methods for abbreviations of strength of the medium with respect to their media. bacteriolytic activity. On the contrary, S. albus strains exhibited heterogeneous bacteriolytic ity of S. aureus strains varied uniformly with activity patterns. In fact, even though the madifferent concentrations of NaCl. S. albus jority ofthe latter strains showed optimal activstrains usually differed from S. aureus regard- ity at NaCl concentrations of 2.25 and 3%, some ing their lytic activity in response to the same were most active at lower concentrations (0.75 parameter; furthermore, they showed marked and 1.5%), or even in a medium without NaCl. differences among themselves. The effect of Moreover, the highest incidence of very large ionic strength appeared to be most pronounced zones of transparency (+++ +) was found at an under conditions of low peptone concentration. NaCl concentration of 4%, which inhibited the The carbon source concentration seemed to be activity of roughly one-third of these strains. of less importance: only under conditions of Finally, in media of optimal ionic strengths adequate peptone and NaCl concentrations did different S. albus strains exhibited marked the glucose concentration prove to be signifi- variability in the size and features of the zones cant. Perhaps the utilization of glucose by of transparency. Incidence of bacteriolytic activity among growing bacteria and the accompanying local reduction of pH interfered with expression of 921 staphylococci. As already stated elsewhere in this report, all strains tested were able to bacteriolytic activity. Further analysis of the effect of sodium produce a detectable bacoriolytic activity on
40
INFECT. IMMUN.
SATTA ET AL.
TABLE 3. Bacteriolytic activity of S. aureus strains on chemically defined media varying in peptone, glucose, and NaCI concentrationsa % of strains showing zones of transparency Medium composition +
++
3.5% peptone
2.2% peptone
1% peptone +++
+
++
No glucose, no NaCl 89 98 0.3% glucose, no NaCl 6 82 6 0.6% glucose, no NaCl 73 No glucose, 1.5% NaCl 91 9 0.3% glucose, 1.5% NaCl 2 11 87 0.6% glucose, 1.5% NaCl 84 16 No glucose, 3% NaCl 98 2 98 0.3% glucose, 3% NaCl 21 78 1 2 0.6% glucose, 3% NaCl 7 92 1 1 a One hundred and thirty-two strains were tested. Symbols +, zones of transparency (see text).
+++
1 74 28 64 2 94 83
+
++
69 23 60 26 72 36
98 1 1 + +, and + + + indicate
4 16
+++
31 68 9 38 77 23 11 89 13 87 2 93 6 95 4 different sizes of
TABLE 4. Bacteriolytic activity of S. albus strains on chemically defined media varying in peptone, glucose, and NaCl concentrations a % of strains showing zones of transparency
Medium composition _
1% peptone ++ ~~~~~+
3.5% peptone
2.2% peptone +++
+
++
+++
+
++
+++
No glucose, no NaCl 1 35 43 45 7 0.3% glucose, no NaCl 15 1 1 26 50 5 1 0.6% glucose, no NaCl 15 22 28 5 1 No glucose, 1.5% NaCl 57 3 72 75 5 16 2 0.3% glucose, 1.5% NaCl 53 5 1 71 23 42 56 2 0.6% glucose, 1.5% NaCl 48 1 65 17 46 47 3 No glucose, 3% NaCl 70 6 71 9 1 71 10 1 0.3% glucose, 3% NaCl 46 44 2 34 40 34 23 49 14 0.6% glucose, 3% NaCl 36 43 3 39 34 30 23 25 34 a +, One hundred and thirty-two strains were tested. Symbols + +, and + + + indicate different sizes of zones of transparency (see text).
one particular medium, even though the latter did not prove optimal for all strains. To confirm these results, a greater number of staphylococci (318 S. aureus and 603 S. albus strains) were tested on a similar medium of the following composition: 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride, 0.9% agar, pH 6.9. The reduced agar content ensured an easier diffusion into the medium of substances produced by growing bacteria. On this medium, all S. aureus strains were active and gave similar zones of transparency; S. albus proved active in over 99% of cases, although producing variable zones of transparency, both for size and features, from strain to strain. These results are summarized in Table 6. S. aureus homogeneity and S. albus heterogeneity regarding bacteriolytic activity were thus confirmed. It was also proved that the activity of virtually all staphylococcal strains could be detected by using a rich medium with
a suitable ionic strength. The very few strains not producing any zone of transparency do not invalidate this statement. It is known, in fact,
that separation of staphylococci from micrococci is sometimes uncertain in the absence of data on deoxyribonucleic acid base composition (3, 21).
DISCUSSION In accordance with data in a preliminary report (31), the present results indicate that the production of bacteriolytic activity is a characteristic common to virtually all Staphylococcus strains. The variable and lower incidences obtained by previous authors, particularly in connection with S. albus, could be explained by the following facts: (i) S. albus strains often require a richer medium than S. aureus strains to show a constantly detectable bacteriolytic activity; (ii) the NaCl concentration capable of ensuring optimal bacteriolytic activity is most often dif-
VOL. 16, 1977
BACTERIOLYTIC ACTIVITY OF STAPHYLOCOCCI
TABLE 5. Bacteriolytic activity of S. aureus and S. albus strains in response to different concentrations of NaCla % of strains showing zone of transparency
+
0 0.75 1.5 2.25 3 4 6 8
S. albus
S. aureus
% NaCl
94 10 2 64 93 41 1
++
85 93 36 7
+++
+
++
+++
5 5
17 40 62 53
2 7 5 44
1 1
60 26
34 36
3
56 29
2
a One hundred and thirty-two S. aureus and 132 S. albus strain were tested. Symbols +, + +, and + + + indicate different sizes of zones of transparency (see text).
TABLE 6. Bacteriolytic activity of 318 S. aureus and 603 S. albus strains on a single appropriate medium Bacteriolytic
No. of strains
% of strains
251 67
79 21
4 327 256 16
