Vol. 15, No. 1 Printed in U.S.A.
INFCTION AND IMMUNITY, Jan. 1977, p. 59-65
Copyright © 1977 American Society for Microbiology
Production and Detection of Staphylococcal Elastase DONALD P. HARTMAN AD RICHARD A. MURPHY* Departments of Microbiology and Oral Diagnosis,* University of Illinois at the Medical Center, Chicago, Illinois 60612
Received for publication 30 July 1976
The optimum conditions were determined for the production and detection of staphylococcal elastase from Staphylococcus epidermidis. Optimum production and recovery took place when the dialysis membrane technique was utilized with brain heart infusion agar incubated for 44 h under 15% CO2 and harvested in physiological saline. Near-optimal production took place by 28 h, and this was found more useful for routine use. Incorporation of elastin into the culture medium or the inoculum did not result in higher levels of elastase production. The detection system consisted of 0.25% particulate elastin suspended in a pH 7.0, 0.05 M tris(hydroxymethyl)aminonuethane-hydrochloride-buffered solidified plated medium, Crude undialyzed elastase was best detected when the medium contained either agarose and 10-3 M calcium or purified agar without additional additives. Crude dialyzed elastase was best detected when the medium contained agarose and 10-3 M disodium ethylenediaminetetraacetic acid.
Staphylococci have long been known to be the etiological agents in a large variety of diseases. In most cases, coagulase-positive staphylococci have been implicated, However, coagulase-negative staphylococci have been increasingly recognized as the etiological agent in a variety of diseases, including bacterial endocarditis (3, 11, 13). Coagulase-negative staphylococci have generally been found to either lack certain virulence factors or possess them in smaller quantities than strains of coagulase-positive staphylococci. This, however, is not true for the staphylococcal elastase, which is produced in greater quantities by strains of Staphylococcus epidermidis than by strains of S. aureus (7). Since its discovery (15), staphylococcal elastase has been implicated in a condition called "perifollicular elastolysis," which is characterized by a selective and almost complete loss of elastic fibers in the immediate vicinity of hair follicles that have been infected with elastaseproducing strains of S. epidermidis (16). It has also been postulated that this enzyme plays a role in lung infections (16) and periodontal disease (7). The determination of the role played by elastase in any infection requires that the elastase be obtained in a purified state so that its activity can be ascertained without interference from other biochemically active substances. Before purification can be carried out, the optimal conditions for detecting elastase activity in cellfree preparations and the optimal conditions for the in vitro production of elastase must be de-
termined. Our investigations concerning these factors are described below. (This work was submitted as part of a thesis submitted by D.P.H. in partial fulfillment of the requirements for the M.S. degree in microbiology and elective requirements of the College of Dentistry, University of Illinois.) MATERIALS AND METHODS Strain of Staphylococcus. Strain 332 of S. epidermidis, which was isolated from the oral cavity, was utilized in this investigation. It fermented glucose; aerobically degraded lactose and maltose but not mannitol; produced acetoin but not coagulase; hemolyzed the erythrocytes of human, horse, rabbit, and sheep blood; and possessed phosphatase, gelatinase, caseinase, staphylokinase, nuclease, lysozyme, lipase, and egg yolk factor, as well as elastase activity. Detection of elastase in culture supernatants. In determining the optimum conditions for detecting elastase activity, preparations of crude elastase were produced by the dialysis membrane technique (8, 9) using brain liver heart agar (BLHA; Difco Laboratories) plates incubated under 15% CO2 at 370C for 24 h. The crude elastase preparation was filter sterilized (0.45 ,um) and stored at 4-C. Elastin agar plates for detecting elastase activity were prepared by incorporating 0.25% (wt/vol) elastin (Sigma Chemical Co.) into each medium before autoclaving and dispensing it into sterile plastic petri plates. The plates were generally maintained overnight at room temperature before use. In preliminary experiments, wells 4, 9, and 12 mm in diameter were punched into the medium and filled with serial twofold dilutions of crude elastase. The 9- and 1259
60
INFECT. IMMUN.
HARTMAN AND MURPHY
mm-diameter wells were equally satisfactory for detecting the smallest concentration of elastase. Wells 9 mm in diameter were thus employed for all future experiments since they utilized less material. Unless mentioned otherwise, after the addition of crude elastase to the wells, plates were maintained at 40C for 4 days, at which time the diameters of the zones of clearing were measured. Although results differed in the various media utilized, preparations active at dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16 generally yielded zone diameters of approximately 10, 14, 17, 23, and 26 mm, respectively. The optimum pH and buffer system for detecting elastase activity were determined by placing samples of crude elastase into buffered plates prepared using 1% (wt/vol) Ionagar no. 2 (Oxoid Ltd). The buffers used and the pH ranges they covered were: citrate, pH 5.0 to 5.8; tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.0 to 9.0; Tris-maleate, pH 5.2 to 8.2; sodium-sodium phosphate, pH 5.6 to 6.6; and potassium-sodium phosphate, pH 6.0 to 8.0. These were formulated according to Long (6). Potassium-sodium phosphate-buffered saline agars were also prepared by adding sodium chloride to the potassium-sodium phosphate buffers such that their final ionic strengths were equivalent to that of 0.15 M NaCl as determined with a conductivity meter. In addition, a commercial desiccated potassium-sodium phosphate-buffered saline of pH 8.3 (hemagglutination buffer, Difco) was utilized. The effect of sodium chloride on elastase detection was determined in unbuffered saline agars containing 0.0, 0.05, 0.1, 0.15, 0.2, 0.5, 1.0, 1.5, and 2.0 M NaCl in 1.0% (wt/vol) Ionagar no. 2. Using a crude elastase preparation obtained by harvesting the cultures in Tris-hydrochloride buffer (pH 7.0), the effects of various agars on elastase detection were determined. Agar (Difco), purified agar (Difco), and Ionagar no. 2 were prepared at a 1.5% concentration, and agarose (Wilson Diagnostics Inc.) was prepared at a 1% concentration in the same Tris-hydrochloride buffer. Plates were incubated at 370C for 24 h and then at 40C for 3 more days before the results were read. This splitincubation procedure was utilized here and in the following experiments when it was noted that the culture supernatant frequently required 2 or 3 days to enter the agar of freshly made plates if they were directly incubated at 4VC. The roles of metal ions and ethylenediaminetetraacetic acid (EDTA) in elastase detection were assessed using a culture filtrate harvested in Trishydrochloride buffer (pH 7.0). This was placed into plates of similarly buffered media containing purified agar (1.5%) or agarose (1%) and either 10-3 M CaCl2, CuCl2, FeCl3, MgCl2 6H20, Zn(C2H302)2, Na2EDTA, or no added salt. A 5-ml sample of this elastase preparation was also exhaustively dialyzed against the Tris buffer and filter sterilized. This preparation was tested in the above media as well as in Tris-hydrochloride-buffered agar (1.5%; Difco) and Ionagar no. 2 (1.5%). Optimal conditions for the production of elastase. Unless mentioned otherwise, studies on
elastase production utilized the dialysis membrane technique with batches of 10 plates each being incubated under 15% CO2 at 37°C for 24 h. Each batch was harvested in 3 ml of sterile saline, filter sterilized (0.45 ,um), and stored at 4°C. All culture filtrates were assayed by placing them in 9-mmdiameter wells in Tris-hydrochloride-buffered (pH 7.0) purified agar (1.5%) containing 0.25% (wt/vol) elastin. Activity was ascertained after 24-h incubation at 37°C in a humidified incubator, followed by 72 h of incubation at 4°C. The following media and cultural conditions were used in determining the best medium for the production of elastase: BLHA (containing 1.5% agar [Difco]), brain heart infusion agar (BHIA; Difco), tryptic soy agar (TSA; Difco), Pharmamedia (Traders Oil Mill Co.), heart infusion agar (HIA; Difco), heart infusion agar with Oxoid yeast extract (2.5%, wt/vol; Oxoid), brain liver heart semisolid agar (contains 0.175% agar [Difco]), and brain heart infusion broth (Difco), all incubated in petri plates, and brain heart infusion broth, incubated with shaking in a flask. Pharmamedia was prepared at concentrations of 2 and 4% (wt/vol) in distilled water containing 0.5% (wt/vol) NaCl and 1.5% (wt/vol) agar (Difco). All media were incubated in an atmosphere of 15% CO2.
RESULTS
Stability of crude elastase preparations. A crude elastase preparation stored at 4°C was tested for enzymatic activity at 1 day and at 1, 3, and 5 weeks after harvesting using Trishydrochloride-buffered (pH 7.0) elastin agar (Ionagar no. 2) plates. The preparation initially produced a zone of elastolysis 24 mm in diameter. This dropped to 20 mm within week 1, with a further decrease to 18 and 16 mm by weeks 3 and 5, respectively. Due to the instability of the elastase preparations, they were generally utilized within 1 week after their production. Effect of incubation time on the detection of elastase in culture supernatants. Using Tris-hydrochloride-buffered (pH 7.0) elastin agar (Ionagar no. 2) plates and serial twofold dilutions of elastase, activity was evident for
the more concentrated preparations within 24 h of placing the material in the wells. Zone diameters around these preparations continued to increase over the entire 8-day period of observation. Dilute preparations required up to 4 days of incubation before traces of activity were evident. Plates were therefore routinely incubated for 4 days before elastase activity was measured. Effect of buffer system and pH on the detection of elastase in culture supernatants. The elastase preparation was active in each of the buffer systems utilized (Fig. 1 and 2). In the plates incubated at 370C (Fig. 1), optimum ac-
VOL. 15, 1977 15*
14-
P0,
K-No
P040.0
NO-No
IM
NoCl
P0,
Tris-HCI
13~~~~~~~~ Tr i3- Moleote
U) 13-
2,
K-No
130 A
Citrate
12-
08E I0-
6
5
7
8
pH
1. Effect of buffer system and pH of staphylococcal elastase at 37'C.
FIG. tection
26-
* K- No
P04
*
P04
K-No
A No -Noa 0
25]
Trio
the de-
on
I M NCI
PO$
MCI
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Tris-MiJeote
A
Citrate
24237
2272172019fl
.rh 02
a
C E
312II
100-
-T-
I 5
6
7
9
pH
FIG. tection
2. Effect of buffer system and pH of staphylococcal elastase at 4C(2
tivity occurred
to
an
Tris-maleate buffer,
equal degree
on
in the
and in both the
pH
the de-
pH
6.0
6.2 and
potassium-sodium phosphate buffers containing 0.1 M NaCl. In the plates incubated at 400 (Fig. 2) the greatest elastase activity was found in the four potassium-sodium phosphate buffers covering the range of pH 6.0 to 7.0. Of almost equal activity were the Tris-maleate buffer at pH 6.8 and the Tris-hydrochloride buffer at pH 7.0O. The addition of 0.1 M Na~l to the potassium-sodium phosphate buffers depressed elastase activity at all pH values in the plates incubated atr40, while generally potentiating activity in the plates incubated at 3700. No one buffer system or single buffer was clearly superior to the others. The potassium-
STAPHYLOCOCCAL ELASTASE 61 sodium phosphate buffer at pH 6.6 and the Trishydrochloride buffer at pH 7.0, both of which yielded high activity, were considered for further studies due to the frequency of use of phosphate and Tris-hydrochloride buffer systems by other investigators. Effect of sodium chloride on the detection of elastase in culture supernatants. Using unbuffered agars containing 0.00, 0.05, 0.1, 0.15, 0.2, 0.5, 1.0, and 2.0 M Na~l, elastase activity was detected at 400 in those agars containing 0.05 to 0.2 M NaCi, with a peak of activity at 0.15 M (Table 1). No elastase activity was seen in any of the agars at 3700. Although the activity seen at optimum NaCl strength was low, it was consistent with the activity of elastase at the reduced pH of these media (pH 5.6 to 5.9). Since the greatest activity was shown in the buffered and NaCl agars at 400, the following experiments all utilized incubation at this temperature. Effect of agars on the detection of elastase in culture supernatants. In assessing the effect of the buffer system, pH, and sodium chloride on the activity of elastase in agar media, Ionagar no. 2 was used as the solidifying agent. Other agars were then tested to determine whether one was superior. The largest zones of activity were obtained with purified agar (26 mm) followed by agar (Difco; 22 mm), Ionagar no. 2 (19 mm), and agarose (14 mm). Purified agar thus appeared to be the solidifying agent of choice for the detection of elastase activity in the crude preparations. Effect of metal ions and EDTA on the detection of elastase in culture supernatants. Metal ions or Na2EDTA were incorporated into purified agar and agarose to determine whether a metallic cofactor would enhance the activity of elastase. Dialyzed and undialyzed elastase preparations were utilized. Because TABLE 1. Effect of different polarities of NaCi on elastolytic activity
6.6
NaCI molarity
Zone diam (MM)a
40C 3rC 0 0 0.0 11 0 0.05 14 0 0.10 0 15 0.15 13 0 0.20 0 0 0.50 0 0 1.00 0 0 1.50 0 0 2.00 a Diameter of zone of elastolysis around a 9-mmdiameter wall.
62
INFECT. IMMUN.
HARTMAN AND MURPHY
calcium, copper, and zinc form insoluble salts with potassium-sodium phosphate buffers, the Tris-hydrochloride buffer at pH 7.0 was utilized. The activity of the undialyzed preparation in purified agar was not enhanced by any of the metal ions, whereas zinc and EDTA partially inhibited and copper completely inhibited the elastase (Table 2). The undialyzed preparation generally showed lower activity in agarose than in purified agar. Calcium completely restored this activity, whereas the presence of iron, magnesium, and EDTA partially restored activity. Copper and zinc completely inhibited activity. The dialyzed preparation was inactive in all purified agar media except that containing EDTA, in which a low level of elastase activity was noted (Table 2). The dialyzed preparation was also inactive in agarose in the absence of metal ions or in the presence of copper or zinc. Slight activity was observed in agarose containing calcium, iron, and magnesium salts. Activity of the dialyzed preparation was greatest in agarose containing EDTA. The dialyzed preparation, when tested in buffered agars made with either agar (Difco) or Ionagar no. 2 and containing no added metallic salts or EDTA, exhibited no elastase activity. Elastin suspended in either Tris-hydrochloride-buffered (0.5 M, pH 7.0) purified agar or in similarly buffered agarose containing 10-3 M Ca appeared to be the best medium for the detection of elastase activity in undialyzed preparations. Thus, for purposes of simplicity and economy, the Tris-hydrochloride-buffered purified agar plates containing elastin were used in the following studies on elastase production. The best medium for the detection of elastase activity in dialyzed preparations conTABLE 2. Effect of the incorporation of metal ions into purified agar and agarose on the elastase activity of crude and dialyzed preparations
sisted of elastin suspended in Tris-hydrochloride-buffered (0.05 M, pH 7.0) agarose containing 10-3 M Na2EDTA. Effect of harvesting fluid on elastase recovery. Using BLHA in the dialysis membrane technique, equal quantities of elastase were recovered when the membranes were harvested in either distilled water, potassium-sodium phosphate buffer (pH 7.0), or 0.15 M NaCl. However, when either water or the potassiumsodium phosphate buffer was used, a cloudiness appeared in the clear culture filtrates after standing 1 or 2 h at 4°C. This cloudiness disappeared when solid sodium chloride was added to either of these fluids, with clearing occurring when the salt concentration reached 0.17 M NaCl. Since this precipitation was not seen when membranes were harvested with saline (0.15 M), saline was chosen as the harvesting fluid. Effect of incubation time on elastase production. At 4-h intervals over a period of 48 h, two samples of 10 plates each were harvested and simultaneously tested for elastase activity. The first detectable elastolytic activity occurred at 12 h (Fig. 3). After 20 h the rate of increase of elastase activity began to taper off, and increases beyond 32 h of incubation were minimal. An incubation time of 28 h yielded nearoptimum levels of elastase. This was thus chosen for future use to make the total time involved in inoculation, incubation, and harvesting more convenient for routine use. With BLHA as the nutrient medium, the dialysis membrane harvests, after centrifugation, became increasingly viscous as the incubation time increased. By 24 h, harvests were so thick that they only passed through 0.45-,um filters with great difficulty. This gelatinous nature of the material offered no difficulties in detecting its elastase activity, but may present difficulties during later purification studies. 24-
Zone diam (mm) witha: Ion
Undialyzed elastase
Purified
22 -
Dialyzed elastase
20
O6
w
,4
0 o E
18-
Purified agar
Agarose
27 27
Agarose 14 28
0 0
0 12
16 It E 14 Cw 12 -
0
0
0
0
26 20 Mg 27 23 Zn 17 0 EDTA 22 25 a See footnote a, Table 1.
0 0 0 14
11 11 0 20
10 -
agar
None Ca Cu Fe
m
.o
_
0-
0
4
8
12 16 20 24 28 32 36 40 44 48 Hours
FIG. 3. Effect of duration of incubation on elastase production.
VOL. 15, 1977
STAPHYLOCOCCAL ELASTASE
Effect of culture medium and growth conditions on elastase production. Various nutrient media were examined for their ability to promote elastase production. All media were harvested at 28 h. In each medium tested, the elastase activity of the resulting preparation appeared directly correlated with the amount of bacterial growth. BLHA and BHIA yielded the highest elastolytic activity (Table 3). HIA, when fortified with yeast extract, yielded the next highest quantity of elastase. Much smaller quantities of elastase were also produced in unfortified HIA and TSA. All other media utilized failed to produce detectable amounts of staphylococcal elastase. The dialysis membrane technique thus appeared necessary for the production of elastase, and either BHIA or BLHA was the medium of choice. The harvest fluid of BHIA was less viscous than that of the BLHA harvest, and for this reason BHIA was chosen for further studies. Effect of atmosphere on elastase production. Plates were incubated for 1, 2, and 7 days in either a humidified incubator, a humidified incubator with the addition of 15% CGO, or under anaerobic conditions produced by the GasPak anaerobic system (BioQuest). Incubation in air alone for 1 day yielded material producing an 11-mm-diameter zone of elastolysis. Longer incubation in air, however, resulted in inactive preparations. Incubation in the presence of C00 yielded material producing an 18-mm-diameter zone of elastolysis after 1 day, 12-mm diameter after 2 days, and inactive preparations after 7 days. No elastolytic activity could be detected using the dialysis membrane technique under anaerobic conditions, TABLE 3. Effect of culture medium and growth conditions on elastase production Culture medium Brain heart infusion agar Brain liver heart agar Tryptic soy agar Heart infusion agar Heart infusion agar with yeast extract Pharmamedia, 2%
Pharmamedia, 4%
Culture
vessel
Zone
diam (mm)a
Plates
22
Plates Plates Plates Plates
21 13 12 18
Plates Plates Plates
0
Brain liver heart semisolid Plates Brain heart infusion broth Flask Brain heart infusion broth a See footnote a, Table 1.
0 0
0
0
63
even after 1 week of incubation. Growth of the culture on a HIA elastin agar plate under anaerobic conditions also failed to show elastase activity even after 6 weeks of incubation. Air enriched with 15% CO2 was thus better than either air alone or anaerobiosis and was considered optimal for elastase production. Inducibility of staphylococcal elastase. Use of the dialysis membrane technique with the various media employed in this investigation indicated that staphylococcal elastase may have been produced constitutively. To determine whether higher levels of enzyme activity could be induced, elastin (0.25%) was incorporated either into BHIA below the dialysis membrane or into the inoculum above the dialysis membrane. Higher levels of elastase were not produced in either case as compared with elastin-free cultural conditions. It appeared, therefore, that S. epidermidis culture 332 could not be induced to produce higher levels of elastase in BHIA.
DISCUSSION The development of an agar substrate plate method for the detection of staphylococcal elastase in cell-free preparations has several advantages over other methods of detection. Colorimetric methods using a chromogenic moiety attached to elastin (10, 12, 14) may not be specific (4) or sufficiently sensitive (5) even in the detection of pancreatic elastase, for which they were developed. The use of synthetic substrates suffers from similar problems (5). Although substrates other than plain particulate elastin may be developed and show specificity in a pancreatic elastase system, great caution must be exercised before ascribing similar specificity when using these materials in the study of bacterial elastases, such as the staphylococcal elastase, which may act in a different manner. For these reasons, an agar substrate plate method, utilizing particulate elastin, was developed for detecting and quantitating staphylococcal elastase. In addition to specificity, this method requires a minimum amount of preparation and many small samples can be easily tested and compared at one time. Crude, undialyzed staphylococcal elastase was best detected in a medium consisting of 0.25% elastin suspended in a pH 7.0, 0.05 M Tris-hydrochloride-buffered medium containing either agarose and 10-3 M calcium or purified agar without additional additives. Activity present in dialyzed crude preparations was best detected in pH 7.0, 0.05 M Tris-hydrochloridebuffered agarose containing 10-3 M Na2EDTA.
64
INFECT. IMMUN.
HARTMAN AND MURPHY
Overnight incubation at 370C followed by 3 days at 40C yielded the optimum activity in these media. These results are similar to those of Varadi and Saqueton (15), who also found optimum elastase activity at pH 7.0 in Tris buffer. They, however, found no inhibition by the inclusion of 0.15 M NaCl into the buffer, whereas we found a slight potentiation by NaCl at 37C and a significant inhibition at 40C. They also found that EDTA almost completely inhibited their crude elastase, whereas we found only a partial inhibition of undialyzed elastase. This might only be a result of a difference in concentration between these preparations and ours. The observed elastase activity appears to be the result of a complex interaction of elastase with metallic cofactors and metallic inhibitors. This was indicated by the loss of activity in dialyzed preparations and its subsequent activation or potentiation not only by the addition of calcium, iron, or magnesium, but also by EDTA, a seemingly contradictory situation. Perhaps our preparation contains more than one elastolytic moiety having different cofactor requirements. The difference in optimum detection mediums, depending on the dialyzed or undialyzed state of the elastase preparation, will be very significant when attempts to purify the elastase are carried out. It is expected that the more purified elastase will behave similar to that in the dialyzed preparation. The production of highly active preparations of staphylococcal elastase was facilitated by the use of the dialysis membrane technique. Maximum levels of elastase were produced using either BLHA or BHIA and incubating the plates in an atmosphere of 15% CO2 for 44 h. -Near optimal yields could be obtained after 28 h of incubation and, when the times involved in inoculation and harvesting were taken into account, this length of time was found most convenient for routine production of elastase. The use of this shorter incubation time, as well as the use of BHIA instead of BLHA, also resulted in a considerable decrease in the viscosity of the resultant elastase preparations. This viscosity is not unique to this strain of Staphylococcus, since we have reported a similar finding for S. aureus 146P (R. A. Murphy and R. Haque, Int. Assoc. Dent. Res. Abstr., p. 216, 1971) and also observed this with many other strains of staphylococci. This viscous material may be identical to the slime layer material reported by others (2) to be produced by strains of S. aureus. In this connection it may be of interest to note that
the material we observed was produced by strains of S. epidermidis as well as strains of S. aureus. A definite relationship between staphylococcal elastase and other staphylococcal proteases cannot be determined as yet. There are similarities between our elastase and the three proteases of S. aureus delineated by Arvidson and co-workers (1). These proteases were all active against casein, and their production was potentiated by the incorporation of yeast extract into the medium, as is the case for our elastase. One protease, however, was completely inactivated by EDTA, and the others were unaffected, whereas our elastase was only slightly inhibited under similar conditions. Although our elastase would appear to be a separate proteolytic entity, as has been mentioned, it is possible that our preparation contained more than one elastase and that one of these was inhibited by EDTA and the other(s) were unaffected. In addition, it may well be that the elastase of S. aureus possesses characteristics different from those identified here and it may behave identically to one ofArvidson's proteases, which were produced by the V8 strain of S. aureus. An explanation of these and other problems must, perhaps, await attempts to purif the staphylococcal elastase. ACKNOWLEDGMENT This investigation was supported by Public Health Service general research support grant 5501-RR-53-3-11 no. 108/
12. LITERATURE CITED 1. Arvidson, S., T. Holme, and B. Lindholm. 1972. The formation of extracellular proteolytic enzymes by Staphylococcus aureus. Acta Pathol. Microbiol. Sand. 80B:835-844. 2. Ekstedt, R. D., and J. M. Bernhard. 1973. Preparation and characterization of a slime layer material produced by Staphylococcus aureus. Proc. Soc. Exp. Biol. Med. 142:86-91. 3. Keys, T. F., and W. L. Hewitt. 1973. Endocarditis due to micrococci and Staphylococcus epidermidis. Arch. Intern. Med. 132:216-220. 4. Lieberman, J. 1972. Digestion of antitrypsin-deficient lung by leukoproteases, p. 189-203. In C. Mittman (ed.), Pulmonary emphysema and proteolysis. Academic Press Inc., New York. 5. Loeven, W. A. 1972. Elastolytic activity in blood, P. 269-274. In C. Mittman (ed.), Pulmonary emphysema and proteolysis. Academic Press Inc., New York. 6. Long, C. 1961. Biochemists' handbook, p. 29-42. E. and F. N. Spon Ltd. London. 7. Murphy, R. A. 1974. Elastase production by oral staphylococci. J. Dent. Res. 53:832-834. 8. Murphy, R. A., and R. Haque. 1967. Purification and properties of staphylococcal delta hemolysin. I. Production of delta hemolysin. J. Bacteriol. 94:13271333.
VOL. 15, 1977 9. Murphy, R. A., and R. Haque. 1974. Large scale production of staphylococcal delta hemolysin by the dialysis membrane technique. Can. J. Microbiol. 20:10611063. 10. Naughton, M. A., and F. Sanger. 1961. Purification and specificity of pancreatic elastase. Biochem. J. 78:156163. 11. Quinn, E. L., F. Cox, and E. H. Drake. 1966. Staphylococcic endocarditis. J. Am. Med. Assoc. 196:815-818. 12. Rinderknecht, H., M. C. Geokas, P. Silverman, Y. Lillard, and B. J. Haverback. 1968. New methods for the determination of elastase. Chin. Chim. Acta 19:327-339.
STAPHYLOCOCCAL ELASTASE
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13. Robbins-Browne, R. M., and H. J. Koornhof. 1973. The aetiology and bacteriological monitoring of bacterial endocarditis at the Johannesburg General Hospital. S. Afr. Med. J. 77:407-412.. 14. Sachar, L. A., K. K. Winter, N. Sicher, and S. Frankel. 1955. Photometric method for estimation of elastase activity. Proc. Soc. Exp. Biol. Med. 90:323-326. 15. Varadi, D. P. and A. C. Saqueton. 1968. Elastase from Staphylococcus epidermidis. Nature (London) 218: 468-470. 16. Varadi, D. P. and A. C. Saqueton. 1970. Perifollicular elastolysis. Br. J. Dermatol. 83:143-150.
INFCTION AND IMMUNITY, Jan. 1977, p. 59-65
Copyright © 1977 American Society for Microbiology
Production and Detection of Staphylococcal Elastase DONALD P. HARTMAN AD RICHARD A. MURPHY* Departments of Microbiology and Oral Diagnosis,* University of Illinois at the Medical Center, Chicago, Illinois 60612
Received for publication 30 July 1976
The optimum conditions were determined for the production and detection of staphylococcal elastase from Staphylococcus epidermidis. Optimum production and recovery took place when the dialysis membrane technique was utilized with brain heart infusion agar incubated for 44 h under 15% CO2 and harvested in physiological saline. Near-optimal production took place by 28 h, and this was found more useful for routine use. Incorporation of elastin into the culture medium or the inoculum did not result in higher levels of elastase production. The detection system consisted of 0.25% particulate elastin suspended in a pH 7.0, 0.05 M tris(hydroxymethyl)aminonuethane-hydrochloride-buffered solidified plated medium, Crude undialyzed elastase was best detected when the medium contained either agarose and 10-3 M calcium or purified agar without additional additives. Crude dialyzed elastase was best detected when the medium contained agarose and 10-3 M disodium ethylenediaminetetraacetic acid.
Staphylococci have long been known to be the etiological agents in a large variety of diseases. In most cases, coagulase-positive staphylococci have been implicated, However, coagulase-negative staphylococci have been increasingly recognized as the etiological agent in a variety of diseases, including bacterial endocarditis (3, 11, 13). Coagulase-negative staphylococci have generally been found to either lack certain virulence factors or possess them in smaller quantities than strains of coagulase-positive staphylococci. This, however, is not true for the staphylococcal elastase, which is produced in greater quantities by strains of Staphylococcus epidermidis than by strains of S. aureus (7). Since its discovery (15), staphylococcal elastase has been implicated in a condition called "perifollicular elastolysis," which is characterized by a selective and almost complete loss of elastic fibers in the immediate vicinity of hair follicles that have been infected with elastaseproducing strains of S. epidermidis (16). It has also been postulated that this enzyme plays a role in lung infections (16) and periodontal disease (7). The determination of the role played by elastase in any infection requires that the elastase be obtained in a purified state so that its activity can be ascertained without interference from other biochemically active substances. Before purification can be carried out, the optimal conditions for detecting elastase activity in cellfree preparations and the optimal conditions for the in vitro production of elastase must be de-
termined. Our investigations concerning these factors are described below. (This work was submitted as part of a thesis submitted by D.P.H. in partial fulfillment of the requirements for the M.S. degree in microbiology and elective requirements of the College of Dentistry, University of Illinois.) MATERIALS AND METHODS Strain of Staphylococcus. Strain 332 of S. epidermidis, which was isolated from the oral cavity, was utilized in this investigation. It fermented glucose; aerobically degraded lactose and maltose but not mannitol; produced acetoin but not coagulase; hemolyzed the erythrocytes of human, horse, rabbit, and sheep blood; and possessed phosphatase, gelatinase, caseinase, staphylokinase, nuclease, lysozyme, lipase, and egg yolk factor, as well as elastase activity. Detection of elastase in culture supernatants. In determining the optimum conditions for detecting elastase activity, preparations of crude elastase were produced by the dialysis membrane technique (8, 9) using brain liver heart agar (BLHA; Difco Laboratories) plates incubated under 15% CO2 at 370C for 24 h. The crude elastase preparation was filter sterilized (0.45 ,um) and stored at 4-C. Elastin agar plates for detecting elastase activity were prepared by incorporating 0.25% (wt/vol) elastin (Sigma Chemical Co.) into each medium before autoclaving and dispensing it into sterile plastic petri plates. The plates were generally maintained overnight at room temperature before use. In preliminary experiments, wells 4, 9, and 12 mm in diameter were punched into the medium and filled with serial twofold dilutions of crude elastase. The 9- and 1259
60
INFECT. IMMUN.
HARTMAN AND MURPHY
mm-diameter wells were equally satisfactory for detecting the smallest concentration of elastase. Wells 9 mm in diameter were thus employed for all future experiments since they utilized less material. Unless mentioned otherwise, after the addition of crude elastase to the wells, plates were maintained at 40C for 4 days, at which time the diameters of the zones of clearing were measured. Although results differed in the various media utilized, preparations active at dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16 generally yielded zone diameters of approximately 10, 14, 17, 23, and 26 mm, respectively. The optimum pH and buffer system for detecting elastase activity were determined by placing samples of crude elastase into buffered plates prepared using 1% (wt/vol) Ionagar no. 2 (Oxoid Ltd). The buffers used and the pH ranges they covered were: citrate, pH 5.0 to 5.8; tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.0 to 9.0; Tris-maleate, pH 5.2 to 8.2; sodium-sodium phosphate, pH 5.6 to 6.6; and potassium-sodium phosphate, pH 6.0 to 8.0. These were formulated according to Long (6). Potassium-sodium phosphate-buffered saline agars were also prepared by adding sodium chloride to the potassium-sodium phosphate buffers such that their final ionic strengths were equivalent to that of 0.15 M NaCl as determined with a conductivity meter. In addition, a commercial desiccated potassium-sodium phosphate-buffered saline of pH 8.3 (hemagglutination buffer, Difco) was utilized. The effect of sodium chloride on elastase detection was determined in unbuffered saline agars containing 0.0, 0.05, 0.1, 0.15, 0.2, 0.5, 1.0, 1.5, and 2.0 M NaCl in 1.0% (wt/vol) Ionagar no. 2. Using a crude elastase preparation obtained by harvesting the cultures in Tris-hydrochloride buffer (pH 7.0), the effects of various agars on elastase detection were determined. Agar (Difco), purified agar (Difco), and Ionagar no. 2 were prepared at a 1.5% concentration, and agarose (Wilson Diagnostics Inc.) was prepared at a 1% concentration in the same Tris-hydrochloride buffer. Plates were incubated at 370C for 24 h and then at 40C for 3 more days before the results were read. This splitincubation procedure was utilized here and in the following experiments when it was noted that the culture supernatant frequently required 2 or 3 days to enter the agar of freshly made plates if they were directly incubated at 4VC. The roles of metal ions and ethylenediaminetetraacetic acid (EDTA) in elastase detection were assessed using a culture filtrate harvested in Trishydrochloride buffer (pH 7.0). This was placed into plates of similarly buffered media containing purified agar (1.5%) or agarose (1%) and either 10-3 M CaCl2, CuCl2, FeCl3, MgCl2 6H20, Zn(C2H302)2, Na2EDTA, or no added salt. A 5-ml sample of this elastase preparation was also exhaustively dialyzed against the Tris buffer and filter sterilized. This preparation was tested in the above media as well as in Tris-hydrochloride-buffered agar (1.5%; Difco) and Ionagar no. 2 (1.5%). Optimal conditions for the production of elastase. Unless mentioned otherwise, studies on
elastase production utilized the dialysis membrane technique with batches of 10 plates each being incubated under 15% CO2 at 37°C for 24 h. Each batch was harvested in 3 ml of sterile saline, filter sterilized (0.45 ,um), and stored at 4°C. All culture filtrates were assayed by placing them in 9-mmdiameter wells in Tris-hydrochloride-buffered (pH 7.0) purified agar (1.5%) containing 0.25% (wt/vol) elastin. Activity was ascertained after 24-h incubation at 37°C in a humidified incubator, followed by 72 h of incubation at 4°C. The following media and cultural conditions were used in determining the best medium for the production of elastase: BLHA (containing 1.5% agar [Difco]), brain heart infusion agar (BHIA; Difco), tryptic soy agar (TSA; Difco), Pharmamedia (Traders Oil Mill Co.), heart infusion agar (HIA; Difco), heart infusion agar with Oxoid yeast extract (2.5%, wt/vol; Oxoid), brain liver heart semisolid agar (contains 0.175% agar [Difco]), and brain heart infusion broth (Difco), all incubated in petri plates, and brain heart infusion broth, incubated with shaking in a flask. Pharmamedia was prepared at concentrations of 2 and 4% (wt/vol) in distilled water containing 0.5% (wt/vol) NaCl and 1.5% (wt/vol) agar (Difco). All media were incubated in an atmosphere of 15% CO2.
RESULTS
Stability of crude elastase preparations. A crude elastase preparation stored at 4°C was tested for enzymatic activity at 1 day and at 1, 3, and 5 weeks after harvesting using Trishydrochloride-buffered (pH 7.0) elastin agar (Ionagar no. 2) plates. The preparation initially produced a zone of elastolysis 24 mm in diameter. This dropped to 20 mm within week 1, with a further decrease to 18 and 16 mm by weeks 3 and 5, respectively. Due to the instability of the elastase preparations, they were generally utilized within 1 week after their production. Effect of incubation time on the detection of elastase in culture supernatants. Using Tris-hydrochloride-buffered (pH 7.0) elastin agar (Ionagar no. 2) plates and serial twofold dilutions of elastase, activity was evident for
the more concentrated preparations within 24 h of placing the material in the wells. Zone diameters around these preparations continued to increase over the entire 8-day period of observation. Dilute preparations required up to 4 days of incubation before traces of activity were evident. Plates were therefore routinely incubated for 4 days before elastase activity was measured. Effect of buffer system and pH on the detection of elastase in culture supernatants. The elastase preparation was active in each of the buffer systems utilized (Fig. 1 and 2). In the plates incubated at 370C (Fig. 1), optimum ac-
VOL. 15, 1977 15*
14-
P0,
K-No
P040.0
NO-No
IM
NoCl
P0,
Tris-HCI
13~~~~~~~~ Tr i3- Moleote
U) 13-
2,
K-No
130 A
Citrate
12-
08E I0-
6
5
7
8
pH
1. Effect of buffer system and pH of staphylococcal elastase at 37'C.
FIG. tection
26-
* K- No
P04
*
P04
K-No
A No -Noa 0
25]
Trio
the de-
on
I M NCI
PO$
MCI
0
Tris-MiJeote
A
Citrate
24237
2272172019fl
.rh 02
a
C E
312II
100-
-T-
I 5
6
7
9
pH
FIG. tection
2. Effect of buffer system and pH of staphylococcal elastase at 4C(2
tivity occurred
to
an
Tris-maleate buffer,
equal degree
on
in the
and in both the
pH
the de-
pH
6.0
6.2 and
potassium-sodium phosphate buffers containing 0.1 M NaCl. In the plates incubated at 400 (Fig. 2) the greatest elastase activity was found in the four potassium-sodium phosphate buffers covering the range of pH 6.0 to 7.0. Of almost equal activity were the Tris-maleate buffer at pH 6.8 and the Tris-hydrochloride buffer at pH 7.0O. The addition of 0.1 M Na~l to the potassium-sodium phosphate buffers depressed elastase activity at all pH values in the plates incubated atr40, while generally potentiating activity in the plates incubated at 3700. No one buffer system or single buffer was clearly superior to the others. The potassium-
STAPHYLOCOCCAL ELASTASE 61 sodium phosphate buffer at pH 6.6 and the Trishydrochloride buffer at pH 7.0, both of which yielded high activity, were considered for further studies due to the frequency of use of phosphate and Tris-hydrochloride buffer systems by other investigators. Effect of sodium chloride on the detection of elastase in culture supernatants. Using unbuffered agars containing 0.00, 0.05, 0.1, 0.15, 0.2, 0.5, 1.0, and 2.0 M Na~l, elastase activity was detected at 400 in those agars containing 0.05 to 0.2 M NaCi, with a peak of activity at 0.15 M (Table 1). No elastase activity was seen in any of the agars at 3700. Although the activity seen at optimum NaCl strength was low, it was consistent with the activity of elastase at the reduced pH of these media (pH 5.6 to 5.9). Since the greatest activity was shown in the buffered and NaCl agars at 400, the following experiments all utilized incubation at this temperature. Effect of agars on the detection of elastase in culture supernatants. In assessing the effect of the buffer system, pH, and sodium chloride on the activity of elastase in agar media, Ionagar no. 2 was used as the solidifying agent. Other agars were then tested to determine whether one was superior. The largest zones of activity were obtained with purified agar (26 mm) followed by agar (Difco; 22 mm), Ionagar no. 2 (19 mm), and agarose (14 mm). Purified agar thus appeared to be the solidifying agent of choice for the detection of elastase activity in the crude preparations. Effect of metal ions and EDTA on the detection of elastase in culture supernatants. Metal ions or Na2EDTA were incorporated into purified agar and agarose to determine whether a metallic cofactor would enhance the activity of elastase. Dialyzed and undialyzed elastase preparations were utilized. Because TABLE 1. Effect of different polarities of NaCi on elastolytic activity
6.6
NaCI molarity
Zone diam (MM)a
40C 3rC 0 0 0.0 11 0 0.05 14 0 0.10 0 15 0.15 13 0 0.20 0 0 0.50 0 0 1.00 0 0 1.50 0 0 2.00 a Diameter of zone of elastolysis around a 9-mmdiameter wall.
62
INFECT. IMMUN.
HARTMAN AND MURPHY
calcium, copper, and zinc form insoluble salts with potassium-sodium phosphate buffers, the Tris-hydrochloride buffer at pH 7.0 was utilized. The activity of the undialyzed preparation in purified agar was not enhanced by any of the metal ions, whereas zinc and EDTA partially inhibited and copper completely inhibited the elastase (Table 2). The undialyzed preparation generally showed lower activity in agarose than in purified agar. Calcium completely restored this activity, whereas the presence of iron, magnesium, and EDTA partially restored activity. Copper and zinc completely inhibited activity. The dialyzed preparation was inactive in all purified agar media except that containing EDTA, in which a low level of elastase activity was noted (Table 2). The dialyzed preparation was also inactive in agarose in the absence of metal ions or in the presence of copper or zinc. Slight activity was observed in agarose containing calcium, iron, and magnesium salts. Activity of the dialyzed preparation was greatest in agarose containing EDTA. The dialyzed preparation, when tested in buffered agars made with either agar (Difco) or Ionagar no. 2 and containing no added metallic salts or EDTA, exhibited no elastase activity. Elastin suspended in either Tris-hydrochloride-buffered (0.5 M, pH 7.0) purified agar or in similarly buffered agarose containing 10-3 M Ca appeared to be the best medium for the detection of elastase activity in undialyzed preparations. Thus, for purposes of simplicity and economy, the Tris-hydrochloride-buffered purified agar plates containing elastin were used in the following studies on elastase production. The best medium for the detection of elastase activity in dialyzed preparations conTABLE 2. Effect of the incorporation of metal ions into purified agar and agarose on the elastase activity of crude and dialyzed preparations
sisted of elastin suspended in Tris-hydrochloride-buffered (0.05 M, pH 7.0) agarose containing 10-3 M Na2EDTA. Effect of harvesting fluid on elastase recovery. Using BLHA in the dialysis membrane technique, equal quantities of elastase were recovered when the membranes were harvested in either distilled water, potassium-sodium phosphate buffer (pH 7.0), or 0.15 M NaCl. However, when either water or the potassiumsodium phosphate buffer was used, a cloudiness appeared in the clear culture filtrates after standing 1 or 2 h at 4°C. This cloudiness disappeared when solid sodium chloride was added to either of these fluids, with clearing occurring when the salt concentration reached 0.17 M NaCl. Since this precipitation was not seen when membranes were harvested with saline (0.15 M), saline was chosen as the harvesting fluid. Effect of incubation time on elastase production. At 4-h intervals over a period of 48 h, two samples of 10 plates each were harvested and simultaneously tested for elastase activity. The first detectable elastolytic activity occurred at 12 h (Fig. 3). After 20 h the rate of increase of elastase activity began to taper off, and increases beyond 32 h of incubation were minimal. An incubation time of 28 h yielded nearoptimum levels of elastase. This was thus chosen for future use to make the total time involved in inoculation, incubation, and harvesting more convenient for routine use. With BLHA as the nutrient medium, the dialysis membrane harvests, after centrifugation, became increasingly viscous as the incubation time increased. By 24 h, harvests were so thick that they only passed through 0.45-,um filters with great difficulty. This gelatinous nature of the material offered no difficulties in detecting its elastase activity, but may present difficulties during later purification studies. 24-
Zone diam (mm) witha: Ion
Undialyzed elastase
Purified
22 -
Dialyzed elastase
20
O6
w
,4
0 o E
18-
Purified agar
Agarose
27 27
Agarose 14 28
0 0
0 12
16 It E 14 Cw 12 -
0
0
0
0
26 20 Mg 27 23 Zn 17 0 EDTA 22 25 a See footnote a, Table 1.
0 0 0 14
11 11 0 20
10 -
agar
None Ca Cu Fe
m
.o
_
0-
0
4
8
12 16 20 24 28 32 36 40 44 48 Hours
FIG. 3. Effect of duration of incubation on elastase production.
VOL. 15, 1977
STAPHYLOCOCCAL ELASTASE
Effect of culture medium and growth conditions on elastase production. Various nutrient media were examined for their ability to promote elastase production. All media were harvested at 28 h. In each medium tested, the elastase activity of the resulting preparation appeared directly correlated with the amount of bacterial growth. BLHA and BHIA yielded the highest elastolytic activity (Table 3). HIA, when fortified with yeast extract, yielded the next highest quantity of elastase. Much smaller quantities of elastase were also produced in unfortified HIA and TSA. All other media utilized failed to produce detectable amounts of staphylococcal elastase. The dialysis membrane technique thus appeared necessary for the production of elastase, and either BHIA or BLHA was the medium of choice. The harvest fluid of BHIA was less viscous than that of the BLHA harvest, and for this reason BHIA was chosen for further studies. Effect of atmosphere on elastase production. Plates were incubated for 1, 2, and 7 days in either a humidified incubator, a humidified incubator with the addition of 15% CGO, or under anaerobic conditions produced by the GasPak anaerobic system (BioQuest). Incubation in air alone for 1 day yielded material producing an 11-mm-diameter zone of elastolysis. Longer incubation in air, however, resulted in inactive preparations. Incubation in the presence of C00 yielded material producing an 18-mm-diameter zone of elastolysis after 1 day, 12-mm diameter after 2 days, and inactive preparations after 7 days. No elastolytic activity could be detected using the dialysis membrane technique under anaerobic conditions, TABLE 3. Effect of culture medium and growth conditions on elastase production Culture medium Brain heart infusion agar Brain liver heart agar Tryptic soy agar Heart infusion agar Heart infusion agar with yeast extract Pharmamedia, 2%
Pharmamedia, 4%
Culture
vessel
Zone
diam (mm)a
Plates
22
Plates Plates Plates Plates
21 13 12 18
Plates Plates Plates
0
Brain liver heart semisolid Plates Brain heart infusion broth Flask Brain heart infusion broth a See footnote a, Table 1.
0 0
0
0
63
even after 1 week of incubation. Growth of the culture on a HIA elastin agar plate under anaerobic conditions also failed to show elastase activity even after 6 weeks of incubation. Air enriched with 15% CO2 was thus better than either air alone or anaerobiosis and was considered optimal for elastase production. Inducibility of staphylococcal elastase. Use of the dialysis membrane technique with the various media employed in this investigation indicated that staphylococcal elastase may have been produced constitutively. To determine whether higher levels of enzyme activity could be induced, elastin (0.25%) was incorporated either into BHIA below the dialysis membrane or into the inoculum above the dialysis membrane. Higher levels of elastase were not produced in either case as compared with elastin-free cultural conditions. It appeared, therefore, that S. epidermidis culture 332 could not be induced to produce higher levels of elastase in BHIA.
DISCUSSION The development of an agar substrate plate method for the detection of staphylococcal elastase in cell-free preparations has several advantages over other methods of detection. Colorimetric methods using a chromogenic moiety attached to elastin (10, 12, 14) may not be specific (4) or sufficiently sensitive (5) even in the detection of pancreatic elastase, for which they were developed. The use of synthetic substrates suffers from similar problems (5). Although substrates other than plain particulate elastin may be developed and show specificity in a pancreatic elastase system, great caution must be exercised before ascribing similar specificity when using these materials in the study of bacterial elastases, such as the staphylococcal elastase, which may act in a different manner. For these reasons, an agar substrate plate method, utilizing particulate elastin, was developed for detecting and quantitating staphylococcal elastase. In addition to specificity, this method requires a minimum amount of preparation and many small samples can be easily tested and compared at one time. Crude, undialyzed staphylococcal elastase was best detected in a medium consisting of 0.25% elastin suspended in a pH 7.0, 0.05 M Tris-hydrochloride-buffered medium containing either agarose and 10-3 M calcium or purified agar without additional additives. Activity present in dialyzed crude preparations was best detected in pH 7.0, 0.05 M Tris-hydrochloridebuffered agarose containing 10-3 M Na2EDTA.
64
INFECT. IMMUN.
HARTMAN AND MURPHY
Overnight incubation at 370C followed by 3 days at 40C yielded the optimum activity in these media. These results are similar to those of Varadi and Saqueton (15), who also found optimum elastase activity at pH 7.0 in Tris buffer. They, however, found no inhibition by the inclusion of 0.15 M NaCl into the buffer, whereas we found a slight potentiation by NaCl at 37C and a significant inhibition at 40C. They also found that EDTA almost completely inhibited their crude elastase, whereas we found only a partial inhibition of undialyzed elastase. This might only be a result of a difference in concentration between these preparations and ours. The observed elastase activity appears to be the result of a complex interaction of elastase with metallic cofactors and metallic inhibitors. This was indicated by the loss of activity in dialyzed preparations and its subsequent activation or potentiation not only by the addition of calcium, iron, or magnesium, but also by EDTA, a seemingly contradictory situation. Perhaps our preparation contains more than one elastolytic moiety having different cofactor requirements. The difference in optimum detection mediums, depending on the dialyzed or undialyzed state of the elastase preparation, will be very significant when attempts to purify the elastase are carried out. It is expected that the more purified elastase will behave similar to that in the dialyzed preparation. The production of highly active preparations of staphylococcal elastase was facilitated by the use of the dialysis membrane technique. Maximum levels of elastase were produced using either BLHA or BHIA and incubating the plates in an atmosphere of 15% CO2 for 44 h. -Near optimal yields could be obtained after 28 h of incubation and, when the times involved in inoculation and harvesting were taken into account, this length of time was found most convenient for routine production of elastase. The use of this shorter incubation time, as well as the use of BHIA instead of BLHA, also resulted in a considerable decrease in the viscosity of the resultant elastase preparations. This viscosity is not unique to this strain of Staphylococcus, since we have reported a similar finding for S. aureus 146P (R. A. Murphy and R. Haque, Int. Assoc. Dent. Res. Abstr., p. 216, 1971) and also observed this with many other strains of staphylococci. This viscous material may be identical to the slime layer material reported by others (2) to be produced by strains of S. aureus. In this connection it may be of interest to note that
the material we observed was produced by strains of S. epidermidis as well as strains of S. aureus. A definite relationship between staphylococcal elastase and other staphylococcal proteases cannot be determined as yet. There are similarities between our elastase and the three proteases of S. aureus delineated by Arvidson and co-workers (1). These proteases were all active against casein, and their production was potentiated by the incorporation of yeast extract into the medium, as is the case for our elastase. One protease, however, was completely inactivated by EDTA, and the others were unaffected, whereas our elastase was only slightly inhibited under similar conditions. Although our elastase would appear to be a separate proteolytic entity, as has been mentioned, it is possible that our preparation contained more than one elastase and that one of these was inhibited by EDTA and the other(s) were unaffected. In addition, it may well be that the elastase of S. aureus possesses characteristics different from those identified here and it may behave identically to one ofArvidson's proteases, which were produced by the V8 strain of S. aureus. An explanation of these and other problems must, perhaps, await attempts to purif the staphylococcal elastase. ACKNOWLEDGMENT This investigation was supported by Public Health Service general research support grant 5501-RR-53-3-11 no. 108/
12. LITERATURE CITED 1. Arvidson, S., T. Holme, and B. Lindholm. 1972. The formation of extracellular proteolytic enzymes by Staphylococcus aureus. Acta Pathol. Microbiol. Sand. 80B:835-844. 2. Ekstedt, R. D., and J. M. Bernhard. 1973. Preparation and characterization of a slime layer material produced by Staphylococcus aureus. Proc. Soc. Exp. Biol. Med. 142:86-91. 3. Keys, T. F., and W. L. Hewitt. 1973. Endocarditis due to micrococci and Staphylococcus epidermidis. Arch. Intern. Med. 132:216-220. 4. Lieberman, J. 1972. Digestion of antitrypsin-deficient lung by leukoproteases, p. 189-203. In C. Mittman (ed.), Pulmonary emphysema and proteolysis. Academic Press Inc., New York. 5. Loeven, W. A. 1972. Elastolytic activity in blood, P. 269-274. In C. Mittman (ed.), Pulmonary emphysema and proteolysis. Academic Press Inc., New York. 6. Long, C. 1961. Biochemists' handbook, p. 29-42. E. and F. N. Spon Ltd. London. 7. Murphy, R. A. 1974. Elastase production by oral staphylococci. J. Dent. Res. 53:832-834. 8. Murphy, R. A., and R. Haque. 1967. Purification and properties of staphylococcal delta hemolysin. I. Production of delta hemolysin. J. Bacteriol. 94:13271333.
VOL. 15, 1977 9. Murphy, R. A., and R. Haque. 1974. Large scale production of staphylococcal delta hemolysin by the dialysis membrane technique. Can. J. Microbiol. 20:10611063. 10. Naughton, M. A., and F. Sanger. 1961. Purification and specificity of pancreatic elastase. Biochem. J. 78:156163. 11. Quinn, E. L., F. Cox, and E. H. Drake. 1966. Staphylococcic endocarditis. J. Am. Med. Assoc. 196:815-818. 12. Rinderknecht, H., M. C. Geokas, P. Silverman, Y. Lillard, and B. J. Haverback. 1968. New methods for the determination of elastase. Chin. Chim. Acta 19:327-339.
STAPHYLOCOCCAL ELASTASE
65
13. Robbins-Browne, R. M., and H. J. Koornhof. 1973. The aetiology and bacteriological monitoring of bacterial endocarditis at the Johannesburg General Hospital. S. Afr. Med. J. 77:407-412.. 14. Sachar, L. A., K. K. Winter, N. Sicher, and S. Frankel. 1955. Photometric method for estimation of elastase activity. Proc. Soc. Exp. Biol. Med. 90:323-326. 15. Varadi, D. P. and A. C. Saqueton. 1968. Elastase from Staphylococcus epidermidis. Nature (London) 218: 468-470. 16. Varadi, D. P. and A. C. Saqueton. 1970. Perifollicular elastolysis. Br. J. Dermatol. 83:143-150.
