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Lipolytic and proteolytic properties of staphylococci
Zbl. Bakt. Hyg., LAbt. Orig. A 254,452-458 (1983)
Department of Bacteriology, National Institute of Hygiene, Warszawa, Poland
Lipolytic and Proteolytic Properties of Staphylococci Lipolytische und proteolytische Aktivitaten von Staphylokokken S. TYSKI, P. CIBOROWSKI, W. HRYNIEWICZ, and
J. JELJASZEWICZ
Received December 11, 1982
Summary Lipolytic and proteolytic activities were estimated in coagulase-positive and coagulasenegative strains of human origin. Several different substrates were used for determination of these activities. Staphylococcus au reus strains were very frequently lipolytic (95%) and proteolytic (87.5%). Among coagulase-negative strains, 85.4% were proteolytic and 10.25% exhibited lipolytic activity.
Zusammenfassung In der vorliegenden Arbeit wurden die lipolytischen und proteolytischen Aktivitaten von koagulasepositiven und -negativen Staphylokokkenstammen menschlicher Herkunft untersucht. Fur diese Tests wurden verschiedene Substrate verwendet. Die iiberpriifrcn Staphylococcus aureus-Stiimme zeigten in hoher Frequenz lipolytische (95%) und proteolytische (87,5%) Eigenschaften. Von den getesteten koagulasenegativen Staphylokokkenstammen erwiesen sich 85,4% als proteolytisch und lediglich 10,25% als lipolytisch.
Staphylococci are capable of producing several extracellular enzymes and toxins, the biological significance of which is not fully understood. There are, however, some indications concerning their possible role in pathogenesis of staphylococcal infection (1,17). The purpose of this study was to estimate the frequency of lipase and protease production by staphylococci (coagulase-positive and coagulase-negative) isolated from various clinical materials. These activities were tested by modern techniques with application of different substrates and related to the production of hemolysins and nuclease.
Lipolytic and Proteolytic Properties of Staphylococci
453
Materials and Methods Strains: 99 strains were isolated from following human sources: 11 - septicemia, 35 skin abscesses, 20 - nasopharynx, 9 - burns, and 24 constituted a variety of materials such as urine, otitis excretions etc. 78 strains were coagulase-, nuclease and clumping factor (CF)-positive, and were classified as Staphylococcus aureus. Two other strains were added to this group which were coagulase-negative, but nuclease and CF-positive. 19 remaining strains were coagulase-, nuclease-, and CF-negative and represented species Staphylococcus epidermidis and Staphylococcus sapropbyticus. They were differentiated on the basis of novobiocin sensitivity test, ability to produce acid phosphatase and to hydrolyse trehalose and mannitol. Eleven strains were classified as S. epidermidis and eight as S. saprophyticus. The strains were kept on nutrient agar slants and reidentified before application. Media: Brain Heart Infusion Broth (Difco) was inoculated with a single bacterial colony from blood agar culture, and incubated for 24 h at 37°C on a shaker. Resulting cultures were centrifuged and supernatants used for enzymatic determinations. Hemolysis: It was checked on sheep blood agar plates after 18 h incubation at 37°C, followed by additional observation after refrigeration at 4 °C for 24 h. Clumping factor: CF was assayed by slide agglutination technique as described by Lipinski et al. (21). Staphylocoagulase: This test was performed according to [eliaszeu/icz (15). Nuclease: A method involving agar plates with DNA and methyl green and proposed by Marker and Gray (22) was used. Proteases: Proteolytic activity was measured according to a modified technique of Kunitz (4,20), using four different substrates: hemoglobin (Difco), bovine albumin (Serva), sodium caseinate (Difco), and gelatin (Difco). 1% solution of the substrate in 0.05 M Tris-HCI buffer of pH 7.4, supplemented with 1 mM cysteine hydrochloride and 1 mM CaCI" was used as a basic system. To each substrate sample (4 rnl), 0.2 ml of a tested culture supernatant was added. The mixture was incubated for 30 min at 37°C in a water bath. The reaction was stopped by addition of 3 ml of 10% HCl0 4 and left for 15 min, followed by recovering of the precipitate by filtration through Whatman No. 42 filter. All samples were run in parallel. One unit of proteolytic activity was defined as an amount of the enzyme resulting in an absorbance of 1.0 at 280 nm of the supernatant after precipitation with perichloric acid and incubation for 30 min. Lipase: Lipolytic activity was measured by two methods with application of five different substrates. 1. Qualitative method: 1% solution of Tween 20, Tween 80 or tributyrin in 0.05 M Tris-HCI buffer of pH 8.0 wirh 0.5% csci, was solidified with 1% agar and plares were prepared. Egg yolk emulsion was prepared according to O'Leary and Weld (24). Lipolytic activity was determined in 24 h culture supernatants poured into wells cut into the agar. When Tween 20 or Tween 80 were used, opacity formation around wells was considered as a positive reaction. With tributyrin as a substrate, clearance zone around wells was accepted as a positive result. Hydrolysis of egg yolk was recorded according to Shah and Wilson (29). 2. Quantitative method: Free fatty acids liberated from olive oil emulsion by lipase present in culture supernatants, were measured by potentiometric titration (33).
Results All strains of S. aureus produced nuclease, while only one coagulase-negative strain (S. sapropbyticus isolated from urinary tract infection) was nuclease-positive. Hemolytic activity was found in 69 (86.25%) of S. aureus strains and none in coagulase-negative staphylococci.
mic cla-
of
SA
SE
Other
sources
7
4
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2
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11 2 1
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1
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30
a a
9
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0
9
2
2
a 1
4
1
4
1
1
1
11
0
30
0
0
8
+
6
1
11
9
a 2
1
0
11
0
30
6
2
a
2
2
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1
4
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2
2
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4
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1
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0
9
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-
-
1
11
9
1
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30
a
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9
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6
2
0
2
2
4
1
4
1
1
a
-
-
1
11
9
1
a
6
2
0
2
2
4
1
11
4
a
1
1
a
30
0
0
9
+
2
4
5
1
0
4
1
6
0
a a
+
5
9
4
2
2
11
a
28
1
1
9
-
1
3
5
4
0
4
8
5
3
1
9
1 6
26
a
1
1
6
8
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a
3
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5
6
7
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7
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2
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25
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1
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7
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7
a
17
1
1
4
+
-
3
6
2
1
2
8
1
17
a a
5
Hemoglobin
Proteolytic substrates
Olive oil Tween 80 Tween 20 Egg yolk Tributyrin Gelatin Albumin Casein
Lipolytic substrates
TOTAL 7
9
11 1
1
70 7
10
10 1
1
70 7
10
10
1
1
70
10 7
10
1
1
70
10 7
10
3
3
19
5
8
61
1
4
26
7
7
54
3
6
28
5
5
52
= Staphylococcus aureus, SE = Staphylococcus epidermidis, SS = Staphylococcus saprophyticus
1
8
0
SS
* SA
2
11
0
SE
69
11
69
SA
3
5
42
5
6
38
SS a 4 0 4 a 4 0 4 0 4 0 4 2 2 a 4 1 3 1 3 ==================================================================================================================
SA
0
SS
Burns
2
3
0
SE
1
pharynx
14
SA
1
Naso-
3
1
0
0
SS*
1
31
0
SE*
1
-
SE
8
SA*
+
Hemolysin
Abscesses SA
Blood
isolation ssification
Taxono-
Source
Table 1. Lipolytic and proteolytic substrate specificity of staphylococcal strains depending of their origin ~
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Lipolytic and Proteolytic Properties of Staphylococci
455
Extensive lipolytic activity was noted in 70 (87.5%) strains of S. aureus (Table 1). Coagulase-negative strains only occasionally exhibited this activity. Only two (10.5%) out of 19 coagulase-negative strains were lipase-positive (one each of S. epidermidis and S. sapropbyticusi, There was no apparent correlation between lipase production and the source of strain. Lipolytic activity was determined in this study by application of five different substrates (Tween 20, Tween 80, tributyrin, egg yolk and olive oil emulsion). It was shown that lipase present in culture supernatants of S. au reus hydrolyses all five substrates or none at all (with one exception, see Table 1). The same was true for coagulase-negative staphylococci. Proteolytic activity was determined with application of four different substrates (casein, albumin, hemoglobin, and gelatin). Seventy six (95%) out of 80 strains of S. aureus exhibited proteolytic activity on at least one of the substrates used (Table 1). High number of protease-positive strains was also found among coagulase-negative strains; 6 out of 8 strains of S. saprophyticus and 9 out of 11 strains of S. epidermidis. This means that about 79% of coagulase-negative strains were proteolytic. It should be noted, however, that only 16 strains (21%) of S. aureus were able to hydrolyse all four substrates. This property was slightly higher with coagulasenegative staphylococci (35.3%). The highest number of protease-positive strains of S. aureus was found using hemoglobin as a substrate. As many as 59 (73.7%) strains were positive. Using casein as a substrate, 49 (62.2%) strains were classified as proteolytic. Six of these strains were active only against casein and did not affect other substrates. Remaining substrates were less susceptible to staphylococcal proteases and 44 (55%) hydrolysed albumin, while 39 (48.7%) were active toward gelatin. No correlation between lipase and protease production by S. aureus was observed (Table 2). Among 76 protease-positive strains of S. aureus (positive with at least one substrate), 10 were lipase-negative and among protease-negative strains four were lipolytic. Table 2. Correlation of lipolytic and proteolytic activities of different strains of S. aureus Proteolytic activity
Lipolytic activity Gelatin +
+
*
+
20.0* 67.5 5.0
Albumin
7.5
Casein
Hemoglobin
+
+
27.5
60.0
33.5
54.0
43.5
43.5
5.0
7.5
2.5
10.0
8.0
5.0
Percentage of strains tested
Discussion It has been demonstrated by the present study that 87.5% of S. aureus strains of human origin are lipolytic, while 95% of them are proteolytic. Among coagulase-
456
S.Tyski, P.Ciborowski, W.Hr yniewicz, and J.]elj aszewicz
negati ve stra ins only about 10% were lipol ytic, but nearl y 89% of th em were proteolytic. There was no significant d ifferen ce bet ween S. epiderm idis and Sc sapr opby ticus stra ins, although a limited number of str ain s was tested. Lip olytic activity is usually related to S. aureus, since it was shown before (2, 9, 27) and was confirmed in th e pre sent study , that onl y few coagulase-negative staph ylococci are lipa se-po sitive. H owever, contradictory findings have been reported by W illis and T urner (34), who found all coa gulase-negative strains investigated by th em as lipase-po sitive. This difference may be due to various substrates used and perha ps different interpretati on of th e results. It is parti cularl y impo rta nt when using tr ibutyrin as a substra te, since so me sterile and not inoc ulated nutrient broth may pr oduce some zo ne of clea ring; thi s false-p ositi ve result may be interpreted as a positive reaction. No cor relation ha s been fo und between the source of strai ns and the ir lipol ytic and proteolytic activities. It is in keeping with the earli er published reports (2, 3, 16,32). No complete correlation was either observed betwe en hemolysis and lipase pr oduction, although these tw o activities seem to be typi cal fo r S. aureus strains (8, 11). Co ncern ing the proteolytic acitvity, our results although simila r are not compa ra ble to the data presented by other authors (5,6,9, 12, 13, 16, 23, 25, 26, 28), since the y investigated strains from not homogenous sources. M or eover, in none of th ese reports, sub strates used in our study were applied at th e sa me time. Our res ults demonstr at e th at pro teolytic activity is not char acteri stic for coa gu lase-positive sta phylococci, and th at th is property is also wid ely dis tributed a mong coagulase-negative stra ins. This is not in keeping with some earlier ob ser vations suggesti ng th at th is pro pert y is attributed mostly to coagulase-p ositive stap hylococci (25,26) . Report of Levy (10) sup po rts our find ings demonstr ating th at proteolytic activity is often ext ensive in coagulase-negative sta phy lococ ci. As stated before, th ese differences may be du e to a difference in the subst ra tes and techniques used. In earlier studies, gelatin was mainly used. It h as been recentl y replaced b y casein and other substra tes were only spor adica lly applied. We have employed four differ ent substra tes. Basing on various spectra of proteolytic activity, it seems that more th an one protease exists. It is imp ossible, however, to conclude at thi s moment how man y different proteolytic enzymes are excreted by staphylococci and how many by a particular single strai n. Early studies on lipase produc tio n by va rious staphy lococc i were pr acti cally limited to egg yolk, and only occasionally Tween 80 was used (2.,3 , 16, 18, 19, 27, 30). O nly few studies repo rte d application of more substra tes (14, 27). We have emp loyed in our study both na tura l fats, synthetic trigl ycerides and other synthetic esters, never used together before in a single investigation. All sta ph ylococcal strains hydrolysed either five substrates or none at all. This finding has been confirmed also with a highly purified staphylococcal lipase (33) and resulted in conclusion that sta phylococci produce one lipolytic enzyme with a broad spectrum of activity. This is in keeping with sugge stion s of Tirun arayanan and Lun dbeck (31) and of Brunner et al. (7), who concluded th at lipase and esterase activ ity reside in th e sa me protein molecule . It has been sho wn in thi s investiga tio n th at both lipol ytic and p roteol ytic activities are widel y distributed among sta phy lococci. Lipa se seems to be an enzyme with a br oad spectru m of activit y and is mainly produced by S. aureus. Proteolytic activity
Lipolytic and Proteolytic Properties of Staphylococci
457
is exhibited by both coagulase-positive and coagulase-negative strains, and results differ depending on the substrate used. Acknowledgement. This investigation was supported by research grant 05-339-C from the U. S. Public Health Service.
References 1. Abramson, c.: Staphylococcal enzymes. In: The Staphylococci, p. 187-248, j. O. Cohen (ed.). Wiley-Interscience, New York (1972) 2. Alder, V. G., W. A. Gillespie, and G. Herdan: Production of opacity in egg-yolk broth by staphylococci from various sources. J. Path. Bact. 66 (1953) 205-210 3. Alder, V. G., W. A. Gillespie, and M. E. M. Thompson: Virulence and phage patterns of antibiotic-resistant staphylococci in a hospital. J. Path. Bact. 70 (1955) 503-511 4. Arvidson, S.: Hydrolysis of casein by three extracellular proteolytic enzymes from Staphylococcus aureus strain V8. Acta path. microbiol. scand. B 81 (1977) 538-544 5. Baird-Parker, A. c.: A classification of micrococci and staphylococci based on physiological and biochemical tests. J. gen. Microbiol. 30 (1963) 409-427 6. Bjorhlind, A. and S. Arvidson: Occurrence of an extracellular serinoprotease among S. aureus strains. Acta path. microbiol. scand. B 85 (1977) 277-280 7. Brunner, H., C. G. Gemmel, H. Hauser, and F.]. Fehrenbach: Chemical and biological properties of Staphylococcus aureus lipase. In: Staphylococci and Staphylococcal Infections, p. 329-333, ].jeljaszewicz (ed.). Gustav Fischer Verlag, Stuttgart (1981) 8. Christie, R. and].]. Graydon: Observations of staphylococcal haemolysins and staphylococcallipase. Aust. J. expo Bioi, med. Sci. 19 (1941) 9-16 9. Delmotte, A.: L'activite lipolytique microbienne decclce par la methode de Sierra avec reference speciale au M. pyogenes. Antonie v. Leeuwenhoek 24 (1958) 309-320 10. Elek, S. D.: Staphylococcus pyogenes and its Relation to Disease, p. 287-289. E. S. Livingstone, Edinburgh - London (1959) 11. Elias, B. and ]. Kafer: Staphylococcus aureus hemolysins and their use in strain typing. Acta microbiol. Acad. Sci. Hung. 27 (1980) 183-190 12. Goldbach, W. und H. Haenel: Ober Lysozym und Proteasebildung bei Staphylokokken. Zbl. Bakt., 1.Abt. Orig. 195 (1964) 201-205 13. Hasche, K. D., W. Schaeg, H. Blobel, and ]. Briichler : Purification of protease from Staphylococcus aureus. Zbl. Bakt. Hyg., 1.Abt. Orig. A 238 (1977) 300-309 14. Hugo, W. B. and E. G. Beveridge: A quantitative and qualitative study of the lipolytic activity of single strains of seven bacterial species. J. appl. Bact. 25 (1962) 72-82 15. [eliaszeioicz, ].: Studies on staphylococcal coagulases. 1. Acta microbiol. Polon. 7 (1958) 17-34 16. [eljaszeioicz, ].: Studies on staphylococcal coagulases. IV. Occurrence of various toxic and biochemical properties among coagulase-negative and coagulase-positive strains of staphylococci. Med. Dosw, Mikrobiol. 12 (1960) 33-41 17. [eliaszeioicz, ]., S. Szmigielshi, and W. Hryniewicz: Biological effects of staphylococcal and streptococcal toxins. In: Bacterial Toxins and Cell Membranes, p. 185-227, j.]eljaszewicz and T. Wadstram (ed.). Academic Press, London (1978) 18. [essen, 0., V. Faber, K. Rcsendal, and K. R. Eriksen: Some properties of Staphylococcus aureus possibly related to pathogenicity. A. A study of 446 strains from different types of human infections. Acta path. microbiol. scand. 47 (1959) 316-326 19. [essen, 0., V. Faber, K. Rosendal, and K. R. Eriksen: Some properties of Staphylococcus aureus possibly related "0 pathogenicity. 2. In vitro properties and origin of the infecting strains correlated to mortality in 190 patients with Staphylococcus au reus bacteremia. Acta path. microbiol. scand. 47 (1959) 327-335 20. Kunitz, M.: Crystalline soybean trypsin inhibitor. J. gen. Physiol. 30 (1947) 291 31 Zbl. Bakt. Hyg., I. Abr, Orig. A 254
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S.Tyski , Pi Ciborow sk i, W.Hryniewi cz, and J.]eljaszewicz
21. Lipillski, B., j. Hatoiger, and]. [eljaszetoicz: Staphylococc al clumping with soluble fibrin monomer substrates. ] . expoM ed . 126 (1967) 979- 988 22. Ma rker, S. C. and E. D. Gray : Simple meth od for the pr ep ar at ion of sta phylococca l nucl ease. Appl. Microbiol. 23 (1972) 368-371 23. Ma rtley, F.G., W .A. ja rvis, D.F. Bacon, and R. C.Lawrence : T ypin g of coagulasepo siti ve staphylococci by p rot eolytic activity on buffered caseinate aga r with special refere nce to bacteriophage nontypabl e str ains . Infect. lm mun. 2 (1970) 439-442 24. O'L eary, W. M . and j. T. Weld: Lipolytic activities of Staphylo coccus aureus. 1. Nature of enzym e pro ducing fr ee fatty aci ds from pla sma lip ids. J. Bact . 88 (1964) 1356- 1363 25. Papaeuangelou, G. and j. Papauassiliou : Comparison of desoxyri bonuclease acti vity to so me other criteria of Staphylococcus pathogeni cit y. Path. Mi crobiol. 30 (1967) 59-63 26. Quie, P. G. and L. W. Wa nnamake r: Staphylo coccal Muller ph enom enon ; relat ion ship to th e plasmi nogen -pl asm in syste m. J. Bact . 82 (1961) 770-783 27. Rosendal, K. and P. BUlow : Staph ylococcus aureus stra ins isol ated in Danish hospitals. Act a path. microbiol. scand. 71 (1967) 422-438 28. Saski, S. et B. Fejgin: Au sujet des pro prieres proteol yriqu e et fibro lytique de sraphylocoques en connexion avec leur po uvoir toxigene. C. R. Soc. BioI. (Pa ris) 126 (1937) 139- 141 29. Shah, D. B. and j.B. Wilson: Egg yolk factor of Staphylococcus aureus. 1. Nature of the substrate and enzyme involved in th e egg yolk opacity reacti on . ]. Bact. 85 (1963) 516521 30. Sinel, H.]. and J. Baumgart : Co mparison of egg yolk containing selective media for the qu antitation and isolation of co agula se-positive staphylococci . Zbl. Bakt., 1. Abt. Orig. 204 (1967) 248-264 31. Ti runarayanan, M . O. and H . Lundbeck: Investigations on the enzy mes and toxins of stap hylococci. 1. H ydrol ysis of triglyceri des and other esters by lipase . Acta path. microbioI. scand. 73 (1968) 437-449 32. Tr ussell, R. E. and L. A. Weed: T he lipol ytic act ion of sta phylococci on so me pure tri glycerides . J. Bact. 33 (1937) 38 1-388 33. T yski, S. : Stap hylo coccal lip ase - p urification and some properti es. In : Stap hyl ococci and Staphylococcal Infection s, p. 335-3 42, J.jeljaszewicz (ed.). Gustav Fischer Verlag, Stuttg ar t (1981) 34. W illis, A. T . and G. C. T urner : Staph ylococcal lipolysis an d pigment at ion . ]. Path. Bact. 84 (1962) 337- 347 Dr. Stefan T yski, Department of Bacteriology, N ati onal Institut e of H ygiene , 24 Chocimska , 00-791 Warszawa , Pol and
Department of Bacteriology, National Institute of Hygiene, Warszawa, Poland
Lipolytic and Proteolytic Properties of Staphylococci Lipolytische und proteolytische Aktivitaten von Staphylokokken S. TYSKI, P. CIBOROWSKI, W. HRYNIEWICZ, and
J. JELJASZEWICZ
Received December 11, 1982
Summary Lipolytic and proteolytic activities were estimated in coagulase-positive and coagulasenegative strains of human origin. Several different substrates were used for determination of these activities. Staphylococcus au reus strains were very frequently lipolytic (95%) and proteolytic (87.5%). Among coagulase-negative strains, 85.4% were proteolytic and 10.25% exhibited lipolytic activity.
Zusammenfassung In der vorliegenden Arbeit wurden die lipolytischen und proteolytischen Aktivitaten von koagulasepositiven und -negativen Staphylokokkenstammen menschlicher Herkunft untersucht. Fur diese Tests wurden verschiedene Substrate verwendet. Die iiberpriifrcn Staphylococcus aureus-Stiimme zeigten in hoher Frequenz lipolytische (95%) und proteolytische (87,5%) Eigenschaften. Von den getesteten koagulasenegativen Staphylokokkenstammen erwiesen sich 85,4% als proteolytisch und lediglich 10,25% als lipolytisch.
Staphylococci are capable of producing several extracellular enzymes and toxins, the biological significance of which is not fully understood. There are, however, some indications concerning their possible role in pathogenesis of staphylococcal infection (1,17). The purpose of this study was to estimate the frequency of lipase and protease production by staphylococci (coagulase-positive and coagulase-negative) isolated from various clinical materials. These activities were tested by modern techniques with application of different substrates and related to the production of hemolysins and nuclease.
Lipolytic and Proteolytic Properties of Staphylococci
453
Materials and Methods Strains: 99 strains were isolated from following human sources: 11 - septicemia, 35 skin abscesses, 20 - nasopharynx, 9 - burns, and 24 constituted a variety of materials such as urine, otitis excretions etc. 78 strains were coagulase-, nuclease and clumping factor (CF)-positive, and were classified as Staphylococcus aureus. Two other strains were added to this group which were coagulase-negative, but nuclease and CF-positive. 19 remaining strains were coagulase-, nuclease-, and CF-negative and represented species Staphylococcus epidermidis and Staphylococcus sapropbyticus. They were differentiated on the basis of novobiocin sensitivity test, ability to produce acid phosphatase and to hydrolyse trehalose and mannitol. Eleven strains were classified as S. epidermidis and eight as S. saprophyticus. The strains were kept on nutrient agar slants and reidentified before application. Media: Brain Heart Infusion Broth (Difco) was inoculated with a single bacterial colony from blood agar culture, and incubated for 24 h at 37°C on a shaker. Resulting cultures were centrifuged and supernatants used for enzymatic determinations. Hemolysis: It was checked on sheep blood agar plates after 18 h incubation at 37°C, followed by additional observation after refrigeration at 4 °C for 24 h. Clumping factor: CF was assayed by slide agglutination technique as described by Lipinski et al. (21). Staphylocoagulase: This test was performed according to [eliaszeu/icz (15). Nuclease: A method involving agar plates with DNA and methyl green and proposed by Marker and Gray (22) was used. Proteases: Proteolytic activity was measured according to a modified technique of Kunitz (4,20), using four different substrates: hemoglobin (Difco), bovine albumin (Serva), sodium caseinate (Difco), and gelatin (Difco). 1% solution of the substrate in 0.05 M Tris-HCI buffer of pH 7.4, supplemented with 1 mM cysteine hydrochloride and 1 mM CaCI" was used as a basic system. To each substrate sample (4 rnl), 0.2 ml of a tested culture supernatant was added. The mixture was incubated for 30 min at 37°C in a water bath. The reaction was stopped by addition of 3 ml of 10% HCl0 4 and left for 15 min, followed by recovering of the precipitate by filtration through Whatman No. 42 filter. All samples were run in parallel. One unit of proteolytic activity was defined as an amount of the enzyme resulting in an absorbance of 1.0 at 280 nm of the supernatant after precipitation with perichloric acid and incubation for 30 min. Lipase: Lipolytic activity was measured by two methods with application of five different substrates. 1. Qualitative method: 1% solution of Tween 20, Tween 80 or tributyrin in 0.05 M Tris-HCI buffer of pH 8.0 wirh 0.5% csci, was solidified with 1% agar and plares were prepared. Egg yolk emulsion was prepared according to O'Leary and Weld (24). Lipolytic activity was determined in 24 h culture supernatants poured into wells cut into the agar. When Tween 20 or Tween 80 were used, opacity formation around wells was considered as a positive reaction. With tributyrin as a substrate, clearance zone around wells was accepted as a positive result. Hydrolysis of egg yolk was recorded according to Shah and Wilson (29). 2. Quantitative method: Free fatty acids liberated from olive oil emulsion by lipase present in culture supernatants, were measured by potentiometric titration (33).
Results All strains of S. aureus produced nuclease, while only one coagulase-negative strain (S. sapropbyticus isolated from urinary tract infection) was nuclease-positive. Hemolytic activity was found in 69 (86.25%) of S. aureus strains and none in coagulase-negative staphylococci.
mic cla-
of
SA
SE
Other
sources
7
4
9
0
2
7
-
11
2 5
11 2 1
9
1
a
11
0
30
a a
9
+
0
9
2
2
a 1
4
1
4
1
1
1
11
0
30
0
0
8
+
6
1
11
9
a 2
1
0
11
0
30
6
2
a
2
2
4
1
4
1
a
2
2
4
1
4
1
1
a
0
9
a 1
+
-
-
1
11
9
1
a
11
0
30
a
0
9
+
6
2
0
2
2
4
1
4
1
1
a
-
-
1
11
9
1
a
6
2
0
2
2
4
1
11
4
a
1
1
a
30
0
0
9
+
2
4
5
1
0
4
1
6
0
a a
+
5
9
4
2
2
11
a
28
1
1
9
-
1
3
5
4
0
4
8
5
3
1
9
1 6
26
a
1
1
6
8
0
a
3
+
-
5
6
7
1
a
5
1
9
1
0
1
+
2
7
2
2
2
10
a
25
0
1
8
4
7
7
2
a
7
a
17
1
1
4
+
-
3
6
2
1
2
8
1
17
a a
5
Hemoglobin
Proteolytic substrates
Olive oil Tween 80 Tween 20 Egg yolk Tributyrin Gelatin Albumin Casein
Lipolytic substrates
TOTAL 7
9
11 1
1
70 7
10
10 1
1
70 7
10
10
1
1
70
10 7
10
1
1
70
10 7
10
3
3
19
5
8
61
1
4
26
7
7
54
3
6
28
5
5
52
= Staphylococcus aureus, SE = Staphylococcus epidermidis, SS = Staphylococcus saprophyticus
1
8
0
SS
* SA
2
11
0
SE
69
11
69
SA
3
5
42
5
6
38
SS a 4 0 4 a 4 0 4 0 4 0 4 2 2 a 4 1 3 1 3 ==================================================================================================================
SA
0
SS
Burns
2
3
0
SE
1
pharynx
14
SA
1
Naso-
3
1
0
0
SS*
1
31
0
SE*
1
-
SE
8
SA*
+
Hemolysin
Abscesses SA
Blood
isolation ssification
Taxono-
Source
Table 1. Lipolytic and proteolytic substrate specificity of staphylococcal strains depending of their origin ~
N
n°
::;:
(l)
N
'-< ~ "];i0
':-'
::l 0..
!'>
:sn° n° J"'
::l
'<
'"'
::r:
~
c:
:s
'"'
0 0
5-'
o
~
~
'<
>--l
-l'-
V,
-l'-
Lipolytic and Proteolytic Properties of Staphylococci
455
Extensive lipolytic activity was noted in 70 (87.5%) strains of S. aureus (Table 1). Coagulase-negative strains only occasionally exhibited this activity. Only two (10.5%) out of 19 coagulase-negative strains were lipase-positive (one each of S. epidermidis and S. sapropbyticusi, There was no apparent correlation between lipase production and the source of strain. Lipolytic activity was determined in this study by application of five different substrates (Tween 20, Tween 80, tributyrin, egg yolk and olive oil emulsion). It was shown that lipase present in culture supernatants of S. au reus hydrolyses all five substrates or none at all (with one exception, see Table 1). The same was true for coagulase-negative staphylococci. Proteolytic activity was determined with application of four different substrates (casein, albumin, hemoglobin, and gelatin). Seventy six (95%) out of 80 strains of S. aureus exhibited proteolytic activity on at least one of the substrates used (Table 1). High number of protease-positive strains was also found among coagulase-negative strains; 6 out of 8 strains of S. saprophyticus and 9 out of 11 strains of S. epidermidis. This means that about 79% of coagulase-negative strains were proteolytic. It should be noted, however, that only 16 strains (21%) of S. aureus were able to hydrolyse all four substrates. This property was slightly higher with coagulasenegative staphylococci (35.3%). The highest number of protease-positive strains of S. aureus was found using hemoglobin as a substrate. As many as 59 (73.7%) strains were positive. Using casein as a substrate, 49 (62.2%) strains were classified as proteolytic. Six of these strains were active only against casein and did not affect other substrates. Remaining substrates were less susceptible to staphylococcal proteases and 44 (55%) hydrolysed albumin, while 39 (48.7%) were active toward gelatin. No correlation between lipase and protease production by S. aureus was observed (Table 2). Among 76 protease-positive strains of S. aureus (positive with at least one substrate), 10 were lipase-negative and among protease-negative strains four were lipolytic. Table 2. Correlation of lipolytic and proteolytic activities of different strains of S. aureus Proteolytic activity
Lipolytic activity Gelatin +
+
*
+
20.0* 67.5 5.0
Albumin
7.5
Casein
Hemoglobin
+
+
27.5
60.0
33.5
54.0
43.5
43.5
5.0
7.5
2.5
10.0
8.0
5.0
Percentage of strains tested
Discussion It has been demonstrated by the present study that 87.5% of S. aureus strains of human origin are lipolytic, while 95% of them are proteolytic. Among coagulase-
456
S.Tyski, P.Ciborowski, W.Hr yniewicz, and J.]elj aszewicz
negati ve stra ins only about 10% were lipol ytic, but nearl y 89% of th em were proteolytic. There was no significant d ifferen ce bet ween S. epiderm idis and Sc sapr opby ticus stra ins, although a limited number of str ain s was tested. Lip olytic activity is usually related to S. aureus, since it was shown before (2, 9, 27) and was confirmed in th e pre sent study , that onl y few coagulase-negative staph ylococci are lipa se-po sitive. H owever, contradictory findings have been reported by W illis and T urner (34), who found all coa gulase-negative strains investigated by th em as lipase-po sitive. This difference may be due to various substrates used and perha ps different interpretati on of th e results. It is parti cularl y impo rta nt when using tr ibutyrin as a substra te, since so me sterile and not inoc ulated nutrient broth may pr oduce some zo ne of clea ring; thi s false-p ositi ve result may be interpreted as a positive reaction. No cor relation ha s been fo und between the source of strai ns and the ir lipol ytic and proteolytic activities. It is in keeping with the earli er published reports (2, 3, 16,32). No complete correlation was either observed betwe en hemolysis and lipase pr oduction, although these tw o activities seem to be typi cal fo r S. aureus strains (8, 11). Co ncern ing the proteolytic acitvity, our results although simila r are not compa ra ble to the data presented by other authors (5,6,9, 12, 13, 16, 23, 25, 26, 28), since the y investigated strains from not homogenous sources. M or eover, in none of th ese reports, sub strates used in our study were applied at th e sa me time. Our res ults demonstr at e th at pro teolytic activity is not char acteri stic for coa gu lase-positive sta phylococci, and th at th is property is also wid ely dis tributed a mong coagulase-negative stra ins. This is not in keeping with some earlier ob ser vations suggesti ng th at th is pro pert y is attributed mostly to coagulase-p ositive stap hylococci (25,26) . Report of Levy (10) sup po rts our find ings demonstr ating th at proteolytic activity is often ext ensive in coagulase-negative sta phy lococ ci. As stated before, th ese differences may be du e to a difference in the subst ra tes and techniques used. In earlier studies, gelatin was mainly used. It h as been recentl y replaced b y casein and other substra tes were only spor adica lly applied. We have employed four differ ent substra tes. Basing on various spectra of proteolytic activity, it seems that more th an one protease exists. It is imp ossible, however, to conclude at thi s moment how man y different proteolytic enzymes are excreted by staphylococci and how many by a particular single strai n. Early studies on lipase produc tio n by va rious staphy lococc i were pr acti cally limited to egg yolk, and only occasionally Tween 80 was used (2.,3 , 16, 18, 19, 27, 30). O nly few studies repo rte d application of more substra tes (14, 27). We have emp loyed in our study both na tura l fats, synthetic trigl ycerides and other synthetic esters, never used together before in a single investigation. All sta ph ylococcal strains hydrolysed either five substrates or none at all. This finding has been confirmed also with a highly purified staphylococcal lipase (33) and resulted in conclusion that sta phylococci produce one lipolytic enzyme with a broad spectrum of activity. This is in keeping with sugge stion s of Tirun arayanan and Lun dbeck (31) and of Brunner et al. (7), who concluded th at lipase and esterase activ ity reside in th e sa me protein molecule . It has been sho wn in thi s investiga tio n th at both lipol ytic and p roteol ytic activities are widel y distributed among sta phy lococci. Lipa se seems to be an enzyme with a br oad spectru m of activit y and is mainly produced by S. aureus. Proteolytic activity
Lipolytic and Proteolytic Properties of Staphylococci
457
is exhibited by both coagulase-positive and coagulase-negative strains, and results differ depending on the substrate used. Acknowledgement. This investigation was supported by research grant 05-339-C from the U. S. Public Health Service.
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21. Lipillski, B., j. Hatoiger, and]. [eljaszetoicz: Staphylococc al clumping with soluble fibrin monomer substrates. ] . expoM ed . 126 (1967) 979- 988 22. Ma rker, S. C. and E. D. Gray : Simple meth od for the pr ep ar at ion of sta phylococca l nucl ease. Appl. Microbiol. 23 (1972) 368-371 23. Ma rtley, F.G., W .A. ja rvis, D.F. Bacon, and R. C.Lawrence : T ypin g of coagulasepo siti ve staphylococci by p rot eolytic activity on buffered caseinate aga r with special refere nce to bacteriophage nontypabl e str ains . Infect. lm mun. 2 (1970) 439-442 24. O'L eary, W. M . and j. T. Weld: Lipolytic activities of Staphylo coccus aureus. 1. Nature of enzym e pro ducing fr ee fatty aci ds from pla sma lip ids. J. Bact . 88 (1964) 1356- 1363 25. Papaeuangelou, G. and j. Papauassiliou : Comparison of desoxyri bonuclease acti vity to so me other criteria of Staphylococcus pathogeni cit y. Path. Mi crobiol. 30 (1967) 59-63 26. Quie, P. G. and L. W. Wa nnamake r: Staphylo coccal Muller ph enom enon ; relat ion ship to th e plasmi nogen -pl asm in syste m. J. Bact . 82 (1961) 770-783 27. Rosendal, K. and P. BUlow : Staph ylococcus aureus stra ins isol ated in Danish hospitals. Act a path. microbiol. scand. 71 (1967) 422-438 28. Saski, S. et B. Fejgin: Au sujet des pro prieres proteol yriqu e et fibro lytique de sraphylocoques en connexion avec leur po uvoir toxigene. C. R. Soc. BioI. (Pa ris) 126 (1937) 139- 141 29. Shah, D. B. and j.B. Wilson: Egg yolk factor of Staphylococcus aureus. 1. Nature of the substrate and enzyme involved in th e egg yolk opacity reacti on . ]. Bact. 85 (1963) 516521 30. Sinel, H.]. and J. Baumgart : Co mparison of egg yolk containing selective media for the qu antitation and isolation of co agula se-positive staphylococci . Zbl. Bakt., 1. Abt. Orig. 204 (1967) 248-264 31. Ti runarayanan, M . O. and H . Lundbeck: Investigations on the enzy mes and toxins of stap hylococci. 1. H ydrol ysis of triglyceri des and other esters by lipase . Acta path. microbioI. scand. 73 (1968) 437-449 32. Tr ussell, R. E. and L. A. Weed: T he lipol ytic act ion of sta phylococci on so me pure tri glycerides . J. Bact. 33 (1937) 38 1-388 33. T yski, S. : Stap hylo coccal lip ase - p urification and some properti es. In : Stap hyl ococci and Staphylococcal Infection s, p. 335-3 42, J.jeljaszewicz (ed.). Gustav Fischer Verlag, Stuttg ar t (1981) 34. W illis, A. T . and G. C. T urner : Staph ylococcal lipolysis an d pigment at ion . ]. Path. Bact. 84 (1962) 337- 347 Dr. Stefan T yski, Department of Bacteriology, N ati onal Institut e of H ygiene , 24 Chocimska , 00-791 Warszawa , Pol and