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 determinationof these activities. Staphylococcus aureus strains were very frequently lipolytic (95%) andproteolytic (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 vonkoagulasepositiven 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 Staphylokokkenstammenerwiesen 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 staphylococcalinfection (1,17).

The purpose of this study was to estimate the frequency of lipase and proteaseproduction by staphylococci (coagulase-positive and coagulase-negative) isolatedfrom various clinical materials. These activities were tested by modern techniqueswith application of different substrates and related to the production of hemolysinsand 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 suchas urine, otitis excretions etc. 78 strains were coagulase-, nuclease and clumping factor(CF)-positive, and were classified as Staphylococcus aureus. Two other strains were addedto this group which were coagulase-negative, but nuclease and CF-positive. 19 remainingstrains were coagulase-, nuclease-, and CF-negative and represented species Staphylococcusepidermidis and Staphylococcus sapropbyticus. They were differentiated on the basis ofnovobiocin sensitivity test, ability to produce acid phosphatase and to hydrolyse trehaloseand 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 colonyfrom blood agar culture, and incubated for 24 h at 37°C on a shaker. Resulting cultureswere 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 byLipinski 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), sodiumcaseinate (Difco), and gelatin (Difco). 1% solution of the substrate in 0.05 M Tris-HCIbuffer of pH 7.4, supplemented with 1 mM cysteine hydrochloride and 1 mM CaCI" wasused 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. Thereaction was stopped by addition of 3 ml of 10% HCl04 and left for 15 min, followed byrecovering of the precipitate by filtration through Whatman No. 42 filter. All samples wererun in parallel. One unit of proteolytic activity was defined as an amount of the enzymeresulting in an absorbance of 1.0 at 280 nm of the supernatant after precipitation withperichloric 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 MTris-HCI buffer of pH 8.0 wirh 0.5% csci, was solidified with 1% agar and plares wereprepared. Egg yolk emulsion was prepared according to O'Leary and Weld (24). Lipolyticactivity 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 consideredas a positive reaction. With tributyrin as a substrate, clearance zone around wells wasaccepted as a positive result. Hydrolysis of egg yolk was recorded according to Shah andWilson (29).

2. Quantitative method: Free fatty acids liberated from olive oil emulsion by lipasepresent in culture supernatants, were measured by potentiometric titration (33).

Results

All strains of S. aureus produced nuclease, while only one coagulase-negativestrain (S. sapropbyticus isolated from urinary tract infection) was nuclease-positive.Hemolytic activity was found in 69 (86.25%) of S. aureus strains and none incoagulase-negative staphylococci.

-l'-

V,

-l'-

Tab

le1.

Lip

olyt

ican

dp

rote

oly

tic

subs

trat

esp

ecif

icit

yof

stap

hylo

cocc

alst

rain

sde

pend

ing

ofth

eir

orig

in

~

So

urce

Ta

xo

no

-L

ipo

lyti

csu

bstr

ate

sP

rote

oly

tic

su

bstr

ate

s>--

l'<

of

mic

cla

-H

em

oly

sin

~+

-O

liv

eo

ilT

wee

n80

Tw

een

20E

gg

yo

lkT

rib

uty

rin

Gela

tin

Alb

um

inC

asein

Hem

og

lob

inis

ola

tio

nssif

ica

tio

n+

-+

-+

-+

-+

-+

-+

-+

+-

~ oa

a5-

'B

loo

dSA

*8

18

19

9a

99

aa

93

61

84

50 '"'

SE

*0

10

1a

10

10

10

1a

1a

10

11

a0 :s

SS

*0

10

1a

1a

1a

10

10

10

11

01

a<

J> c:A

bscesses

SA31

330

430

430

430

430

46

288

269

2517

17~

SE0

10

10

10

10

1a

11

aa

11

aa

1::r: '"''< ::l

Na

so-

SA14

111

411

411

411

411

44

116

95

107

8n° :s

ph

ary

nx

SE0

2a

2a

20

2a

2a

20

21

1a

2a

2n°

SS0

31

21

21

21

21

21

20

31

22

1J"

'!'> ::l 0.

.B

urn

sSA

72

90

9a

9a

90

90

54

45

72

72

':-'

'-<

Oth

er

SA9

411

211

211

211

211

24

95

86

77

6~ "];i0

72

56

66

<J>

sou

rces

SE0

11

11

62

53

45

24

3N (l

)

SSa

40

4a

40

40

40

42

21

31

3a

4::;: n°

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

N

TO

TA

LSA

6911

6911

7010

7010

7010

7010

1961

2654

2852

4238

SE0

112

91

101

101

101

103

84

76

55

6

SS0

81

71

71

71

71

73

51

73

53

5

*SA

=S

tap

hy

loco

ccu

sau

reu

s,

SE=

Sta

ph

ylo

co

ccu

sep

iderm

idis

,SS

=S

tap

hy

loco

ccu

ssap

rop

hy

ticu

s

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 ofS. epidermidis and S. sapropbyticusi, There was no apparent correlation betweenlipase production and the source of strain. Lipolytic activity was determined in thisstudy 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. aureus 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 ofS. 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 strainsof S. epidermidis. This means that about 79% of coagulase-negative strains wereproteolytic.

It should be noted, however, that only 16 strains (21%) of S. aureus were ableto 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 usinghemoglobin as a substrate. As many as 59 (73.7%) strains were positive. Usingcasein as a substrate, 49 (62.2%) strains were classified as proteolytic. Six of thesestrains were active only against casein and did not affect other substrates. Remainingsubstrates 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 atleast one substrate), 10 were lipase-negative and among protease-negative strainsfour were lipolytic.

Table 2. Correlation of lipolytic and proteolytic activities of different strains of S.aureus

Lipolytic activity

+

Proteolytic activity

Gelatin Albumin Casein Hemoglobin

+ + + +

20.0* 67.5 27.5 60.0 33.5 54.0 43.5 43.55.0 7.5 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 ofhuman 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 nearly 89% of them wereproteolytic. There was no significant d ifference bet ween S. epidermidis and Sc sapro­pby ticus stra ins, although a limited number of str ain s was tested.

Lipolytic activity is usually related to S. aureus, since it was shown before (2, 9,27) and was confirmed in th e present study, that onl y few coagulase-negative sta­ph ylococci are lipa se-po sitive. H owever, contradictory findings have been reportedby Willis and T urner (34), who found all coagulase-negative strains investigated byth em as lipase-po sitive. This difference may be due to various substrates used andperha ps different interpretati on of th e results. It is parti cularl y impo rta nt whenusing tr ibutyrin as a substra te, since some sterile and not inoc ulated nutrient brothmay produce some zone of clearing; thi s false-positive result may be interpreted asa positive reaction.

No cor relation has been found between the source of strains and the ir lipol yticand proteolytic activities. It is in keeping with the earli er published reports (2, 3,16,32). No complete correlat ion was either observed betwe en hemolysis and lipaseproduction, although these tw o activities seem to be typi cal for S. aureus strains(8, 11).

Concern ing the proteolytic acitvity, our results although similar are not com­pa rable 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. Mor eover, in noneof th ese reports, sub strates used in our study were applied at th e same time.

Our results demonstr at e th at pro teolytic activity is not characteri stic for coagu­lase-positive sta phylococci, and that th is property is also wid ely dis tributed amongcoagulase-negative stra ins. This is not in keeping with some earlier ob ser vationssuggesting th at th is propert y is attributed mostly to coagulase-p ositive staphylo­cocci (25,26) . Report of Levy (10) suppo rts our findings demonstr ating th at proteo­lytic activity is often ext ensive in coagulase-negative sta phylococ 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, gelat in was mainly used. It has been recentl y replaced by caseinand other substra tes were only spor adica lly applied. We have employed four dif­ferent substra tes. Basing on various spectra of proteolytic activity, it seems thatmore th an one protease exists. It is imp ossible, however, to conclude at thi s momenthow man y different proteolytic enzymes are excreted by staphylococci and howmany by a particular single strain.

Early studies on lipase produc tio n by various staphylococc i were pr acti callylimited to egg yolk, and only occasionally Tween 80 was used (2.,3 , 16, 18, 19, 27,30). Only few studies repo rted application of more substra tes (14, 27). We haveemployed in our study both na tura l fats, synthetic trigl ycerides and other syntheticesters, never used together before in a single investigation. All sta phylococcal strainshydrolysed either five substrates or none at all. This finding has been confirmed alsowith a highly purified staphylococcal lipase (33) and resulted in conclusion thatsta phylococci produce one lipolytic enzyme with a broad spectrum of activity. Thisis in keeping with suggestion s of Tirunarayanan and Lun dbeck (31) and of Brunneret al. (7), who concluded th at lipase and esterase activ ity reside in th e sa me proteinmolecule .

It has been sho wn in thi s investiga tio n th at both lipol ytic and proteolytic activitiesare widel y distributed among sta phylococci. Lipa se seems to be an enzyme with abr oad spectru m of act ivity 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 resultsdiffer depending on the substrate used.

Acknowledgement. This investigation was supported by research grant 05-339-C fromthe 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 brothby 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 ofantibiotic-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 amongS. aureus strains. Acta path. microbiol. scand. B 85 (1977) 277-280

7. Brunner, H., C. G. Gemmel, H. Hauser, and F.]. Fehrenbach: Chemical and biologicalproperties 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 avecreference 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 lipolyticactivity 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 toxicand biochemical properties among coagulase-negative and coagulase-positive strains ofstaphylococci. Med. Dosw, Mikrobiol. 12 (1960) 33-41

17. [eliaszeioicz, ]., S.Szmigielshi, and W. Hryniewicz: Biological effects of staphylococcaland 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 Staphylococcusaureus possibly related to pathogenicity. A. A study of 446 strains from different typesof human infections. Acta path. microbiol. scand. 47 (1959) 316-326

19. [essen, 0., V. Faber, K. Rosendal, and K. R. Eriksen: Some properties of Staphylococcusaureus possibly related "0 pathogenicity. 2. In vitro properties and origin of the infectingstrains 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

458 S.Tyski , Pi Ciborow ski, W.Hryniewi cz, and J.]eljaszewicz

21. Lipillski, B., j. Hatoiger, and]. [eljaszetoicz: Staphylococc al clumping with soluble fibrinmonomer substrates. ] . expoM ed. 126 (1967) 979- 988

22. Ma rker, S. C. and E.D. Gray : Simple meth od for the prep arat ion of staphylococca lnucl 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 coagulase­positi ve staphylococci by prot eolytic activity on buffered caseinate aga r with specialreference to bacteriophage nontypabl e str ains . Infect. lm mun. 2 (1970) 439-442

24. O'Leary, W. M . and j. T. Weld: Lipolytic activities of Staphylococcus aureus. 1. Natureof enzyme pro ducing free fatty acids from plasma lip ids. J.Bact . 88 (1964) 1356- 1363

25. Papaeuangelou, G. and j. Papauassiliou : Comparison of desoxyribonuclease acti vity to

so me other criteria of Staphylococcus pathogenicity. Path. Mi crobiol. 30 (1967) 59-6326. Quie, P. G. and L. W. Wa nnamaker: Staphylococcal Muller phenomenon ; relat ionship

to the plasmi nogen -plasmin system. J. Bact . 82 (1961) 770-78327. Rosendal, K. and P. BUlow : Staph ylococcus aureus stra ins isolated in Danish hospitals.

Act a path. microbiol. scand. 71 (1967) 422-43828. Saski, S. et B.Fejgin: Au sujet des proprieres proteolyriqu e et fibro lytique de sraphylo­

coques en connexion avec leur po uvoir toxigene. C. R. Soc. BioI. (Paris) 126 (1937)139- 141

29. Shah, D. B. and j.B. Wilson: Egg yolk factor of Staphylococcus aureus. 1. Nature of thesubstrate and enzyme involved in th e egg yolk opacity reaction . ]. Bact. 85 (1963) 516­521

30. Sinel, H.]. and J. Baumgart : Co mparison of egg yolk containing selective media for thequ 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 enzymes and toxins ofstap 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 some pure tri ­glycerides . J. Bact. 33 (1937) 381-388

33. T yski , S. : Staphylococcal lipase - purification and some properti es. In : Staphylococciand Staphylococcal Infections, p. 335-3 42, J.jeljaszewicz (ed.). Gustav Fischer Verlag,Stuttg ar t (1981)

34. Willis, A. T . and G. C. T urner: Staphylococcal lipolysis an d pigment at ion . ]. Path. Bact.84 (1962) 337- 347

Dr. Stefan Tyski, Department of Bacteriology, National Institute of H ygiene , 24 Cho­cimska , 00-791 Warszawa, Poland