JOURNAL OF CLINICAL MICROBIOLOGY, May 1983, p. 759-7670095-1137/83/050759-09$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 17, No. 5

Fibrinogenolytic and Fibrinolytic Activity in OralMicroorganisms

MAUDE B. WIKSTROM,1* GUNNAR DAHLtN,1 AND ANDERS LINDE2Department of Oral Microbiology, Institute of Medical Microbiology,' and Laboratory of Oral Biology,

Department of Histology,2 University of Goteborg, S-413 46 Goteborg, Sweden

Received 25 October 1982/Accepted 21 January 1983

Samples were taken from blood accumulated in dental alveoli after surgicalremoval of mandibular third molars, from subgingival plaque of teeth withadvanced periodontal destructions, from teeth with infected necrotic pulps, andfrom subjects suffering from angular cheilitis. Of the microorganisms subculturedfrom these samples, 116 strains were assayed for enzymes degrading fibrinogenand fibrin. Enzymes degrading fibrinogen were assayed with the thin-layerenzyme assay cultivation technique. This assay involves the cultivation ofmicroorganisms on culture agars applied over fibrinogen-coated polystyrenesurfaces. Enzymes degrading fibrin were assayed with both a plate assay and atube assay, in which fibrin was mixed with a microbial culture medium. Microor-ganisms degrading fibrinogen or fibrin or both were isolated from all samplingsites. Activity was mainly detected in strains of Actinomyces, Bacteroides,Fusobacterium, Peptococcus, Propionibacterium, and Staphylococcus aureus.Most Fusobacterium strains degraded fibrinogen only. Enzymes degrading fibrin-ogen as well as enzymes degrading fibrin via activation of plasminogen wererevealed in strains of Clostridium, S. aureus, and Streptococcus pyogenes. It wasgenerally found that fibrinogen was degraded by more strains than was fibrin,which indicates that different proteases may be involved.

The deposition of fibrin in wounds protectsthe surrounding tissues from invasion by micro-organisms. Fibrinogen, the precursor of fibrin,has also been considered to be of importance inthe defense system against microorganisms,since high levels of fibrinogen in blood are oftenfound in patients with infectious diseases (3). Itis known that split products of fibrinogen mayimpede the polymerization of fibrin and increasethe tendency to bleed (3). Furthermore, the netof fibrin formed in the presence of such frag-ments seems to be weaker (1). Thus, a produc-tion of fibrinogen- and fibrin-degrading enzymesby microorganisms should be of significance fortheir virulence.

Degradation of fibrin by microorganisms oforal origin has been studied in several investiga-tions. Strains of Staphylococcus aureus and of,B-hemolytic streptococci with fibrinolytic activi-ty were isolated in samples from the nose andthroat of patients with epistaxis (29). Fibrinolyt-ic activity in strains of Bacteroides asaccharo-lyticus (now classified as B. gingivalis [10]) andTreponema denticola, both associated with peri-odontal diseases and with acute necrotizing ul-cerative gingivitis, has also been demonstrated(27). On the other hand, no fibrinolytic activitycould be found in microorganisms isolated from

dental fibrinolytic alveolitis ("dry socket") (2)or from saliva collected from patients with in-creased fibrinolytic activity in mixed saliva (22).However, there is still a lack of information as

to production of fibrinogen-degrading enzymesby oral microorganisms. Another importantquestion is whether microbial fibrinogen-degrad-ing capacity is necessarily concurrent with afibrin-degrading capacity. These are the ques-tions which have been addressed in this study.

MATERIALS AND METHODSSampling and isolation of bacteria. None of the

patients were subjected to antimicrobial treatmentbefore sampling.

Dental alveolar blood. In connection with surgicalremoval of mandibular third molars, samples of theblood which accumulated in the dental alveoli werecollected immediately before suturing. Surgical proce-dures were carried out according to routine methods,including a final rinsing of the alveolus with 60 ml of aO.9o NaCl solution. The ambient atmosphere over thedental alveoli was displaced with oxygen-free C02,and precautions were taken to avoid contaminationduring the sampling procedure. The blood sample,about 0.1 ml, was aspirated through a sterile Teflontube attached to a syringe. The sample was transferredto a transport medium, VMGA III (i.e., VMG III [26]anaerobically prepared and stored), and brought to thelaboratory within 1 h.

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760 WIKSTROM, DAHLEN, AND LINDE

All handling of the sample was performed in ananaerobic glove box (85% N2, 10% H2, 5% C02). Thesample bottle was shaken for 20 s in a whirling mixer.A 0.1-ml volume of the transport medium was spreadon a prereduced horse blood agar plate, and 1 ml wasinoculated in a tube with culture medium HCMGA-Sula (i.e., HCMG-SuIa [26] anaerobically preparedand stored). HCMG medium is an egg white-contain-ing meat-peptone mixture with 1% (wt/vol) glucose,3% (wt/vol) yeast extract, 0.3% (wt/vol) cysteine HCI,and 2% (wt/vol) agar added. Sula is a supplementconsisting of liver extract, yeast autolysate, inactivat-ed horse serum, and a piece of raw potato. Both aredescribed in detail by Moller (26). The media wereincubated at 36°C in a special compartment of theglove box for at least 16 days. In addition, 0.1 ml of thetransport medium was inoculated on a horse bloodagar plate and incubated at 36°C under aerobic condi-tions for 3 to 4 days. If growth in the tubes was found,the tubes were shaken in a whirling mixer, and 0.2 mlof the tube contents was inoculated on blood agarplates and incubated in aerobic and anaerobic condi-tions as described above. Colonies and cells withdifferent morphologies, Gram stains, and oxygen sen-sitivities were subcultured. Fifty-four strains wereisolated from five subjects and tested for fibrinogeno-lytic activity. Only strains degrading fibrinogen werefurther examined for fibrinolytic activity.Deep periodontal pockets. Human subgingival plaque

was obtained from teeth not previously subjected toperiodontal treatment and with pocket depths exceed-ing 6 mm. An acrylic chamber was applied over theactual teeth (12) and tightly adapted to mucosa andteeth with impression material. The chamber was filledwith oxygen-free carbon dioxide and covered withplastic foil. The subgingival plaque sample was takenwith a curette and transferred to transport mediumVMGA I (i.e., VMG I [26] anaerobically prepared andstored) kept in a vial inside the acrylic chamber. Thesample (delivered to the laboratory within 1 h) wasdispersed, diluted, and incubated, as previously de-scribed (12), on horse blood agar plates and brucellaagar (no. 11086 BBL; Microbiology Systems, Cock-eysville, Md.) plates enriched with 5% (vol/vol) defi-brinated horse blood, 0.5% (vol/vol) hemolyzed horseblood, 5 jig of menadione per ml prepared according tothe Virginia Polytechnic Institute manual (17), and 2%(vol/vol) potato juice (26) anaerobically prepared. Bac-teria that occurred in a frequency of 5% or more of thetotal CFU count were subcultured for further charac-terization and identification. The predominatingstrains isolated from pockets offour teeth from each oftwo subjects were tested for fibrinogenolytic and fibri-nolytic, activity.

Necrotic dental pulp. Sampling procedures and labo-ratory handling of the samples from root canals ofnecrotic teeth were carried out with principles outlinedby Moller (26). The vitality of the teeth was lost due totrauma, and the pulps had not been exposed to the oralcavity before sampling. Further details of the clinicalmaterial have been described previously (11). Afterpreparation and sterility control of the operation field,the pulp chamber was opened with sterile burrs. Aftermechanical debridement of the canal walls with Hed-strom files, samples were taken with charcoal-impreg-nated paper points. Special precautions were taken toreach the apical region of the root canal (26). The

samples were transferred to transport medium VMGAIII. Inoculation and cultivation were made as de-scribed previously (11) on aerobic and anaerobic horseblood agar plates and in HCMGA-SuIa and HCMGA-Sull (Sull is a supplement consisting of liver extractand inactivated horse serum) (26). All isolated strainswere tested for fibrinogenolytic and fibrinolytic activi-ty.Angular cheilitis. Some clinical and microbial data of

the patients with angular cheilitis have been reported(S.-Ch. Ohman and G. Dahldn, Scand. Div. Int. As-soc. Dent. Res. Abstr. no. 71, 1982) and furtherinformation will be given in a separate publication(S.-C. Ohman and G. Dahldn, manuscript in prepara-tion). The samples were taken before any treatment ofthe disease had been initiated. The sampling fromangulus was performed by use of a sharp carver thatwas scraped with firm pressure over the actual lesion.The samples were transferred to transport mediumVMGA III. At the laboratory they were dispersed andinoculated (13) onto horse blood agar plates, prere-duced horse blood agar plates to which menadione andhemolyzed blood had been added (17), staphylococcusagar (no. 0297; Difco Laboratories, Detroit, Mich.)plates, gentian violet plates (9), and Sabouraud-glu-cose agar (Difco 0109) plates with tetrazolium chlo-ride. The microorganisms were identified and semi-quantified according to a method previously described(13). One or two of the most predominant strains(except for a-streptococci) were selected for the pres-ent study. Samples were also taken as scrapings fromthe nose and, in cases of denture wearers, also fromthe palate. In these samples special attention wasgiven to the presence of S. aureus, Streptococcuspyogenes, and fungi, which were also selected for thepresent study.

Identification of microorganisms. Identification ofanaerobes to species was made according to the Vir-ginia Polytechnic Institute anaerobe manual (17) bybiochemical tests and by gas chromatographic analy-ses of metabolic end products. The gas chromatograph(The Perkin-Elmer Corp., Norwalk, Conn.; Sigma 2B)was mounted with a flame ionization detector. Columnpacking was 5% free fatty phase on Chromosorb G-AW-DMCS, 80/100 mesh. The carrier gas was N2. Theinjection port and detector temperature was 150°C,and the oven temperature was 120°C. Facultativelyanaerobic strains were identified according to Ber-gey's Manual (6). Yeasts were identified by use of theAPI 20 Candida auxanogram (API Systems, Monta-lieu-Vercieu, France).Enzyme assays. (i) Fibrinogenolytic activity. Fibrino-

genolytic activity was tested by the thin-layer enzymeassay (TEA) cultivation technique (33). The assay isbased on the finding that the wettability of a protein-coated polystyrene surface is decreased, i.e., thehydrophobicity is increased, after degradation of theprotein (35). Polystyrene petri dishes (Nunc, Roskilde,Denmark) were coated with human fibrinogen (Kabi,Stockholm, Sweden) (35). The microorganisms wereinoculated as spots on a culture agar medium appliedover the fibrinogen-coated surfaces. The medium usedwas HCMG agar base (26) with the glucose concentra-tion reduced to 0.05% (wt/vol) and with 0.05% CaSO4(wt/vol) added (33). The plates were incubated at 36°Cin an appropriate atmosphere for the microorganismsuntil a heavy growth had been obtained. The culture

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FIBRINOGENOLYTIC AND FIBRINOLYTIC ACTIVITY 761

TABLE 1. Sensitivity of the assays'Fibrin plates with Fibrin clot in tube with

Trypsin Fibrinogen-coated plasminogen: plasminogen:(U/liter) ~polystyrene(U/iter) ysurfaces (MM)b Not added Added Not added Added

(MM)b (mm)" o de de

1,750 17.8 17.9 17.7 + +350 13.7 13.9 13.8 + +175 12.6 12.9 12.8 + +35 9.0 9.1 9.0 + +17.5 7.5 7.3 7.1 + +3.5 3.8 3.7 3.5 -

a Plates (n = 5) and tubes (n = 3) were incubated with trypsin for 18 h at 36°C. The plates were tested with theradial diffusion in agar gel technique (29). The mean values of the diameters of the enzyme-affected areas aregiven.

b Standard deviation ranged between 0.0 and 0.3. The correlation coefficients between the diameters of theenzyme-affected areas and the logarithmic value of enzyme concentrations were 0.99.

medium was then removed. Fibrinogenolytic activityof the microorganisms was detected in the form ofreduced wettability of the fibrinogen-coated surfacesafter condensation of water vapor on the surface.

(ii) Fibrinolytic activity. Both direct fibrinolytic ac-tivity and activity via activation of plasminogen (indi-rect activity) of the microorganisms were tested. Forscreening of direct activity the plasminogen content ofthe fibrinogen, used for preparation of the fibrin, wasremoved by affinity chromatography on lysine-Sepha-rose 4B (Pharmacia Fine Chemicals, Uppsala, Swe-den). The gel was washed and packed in the column asdescribed by Deutsch and Mertz (14), and the separa-tion procedure was performed as outlined by Noren etal. (28). Human plasminogen (Kabi) was added to theplasminogen-free fibrinogen solution for screening of

indirect activity on fibrin. Bovine thrombin (E. MerckAG, Darmstadt, West Germany) was used for conver-sion of fibrinogen to fibrin. The methods used forscreening of fibrinolytic activity were slightly modifiedversions of the fibrin agar plate assay described byNoren et al. (28) and the fibrin clot tube assay de-scribed by Jeffries and Buckley (18). HCMG agar basewas substituted for agarose in the fibrin agar plates.The plates were stored overnight at 4°C for stabiliza-tion and were prewarmed to room temperature beforeuse. The microorganisms were inoculated as spots onthe surface of the culture agar medium and thenincubated in appropriate conditions. The degradationof fibrin was detected as a clear zone around themicrobial colonies.HCMG medium without addition of agar was used

TABLE 2. Bacteria isolated from alveolar blood after surgical removal of mandibular third molarsaNo. of Fibrinolytic activity'

Subject strains Fibrinogenolytic strainsisolated Plate assay Tube assay

E 11 Peptostreptococcus intermedius - -Actinomyces viscosus +WEubacterium tenueFusobacterium sp. ND ND

S 11 Peptococcus magnusActinomyces bovisBacteroides sp. (pigmented) ND ND

U 7 Propionibacterium acnes +W +

Gram-negative anaerobic rod ND ND

M 12 Propionibacterium acnes +W

Propionibacterium acnes - -

A 13 Peptostreptococcus intermedius +Propionibacterium acnes +Actinomyces sp. ND NDBacteroides sp. (pigmented) ND ND

a Test samples were collected immediately before suturing of the alveoli. Only bacteria with fibrinogenolyticactivity were selected and assayed for fibrinolytic activity.

b ND, Not determined; w, weak reaction.c Activity revealed only with plasminogen-containing substrate.

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TABLE 3. Fibrinogenolytic and fibrinolytic activity in bacteria isolated from subgingival plaque of teeth withpocket depths exceeding 6 mm'

Strains

E 14 Bacteroides ureolyticusFusobacterium gonidiaformans

24 Propionibacterium acnesFusobacterium naviformeBacteroides sp. (pigmented)

45 Clostridium sp.Fusobacterium nucleatumFusobacterium nav,iformeBacteroides melaninogenicus

subsp. intermedius

41 Veillonella parvulaActinomyces siscosus

F 45 Clostridium cochleariumFusobacterium naviformeBacteroides fragilis, otherBacteroides sp. (pigmented)

11 Peptococcus microsBacteroides ureolyticus

42 Peptococcus variabilisActinomyces sp.Clostridium bifermentans

34 Peptococcus prevotiiEubacterium ventriosum

Fibrinogeno- Fibrinolytic activitylytic activity Plate assay Tube assay

+

+

+

+

+

+

a Strains in a frequency of 5% or more were assayed.b Activity revealed only with plasminogen-containing substrate.C A saccharolytic strain not possible to identify to species by the diagnostic

Bacteroides given by Harding et al. (16).

for the fibrin clot tube assay. The fibrin clot tubesintended for anaerobic microorganisms were preparedin anaerobic conditions in a glove box. Degradation offibrin was detected by a liquefaction of the clot.

Both the plates and the tubes were examined dailyfor 1 week for lysis of the fibrin when incubated withaerobic strains and once weekly for 3 weeks whenincubated with anaerobic strains. Indirect enzymaticactivity on fibrin could not be detected if the strainsalso produced enzymes with direct activity on fibrin.

Sensitivity of the assays. The sensitivity of the assays

was tested with trypsin (3.5 U/mg; no. 25479, Merck)which was serially diluted (Table 1) in 9 mM universalbuffer (5), pH 7.8.The TEA, with fibrinogen as substrate, was tested

as described by Wikstrom et al. (34) with Bacto-Agar(no. 0140, Difco) as diffusion agar.

The tests of the fibrin plate assay were performed as

described by Noren et al. (28) except that 2% (wt/vol)Bacto-Agar in 28.5 mM universal buffer, pH 7.8, was

used instead of agarose. Both plasminogen-free fibrinplates and fibrin plates to which plasminogen had beenadded were tested.The fibrin clot tube assay was tested by incubation

characteristics of pigmented

of a 1-ml fibrin clot with 0.05 ml of trypsin solution.The clots were prepared as described by Jeffries andBuckley (18) except that 9 mM universal buffer, pH7.8, was used instead of a culture medium.

RESULTS

The plate assays, the TEA for detection offibrinogenolytic activity, and the fibrin plateassay (both with and without plasminogen)showed equal sensitivity to trypsin (Table 1).The fibrin clot tube assay was slightly lesssensitive to trypsin than the fibrin plate assay

(Table 1).In all, 116 microbial strains were examined.

Fibrinogenolytic or fibrinolytic activity or bothwas detected in 15 of 54 bacterial strains isolatedfrom dental alveolar blood (Table 2), in 14 of 22strains isolated from subgingival plaque (Table3), in 6 of 19 strains isolated from teeth withnecrotic pulps (Table 4), and in 12 of 21 strainsisolated from subjects with angular cheilitis (Ta-ble 5).

Subject Tooth

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FIBRINOGENOLYTIC AND FIBRINOLYTIC ACTIVITY 763

TABLE 4. Fibrinogenolytic andin bacteria isolated from the rool

teeth with intact cr

Fi

Tootha Strain 9

1 Veillonella parvulabFusobacterium sp.Bacteroides oralis

2 Bacteroides fragilis,other

3 Fusobacterium sp.

4 Bacteroides sp.

5 Bacteroides furco-sus

6 Bacteroides fragilis,other

Bacteroides sp.

7 Streptococcus mi-tior

Peptostreptococcusproductus

Peptostreptococcusintermedius

Peptococcus sac-charolyticus

fibrinolytic activity However, all strains within the same species didt canals of necrotic not always show the same result. Five of the 15,owns strains of Bacteroides exhibited fibrinogenolytic

ibrino- Fibrinolytic activity. Three of the positive strains were fur-geno- activity ther tested for fibrinolytic activity and all re-lytic Plate Tube vealed such activity. It should be noted thatOlivity assay assay most of these strains were pigmented strains of

+ Bacteroides. One offour strains tested of Bacte-+ + roides fragilis (other) degraded solely fibrin.

- _ _ Other strains with concurrent fibrinogenolyticand fibrinolytic activity belonged to the species

- - - Actinomyces viscosus, Clostridium sp., Pepto-coccus intermedius, Peptococcus variabilis,Propionibacterium acnes, and S. aureus. None

+ - - of the Fusobacterium strains degraded both fi-brinogen and fibrin. Of the seven strains testedfor both fibrinogen and fibrin activity, six de-graded fibrinogen only and one degraded fibrinonly (Table 6). The activity on fibrin, shown instrains of S. aureus, was mediated by activation

- - - of plasminogen. Other bacteria showing activa-tor activity were the Streptococcus pyogenes

+ + - strain and one of the Clostridium strains.When comparing the results with the two

assays for detection of activity on fibrin, it wasfound that more strains revealed fibrinolyticactivity with the plate assay than with the tube

- - - assay. Fourteen strains were positive with bothassays; nine, with only the plate assay; and

- - - three, with the tube assay.

8 Propionibacteriumacnes

Eubacterium lentumStreptococcus mil-

leriBacteroides fragilis,

otherVeillonella alcales-censd

Lactobacillus del-brueckii

a One tooth per subject.b Catalase negative.c Activity revealed only with p

ing substrate.d Catalase positive.

Of 38 fibrinogenolytic strained a concurrent fibrinolytic ac'four strains activity was onfibrin as substrate (Table 3, 4fibrinogen-degrading capacitynisms is not necessarily concudegrading capacity.

Fibrinogenolytic activity w,in strains belonging to the gerBacteroides, Fusobacterium, ipionibacterium, and Staphylo

+ + - DISCUSSIONIn the present study it was possible to demon-

strate a production of enzymes degrading fibrin-ogen as well as fibrin by microorganisms isolat-

- - + ed from blood accumulated in dental alveoliafter surgical removal of mandibular third mo-

- - - lars, from subgingival plaque of teeth with ad-vanced periodontal destructions, from teethwith infected necrotic pulps, and from patientswith angular cheilitis. Although most strainsdegraded both fibrinogen and fibrin, there werealso several strains which degraded only one or

ilasminogen-contain- the other.It has been shown that microorganisms pro-

duce fibrinolytic enzymes belonging to the ser-ine and the thiol as well as the metallo groups ofproteases (18). By using fibrinogen and fibrin as

is, 21 also exhibit- enzyme substrates, as was done in this study,tivity (Table 6). In proteases of all of these groups should be possi-fly detected with ble to detect.t, and 6). Thus, a The method used for detection of fibrinogeno-of the microorga- lytic activity, the TEA, provides a sensitive andrrent with a fibrin- simple method for determination of proteolytic

activity (33-35). Data available about adsorptionas mainly noticed of fibrinogen to hydrophobic surfaces (e.g., aiera Actinomyces, polystyrene surface) indicate that fibrinogen isPeptococcus, Pro- adsorbed in a monomolecular layer and that thecoccus (Table 6). molecules retain a native conformation (4; P. A.

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764 WIKSTROM, DAHLtN, AND LINDE

TABLE 5. Fibrinogenolytic and fibrinolytic activity microorganisms isolated in subjects with angularcheilitisa

SubecSmpesFibrinogeno- Fibrinolytic activitySubject Sample site Strains lytic activity Plate Tube

assay assay

1 Angulus Staphylococcus aureus + +b +bVeillonella parvula - - -

2 Angulus Staphylococcus aureus +Palatal mucosa Staphylococcus aureus + +b + b

3 Angulus Staphylococcus epidermidis - - -

Palatal mucosa Veillonella alcalescens - - -

Candida albicans - -

Candida tropicalis - -

4 Angulus Staphylococcus aureus + +b +bNose Staphylococcus aureus + + b +b

5 Angulus Staphylococcus epidermidisVeillonella alcalescens +

6 Angulus Staphylococcus aureus + +b +bCandida albicans

7 Angulus Staphylococcus aureus + +b +b

8 Angulus Staphylococcus aureus + +b +bPalatal mucosa Candida albicansNose Staphylococcus aureus + +b +b

Streptococcus pyogenes + +b +b

9 Angulus Staphylococcus aureus + +b +bHaemophilus parainfluenzae

a Predominant strains were assayed (except for streptococci of the viridans group).b Activity revealed only with plasminogen-containing substrate.

Cuypers, Ph.D. thesis, Medical University Lim-burg, Maastricht, The Netherlands, 1976). Theadsorbed fibrinogen also retains its predictedsensitivity to degradation by trypsin and pronase(pronase P from Streptomyces griseus) (33, 35).Thus, TEA seems to be well suited for studies offibrinogenolytic activity. It should be men-tioned, however, that the fibrinogenolytic activi-ty detected with TEA is probably a direct activi-ty on fibrinogen. No effect of streptokinase(which acts via activation of plasminogen) couldbe detected on fibrinogen-coated polystyrenesurfaces (data not shown).For detection of fibrinolytic activity a fibrin

agar plate assay, performed essentially as de-scribed by Noren et al. (28), and a fibrin clottube assay (18) were used.

In previous fibrin agar plate assays the fibrinplates have been heated to destroy their plas-minogen content (20). This was performed tomake it possible to discriminate between directand activator activity via plasminogen (indirectactivity) on fibrin. However, heating may causean altered reactivity of the fibrin (30). This step

is not necessary in the assay elaborated byNoren et al. (28), because the fibrinogen used forpreparation of the fibrin plates is purified fromplasminogen by affinity chromatography. Fordetermination of indirect activity on fibrin aconstant amount of plasminogen is added to thefibrinogen before preparation of the plates. Forthis reason the fibrin used throughout the pres-ent study was prepared as described by Noren etal. (28).

It is well known that cultivation conditionsmay influence microbial enzyme production (8).To be able to exclude the culture medium as afactor responsible for divergent results, thesame medium was used in all assays done here.HCMG medium (26) was chosen to fit the re-quirements of the fibrin plate assay of a nonpig-mented diffusion agar and to be suitable forcultivation of the fastidious microorganisms tobe examined.With the techniques used, it was possible for

the enzymes produced by the microorganismsduring growth to act directly on the enzymesubstrates. This endows the assays with a high

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FIBRINOGENOLYTIC AND FIBRINOLYTIC ACTIVITY 765

TABLE 6. Frequency of isolated strains of thespecies revealing fibrinogenolytic or fibrinolytic

activity or both

No. of strains revealingNo. of activity

Species strain On Onl With bothsassayedwith with fibrin-

fibrin- fibrn ogenogen firnand fibrin

Staphylococus au-reus

Streptococcus py-ogenes

Peptococcus mag-nus

Peptococcus microsPeptococcus prevo-

tiiPeptococcus varia-

bilisPeptostreptococcus

intermediusActinomyces bovisActinomyces visco-

sus

Actinomyces sp.Clostridium sp.Clostridium coch-

leariumEubacterium tenuePropionibacterium

acnesVeillonella alcales-cens

Veillonella parvulaBacteroides sp.Bacteroides sp.

(pigmented)Bacteroides fragilis,

otherFusobacterium sp.Fusobacterium go-

nidiaformansFusobacterium na-

viformeFusobacterium nu-

cleatumGram-negative an-

aerobic rodd

10

1

1

11

1

3

12

21

1

1

6

3

3

2

4

4

3

1

3

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2C

1

3

1

lb

a Activity revealed only with plasminogen-contain-ing substrate.

b Assayed only for fibrinogenolytic activity.c One strain assayed only for fibrinogenolytic activi-

ty.d Not identified.

detection capacity and makes them simple andrapid to perform.

Despite the fact that the fibrinogen and thefibrin plate assays are almost equally sensitive totrypsin (Table 1), more strains exhibited fibrino-genolytic than fibrinolytic activity (Table 6). In a

few strains only fibrinogen activity was found.

Thus, microbial production of proteolytic en-zymes degrading only one of the proteins cannotbe excluded.

Six of the seven Fusobacterium strains testedfor both fibrinogenolytic and fibrinolytic activitydegraded fibrinogen only. One strain degradedfibrin only. Three of the 13 Bacteroides strainstested for fibrinogenolytic and fibrinolytic activi-ty degraded both, and 1 strain degraded fibrinonly. This is in agreement with the results ob-tained by Nitzan et al. (27), who used fibrin asthe enzyme substrate. These investigators tested10 strains of Fusobacterium without reportingany positive strain and 20 strains of Bacteroides,of which 6 strains showed direct activity onfibrin. The fibrinolytic Bacteroides strains in thestudy of Nitzan et al. (27) were all B. melanino-genicus subsp. asaccharolyticus, now classifiedas B. asaccharolyticus (13). Proteolytic enzymeactivity has beeq shown to be a reliable charac-teristic of B. gingivalis (oral strains of black-pigmented asaccharolytic Bacteroides) (21, 25).Thus, three of the four black-pigmented Bacte-roides strains, which were not identified to spe-cies in the present study (Table 6), could bestrains of B. gingivalis due to their proteolyticactivity. The fourth strain that also showed aproteolytic activity could not be classified as B.gingivalis due to saccharolytic activity. Theexact classification of this strain was, however,uncertain. The indirect activity on fibrin of S.aureus, Clostridium species, and beta-hemolyticstreptococci is well documented (7). In the pres-ent study strains of these species also revealedfibrinogenolytic activity.

In some cases the two assays used for detec-tion of fibrinolytic activity gave divergent re-sults. This may be due to the fibrin plate assaybeing slightly more sensitive than the fibrin clottube assay. However, it may also be due todifferent abilities of the microorganisms to growand to produce enzymes on an agar surface(plate assay) and in an agar gel (tube assay).Judging from the results obtained in the presentstudy, the fibrin plate assay seems to be themethod of choice for screening fibrinolytic activ-ity. In our experience, the main advantages ofthe tube assay are the easy detection of fibrino-lytic activity and the small amount of fibrinrequired.The microbial species isolated and assayed for

fibrinogenolytic and fibrinolytic activity in thepresent study have been found in the samelocations by others. MacGregor and Hart (23,24) reported a predominance of Fusobacteriumand Bacteroides strains among the anaerobicmicroorganisms in dental alveolar blood collect-ed after tooth extractions. In addition, theyisolated strains of Actinomyces species. Severalstrains of these species were isolated from den-

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766 WIKSTROM, DAHLEN, AND LINDE

tal alveolar blood also in the present study(Table 2). Microorganisms of Fusobacterium,Bacteroides, Actinomyces, and Clostridium spe-cies are all found in increased numbers in peri-odontitis (31, 32). In the present study also,these constituted the main group of microorga-nisms found in subgingival plaque of teeth withadvanced periodontal destructions (Table 3).The frequent occurrence of Fusobacterium andBacteroides strains in samples from teeth withnecrotic pulps (Table 4) is also well established(19, 36; G. Sundqvist, Ph.D. thesis, Universityof Umea, Umea, Sweden, 1976). S. aureus andCandida albicans microorganisms comprisedmost of the tested strains from patients withangular cheilitis in the present study (Table 5).In a previous study strains of these species wereshown (Ohman and Dahlen, Scand. Div. Int.Assoc. Dent. Res. abstr. no. 71, 1982) to fre-quently occur in such infections.

In conclusion, strains producing fibrinogen-and fibrin-degrading enzymes were found in allof the various sampling sites. Strains of thespecies most frequently found also most fre-quently revealed such activity. It was also foundthat fibrinogenolytic activity is not necessarilyconcurrent with fibrinolytic activity or viceversa. It seems reasonable to suggest that micro-bial production of fibrinogenolytic and fibrino-lytic enzymes is a significant virulence factor.

ACKNOWLEDGMENTSWe thank Arne Tjernberg for sampling of dental alveolus

blood.This work was supported by grants from Goteborgs Tandla-

kare-Sallskap, the Faculty of Odontology, University of Gote-borg, the Swedish Medical Research Council (grant 2789), andthe Swedish Board for Technical Development.

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