detection and preliminary studies on dextranase-producing

INFECTION AND IMMUNITY, Dec. 1973, p. 1009-1016Copyright i 1973 American Society for Microbiology

Vol. 8. No. 6Printed in U.S.A.

Detection and Preliminary Studies on

Dextranase-Producing Microorganisms fromHuman Dental Plaque

ROBERT H. STAAT, THOMAS H. GAWRONSKI, AND CHARLES F. SCHACHTELE

Microbiology Research Laboratories, School of Dentistry, University of Minnesota, Minneapolis, Minnesota55455

Received for publication 26 July 1973

An enriched nutrient agar medium containing blue dextran has been utilizedfor the detection of dextranase-producing microorganisms in human dentalplaque. When compared with the total viable anaerobic plaque flora, theproportion of these microbes in supragingival plaque from different individualsvaried over a wide range. Preliminary characterization of some of the dextranase-producing microorganisms revealed a heterogeneous mixture of cell types withvarying morphological and biochemical characteristics. Several bacterial isolateswere tentatively identified as being members of the genus Actinomyces. Anadditional isolate appeared to belong to the genus Bacteroides. The dextran-degrading enzymes produced by these bacteria are extracellular, and a cell-freepreparation from one of the isolates has been shown to cause extensiveendohydrolytic cleavage of high-molecular-weight dextrans.

Extracellular dextrans (glucans) producedfrom sucrose by microorganisms found in dentalplaques appear to play at least two roles in theformation of dental caries. First, studies withthe bacterium Streptococcus mutans havedemonstrated that the cariogenic potential ofthis microorganism is dependent on the produc-tion of dextrans which can initiate cell aggrega-tion and plaque formation (18-21). Second,glucans have been theorized to be important asa part of the stable intracellular plaque matrix(18, 24, 26). Extracellular plaque dextrans havebeen shown to be both morphologically (26, 37)and chemically (24, 29) heterogeneous, andprevious work indicated that they are not read-ily degraded by oral bacteria (19, 21, 25) ormixed plaque suspensions (14, 46). However,recent studies have strengthened the proposalthat there are indigenous dextranolytic enzymesin plaque which are capable of attacking at leasta portion of these polysaccharides (29, 45).

In this communication we demonstrate thepresence of varying proportions of dextranase-producing microorganisms in human dentalplaque samples and present preliminary dataon the characterization of several such microbesand their dextran-degrading enzymes.

MATERIALS AND METHODSPlaque samples. Human supragingival plaque

samples were obtained from laboratory personnel witha sterile curette. Care was taken to avoid touching thegingival tissues or contaminating the sample withsubgingival plaque. The plaque from several toothsurfaces obtained from a single individual was sus-pended in a reduced transport medium (33).The plaque samples were disrupted by using soni-

cation for two 15-s bursts at 30 W with a BransonW-140E sonicator equipped with a microtip (HeatSystems-Ultrasonics Inc., Plainview, N.Y.). Plaquewas quantitated turbidometrically at a wavelength of600 nm as described previously (17).Enumeration of plaque flora. Sonically treated

plaque samples were diluted in 0.7 M potassiumphosphate buffer (pH 6.8) and plated on tryptic soyagar (TSA) (Difco, Detroit, Mich.) supplementedwith 5% sterile defibrinated sheep blood. Plates wereincubated in an atmosphere of 80% N2, 10% CO2, and10% H2 at 37 C for 72 h.

Detection of dextranase-producing microorga-nisms. Diluted plaque suspensions were plated ontoTSA which contained 0.5% blue dextran 2000 (Phar-macia, Uppsala, Sweden), 0.5% dextran T40 (Phar-macia), 0.2% glucose, and 0.1% yeast extract. Afteranaerobic incubation of the plates as described above,dextranase-producing microorganisms were readilyidentified by the presence of a decolorized zonearound a colony (see Fig. 1). The reliability of this

1009

STAAT, GAWRONSKI, AND SCHACHTELE

technique for detecting dextran hydrolysis was shownby precipitating the dextrans with absolute ethanol asdescribed by Simonson et al. (41). The areas in whichdextrans failed to visibly precipitate were identical tothe decolorized zone, indicating that color reductionwas equivalent to dextran hydrolysis.

Isolation and characterization of microorga-nisms. Dextranase-producing colonies were picked,streaked for isolation on the TSA blood agar medium,and incubated anaerobically at 37 C for 72 h. Eachcolony type was then restreaked on the blue dextranmedium for pure culture isolation of dextranase-pro-ducing microorganisms. The physiological parameterslisted in Table 2 were determined by using the generalprocedures described in the Manual of Microbiologi-cal Methods (13) except that tryptic soy (TS) brothwithout glucose was used as the basal medium for thecarbohydrate fermentation studies. The sulfide, in-dole, and motility tests were determined in SIM agar(Difco). Cellular morphology was determined fromGram-stain smears of actively growing pure cultures.Crude enzyme preparation. Enzymes were pre-

pared by anaerobically growing the microorganisms in500 ml of TS broth supplemented with 0.5% dextranT40 and 0.1% yeast extract for 72 to 96 h at 37 C. Thecultures were chilled to 4 C and then cleared of cellsby centrifugation (8,000 x g, 10 min). The cell-freesupernatant was concentrated by using an Amicon(Amicon Corp., Lexington, Mass.) ultrafiltration sys-tem with a PM10 membrane at a pressure of 50 lb/in2.The concentrated preparation was dialyzed in thecold against distilled water for 12 h, followed bydialysis against 0.01 M acetate buffer (pH 4.5). Thecrude enzyme preparation was clarified by centrifuga-tion (10,000 x g, 10 min) and stored at 4 C. Thedextranolytic activity of these preparations is stablefor at least 2 months under these conditions. Proteinwas determined by the method of Lowry et al. (34).Dextranase assays. Enzyme activity was mea-

sured by monitoring the release of reducing sugarduring incubation of the enzyme and substrate at37 C. A typical reaction mixture contained 1.3 ml of0.1 M sodium citrate buffer (pH 5.65), 0.5 ml ofdextran T40 (20 mg/ml), and 0.2 ml of enzyme.Reducing sugar was measured by using the Nelson-Somogyi method (42) on 0.1-ml samples of the reac-tion mixtures. An alternate dextranase assay involvedhydrolysis of the blue dextran incorporated into thenutrient agar medium discussed above. Enzyme solu-tions were placed in wells cut into the agar andincubation was for 24 h at 37 C. A visible, clear zone

around the well resulted from diffusion of the enzymeand hydrolysis of the blue dextran. An enzymaticallyactive dextranase preparation was required for hy-drolysis (see Fig. 2). The assay is similar to thetechnique used by Ceska (7, 8) for studies on thehydrolysis of blue starch fixed in agar by the enzymea-amylase. Penicillium dextranase (a-1, 6-glucan6-glucanohydrolase, EC 3.2.1.11, Worthington Bio-chemical Corp., Freehold, N.J.) was used in controlexperiments.

Analysis of dextranase reaction products. Thesize of the enzyme reaction products was determinedby gel chromatography by using Bio-Gel P6 (Bio-RadLaboratories, Richmond, Calif.). A 1-ml sample of a

24-h reaction mixture was placed on a column (1.5 by25 cm) which had been prewashed with 0.1 M sodiumacetate (pH 4.5). Carbohydrates were eluted with thesame buffer, and the content of each fraction wasdetermined by using the phenol-sulphuric acid assay(16). Maltose and dextran T40 at a concentration of 1mg/ml were used to calibrate the column.

RESULTSDetection of dextranase-producing micro-

organisms in human dental plaque. We havetaken advantage of the observation originallymade by Mencier (36) that microorganismsfrom soil which produced extracellular enzymeactivity capable of degrading dextrans caused adecolorization of agar media containing bluedextran. Our modification of this medium hasenabled us to isolate and enumerate dextra-nase-producing microorganisms from humandental plaque. A typical dental plaque suspen-sion plated on the differential medium is shownin Fig. 1. Within most of the decolorized zonesone can readily determine the centrally locatedcolony which appears to be responsible fordextranase production.

In order to evaluate the ubiquitousness ofdextranase-producing microorganisms inhuman dental plaques, we obtained plaquesamples from six laboratory employees anddetermined the proportion of these organismsrelative to the total viable anaerobic flora(Table 1). Dextranase-positive organisms werepresent in each subject's plaque, and the quan-tity of these microorganisms varied between0.16 and 3.5%. These variations do not appearto correlate with the microbial density of theindividual plaque samples. The great variationin colony morphology observed among the dex-tranase-producing microorganisms indicatedthat a wide range of microbial types was capa-ble of producing the enzyme. However, for theplaque from any specific individual, one or twoparticular colony types usually predominated.Characterization of microbial isolates. As

indicated above, the plaque microorganismscapable of producing dextranolytic enzymes are

TABLE 1. Enumeration of dextranase-producingmicroorganisms in human dental plaque samples

Viable micro- Dextranase-Plaque sample organisms/mg of producing

plaque (wet wt) colonies (%)

1 5.8 x 108 0.452 1.2 x 108 0.293 4.3 x 107 0.164 5.0 x 101 0.325 8.5 x 107 0.786 5.7 x 10' 3.5

1010 INFECT. IMMUNITY

MICROBIAL DEXTRANASES IN DENTAL PLAQUE

FIG. 1. Visualization of dextranase-producing colonies on enriched blue dextran agar medium. A dilutedhuman dental plaque suspension was spread on the plate and incubation was carried out as described inMaterials and Methods. Note that within each decolorized zone there is usually a centrally located colonywhich can be readily obtained for additional testing.

FIG. 2. Hydrolysis of blue dextran by dextranases. Enzyme solutions (50 jiliters) were placed in wells whichhad been cut out of the blue dextran medium, and the plates were incubated aerobically at 37 C for 24 h.Abbreviations: Gl, G2, G3, cell-free culture supernatants derived from the appropriate plaque isolate; W,commercially available Penicillium dextranase (100 U/ml); K, heat-inactivated (95 C, 20 min) preparation.

heterogeneous and vary depending upon the we were detecting by our blue dextran platingindividual from whom the plaque was obtained. method, we selected four isolates which wereTo gain some insight into the type of microbes readily cultivable. Table 2 presents a summary

VOL. 8, 1973 1011


TABLE 2. Characteristics of some dextranase-producing microorganisms isolated from human dental plaquesamples

IsolateCharacteristic

Gi G2 G3 G6

Cellular morphology Irregular Irregular Irregular Filamentousrods rods rods rods

Gram reaction + + +Oxygen relationship Anaerobe Anaerobe Anaerobe AnaerobeMotility - - - -

Catalase test - - - -

Indole production - - - -

Nitrate reduction - - - -

Gelatin liquefication - - - NDaH2S production - - -

Red blood cell hemolysisb 4 4Carbohydrate fermentation:c

Glucose + + + 4

Mannose + + + 4Maltose + + + 4

Starch 4 4 4 4Dextran (T40) - - - 4Esculin 4- + -

Lactose + 4 4 4

a ND, Not done.° ± denotes weak hemolysis after prolonged incubation (6 days).c + denotes pH 4.0 to 5.0, + denotes pH 5.0 to 6.0, and - denotes pH > 6.0. All of the carbohydrates were

present at a final concentration of 1% except for esculin, which was used at a concentration of 0.025%.

of some characteristics of these microorganisms.Isolates Gl, G2, and G3 were anaerobic, irregu-larly shaped, gram-positive rods. They did notexhibit detectable branching or hemolytic ac-tivity after 3 days of incubation on blood agarplates. In older cultures Y-shaped organismswere observed. The carbohydrate fermentationpatterns of these organisms were similar, al-though none of the microorganisms could pro-duce acid from high-molecular-weight dextran.In spite of the apparent similarities betweenthese organisms, they had markedly differentcolonial morphologies. Gl colonies were rough,"heaped-up," irregularly shaped, and white.Colonies of isolates G2 and G3 were smooth,circular, convex, and white. Isolate G6 was agram-negative, anaerobic, filamentous rod. Onblood agar this organism produced flat, irregu-larly shaped colonies which were pink-brown incolor. Of special interest is the fact that thisisolate could produce acic from high-molecular-weight dextran.

Studies on the dextranases from plaquemicroorganisms. Since the level of enzymeactivity in cell-free culture supernatants ob-tained from glucose-grown cultures of severalisolates was low and the residual reducing sugarcontent of the medium was high, we could notmeasure dextran hydrolysis by the reducingsugar assay. However, the blue dextran agar-

diffusion method was sensitive enough to allowevaluation of enzyme activities in crude enzymepreparations. Figure 2 presents a study in whichthe dextranase activity in cell-free culture su-pernatants from isolates Gl, G2, and G3 wastested along with a commercially availablePenicillium dextranase. It is clear that each ofthe preparations contained dextranolytic activ-ity, and these results confirm that the dextra-nases from the plaque isolates were extracellu-lar. We have not examined these microbes forcell-bound or intracellular dextranase activity.

In order to clearly demonstrate that theactivities being studied actually involved degra-dation of dextran, a concentrated enzyme prep-aration from isolate Gl was incubated withdextran T40, and the increase in reducing sugarwas monitored. The data presented in Fig. 3illustrate that, in comparison with a heat-inac-tivated enzyme preparation (closed circles), theGl enzyme rapidly initiated hydrolysis of thedextran (open circles).Microbial dextranases have been reported

which hydrolyze dextran via an exo-type mech-anism, and the end product is often the disac-charide isomaltose (43). There are also endohy-drolytic dextranases which degrade the sub-strate to oligosaccharides (28). In order toevaluate the type of mechanism by which theGl dextranase hydrolyzed the high-molecular-



0.4

0.3

ocu

o -Ss

D-%.O 0.2

0.1I

0 2 3 4

Time(hours)

FIG. 3. Release of reducing sugar from dextran byan enzyme preparation from isolate Gl. The crudeenzyme and T40 dextran were at a concentration of9.7 and 5.0 mg/ml, respectively. Symbols: 0, activeenzyme; 0, heat-inactivated (95 C, 20 min) enzyme.

weight dextran T40, we chromatographed onBio-Gel P6 a reaction mixture similar to thatobtained from the experiment in Fig. 3 (Fig. 4).There was no detectable intact dextran remain-ing in the reaction mixture and essentially no

saccharides with molecular weights less than1,000. The majority of the carbohydrate was inthe form of oligosaccharides with a molecularweight range of 2,000 to 5,000. This is in closeagreement with the polymer size (molecularweight 3,100) calculated from the reducingsugar ratio for endohydrolytic cleavage of all ofthe T40 dextran as demonstrated in Fig. 4. Theend products of the reaction or the bond speci-ficities of this enzyme have not been furthercharacterized at this time.

DISCUSSIONUse of an enriched agar medium containing

blue dextran has allowed us to demonstrate that

a significant proportion of the anaerobic mi-crobial flora in human dental plaques have thecapability of producing dextran-degrading en-

zymes. The density of dextran-degrading micro-organisms varied from sample to sample (Table1). We have encountered two problems in our

attempts to obtain an exact evaluation of theproportion of these microbes. First, althoughour data on the total viable anaerobic flora arecomparable to the results of others (see 33), theefficiency of plating on TSA-blood and bluedextran plates differs. Total counts on the latterwere usually 20 to 50% of the TSA-blood plates.Thus, the percentages presented in Table 1 maybe low since some dextran-producing microor-ganisms may be among those organisms thatrequire the richer TSA-blood medium. Thesecond problem which we have encountered inattempts to quantitate dextranase producers isthat some colonies which gave clearing of bluedextran on our initial plating are refractory tosubculturing. Such fastidious organisms wouldnot grow on TSA-blood agar or in TS brothwhen removed from the initial plates. Thus, we

were occasionally unable to confirm that a

microorganism which appeared to be a dextra-nase producer was really capable of yieldingsuch activity. In spite of these difficulties, thereis no doubt that the level of dextranase-produc-ing microbes varies greatly in plaque fromdifferent individuals. The highest level we haveobserved is the 3.5% presented in Table 1. Thelowest level of these organisms was found inplaque from a school-age child (less than 0.01%,data not shown).Anaerobic gram-positive rods have been.

shown to make up approximately 20% of thecultivable microorganisms present on the sur-faces of human teeth (22, 23). A signlificant

0.5l

0.4-

0)1

U

c~ 0.3 F-

D0 -3 Vex/ron 740

0.2 Mo/tose

Fraction Number

FIG. 4. Bio-Gel P-6 column chromatography of thereaction products resulting from degradation of dex-tran T40 with a concentrated enzyme preparationfrom isolate Gl. Symbols: 0, intact dextran T40 andmaltose; 0, reaction products.

VOL. 8, 1973 1013


number of these oral "diphtheroides" have beenassigned into the genera Actinomyces, Coryne-bacterium, and Propionibacterium by Rasmus-sen et al. (38). Based on these workers' results,we have tentatively identified isolates Gl, G2,and G3 (Table 2) as belonging to the genusActinomyces. However, the heterogeneity of thecharacteristics of these types of microorganisms(38) will require that metabolic end-productanalysis and antigenic studies be performedbefore a more conclusive identification of ourisolates can be accomplished.Anaerobic gram-negative rods have been

found in considerable quantities in the gingivalcrevice (23) and can be placed into at least fourgenera (Bacteroides, Fusobacterium, Spiril-lum, and Vibrio) as discussed by Loesche andGibbons (32). The data presented in Table 2would indicate placement of isolate G6 in thegenus Bacteroides. However, additional studieson this strain will be necessary for conclusiveidentification. Hehre and Sery (27) studieddextranase-producing anaerobic bacteria fromthe human intestine and found that thesemicrobes constitute an appreciable portion ofthe normal fecal flora. The predominant type ofbacteria capable of producing dextranase inthese studies was placed in the genus Bacte-roides (27).

It is important to emphasize that we havelooked at only a limited number of microbes inplaque which appear capable of producing dex-tranase. Walker (45) has recently presenteddata on two streptococci from plaque whichproduce dextran-degrading enzymes. We havedetected gram-positive cocci among our iso-lates, and we are currently characterizing sev-eral of these bacteria. We have not as yetattempted to utilize dextran in broth to enrichfor microorganisms in plaque which are capableof utilizing this carbohydrate as a carbonsource.The dextranases produced by isolates Gl,

G2, and G3 are extracellular enzymes since theyare excreted from isolated colonies of eachorganism and are found in cell-free culturesupernatants (Fig. 2). Most fungal dextranasesthat have been investigated are extracellular (5,6, 10), although an intracellular fungal dextra-nase has been studied (30). Bifidobacteriumbifidus produces an extracellular dextranase(1-3, 12), and a soil bacterium has been isolatedwhich produces at least two extracellular dex-tranases and some intracellular dextranase ac-tivity (11, 39).Our preliminary studies on the action pattern

of the Gl dextranase indicates that it is anendo-type enzyme resulting in the production ofoligosaccharides from high-molecular-weight

dextran (Fig. 4). The observation that thisisolate will not ferment dextran to acid suggeststhat little, if any, exo-type enzyme activity isproduced under our growth conditions (Table2). The observation that isolate G6 will produceacid from high-molecular-weight dextran indi-cates that this strain may produce exo-typeenzyme activity (Table 2). Interestingly, theonly other bacterium which has been reportedto produce an extracellular exo-type enzymecapable of degrading dextrans also belongs tothe genus Bacteroides (40). Intracellular exo-type dextranase activities have been reported(11, 47), and it has been suggested that the invivo function of such enzymes is to degrade theoligosaccharides formed by extracellular dex-tranases to D-glucose (11).Dextranases (EC 3.2.1.11) have been defined

as enzymes capable of degrading the a-(16)-glucopyranosyl linkage in dextrans (15). Thecommercial dextrans (T40 and blue dextran)used in our studies were derived from nativedextran produced by Leuconostoc mesenter-oides strain B-512. These dextrans containabout 95% a-(1 - 6)-linkages in the primaryand side chains (44). The side chains areconnected through a-(1 - 3)-linkages and aremore than one glucose unit long (31). Bluedextran results from attachment of a polycyclicchromophore to high-molecular-weight dextran(4), and hydrolysis of a-(1 6)-linkages in thisdextran results in release of the dye complex (9,35). Based on these facts, our technique for thedetection of dextranase-producing microbeswould presumably select for microorganismscapable of degrading a-(1 _ 6)-linkages. How-ever, the fact that many microorganisms pro-duce a variety of extracellular glucanohydrolaseactivities (28, 39) indicates that our selectiontechnique may also allow us to detect organismsthat have multiple enzymes with different link-age specificities.Recent studies have emphasized the role of

the water-insoluble, highly branched extracel-lular glucans from S. mutans as a major deter-minant in the cariogenicity of this bacterium(24-26). The large proportion of a-(1 - 3)-link-ages in the S. mutans glucan makes this poly-saccharide resistant to dextranases with a-(16) -linkage specificity. However, Walker (45) haspresented studies which indicate a possible rolefor plaque dextranases with a-(1 - 6)-bondspecificity in the regulation of water-insolubleglucan production by oral streptococci. Thesynthesis of streptococcal glucans containing ahigh proportion of a-(1 - 3)-linkages is sensitiveto the presence of dextranases which are specificfor a-(1 -- 6)-linkages (24, 25, 45). Thus, theproduction of water-insoluble plaque glucans



may be greatly affected by the presence ofdextranases with a-(1 - 6)-linkage specificity.Our findings that there are quantitative andqualitative differences in the microbes capableof producing dextranases in human dentalplaque indicate that the level of indigenousdextranase activity may play a significant rolein the formation, metabolism, and pathogenic-ity of plaque.

ACKNOWLEDGMENTS

We thank Robert W. Oman for his excellent technicalassistance and C. J. Witkop for the use of his laboratoryfacilities.

This work was supported by funds from the University ofMinnesota Graduate School and in part by Public HealthService contract no. NIH-71-2331 and grant DE 03654 fromthe National Institute of Dental Research.

C. F. S. was the recipient of Public Health Service CareerDevelopment Award K4-DE-42,859.

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detection and preliminary studies on dextranase-producing

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