Detection and Preliminary Studies on

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1 INFECTION AND IMMUNITY, Dec. 1973, p Copyright i 1973 American Society for Microbiology Vol. 8. No. 6 Printed in U.S.A. Detection and Preliminary Studies on Dextranase-Producing Microorganisms from Human Dental Plaque ROBERT H. STAAT, THOMAS H. GAWRONSKI, AND CHARLES F. SCHACHTELE Microbiology Research Laboratories, School of Dentistry, University of Minnesota, Minneapolis, Minnesota Received for publication 26 July 1973 An enriched nutrient agar medium containing blue dextran has been utilized for the detection of dextranase-producing microorganisms in human dental plaque. When compared with the total viable anaerobic plaque flora, the proportion of these microbes in supragingival plaque from different individuals varied over a wide range. Preliminary characterization of some of the dextranaseproducing microorganisms revealed a heterogeneous mixture of cell types with varying morphological and biochemical characteristics. Several bacterial isolates were tentatively identified as being members of the genus Actinomyces. An additional isolate appeared to belong to the genus Bacteroides. The dextrandegrading enzymes produced by these bacteria are extracellular, and a cell-free preparation from one of the isolates has been shown to cause extensive endohydrolytic cleavage of high-molecular-weight dextrans. Extracellular dextrans (glucans) produced from sucrose by microorganisms found in dental plaques appear to play at least two roles in the formation of dental caries. First, studies with the bacterium Streptococcus mutans have demonstrated that the cariogenic potential of this microorganism is dependent on the production of dextrans which can initiate cell aggregation and plaque formation (18-21). Second, glucans have been theorized to be important as a part of the stable intracellular plaque matrix (18, 24, 26). Extracellular plaque dextrans have been shown to be both morphologically (26, 37) and chemically (24, 29) heterogeneous, and previous work indicated that they are not readily degraded by oral bacteria (19, 21, 25) or mixed plaque suspensions (14, 46). However, recent studies have strengthened the proposal that there are indigenous dextranolytic enzymes in plaque which are capable of attacking at least a portion of these polysaccharides (29, 45). In this communication we demonstrate the presence of varying proportions of dextranaseproducing microorganisms in human dental plaque samples and present preliminary data on the characterization of several such microbes and their dextran-degrading enzymes. MATERIALS AND METHODS Plaque samples. Human supragingival plaque samples were obtained from laboratory personnel with a sterile curette. Care was taken to avoid touching the gingival tissues or contaminating the sample with subgingival plaque. The plaque from several tooth surfaces obtained from a single individual was suspended in a reduced transport medium (33). The plaque samples were disrupted by using sonication for two 15-s bursts at 30 W with a Branson W-140E sonicator equipped with a microtip (Heat Systems-Ultrasonics Inc., Plainview, N.Y.). Plaque was quantitated turbidometrically at a wavelength of 600 nm as described previously (17). Enumeration of plaque flora. Sonically treated plaque samples were diluted in 0.7 M potassium phosphate buffer (ph 6.8) and plated on tryptic soy agar (TSA) (Difco, Detroit, Mich.) supplemented with 5% sterile defibrinated sheep blood. Plates were incubated in an atmosphere of 80% N2, 10% CO2, and 10% H2 at 37 C for 72 h. Detection of dextranase-producing microorganisms. Diluted plaque suspensions were plated onto TSA which contained 0.5% blue dextran 2000 (Pharmacia, Uppsala, Sweden), 0.5% dextran T40 (Pharmacia), 0.2% glucose, and 0.1% yeast extract. After anaerobic incubation of the plates as described above, dextranase-producing microorganisms were readily identified by the presence of a decolorized zone around a colony (see Fig. 1). The reliability of this 1009

2 1010 STAAT, GAWRONSKI, AND SCHACHTELE INFECT. IMMUNITY technique for detecting dextran hydrolysis was shown by precipitating the dextrans with absolute ethanol as described by Simonson et al. (41). The areas in which dextrans failed to visibly precipitate were identical to the decolorized zone, indicating that color reduction was equivalent to dextran hydrolysis. Isolation and characterization of microorganisms. Dextranase-producing colonies were picked, streaked for isolation on the TSA blood agar medium, and incubated anaerobically at 37 C for 72 h. Each colony type was then restreaked on the blue dextran medium for pure culture isolation of dextranase-producing microorganisms. The physiological parameters listed in Table 2 were determined by using the general procedures described in the Manual of Microbiological Methods (13) except that tryptic soy (TS) broth without glucose was used as the basal medium for the carbohydrate fermentation studies. The sulfide, indole, and motility tests were determined in SIM agar (Difco). Cellular morphology was determined from Gram-stain smears of actively growing pure cultures. Crude enzyme preparation. Enzymes were prepared by anaerobically growing the microorganisms in 500 ml of TS broth supplemented with 0.5% dextran T40 and 0.1% yeast extract for 72 to 96 h at 37 C. The cultures were chilled to 4 C and then cleared of cells by centrifugation (8,000 x g, 10 min). The cell-free supernatant was concentrated by using an Amicon (Amicon Corp., Lexington, Mass.) ultrafiltration system with a PM10 membrane at a pressure of 50 lb/in2. The concentrated preparation was dialyzed in the cold against distilled water for 12 h, followed by dialysis against 0.01 M acetate buffer (ph 4.5). The crude enzyme preparation was clarified by centrifugation (10,000 x g, 10 min) and stored at 4 C. The dextranolytic activity of these preparations is stable for at least 2 months under these conditions. Protein was determined by the method of Lowry et al. (34). Dextranase assays. Enzyme activity was measured by monitoring the release of reducing sugar during incubation of the enzyme and substrate at 37 C. A typical reaction mixture contained 1.3 ml of 0.1 M sodium citrate buffer (ph 5.65), 0.5 ml of dextran 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 reaction mixtures. An alternate dextranase assay involved hydrolysis of the blue dextran incorporated into the nutrient agar medium discussed above. Enzyme solutions were placed in wells cut into the agar and incubation was for 24 h at 37 C. A visible, clear zone around the well resulted from diffusion of the enzyme and hydrolysis of the blue dextran. An enzymatically active dextranase preparation was required for hydrolysis (see Fig. 2). The assay is similar to the technique used by Ceska (7, 8) for studies on the hydrolysis of blue starch fixed in agar by the enzyme a-amylase. Penicillium dextranase (a-1, 6-glucan 6-glucanohydrolase, EC , Worthington Biochemical Corp., Freehold, N.J.) was used in control experiments. Analysis of dextranase reaction products. The size of the enzyme reaction products was determined by gel chromatography by using Bio-Gel P6 (Bio-Rad Laboratories, Richmond, Calif.). A 1-ml sample of a 24-h reaction mixture was placed on a column (1.5 by 25 cm) which had been prewashed with 0.1 M sodium acetate (ph 4.5). Carbohydrates were eluted with the same buffer, and the content of each fraction was determined by using the phenol-sulphuric acid assay (16). Maltose and dextran T40 at a concentration of 1 mg/ml were used to calibrate the column. RESULTS Detection of dextranase-producing microorganisms in human dental plaque. We have taken advantage of the observation originally made by Mencier (36) that microorganisms from soil which produced extracellular enzyme activity capable of degrading dextrans caused a decolorization of agar media containing blue dextran. Our modification of this medium has enabled us to isolate and enumerate dextranase-producing microorganisms from human dental plaque. A typical dental plaque suspension plated on the differential medium is shown in Fig. 1. Within most of the decolorized zones one can readily determine the centrally located colony which appears to be responsible for dextranase production. In order to evaluate the ubiquitousness of dextranase-producing microorganisms in human dental plaques, we obtained plaque samples from six laboratory employees and determined the proportion of these organisms relative to the total viable anaerobic flora (Table 1). Dextranase-positive organisms were present in each subject's plaque, and the quantity of these microorganisms varied between 0.16 and 3.5%. These variations do not appear to correlate with the microbial density of the individual plaque samples. The great variation in colony morphology observed among the dextranase-producing microorganisms indicated that a wide range of microbial types was capable of producing the enzyme. However, for the plaque from any specific individual, one or two particular colony types usually predominated. Characterization of microbial isolates. As indicated above, the plaque microorganisms capable of producing dextranolytic enzymes are TABLE 1. Enumeration of dextranase-producing microorganisms in human dental plaque samples Viable micro- Dextranase- Plaque sample organisms/mg of producing plaque (wet wt) colonies (%) x x x x x x 10' 3.5

3 VOL. 8, 1973 MICROBIAL DEXTRANASES IN DENTAL PLAQUE 1011 Downloaded from FIG. 1. Visualization of dextranase-producing colonies on enriched blue dextran agar medium. A diluted human dental plaque suspension was spread on the plate and incubation was carried out as described in Materials and Methods. Note that within each decolorized zone there is usually a centrally located colony which can be readily obtained for additional testing. FIG. 2. Hydrolysis of blue dextran by dextranases. Enzyme solutions (50 jiliters) were placed in wells which had 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 plating individual from whom the plaque was obtained. method, we selected four isolates which were To gain some insight into the type of microbes readily cultivable. Table 2 presents a summary on September 22, 2018 by guest

4 1012 STAAT, GAWRONSKI, AND SCHACHTELE INFECT. IMMUNITY TABLE 2. Characteristics of some dextranase-producing microorganisms isolated from human dental plaque samples Characteristic Isolate Gi G2 G3 G6 Cellular morphology Irregular Irregular Irregular Filamentous rods rods rods rods Gram reaction Oxygen relationship Anaerobe Anaerobe Anaerobe Anaerobe Motility Catalase test Indole production Nitrate reduction Gelatin liquefication NDa H2S production Red blood cell hemolysisb 4 4 Carbohydrate fermentation:c Glucose Mannose Maltose Starch Dextran (T40) Esculin Lactose 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, irregularly shaped, gram-positive rods. They did not exhibit detectable branching or hemolytic activity after 3 days of incubation on blood agar plates. In older cultures Y-shaped organisms were observed. The carbohydrate fermentation patterns of these organisms were similar, although none of the microorganisms could produce acid from high-molecular-weight dextran. In spite of the apparent similarities between these organisms, they had markedly different colonial 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 a gram-negative, anaerobic, filamentous rod. On blood agar this organism produced flat, irregularly shaped colonies which were pink-brown in color. Of special interest is the fact that this isolate could produce acic from high-molecularweight dextran. Studies on the dextranases from plaque microorganisms. Since the level of enzyme activity in cell-free culture supernatants obtained from glucose-grown cultures of several isolates was low and the residual reducing sugar content of the medium was high, we could not measure dextran hydrolysis by the reducing sugar assay. However, the blue dextran agardiffusion method was sensitive enough to allow evaluation of enzyme activities in crude enzyme preparations. Figure 2 presents a study in which the dextranase activity in cell-free culture supernatants from isolates Gl, G2, and G3 was tested along with a commercially available Penicillium dextranase. It is clear that each of the preparations contained dextranolytic activity, and these results confirm that the dextranases from the plaque isolates were extracellular. We have not examined these microbes for cell-bound or intracellular dextranase activity. In order to clearly demonstrate that the activities being studied actually involved degradation of dextran, a concentrated enzyme preparation from isolate Gl was incubated with dextran T40, and the increase in reducing sugar was monitored. The data presented in Fig. 3 illustrate that, in comparison with a heat-inactivated enzyme preparation (closed circles), the Gl enzyme rapidly initiated hydrolysis of the dextran (open circles). Microbial dextranases have been reported which hydrolyze dextran via an exo-type mechanism, and the end product is often the disaccharide isomaltose (43). There are also endohydrolytic dextranases which degrade the substrate to oligosaccharides (28). In order to evaluate the type of mechanism by which the Gl dextranase hydrolyzed the high-molecular-

5 VOL. 8, 1973 MICROBIAL DEXTRANASES IN DENTAL PLAQUE 1013 ocu o -S s D- %.O I Time (hours) FIG. 3. Release of reducing sugar from dextran by an enzyme preparation from isolate Gl. The crude enzyme and T40 dextran were at a concentration of 9.7 and 5.0 mg/ml, respectively. Symbols: 0, active enzyme; 0, heat-inactivated (95 C, 20 min) enzyme. weight dextran T40, we chromatographed on Bio-Gel P6 a reaction mixture similar to that obtained from the experiment in Fig. 3 (Fig. 4). There was no detectable intact dextran remaining in the reaction mixture and essentially no saccharides with molecular weights less than 1,000. The majority of the carbohydrate was in the form of oligosaccharides with a molecular weight range of 2,000 to 5,000. This is in close agreement with the polymer size (molecular weight 3,100) calculated from the reducing sugar ratio for endohydrolytic cleavage of all of the T40 dextran as demonstrated in Fig. 4. The end products of the reaction or the bond specificities of this enzyme have not been further characterized at this time. DISCUSSION Use of an enriched agar medium containing blue dextran has allowed us to demonstrate that a significant proportion of the anaerobic microbial flora in human dental plaques have the capability of producing dextran-degrading enzymes. The density of dextran-degrading microorganisms varied from sample to sample (Table 1). We have encountered two problems in our attempts to obtain an exact evaluation of the proportion of these microbes. First, although our data on the total viable anaerobic flora are comparable to the results of others (see 33), the efficiency of plating on TSA-blood and blue dextran plates differs. Total counts on the latter were usually 20 to 50% of the TSA-blood plates. Thus, the percentages presented in Table 1 may be low since some dextran-producing microorganisms may be among those organisms that require the richer TSA-blood medium. The second problem which we have encountered in attempts to quantitate dextranase producers is that some colonies which gave clearing of blue dextran on our initial plating are refractory to subculturing. Such fastidious organisms would not grow on TSA-blood agar or in TS broth when removed from the initial plates. Thus, we were occasionally unable to confirm that a microorganism which appeared to be a dextranase producer was really capable of yielding such activity. In spite of these difficulties, there is no doubt that the level of dextranase-producing microbes varies greatly in plaque from different individuals. The highest level we have observed is the 3.5% presented in Table 1. The lowest level of these organisms was found in plaque 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 the cultivable microorganisms present on the surfaces of human teeth (22, 23). A signlificant 0.5l 0.4-0)1 U c~ 0.3 F- D0-3 Vex/ron Mo/tose Fraction Number FIG. 4. Bio-Gel P-6 column chromatography of the reaction products resulting from degradation of dextran T40 with a concentrated enzyme preparation from isolate Gl. Symbols: 0, intact dextran T40 and maltose; 0, reaction products.

6 1014 STAAT, GAWRONSKI, AND SCHACHTELE INFECT. IMMUNITY number of these oral "diphtheroides" have been assigned into the genera Actinomyces, Corynebacterium, and Propionibacterium by Rasmussen et al. (38). Based on these workers' results, we have tentatively identified isolates Gl, G2, and G3 (Table 2) as belonging to the genus Actinomyces. However, the heterogeneity of the characteristics of these types of microorganisms (38) will require that metabolic end-product analysis and antigenic studies be performed before a more conclusive identification of our isolates can be accomplished. Anaerobic gram-negative rods have been found in considerable quantities in the gingival crevice (23) and can be placed into at least four genera (Bacteroides, Fusobacterium, Spirillum, and Vibrio) as discussed by Loesche and Gibbons (32). The data presented in Table 2 would indicate placement of isolate G6 in the genus Bacteroides. However, additional studies on this strain will be necessary for conclusive identification. Hehre and Sery (27) studied dextranase-producing anaerobic bacteria from the human intestine and found that these microbes constitute an appreciable portion of the normal fecal flora. The predominant type of bacteria capable of producing dextranase in these studies was placed in the genus Bacteroides (27). It is important to emphasize that we have looked at only a limited number of microbes in plaque which appear capable of producing dextranase. Walker (45) has recently presented data on two streptococci from plaque which produce dextran-degrading enzymes. We have detected gram-positive cocci among our isolates, and we are currently characterizing several of these bacteria. We have not as yet attempted to utilize dextran in broth to enrich for microorganisms in plaque which are capable of utilizing this carbohydrate as a carbon source. The dextranases produced by isolates Gl, G2, and G3 are extracellular enzymes since they are excreted from isolated colonies of each organism and are found in cell-free culture supernatants (Fig. 2). Most fungal dextranases that have been investigated are extracellular (5, 6, 10), although an intracellular fungal dextranase has been studied (30). Bifidobacterium bifidus produces an extracellular dextranase (1-3, 12), and a soil bacterium has been isolated which produces at least two extracellular dextranases and some intracellular dextranase activity (11, 39). Our preliminary studies on the action pattern of the Gl dextranase indicates that it is an endo-type enzyme resulting in the production of oligosaccharides from high-molecular-weight dextran (Fig. 4). The observation that this isolate will not ferment dextran to acid suggests that little, if any, exo-type enzyme activity is produced under our growth conditions (Table 2). The observation that isolate G6 will produce acid from high-molecular-weight dextran indicates that this strain may produce exo-type enzyme activity (Table 2). Interestingly, the only other bacterium which has been reported to produce an extracellular exo-type enzyme capable of degrading dextrans also belongs to the genus Bacteroides (40). Intracellular exotype dextranase activities have been reported (11, 47), and it has been suggested that the in vivo function of such enzymes is to degrade the oligosaccharides formed by extracellular dextranases to D-glucose (11). Dextranases (EC ) have been defined as enzymes capable of degrading the a-(1 6)-glucopyranosyl linkage in dextrans (15). The commercial dextrans (T40 and blue dextran) used in our studies were derived from native dextran produced by Leuconostoc mesenteroides strain B-512. These dextrans contain about 95% a-(1-6)-linkages in the primary and side chains (44). The side chains are connected through a-(1-3)-linkages and are more than one glucose unit long (31). Blue dextran results from attachment of a polycyclic chromophore to high-molecular-weight dextran (4), and hydrolysis of a-(1 6)-linkages in this dextran results in release of the dye complex (9, 35). Based on these facts, our technique for the detection of dextranase-producing microbes would presumably select for microorganisms capable of degrading a-(1 _ 6)-linkages. However, the fact that many microorganisms produce a variety of extracellular glucanohydrolase activities (28, 39) indicates that our selection technique may also allow us to detect organisms that have multiple enzymes with different linkage specificities. Recent studies have emphasized the role of the water-insoluble, highly branched extracellular glucans from S. mutans as a major determinant in the cariogenicity of this bacterium (24-26). The large proportion of a-(1-3)-linkages in the S. mutans glucan makes this polysaccharide resistant to dextranases with a-(1 6) -linkage specificity. However, Walker (45) has presented studies which indicate a possible role for plaque dextranases with a-(1-6)-bond specificity in the regulation of water-insoluble glucan production by oral streptococci. The synthesis of streptococcal glucans containing a high proportion of a-(1-3)-linkages is sensitive to the presence of dextranases which are specific for a-(1 -- 6)-linkages (24, 25, 45). Thus, the production of water-insoluble plaque glucans

7 VOL. 8, 1973 MICROBIAL DEXTRANASES IN DENTAL PLAQUE 1015 may be greatly affected by the presence of dextranases with a-(1-6)-linkage specificity. Our findings that there are quantitative and qualitative differences in the microbes capable of producing dextranases in human dental plaque indicate that the level of indigenous dextranase activity may play a significant role in the formation, metabolism, and pathogenicity of plaque. ACKNOWLEDGMENTS We thank Robert W. Oman for his excellent technical assistance and C. J. Witkop for the use of his laboratory facilities. This work was supported by funds from the University of Minnesota Graduate School and in part by Public Health Service contract no. NIH and grant DE from the National Institute of Dental Research. C. F. S. was the recipient of Public Health Service Career Development Award K4-DE-42,859. LITERATURE CITED 1. Bailey, R. W., and R. T. J. Clarke A bacterial dextranase. Biochem. J. 72: Bailey, R. W., D. H. Hutson, and H. 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