sanguis from Human Tooth Surfaces - Journal of Clinical Microbiology

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1980, p. 243-249 0095-1137/80/08-0243/07$02.00/0

Vol. 12, No. 2

Isolation and Immunobiological Classification of Streptococcus sanguis from Human Tooth Surfaces SHIGEYUKI HAMADA,'* MITSUO TORII,2 YASUHIKO TSUCHITANI,2 AND SHOZO KOTANI' Department of Oral Microbiology' and Department of Operative Dentistry,2 Osaka University Dental School, Nakanoshima, Kita-ku, Osaka, 530 Japan

A total of 113 pure cultures of Streptococcus sanguis were obtained from dental plaque samples of 64 subjects. Ail isolates synthesized glucan from sucrose, elaborated peroxide, and were alpha-hemolytic. Two biotypes and four serotypes were differentiated within the species. Biotype A (95 isolates) fermented salicin and inulin and hydrolyzed arginine and esculin, whereas biotype B (18 isolates) did not possess these activities. The isolates were serotyped with autoclaved extracts against whole-cell antiserum to strains ATCC 10556 or ST3 (serotype I), ATCC 10557 (serotype II), ATCC 10558 (serotype III), and ST7 (serotype IV), by the capillary precipitin test. Serotypes I, II, III, and IV were found to consist of 24, 16, 37, and 15 isolates. Type IV was demonstrated anew in this study. The remaining 21 isolates were not typed because of either multiple reactions or nonreactivity against the standardized typing sera. Ail isolates of serotype Il belonged to biotype B, which resembles Streptococcus mitior physiologically. Five isolates representing four serotypes and an untypable strain were examined for their cariogenicity against specific-pathogen-free Sprague-Dawley rats fed high sucrose diet no. 2000. Organisms of each isolate were established in the mouths of the rats, but only three isolates induced weak caries that were restricted to pits and fissures of occlusal surfaces of the teeth.

Streptococcus sanguis was first isolated and identified as an independent species by White and Niven (40). Since then, the organism has been isolated frequently from the blood of patients with subacute bacterial endocarditis and has been suspected of being a main causative agent of the disease. It has been demonstrated that S. sanguis constitutes a significant proportion of the streptococcal flora in dental plaque (1, 2). Investigations have indicated that S. sanguis is among the earliest bacteria to colonize human tooth surfaces (3, 38). Furthermore, S. sanguis has been reported to have an affinity for certain salivary glycoproteins (14, 27) and some oral filamentous bacteria (4, 29). In addition, S. sanguis synthesizes glucans from sucrose, as do Streptococcus mutans and certain strains of Streptococcus mitior (2, 11). These results strongly indicated that S. sanguis is involved in dental plaque formation on the tooth surfaces. S. sanguis can be easily separated from S. mutans with respect to fermentation of mannitol and sorbitol, colony morphologies on mitis salivarius agar, and adherence to solid surfaces in the presence of sucrose (2, 11, 32). Although some attempts to subclassify S. sanguis have been reported, a generalized agreement concerning the classification of S. sanguis has not been

reached. More information is required to clarify the immunological and biochemical properties of oral S. sanguis. In the present communication, we attempt to isolate and classify S. sanguis strains from human tooth surfaces. MATERIALS AND METHODS Reference bacterial strains. Reference strains of S. sanguis 10556, 10557, and 10558, and S. mitior (mitis) strains 903, 6249, and 9811 were obtained from the American Type Culture Collection (Rockville, Md.). Other reference strains were kindly provided by the following investigators: S. sanguis/group H Streptococcus 804 (E. Newbrun), M5 (B. Rosan), Challis (M. Yoshioka), 34 (F. C. McIntire), Blackburn and Perryer (J. Eriksen), Channon and K208 (R. R. Facklam), Streptococcus milleri NCTC 10708 and Streptococcus MG ATCC 9895 (S. Edwardson), Streptococcus faecalis SS-499 (R. R. Facklam), Streptococcus bovis 8177 (H. D. Slade), and DS96 (W. G. Iverson), Streptococcus salivarius HT104 and HT129 (own isolates), and S. mitior J5 and HI (own isolates). Reference strains of S. mutans and groups A and E streptococci were selected from our culture collection. These strains were maintained on brain heart infusion (Difco) agar slants at 4°C and transferred biweekly for routine use. They were also lyophilized immediately after their properties were confirmed without repeated laboratory transfers. Isolation of S. sanguis from human teeth. Den243

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tal plaque samples were obtained from each of 47 used for immunization were grown overnight at 37°C subjects, 5 years old, in a nursery located in the in Todd-Hewitt broth (Difco) supplemented with 0.5% suburbs of Osaka, and 17 subjects, aged 17 to 45 years, glucose. Cells were collected by centrifugation and who came to receive treatment for dental caries at washed twice with sterile saline. The cells were resusOsaka University Dental Hospital. Samples were col- pended to give an optical density of 1.0 at a wavelength lected using spoon excavators from dental plaque of of 550 nm in a Shimadzu Bausch-Lomb Spectronic 20 the labial smooth surface of upper incisors in which spectrophotometer. The suspension was made with no detectable dental caries was observed. After these sterile saline containing 0.01% merthiolate and kept at plaque samples were dispersed in 1.0 ml of sterile 4°C until use. Rabbits (ca. 3 kg) were immunized with daily intrasaline with a Tomy model UR-150R ultrasonic oscillator at a maximum amplitude for 30 s, they were venous injections for 6 days of each week for a total of diluted in 10-fold serial steps and inoculated on mitis 4 weeks: 0.5 ml during week 1, 1.0 ml for week 2, and salivarius agar plates (Difco). The plates were aerobi- 2.0 ml for weeks 3 and 4. After 1 week of rest, blood cally incubated at 37°C for 2 days. A few colonies with samples were taken, and the serum was separated and consistency on mitis salivarius agar which were con- kept at -20°C. Standardized serotyping antisera were sidered to resemble S. sanguis (1) were isolated from prepared by adsorbing antisera (1.0 ml) with the whole each subject, and a pure culture of each isolate was cells (20 mg, dry weight) of cross-reacting strains. Adsorption was done by incubating the mixture of obtained by repeated passage on blood agar plates. Cultural and biochemical characterization. antisera and whole cells at 37°C for 1 h, followed by The isolated strains were examined for selected cul- overnight incubation at 4°C. Preparation of antigen. Reference and clinically tural and biochemical characteristics by methods described in previous reports (2, 6, 8). The fermentation isolated strains of S. sanguis were grown in Toddof sorbitol, mannitol, salicin, and inulin was tested Hewitt broth supplemented with 0.5% glucose. The with phenol red broth base (Difco) containing 1% of cells were collected by centrifugation, washed twice the sugar examined. Acid production was recorded with water, and lyophilized. Antigens were extracted after 48 h of incubation at 37°C. Glucan synthesis was from lyophilized cells. Cells (20 mg/ml of saline) were tested in Trypticase soy broth (BBL Microbiology autoclaved at 120°C for 20 min. After centrifugation, Systems) containing 5% sucrose. After 48 h of incu- the cell-free supernatant was employed as an antigen, bation at 37°C, cultures were centrifuged and an equal as described previously (18). Immunochemical reactions. Capillary precipitin volume of ethanol was added to the centrifuged supernatant. Formation of a white precipitate or turbid- tests were carried out by the procedure of Lancefield ity indicated the positive production of glucan. How- (26), and precipitin reactions were graded from - to ever, certain strains produced viscous cultures when 4+. Immunodiffusion was performed in the usual mangrown as described above. This property was recorded ner with 1% Noble agar (Difco) in 0.05 M barbitalhydrochloride buffer (pH 8.2). as gel formation. Caries induction in rats by selected strains of Hydrolysis (deamination) of L-arginine was carried out by the description of Niven et al. (30). Hydrolysis S. sanguis. Clinically isolated strains ST3, ST102, of esculin was tested with a broth containing 1% Tryp- ST183, ST7, and ST160, representing different proticase peptone (BBL Microbiology Systems), 0.5% posed serotypes (see below) were made streptomycin yeast extract (BBL Microbiology Systems), 0.05% fer- resistant (1.5 mg/ml of culture medium) to facilitate ric citrate, 0.5% NaCI, and 0.1% esculin, pH 6.5 (8). recovery and identification of the implanted orgaBlackening of the culture medium after incubation for nisms. The suffix R referred to streptomycin-resistant 7 days at 37°C was defined as a positive hydrolysis of strains. The caries test employed was essentially the esculin. Hemolytic activity of each isolate was exam- same as that reported in a previous paper (17). The ined after culturing it on a Trypticase soy agar plate protocol for the animal experiment is summarized in containing 5% (vol/vol) defibrinated sheep blood for Fig. 1. Briefly, specific-pathogen-free Sprague-Dawley 2 days at 37°C in air. Peroxide formation was detected rats free of microbiologically detectable S. sanguis on blood agar plates containing 0.1% benzidine dihy- and S. mutans were purchased from Clea Japan Ltd. drochloride incubated aerobically (25). Peroxide pro- (Osaka), and were infected repeatedly with a test strain (ca. 10" cells) after the indigenous flora of the ducers developed a black halo around the colonies. was suppressed by administration of penicillin G Preparation of typing antisera. Antisera were rats mg/ml of drinking water) and tetracycline (4 mg/ prepared in rabbits with merthiolate-killed cell sus- g(2.5 diet) for 3 days. Rats were fed caries-inducing diet pensions of S. sanguis. Strains ATCC 10556 (serotype no.of2000 and sterile water ad libitum from 19 days 1), ATCC 10557 (serotype 11), ATCC 10558 (serotype of age to(23) the end of the 3-month experiment (17). III), and clinically isolated strains ST3 and ST7 were used as immunogens. Strains 10556, 10557, and 10558 Caries scores were assessed by the method of Keyes were originally designated as serotypes 1, II, and I/II, (22). respectively (39). In this paper, serotype I/Il was RESULTS redesignated as an independent serotype III. PrelimiBiological properties of clinical isolates nary experiments revealed that immunological specificity of strain ST3 was essentially identical with that of S. sanguis. A total of 113 pure cultures were of strain 10556, and strain ST7 possessed a unique obtained from dental plaque samples of 64 subantigen that was different from serotype 1, II, and III jects with mitis salivarius agar plates. As sumantigens and was defined as serotype IV. Organisms marized in Table 1, all the isolates produced

TYPING OF HUMAN ISOLATES OF S. SANGUIS

VOL. 12, 1980 Age of rat

0

[Experiment]

îI

Conventional powdered diet I

Breast

feeding

k

Treatment

I

.

[9]

[°]

Di et

105 -10D9

28

18 19

15

245

day [86-'90]

Cariogenic diet #2000

t1 t1 t t

t t1 t t1

Inoculation of test strain

Antibiotics

FIG. 1. Timetable for testing cariogenicity of representative isolates of S. sanguis with specific-pathogenfree Sprague-Dawley rats. TABLE 1. Biological properties of clinical isolates of S. sanguis from human tooth surfaces Hydrolysis Fermentation of: Glucan Hemolysison No. of H202 sheep blood producisolates tiorma Biotype from Esculin tion agar Salicin Inulin Marul Sorbitol Argexamined tion sucrose

A B

95 18

+ +

Alpha Alpha

+ +

glucan from sucrose, and 31 isolates increased the viscosity of the sucrose-containing broth, forming a visible gel. All isolates also elaborated hydrogen peroxide when grown aerobically and were alpha-hemolytic in sheep blood agar plates. However, these isolates were differentiated into two groups in terms of their abiity to ferment salicin and inulin and to hydrolyze arginine and esculin. Ninety-five isolates fermented salicin and inulin and were designated as biotype A. The rest of the isolates tested (18 isolates) were found to lack these abilities and were designated as biotype B. Among biotype A isolates, 13 isolates fermented sorbitol and 3 fermented mannitol. Only one isolate fermented both sorbitol and mannitol. No biotype B isolates fermented sorbitol or mannitol. In this connection, most reference strains received as S. sanguis or group H Streptococcus except strains ATCC 10557, and 34 were found to belong to biotype A. Strains of S. mitior were defined as biotype B. Immunological properties of clinical isolates of S. sanguis. Antisera against reference strains 10557 and 10558 were found to produce a heavy precipitate with homologous antigens. These antisera were used as typing sera for serotype II and III, respectively. Antisera prepared against serotype I reference strain 10556 were not suitable for the purpose of serotyping, because they produced only a slight precipitate with the homologous antigen. However, a clinical isolate ST3 was found to possess a very similar antigenic specificity, as depicted in Fig. 2. ST3 cells were therefore used to immunize rabbits for serotype I specificity. An ST3 antiserum which produced a heavy precipitate with ST3 and 10556 antigens was obtained. The antiST3 serum was used as type I antiserum.

+

+

ST3

toi -

mne

-

10556

+

+

ST3

o o o ANTI--ST

ANrI-10556 FIG. 2. Immunological identity between S. sanguis strains ATCC 10556 and ST3, as demonstrated by immunodiffusion test. Upper wells: autoclaved antigen extracts from strains ST3 and ATCC 10556. Lower wells: antiserum against whole cells of S. sanguis ST3 and ATCC 10556.

Although these antisera showed marked positive capillary precipitin reactions with antigens of homologous ceils, they also reacted with reference antigens of other serotypes of S. sanguis. Therefore, each antiserum was adsorbed with whole cells of cross-reacting strain(s) (Table 2). The immunological specificities of the serotype antigens were also demonstrated by immunodiffusion tests (Fig. 3). Serotype I and II antisera adsorbed with whole cells of serotype III strain Challis no longer reacted with antigens of heterologous serotypes. Serotype III antiserum adsorbed with ST3 cells of serotype I reacted strongly with homologous serotype III strains, although it continued to show positive precipitin reactions with other serotype I strains, 10556 and 804. Therefore, serotype III strains were defined as those exclusively reactive with anti10558 which had been adsorbed with ST3 cells. On the other hand, serotype I strains were reactive with both anti-ST3 and anti-10558 sera adsorbed with Challis and ST3 cells, respec-

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HAMADA ET AL.

TABLE 2. Preparation of typing antiserum adsorbed with whole cells of cross-reacting strains and the serological specificity by capillary precipitin reactions Typing antiseruma

AniIAt-lAntiAntiI AntiII Ann III

Antigen

AntiIV

S. sanguis ST3 (I)b 2+c ± 2+ 2+ 10556 (I) 3+ 804 (I) 2+ ± + + 3+ 10557 (II) 10558 (III) 3+ M5 (III) 3+ Challis (III) 3+ + ST7 (IV) 3+ ST6 (IV) + S. mitior 903 2+ 6249 9811 J5 Hl Anti-I, Anti-ST3 adsorbed with cells of strain Challis; Anti-II, anti-10557 adsorbed with Challis cells; Anti-III, anti-10558 adsorbed with ST3 cells (adsorption with either 10556 or 804 cells resulted in complete loss of precipitin activity against the homologous antigen); anti-IV, anti-ST7 adsorbed with cells of ST3 and 10558. 8Serotype of S. sanguis. 105570 ST7

o Challis FIG. 3.

ST7

J. CLIN. M1CROBIOL.

Q

ST7

ST70

Precipitin bands in

0804

10557

d.

O0558 ~~~Challis O

OOM5ST7

oST3

O 10558

0 o

\'

010558 /

o

ST3

ST6

agar

gel immunodif-

fusion tests. Peripheral wells contain an antigen extract from each strain described. Center wells contain typing antiserum of serotype I (a), II (b), III (c), and IV (d). Adsorption of each antiserum was carried out as shown in Table 3.

Cariogenicity of selected clinical isolates. Three strains, ST183R, ST7R, and ST160R, out of five clinical isolates significantly induced pitand-fissure-type caries in specific-pathogen-free Sprague-Dawley rats as compared with uninoculated control rats (Table 4, Fig. 4). No smooth surface caries were observed in all rats of the test groups. No significant increase in dental caries developed in rats inoculated with strains ST3R and ST102R. Dental plaque accumulated slightly in the fissures of the occlusal surface, but not on smooth surfaces of rat molars.

DISCUSSION S. sanguis has been classified by several investigators based on the immunological, biological, or genetic specificities. Two serotypes (I and II) were first proposed with 42 isolates from human blood with subacute bacterial endocarditis (39). However, a significant number of strains were found to possess both I and II antigenic specificities and were designated as not react with standardized typing antisera (Ta- serotype I/II (39). ble 2). It has been suggested that many dextran-proAs tabulated in Table 3, 24, 16, 37, and 15 ducing strains isolated from patients with endoisolates out of 113 isolates tested were classified carditis reacted with antiserum specific for group as serotype 1, II, III, and IV, respectively. The H streptococci (6, 12, 19, 33, 34), although the remaining 21 isolates were not typed because of definition of group H antigen is uncertain as to either multiple or nonreactivity against the stan- being synonymous with that of S. sanguis (5, dardized typing sera described above. 37). Rosan (35) has reported that S. sanguis was It was also elucidated that ail isolates of se- separated into two major serotypes and a hetrotype Il belonged to biotype B, whereas others, erogeneous group based on immunoelectrophoincluding serotypes 1, II, III, and nontypable retic analysis. Subsequently, a glycerol teichoic ones belonged to biotype A. acid antigen was strongly suggested to be Lance-

tively, by the capillary precipitin tests (Table 2). Some clinical isolates were found to be reactive with none of the typing antisera. Anti-ST7 serum adsorbed with ST3 and 10558 ceils was used as serotype IV antiserum. The isolates whose antigen extracts were reactive with only this antiserum were classified anew as serotype IV. Seventeen antigens from various streptococcal species other than S. sanguis/S. mitior did


VOL. 12, 1980

247

TABLE 3. Serotypes and biologicalproperties of clinical isolates of S. sanguis No. of isolates exhibiting: No. of isolates

Serotype

I II IV

24 16 37 15

Untypable Multireactive Nonreactive

12 9

III

Fermentation of: Sorbitol 1 0

ManniSalicin toi

Hydrolysis of: Inulin

Argifine

GeilEscu- forma- mg

il 0

1 0 3 0

23 0 35 15

20 0 33 13

24 0 37 15

lin 20 0 32 15

2 0

0 0

il 7

12 8

12 8

12 7

TABLE 4. Caries induction in specific-pathogen-free Sprague-Dawley rats infected with isolates of S. sanguis

Glucan

Alphahmheol

H202

pro

tion 24 16 37 15

7 0 14 1

24 16 37 15

duction 4 16 37 15

12 9

8 1

12 9

12 9

anti-10558 (serotype III) serum was adsorbed with either 10556 or 804 (serotype 1) cells (data not shown, cf. Table 2). No special attention has Caries score No. of Period been paid to the relatedness with "group H" Strain rats (days) (mean ± SE)a specificity, but group H strains Blackburn, 8 90 6.8 ± 0.8 Noninfected Channon, and Perryer were found to belong to 90 8.0 + 2.2 8 ST3R (I/A)b serotype III, III, and I, respectively, accordour 8 90 9.3 + 1.9 ST102R (II/B) ing to our typing scheme (data not shown). In 86 12.5 ± 1.4c 8 ST183R (III/A) this connection, "American group H" strain 8 90 16.7 ± 3.5d ST7R (IV/A) 12.2 ± 1.8d 8 86 K208 (5, 36) was not reactive with any of our ST160R (?/A) typing antisera. a Mean ± standard error (SE). Based on their abilities to ferment various b Serotype/biotype of S. sanguis. c Statistical significance (Student's t test) between sugars, including salicin and inulin, and to hycontrol and the test groups, P < 0.01. drolyze arginine and esculin, clinical isolates of d Statistical significance, P < 0.05. S. sanguis were classified into two biotypes, A and B. According to the original description of S. sanguis by Niven et al. (31), biotype A, which ,.Y can split arginine and esculin and synthesize glucan from sucrose, may be typical S. sanguis. w N~~~~~~~~!. Glucan-producing S. mitior strain ATCC 9811 was found to be very similar to biotype B S. iwe sanguis ATCC 10557, as was reported previously by Coykendall and Specht (8). Among biotype A S. sanguis, 17 isolates did not adhere significantly to glass surfaces in sucrose broth unlike S. mutans. No antigen extracts of the isolates FIG. 4. Fissure caries lesions typical of those ob- reacted with antisera against 7 recognized seroserved in SPF rats infected with S. sanguis strain types including serotype b of S. mutans by imST7. munodiffusion test. Related to this, only serotype b S. mutans has been found to deaminate field group H antigen (36). In our hands, however, immunoelectropho- arginine (32), and no serotype b S. mutans has retic patterns of reference antigens against an- been isolated from Japanese children (16, 28). tiserum prepared for S. sanguis M5 were differ- Furthermore, Streptococcus bovis, another gluent from those of Rosan (35, 36). Therefore, we can-forming streptococcal species, does not hyessentially followed the serotyping scheme orig- drolyze arginine (9). These results indicate that inally proposed by Washburn et al. (39). Their these 17 isolates can be identified as S. sanguis type I/II was considered as an independent se- irrespective of their abberant sugar fermentation rotype III in this study, although a close relation abilities. We demonstrated the presence of a new sebetween serotype I and III was demonstrated by the fact that no precipitin activity remained rotype IV as well as known serotypes I, II, and III in our clinical isolates of S. sanguis (Table even with homologous serotype III antigens if ....

7k:~~~~~~~~~~~~* e w!!l

...,

'71,

%

248

J. CLIN. MICROBIOL.

HAMADA ET AL.

3). Serotype III, I, Il, and IV consisted of 33, 21, 14, and 13% of the total clinical isolates examined in the order of prevalence, and 19% of the isolates could not be classified serologicaily. The proportion of serotype III was significantly higher than any other serotypes. It was also found that most reference strains of S. sanguis were defined as serotype III. On the other hand, Forsum and Holmberg (13) reported that by using fluorescent-antibody technique serotype I was observed most frequently in smear preparation of human dental plaque and serotype III was not found at ail, which is different from the present result. Carlsson (2) also did not find serotype III in his isolates in Sweden, where serotype II may not exist. Of 21 untypable isolates, 12 were reactive with plural typing antisera, indicating the antigenic complexity of these isolates. In contrast, nine isolates were found to be nonreactive with any of the typing antisera through serotypes I to IV. In this connection, it was found that a precipitin band was formed between our serotype IV antiserum (Table 2) and autoclaved antigen extract of recently proposed group W Streptococcus (20) strain 10043. However, the precipitin band had no immunological relatedness with that formed between homologous type IV antigen and antiserum (data not shown). These results indicate that the immunological specificity of serotype IV antigen is different from that of group W Streptococcus, although group W strains have been reported to possess biological properties similar to those of S. sanguis (20, 21). It is noted that serotype Il included our biotype B and Rosan's heterogenous strains (35). Coykendall and Specht (8) classified S. sanguis mainly based on deoxyribonucleic acid base composition and deoxyribonucleic acid-deoxyri-

bonucleic acid homologies. They demonstrated that strains fermenting salicin and inulin and hydrolyzing arginine and esculin (biotype A, Table 1) were separated into two genospecies, which coincided with serotype I and III, whereas strains not fermenting or hydrolyzing the above agents that were represented by strain 10557 included biotype B/serotype Il strains in our system. Henriksen and Henrichsen (21) also reported that serotype Il antigens differed from serotype I and III (their serotype I/Il), and only the homologous strains reacted with anti-type II serum. It is more likely that biotype B/serotype II S. sanguis resembles glucan-producing S. mitior with respect to its biochemical and genetic properties (7, 8). However, Carlsson (2), Facklam (11), and we ourselves have classified biotype B/serotype Il strains as S. sanguis, setting a high value on the ability of glucan synthesis from sucrose by these strains. For comparison, relationships among various typing schemes reported by several investigators are summarized in Table 5. S. sanguis has been established to be a predominant streptococcal species in human dental plaque flora (2), although it was originally isolated from the blood of patients with subacute bacterial endocarditis (40). Several studies have examined the etiological role of S. sanguis strains in dental caries development. S. sanguis strains 10556 and Hockley (I. L. Shklair, S. Hoffman, and H. D. Tow, Abstr. Int. Assoc. Dent. Res., p. 54, 1970, Abstr. no. 24) and strain OMZ9 were demonstrated to be cariogenic in rats whose normal flora had been suppressed by antibiotics throughout the experimental period (15). In general, our present study also showed that the cariogenicity of S. sanguis isolates was

TABLE 5. Comparison of proposed classification schemes of S. sanguis and closely related species of streptococci Designation Reference

Grouping

Serotype

1, biotype Serotype II, biotype B Aa ~ ~

39

12, 21, 33

Serotype Serotype

I I H I:B 3

II II

Serotype III, bio-

Serotype IV biotype A

~ ~~~ye typeA I/Il

I/II Lancefield group H 2 Biotype I:A 8 Genotype 2 1 35 2 Serotype Heterogeneous 2 il I Biotype I II a Subheadings are classification scheme used in this paper. h Some strains may belong to group W proposed by Henriksen and Eriksen (20).

Serotype t biotype A

_b

VOL. 12, 1980


not significant, which is in sharp contrast to the cariogenicity of S. mutans ( 17, 24). ACKNOWLEDGMENT We thank Rosemary Linzer (State University of New York at Buffalo) for assistance with the English version of the manuscript.

LITERATURE CITED 1. Carlsson, J. 1965. Zooglea-forming streptococci,

2.

3. 4.

5.

6.

7.

8. 9.

10.

resem-

bling Streptococcus sanguis, isolated from dental plaque in man. Odontol. Revy 16:348-358. Carlsson, J. 1968. A numerical taxonomic study of human oral streptococci. Odontol. Revy 19:137-160. Carlsson, J., H. Grahnen, C. Jonsson, and S. Winkler. 1970. Establishment of Streptococcus sanguis in the mouths of infants. Arch. Oral Biol. 15:1143-1148. Cisar, J. O., P. E. Kolenbrander, and F. C. MeIntire. 1979. Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii. Infect. Immun. 24: 742-752. Cole, R. M., G. B. Calandra, E. Huff, and K. M. Nugent. 1976. Attributes of potential utility in differentiating among "group H" streptococci or Streptococcus sanguis. J. Dent. Res. 55:A142-A153. Colman, G., and R. E. 0. Williams. 1972. Taxonomy of some human viridans streptococci, p. 281-299. In L. W. Wannamaker, and J. M. Matson (ed.), Streptococci and streptococcal disease. Academic Press Inc., New York. Coykendall, A. L., and A. J. Munzenmaier. 1978. Deoxyribonucleic acid base sequence studies on glucanproducing and glucan-negative strains of Streptococcus mitior. Int. J. Syst. Bacteriol. 28:511-515. Coykendall, A. L., and P. A. Specht. 1975. DNA base sequence homologies among strains of Streptococcus sanguis. J. Gen. Microbiol. 91:92-98. Deibel, R. H. 1964. The group D streptococci. Bacteriol. Rev. 28:330-366. Ellen, R. P., and I. B. Balcerzak-Raczkowski. 1977. Interbacterial agregation of Actinomyces naeslundii and dental plaque streptococci. J. Periodont. Res. 12:

11-20. 11. Facklam, R. R. 1977. Physiological differentiation of viridans streptococci. J. Clin. Microbiol. 5:184-201. 12. Farmer, F. D. 1954. Serological subdivisions among the Lancefield group H streptococci. J. Gen. Microbiol. 11: 131-138. 13. Forsum, U., and K. Holmberg. 1974. Identification of Streptococcus sanguis by defined immunofluorescence. Caries Res. 8:105-112. 14. Gibbons, R. J., and J. van Houte. 1973. On the formation of dental plaques. J. Periodontol. 44:347-360. 15. Guggenheim, B. 1968. Streptococci of dental plaques. Caries Res. 2:147-163. 16. Hamada, S., N. Masuda, T. Ooshima, S. Sobue, and S. Kotani. 1976. Epidemiological survey of Streptococcus mutans among Japanese children. Identification and serological typing of the isolated strains. Jpn. J. Microbiol. 20:33-44. 17. Hamada, S., T. Ooshima, M. Torii, H. Imanishi, N. Masuda, S. Sobue, and S. Kotani. 1978. Dental caries induction in experimental animal by clinical strains of Streptococcus mutans isolated from Japanese children. Microbiol. Immunol. 22:301-314. 18. Hamada, S., S. Tai, and H. D. Slade. 1976. Selective

adsorption of heterophile polyglycerophosphate antigen from antigen extracts of Streptococcus mutans and other gram-positive bacteria. Infect. Immun. 14:903910.

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